A versatile modular bioreactor platform for Tissue Engineering.

PubMed

Schuerlein, Sebastian; Schwarz, Thomas; Krziminski, Steffan; Gätzner, Sabine; Hoppensack, Anke; Schwedhelm, Ivo; Schweinlin, Matthias; Walles, Heike; Hansmann, Jan

2017-02-01

Tissue Engineering (TE) bears potential to overcome the persistent shortage of donor organs in transplantation medicine. Additionally, TE products are applied as human test systems in pharmaceutical research to close the gap between animal testing and the administration of drugs to human subjects in clinical trials. However, generating a tissue requires complex culture conditions provided by bioreactors. Currently, the translation of TE technologies into clinical and industrial applications is limited due to a wide range of different tissue-specific, non-disposable bioreactor systems. To ensure a high level of standardization, a suitable cost-effectiveness, and a safe graft production, a generic modular bioreactor platform was developed. Functional modules provide robust control of culture processes, e.g. medium transport, gas exchange, heating, or trapping of floating air bubbles. Characterization revealed improved performance of the modules in comparison to traditional cell culture equipment such as incubators, or peristaltic pumps. By combining the modules, a broad range of culture conditions can be achieved. The novel bioreactor platform allows using disposable components and facilitates tissue culture in closed fluidic systems. By sustaining native carotid arteries, engineering a blood vessel, and generating intestinal tissue models according to a previously published protocol the feasibility and performance of the bioreactor platform was demonstrated. © 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recent Advances in Cartilage Tissue Engineering: From the Choice of Cell Sources to the Use of Bioreactors

NASA Astrophysics Data System (ADS)

Martin, Ivan; Démarteau, Olivier; Braccini, Alessandra

Grafting engineered cartilage tissues represents a promising approach for the repair of joint injuries. Recent animal experiments have demonstrated that tissues engineered by culturing chondrocytes on 3D scaffolds in bioreactors provide functional templates for orderly repair of large osteochondral lesions. To date, however, a reproducible generation of uniform cartilage tissues of predefined size starting from adult human cells has not been achieved. In this paper we review some of the recent advances and challenges ahead in the identification of appropriate (i) cell sources, (ii) bioactive factors, (iii) 3D scaffolds and (iv) bioreactors for human cartilage tissue engineering. We also present an example of how integrated efforts in these different areas can help addressing fundamental questions and advancing the field of cartilage tissue engineering towards clinical use. The presented experiment demonstrates that human nasal chondrocytes are responsive to dynamic loading and thus could be further investigated as a cell source for implantation in a joint environment.

Development of a Bioreactor to Culture Tissue Engineered Ureters Based on the Application of Tubular OPTIMAIX 3D Scaffolds.

PubMed

Seifarth, Volker; Gossmann, Matthias; Janke, Heinz Peter; Grosse, Joachim O; Becker, Christoph; Heschel, Ingo; Artmann, Gerhard M; Temiz Artmann, Aysegül

2015-01-01

Regenerative medicine, tissue engineering and biomedical research give hope to many patients who need bio-implants. Tissue engineering applications have already been developed based on bioreactors. Physiological ureter implants, however, do not still function sufficiently, as they represent tubular hollow structures with very specific cellular structures and alignments consisting of several cell types. The aim of this study was to a develop a new bioreactor system based on seamless, collagenous, tubular OPTIMAIX 3D prototype sponge as scaffold material for ex-vivo culturing of a tissue engineered ureter replacement for future urological applications. Particular emphasis was given to a great extent to mimic the physiological environment similar to the in vivo situation of a ureter. NIH-3T3 fibroblasts, C2C12, Urotsa and primary genitourinary tract cells were applied as co-cultures on the scaffold and the penetration of cells into the collagenous material was followed. By the end of this study, the bioreactor was functioning, physiological parameter as temperature and pH and the newly developed BIOREACTOR system is applicable to tubular scaffold materials with different lengths and diameters. The automatized incubation system worked reliably. The tubular OPTIMAIX 3D sponge was a suitable scaffold material for tissue engineering purposes and co-cultivation procedures. © 2015 S. Karger AG, Basel.

Dynamic Bioreactor Culture of High Volume Engineered Bone Tissue

PubMed Central

Nguyen, Bao-Ngoc B.; Ko, Henry; Moriarty, Rebecca A.; Etheridge, Julie M.

2016-01-01

Within the field of tissue engineering and regenerative medicine, the fabrication of tissue grafts of any significant size—much less a whole organ or tissue—remains a major challenge. Currently, tissue-engineered constructs cultured in vitro have been restrained in size primarily due to the diffusion limit of oxygen and nutrients to the center of these grafts. Previously, we developed a novel tubular perfusion system (TPS) bioreactor, which allows the dynamic culture of bead-encapsulated cells and increases the supply of nutrients to the entire cell population. More interestingly, the versatility of TPS bioreactor allows a large range of engineered tissue volumes to be cultured, including large bone grafts. In this study, we utilized alginate-encapsulated human mesenchymal stem cells for the culture of a tissue-engineered bone construct in the size and shape of the superior half of an adult human femur (∼200 cm3), a 20-fold increase over previously reported volumes of in vitro engineered bone grafts. Dynamic culture in TPS bioreactor not only resulted in high cell viability throughout the femur graft, but also showed early signs of stem cell differentiation through increased expression of osteogenic genes and proteins, consistent with our previous models of smaller bone constructs. This first foray into full-scale bone engineering provides the foundation for future clinical applications of bioengineered bone grafts. PMID:26653703

A versatile modular bioreactor platform for Tissue Engineering

PubMed Central

Schuerlein, Sebastian; Schwarz, Thomas; Krziminski, Steffan; Gätzner, Sabine; Hoppensack, Anke; Schwedhelm, Ivo; Schweinlin, Matthias; Walles, Heike

2016-01-01

Abstract Tissue Engineering (TE) bears potential to overcome the persistent shortage of donor organs in transplantation medicine. Additionally, TE products are applied as human test systems in pharmaceutical research to close the gap between animal testing and the administration of drugs to human subjects in clinical trials. However, generating a tissue requires complex culture conditions provided by bioreactors. Currently, the translation of TE technologies into clinical and industrial applications is limited due to a wide range of different tissue‐specific, non‐disposable bioreactor systems. To ensure a high level of standardization, a suitable cost‐effectiveness, and a safe graft production, a generic modular bioreactor platform was developed. Functional modules provide robust control of culture processes, e.g. medium transport, gas exchange, heating, or trapping of floating air bubbles. Characterization revealed improved performance of the modules in comparison to traditional cell culture equipment such as incubators, or peristaltic pumps. By combining the modules, a broad range of culture conditions can be achieved. The novel bioreactor platform allows using disposable components and facilitates tissue culture in closed fluidic systems. By sustaining native carotid arteries, engineering a blood vessel, and generating intestinal tissue models according to a previously published protocol the feasibility and performance of the bioreactor platform was demonstrated. PMID:27492568

Computational study of culture conditions and nutrient supply in a hollow membrane sheet bioreactor for large-scale bone tissue engineering.

PubMed

Khademi, Ramin; Mohebbi-Kalhori, Davod; Hadjizadeh, Afra

2014-03-01

Successful bone tissue culture in a large implant is still a challenge. We have previously developed a porous hollow membrane sheet (HMSh) for tissue engineering applications (Afra Hadjizadeh and Davod Mohebbi-Kalhori, J Biomed. Mater. Res. Part A [2]). This study aims to investigate culture conditions and nutrient supply in a bioreactor made of HMSh. For this purpose, hydrodynamic and mass transport behavior in the newly proposed hollow membrane sheet bioreactor including a lumen region and porous membrane (scaffold) for supporting and feeding cells with a grooved section for accommodating gel-cell matrix was numerically studied. A finite element method was used for solving the governing equations in both homogenous and porous media. Furthermore, the cell resistance and waste production have been included in a 3D mathematical model. The influences of different bioreactor design parameters and the scaffold properties which determine the HMSh bioreactor performance and various operating conditions were discussed in detail. The obtained results illustrated that the novel scaffold can be employed in the large-scale applications in bone tissue engineering.

Designing a 'neotissue' using the principles of biology, chemistry and engineering.

PubMed

Nannaparaju, Madhusudhan; Oragui, Emeka; Khan, Wasim S

2012-01-01

The traditional methods of treating musculoskeletal injuries and disorders are not completely effective and have several limitations. Tissue engineering involves using the principles of biology, chemistry and engineering to design a 'neotissue' that augments a malfunctioning in vivo tissue. The main requirements for functional engineered tissue include reparative cellular components that proliferate on a scaffold grown within a bioreactor that provides specific biochemical and physical signals to regulate cell differentiation and tissue assembly. In this review we provide an overview of the biology of common musculoskeletal tissue and discuss their common pathologies. We also describe the commonly used stem cells, scaffolds and bioreactors and evaluate their role in issue engineering.

Multifunctional Bioreactor System for Human Intestine Tissues

PubMed Central

2017-01-01

The three-dimensional (3D) cultivation of intestinal cells and tissues in dynamic bioreactor systems to represent in vivo intestinal microenvironments is essential for developing regenerative medicine treatments for intestinal diseases. We have previously developed in vitro human intestinal tissue systems using a 3D porous silk scaffold system with intestinal architectures and topographical features for the adhesion, growth, and differentiation of intestinal cells under static culture conditions. In this study, we designed and fabricated a multifunctional bioreactor system that incorporates pre-epithelialized 3D silk scaffolds in a dynamic culture environment for in vitro engineering of human intestine tissues. The bioreactor system allows for control of oxygen levels in perfusion fluids (aerobic simulated intestinal fluid (SIF), microaerobic SIF, and anaerobic SIF), while ensuring control over the mechanical and chemical microenvironments present in native human intestines. The bioreactor system also enables 3D cell culture with spatial separation and cultivation of cocultured epithelial and stromal cells. Preliminary functional analysis of tissues housed in the bioreactor demonstrated that the 3D tissue constructs survived and maintained typical phenotypes of intestinal epithelium, including epithelial tight junction formation, intestinal biomarker expression, microvilli formation, and mucus secretion. The unique combination of a dynamic bioreactor and 3D intestinal constructs offers utility for engineering human intestinal tissues for the study of intestinal diseases and discovery options for new treatments. PMID:29333491

In vitro osteogenesis of human stem cells by using a three-dimensional perfusion bioreactor culture system: a review.

PubMed

Ceccarelli, Gabriele; Bloise, Nora; Vercellino, Marco; Battaglia, Rosalia; Morgante, Lucia; De Angelis, Maria Gabriella Cusella; Imbriani, Marcello; Visai, Livia

2013-04-01

Tissue engineering (by culturing cells on appropriate scaffolds, and using bioreactors to drive the correct bone structure formation) is an attractive alternative to bone grafting or implantation of bone substitutes. Osteogenesis is a biological process that involves many molecular intracellular pathways organized to optimize bone modeling. The use of bioreactor systems and especially the perfusion bioreactor, provides both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In this mini-review all the characteristics for the production of an appropriate bone construct are analyzed: the stem cell source, scaffolds useful for the seeding of pre-osteoblastic cells and the effects of fluid flow on differentiation and proliferation of bone precursor cells. By automating and standardizing tissue manufacture in controlled closed systems, engineered tissues may reduce the gap between the process of bone formation in vitro and subsequent graft of bone substitutes in vivo.

Discovery of Hyperpolarized Molecular Imaging Biomarkers in a Novel Prostate Tissue Slice Culture Model

DTIC Science & Technology

2013-06-01

research is to optimize an MRS-compatible, 3D Tissue Culture Bioreactor for use with primary human prostate tissue cultures (TSCs) and use it to...Tissue Culture Bioreactor ” to be submitted to the Journal Magnetic Resonance in Medicine. CONCLUSIONS: We have engineered a robust MR compatible 3D ...loss of structure, function or metabolism within a NMR compatible 3-D tissue culture bioreactor , and that magnetic resonance spectroscopy studies of

Heart tissue grown in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2001-01-01

Lisa Freed and Gordana Vunjak-Novakovic, both of the Massachusetts Institute of Technology (MIT), have taken the first steps toward engineering heart muscle tissue that could one day be used to patch damaged human hearts. Cells isolated from very young animals are attached to a three-dimensional polymer scaffold, then placed in a NASA bioreactor. The cells do not divide, but after about a week start to cornect to form a functional piece of tissue. Here, a transmission electron micrograph of engineered tissue shows a number of important landmarks present in functional heart tissue: (A) well-organized myofilaments (Mfl), z-lines (Z), and abundant glycogen granules (Gly); and (D) intercalcated disc (ID) and desmosomes (DES). The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Credit: MIT

A novel perfused rotary bioreactor for cardiomyogenesis of embryonic stem cells.

PubMed

Teo, Ailing; Mantalaris, Athanasios; Song, Kedong; Lim, Mayasari

2014-05-01

Developments in bioprocessing technology play an important role for overcoming challenges in cardiac tissue engineering. To this end, our laboratory has developed a novel rotary perfused bioreactor for supporting three-dimensional cardiac tissue engineering. The dynamic culture environments provided by our novel perfused rotary bioreactor and/or the high-aspect rotating vessel produced constructs with higher viability and significantly higher cell numbers (up to 4 × 10(5) cells/bead) than static tissue culture flasks. Furthermore, cells in the perfused rotary bioreactor showed earlier gene expressions of cardiac troponin-T, α- and β-myosin heavy chains with higher percentages of cardiac troponin-I-positive cells and better uniformity of sacromeric α-actinin expression. A dynamic and perfused environment, as provided by this bioreactor, provides a superior culture performance in cardiac differentiation for embryonic stem cells particularly for larger 3D constructs.

Design of a flow perfusion bioreactor system for bone tissue-engineering applications.

PubMed

Bancroft, Gregory N; Sikavitsas, Vassilios I; Mikos, Antonios G

2003-06-01

Several different bioreactors have been investigated for tissue-engineering applications. Among these bioreactors are the spinner flask and the rotating wall vessel reactor. In addition, a new type of culture system has been developed and investigated, the flow perfusion culture bioreactor. Flow perfusion culture offers several advantages, notably the ability to mitigate both external and internal diffusional limitations as well as to apply mechanical stress to the cultured cells. For such investigation, a flow perfusion culture system was designed and built. This design is the outgrowth of important design requirements and incorporates features crucial to successful experimentation with such a system.

Bioreactor-induced mesenchymal progenitor cell differentiation and elastic fiber assembly in engineered vascular tissues.

PubMed

Lin, Shigang; Mequanint, Kibret

2017-09-01

In vitro maturation of engineered vascular tissues (EVT) requires the appropriate incorporation of smooth muscle cells (SMC) and extracellular matrix (ECM) components similar to native arteries. To this end, the aim of the current study was to fabricate 4mm inner diameter vascular tissues using mesenchymal progenitor cells seeded into tubular scaffolds. A dual-pump bioreactor operating either in perfusion or pulsatile perfusion mode was used to generate physiological-like stimuli to promote progenitor cell differentiation, extracellular elastin production, and tissue maturation. Our data demonstrated that pulsatile forces and perfusion of 3D tubular constructs from both the lumenal and ablumenal sides with culture media significantly improved tissue assembly, effectively inducing mesenchymal progenitor cell differentiation to SMCs with contemporaneous elastin production. With bioreactor cultivation, progenitor cells differentiated toward smooth muscle lineage characterized by the expression of smooth muscle (SM)-specific markers smooth muscle alpha actin (SM-α-actin) and smooth muscle myosin heavy chain (SM-MHC). More importantly, pulsatile perfusion bioreactor cultivation enhanced the synthesis of tropoelastin and its extracellular cross-linking into elastic fiber compared with static culture controls. Taken together, the current study demonstrated progenitor cell differentiation and vascular tissue assembly, and provides insights into elastin synthesis and assembly to fibers. Incorporation of elastin into engineered vascular tissues represents a critical design goal for both mechanical and biological functions. In the present study, we seeded porous tubular scaffolds with multipotent mesenchymal progenitor cells and cultured in dual-pump pulsatile perfusion bioreactor. Physiological-like stimuli generated by bioreactor not only induced mesenchymal progenitor cell differentiation to vascular smooth muscle lineage but also actively promoted elastin synthesis and fiber assembly. Gene expression and protein synthesis analyses coupled with histological and immunofluorescence staining revealed that elastin-containing vascular tissues were fabricated. More importantly, co-localization and co-immunoprecipitation experiments demonstrated that elastin and fibrillin-1 were abundant throughout the cross-section of the tissue constructs suggesting a process of elastin protein crosslinking. This study paves a way forward to engineer elastin-containing functional vascular substitutes from multipotent progenitor cells in a bioreactor. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Microgravity

NASA Image and Video Library

2000-12-15

Paul Ducheyne, a principal investigator in the microgravity materials science program and head of the University of Pernsylvania's Center for Bioactive Materials and Tissue Engineering, is leading the trio as they use simulated microgravity to determine the optimal characteristics of tiny glass particles for growing bone tissue. The result could make possible a much broader range of synthetic bone-grafting applications. Even in normal gravity, bioactive glass particles enhance bone growth in laboratory tests with flat tissue cultures. Ducheyne and his team believe that using the bioactive microcarriers in a rotating bioreactor in microgravity will produce improved, three-dimensional tissue cultures. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: NASA and University of Pennsylvania Center for Bioactive Materials and Tissue Engineering.

Reconstruction of thin fluorophore-filled capillaries in thick scattering medium using fluorescence diffuse optical tomography within the diffusion approximation

NASA Astrophysics Data System (ADS)

Desrochers, Johanne; Vermette, Patrick; Fontaine, Réjean; Bérubé-Lauzière, Yves

2009-02-01

Current efforts in tissue engineering target the growth of 3D volumes of tissue cultures in bioreactor conditions. Fluorescence optical tomography has the potential to monitor cells viability and tissue growth non-destructively directly within the bioreactor via bio-molecular fluorescent labelling strategies. We currently work on developing the imaging instrumentation for tissue cultures in bioreactor conditions. Previously, we localized in 3D thin fluorescent-labelled capillaries in a cylindrically shaped bioreactor phantom containing a diffusive medium with our time-of-flight localization technique. Here, we present our first reconstruction results of the spatial distribution of fluorophore concentrations for labelled capillaries embedded in a bioreactor phantom.

Heart tissue grown in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2001-01-01

Lisa Freed and Gordana Vunjak-Novakovic, both of the Massachusetts Institute of Technology (MIT), have taken the first steps toward engineering heart muscle tissue that could one day be used to patch damaged human hearts. Cells isolated from very young animals are attached to a three-dimensional polymer scaffold, then placed in a NASA bioreactor. The cells do not divide, but after about a week start to cornect to form a functional piece of tissue. Functionally connected heart cells that are capable of transmitting electrical signals are the goal for Freed and Vunjak-Novakovic. Electrophysiological recordings of engineered tissue show spontaneous contractions at a rate of 70 beats per minute (a), and paced contractions at rates of 80, 150, and 200 beats per minute respectively (b, c, and d). The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Credit: NASA and MIT.

Nanotechnology in vascular tissue engineering: from nanoscaffolding towards rapid vessel biofabrication.

PubMed

Mironov, Vladimir; Kasyanov, Vladimir; Markwald, Roger R

2008-06-01

The existing methods of biofabrication for vascular tissue engineering are still bioreactor-based, extremely expensive, laborious and time consuming and, furthermore, not automated, which would be essential for an economically successful large-scale commercialization. The advances in nanotechnology can bring additional functionality to vascular scaffolds, optimize internal vascular graft surface and even help to direct the differentiation of stem cells into the vascular cell phenotype. The development of rapid nanotechnology-based methods of vascular tissue biofabrication represents one of most important recent technological breakthroughs in vascular tissue engineering because it dramatically accelerates vascular tissue assembly and, importantly, also eliminates the need for a bioreactor-based scaffold cellularization process.

Numerical simulation of fluid field and in vitro three-dimensional fabrication of tissue-engineered bones in a rotating bioreactor and in vivo implantation for repairing segmental bone defects.

PubMed

Song, Kedong; Wang, Hai; Zhang, Bowen; Lim, Mayasari; Liu, Yingchao; Liu, Tianqing

2013-03-01

In this paper, two-dimensional flow field simulation was conducted to determine shear stresses and velocity profiles for bone tissue engineering in a rotating wall vessel bioreactor (RWVB). In addition, in vitro three-dimensional fabrication of tissue-engineered bones was carried out in optimized bioreactor conditions, and in vivo implantation using fabricated bones was performed for segmental bone defects of Zelanian rabbits. The distribution of dynamic pressure, total pressure, shear stress, and velocity within the culture chamber was calculated for different scaffold locations. According to the simulation results, the dynamic pressure, velocity, and shear stress around the surface of cell-scaffold construction periodically changed at different locations of the RWVB, which could result in periodical stress stimulation for fabricated tissue constructs. However, overall shear stresses were relatively low, and the fluid velocities were uniform in the bioreactor. Our in vitro experiments showed that the number of cells cultured in the RWVB was five times higher than those cultured in a T-flask. The tissue-engineered bones grew very well in the RWVB. This study demonstrates that stress stimulation in an RWVB can be beneficial for cell/bio-derived bone constructs fabricated in an RWVB, with an application for repairing segmental bone defects.

Strategies for Enhancing the Accumulation and Retention of Extracellular Matrix in Tissue-Engineered Cartilage Cultured in Bioreactors

PubMed Central

Shahin, Kifah; Doran, Pauline M.

2011-01-01

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min−1) and gradually increasing (0.075–0.2 mL min−1) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0–4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8–5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min−1. GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention. PMID:21858004

Smooth muscle cell seeding of decellularized scaffolds: the importance of bioreactor preconditioning to development of a more native architecture for tissue-engineered blood vessels.

PubMed

Yazdani, Saami K; Watts, Benjamin; Machingal, Masood; Jarajapu, Yagna P R; Van Dyke, Mark E; Christ, George J

2009-04-01

Vascular smooth muscle cells (VSMCs) impart important functional characteristics in the native artery and, therefore, should logically be incorporated in the development of tissue-engineered blood vessels. However, the native architecture and low porosity of naturally derived biomaterials (i.e., decellularized vessels) have impeded efforts to seed and incorporate a VSMC layer in tissue-engineered blood vessels. To this end, the goal of this study was to develop improved methods for seeding, proliferation, and maturation of VSMCs on decellularized porcine carotid arteries. Decellularized vessels were prepared in the absence and presence of the adventitial layer, and statically seeded with a pipette containing a suspension of rat aortic VSMCs. After cell seeding, recellularized engineered vessels were placed in a custom bioreactor system for 1-2 weeks to enhance cellular proliferation, alignment, and maturation. Initial attachment of VSMCs was dramatically enhanced by removing the adventitial layer of the decellularized porcine artery. Moreover, cyclic bioreactor conditioning (i.e., flow and pressure) resulted in increased VSMC proliferation and accelerated formation of a muscularized medial layer in the absence of the adventitial layer during the first week of preconditioning. Fura-2-based digital imaging microscopy revealed marked and reproducible depolarization-induced calcium mobilization after bioreactor preconditioning in the absence, but not in the presence, of the adventitia. The major finding of this investigation is that bioreactor preconditioning accelerates the formation of a significant muscular layer on decellularized scaffolds, in particular on adventitia-denuded scaffolds. Further, the VSMC layer of bioreactor-preconditioned vessels was capable of mobilizing calcium in response to cellular depolarization. These findings represent an important first step toward the development of tissue-engineered vascular grafts that more closely mimic native vasculature.

A miniaturized, optically accessible bioreactor for systematic 3D tissue engineering research.

PubMed

Laganà, Matteo; Raimondi, Manuela T

2012-02-01

Perfusion bioreactors are widely used in tissue engineering and pharmaceutical research to provide reliable models of tissue growth under controlled conditions. Destructive assays are not able to follow the evolution of the growing tissue on the same construct, so it is necessary to adopt non-destructive analysis. We have developed a miniaturized, optically accessible bioreactor for interstitial perfusion of 3D cell-seeded scaffolds. The scaffold adopted was optically transparent, with highly defined architecture. Computational fluid dynamics (CFD) analysis was useful to predict the flow behavior in the bioreactor scaffold chamber (that was laminar flow, Re = 0.179, with mean velocity equal to 100 microns/s). Moreover, experimental characterization of the bioreactor performance gave that the maximum allowable pressure was 0.06 MPa and allowable flow rate up to 25 ml/min. A method, to estimate quantitatively and non destructively the cell proliferation (from 15 to 43 thousand cells) and tissue growth (from 2% to 43%) during culture time, was introduced and validated. An end point viability test was performed to check the experimental set-up overall suitability for cell culture with successful results. Morphological analysis was performed at the end time point to show the complex tridimensional pattern of the biological tissue growth. Our system, characterized by controlled conditions in a wide range of allowable flow rate and pressure, permits to systematically study the influence of several parameters on engineered tissue growth, using viable staining and a standard fluorescence microscope.

Fabrication, characterization, and in vitro evaluation of poly(lactic acid glycolic acid)/nano-hydroxyapatite composite microsphere-based scaffolds for bone tissue engineering in rotating bioreactors.

PubMed

Lv, Qing; Nair, Lakshmi; Laurencin, Cato T

2009-12-01

Dynamic flow culture bioreactor systems have been shown to enhance in vitro bone tissue formation by facilitating mass transfer and providing mechanical stimulation. Our laboratory has developed a biodegradable poly (lactic acid glycolic acid) (PLAGA) mixed scaffold consisting of lighter-than-water (LTW) and heavier-than-water (HTW) microspheres as potential matrices for engineering tissue using a high aspect ratio vessel (HARV) rotating bioreactor system. We have demonstrated enhanced osteoblast differentiation and mineralization on PLAGA scaffolds in the HARV rotating bioreactor system when compared with static culture. The objective of the present study is to improve the mechanical properties and bioactivity of polymeric scaffolds by designing LTW polymer/ceramic composite scaffolds suitable for dynamic culture using a HARV bioreactor. We employed a microsphere sintering method to fabricate three-dimensional PLAGA/nano-hydroxyapatite (n-HA) mixed scaffolds composed of LTW and HTW composite microspheres. The mechanical properties, pore size and porosity of the composite scaffolds were controlled by varying parameters, such as sintering temperature, sintering time, and PLAGA/n-HA ratio. The PLAGA/n-HA (4:1) scaffold sintered at 90 degrees C for 3 h demonstrated the highest mechanical properties and an appropriate pore structure for bone tissue engineering applications. Furthermore, evaluation human mesenchymal stem cells (HMSCs) response to PLAGA/n-HA scaffolds was performed. HMSCs on PLAGA/n-HA scaffolds demonstrated enhanced proliferation, differentiation, and mineralization when compared with those on PLAGA scaffolds. Therefore, PLAGA/n-HA mixed scaffolds are promising candidates for HARV bioreactor-based bone tissue engineering applications. Copyright 2008 Wiley Periodicals, Inc.

Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors.

PubMed

Sart, Sébastien; Agathos, Spiros N; Li, Yan; Ma, Teng

2016-01-01

Human mesenchymal stem cells (hMSCs) have emerged as an important cell type in cell therapy and tissue engineering. In these applications, maintaining the therapeutic properties of hMSCs requires tight control of the culture environments and the structural cell organizations. Bioreactor systems are essential tools to achieve these goals in the clinical-scale expansion and tissue engineering applications. This review summarizes how different bioreactors provide cues to regulate the structure and the chemico-mechanical microenvironment of hMSCs with a focus on 3D organization. In addition to conventional bioreactors, recent advances in microfluidic bioreactors as a novel approach to better control the hMSC microenvironment are also discussed. These advancements highlight the key role of bioreactor systems in preserving hMSC's functional properties by providing dynamic and temporal regulation of in vitro cellular microenvironment. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

In vitro fabrication of a tissue engineered human cardiovascular patch for future use in cardiovascular surgery.

PubMed

Yang, Chao; Sodian, Ralf; Fu, Ping; Lüders, Cora; Lemke, Thees; Du, Jing; Hübler, Michael; Weng, Yuguo; Meyer, Rudolf; Hetzer, Roland

2006-01-01

One approach to tissue engineering has been the development of in vitro conditions for the fabrication of functional cardiovascular structures intended for implantation. In this experiment, we developed a pulsatile flow system that provides biochemical and biomechanical signals in order to regulate autologous, human patch-tissue development in vitro. We constructed a biodegradable patch scaffold from porous poly-4-hydroxy-butyrate (P4HB; pore size 80 to 150 microm). The scaffold was seeded with pediatric aortic cells. The cell-seeded patch constructs were placed in a self-developed bioreactor for 7 days to observe potential tissue formation under dynamic cell culture conditions. As a control, cell-seeded scaffolds were not conditioned in the bioreactor system. After maturation in vitro, the analysis of the tissue engineered constructs included biochemical, biomechanical, morphologic, and immunohistochemical examination. Macroscopically, all tissue engineered constructs were covered by cells. After conditioning in the bioreactor, the cells were mostly viable, had grown into the pores, and had formed tissue on the patch construct. Electron microscopy showed confluent smooth surfaces. Additionally, we demonstrated the capacity to generate collagen and elastin under in vitro pulsatile flow conditions in biochemical examination. Biomechanical testing showed mechanical properties of the tissue engineered human patch tissue without any statistical differences in strength or resistance to stretch between the static controls and the conditioned patches. Immunohistochemical examination stained positive for alpha smooth muscle actin, collagen type I, and fibronectin. There was minor tissue formation in the nonconditioned control samples. Porous P4HB may be used to fabricate a biodegradable patch scaffold. Human vascular cells attached themselves to the polymeric scaffold, and extracellular matrix formation was induced under controlled biomechanical and biodynamic stimuli in a self-developed pulsatile bioreactor system.

A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering.

PubMed

Zhang, Zhi-Yong; Teoh, Swee Hin; Teo, Erin Yiling; Khoon Chong, Mark Seow; Shin, Chong Woon; Tien, Foo Toon; Choolani, Mahesh A; Chan, Jerry K Y

2010-11-01

Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p < 0.05) in large 785 mm(3) macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6×, p < 0.01) and calcium deposition (1.3-2.3×, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds. Copyright © 2010 Elsevier Ltd. All rights reserved.

Nano-ceramic composite scaffolds for bioreactor-based bone engineering.

PubMed

Lv, Qing; Deng, Meng; Ulery, Bret D; Nair, Lakshmi S; Laurencin, Cato T

2013-08-01

Composites of biodegradable polymers and bioactive ceramics are candidates for tissue-engineered scaffolds that closely match the properties of bone. We previously developed a porous, three-dimensional poly (D,L-lactide-co-glycolide) (PLAGA)/nanohydroxyapatite (n-HA) scaffold as a potential bone tissue engineering matrix suitable for high-aspect ratio vessel (HARV) bioreactor applications. However, the physical and cellular properties of this scaffold are unknown. The present study aims to evaluate the effect of n-HA in modulating PLAGA scaffold properties and human mesenchymal stem cell (HMSC) responses in a HARV bioreactor. By comparing PLAGA/n-HA and PLAGA scaffolds, we asked whether incorporation of n-HA (1) accelerates scaffold degradation and compromises mechanical integrity; (2) promotes HMSC proliferation and differentiation; and (3) enhances HMSC mineralization when cultured in HARV bioreactors. PLAGA/n-HA scaffolds (total number = 48) were loaded into HARV bioreactors for 6 weeks and monitored for mass, molecular weight, mechanical, and morphological changes. HMSCs were seeded on PLAGA/n-HA scaffolds (total number = 38) and cultured in HARV bioreactors for 28 days. Cell migration, proliferation, osteogenic differentiation, and mineralization were characterized at four selected time points. The same amount of PLAGA scaffolds were used as controls. The incorporation of n-HA did not alter the scaffold degradation pattern. PLAGA/n-HA scaffolds maintained their mechanical integrity throughout the 6 weeks in the dynamic culture environment. HMSCs seeded on PLAGA/n-HA scaffolds showed elevated proliferation, expression of osteogenic phenotypic markers, and mineral deposition as compared with cells seeded on PLAGA scaffolds. HMSCs migrated into the scaffold center with nearly uniform cell and extracellular matrix distribution in the scaffold interior. The combination of PLAGA/n-HA scaffolds with HMSCs in HARV bioreactors may allow for the generation of engineered bone tissue. In cases of large bone voids (such as bone cancer), tissue-engineered constructs may provide alternatives to traditional bone grafts by culturing patients' own MSCs with PLAGA/n-HA scaffolds in a HARV culture system.

Microgravity

NASA Image and Video Library

2001-05-15

Lisa Freed and Gordana Vunjak-Novakovic, both of the Massachusetts Institute of Technology (MIT), have taken the first steps toward engineering heart muscle tissue that could one day be used to patch damaged human hearts. Cells isolated from very young animals are attached to a three-dimensional polymer scaffold, then placed in a NASA bioreactor. The cells do not divide, but after about a week start to cornect to form a functional piece of tissue. Here, a transmission electron micrograph of engineered tissue shows a number of important landmarks present in functional heart tissue: (A) well-organized myofilaments (Mfl), z-lines (Z), and abundant glycogen granules (Gly); and (D) intercalcated disc (ID) and desmosomes (DES). The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Credit: MIT

Biomimetic fetal rotation bioreactor for engineering bone tissues-Effect of cyclic strains on upregulation of osteogenic gene expression.

PubMed

Ravichandran, Akhilandeshwari; Wen, Feng; Lim, Jing; Chong, Mark Seow Khoon; Chan, Jerry K Y; Teoh, Swee-Hin

2018-04-01

Cells respond to physiological mechanical stresses especially during early fetal development. Adopting a biomimetic approach, it is necessary to develop bioreactor systems to explore the effects of physiologically relevant mechanical strains and shear stresses for functional tissue growth and development. This study introduces a multimodal bioreactor system that allows application of cyclic compressive strains on premature bone grafts that are cultured under biaxial rotation (chamber rotation about 2 axes) conditions for bone tissue engineering. The bioreactor is integrated with sensors for dissolved oxygen levels and pH that allow real-time, non-invasive monitoring of the culture parameters. Mesenchymal stem cells-seeded polycaprolactone-β-tricalcium phosphate scaffolds were cultured in this bioreactor over 2 weeks in 4 different modes-static, cyclic compression, biaxial rotation, and multimodal (combination of cyclic compression and biaxial rotation). The multimodal culture resulted in 1.8-fold higher cellular proliferation in comparison with the static controls within the first week. Two weeks of culture in the multimodal bioreactor utilizing the combined effects of optimal fluid flow conditions and cyclic compression led to the upregulation of osteogenic genes alkaline phosphatase (3.2-fold), osteonectin (2.4-fold), osteocalcin (10-fold), and collagen type 1 α1 (2-fold) in comparison with static cultures. We report for the first time, the independent and combined effects of mechanical stimulation and biaxial rotation for bone tissue engineering using a bioreactor platform with non-invasive sensing modalities. The demonstrated results show leaning towards the futuristic vision of using a physiologically relevant bioreactor system for generation of autologous bone grafts for clinical implantation. Copyright © 2018 John Wiley & Sons, Ltd.

Bare Bones of Bioactive Glass

NASA Technical Reports Server (NTRS)

2000-01-01

Paul Ducheyne, a principal investigator in the microgravity materials science program and head of the University of Pernsylvania's Center for Bioactive Materials and Tissue Engineering, is leading the trio as they use simulated microgravity to determine the optimal characteristics of tiny glass particles for growing bone tissue. The result could make possible a much broader range of synthetic bone-grafting applications. Even in normal gravity, bioactive glass particles enhance bone growth in laboratory tests with flat tissue cultures. Ducheyne and his team believe that using the bioactive microcarriers in a rotating bioreactor in microgravity will produce improved, three-dimensional tissue cultures. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: NASA and University of Pennsylvania Center for Bioactive Materials and Tissue Engineering.

Analysis of type II diabetes mellitus adipose-derived stem cells for tissue engineering applications

PubMed Central

Minteer, Danielle Marie; Young, Matthew T; Lin, Yen-Chih; Over, Patrick J; Rubin, J Peter; Gerlach, Jorg C

2015-01-01

To address the functionality of diabetic adipose-derived stem cells in tissue engineering applications, adipose-derived stem cells isolated from patients with and without type II diabetes mellitus were cultured in bioreactor culture systems. The adipose-derived stem cells were differentiated into adipocytes and maintained as functional adipocytes. The bioreactor system utilizes a hollow fiber–based technology for three-dimensional perfusion of tissues in vitro, creating a model in which long-term culture of adipocytes is feasible, and providing a potential tool useful for drug discovery. Daily metabolic activity of the adipose-derived stem cells was analyzed within the medium recirculating throughout the bioreactor system. At experiment termination, tissues were extracted from bioreactors for immunohistological analyses in addition to gene and protein expression. Type II diabetic adipose-derived stem cells did not exhibit significantly different glucose consumption compared to adipose-derived stem cells from patients without type II diabetes (p > 0.05, N = 3). Expression of mature adipocyte genes was not significantly different between diabetic/non-diabetic groups (p > 0.05, N = 3). Protein expression of adipose tissue grown within all bioreactors was verified by Western blotting.The results from this small-scale study reveal adipose-derived stem cells from patients with type II diabetes when removed from diabetic environments behave metabolically similar to the same cells of non-diabetic patients when cultured in a three-dimensional perfusion bioreactor, suggesting that glucose transport across the adipocyte cell membrane, the hindrance of which being characteristic of type II diabetes, is dependent on environment. The presented observation describes a tissue-engineered tool for long-term cell culture and, following future adjustments to the culture environment and increased sample sizes, potentially for anti-diabetic drug testing. PMID:26090087

A Novel Human Adipocyte-derived Basement Membrane for Tissue Engineering Applications

NASA Astrophysics Data System (ADS)

Damm, Aaron

Tissue engineering strategies have traditionally focused on the use of synthetic polymers as support scaffolds for cell growth. Recently, strategies have shifted towards a natural biologically derived scaffold, with the main focus on decellularized organs. Here, we report the development and engineering of a scaffold naturally secreted by human preadipocytes during differentiation. During this differentiation process, the preadipocytes remodel the extracellular matrix by releasing new extracellular proteins. Finally, we investigated the viability of the new basement membrane as a scaffold for tissue engineering using human pancreatic islets, and as a scaffold for soft tissue repair. After identifying the original scaffold material, we sought to improve the yield of material, treating the cell as a bioreactor, through various nutritional and cytokine stimuli. The results suggest that adipocytes can be used as bioreactors to produce a designer-specified engineered human extracellular matrix scaffold for specific tissue engineering applications.

A Dual-Mode Bioreactor System for Tissue Engineered Vascular Models.

PubMed

Bono, N; Meghezi, S; Soncini, M; Piola, M; Mantovani, D; Fiore, Gianfranco Beniamino

2017-06-01

In the past decades, vascular tissue engineering has made great strides towards bringing engineered vascular tissues to the clinics and, in parallel, obtaining in-lab tools for basic research. Herein, we propose the design of a novel dual-mode bioreactor, useful for the fabrication (construct mode) and in vitro stimulation (culture mode) of collagen-based tubular constructs. Collagen-based gels laden with smooth muscle cells (SMCs) were molded directly within the bioreactor culture chamber. Based on a systematic characterization of the bioreactor culture mode, constructs were subjected to 10% cyclic strain at 0.5 Hz for 5 days. The effects of cyclic stimulation on matrix re-arrangement and biomechanical/viscoelastic properties were examined and compared vs. statically cultured constructs. A thorough comparison of cell response in terms of cell localization and expression of contractile phenotypic markers was carried out as well. We found that cyclic stimulation promoted cell-driven collagen matrix bi-axial compaction, enhancing the mechanical strength of strained samples with respect to static controls. Moreover, cyclic strain positively affected SMC behavior: cells maintained their contractile phenotype and spread uniformly throughout the whole wall thickness. Conversely, static culture induced a noticeable polarization of cell distribution to the outer rim of the constructs and a sharp reduction in total cell density. Overall, coupling the use of a novel dual-mode bioreactor with engineered collagen-gel-based tubular constructs demonstrated to be an interesting technology to investigate the modulation of cell and tissue behavior under controlled mechanically conditioned in vitro maturation.

Microgravity

NASA Image and Video Library

2001-05-15

Lisa Freed and Gordana Vunjak-Novakovic, both of the Massachusetts Institute of Technology (MIT), have taken the first steps toward engineering heart muscle tissue that could one day be used to patch damaged human hearts. Cells isolated from very young animals are attached to a three-dimensional polymer scaffold, then placed in a NASA bioreactor. The cells do not divide, but after about a week start to cornect to form a functional piece of tissue. Functionally connected heart cells that are capable of transmitting electrical signals are the goal for Freed and Vunjak-Novakovic. Electrophysiological recordings of engineered tissue show spontaneous contractions at a rate of 70 beats per minute (a), and paced contractions at rates of 80, 150, and 200 beats per minute respectively (b, c, and d). The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Credit: NASA and MIT.

Enhancement of matrix production and cell proliferation in human annulus cells under bioreactor culture.

PubMed

Yang, Xinlin; Wang, Daidong; Hao, Jianrong; Gong, Meiqing; Arlet, Vincent; Balian, Gary; Shen, Francis H; Li, Xudong Joshua

2011-06-01

Tissue engineering is a promising approach for treatment of disc degeneration. Herein, we evaluated effects of rotating bioreactor culture on the extracellular matrix production and proliferation of human annulus fibrosus (AF) cells. AF cells were embedded into alginate beads, and then cultured up to 3 weeks in a rotating wall vessel bioreactor or a static vessel. By real-time reverse transcription-polymerase chain reaction, expression of aggrecan, collagen type I and type II, and collagen prolyl 4-hydroxylase II was remarkably elevated, whereas expression of matrix metalloproteinase 3 and a disintegrin and metalloproteinase with thrombospondin motifs 5 was significantly decreased under bioreactor. Biochemical analysis revealed that the levels of the whole cell-associated proteoglycan and collagen were approximately five- and twofolds in rotating bioreactor, respectively, compared to those in static culture. Moreover, AF cell proliferation was augmented in rotating bioreactor. DNA contents were threefolds higher in rotating bioreactor than that in static culture. Expression of the proliferating cell nuclear antigen was robustly enhanced in rotating bioreactor as early as 1 week. Our findings suggested that rotating bioreactor culture would be an effective technique for expansion of human annulus cells for tissue engineering driven treatment of disc degeneration.

Visualizing feasible operating ranges within tissue engineering systems using a "windows of operation" approach: a perfusion-scaffold bioreactor case study.

PubMed

McCoy, Ryan J; O'Brien, Fergal J

2012-12-01

Tissue engineering approaches to developing functional substitutes are often highly complex, multivariate systems where many aspects of the biomaterials, bio-regulatory factors or cell sources may be controlled in an effort to enhance tissue formation. Furthermore, success is based on multiple performance criteria reflecting both the quantity and quality of the tissue produced. Managing the trade-offs between different performance criteria is a challenge. A "windows of operation" tool that graphically represents feasible operating spaces to achieve user-defined levels of performance has previously been described by researchers in the bio-processing industry. This paper demonstrates the value of "windows of operation" to the tissue engineering field using a perfusion-scaffold bioreactor system as a case study. In our laboratory, perfusion bioreactor systems are utilized in the context of bone tissue engineering to enhance the osteogenic differentiation of cell-seeded scaffolds. A key challenge of such perfusion bioreactor systems is to maximize the induction of osteogenesis but minimize cell detachment from the scaffold. Two key operating variables that influence these performance criteria are the mean scaffold pore size and flow-rate. Using cyclooxygenase-2 and osteopontin gene expression levels as surrogate indicators of osteogenesis, we employed the "windows of operation" methodology to rapidly identify feasible operating ranges for the mean scaffold pore size and flow-rate that achieved user-defined levels of performance for cell detachment and differentiation. Incorporation of such tools into the tissue engineer's armory will hopefully yield a greater understanding of the highly complex systems used and help aid decision making in future translation of products from the bench top to the market place. Copyright © 2012 Wiley Periodicals, Inc.

Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization

PubMed Central

Uzarski, Joseph S.; Bijonowski, Brent M.; Wang, Bo; Ward, Heather H.; Wandinger-Ness, Angela

2015-01-01

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems. PMID:25929317

Dual-Purpose Bioreactors to Monitor Noninvasive Physical and Biochemical Markers of Kidney and Liver Scaffold Recellularization.

PubMed

Uzarski, Joseph S; Bijonowski, Brent M; Wang, Bo; Ward, Heather H; Wandinger-Ness, Angela; Miller, William M; Wertheim, Jason A

2015-10-01

Analysis of perfusion-based bioreactors for organ engineering and a detailed evaluation of physical and biochemical parameters that measure dynamic changes within maturing cell-laden scaffolds are critical components of ex vivo tissue development that remain understudied topics in the tissue and organ engineering literature. Intricately designed bioreactors that house developing tissue are critical to properly recapitulate the in vivo environment, deliver nutrients within perfused media, and monitor physiological parameters of tissue development. Herein, we provide an in-depth description and analysis of two dual-purpose perfusion bioreactors that improve upon current bioreactor designs and enable comparative analyses of ex vivo scaffold recellularization strategies and cell growth performance during long-term maintenance culture of engineered kidney or liver tissues. Both bioreactors are effective at maximizing cell seeding of small-animal organ scaffolds and maintaining cell survival in extended culture. We further demonstrate noninvasive monitoring capabilities for tracking dynamic changes within scaffolds as the native cellular component is removed during decellularization and model human cells are introduced into the scaffold during recellularization and proliferate in maintenance culture. We found that hydrodynamic pressure drop (ΔP) across the retained scaffold vasculature is a noninvasive measurement of scaffold integrity. We further show that ΔP, and thus resistance to fluid flow through the scaffold, decreases with cell loss during decellularization and correspondingly increases to near normal values for whole organs following recellularization of the kidney or liver scaffolds. Perfused media may be further sampled in real time to measure soluble biomarkers (e.g., resazurin, albumin, or kidney injury molecule-1) that indicate degree of cellular metabolic activity, synthetic function, or engraftment into the scaffold. Cell growth within bioreactors is validated for primary and immortalized cells, and the design of each bioreactor is scalable to accommodate any three-dimensional scaffold (e.g., synthetic or naturally derived matrix) that contains conduits for nutrient perfusion to deliver media to growing cells and monitor noninvasive parameters during scaffold repopulation, broadening the applicability of these bioreactor systems.

A novel axial-stress bioreactor system combined with a substance exchanger for tissue engineering of 3D constructs.

PubMed

Li, Song-Tao; Liu, Yong; Zhou, Qiang; Lue, Ren-Fa; Song, Lei; Dong, Shi-Wu; Guo, Ping; Kopjar, Branko

2014-03-01

This study introduced a prototype of an axial-stress bioreactor system that supports long-term growth and development of engineered tissues. The main features of this bioreactor are an integrated substance exchanger and feedback control of pH and PO₂. A 21-day study was conducted to validate the system's ability to maintain a stable environment, while remaining sterile. Our results showed that the pH, PO₂, and nutrient (glucose) remained balanced at appropriate levels, while metabolic waste (lactic acid) was removed. No bacteria or fungi were detected in the system or tissue; thus, demonstrating that it was sterile. These data indicate the bioreactor's strong potential for long-term tissue culture. To explore this idea, the effect of dynamic culture, including cyclic compression and automatic substance exchange, on mouse bone-marrow mesenchymal stem cells (BMSCs) seeded in decalcified bone matrix was studied using the bioreactor prototype. Histological sections of the engineered tissues showed higher cell densities in scaffolds in dynamic culture compared to those in static culture, while cell cycle analysis showed that dynamic culture promoted BMSC proliferation (proliferation index, PI=34.02±1.77) more effectively than static culture (PI=26.66±1.81). The results from a methyl thiazolyl tetrazolium assay were consistent with the loading experimental data. Furthermore, elevated alkaline phosphatase activity and calcium content were observed in dynamic condition compared to static culture. In conclusion, this bioreactor system supplies a method of modulating the pH and PO₂ in defined ranges with only small fluctuations; it can be used as a physiological or pathological analog. Automatic control of the environment is a practical solution for long-term, steady-state culture for future commercialization.

Characterization of a novel bioreactor system for 3D cellular mechanobiology studies.

PubMed

Cook, Colin A; Huri, Pinar Y; Ginn, Brian P; Gilbert-Honick, Jordana; Somers, Sarah M; Temple, Joshua P; Mao, Hai-Quan; Grayson, Warren L

2016-08-01

In vitro engineering systems can be powerful tools for studying tissue development in response to biophysical stimuli as well as for evaluating the functionality of engineered tissue grafts. It has been challenging, however, to develop systems that adequately integrate the application of biomimetic mechanical strain to engineered tissue with the ability to assess functional outcomes in real time. The aim of this study was to design a bioreactor system capable of real-time conditioning (dynamic, uniaxial strain, and electrical stimulation) of centimeter-long 3D tissue engineered constructs simultaneously with the capacity to monitor local strains. The system addresses key limitations of uniform sample loading and real-time imaging capabilities. Our system features an electrospun fibrin scaffold, which exhibits physiologically relevant stiffness and uniaxial alignment that facilitates cell adhesion, alignment, and proliferation. We have demonstrated the capacity for directly incorporating human adipose-derived stromal/stem cells into the fibers during the electrospinning process and subsequent culture of the cell-seeded constructs in the bioreactor. The bioreactor facilitates accurate pre-straining of the 3D constructs as well as the application of dynamic and static uniaxial strains while monitoring bulk construct tensions. The incorporation of fluorescent nanoparticles throughout the scaffolds enables in situ monitoring of local strain fields using fluorescent digital image correlation techniques, since the bioreactor is imaging compatible, and allows the assessment of local sample stiffness and stresses when coupled with force sensor measurements. In addition, the system is capable of measuring the electromechanical coupling of skeletal muscle explants by applying an electrical stimulus and simultaneously measuring the force of contraction. The packaging of these technologies, biomaterials, and analytical methods into a single bioreactor system has produced a powerful tool that will enable improved engineering of functional 3D ligaments, tendons, and skeletal muscles. Biotechnol. Bioeng. 2016;113: 1825-1837. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Modeling and design of optimal flow perfusion bioreactors for tissue engineering applications.

PubMed

Hidalgo-Bastida, L Araida; Thirunavukkarasu, Sundaramoorthy; Griffiths, Sarah; Cartmell, Sarah H; Naire, Shailesh

2012-04-01

Perfusion bioreactors have been used in different tissue engineering applications because of their consistent distribution of nutrients and flow-induced shear stress within the tissue-engineering scaffold. A widely used configuration uses a scaffold with a circular cross-section enclosed within a cylindrical chamber and inlet and outlet pipes which are connected to the chamber on either side through which media is continuously circulated. However, fluid-flow experiments and simulations have shown that the majority of the flow perfuses through the center. This pattern creates stagnant zones in the peripheral regions as well as in those of high flow rate near the inlet and outlet. This non-uniformity of flow and shear stress, owing to a circular design, results in limited cell proliferation and differentiation in these areas. The focus of this communication is to design an optimized perfusion system using computational fluid dynamics as a mathematical tool to overcome the time-consuming trial and error experimental method. We compared the flow within a circular and a rectangular bioreactor system. Flow simulations within the rectangular bioreactor are shown to overcome the limitations in the circular design. This communication challenges the circular cross-section bioreactor configuration paradigm and provides proof of the advantages of the new design over the existing one. Copyright © 2011 Wiley Periodicals, Inc.

Human embryonic stem cell-derived mesodermal progenitors display substantially increased tissue formation compared to human mesenchymal stem cells under dynamic culture conditions in a packed bed/column bioreactor.

PubMed

de Peppo, Giuseppe Maria; Sladkova, Martina; Sjövall, Peter; Palmquist, Anders; Oudina, Karim; Hyllner, Johan; Thomsen, Peter; Petite, Hervé; Karlsson, Camilla

2013-01-01

Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.

Large Animal Models of an In Vivo Bioreactor for Engineering Vascularized Bone.

PubMed

Akar, Banu; Tatara, Alexander M; Sutradhar, Alok; Hsiao, Hui-Yi; Miller, Michael; Cheng, Ming-Huei; Mikos, Antonios G; Brey, Eric M

2018-04-12

Reconstruction of large skeletal defects is challenging due to the requirement for large volumes of donor tissue and the often complex surgical procedures. Tissue engineering has the potential to serve as a new source of tissue for bone reconstruction, but current techniques are often limited in regards to the size and complexity of tissue that can be formed. Building tissue using an in vivo bioreactor approach may enable the production of appropriate amounts of specialized tissue, while reducing issues of donor site morbidity and infection. Large animals are required to screen and optimize new strategies for growing clinically appropriate volumes of tissues in vivo. In this article, we review both ovine and porcine models that serve as models of the technique proposed for clinical engineering of bone tissue in vivo. Recent findings are discussed with these systems, as well as description of next steps required for using these models, to develop clinically applicable tissue engineering applications.

The design and development of a high-throughput magneto-mechanostimulation device for cartilage tissue engineering.

PubMed

Brady, Mariea A; Vaze, Reva; Amin, Harsh D; Overby, Darryl R; Ethier, C Ross

2014-02-01

To recapitulate the in vivo environment and create neo-organoids that replace lost or damaged tissue requires the engineering of devices, which provide appropriate biophysical cues. To date, bioreactors for cartilage tissue engineering have focused primarily on biomechanical stimulation. There is a significant need for improved devices for articular cartilage tissue engineering capable of simultaneously applying multiple biophysical (electrokinetic and mechanical) stimuli. We have developed a novel high-throughput magneto-mechanostimulation bioreactor, capable of applying static and time-varying magnetic fields, as well as multiple and independently adjustable mechanical loading regimens. The device consists of an array of 18 individual stations, each of which uses contactless magnetic actuation and has an integrated Hall Effect sensing system, enabling the real-time measurements of applied field, force, and construct thickness, and hence, the indirect measurement of construct mechanical properties. Validation tests showed precise measurements of thickness, within 14 μm of gold standard calliper measurements; further, applied force was measured to be within 0.04 N of desired force over a half hour dynamic loading, which was repeatable over a 3-week test period. Finally, construct material properties measured using the bioreactor were not significantly different (p=0.97) from those measured using a standard materials testing machine. We present a new method for articular cartilage-specific bioreactor design, integrating combinatorial magneto-mechanostimulation, which is very attractive from functional and cost viewpoints.

Rotating three-dimensional dynamic culture of adult human bone marrow-derived cells for tissue engineering of hyaline cartilage.

PubMed

Sakai, Shinsuke; Mishima, Hajime; Ishii, Tomoo; Akaogi, Hiroshi; Yoshioka, Tomokazu; Ohyabu, Yoshimi; Chang, Fei; Ochiai, Naoyuki; Uemura, Toshimasa

2009-04-01

The method of constructing cartilage tissue from bone marrow-derived cells in vitro is considered a valuable technique for hyaline cartilage regenerative medicine. Using a rotating wall vessel (RWV) bioreactor developed in a NASA space experiment, we attempted to efficiently construct hyaline cartilage tissue from human bone marrow-derived cells without using a scaffold. Bone marrow aspirates were obtained from the iliac crest of nine patients during orthopedic operation. After their proliferation in monolayer culture, the adherent cells were cultured in the RWV bioreactor with chondrogenic medium for 2 weeks. Cells from the same source were cultured in pellet culture as controls. Histological and immunohistological evaluations (collagen type I and II) and quantification of glycosaminoglycan were performed on formed tissues and compared. The engineered constructs obtained using the RWV bioreactor showed strong features of hyaline cartilage in terms of their morphology as determined by histological and immunohistological evaluations. The glycosaminoglycan contents per microg DNA of the tissues were 10.01 +/- 3.49 microg/microg DNA in the case of the RWV bioreactor and 6.27 +/- 3.41 microg/microg DNA in the case of the pellet culture, and their difference was significant. The RWV bioreactor could provide an excellent environment for three-dimensional cartilage tissue architecture that can promote the chondrogenic differentiation of adult human bone marrow-derived cells.

Regeneration and Maintenance of Intestinal Smooth Muscle Phenotypes

NASA Astrophysics Data System (ADS)

Walthers, Christopher M.

Tissue engineering is an emerging field of biomedical engineering that involves growing artificial organs to replace those lost to disease or injury. Within tissue engineering, there is a demand for artificial smooth muscle to repair tissues of the digestive tract, bladder, and vascular systems. Attempts to develop engineered smooth muscle tissues capable of contracting with sufficient strength to be clinically relevant have so far proven unsatisfactory. The goal of this research was to develop and sustain mature, contractile smooth muscle. Survival of implanted SMCs is critical to sustain the benefits of engineered smooth muscle. Survival of implanted smooth muscle cells was studied with layered, electrospun polycaprolactone implants with lasercut holes ranging from 0--25% porosity. It was found that greater angiogenesis was associated with increased survival of implanted cells, with a large increase at a threshold between 20% and 25% porosity. Heparan sulfate coatings improved the speed of blood vessel infiltration after 14 days of implantation. With these considerations, thicker engineered tissues may be possible. An improved smooth muscle tissue culture technique was utilized. Contracting smooth muscle was produced in culture by maintaining the native smooth muscle tissue organization, specifically by sustaining intact smooth muscle strips rather than dissociating tissue in to isolated smooth muscle cells. Isolated cells showed a decrease in maturity and contained fewer enteric neural and glial cells. Muscle strips also exhibited periodic contraction and regular fluctuation of intracellular calclium. The muscle strip maturity persisted after implantation in omentum for 14 days on polycaprolactone scaffolds. A low-cost, disposable bioreactor was developed to further improve maturity of cultured smooth muscle cells in an environment of controlled cyclical stress.The bioreactor consistently applied repeated mechanical strain with controllable inputs for strain, frequency, and duty cycle. Cells grown on protein-conjugated silicone membranes showed a morphological change while undergoing bioreactor stress. Analyzing change in muscle strips undergoing bioreactor stress is an area for future research. The overall goal of this research was to move engineered smooth muscle towards tissues capable of contracting with physiologically relevant strength and frequency. This approach first increased survival of smooth muscle constructs, and then sought to improve contractile ability of smooth muscle cells.

Hydrofocusing Bioreactor for Three-Dimensional Cell Culture

NASA Technical Reports Server (NTRS)

Gonda, Steve R.; Spaulding, Glenn F.; Tsao, Yow-Min D.; Flechsig, Scott; Jones, Leslie; Soehnge, Holly

2003-01-01

The hydrodynamic focusing bioreactor (HFB) is a bioreactor system designed for three-dimensional cell culture and tissue-engineering investigations on orbiting spacecraft and in laboratories on Earth. The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear culture environment simultaneously with the "herding" of suspended cells, tissue assemblies, and air bubbles. Under development for use in the Biotechnology Facility on the International Space Station, the HFB has successfully grown large three-dimensional, tissuelike assemblies from anchorage-dependent cells and grown suspension hybridoma cells to high densities. The HFB, based on the principle of hydrodynamic focusing, provides the capability to control the movement of air bubbles and removes them from the bioreactor without degrading the low-shear culture environment or the suspended three-dimensional tissue assemblies. The HFB also provides unparalleled control over the locations of cells and tissues within its bioreactor vessel during operation and sampling.

Rapid Engineering of Three-Dimensional, Multicellular Tissues With Polymeric Scaffolds

NASA Technical Reports Server (NTRS)

Gonda, Steve R.; Jordan, Jacqueline; Fraga, Denise N.

2007-01-01

A process has been developed for the rapid tissue engineering of multicellular-tissue-equivalent assemblies by the controlled enzymatic degradation of polymeric beads in a low-fluid-shear bioreactor. In this process, the porous polymeric beads serve as temporary scaffolds to support the assemblies of cells in a tissuelike 3D configuration during the critical initial growth phases of attachment of anchorage-dependent cells, aggregation of the cells, and formation of a 3D extracellular matrix. Once the cells are assembled into a 3D array and enmeshed in a structural supportive 3D extracellular matrix (ECM), the polymeric scaffolds can be degraded in the low-fluid-shear environment of the NASA-designed bioreactor. The natural 3D tissuelike assembly, devoid of any artificial support structure, is maintained in the low-shear bioreactor environment by the newly formed natural cellular/ECM. The elimination of the artificial scaffold allows normal tissue structure and function.

Portable bioreactor for perfusion and electrical stimulation of engineered cardiac tissue.

PubMed

Tandon, Nina; Taubman, Alanna; Cimetta, Elisa; Saccenti, Laetitia; Vunjak-Novakovic, Gordana

2013-01-01

Cardiac tissue engineering aims to create functional tissue constructs that can reestablish the structure and function of injured myocardium. Although bioreactors have facilitated the engineering of cardiac patches of clinically relevant size in vitro, a major drawback remains the transportation of the engineered tissues from a production facility to a medical operation facility while maintaining tissue viability and preventing contamination. Furthermore, after implantation, most of the cells are endangered by hypoxic conditions that exist before vascular flow is established. We developed a portable device that provides the perfusion and electrical stimulation necessary to engineer cardiac tissue in vitro, and to transport it to the site where it will be implantated. The micropump-powered perfusion apparatus may additionally function as an extracorporeal active pumping system providing nutrients and oxygen supply to the graft post-implantation. Such a system, through perfusion of oxygenated media and bioactive molecules (e.g. growth factors), could transiently support the tissue construct until it connects to the host vasculature and heart muscle, after which it could be taken away or let biodegrade.

Challenges in engineering osteochondral tissue grafts with hierarchical structures.

PubMed

Gadjanski, Ivana; Vunjak-Novakovic, Gordana

2015-01-01

A major hurdle in treating osteochondral (OC) defects is the different healing abilities of two types of tissues involved - articular cartilage and subchondral bone. Biomimetic approaches to OC-construct engineering, based on recapitulation of biological principles of tissue development and regeneration, have potential for providing new treatments and advancing fundamental studies of OC tissue repair. This review on state of the art in hierarchical OC tissue graft engineering is focused on tissue engineering approaches designed to recapitulate the native milieu of cartilage and bone development. These biomimetic systems are discussed with relevance to bioreactor cultivation of clinically sized, anatomically shaped human cartilage/bone constructs with physiologic stratification and mechanical properties. The utility of engineered OC tissue constructs is evaluated for their use as grafts in regenerative medicine, and as high-fidelity models in biological research. A major challenge in engineering OC tissues is to generate a functionally integrated stratified cartilage-bone structure starting from one single population of mesenchymal cells, while incorporating perfusable vasculature into the bone, and in bone-cartilage interface. To this end, new generations of advanced scaffolds and bioreactors, implementation of mechanical loading regimens and harnessing of inflammatory responses of the host will likely drive the further progress.

Engineering large cartilage tissues using dynamic bioreactor culture at defined oxygen conditions.

PubMed

Daly, Andrew C; Sathy, Binulal N; Kelly, Daniel J

2018-01-01

Mesenchymal stem cells maintained in appropriate culture conditions are capable of producing robust cartilage tissue. However, gradients in nutrient availability that arise during three-dimensional culture can result in the development of spatially inhomogeneous cartilage tissues with core regions devoid of matrix. Previous attempts at developing dynamic culture systems to overcome these limitations have reported suppression of mesenchymal stem cell chondrogenesis compared to static conditions. We hypothesize that by modulating oxygen availability during bioreactor culture, it is possible to engineer cartilage tissues of scale. The objective of this study was to determine whether dynamic bioreactor culture, at defined oxygen conditions, could facilitate the development of large, spatially homogeneous cartilage tissues using mesenchymal stem cell laden hydrogels. A dynamic culture regime was directly compared to static conditions for its capacity to support chondrogenesis of mesenchymal stem cells in both small and large alginate hydrogels. The influence of external oxygen tension on the response to the dynamic culture conditions was explored by performing the experiment at 20% O 2 and 3% O 2 . At 20% O 2 , dynamic culture significantly suppressed chondrogenesis in engineered tissues of all sizes. In contrast, at 3% O 2 dynamic culture significantly enhanced the distribution and amount of cartilage matrix components (sulphated glycosaminoglycan and collagen II) in larger constructs compared to static conditions. Taken together, these results demonstrate that dynamic culture regimes that provide adequate nutrient availability and a low oxygen environment can be employed to engineer large homogeneous cartilage tissues. Such culture systems could facilitate the scaling up of cartilage tissue engineering strategies towards clinically relevant dimensions.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Exterior view of the NASA Bioreactor Engineering Development Unit flown on Mir. The rotating wall vessel is behind the window on the face of the large module. Control electronics are in the module at left; gas supply and cooling fans are in the module at back. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Biocompatible, biodegradable polymer-based, lighter than or light as water scaffolds for tissue engineering and methods for preparation and use thereof

NASA Technical Reports Server (NTRS)

Khan, Mohammed Yusuf (Inventor); Laurencin, Cato T. (Inventor); Lu, Helen H. (Inventor); Botchwey, Edward (Inventor); Pollack, Solomon R. (Inventor); Levine, Elliot (Inventor)

2012-01-01

Scaffolds for tissue engineering prepared from biocompatible, biodegradable polymer-based, lighter than or light as water microcarriers and designed for cell culturing in vitro in a rotating bioreactor are provided. Methods for preparation and use of these scaffolds as tissue engineering devices are also provided.

Mechanical stretching for tissue engineering: two-dimensional and three-dimensional constructs.

PubMed

Riehl, Brandon D; Park, Jae-Hong; Kwon, Il Keun; Lim, Jung Yul

2012-08-01

Mechanical cell stretching may be an attractive strategy for the tissue engineering of mechanically functional tissues. It has been demonstrated that cell growth and differentiation can be guided by cell stretch with minimal help from soluble factors and engineered tissues that are mechanically stretched in bioreactors may have superior organization, functionality, and strength compared with unstretched counterparts. This review explores recent studies on cell stretching in both two-dimensional (2D) and three-dimensional (3D) setups focusing on the applications of stretch stimulation as a tool for controlling cell orientation, growth, gene expression, lineage commitment, and differentiation and for achieving successful tissue engineering of mechanically functional tissues, including cardiac, muscle, vasculature, ligament, tendon, bone, and so on. Custom stretching devices and lab-specific mechanical bioreactors are described with a discussion on capabilities and limitations. While stretch mechanotransduction pathways have been examined using 2D stretch, studying such pathways in physiologically relevant 3D environments may be required to understand how cells direct tissue development under stretch. Cell stretch study using 3D milieus may also help to develop tissue-specific stretch regimens optimized with biochemical feedback, which once developed will provide optimal tissue engineering protocols.

Mechanical Stretching for Tissue Engineering: Two-Dimensional and Three-Dimensional Constructs

PubMed Central

Riehl, Brandon D.; Park, Jae-Hong; Kwon, Il Keun

2012-01-01

Mechanical cell stretching may be an attractive strategy for the tissue engineering of mechanically functional tissues. It has been demonstrated that cell growth and differentiation can be guided by cell stretch with minimal help from soluble factors and engineered tissues that are mechanically stretched in bioreactors may have superior organization, functionality, and strength compared with unstretched counterparts. This review explores recent studies on cell stretching in both two-dimensional (2D) and three-dimensional (3D) setups focusing on the applications of stretch stimulation as a tool for controlling cell orientation, growth, gene expression, lineage commitment, and differentiation and for achieving successful tissue engineering of mechanically functional tissues, including cardiac, muscle, vasculature, ligament, tendon, bone, and so on. Custom stretching devices and lab-specific mechanical bioreactors are described with a discussion on capabilities and limitations. While stretch mechanotransduction pathways have been examined using 2D stretch, studying such pathways in physiologically relevant 3D environments may be required to understand how cells direct tissue development under stretch. Cell stretch study using 3D milieus may also help to develop tissue-specific stretch regimens optimized with biochemical feedback, which once developed will provide optimal tissue engineering protocols. PMID:22335794

Recent Advances in Tissue Engineering Strategies for the Treatment of Joint Damage.

PubMed

Stephenson, Makeda K; Farris, Ashley L; Grayson, Warren L

2017-08-01

While the clinical potential of tissue engineering for treating joint damage has yet to be realized, research and commercialization efforts in the field are geared towards overcoming major obstacles to clinical translation, as well as towards achieving engineered grafts that recapitulate the unique structures, function, and physiology of the joint. In this review, we describe recent advances in technologies aimed at obtaining biomaterials, stem cells, and bioreactors that will enable the development of effective tissue-engineered treatments for repairing joint damage. 3D printing of scaffolds is aimed at improving the mechanical structure and microenvironment necessary for bone regeneration within a damaged joint. Advances in our understanding of stem cell biology and cell manufacturing processes are informing translational strategies for the therapeutic use of allogeneic and autologous cells. Finally, bioreactors used in combination with cells and biomaterials are promising strategies for generating large tissue grafts for repairing damaged tissues in pre-clinical models. Together, these advances along with ongoing research directions are making tissue engineering increasingly viable for the treatment of joint damage.

Large Scale Expansion of Human Umbilical Cord Cells in a Rotating Bed System Bioreactor for Cardiovascular Tissue Engineering Applications

PubMed Central

Reichardt, Anne; Polchow, Bianca; Shakibaei, Mehdi; Henrich, Wolfgang; Hetzer, Roland; Lueders, Cora

2013-01-01

Widespread use of human umbilical cord cells for cardiovascular tissue engineering requires production of large numbers of well-characterized cells under controlled conditions. In current research projects, the expansion of cells to be used to create a tissue construct is usually performed in static cell culture systems which are, however, often not satisfactory due to limitations in nutrient and oxygen supply. To overcome these limitations dynamic cell expansion in bioreactor systems under controllable conditions could be an important tool providing continuous perfusion for the generation of large numbers of viable pre-conditioned cells in a short time period. For this purpose cells derived from human umbilical cord arteries were expanded in a rotating bed system bioreactor for up to 9 days. For a comparative study, cells were cultivated under static conditions in standard culture devices. Our results demonstrated that the microenvironment in the perfusion bioreactor was more favorable than that of the standard cell culture flasks. Data suggested that cells in the bioreactor expanded 39 fold (38.7 ± 6.1 fold) in comparison to statically cultured cells (31.8 ± 3.0 fold). Large-scale production of cells in the bioreactor resulted in more than 3 x 108 cells from a single umbilical cord fragment within 9 days. Furthermore cell doubling time was lower in the bioreactor system and production of extracellular matrix components was higher. With this study, we present an appropriate method to expand human umbilical cord artery derived cells with high cellular proliferation rates in a well-defined bioreactor system under GMP conditions. PMID:23847691

Mechanostimulation protocols for cardiac tissue engineering.

PubMed

Govoni, Marco; Muscari, Claudio; Guarnieri, Carlo; Giordano, Emanuele

2013-01-01

Owing to the inability of self-replacement by a damaged myocardium, alternative strategies to heart transplantation have been explored within the last decades and cardiac tissue engineering/regenerative medicine is among the present challenges in biomedical research. Hopefully, several studies witness the constant extension of the toolbox available to engineer a fully functional, contractile, and robust cardiac tissue using different combinations of cells, template bioscaffolds, and biophysical stimuli obtained by the use of specific bioreactors. Mechanical forces influence the growth and shape of every tissue in our body generating changes in intracellular biochemistry and gene expression. That is why bioreactors play a central role in the task of regenerating a complex tissue such as the myocardium. In the last fifteen years a large number of dynamic culture devices have been developed and many results have been collected. The aim of this brief review is to resume in a single streamlined paper the state of the art in this field.

Mechanostimulation Protocols for Cardiac Tissue Engineering

PubMed Central

Govoni, Marco; Muscari, Claudio; Guarnieri, Carlo; Giordano, Emanuele

2013-01-01

Owing to the inability of self-replacement by a damaged myocardium, alternative strategies to heart transplantation have been explored within the last decades and cardiac tissue engineering/regenerative medicine is among the present challenges in biomedical research. Hopefully, several studies witness the constant extension of the toolbox available to engineer a fully functional, contractile, and robust cardiac tissue using different combinations of cells, template bioscaffolds, and biophysical stimuli obtained by the use of specific bioreactors. Mechanical forces influence the growth and shape of every tissue in our body generating changes in intracellular biochemistry and gene expression. That is why bioreactors play a central role in the task of regenerating a complex tissue such as the myocardium. In the last fifteen years a large number of dynamic culture devices have been developed and many results have been collected. The aim of this brief review is to resume in a single streamlined paper the state of the art in this field. PMID:23936858

Improved repair of bone defects with prevascularized tissue-engineered bones constructed in a perfusion bioreactor.

PubMed

Li, De-Qiang; Li, Ming; Liu, Pei-Lai; Zhang, Yuan-Kai; Lu, Jian-Xi; Li, Jian-Min

2014-10-01

Vascularization of tissue-engineered bones is critical to achieving satisfactory repair of bone defects. The authors investigated the use of prevascularized tissue-engineered bone for repairing bone defects. The new bone was greater in the prevascularized group than in the non-vascularized group, indicating that prevascularized tissue-engineered bone improves the repair of bone defects. [Orthopedics. 2014; 37(10):685-690.]. Copyright 2014, SLACK Incorporated.

[Engineering a bone free flap for maxillofacial reconstruction: technical restrictions].

PubMed

Raoul, G; Myon, L; Chai, F; Blanchemain, N; Ferri, J

2011-09-01

Vascularisation is a key for success in bone tissue engineering. Creating a functional vascular network is an important concern so as to ensure vitality in regenerated tissues. Many strategies were developed to achieve this goal. One of these is cellular growth technique by perfusion bioreactor chamber. These new technical requirements came along with improved media and chamber receptacles: bioreactors (chapter 2). Some bone tissue engineering processes already have clinical applications but for volumes limited by the lack of vascularisation. Resorbable or non-resorbable membranes are an example. They are used separately or in association with bone grafts and they protect the graft during the revascularization process. Potentiated osseous regeneration uses molecular or cellular adjuvants (BMPs and autologous stem cells) to improve osseous healing. Significant improvements were made: integration of specific sequences, which may guide and enhance cells differentiation in scaffold; nano- or micro-patterned cell containing scaffolds. Finally, some authors consider the patient body as an ideal bioreactor to induce vascularisation in large volumes of grafted tissues. "Endocultivation", i.e., cellular culture inside the human body was proven to be feasible and safe. The properties of regenerated bone in the long run remain to be assessed. The objective to reach remains the engineering of an "in vitro" osseous free flap without morbidity. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

NASA Bioreactor tissue culture

NASA Technical Reports Server (NTRS)

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue

PubMed Central

Lee, Eun Jung; Luo, Jianwen; Duan, Yi; Yeager, Keith; Konofagou, Elisa; Vunjak-Novakovic, Gordana

2012-01-01

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue, but due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation, and unconstrained (i.e., not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate in concert these three key factors. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modeling studies. We then culture cardiac cells obtained from neonatal rats in porous, channeled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After eight days of culture, constructs grown with the simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23±0.10% vs. 0.14±0.05, 0.13±0.08, or 0.09±0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization than either control group. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs. PMID:22170772

Role of Bioreactor Technology in Tissue Engineering for Clinical Use and Therapeutic Target Design.

PubMed

Selden, Clare; Fuller, Barry

2018-04-24

Micro and small bioreactors are well described for use in bioprocess development in pre-production manufacture, using ultra-scale down and microfluidic methodology. However, the use of bioreactors to understand normal and pathophysiology by definition must be very different, and the constraints of the physiological environment influence such bioreactor design. This review considers the key elements necessary to enable bioreactors to address three main areas associated with biological systems. All entail recreation of the in vivo cell niche as faithfully as possible, so that they may be used to study molecular and cellular changes in normal physiology, with a view to creating tissue-engineered grafts for clinical use; understanding the pathophysiology of disease at the molecular level; defining possible therapeutic targets; and enabling appropriate pharmaceutical testing on a truly representative organoid, thus enabling better drug design, and simultaneously creating the potential to reduce the numbers of animals in research. The premise explored is that not only cellular signalling cues, but also mechano-transduction from mechanical cues, play an important role.

Microgravity

NASA Image and Video Library

1996-01-01

Exterior view of the NASA Bioreactor Engineering Development Unit flown on Mir. The rotating wall vessel is behind the window on the face of the large module. Control electronics are in the module at left; gas supply and cooling fans are in the module at back. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Cytoskeletal and functional changes in bioreactor assembled thyroid tissue organoids exposed to gamma radiation

NASA Technical Reports Server (NTRS)

Green, Lora M.; Patel, Zarana; Murray, Deborah K.; Rightnar, Steven; Burell, Cheryl G.; Gridley, Daila S.; Nelson, Gregory A.

2002-01-01

Fischer rat thyroid cells were grown under low-shear stress in a bioreactor to a stage of organization composed of integrated follicles resembling small thyroid glands prior to exposure to 3 Gray-gamma radiation. Bioreactor tissues and controls (both irradiated and non-irradiated) were harvested at 24, 48, 96 and 144 hours post-exposure. Tissue samples were fixed and fluorescently labeled for actin and microtubules. Tissues were assessed for changes in cytoskeletal components induced by radiation and quantified by laser scanning cytometry. ELISA's were used to quantify transforming growth factor-beta and thyroxin released from cells to the culture supernatant. Tissue architecture was disrupted by exposure to radiation with the structural organization of actin and loss of follicular content the most obviously affected. With time post-irradiation the actin appeared disordered and the levels of fluorescence associated with filamentous-actin and microtubules cycled in the tissue analogs, but not in the flask-grown cultures. Active transforming growth factor-beta was higher in supernatants from the irradiated bioreactor tissue. Thyroxin release paralleled cell survival in the bioreactors and control cultures. Thus, the engineered tissue responses to radiation differed from those of conventional tissue culture making it a potentially better mimic of the in vivo situation.

Challenges in engineering osteochondral tissue grafts with hierarchical structures Ivana Gadjanski, Gordana Vunjak Novakovic

PubMed Central

Gadjanski, Ivana; Vunjak-Novakovic, Gordana

2015-01-01

Introduction A major hurdle in treating osteochondral (OC) defects are the different healing abilities of two types of tissues involved - articular cartilage and subchondral bone. Biomimetic approaches to OC-construct-engineering, based on recapitulation of biological principles of tissue development and regeneration, have potential for providing new treatments and advancing fundamental studies of OC tissue repair. Areas covered This review on state of the art in hierarchical OC tissue graft engineering is focused on tissue engineering approaches designed to recapitulate the native milieu of cartilage and bone development. These biomimetic systems are discussed with relevance to bioreactor cultivation of clinically sized, anatomically shaped human cartilage/bone constructs with physiologic stratification and mechanical properties. The utility of engineered OC tissue constructs is evaluated for their use as grafts in regenerative medicine, and as high-fidelity models in biological research. Expert opinion A major challenge in engineering OC tissues is to generate a functionally integrated stratified cartilage-bone structure starting from one single population of mesenchymal cells, while incorporating perfusable vasculature into the bone, and in bone-cartilage interface. To this end, new generations of advanced scaffolds and bioreactors, implementation of mechanical loading regimens, and harnessing of inflammatory responses of the host will likely drive the further progress. PMID:26195329

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain.

PubMed

Heher, Philipp; Maleiner, Babette; Prüller, Johanna; Teuschl, Andreas Herbert; Kollmitzer, Josef; Monforte, Xavier; Wolbank, Susanne; Redl, Heinz; Rünzler, Dominik; Fuchs, Christiane

2015-09-01

The generation of functional biomimetic skeletal muscle constructs is still one of the fundamental challenges in skeletal muscle tissue engineering. With the notion that structure strongly dictates functional capabilities, a myriad of cell types, scaffold materials and stimulation strategies have been combined. To further optimize muscle engineered constructs, we have developed a novel bioreactor system (MagneTissue) for rapid engineering of skeletal muscle-like constructs with the aim to resemble native muscle in terms of structure, gene expression profile and maturity. Myoblasts embedded in fibrin, a natural hydrogel that serves as extracellular matrix, are subjected to mechanical stimulation via magnetic force transmission. We identify static mechanical strain as a trigger for cellular alignment concomitant with the orientation of the scaffold into highly organized fibrin fibrils. This ultimately yields myotubes with a more mature phenotype in terms of sarcomeric patterning, diameter and length. On the molecular level, a faster progression of the myogenic gene expression program is evident as myogenic determination markers MyoD and Myogenin as well as the Ca(2+) dependent contractile structural marker TnnT1 are significantly upregulated when strain is applied. The major advantage of the MagneTissue bioreactor system is that the generated tension is not exclusively relying on the strain generated by the cells themselves in response to scaffold anchoring but its ability to subject the constructs to individually adjustable strain protocols. In future work, this will allow applying mechanical stimulation with different strain regimes in the maturation process of tissue engineered constructs and elucidating the role of mechanotransduction in myogenesis. Mechanical stimulation of tissue engineered skeletal muscle constructs is a promising approach to increase tissue functionality. We have developed a novel bioreactor-based 3D culture system, giving the user the possibility to apply different strain regimes like static, cyclic or ramp strain to myogenic precursor cells embedded in a fibrin scaffold. Application of static mechanical strain leads to alignment of fibrin fibrils along the axis of strain and concomitantly to highly aligned myotube formation. Additionally, the pattern of myogenic gene expression follows the temporal progression observed in vivo with a more thorough induction of the myogenic program when static strain is applied. Ultimately, the strain protocol used in this study results in a higher degree of muscle maturity demonstrated by enhanced sarcomeric patterning and increased myotube diameter and length. The introduced bioreactor system enables new possibilities in muscle tissue engineering as longer cultivation periods and different strain applications will yield tissue engineered muscle-like constructs with improved characteristics in regard to functionality and biomimicry. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Comparison of Simulated Microgravity and Hydrostatic Pressure for Chondrogenesis of hASC.

PubMed

Mellor, Liliana F; Steward, Andrew J; Nordberg, Rachel C; Taylor, Michael A; Loboa, Elizabeth G

2017-04-01

Cartilage tissue engineering is a growing field due to the lack of regenerative capacity of native tissue. The use of bioreactors for cartilage tissue engineering is common, but the results are controversial. Some studies suggest that microgravity bioreactors are ideal for chondrogenesis, while others show that mimicking hydrostatic pressure is crucial for cartilage formation. A parallel study comparing the effects of loading and unloading on chondrogenesis has not been performed. The goal of this study was to evaluate chondrogenesis of human adipose-derived stem cells (hASC) under two different mechanical stimuli relative to static culture: microgravity and cyclic hydrostatic pressure (CHP). Pellets of hASC were cultured for 14 d under simulated microgravity using a rotating wall vessel bioreactor or under CHP (7.5 MPa, 1 Hz, 4 h · d-1) using a hydrostatic pressure vessel. We found that CHP increased mRNA expression of Aggrecan, Sox9, and Collagen II, caused a threefold increase in sulfated glycosaminoglycan production, and resulted in stronger vimentin staining intensity and organization relative to microgravity. In addition, Wnt-signaling patterns were altered in a manner that suggests that simulated microgravity decreases chondrogenic differentiation when compared to CHP. Our goal was to compare chondrogenic differentiation of hASC using a microgravity bioreactor and a hydrostatic pressure vessel, two commonly used bioreactors in cartilage tissue engineering. Our results indicate that CHP promotes hASC chondrogenesis and that microgravity may inhibit hASC chondrogenesis. Our findings further suggest that cartilage formation and regeneration might be compromised in space due to the lack of mechanical loading.Mellor LF, Steward AJ, Nordberg RC, Taylor MA, Loboa EG. Comparison of simulated microgravity and hydrostatic pressure for chondrogenesis of hASC. Aerosp Med Hum Perform. 2017; 88(4):377-384.

Design of a novel bioreactor and application in vascular tissue engineering

NASA Astrophysics Data System (ADS)

Zhang, Zhi-Xiong; Xi, Ting-Fei; Wang, Ying-Jun; Chen, Xiao-Song; Zhang, Jian; Wang, Chun-Ren; Gu, Yong-Quan; Chen, Liang; Li, Jian-Xin; Chen, Bing

2008-11-01

Endothelial cells (ECs) detachment under high shear stress at the early period of transplantation resulted in thrombosis and occlusion. To solve this problem, we developed a novel bioreactor. The bioreactor mimicked the formation of pulsatile flow in physiological conditions. Human umbilical vein ECs were seeded onto the lumen of living tissue conduits grown within dog peritoneal cavity. The shear stress generated by the bioreactor was increased step by step from 1.5 ± 0.8 dyn/cm 2 to 5.3 ± 2.4 dyn/cm 2, and was applied to ECs after static culture for 2 days. The results showed that completely confluent monolayer ECs were elongated, and were oriented parallel to the flow direction. The bioreactor could provide good environment for formation of endothelium. Stepwise increase shear stress could strengthen cell-cell and cell-extracellular matrix. The flow conditions of the bioreactor play a key role to determine the quality of the ECs lining.

Novel technique for online characterization of cartilaginous tissue properties.

PubMed

Yuan, Tai-Yi; Huang, Chun-Yuh; Yong Gu, Wei

2011-09-01

The goal of tissue engineering is to use substitutes to repair and restore organ function. Bioreactors are an indispensable tool for monitoring and controlling the unique environment for engineered constructs to grow. However, in order to determine the biochemical properties of engineered constructs, samples need to be destroyed. In this study, we developed a novel technique to nondestructively online-characterize the water content and fixed charge density of cartilaginous tissues. A new technique was developed to determine the tissue mechano-electrochemical properties nondestructively. Bovine knee articular cartilage and lumbar annulus fibrosus were used in this study to demonstrate that this technique could be used on different types of tissue. The results show that our newly developed method is capable of precisely predicting the water volume fraction (less than 3% disparity) and fixed charge density (less than 16.7% disparity) within cartilaginous tissues. This novel technique will help to design a new generation of bioreactors which are able to actively determine the essential properties of the engineered constructs, as well as regulate the local environment to achieve the optimal conditions for cultivating constructs.

Bioreactors for guiding muscle tissue growth and development.

PubMed

Dennis, R G; Smith, B; Philp, A; Donnelly, K; Baar, K

2009-01-01

Muscle tissue bioreactors are devices which are employed to guide and monitor the development of engineered muscle tissue. These devices have a modern history that can be traced back more than a century, because the key elements of muscle tissue bioreactors have been studied for a very long time. These include barrier isolation and culture of cells, tissues and organs after isolation from a host organism; the provision of various stimuli intended to promote growth and maintain the muscle, such as electrical and mechanical stimulation; and the provision of a perfusate such as culture media or blood derived substances. An accurate appraisal of our current progress in the development of muscle bioreactors can only be made in the context of the history of this endeavor. Modern efforts tend to focus more upon the use of computer control and the application of mechanical strain as a stimulus, as well as substrate surface modifications to induce cellular organization at the early stages of culture of isolated muscle cells.

Tissue-engineered bone constructed in a bioreactor for repairing critical-sized bone defects in sheep.

PubMed

Li, Deqiang; Li, Ming; Liu, Peilai; Zhang, Yuankai; Lu, Jianxi; Li, Jianmin

2014-11-01

Repair of bone defects, particularly critical-sized bone defects, is a considerable challenge in orthopaedics. Tissue-engineered bones provide an effective approach. However, previous studies mainly focused on the repair of bone defects in small animals. For better clinical application, repairing critical-sized bone defects in large animals must be studied. This study investigated the effect of a tissue-engineered bone for repairing critical-sized bone defect in sheep. A tissue-engineered bone was constructed by culturing bone marrow mesenchymal-stem-cell-derived osteoblast cells seeded in a porous β-tricalcium phosphate ceramic (β-TCP) scaffold in a perfusion bioreactor. A critical-sized bone defect in sheep was repaired with the tissue-engineered bone. At the eighth and 16th week after the implantation of the tissue-engineered bone, X-ray examination and histological analysis were performed to evaluate the defect. The bone defect with only the β-TCP scaffold served as the control. X-ray showed that the bone defect was successfully repaired 16 weeks after implantation of the tissue-engineered bone; histological sections showed that a sufficient volume of new bones formed in β-TCP 16 weeks after implantation. Eight and 16 weeks after implantation, the volume of new bones that formed in the tissue-engineered bone group was more than that in the β-TCP scaffold group (P < 0.05). Tissue-engineered bone improved osteogenesis in vivo and enhanced the ability to repair critical-sized bone defects in large animals.

Biofabrication of customized bone grafts by combination of additive manufacturing and bioreactor knowhow.

PubMed

Costa, Pedro F; Vaquette, Cédryck; Baldwin, Jeremy; Chhaya, Mohit; Gomes, Manuela E; Reis, Rui L; Theodoropoulos, Christina; Hutmacher, Dietmar W

2014-09-01

This study reports on an original concept of additive manufacturing for the fabrication of tissue engineered constructs (TEC), offering the possibility of concomitantly manufacturing a customized scaffold and a bioreactor chamber to any size and shape. As a proof of concept towards the development of anatomically relevant TECs, this concept was utilized for the design and fabrication of a highly porous sheep tibia scaffold around which a bioreactor chamber of similar shape was simultaneously built. The morphology of the bioreactor/scaffold device was investigated by micro-computed tomography and scanning electron microscopy confirming the porous architecture of the sheep tibiae as opposed to the non-porous nature of the bioreactor chamber. Additionally, this study demonstrates that both the shape, as well as the inner architecture of the device can significantly impact the perfusion of fluid within the scaffold architecture. Indeed, fluid flow modelling revealed that this was of significant importance for controlling the nutrition flow pattern within the scaffold and the bioreactor chamber, avoiding the formation of stagnant flow regions detrimental for in vitro tissue development. The bioreactor/scaffold device was dynamically seeded with human primary osteoblasts and cultured under bi-directional perfusion for two and six weeks. Primary human osteoblasts were observed homogenously distributed throughout the scaffold, and were viable for the six week culture period. This work demonstrates a novel application for additive manufacturing in the development of scaffolds and bioreactors. Given the intrinsic flexibility of the additive manufacturing technology platform developed, more complex culture systems can be fabricated which would contribute to the advances in customized and patient-specific tissue engineering strategies for a wide range of applications.

Design and Fabrication of Anatomical Bioreactor Systems Containing Alginate Scaffolds for Cartilage Tissue Engineering

PubMed Central

Gharravi, Anneh Mohammad; Orazizadeh, Mahmoud; Ansari-Asl, Karim; Banoni, Salem; Izadi, Sina; Hashemitabar, Mahmoud

2012-01-01

The aim of the present study was to develop a tissue-engineering approach through alginate gel molding to mimic cartilage tissue in a three-dimensional culture system. The perfusion biomimetic bioreactor was designed to mimic natural joint. The shear stresses exerting on the bioreactor chamber were calculated by Computational Fluid Dynamic (CFD). Several alginate/bovine chondrocyte constructs were prepared, and were cultured in the bioreactor. Histochemical and immunohistochemical staining methods for the presence of glycosaminoglycan(GAG), overall matrix production and type II collagen protein were performed, respectively. The dynamic mechanical device applied a linear mechanical displacement of 2 mm to 10 mm. The CFD modeling indicated peak velocity and maximum wall shear stress were 1.706×10−3 m/s and 0.02407 dyne/cm 2, respectively. Histochemical and immunohistochemical analysis revealed evidence of cartilage-like tissue with lacunas similar to those of natural cartilage and the production of sulfated GAG of matrix by the chondrons, metachromatic territorial matrix-surrounded cells and accumulation of type II collagen around the cells. The present study indicated that when chondrocytes were seeded in alginate hydrogel and cultured in biomimetic cell culture system, cells survived well and secreted newly synthesized matrix led to improvement of chondrogenesis. PMID:23408660

In Vitro Engineering of Vascularized Tissue Surrogates

PubMed Central

Sakaguchi, Katsuhisa; Shimizu, Tatsuya; Horaguchi, Shigeto; Sekine, Hidekazu; Yamato, Masayuki; Umezu, Mitsuo; Okano, Teruo

2013-01-01

In vitro scaling up of bioengineered tissues is known to be limited by diffusion issues, specifically a lack of vasculature. Here, we report a new strategy for preserving cell viability in three-dimensional tissues using cell sheet technology and a perfusion bioreactor having collagen-based microchannels. When triple-layer cardiac cell sheets are incubated within this bioreactor, endothelial cells in the cell sheets migrate to vascularize in the collagen gel, and finally connect with the microchannels. Medium readily flows into the cell sheets through the microchannels and the newly developed capillaries, while the cardiac construct shows simultaneous beating. When additional triple-layer cell sheets are repeatedly layered, new multi-layer construct spontaneously integrates and the resulting construct becomes a vascularized thick tissue. These results confirmed our method to fabricate in vitro vascularized tissue surrogates that overcomes engineered-tissue thickness limitations. The surrogates promise new therapies for damaged organs as well as new in vitro tissue models. PMID:23419835

Transport Advances in Disposable Bioreactors for Liver Tissue Engineering

NASA Astrophysics Data System (ADS)

Catapano, Gerardo; Patzer, John F.; Gerlach, Jörg Christian

Acute liver failure (ALF) is a devastating diagnosis with an overall survival of approximately 60%. Liver transplantation is the therapy of choice for ALF patients but is limited by the scarce availability of donor organs. The prognosis of ALF patients may improve if essential liver functions are restored during liver failure by means of auxiliary methods because liver tissue has the capability to regenerate and heal. Bioartificial liver (BAL) approaches use liver tissue or cells to provide ALF patients with liver-specific metabolism and synthesis products necessary to relieve some of the symptoms and to promote liver tissue regeneration. The most promising BAL treatments are based on the culture of tissue engineered (TE) liver constructs, with mature liver cells or cells that may differentiate into hepatocytes to perform liver-specific functions, in disposable continuous-flow bioreactors. In fact, adult hepatocytes perform all essential liver functions. Clinical evaluations of the proposed BALs show that they are safe but have not clearly proven the efficacy of treatment as compared to standard supportive treatments. Ambiguous clinical results, the time loss of cellular activity during treatment, and the presence of a necrotic core in the cell compartment of many bioreactors suggest that improvement of transport of nutrients, and metabolic wastes and products to or from the cells in the bioreactor is critical for the development of therapeutically effective BALs. In this chapter, advanced strategies that have been proposed over to improve mass transport in the bioreactors at the core of a BAL for the treatment of ALF patients are reviewed.

Biomimetic perfusion and electrical stimulation applied in concert improved the assembly of engineered cardiac tissue.

PubMed

Maidhof, Robert; Tandon, Nina; Lee, Eun Jung; Luo, Jianwen; Duan, Yi; Yeager, Keith; Konofagou, Elisa; Vunjak-Novakovic, Gordana

2012-11-01

Maintenance of normal myocardial function depends intimately on synchronous tissue contraction, driven by electrical activation and on adequate nutrient perfusion in support thereof. Bioreactors have been used to mimic aspects of these factors in vitro to engineer cardiac tissue but, due to design limitations, previous bioreactor systems have yet to simultaneously support nutrient perfusion, electrical stimulation and unconstrained (i.e. not isometric) tissue contraction. To the best of our knowledge, the bioreactor system described herein is the first to integrate these three key factors in concert. We present the design of our bioreactor and characterize its capability in integrated experimental and mathematical modelling studies. We then cultured cardiac cells obtained from neonatal rats in porous, channelled elastomer scaffolds with the simultaneous application of perfusion and electrical stimulation, with controls excluding either one or both of these two conditions. After 8 days of culture, constructs grown with simultaneous perfusion and electrical stimulation exhibited substantially improved functional properties, as evidenced by a significant increase in contraction amplitude (0.23 ± 0.10% vs 0.14 ± 0.05%, 0.13 ± 0.08% or 0.09 ± 0.02% in control constructs grown without stimulation, without perfusion, or either stimulation or perfusion, respectively). Consistently, these constructs had significantly improved DNA contents, cell distribution throughout the scaffold thickness, cardiac protein expression, cell morphology and overall tissue organization compared to control groups. Thus, the simultaneous application of medium perfusion and electrical conditioning enabled by the use of the novel bioreactor system may accelerate the generation of fully functional, clinically sized cardiac tissue constructs. Copyright © 2011 John Wiley & Sons, Ltd.

Oscillating Cell Culture Bioreactor

NASA Technical Reports Server (NTRS)

Freed, Lisa E.; Cheng, Mingyu; Moretti, Matteo G.

2010-01-01

To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies.

A multiphysics 3D model of tissue growth under interstitial perfusion in a tissue-engineering bioreactor.

PubMed

Nava, Michele M; Raimondi, Manuela T; Pietrabissa, Riccardo

2013-11-01

The main challenge in engineered cartilage consists in understanding and controlling the growth process towards a functional tissue. Mathematical and computational modelling can help in the optimal design of the bioreactor configuration and in a quantitative understanding of important culture parameters. In this work, we present a multiphysics computational model for the prediction of cartilage tissue growth in an interstitial perfusion bioreactor. The model consists of two separate sub-models, one two-dimensional (2D) sub-model and one three-dimensional (3D) sub-model, which are coupled between each other. These sub-models account both for the hydrodynamic microenvironment imposed by the bioreactor, using a model based on the Navier-Stokes equation, the mass transport equation and the biomass growth. The biomass, assumed as a phase comprising cells and the synthesised extracellular matrix, has been modelled by using a moving boundary approach. In particular, the boundary at the fluid-biomass interface is moving with a velocity depending from the local oxygen concentration and viscous stress. In this work, we show that all parameters predicted, such as oxygen concentration and wall shear stress, by the 2D sub-model with respect to the ones predicted by the 3D sub-model are systematically overestimated and thus the tissue growth, which directly depends on these parameters. This implies that further predictive models for tissue growth should take into account of the three dimensionality of the problem for any scaffold microarchitecture.

A factorial design to identify process parameters affecting whole mechanically disrupted rat pancreata in a perfusion bioreactor.

PubMed

Sharp, Jamie; Spitters, Tim Wgm; Vermette, Patrick

2018-03-01

Few studies report whole pancreatic tissue culture, as it is a difficult task using traditional culture methods. Here, a factorial design was used to investigate the singular and combinational effects of flow, dissolved oxygen concentration (D.O.) and pulsation on whole mechanically disrupted rat pancreata in a perfusion bioreactor. Whole rat pancreata were cultured for 72 h under defined bioreactor process conditions. Secreted insulin was measured and histological (haematoxylin and eosin (H&E)) as well as immunofluorescent insulin staining were performed and quantified. The combination of flow and D.O. had the most significant effect on secreted insulin at 5 h and 24 h. The D.O. had the biggest effect on tissue histological quality, and pulsation had the biggest effect on the number of insulin-positive structures. Based on the factorial design analysis, bioreactor conditions using high flow, low D.O., and pulsation were selected to further study glucose-stimulated insulin secretion. Here, mechanically disrupted rat pancreata were cultured for 24 h under these bioreactor conditions and were then challenged with high glucose concentration for 6 h and high glucose + IBMX (an insulin secretagogue) for a further 6 h. These cultures secreted insulin in response to high glucose concentration in the first 6 h, however stimulated-insulin secretion was markedly weaker in response to high glucose concentration + IBMX thereafter. After this bioreactor culture period, higher tissue metabolic activity was found compared to that of non-bioreacted static controls. More insulin- and glucagon-positive structures, and extensive intact endothelial structures were observed compared to non-bioreacted static cultures. H&E staining revealed more intact tissue compared to static cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:432-444, 2018. © 2017 American Institute of Chemical Engineers.

Design and efficacy of a single-use bioreactor for heart valve tissue engineering.

PubMed

Converse, Gabriel L; Buse, Eric E; Neill, Kari R; McFall, Christopher R; Lewis, Holley N; VeDepo, Mitchell C; Quinn, Rachael W; Hopkins, Richard A

2017-02-01

Heart valve tissue engineering offers the promise of improved treatments for congenital heart disorders; however, widespread clinical availability of a tissue engineered heart valve (TEHV) has been hindered by scientific and regulatory concerns, including the lack of a disposable, bioreactor system for nondestructive valve seeding and mechanical conditioning. Here we report the design for manufacture and the production of full scale, functional prototypes of such a system. To evaluate the efficacy of this bioreactor as a tool for seeding, ovine aortic valves were decellularized and subjected to seeding with human mesenchymal stem cells (hMSC). The effects of pulsatile conditioning using cyclic waveforms tuned to various negative and positive chamber pressures were evaluated, with respect to the seeding of cells on the decellularized leaflet and the infiltration of seeded cells into the interstitium of the leaflet. Infiltration of hMSCs into the aortic valve leaflet was observed following 72 h of conditioning under negative chamber pressure. Additional conditioning under positive pressure improved cellular infiltration, while retaining gene expression within the MSC-valve interstitial cell phenotype lineage. This protocol resulted in a subsurface pilot population of cells, not full tissue recellularization. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 249-259, 2017. © 2015 Wiley Periodicals, Inc.

Design and Use of a Novel Bioreactor for Regeneration of Biaxially Stretched Tissue-Engineered Vessels

PubMed Central

Huang, Angela Hai; Lee, Yong-Ung; Calle, Elizabeth A.; Boyle, Michael; Starcher, Barry C.; Humphrey, Jay D.

2015-01-01

Conventional bioreactors are used to enhance extracellular matrix (ECM) production and mechanical strength of tissue-engineered vessels (TEVs) by applying circumferential strain, which is uniaxial stretching. However, the resulting TEVs still suffer from inadequate mechanical properties, where rupture strengths and compliance values are still very different from native arteries. The biomechanical milieu of native arteries consists of both circumferential and axial loading. Therefore, to better simulate the physiological stresses acting on native arteries, we built a novel bioreactor system to enable biaxial stretching of engineered arteries during culture. This new bioreactor system allows for independent control of circumferential and axial stretching parameters, such as displacement and beat rate. The assembly and setup processes for this biaxial bioreactor system are reliable with a success rate greater than 75% for completion of long-term sterile culture. This bioreactor also supports side-by-side assessments of TEVs that are cultured under three types of mechanical conditions (static, uniaxial, and biaxial), all within the same biochemical environment. Using this bioreactor, we examined the impact of biaxial stretching on arterial wall remodeling of TEVs. Biaxial TEVs developed the greatest wall thickness compared with static and uniaxial TEVs. Unlike uniaxial loading, biaxial loading led to undulated collagen fibers that are commonly found in native arteries. More importantly, the biaxial TEVs developed the most mature elastin in the ECM, both qualitatively and quantitatively. The presence of mature extracellular elastin along with the undulated collagen fibers may contribute to the observed vascular compliance in the biaxial TEVs. The current work shows that biaxial stretching is a novel and promising means to improve TEV generation. Furthermore, this novel system allows us to optimize biomechanical conditioning by unraveling the interrelationships among the applied mechanical stress, the resulting ECM properties, and the mechanics of TEVs. PMID:25669988

Perfusion properties of scaffolds: A new approach to tissue engineering designs for bone regeneration

NASA Astrophysics Data System (ADS)

Larionov, P. M.; Maslov, N. A.; Papaeva, E. O.; Yunoshev, A. S.; Filipenko, M. L.; Bogachev, S. S.; Proskurina, A. S.; Samokhin, A. G.; Kudrov, G. A.; Tereshchenko, V. P.; Pavlov, V. V.; Mihailovsky, M. V.; Prohorenko, V. M.; Titov, A. T.; Mamonova, E. V.; Sadovoy, M. A.

2017-09-01

The main approach to tissue engineering involves the use of scaffolds seeded with cells, followed by culturing in a bioreactor. However, the effective use of a bioreactor requires adaptation of the scaffold at the stage of its design. In our opinion, this means assessment of the perfusion properties of the scaffold. Transverse and longitudinal perfusion under hydrostatic pressure of 5, 10, and 15 mmHg, as well as the significance of electrospinning parameters for fabrication of a scaffold sheet and the composition of composite material—11% w/v polycaprolactone with gelatinization of 0.5%, 2%, and 4%, were demonstrated.

A Bioreactor to Identify the Driving Mechanical Stimuli of Tissue Growth and Remodeling.

PubMed

van Kelle, Mathieu A J; Oomen, Pim J A; Bulsink, Jurgen A; Janssen-van den Broek, Marloes W J T; Lopata, Richard G P; Rutten, Marcel C M; Loerakker, Sandra; Bouten, Carlijn V C

2017-06-01

Tissue growth and remodeling are essential processes that should ensure long-term functionality of tissue-engineered (TE) constructs. Even though it is widely recognized that these processes strongly depend on mechanical stimuli, the underlying mechanisms of mechanically induced growth and remodeling are only partially understood. It is generally accepted that cells sense mechanical changes and respond by altering their surroundings, by means of extracellular matrix growth and remodeling, in an attempt to return to a certain preferred mechanical homeostatic state. However, the exact mechanical cues that trigger cells to synthesize and remodel their environment remain unclear. To identify the driving mechanical stimuli of these processes, it is critical to be able to temporarily follow the mechanical state of developing tissues under physiological loading conditions. Therefore, a novel "versatile tissue growth and remodeling" (Vertigro) bioreactor was developed that is capable of tissue culture and mechanical stimulation for a prolonged time period, while simultaneously performing mechanical testing. The Vertigro's unique two-chamber design allows easy, sterile handling of circular 3D TE constructs in a dedicated culture chamber, while a separate pressure chamber facilitates a pressure-driven dynamic loading regime during culture. As a proof-of-concept, temporal changes in the mechanical state of cultured tissues were quantified using nondestructive mechanical testing by means of a classical bulge test, in which the tissue displacement was tracked using ultrasound imaging. To demonstrate the successful development of the bioreactor system, compositional, structural, and geometrical changes were qualitatively and quantitatively assessed using a series of standard analysis techniques. With this bioreactor and associated mechanical analysis technique, a powerful toolbox has been developed to quantitatively study and identify the driving mechanical stimuli of engineered tissue growth and remodeling.

Cardiac tissue engineering using perfusion bioreactor systems

PubMed Central

Radisic, Milica; Marsano, Anna; Maidhof, Robert; Wang, Yadong; Vunjak-Novakovic, Gordana

2009-01-01

This protocol describes tissue engineering of synchronously contractile cardiac constructs by culturing cardiac cell populations on porous scaffolds (in some cases with an array of channels) and bioreactors with perfusion of culture medium (in some cases supplemented with an oxygen carrier). The overall approach is ‘biomimetic’ in nature as it tends to provide in vivo-like oxygen supply to cultured cells and thereby overcome inherent limitations of diffusional transport in conventional culture systems. In order to mimic the capillary network, cells are cultured on channeled elastomer scaffolds that are perfused with culture medium that can contain oxygen carriers. The overall protocol takes 2–4 weeks, including assembly of the perfusion systems, preparation of scaffolds, cell seeding and cultivation, and on-line and end-point assessment methods. This model is well suited for a wide range of cardiac tissue engineering applications, including the use of human stem cells, and high-fidelity models for biological research. PMID:18388955

Model-based cell number quantification using online single-oxygen sensor data for tissue engineering perfusion bioreactors.

PubMed

Lambrechts, T; Papantoniou, I; Sonnaert, M; Schrooten, J; Aerts, J-M

2014-10-01

Online and non-invasive quantification of critical tissue engineering (TE) construct quality attributes in TE bioreactors is indispensable for the cost-effective up-scaling and automation of cellular construct manufacturing. However, appropriate monitoring techniques for cellular constructs in bioreactors are still lacking. This study presents a generic and robust approach to determine cell number and metabolic activity of cell-based TE constructs in perfusion bioreactors based on single oxygen sensor data in dynamic perfusion conditions. A data-based mechanistic modeling technique was used that is able to correlate the number of cells within the scaffold (R(2)  = 0.80) and the metabolic activity of the cells (R(2)  = 0.82) to the dynamics of the oxygen response to step changes in the perfusion rate. This generic non-destructive measurement technique is effective for a large range of cells, from as low as 1.0 × 10(5) cells to potentially multiple millions of cells, and can open-up new possibilities for effective bioprocess monitoring. © 2014 Wiley Periodicals, Inc.

Three-dimensional spatial localization of thin fluorophore-filled capillaries in thick scattering media

NASA Astrophysics Data System (ADS)

Desrochers, Johanne; Vermette, Patrick; Fontaine, Réjean; Bérubé-Lauzière, Yves

2008-06-01

Fluorescence optical diffuse tomography (fDOT) is of much interest in molecular imaging to retrieve information from fluorescence signals emitted from specifically targeted bioprocesses deep within living tissues. An exciting application of fDOT is in the growing field of tissue engineering, where 3D non-invasive imaging techniques are required to ultimately grow 3D engineered tissues. Via appropriate labelling strategies and fluorescent probes, fDOT has the potential to monitor culture environment and cells viability non-destructively directly within the bioreactor environment where tissues are to be grown. Our ultimate objective is to image the formation of blood vessels in bioreactor conditions. Herein, we use a non-contact setup for small animal fDOT imaging designed for 3D light collection around the sample. We previously presented a time of flight approach using a numerical constant fraction discrimination technique to assign an early photons arrival time to every fluorescence time point-spread function collected around the sample. Towards bioreactor in-situ imaging, we have shown the capability of our approach to localize a fluorophore-filled 500 μm capillary immersed coaxially in a cylindrically shaped bioreactor phantom containing an absorbing/scattering medium representative of experiments on real tissue cultures. Here, we go one step further, and present results for the 3D localization of thinner indocyanine green labelled capillaries (250 μm and 360 μm inner diameter) immersed in the same phantom conditions and geometry but with different spatial configurations (10° and 30° capillary inclination).

Mechanical Stimulation Protocols of Human Derived Cells in Articular Cartilage Tissue Engineering - A Systematic Review.

PubMed

Khozoee, Baktash; Mafi, Pouya; Mafi, Reza; Khan, Wasim S

2017-01-01

Mechanical stimulation is a key factor in articular cartilage generation and maintenance. Bioreactor systems have been designed and built in order to deliver specific types of mechanical stimulation. The focus has been twofold, applying a type of preconditioning in order to stimulate cell differentiation, and to simulate in vivo conditions in order to gain further insight into how cells respond to different stimulatory patterns. Due to the complex forces at work within joints, it is difficult to simulate mechanical conditions using a bioreactor. The aim of this review is to gain a deeper understanding of the complexities of mechanical stimulation protocols by comparing those employed in bioreactors in the context of tissue engineering for articular cartilage, and to consider their effects on cultured cells. Allied and Complementary Medicine 1985 to 2016, Ovid MEDLINE[R] 1946 to 2016, and Embase 1974 to 2016 were searched using key terms. Results were subject to inclusion and exclusion criteria, key findings summarised into a table and subsequently discussed. Based on this review it is overwhelmingly clear that mechanical stimulation leads to increased chondrogenic properties in the context of bioreactor articular cartilage tissue engineering using human cells. However, given the variability and lack of controlled factors between research articles, results are difficult to compare, and a standardised method of evaluating stimulation protocols proved challenging. With improved standardisation in mechanical stimulation protocol reporting, bioreactor design and building processes, along with a better understanding of joint behaviours, we hope to perform a meta-analysis on stimulation protocols and methods. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs

PubMed Central

Salazar, Betsy H.; Cashion, Avery T.; Dennis, Robert G.; Birla, Ravi K.

2015-01-01

Purpose The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. Methods The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-hour stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. Results An increase in contractile force was observed after the strain protocol of 10% stretch at 1Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. Conclusion In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue. PMID:26577484

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs.

PubMed

Salazar, Betsy H; Cashion, Avery T; Dennis, Robert G; Birla, Ravi K

2015-12-01

The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-h stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. An increase in contractile force was observed after the strain protocol of 10% stretch at 1 Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue.

Electrical and mechanical stimulation of cardiac cells and tissue constructs.

PubMed

Stoppel, Whitney L; Kaplan, David L; Black, Lauren D

2016-01-15

The field of cardiac tissue engineering has made significant strides over the last few decades, highlighted by the development of human cell derived constructs that have shown increasing functional maturity over time, particularly using bioreactor systems to stimulate the constructs. However, the functionality of these tissues is still unable to match that of native cardiac tissue and many of the stem-cell derived cardiomyocytes display an immature, fetal like phenotype. In this review, we seek to elucidate the biological underpinnings of both mechanical and electrical signaling, as identified via studies related to cardiac development and those related to an evaluation of cardiac disease progression. Next, we review the different types of bioreactors developed to individually deliver electrical and mechanical stimulation to cardiomyocytes in vitro in both two and three-dimensional tissue platforms. Reactors and culture conditions that promote functional cardiomyogenesis in vitro are also highlighted. We then cover the more recent work in the development of bioreactors that combine electrical and mechanical stimulation in order to mimic the complex signaling environment present in vivo. We conclude by offering our impressions on the important next steps for physiologically relevant mechanical and electrical stimulation of cardiac cells and engineered tissue in vitro. Copyright © 2015 Elsevier B.V. All rights reserved.

Perfusion flow bioreactor for 3D in situ imaging: investigating cell/biomaterials interactions.

PubMed

Stephens, J S; Cooper, J A; Phelan, F R; Dunkers, J P

2007-07-01

The capability to image real time cell/material interactions in a three-dimensional (3D) culture environment will aid in the advancement of tissue engineering. This paper describes a perfusion flow bioreactor designed to hold tissue engineering scaffolds and allow for in situ imaging using an upright microscope. The bioreactor can hold a scaffold of desirable thickness for implantation (>2 mm). Coupling 3D culture and perfusion flow leads to the creation of a more biomimetic environment. We examined the ability of the bioreactor to maintain cell viability outside of an incubator environment (temperature and pH stability), investigated the flow features of the system (flow induced shear stress), and determined the image quality in order to perform time-lapsed imaging of two-dimensional (2D) and 3D cell culture. In situ imaging was performed on 2D and 3D, culture samples and cell viability was measured under perfusion flow (2.5 mL/min, 0.016 Pa). The visualization of cell response to their environment, in real time, will help to further elucidate the influences of biomaterial surface features, scaffold architectures, and the influence of flow induced shear on cell response and growth of new tissue. (c) 2006 Wiley Periodicals, Inc.

Distribution and Viability of Fetal and Adult Human Bone Marrow Stromal Cells in a Biaxial Rotating Vessel Bioreactor after Seeding on Polymeric 3D Additive Manufactured Scaffolds

PubMed Central

Leferink, Anne M.; Chng, Yhee-Cheng; van Blitterswijk, Clemens A.; Moroni, Lorenzo

2015-01-01

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation. PMID:26557644

Distribution and Viability of Fetal and Adult Human Bone Marrow Stromal Cells in a Biaxial Rotating Vessel Bioreactor after Seeding on Polymeric 3D Additive Manufactured Scaffolds.

PubMed

Leferink, Anne M; Chng, Yhee-Cheng; van Blitterswijk, Clemens A; Moroni, Lorenzo

2015-01-01

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.

Enhanced elastin synthesis and maturation in human vascular smooth muscle tissue derived from induced-pluripotent stem cells.

PubMed

Eoh, Joon H; Shen, Nian; Burke, Jacqueline A; Hinderer, Svenja; Xia, Zhiyong; Schenke-Layland, Katja; Gerecht, Sharon

2017-04-01

Obtaining vascular smooth muscle tissue with mature, functional elastic fibers is a key obstacle in tissue-engineered blood vessels. Poor elastin secretion and organization leads to a loss of specialization in contractile smooth muscle cells, resulting in over proliferation and graft failure. In this study, human induced-pluripotent stem cells (hiPSCs) were differentiated into early smooth muscle cells, seeded onto a hybrid poly(ethylene glycol) dimethacrylate/poly (l-lactide) (PEGdma-PLA) scaffold and cultured in a bioreactor while exposed to pulsatile flow, towards maturation into contractile smooth muscle tissue. We evaluated the effects of pulsatile flow on cellular organization as well as elastin expression and assembly in the engineered tissue compared to a static control through immunohistochemistry, gene expression and functionality assays. We show that culturing under pulsatile flow resulted in organized and functional hiPSC derived smooth muscle tissue. Immunohistochemistry analysis revealed hiPSC-smooth muscle tissue with robust, well-organized cells and elastic fibers and the supporting microfibril proteins necessary for elastic fiber assembly. Through qRT-PCR analysis, we found significantly increased expression of elastin, fibronectin, and collagen I, indicating the synthesis of necessary extracellular matrix components. Functionality assays revealed that hiPSC-smooth muscle tissue cultured in the bioreactor had an increased calcium signaling and contraction in response to a cholinergic agonist, significantly higher mature elastin content and improved mechanical properties in comparison to the static control. The findings presented here detail an effective approach to engineering elastic human vascular smooth muscle tissue with the functionality necessary for tissue engineering and regenerative medicine applications. Obtaining robust, mature elastic fibers is a key obstacle in tissue-engineered blood vessels. Human induced-pluripotent stem cells have become of interest due to their ability to supplement tissue engineered scaffolds. Their ability to differentiate into cells of vascular lineages with defined phenotypes serves as a potential solution to a major cause of graft failure in which phenotypic shifts in smooth muscle cells lead to over proliferation and occlusion of the graft. Herein, we have differentiated human induced-pluripotent stem cells in a pulsatile flow bioreactor, resulting in vascular smooth muscle tissue with robust elastic fibers and enhanced functionality. This study highlights an effective approach to engineering elastic functional vascular smooth muscle tissue for tissue engineering and regenerative medicine applications. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Evaluation of the Growth Environment of a Hydrostatic Force Bioreactor for Preconditioning of Tissue-Engineered Constructs

PubMed Central

Reinwald, Yvonne; Leonard, Katherine H.L.; Henstock, James R.; Whiteley, Jonathan P.; Osborne, James M.; Waters, Sarah L.; Levesque, Philippe

2015-01-01

Bioreactors have been widely acknowledged as valuable tools to provide a growth environment for engineering tissues and to investigate the effect of physical forces on cells and cell-scaffold constructs. However, evaluation of the bioreactor environment during culture is critical to defining outcomes. In this study, the performance of a hydrostatic force bioreactor was examined by experimental measurements of changes in dissolved oxygen (O2), carbon dioxide (CO2), and pH after mechanical stimulation and the determination of physical forces (pressure and stress) in the bioreactor through mathematical modeling and numerical simulation. To determine the effect of hydrostatic pressure on bone formation, chick femur skeletal cell-seeded hydrogels were subjected to cyclic hydrostatic pressure at 0–270 kPa and 1 Hz for 1 h daily (5 days per week) over a period of 14 days. At the start of mechanical stimulation, dissolved O2 and CO2 in the medium increased and the pH of the medium decreased, but remained within human physiological ranges. Changes in physiological parameters (O2, CO2, and pH) were reversible when medium samples were placed in a standard cell culture incubator. In addition, computational modeling showed that the distribution and magnitude of physical forces depends on the shape and position of the cell-hydrogel constructs in the tissue culture format. Finally, hydrostatic pressure was seen to enhance mineralization of chick femur skeletal cell-seeded hydrogels. PMID:24967717

Evaluation of the growth environment of a hydrostatic force bioreactor for preconditioning of tissue-engineered constructs.

PubMed

Reinwald, Yvonne; Leonard, Katherine H L; Henstock, James R; Whiteley, Jonathan P; Osborne, James M; Waters, Sarah L; Levesque, Philippe; El Haj, Alicia J

2015-01-01

Bioreactors have been widely acknowledged as valuable tools to provide a growth environment for engineering tissues and to investigate the effect of physical forces on cells and cell-scaffold constructs. However, evaluation of the bioreactor environment during culture is critical to defining outcomes. In this study, the performance of a hydrostatic force bioreactor was examined by experimental measurements of changes in dissolved oxygen (O2), carbon dioxide (CO2), and pH after mechanical stimulation and the determination of physical forces (pressure and stress) in the bioreactor through mathematical modeling and numerical simulation. To determine the effect of hydrostatic pressure on bone formation, chick femur skeletal cell-seeded hydrogels were subjected to cyclic hydrostatic pressure at 0-270 kPa and 1 Hz for 1 h daily (5 days per week) over a period of 14 days. At the start of mechanical stimulation, dissolved O2 and CO2 in the medium increased and the pH of the medium decreased, but remained within human physiological ranges. Changes in physiological parameters (O2, CO2, and pH) were reversible when medium samples were placed in a standard cell culture incubator. In addition, computational modeling showed that the distribution and magnitude of physical forces depends on the shape and position of the cell-hydrogel constructs in the tissue culture format. Finally, hydrostatic pressure was seen to enhance mineralization of chick femur skeletal cell-seeded hydrogels.

Noninvasive metabolic imaging of engineered 3D human adipose tissue in a perfusion bioreactor.

PubMed

Ward, Andrew; Quinn, Kyle P; Bellas, Evangelia; Georgakoudi, Irene; Kaplan, David L

2013-01-01

The efficacy and economy of most in vitro human models used in research is limited by the lack of a physiologically-relevant three-dimensional perfused environment and the inability to noninvasively quantify the structural and biochemical characteristics of the tissue. The goal of this project was to develop a perfusion bioreactor system compatible with two-photon imaging to noninvasively assess tissue engineered human adipose tissue structure and function in vitro. Three-dimensional (3D) vascularized human adipose tissues were engineered in vitro, before being introduced to a perfusion environment and tracked over time by automated quantification of endogenous markers of metabolism using two-photon excited fluorescence (TPEF). Depth-resolved image stacks were analyzed for redox ratio metabolic profiling and compared to prior analyses performed on 3D engineered adipose tissue in static culture. Traditional assessments with H&E staining were used to qualitatively measure extracellular matrix generation and cell density with respect to location within the tissue. The distribution of cells within the tissue and average cellular redox ratios were different between static and perfusion cultures, while the trends of decreased redox ratio and increased cellular proliferation with time in both static and perfusion cultures were similar. These results establish a basis for noninvasive optical tracking of tissue structure and function in vitro, which can be applied to future studies to assess tissue development or drug toxicity screening and disease progression.

It's all in the timing: modeling isovolumic contraction through development and disease with a dynamic dual electromechanical bioreactor system.

PubMed

Morgan, Kathy Ye; Black, Lauren Deems

2014-01-01

This commentary discusses the rationale behind our recently reported work entitled "Mimicking isovolumic contraction with combined electromechanical stimulation improves the development of engineered cardiac constructs," introduces new data supporting our hypothesis, and discusses future applications of our bioreactor system. The ability to stimulate engineered cardiac tissue in a bioreactor system that combines both electrical and mechanical stimulation offers a unique opportunity to simulate the appropriate dynamics between stretch and contraction and model isovolumic contraction in vitro. Our previous study demonstrated that combined electromechanical stimulation that simulated the timing of isovolumic contraction in healthy tissue improved force generation via increased contractile and calcium handling protein expression and improved hypertrophic pathway activation. In new data presented here, we further demonstrate that modification of the timing between electrical and mechanical stimulation to mimic a non-physiological process negatively impacts the functionality of the engineered constructs. We close by exploring the various disease states that have altered timing between the electrical and mechanical stimulation signals as potential future directions for the use of this system.

Medical Research System

NASA Technical Reports Server (NTRS)

1993-01-01

Based on Johnson Space Flight Center's development of a rotating bioreactor cell culture apparatus for Space Shuttle medical research, Johnson Space Flight Center engineers who worked on the original project formed a company called Synthecon, with the intention of commercializing the bioreactor technology. Synthecon grows three dimensional tissues in the bioreactor. These are superior to previous two-dimensional tissue samples in the study of human cell growth. A refined version of the Johnson Space Center technology, Synthecon's Rotary Cell Culture System includes a cell culture chamber that rotates around a horizontal axis. The cells establish an orbit that approximates free fall through the liquid medium in the chamber. The technology has significant applications for cancer research and treatment as well as AIDS research.

Suivi in situ de cultures tridimensionnelles en bioreacteur a perfusion grace a la tomographie d'emission par positrons

NASA Astrophysics Data System (ADS)

Chouinard, Julie

The continuous assessment of developing tissue substitutes is crucial to understand their evolution over time. However, this represents quite a challenge when thick samples must be evaluated with standard microscopy techniques. Common characterization methods are time consuming and usually result in the destruction of the culture. Real-time, in situ, non-invasive and non-destructives methods are needed to monitor the growth of large non-transparent constructs in tissue engineering. Medical imaging modalities, which can provide information on the structure and function of internal organs and tissues in living organisms, have the potential of allowing repetitive monitoring of these 3D cultures in vitro. The working hypothesis of this thesis was to establish standard noninvasive and nondestructive real-time bioreactor imaging protocols for in situ monitoring of the viability and metabolism of endothelial cells when grown in perfused 3D fibrin gel scaffolds. To achieve this goal, a culture chamber with hollow fibers was designed and a pulsatile perfusion bioreactor system, able to promote cell survival and proliferation, was constructed and validated. Standard imaging protocols in Positron Emission Tomography (PET) are not adapted to image bioreactor systems. A suitable method had to be devised using the well-known radiotracer 18F-fluorodeoxyglucose ( 18FDG), a marker of glucose metabolism. Optimal uptake conditions were determined using cell monolayers and the best parameters were then applied on perfused 3D cultures to evaluate perfusion, cell viability and emerging cell structures. After only 12 hours of culture, the cell density could be estimated and cell structures were localized within the fibrin gels after 1-2 weeks of culture. PET is a promising tool for tissue engineering with many specific tracers available that might eventually be able to reveal new information on tissue development. Key words: Endothelial cells, Perfusion bioreactor, Positron Emission Tomography (PET), 18F-fluorodeoxyglucose ( 18FDG), Tissue Engineering, 3D cultures, Fibrin.

Microgravity

NASA Image and Video Library

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Tunable osteogenic differentiation of hMPCs in tubular perfusion system bioreactor.

PubMed

Nguyen, Bao-Ngoc B; Ko, Henry; Fisher, John P

2016-08-01

The use of bioreactors for bone tissue engineering has been widely investigated. While the benefits of shear stress on osteogenic differentiation are well known, the underlying effects of dynamic culture on subpopulations within a bioreactor are less evident. In this work, we explore the influence of applied flow in the tubular perfusion system (TPS) bioreactor on the osteogenic differentiation of human mesenchymal progenitor cells (hMPCs), specifically analyzing the effects of axial position along the growth chamber. TPS bioreactor experiments conducted with unidirectional flow demonstrated enhanced expression of osteogenic markers in cells cultured downstream from the inlet flow. We utilized computational fluid dynamic modeling to confirm uniform shear stress distribution on the surface of the scaffolds and along the length of the growth chamber. The concept of paracrine signaling between cell populations was validated with the use of alternating flow, which diminished the differences in osteogenic differentiation between cells cultured at the inlet and outlet of the growth chamber. After the addition of controlled release of bone morphogenic protein-2 (BMP-2) into the system, osteogenic differentiation among subpopulations along the growth chamber was augmented, yet remained homogenous. These results allow for greater understanding of axial bioreactor cultures, their microenvironment, and how well-established parameters of osteogenic differentiation affect bone tissue development. With this work, we have demonstrated the capability of tuning osteogenic differentiation of hMPCs through the application of fluid flow and the addition of exogenous growth factors. Such precise control allows for the culture of distinct subpopulation within one dynamic system for the use of complex engineered tissue constructs. Biotechnol. Bioeng. 2016;113: 1805-1813. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Microgravity cultivation of cells and tissues

NASA Technical Reports Server (NTRS)

Freed, L. E.; Pellis, N.; Searby, N.; de Luis, J.; Preda, C.; Bordonaro, J.; Vunjak-Novakovic, G.

1999-01-01

In vitro studies of cells and tissues in microgravity, either simulated by cultivation conditions on earth or actual, during spaceflight, are expected to help identify mechanisms underlying gravity sensing and transduction in biological organisms. In this paper, we review rotating bioreactor studies of engineered skeletal and cardiovascular tissues carried out in unit gravity, a four month long cartilage tissue engineering study carried out aboard the Mir Space Station, and the ongoing laboratory development and testing of a system for cell and tissue cultivation aboard the International Space Station.

Design and evaluation of a novel subatmospheric pressure bioreactor for the preconditioning of tissue-engineered vascular constructs.

PubMed

Coakley, Daniel N; Shaikh, Faisal M; O'Sullivan, Kathleen; Kavanagh, Eamon G; Grace, Pierce A; McGloughlin, Tim M

2016-02-01

The pre-conditioning of tissue-engineered vascular scaffolds with mechanical stimuli is being recognised as an essential step in producing a functional vascular construct. In this study we design and evaluate a novel bioreactor, which exerts a mechanical strain on developing vascular scaffolds via subatmospheric pressure. We design and construct a bioreactor, which exerts subatmospheric pressure via a vacuum assisted closure unit. Vascular scaffolds seeded with human umbilical endothelial cells were evaluated for structural integrity, microbial contamination, cellular viability, von Willebrand factor (VWF) production, cell proliferation and morphology under a range of subatmospheric pressures (75-200mmHg). The bioreactor produced sustained subatmospheric pressures, which exerted a mechanical strain on the vascular scaffold. No microbial contamination was found during the study. The structural integrity of the vascular construct was maintained. There was no difference in cellular viability between control or subatmospheric pressure groups (p = 0.817). Cells continued to produce VWF under a range of subatmospheric pressures. Cells subjected to subatmospheric pressures of 125mmHg and 200mmHg exhibited higher levels of growth than cells in atmospheric pressure at 24 (p≤0.016) and 48 hour (p≤0.001). Negative pressure affected cellular morphology, which were more organised, elongated and expanded when exposed to subatmospheric pressure. We have constructed and validated a novel subatmospheric bioreactor. The bioreactor maintained a continuous subatmospheric pressure to the vascular scaffolds in a stable, sterile and constant environment. The bioreactor exerted a strain on the vascular sheets, which was shown to alter cellular morphology and enhance cellular proliferation.

Isolation of Resistance-Bearing Microorganisms

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri, J.; Probst, Alexander; Vaishampayan, Parang A.; Ghosh, Sudeshna; Osman, Shariff

2010-01-01

To better exploit the principles of gas transport and mass transport during the processes of cell seeding of 3D scaffolds and in vitro culture of 3D tissue engineered constructs, the oscillatory cell culture bioreactor provides a flow of cell suspensions and culture media directly through a porous 3D scaffold (during cell seeding) and a 3D construct (during subsequent cultivation) within a highly gas-permeable closed-loop tube. This design is simple, modular, and flexible, and its component parts are easy to assemble and operate, and are inexpensive. Chamber volume can be very low, but can be easily scaled up. This innovation is well suited to work with different biological specimens, particularly with cells having high oxygen requirements and/or shear sensitivity, and different scaffold structures and dimensions. The closed-loop changer is highly gas permeable to allow efficient gas exchange during the cell seeding/culturing process. A porous scaffold, which may be seeded with cells, is fixed by means of a scaffold holder to the chamber wall with scaffold/construct orientation with respect to the chamber determined by the geometry of the scaffold holder. A fluid, with/without biological specimens, is added to the chamber such that all, or most, of the air is displaced (i.e., with or without an enclosed air bubble). Motion is applied to the chamber within a controlled environment (e.g., oscillatory motion within a humidified 37 C incubator). Movement of the chamber induces relative motion of the scaffold/construct with respect to the fluid. In case the fluid is a cell suspension, cells will come into contact with the scaffold and eventually adhere to it. Alternatively, cells can be seeded on scaffolds by gel entrapment prior to bioreactor cultivation. Subsequently, the oscillatory cell culture bioreactor will provide efficient gas exchange (i.e., of oxygen and carbon dioxide, as required for viability of metabolically active cells) and controlled levels of fluid dynamic shear (i.e., as required for viability of shear-sensitive cells) to the developing engineered tissue construct. This bioreactor was recently utilized to show independent and interactive effects of a growth factor (IGF-I) and slow bidirectional perfusion on the survival, differentiation, and contractile performance of 3D tissue engineering cardiac constructs. The main application of this system is within the tissue engineering industry. The ideal final application is within the automated mass production of tissue- engineered constructs. Target industries could be both life sciences companies as well as bioreactor device producing companies.

Engineering of arteries in vitro

PubMed Central

Huang, Angela H.; Niklason, Laura E.

2014-01-01

This review will focus on two elements that are essential for functional arterial regeneration in vitro: the mechanical environment and the bioreactors used for tissue growth. The importance of the mechanical environment to embryological development, vascular functionality, and vascular graft regeneration will be discussed. Bioreactors generate mechanical stimuli to simulate the biomechanical environment of the arterial system. This system has been used to reconstruct arterial grafts with appropriate mechanical strength for implantation by controlling the chemical and mechanical environments in which the grafts are grown. Bioreactors are powerful tools to study the effect of mechanical stimuli on extracellular matrix (ECM) architecture and the mechanical properties of engineered vessels. Hence biomimetic systems enable us to optimize chemo-biomechanical culture conditions to regenerate engineered vessels with physiological properties similar to those of native arterial vessels. In addition, this review will introduce and examine various approaches and techniques that have been used to engineer biologically-based vascular grafts, including collagen-based grafts, fibrin-gel grafts, cell sheet engineering, biodegradable polymers, and decellularization of native vessels. PMID:24399290

Three-dimensional culture in a microgravity bioreactor improves the engraftment efficiency of hepatic tissue constructs in mice.

PubMed

Zhang, Shichang; Zhang, Bo; Chen, Xia; Chen, Li; Wang, Zhengguo; Wang, Yingjie

2014-12-01

Tissue-engineered liver using primary hepatocytes has been considered a valuable new therapeutic modality as an alternative to whole organ liver transplantation for different liver diseases. The development of clinically feasible liver tissue engineering approaches, however, has been hampered by the poor engraftment efficiency of hepatocytes. We developed a three-dimensional (3D) culture system using a microgravity bioreactor (MB), biodegradable scaffolds and growth-factor-reduced Matrigel to construct a tissue-engineered liver for transplantation into the peritoneal cavity of non-obese diabetic severe combined immunodeficient mice. The number of viable cells in the hepatic tissue constructs was stably maintained in the 3D MB culture system. Hematoxylin-eosin staining and zonula occludens-1 expression revealed that neonatal mouse liver cells were reorganized to form tissue-like structures during MB culture. Significantly upregulated hepatic functions (albumin secretion, urea production and cytochrome P450 activity) were observed in the MB culture group. Post-transplantation analysis indicated that the engraftment efficiency of the hepatic tissue constructs prepared in MB cultures was higher than that of those prepared in the static cultures. Higher level of hepatic function in the implants was confirmed by the expression of albumin. These findings suggest that 3D MB culture systems may offer an improved method for creating tissue-engineered liver because of the higher engraftment efficiency and the reduction of the initial cell function loss.

Advances in Application of Mechanical Stimuli in Bioreactors for Cartilage Tissue Engineering.

PubMed

Li, Ke; Zhang, Chunqiu; Qiu, Lulu; Gao, Lilan; Zhang, Xizheng

2017-08-01

Articular cartilage (AC) is the weight-bearing tissue in diarthroses. It lacks the capacity for self-healing once there are injuries or diseases due to its avascularity. With the development of tissue engineering, repairing cartilage defects through transplantation of engineered cartilage that closely matches properties of native cartilage has become a new option for curing cartilage diseases. The main hurdle for clinical application of engineered cartilage is how to develop functional cartilage constructs for mass production in a credible way. Recently, impressive hyaline cartilage that may have the potential to provide capabilities for treating large cartilage lesions in the future has been produced in laboratories. The key to functional cartilage construction in vitro is to identify appropriate mechanical stimuli. First, they should ensure the function of metabolism because mechanical stimuli play the role of blood vessels in the metabolism of AC, for example, acquiring nutrition and removing wastes. Second, they should mimic the movement of synovial joints and produce phenotypically correct tissues to achieve the adaptive development between the micro- and macrostructure and function. In this article, we divide mechanical stimuli into three types according to forces transmitted by different media in bioreactors, namely forces transmitted through the liquid medium, solid medium, or other media, then we review and summarize the research status of bioreactors for cartilage tissue engineering (CTE), mainly focusing on the effects of diverse mechanical stimuli on engineered cartilage. Based on current researches, there are several motion patterns in knee joints; but compression, tension, shear, fluid shear, or hydrostatic pressure each only partially reflects the mechanical condition in vivo. In this study, we propose that rolling-sliding-compression load consists of various stimuli that will represent better mechanical environment in CTE. In addition, engineers often ignore the importance of biochemical factors to the growth and development of engineered cartilage. In our point of view, only by fully considering synergistic effects of mechanical and biochemical factors can we find appropriate culture conditions for functional cartilage constructs. Once again, rolling-sliding-compression load under appropriate biochemical conditions may be conductive to realize the adaptive development between the structure and function of engineered cartilage in vitro.

Cell viability viscoelastic measurement in a rheometer used to stress and engineer tissues at low sonic frequencies.

PubMed

Klemuk, Sarah A; Jaiswal, Sanyukta; Titze, Ingo R

2008-10-01

Effects of vibration on human vocal fold extracellular matrix composition and the resultant tissue viscoelastic properties are difficult to study in vivo. Therefore, an in vitro bioreactor, simulating the in vivo physiological environment, was explored. A stress-controlled commercial rheometer was used to administer shear vibrations to living tissues at stresses and frequencies corresponding to male phonation, while simultaneously measuring tissue viscoelastic properties. Tissue environment was evaluated and adjustments made in order to sustain cell life for short term experimentation up to 6 h. Cell nutrient medium evaporation, osmolality, pH, and cell viability of cells cultured in three-dimensional synthetic scaffolds were quantified under comparably challenging environments to the rheometer bioreactor for 4 or 6 h. The functionality of the rheometer bioreactor was demonstrated by applying three vibration regimes to cell-seeded three-dimensional substrates for 2 h. Resulting strain was quantified throughout the test period. Rheologic data and cell viability are reported for each condition, and future improvements are discussed.

Fluid and mass transport modelling to drive the design of cell-packed hollow fibre bioreactors for tissue engineering applications.

PubMed

Shipley, Rebecca J; Waters, Sarah L

2012-12-01

A model for fluid and mass transport in a single module of a tissue engineering hollow fibre bioreactor (HFB) is developed. Cells are seeded in alginate throughout the extra-capillary space (ECS), and fluid is pumped through a central lumen to feed the cells and remove waste products. Fluid transport is described using Navier-Stokes or Darcy equations as appropriate; this is overlaid with models of mass transport in the form of advection-diffusion-reaction equations that describe the distribution and uptake/production of nutrients/waste products. The small aspect ratio of a module is exploited and the option of opening an ECS port is explored. By proceeding analytically, operating equations are determined that enable a tissue engineer to prescribe the geometry and operation of the HFB by ensuring the nutrient and waste product concentrations are consistent with a functional cell population. Finally, results for chondrocyte and cardiomyocyte cell populations are presented, typifying two extremes of oxygen uptake rates.

Estimation of the physiological mechanical conditioning in vascular tissue engineering by a predictive fluid-structure interaction approach.

PubMed

Tresoldi, Claudia; Bianchi, Elena; Pellegata, Alessandro Filippo; Dubini, Gabriele; Mantero, Sara

2017-08-01

The in vitro replication of physiological mechanical conditioning through bioreactors plays a crucial role in the development of functional Small-Caliber Tissue-Engineered Blood Vessels. An in silico scaffold-specific model under pulsatile perfusion provided by a bioreactor was implemented using a fluid-structure interaction (FSI) approach for viscoelastic tubular scaffolds (e.g. decellularized swine arteries, DSA). Results of working pressures, circumferential deformations, and wall shear stress on DSA fell within the desired physiological range and indicated the ability of this model to correctly predict the mechanical conditioning acting on the cells-scaffold system. Consequently, the FSI model allowed us to a priori define the stimulation pattern, driving in vitro physiological maturation of scaffolds, especially with viscoelastic properties.

Designing electrical stimulated bioreactors for nerve tissue engineering

NASA Astrophysics Data System (ADS)

Sagita, Ignasius Dwi; Whulanza, Yudan; Dhelika, Radon; Nurhadi, Ibrahim

2018-02-01

Bioreactor provides a biomimetic ecosystem that is able to culture cells in a physically controlled system. In general, the controlled-parameters are temperature, pH, fluid flow, nutrition flow, etc. In this study, we develop a bioreactor that specifically targeted to culture neural stem cells. This bioreactor could overcome some limitations of conventional culture technology, such as petri dish, by providing specific range of observation area and a uniform treatment. Moreover, the microfluidic bioreactor, which is a small-controlled environment, is able to observe as small number of cells as possible. A perfusion flow is applied to mimic the physiological environment in human body. Additionally, this bioreactor also provides an electrical stimulation which is needed by neural stem cells. In conclusion, we found the correlation between the induced shear stress with geometric parameters of the bioreactor. Ultimately, this system shall be used to observe the interaction between stimulation and cell growth.

Informing Stem Cell-Based Tendon Tissue Engineering Approaches with Embryonic Tendon Development.

PubMed

Okech, William; Kuo, Catherine K

Adult tendons fail to regenerate normal tissue after injury, and instead form dysfunctional scar tissue with abnormal mechanical properties. Surgical repair with grafts is the current standard to treat injuries, but faces significant limitations including pain and high rates of re-injury. To address this, we aim to regenerate new, normal tendons to replace dysfunctional tendons. A common approach to tendon tissue engineering is to design scaffolds and bioreactors based on adult tendon properties that can direct adult stem cell tenogenesis. Despite significant progress, advances have been limited due, in part, to a need for markers and potent induction cues. Our goal is to develop novel tendon tissue engineering approaches informed by embryonic tendon development. We are characterizing structure-property relationships of embryonic tendon to identify design parameters for three-dimensional scaffolds and bioreactor mechanical loading systems to direct adult stem cell tenogenesis. We will review studies in which we quantified changes in the mechanical and biochemical properties of tendon during embryonic development and elucidated specific mechanisms of functional property elaboration. We then examined the effects of these mechanical and biochemical factors on embryonic tendon cell behavior. Using custom-designed bioreactors, we also examined the effects of dynamic mechanical loading and growth factor treatment on embryonic tendon cells. Our findings have established cues to induce tenogenesis as well as metrics to evaluate differentiation. We finish by discussing how we have evaluated the tenogenic differentiation potential of adult stem cells by comparing their responses to that of embryonic tendon cells in these culture systems.

Bioreactor System Using Noninvasive Imaging and Mechanical Stretch for Biomaterial Screening

PubMed Central

Kluge, Jonathan A.; Leisk, Gary G.; Cardwell, Robyn S.; Fernandes, Alexander P.; House, Michael; Ward, Andrew; Dorfmann, A. Luis; Kaplan, David L.

2012-01-01

Screening biomaterial and tissue systems in vitro, for guidance of performance in vivo, remains a major requirement in the field of tissue engineering. It is critical to understand how culture stimulation affects both tissue construct maturation and function, with the goal of eliminating resource-intensive trial-and-error screening and better matching specifications for various in vivo needs. We present a multifunctional and robust bioreactor design that addresses this need. The design enables a range of mechanical inputs, durations, and frequencies to be applied in coordination with noninvasive optical assessments. A variety of biomaterial systems, including micro- and nano-fiber and porous sponge biomaterials, as well as cell-laden tissue engineering constructs were used in validation studies in order to demonstrate the versatility and utility of this new bioreactor design. The silk-based biomaterials highlighted in these studies offered several unique optical signatures for use in label-free nondestructive imaging that allowed for sequential profiling. Both short- and long-term culture studies were conducted to evaluate several practical scenarios of usage: on a short-term basis, we demonstrate that construct cellularity can be monitored by usage of nonpermanent dyes; on a more long-term basis, we show that cell ingrowth can be monitored by GFP-labeling and construct integrity probed with concurrent load/displacement data. The ability to nondestructively track cells, biomaterials, and new matrix formation without harvesting designated samples at each time point will lead to less resource-intensive studies and should enhance our understanding and the discovery of biomaterial designs related to functional tissue engineering. PMID:21298345

In vitro physical stimulation of tissue-engineered and native cartilage.

PubMed

Li, Kelvin W; Klein, Travis J; Chawla, Kanika; Nugent, Gayle E; Bae, Won C; Sah, Robert L

2004-01-01

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered cartilaginous constructs that approach those of native cartilage has not been previously achieved when constructs are cultured under free-swelling conditions. One approach toward stimulating the development of constructs that are mechanically more robust is to expose them to physical environments that are similar, in certain ways, to those encountered by native cartilage. This is a strategy motivated by observations in numerous short-term experiments that certain mechanical signals are potent stimulators of cartilage metabolism. On the other hand, excess mechanical loading can have a deleterious effect on cartilage. Culture conditions that include a physical stimulation component are made possible by the use of specialized bioreactors. This chapter addresses some of the issues involved in using bioreactors as integral components of cartilage tissue engineering and in studying the physical regulation of cartilage. We first consider the generation of cartilaginous constructs in vitro. Next we describe the rationale and design of bioreactors that can impart either mechanical deformation or fluid-induced mechanical signals.

Use of perfusion bioreactors and large animal models for long bone tissue engineering.

PubMed

Gardel, Leandro S; Serra, Luís A; Reis, Rui L; Gomes, Manuela E

2014-04-01

Tissue engineering and regenerative medicine (TERM) strategies for generation of new bone tissue includes the combined use of autologous or heterologous mesenchymal stem cells (MSC) and three-dimensional (3D) scaffold materials serving as structural support for the cells, that develop into tissue-like substitutes under appropriate in vitro culture conditions. This approach is very important due to the limitations and risks associated with autologous, as well as allogenic bone grafiting procedures currently used. However, the cultivation of osteoprogenitor cells in 3D scaffolds presents several challenges, such as the efficient transport of nutrient and oxygen and removal of waste products from the cells in the interior of the scaffold. In this context, perfusion bioreactor systems are key components for bone TERM, as many recent studies have shown that such systems can provide dynamic environments with enhanced diffusion of nutrients and therefore, perfusion can be used to generate grafts of clinically relevant sizes and shapes. Nevertheless, to determine whether a developed tissue-like substitute conforms to the requirements of biocompatibility, mechanical stability and safety, it must undergo rigorous testing both in vitro and in vivo. Results from in vitro studies can be difficult to extrapolate to the in vivo situation, and for this reason, the use of animal models is often an essential step in the testing of orthopedic implants before clinical use in humans. This review provides an overview of the concepts, advantages, and challenges associated with different types of perfusion bioreactor systems, particularly focusing on systems that may enable the generation of critical size tissue engineered constructs. Furthermore, this review discusses some of the most frequently used animal models, such as sheep and goats, to study the in vivo functionality of bone implant materials, in critical size defects.

An automated perfusion bioreactor for the streamlined production of engineered osteogenic grafts.

PubMed

Ding, Ming; Henriksen, Susan S; Wendt, David; Overgaard, Søren

2016-04-01

A computer-controlled perfusion bioreactor was developed for the streamlined production of engineered osteogenic grafts. This system automated the required bioprocesses, from the initial filling of the system through the phases of cell seeding and prolonged cell/tissue culture. Flow through chemo-optic micro-sensors allowed to non-invasively monitor the levels of oxygen and pH in the perfused culture medium throughout the culture period. To validate its performance, freshly isolated ovine bone marrow stromal cells were directly seeded on porous scaffold granules (hydroxyapatite/β-tricalcium-phosphate/poly-lactic acid), bypassing the phase of monolayer cell expansion in flasks. Either 10 or 20 days after culture, engineered cell-granule grafts were implanted in an ectopic mouse model to quantify new bone formation. After four weeks of implantation, histomorphometry showed more bone in bioreactor-generated grafts than cell-free granule controls, while bone formation did not show significant differences between 10 days and 20 days of incubation. The implanted granules without cells had no bone formation. This novel perfusion bioreactor has revealed the capability of activation larger viable bone graft material, even after shorter incubation time of graft material. This study has demonstrated the feasibility of engineering osteogenic grafts in an automated bioreactor system, laying the foundation for a safe, regulatory-compliant, and cost-effective manufacturing process. © 2015 Wiley Periodicals, Inc.

Development of bioengineering system for stem cell proliferation

NASA Astrophysics Data System (ADS)

Park, H. S.; Shah, R.; Shah, C.

2016-08-01

From last decades, intensive research in the field of stem cells proliferation had been promoted due to the unique property of stem cells to self-renew themselves into multiples and has potential to replicate into an organ or tissues and so it's highly demanding though challenging. Bioreactor, a mechanical device, works as a womb for stem cell proliferation by providing nutritious environment for the proper growth of stem cells. Various factors affecting stem cells growth are the bioreactor mechanism, feeding of continuous nutrients, healthy environment, etc., but it always remains a challenge for controlling biological parameters. The present paper unveils the design of mechanical device commonly known as bioreactor in tissues engineering and biotech field, use for proliferation of stem cells and imparts the proper growing condition for stem cells. This high functional bioreactor provides automation mixing of cell culture and stem cells. This design operates in conjunction with mechanism of reciprocating motion. Compare to commercial bioreactors, this proposed design is more convenient, easy to operate and less maintenance is required as bioreactor culture bag is made of polyethylene which is single use purpose. Development of this bioengineering system will be beneficial for better growth and expansion of stem cell

NASA Classroom Bioreactor

NASA Technical Reports Server (NTRS)

Scully, Robert

2004-01-01

Exploration of space provides a compelling need for cell-based research into the basic mechanisms that underlie the profound changes that occur in terrestrial life that is transitioned to low gravity environments. Toward that end, NASA developed a rotating bioreactor in which cells are cultured while continuously suspended in a cylinder in which the culture medium rotates with the cylinder. The randomization of the gravity vector accomplished by the continuous rotation, in a low shear environment, provides an analog of microgravity. Because cultures grown in bioreactors develop structures and functions that are much closer to those exhibited by native tissue than can be achieved with traditional culture methods, bioreactors have contributed substantially to advancing research in the fields of cancer, diabetes, infectious disease modeling for vaccine production, drug efficacy, and tissue engineering. NASA has developed a Classroom Bioreactor (CB) that is built from parts that are easily obtained and assembled, user-friendly and versatile. It can be easily used in simple school settings to examine the effect cultures of seeds or cells. An educational brief provides assembly instructions and lesson plans that describes activities in science, math and technology that explore free fall, microgravity, orbits, bioreactors, structure-function relationships and the scientific method.

A fiber-optic-based imaging system for nondestructive assessment of cell-seeded tissue-engineered scaffolds.

PubMed

Hofmann, Matthias C; Whited, Bryce M; Criswell, Tracy; Rylander, Marissa Nichole; Rylander, Christopher G; Soker, Shay; Wang, Ge; Xu, Yong

2012-09-01

A major limitation in tissue engineering is the lack of nondestructive methods that assess the development of tissue scaffolds undergoing preconditioning in bioreactors. Due to significant optical scattering in most scaffolding materials, current microscope-based imaging methods cannot "see" through thick and optically opaque tissue constructs. To address this deficiency, we developed a fiber-optic-based imaging method that is capable of nondestructive imaging of fluorescently labeled cells through a thick and optically opaque scaffold, contained in a bioreactor. This imaging modality is based on the local excitation of fluorescent cells, the acquisition of fluorescence through the scaffold, and fluorescence mapping based on the position of the excitation light. To evaluate the capability and accuracy of the imaging system, human endothelial cells (ECs), stably expressing green fluorescent protein (GFP), were imaged through a fibrous scaffold. Without sacrificing the scaffolds, we nondestructively visualized the distribution of GFP-labeled cells through a ~500 μm thick scaffold with cell-level resolution and distinct localization. These results were similar to control images obtained using an optical microscope with direct line-of-sight access. Through a detailed quantitative analysis, we demonstrated that this method achieved a resolution on the order of 20-30 μm, with 10% or less deviation from standard optical microscopy. Furthermore, we demonstrated that the penetration depth of the imaging method exceeded that of confocal laser scanning microscopy by more than a factor of 2. Our imaging method also possesses a working distance (up to 8 cm) much longer than that of a standard confocal microscopy system, which can significantly facilitate bioreactor integration. This method will enable the nondestructive monitoring of ECs seeded on the lumen of a tissue-engineered vascular graft during preconditioning in vitro, as well as for other tissue-engineered constructs in the future.

Decoupling the effect of shear stress and stretch on tissue growth & remodeling in a vascular graft.

PubMed

van Haaften, Eline E; Wissing, Tamar B; Rutten, Marcel; Bulsink, Jurgen A; Gashi, Kujtim; van Kelle, Mathieu A J; Smits, Anthal; Bouten, Carlijn; Kurniawan, Nicholas A

2018-06-07

The success of cardiovascular tissue engineering strategies largely depends on the mechanical environment in which cells develop a neo-tissue via growth and remodeling processes. This mechanical environment is defined by the local scaffold architecture to which cells adhere, i.e., the micro-environment, and by external mechanical cues to which cells respond, i.e., hemodynamic loading. The hemodynamic environment of early-developing blood vessels consists of both shear stress (due to blood flow) and circumferential stretch (due to blood pressure). Experimental platforms that recapitulate this mechanical environment in a controlled and tunable manner are thus critical for investigating cardiovascular tissue engineering. In traditional perfusion bioreactors, however, shear stress and stretch are coupled, hampering a clear delineation of their effects on cell and tissue response. Here, we uniquely designed a bioreactor that independently combines these two types of mechanical cues in eight parallel vascular grafts. The system is computationally and experimentally validated, through finite element analysis and culture of tissue constructs respectively, to distinguish various levels of shear stress (up to 5 Pa) and cyclic stretch (up to 1.10). To illustrate the usefulness of the system, we investigated the relative contribution of cyclic stretch (1.05 at 0.5 Hz) and shear stress (1 Pa) to tissue development. Both types of hemodynamic loading contributed to cell alignment, but the contribution of shear stress overruled stretch-induced cell proliferation and matrix (i.e., collagen and glycosaminoglycan) production. At a macroscopic level, cyclic stretching led to the most linear stress-stretch response, which was not related to the presence of shear stress. In conclusion, we have developed a bioreactor that is particularly suited to further unravel the interplay between hemodynamics and in situ tissue engineering processes. Using the new system, the present work highlights the importance of hemodynamic loading to the study of developing vascular tissues.

The role of mechanical loading in ligament tissue engineering.

PubMed

Benhardt, Hugh A; Cosgriff-Hernandez, Elizabeth M

2009-12-01

Tissue-engineered ligaments have received growing interest as a promising alternative for ligament reconstruction when traditional transplants are unavailable or fail. Mechanical stimulation was recently identified as a critical component in engineering load-bearing tissues. It is well established that living tissue responds to altered loads through endogenous changes in cellular behavior, tissue organization, and bulk mechanical properties. Without the appropriate biomechanical cues, new tissue formation lacks the necessary collagenous organization and alignment for sufficient load-bearing capacity. Therefore, tissue engineers utilize mechanical conditioning to guide tissue remodeling and improve the performance of ligament grafts. This review provides a comparative analysis of the response of ligament and tendon fibroblasts to mechanical loading in current bioreactor studies. The differential effect of mechanical stimulation on cellular processes such as protease production, matrix protein synthesis, and cell proliferation is examined in the context of tissue engineering design.

Optimal flow conditions of a tracheobronchial model to reengineer lung structures

NASA Astrophysics Data System (ADS)

Casarin, Stefano; Aletti, Federico; Baselli, Giuseppe; Garbey, Marc

2017-04-01

The high demand for lung transplants cannot be matched by an adequate number of lungs from donors. Since fully ex-novo lungs are far from being feasible, tissue engineering is actively considering implantation of engineered lungs where the devitalized structure of a donor is used as scaffold to be repopulated by stem cells of the receiving patient. A decellularized donated lung is treated inside a bioreactor where transport through the tracheobronchial tree (TBT) will allow for both deposition of stem cells and nourishment for their subsequent growth, thus developing new lung tissue. The key concern is to set optimally the boundary conditions to utilize in the bioreactor. We propose a predictive model of slow liquid ventilation, which combines a one-dimensional (1-D) mathematical model of the TBT and a solute deposition model strongly dependent on fluid velocity across the tree. With it, we were able to track and drive the concentration of a generic solute across the airways, looking for its optimal distribution. This was given by properly adjusting the pumps' regime serving the bioreactor. A feedback system, created by coupling the two models, allowed us to derive the optimal pattern. The TBT model can be easily invertible, thus yielding a straightforward flow/pressure law at the inlet to optimize the efficiency of the bioreactor.

Three-dimensional transgenic cell model to quantify genotoxic effects of space environment

NASA Astrophysics Data System (ADS)

Gonda, S. R.; Wu, H.; Pingerelli, P. L.; Glickman, B. W.

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of multiple copies of defined target genes for genotoxic assessment. Rat 2λ fibroblasts, genetically engineered to contain high-density target genes for mutagenesis (Stratagene, Inc., Austin, TX), were cocultured with human epithelial cells on Cytodex beads in the High Aspect Ratio Bioreactor (Synthecon, Inc, Houston, TX). Multi-bead aggregates were formed by day 5 following the complete covering of the beads by fibroblasts. Cellular retraction occurred 8-14 days after coculture initiation culminating in spheroids retaining few or no beads. Analysis of the resulting tissue assemblies revealed: multicellular spheroids, fibroblasts synthesized collagen, and cell viability was retained for the 30-day test period after removal from the bioreactor. Quantification of mutation at the LacI gene in Rat 2λ fibroblasts in spheroids exposed to 0-2 Gy neon using the Big Blue color assay (Stratagene, Inc.), revealed a linear dose-response for mutation induction. Limited sequencing analysis of mutant clones from 0.25 or 1 Gy exposures revealed a higher frequency of deletions and multiple base sequencing changes with increasing dose. These results suggest that the three-dimensional, multicellular tissue assembly model produced in NASA bioreactors are applicable to a wide variety of studies involving the quantification and identification of genotocity including measurement of the inherent damage incurred in Space.

A strategy to determine operating parameters in tissue engineering hollow fiber bioreactors

PubMed Central

Shipley, RJ; Davidson, AJ; Chan, K; Chaudhuri, JB; Waters, SL; Ellis, MJ

2011-01-01

The development of tissue engineering hollow fiber bioreactors (HFB) requires the optimal design of the geometry and operation parameters of the system. This article provides a strategy for specifying operating conditions for the system based on mathematical models of oxygen delivery to the cell population. Analytical and numerical solutions of these models are developed based on Michaelis–Menten kinetics. Depending on the minimum oxygen concentration required to culture a functional cell population, together with the oxygen uptake kinetics, the strategy dictates the model needed to describe mass transport so that the operating conditions can be defined. If cmin ≫ Km we capture oxygen uptake using zero-order kinetics and proceed analytically. This enables operating equations to be developed that allow the user to choose the medium flow rate, lumen length, and ECS depth to provide a prescribed value of cmin. When , we use numerical techniques to solve full Michaelis–Menten kinetics and present operating data for the bioreactor. The strategy presented utilizes both analytical and numerical approaches and can be applied to any cell type with known oxygen transport properties and uptake kinetics. PMID:21370228

Engineering bone tissue substitutes from human induced pluripotent stem cells.

PubMed

de Peppo, Giuseppe Maria; Marcos-Campos, Iván; Kahler, David John; Alsalman, Dana; Shang, Linshan; Vunjak-Novakovic, Gordana; Marolt, Darja

2013-05-21

Congenital defects, trauma, and disease can compromise the integrity and functionality of the skeletal system to the extent requiring implantation of bone grafts. Engineering of viable bone substitutes that can be personalized to meet specific clinical needs represents a promising therapeutic alternative. The aim of our study was to evaluate the utility of human-induced pluripotent stem cells (hiPSCs) for bone tissue engineering. We first induced three hiPSC lines with different tissue and reprogramming backgrounds into the mesenchymal lineages and used a combination of differentiation assays, surface antigen profiling, and global gene expression analysis to identify the lines exhibiting strong osteogenic differentiation potential. We then engineered functional bone substitutes by culturing hiPSC-derived mesenchymal progenitors on osteoconductive scaffolds in perfusion bioreactors and confirmed their phenotype stability in a subcutaneous implantation model for 12 wk. Molecular analysis confirmed that the maturation of bone substitutes in perfusion bioreactors results in global repression of cell proliferation and an increased expression of lineage-specific genes. These results pave the way for growing patient-specific bone substitutes for reconstructive treatments of the skeletal system and for constructing qualified experimental models of development and disease.

Construction and Characterization of a Novel Vocal Fold Bioreactor

PubMed Central

Zerdoum, Aidan B.; Tong, Zhixiang; Bachman, Brendan; Jia, Xinqiao

2014-01-01

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues. PMID:25145349

Construction and characterization of a novel vocal fold bioreactor.

PubMed

Zerdoum, Aidan B; Tong, Zhixiang; Bachman, Brendan; Jia, Xinqiao

2014-08-01

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 µm. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

Tissue-Engineered Autologous Grafts for Facial Bone Reconstruction

PubMed Central

Bhumiratana, Sarindr; Bernhard, Jonathan C.; Alfi, David M.; Yeager, Keith; Eton, Ryan E.; Bova, Jonathan; Shah, Forum; Gimble, Jeffrey M.; Lopez, Mandi J.; Eisig, Sidney B.; Vunjak-Novakovic, Gordana

2016-01-01

Facial deformities require precise reconstruction of the appearance and function of the original tissue. The current standard of care—the use of bone harvested from another region in the body—has major limitations, including pain and comorbidities associated with surgery. We have engineered one of the most geometrically complex facial bones by using autologous stromal/stem cells, without bone morphogenic proteins, using native bovine bone matrix and a perfusion bioreactor for the growth and transport of living grafts. The ramus-condyle unit (RCU), the most eminent load-bearing bone in the skull, was reconstructed using an image-guided personalized approach in skeletally mature Yucatan minipigs (human-scale preclinical model). We used clinically approved decellularized bovine trabecular bone as a scaffolding material, and crafted it into an anatomically correct shape using image-guided micromilling, to fit the defect. Autologous adipose-derived stromal/stem cells were seeded into the scaffold and cultured in perfusion for 3 weeks in a specialized bioreactor to form immature bone tissue. Six months after implantation, the engineered grafts maintained their anatomical structure, integrated with native tissues, and generated greater volume of new bone and greater vascular infiltration than either non-seeded anatomical scaffolds or untreated defects. This translational study demonstrates feasibility of facial bone reconstruction using autologous, anatomically shaped, living grafts formed in vitro, and presents a platform for personalized bone tissue engineering. PMID:27306665

Combination of biochemical and mechanical cues for tendon tissue engineering.

PubMed

Testa, Stefano; Costantini, Marco; Fornetti, Ersilia; Bernardini, Sergio; Trombetta, Marcella; Seliktar, Dror; Cannata, Stefano; Rainer, Alberto; Gargioli, Cesare

2017-11-01

Tendinopathies negatively affect the life quality of millions of people in occupational and athletic settings, as well as the general population. Tendon healing is a slow process, often with insufficient results to restore complete endurance and functionality of the tissue. Tissue engineering, using tendon progenitors, artificial matrices and bioreactors for mechanical stimulation, could be an important approach for treating rips, fraying and tissue rupture. In our work, C3H10T1/2 murine fibroblast cell line was exposed to a combination of stimuli: a biochemical stimulus provided by Transforming Growth Factor Beta (TGF-β) and Ascorbic Acid (AA); a three-dimensional environment represented by PEGylated-Fibrinogen (PEG-Fibrinogen) biomimetic matrix; and a mechanical induction exploiting a custom bioreactor applying uniaxial stretching. In vitro analyses by immunofluorescence and mechanical testing revealed that the proposed combined approach favours the organization of a three-dimensional tissue-like structure promoting a remarkable arrangement of the cells and the neo-extracellular matrix, reflecting into enhanced mechanical strength. The proposed method represents a novel approach for tendon tissue engineering, demonstrating how the combined effect of biochemical and mechanical stimuli ameliorates biological and mechanical properties of the artificial tissue compared to those obtained with single inducement. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Tissue engineering of heart valves: in vitro experiences.

PubMed

Sodian, R; Hoerstrup, S P; Sperling, J S; Daebritz, S H; Martin, D P; Schoen, F J; Vacanti, J P; Mayer, J E

2000-07-01

Tissue engineering is a new approach, whereby techniques are being developed to transplant autologous cells onto biodegradable scaffolds to ultimately form new functional tissue in vitro and in vivo. Our laboratory has focused on the tissue engineering of heart valves, and we have fabricated a trileaflet heart valve scaffold from a biodegradable polymer, a polyhydroxyalkanoate. In this experiment we evaluated the suitability of this scaffold material as well as in vitro conditioning to create viable tissue for tissue engineering of a trileaflet heart valve. We constructed a biodegradable and biocompatible trileaflet heart valve scaffold from a porous polyhydroxyalkanoate (Meatabolix Inc, Cambridge, MA). The scaffold consisted of a cylindrical stent (1 x 15 x 20 mm inner diameter) and leaflets (0.3 mm thick), which were attached to the stent by thermal processing techniques. The porous heart valve scaffold (pore size 100 to 240 microm) was seeded with vascular cells grown and expanded from an ovine carotid artery and placed into a pulsatile flow bioreactor for 1, 4, and 8 days. Analysis of the engineered tissue included biochemical examination, enviromental scanning electron microscopy, and histology. It was possible to create a trileaflet heart valve scaffold from polyhydroxyalkanoate, which opened and closed synchronously in a pulsatile flow bioreactor. The cells grew into the pores and formed a confluent layer after incubation and pulsatile flow exposure. The cells were mostly viable and formed connective tissue between the inside and the outside of the porous heart valve scaffold. Additionally, we demonstrated cell proliferation (DNA assay) and the capacity to generate collagen as measured by hydroxyproline assay and movat-stained glycosaminoglycans under in vitro pulsatile flow conditions. Polyhydroxyalkanoates can be used to fabricate a porous, biodegradable heart valve scaffold. The cells appear to be viable and extracellular matrix formation was induced after pulsatile flow exposure.

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture.

PubMed

Sun, T; Donoghue, P S; Higginson, J R; Gadegaard, N; Barnett, S C; Riehle, M O

2012-12-01

In tissue engineering, chemical and topographical cues are normally developed using static cell cultures but then applied directly to tissue cultures in three dimensions (3D) and under perfusion. As human cells are very sensitive to changes in the culture environment, it is essential to evaluate the performance of any such cues in a perfused environment before they are applied to tissue engineering. Thus, the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scaled-down bioreactor system, which allows evaluation of the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (a) quick and firm cell adhesion in the 3D scaffold was critical for cell survival in perfusion culture compared with static culture; thus, cell-seeding procedures for static cultures might not be applicable, therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures; (b) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion; (c) micro-grooves still maintained their influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture. Copyright © 2011 John Wiley & Sons, Ltd.

Cartilage constructs engineered from chondrocytes overexpressing IGF-I improve the repair of osteochondral defects in a rabbit model.

PubMed

Madry, H; Kaul, G; Zurakowski, D; Vunjak-Novakovic, G; Cucchiarini, M

2013-04-16

Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes overexpressing a human insulin-like growth factor I (IGF-I) gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-overexpressing chondrocytes markedly improved osteochondral repair compared with control (lacZ) constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects.

CARTILAGE CONSTRUCTS ENGINEERED FROM CHONDROCYTES OVEREXPRESSING IGF-I IMPROVE THE REPAIR OF OSTEOCHONDRAL DEFECTS IN A RABBIT MODEL

PubMed Central

Madry, Henning; Kaul, Gunter; Zurakowski, David; Vunjak-Novakovic, Gordana; Cucchiarini, Magali

2015-01-01

Tissue engineering combined with gene therapy is a promising approach for promoting articular cartilage repair. Here, we tested the hypothesis that engineered cartilage with chondrocytes over expressing a human insulin-like growth factor I (IGF-I) gene can enhance the repair of osteochondral defects, in a manner dependent on the duration of cultivation. Genetically modified chondrocytes were cultured on biodegradable polyglycolic acid scaffolds in dynamic flow rotating bioreactors for either 10 or 28 d. The resulting cartilaginous constructs were implanted into osteochondral defects in rabbit knee joints. After 28 weeks of in vivo implantation, immunoreactivity to ß-gal was detectable in the repair tissue of defects that received lacZ constructs. Engineered cartilaginous constructs based on IGF-I-over expressing chondrocytes markedly improved osteochondral repair compared with control (lacZ) constructs. Moreover, IGF-I constructs cultivated for 28 d in vitro significantly promoted osteochondral repair vis-à-vis similar constructs cultivated for 10 d, leading to significantly decreased osteoarthritic changes in the cartilage adjacent to the defects. Hence, the combination of spatially defined overexpression of human IGF-I within a tissue-engineered construct and prolonged bioreactor cultivation resulted in most enhanced articular cartilage repair and reduction of osteoarthritic changes in the cartilage adjacent to the defect. Such genetically enhanced tissue engineering provides a versatile tool to evaluate potential therapeutic genes in vivo and to improve our comprehension of the development of the repair tissue within articular cartilage defects. Insights gained with additional exploration using this model may lead to more effective treatment options for acute cartilage defects. PMID:23588785

Development of large engineered cartilage constructs from a small population of cells.

PubMed

Brenner, Jillian M; Kunz, Manuela; Tse, Man Yat; Winterborn, Andrew; Bardana, Davide D; Pang, Stephen C; Waldman, Stephen D

2013-01-01

Confronted with articular cartilage's limited capacity for self-repair, joint resurfacing techniques offer an attractive treatment for damaged or diseased tissue. Although tissue engineered cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of large defects. As routine cell expansion methods tend to elicit negative effects on chondrocyte function, we have developed an approach to generate phenotypically stable, large-sized engineered constructs (≥3 cm(2) ) directly from a small amount of donor tissue or cells (as little as 20,000 cells to generate a 3 cm(2) tissue construct). Using rabbit donor tissue, the bioreactor-cultivated constructs were hyaline-like in appearance and possessed a biochemical composition similar to native articular cartilage. Longer bioreactor cultivation times resulted in increased matrix deposition and improved mechanical properties determined over a 4 week period. Additionally, as the anatomy of the joint will need to be taken in account to effectively resurface large affected areas, we have also explored the possibility of generating constructs matched to the shape and surface geometry of a defect site through the use of rapid-prototyped defect tissue culture molds. Similar hyaline-like tissue constructs were developed that also possessed a high degree of shape correlation to the original defect mold. Future studies will be aimed at determining the effectiveness of this approach to the repair of cartilage defects in an animal model and the creation of large-sized osteochondral constructs. Copyright © 2012 American Institute of Chemical Engineers (AIChE).

In vitro fabrication of functional three-dimensional tissues with perfusable blood vessels

PubMed Central

Sekine, Hidekazu; Shimizu, Tatsuya; Sakaguchi, Katsuhisa; Dobashi, Izumi; Wada, Masanori; Yamato, Masayuki; Kobayashi, Eiji; Umezu, Mitsuo; Okano, Teruo

2013-01-01

In vitro fabrication of functional vascularized three-dimensional tissues has been a long-standing objective in the field of tissue engineering. Here we report a technique to engineer cardiac tissues with perfusable blood vessels in vitro. Using resected tissue with a connectable artery and vein as a vascular bed, we overlay triple-layer cardiac cell sheets produced from coculture with endothelial cells, and support the tissue construct with media perfused in a bioreactor. We show that endothelial cells connect to capillaries in the vascular bed and form tubular lumens, creating in vitro perfusable blood vessels in the cardiac cell sheets. Thicker engineered tissues can be produced in vitro by overlaying additional triple-layer cell sheets. The vascularized cardiac tissues beat and can be transplanted with blood vessel anastomoses. This technique may create new opportunities for in vitro tissue engineering and has potential therapeutic applications. PMID:23360990

Cell growth and differentiation on feeder layers is predicted to be influenced by bioreactor geometry.

PubMed

Peng, C A; Palsson, B Ø

1996-06-05

Tissue function is comprised of a complex interplay between biological and physicochemical rate processes. The design of bioreactors for tissue engineering must account for these processes simultaneously in order to obtain a bioreactor that provides a uniform environment for tissue growth and development. In the present study we consider the effects of fluid flow and mass transfer on the growth of a tissue in a parallel-plate bioreactor configuration. The parenchymal cells grow on a preformed stromal (feeder) layer that secretes a growth factor that stimulates parenchymal stem cell replication and differentiation. The biological dynamics are described by a unilineage model that describes the replication and differentiation of the tissue stem cell. The physicochemical rates are described by the Navier-Stokes and convective-diffusion equations. The model equations are solved by a finite element method. Two dimensionless groups govern the behavior of the solution. One is the Graetz number (Gz) that describes the relative rates of convection and diffusion, and the other a new dimensionless ratio (designated by P) that describes the interplay of the growth factor production, diffusion, and stimulation. Four geometries (slab, gondola, diamond, and radial shapes) for the parallel-plate bioreactor are analyzed. The uniformity of cell growth is measured by a two-dimensional coefficient of variance. The concentration distribution of the stroma-derived growth factor was computed first based on fluid flow and bioreactor geometry. Then the concomitant cell density distribution was obtained by integrating the calculated growth factor concentration with the parenchymal cell growth and unilineage differentiation process. The spatiotemporal cell growth patterns in four different bioreactor configurations were investigated under a variety of combinations of Gz (10(-1), 10(0), and 10(1)) and P(10(-2), 10(-1), 10(0), 10(1), and 10(2)). The results indicate high cell density and uniformity can be achieved for parameter values of P = 0.01, ..., 0.1 and Gz = 0.1, ..., 1.0. Among the four geometries investigated the radial-flow-type bioreactor provides the most uniform environment in which parenchymal cells can grow and differentiate ex vivo due to the absence of walls that are parallel to the flow paths creating slow flowing regions.

Three dimensional multi-cellular muscle-like tissue engineering in perfusion-based bioreactors.

PubMed

Cerino, Giulia; Gaudiello, Emanuele; Grussenmeyer, Thomas; Melly, Ludovic; Massai, Diana; Banfi, Andrea; Martin, Ivan; Eckstein, Friedrich; Grapow, Martin; Marsano, Anna

2016-01-01

Conventional tissue engineering strategies often rely on the use of a single progenitor cell source to engineer in vitro biological models; however, multi-cellular environments can better resemble the complexity of native tissues. Previous described co-culture models used skeletal myoblasts, as parenchymal cell source, and mesenchymal or endothelial cells, as stromal component. Here, we propose instead the use of adipose tissue-derived stromal vascular fraction cells, which include both mesenchymal and endothelial cells, to better resemble the native stroma. Percentage of serum supplementation is one of the crucial parameters to steer skeletal myoblasts toward either proliferation (20%) or differentiation (5%) in two-dimensional culture conditions. On the contrary, three-dimensional (3D) skeletal myoblast culture often simply adopts the serum content used in monolayer, without taking into account the new cell environment. When considering 3D cultures of mm-thick engineered tissues, homogeneous and sufficient oxygen supply is paramount to avoid formation of necrotic cores. Perfusion-based bioreactor culture can significantly improve the oxygen access to the cells, enhancing the viability and the contractility of the engineered tissues. In this study, we first investigated the influence of different serum supplementations on the skeletal myoblast ability to proliferate and differentiate during 3D perfusion-based culture. We tested percentages of serum promoting monolayer skeletal myoblast-proliferation (20%) and differentiation (5%) and suitable for stromal cell culture (10%) with a view to identify the most suitable condition for the subsequent co-culture. The 10% serum medium composition resulted in the highest number of mature myotubes and construct functionality. Co-culture with stromal vascular fraction cells at 10% serum also supported the skeletal myoblast differentiation and maturation, hence providing a functional engineered 3D muscle model that resembles the native multi-cellular environment. © 2015 Wiley Periodicals, Inc.

Optimization of Electrical Stimulation Parameters for Cardiac Tissue Engineering

PubMed Central

Tandon, Nina; Marsano, Anna; Maidhof, Robert; Wan, Leo; Park, Hyoungshin; Vunjak-Novakovic, Gordana

2010-01-01

In vitro application of pulsatile electrical stimulation to neonatal rat cardiomyocytes cultured on polymer scaffolds has been shown to improve the functional assembly of cells into contractile cardiac tissue constrcuts. However, to date, the conditions of electrical stimulation have not been optimized. We have systematically varied the electrode material, amplitude and frequency of stimulation, to determine the conditions that are optimal for cardiac tissue engineering. Carbon electrodes, exhibiting the highest charge-injection capacity and producing cardiac tissues with the best structural and contractile properties, and were thus used in tissue engineering studies. Cardiac tissues stimulated at 3V/cm amplitude and 3Hz frequency had the highest tissue density, the highest concentrations of cardiac troponin-I and connexin-43, and the best developed contractile behavior. These findings contribute to defining bioreactor design specifications and electrical stimulation regime for cardiac tissue engineering. PMID:21604379

A comprehensive review of cryogels and their roles in tissue engineering applications.

PubMed

Hixon, Katherine R; Lu, Tracy; Sell, Scott A

2017-10-15

The extracellular matrix is fundamental in providing an appropriate environment for cell interaction and signaling to occur. Replicating such a matrix is advantageous in the support of tissue ingrowth and regeneration through the field of tissue engineering. While scaffolds can be fabricated in many ways, cryogels have recently become a popular approach due to their macroporous structure and durability. Produced through the crosslinking of gel precursors followed by a subsequent controlled freeze/thaw cycle, the resulting cryogel provides a unique, sponge-like structure. Therefore, cryogels have proven advantageous for many tissue engineering applications including roles in bioreactor systems, cell separation, and scaffolding. Specifically, the matrix has been demonstrated to encourage the production of various molecules, such as antibodies, and has also been used for cryopreservation. Cryogels can pose as a bioreactor for the expansion of cell lines, as well as a vehicle for cell separation. Lastly, this matrix has shown excellent potential as a tissue engineered scaffold, encouraging regrowth at numerous damaged tissue sites in vivo. This review will briefly discuss the fabrication of cryogels, with an emphasis placed on their application in various facets of tissue engineering to provide an overview of this unique scaffold's past and future roles. Cryogels are unique scaffolds produced through the controlled freezing and thawing of a polymer solution. There is an ever-growing body of literature that demonstrates their applicability in the realm of tissue engineering as extracellular matrix analogue scaffolds; with extensive information having been provided regarding the fabrication, porosity, and mechanical integrity of the scaffolds. Additionally, cryogels have been reviewed with respect to their role in bioseparation and as cellular incubators. This all-inclusive view of the roles that cryogels can play is critical to advancing the technology and expanding its niche within biomaterials and tissue engineering research. To the best of the authors' knowledge, this is the first comprehensive review of cryogel applications in tissue engineering that includes specific looks at their growing roles as extracellular matrix analogues, incubators, and in bioseparation processes. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Salmonella Typhimurium grown in a rotating wall bioreactor

NASA Technical Reports Server (NTRS)

2003-01-01

Salmonella typhimurium appears green in on human intestinal tissue (stained red) cultured in a NASA rotating wall bioreactor. Dr. Cheryl Nickerson of Tulane University is studying the effects of simulated low-g on a well-known pathogen, Salmonella typhimurium, a bacterium that causes two to four million cases of gastrointestinal illness in the United States each year. While most healthy people recover readily, S. typhimurium can kill people with weakened immune systems. Thus, a simple case of food poisoning could disrupt a space mission. Using the NASA rotating-wall bioreactor, Nickerson cultured S. typhimurium in modeled microgravity. Mice infected with the bacterium died an average of three days faster than the control mice, indicating that S. typhimurium's virulence was enhanced by the bioreactor. Earlier research showed that 3 percent of the genes were altered by exposure to the bioreactor. Nickerson's work earned her a 2001 Presidential Early Career Award for Scientists and Engineers.

Design of electrical stimulation bioreactors for cardiac tissue engineering.

PubMed

Tandon, N; Marsano, A; Cannizzaro, C; Voldman, J; Vunjak-Novakovic, G

2008-01-01

Electrical stimulation has been shown to improve functional assembly of cardiomyocytes in vitro for cardiac tissue engineering. Carbon electrodes were found in past studies to have the best current injection characteristics. The goal of this study was to develop rational experimental design principles for the electrodes and stimulation regime, in particular electrode configuration, electrode ageing, and stimulation amplitude. Carbon rod electrodes were compared via electrochemical impedance spectroscopy (EIS) and we identified a safety range of 0 to 8 V/cm by comparing excitation thresholds and maximum capture rates for neonatal rat cardiomyocytes cultured with electrical stimulation. We conclude with recommendations for studies involving carbon electrodes for cardiac tissue engineering.

Microvascular Guidance: A Challenge to Support the Development of Vascularised Tissue Engineering Construct

PubMed Central

Sukmana, Irza

2012-01-01

The guidance of endothelial cell organization into a capillary network has been a long-standing challenge in tissue engineering. Some research efforts have been made to develop methods to promote capillary networks inside engineered tissue constructs. Capillary and vascular networks that would mimic blood microvessel function can be used to subsequently facilitate oxygen and nutrient transfer as well as waste removal. Vascularization of engineering tissue construct is one of the most favorable strategies to overpass nutrient and oxygen supply limitation, which is often the major hurdle in developing thick and complex tissue and artificial organ. This paper addresses recent advances and future challenges in developing three-dimensional culture systems to promote tissue construct vascularization allowing mimicking blood microvessel development and function encountered in vivo. Bioreactors systems that have been used to create fully vascularized functional tissue constructs will also be outlined. PMID:22623881

Design modification and optimisation of the perfusion system of a tri-axial bioreactor for tissue engineering.

PubMed

Hussein, Husnah; Williams, David J; Liu, Yang

2015-07-01

A systematic design of experiments (DOE) approach was used to optimize the perfusion process of a tri-axial bioreactor designed for translational tissue engineering exploiting mechanical stimuli and mechanotransduction. Four controllable design parameters affecting the perfusion process were identified in a cause-effect diagram as potential improvement opportunities. A screening process was used to separate out the factors that have the largest impact from the insignificant ones. DOE was employed to find the settings of the platen design, return tubing configuration and the elevation difference that minimise the load on the pump and variation in the perfusion process and improve the controllability of the perfusion pressures within the prescribed limits. DOE was very effective for gaining increased knowledge of the perfusion process and optimizing the process for improved functionality. It is hypothesized that the optimized perfusion system will result in improved biological performance and consistency.

Electrical stimulation: a novel tool for tissue engineering.

PubMed

Balint, Richard; Cassidy, Nigel J; Cartmell, Sarah H

2013-02-01

New advances in tissue engineering are being made through the application of different types of electrical stimuli to influence cell proliferation and differentiation. Developments made in the last decade have allowed us to improve the structure and functionality of tissue-engineered products through the use of growth factors, hormones, drugs, physical stimuli, bioreactor use, and two-dimensional (2-D) and three-dimensional (3-D) artificial extracellular matrices (with various material properties and topography). Another potential type of stimulus is electricity, which is important in the physiology and development of the majority of all human tissues. Despite its great potential, its role in tissue regeneration and its ability to influence cell migration, orientation, proliferation, and differentiation has rarely been considered in tissue engineering. This review highlights the importance of endogenous electrical stimulation, gathering the current knowledge on its natural occurrence and role in vivo, discussing the novel methods of delivering this stimulus and examining its cellular and tissue level effects, while evaluating how the technique could benefit the tissue engineering discipline in the future.

Application of an acoustofluidic perfusion bioreactor for cartilage tissue engineering.

PubMed

Li, Siwei; Glynne-Jones, Peter; Andriotis, Orestis G; Ching, Kuan Y; Jonnalagadda, Umesh S; Oreffo, Richard O C; Hill, Martyn; Tare, Rahul S

2014-12-07

Cartilage grafts generated using conventional static tissue engineering strategies are characterised by low cell viability, suboptimal hyaline cartilage formation and, critically, inferior mechanical competency, which limit their application for resurfacing articular cartilage defects. To address the limitations of conventional static cartilage bioengineering strategies and generate robust, scaffold-free neocartilage grafts of human articular chondrocytes, the present study utilised custom-built microfluidic perfusion bioreactors with integrated ultrasound standing wave traps. The system employed sweeping acoustic drive frequencies over the range of 890 to 910 kHz and continuous perfusion of the chondrogenic culture medium at a low-shear flow rate to promote the generation of three-dimensional agglomerates of human articular chondrocytes, and enhance cartilage formation by cells of the agglomerates via improved mechanical stimulation and mass transfer rates. Histological examination and assessment of micromechanical properties using indentation-type atomic force microscopy confirmed that the neocartilage grafts were analogous to native hyaline cartilage. Furthermore, in the ex vivo organ culture partial thickness cartilage defect model, implantation of the neocartilage grafts into defects for 16 weeks resulted in the formation of hyaline cartilage-like repair tissue that adhered to the host cartilage and contributed to significant improvements to the tissue architecture within the defects, compared to the empty defects. The study has demonstrated the first successful application of the acoustofluidic perfusion bioreactors to bioengineer scaffold-free neocartilage grafts of human articular chondrocytes that have the potential for subsequent use in second generation autologous chondrocyte implantation procedures for the repair of partial thickness cartilage defects.

Optical Spectroscopy and Imaging for the Noninvasive Evaluation of Engineered Tissues

PubMed Central

Rice, William L.; Hronik-Tupaj, Marie; Kaplan, David L.

2008-01-01

Optical spectroscopy and imaging approaches offer the potential to noninvasively assess different aspects of the cellular, extracellular matrix, and scaffold components of engineered tissues. In addition, the combination of multiple imaging modalities within a single instrument is highly feasible, allowing acquisition of complementary information related to the structure, organization, biochemistry, and physiology of the sample. The ability to characterize and monitor the dynamic interactions that take place as engineered tissues develop promises to enhance our understanding of the interdependence of processes that ultimately leads to functional tissue outcomes. It is expected that this information will impact significantly upon our abilities to optimize the design of biomaterial scaffolds, bioreactors, and cell systems. Here, we review the principles and performance characteristics of the main methodologies that have been exploited thus far, and we present examples of corresponding tissue engineering studies. PMID:18844604

Development and Translation of a Tissue-Engineered Disc in a Preclinical Rodent Model

DTIC Science & Technology

2012-10-29

PCL nanofiber AF and a hyaluronic acid hydrogel NP, and designed and commencement of construction of a multi-axis bioreactor that will be used to...of a PCL nanofiber AF and a hyaluronic acid hydrogel NP, and design and commencement of construction of a novel multi-axis bioreactor that will be... nanofiber AF and a hyaluronic acid hydrogel NP We have commenced fabrication and in vitro pre-culture of composite DAPS constructs. An AF region

Bioreactor Based Bone Tissue Engineering: Influence of Wall Collision on Osteoblast Cultured on Polymeric Microcarrier Scaffolds in Rotating Bioreactors

DTIC Science & Technology

2005-01-01

heavier than water (HTW; density > I g/cm 3) scaffolds were fabricated by sintering HTW microspheres of 85:15 poly (lactide-co-glycolide) ( PLAGA ), and...mixed scaffolds were designed by mixing lighter than water (LTW; density < 1 g/cm 3) and HTW microspheres of PLAGA . We quantified average velocities of...differentiation. In previous studies, we have described the development of novel poly(lactide-co-glycolide) ( PLAGA ) microsphere based mixed scaffolds that

Bioreactors to Influence Stem Cell Fate: Augmentation of Mesenchymal Stem Cell Signaling Pathways via Dynamic Culture Systems

PubMed Central

Yeatts, Andrew B.; Choquette, Daniel T.; Fisher, John P.

2012-01-01

Background Mesenchymal stem cells (MSCs) are a promising cell source for bone and cartilage tissue engineering as they can be easily isolated from the body and differentiated into osteoblasts and chondrocytes. A cell based tissue engineering strategy using MSCs often involves the culture of these cells on three-dimensional scaffolds; however the size of these scaffolds and the cell population they can support can be restricted in traditional static culture. Thus dynamic culture in bioreactor systems provides a promising means to culture and differentiate MSCs in vitro. Scope of Review This review seeks to characterize key MSC differentiation signaling pathways and provides evidence as to how dynamic culture is augmenting these pathways. Following an overview of dynamic culture systems, discussion will be provided on how these systems can effectively modify and maintain important culture parameters including oxygen content and shear stress. Literature is reviewed for both a highlight of key signaling pathways and evidence for regulation of these signaling pathways via dynamic culture systems. Major Conclusions The ability to understand how these culture systems are affecting MSC signaling pathways could lead to a shear or oxygen regime to direct stem cell differentiation. In this way the efficacy of in vitro culture and differentiation of MSCs on three-dimensional scaffolds could be greatly increased. General Significance Bioreactor systems have the ability to control many key differentiation stimuli including mechanical stress and oxygen content. The further integration of cell signaling investigations within dynamic culture systems will lead to a quicker realization of the promise of tissue engineering and regenerative medicine. PMID:22705676

Cell laden hydrogel construct on-a-chip for mimicry of cardiac tissue in-vitro study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghiaseddin, Ali; Pouri, Hossein; Soleimani, Masoud

Since the leading cause of death are cardiac diseases, engineered heart tissue (EHT) is one of the most appealing topics defined in tissue engineering and regenerative medicine fields. The importance of EHT is not only for heart regeneration but also for in vitro developing of cardiology. Cardiomyocytes could grow and commit more naturally in their microenvironment rather than traditional cultivation. Thus, this research tried to develop a set up on-a-chip to produce EHT based on chitosan hydrogel. Micro-bioreactor was hydrodynamically designed and simulated by COMSOL and produced via soft lithography process. Chitosan hydrogel was also prepared, adjusted, and assessed by XRD,more » FTIR and also its degradation rate and swelling ratio were determined. Finally, hydrogels in which mice cardiac progenitor cells (CPC) were loaded were injected into the micro-device chambers and cultured. Each EHT in every chamber was evaluated separately. Prepared EHTs showed promising results that expanded in them CPCs and work as an integrated syncytium. High cell density culture was the main accomplishment of this study. - Highlights: • An engineered heart tissue in its microenvironment at a perfused micro-bioreactor is proposed. • Cell proliferation of cardiac cells in high cell density is achievable in setup while sacrificing hydrogel is degrading. • 16 distinct heart tissue constructs in each run reduce the time and cost and increase the test results accuracy.« less

Hydrostatic Pressure in Articular Cartilage Tissue Engineering: From Chondrocytes to Tissue Regeneration

PubMed Central

Elder, Benjamin D.

2009-01-01

Cartilage has a poor intrinsic healing response, and neither the innate healing response nor current clinical treatments can restore its function. Therefore, articular cartilage tissue engineering is a promising approach for the regeneration of damaged tissue. Because cartilage is exposed to mechanical forces during joint loading, many tissue engineering strategies use exogenous stimuli to enhance the biochemical or biomechanical properties of the engineered tissue. Hydrostatic pressure (HP) is emerging as arguably one of the most important mechanical stimuli for cartilage, although no optimal treatment has been established across all culture systems. Therefore, this review evaluates prior studies on articular cartilage involving the use of HP, with a particular emphasis on the treatments that appear promising for use in future studies. Additionally, this review addresses HP bioreactor design, chondroprotective effects of HP, the use of HP for chondrogenic differentiation, the effects of high pressures, and HP mechanotransduction. PMID:19196119

Hydrostatic pressure in articular cartilage tissue engineering: from chondrocytes to tissue regeneration.

PubMed

Elder, Benjamin D; Athanasiou, Kyriacos A

2009-03-01

Cartilage has a poor intrinsic healing response, and neither the innate healing response nor current clinical treatments can restore its function. Therefore, articular cartilage tissue engineering is a promising approach for the regeneration of damaged tissue. Because cartilage is exposed to mechanical forces during joint loading, many tissue engineering strategies use exogenous stimuli to enhance the biochemical or biomechanical properties of the engineered tissue. Hydrostatic pressure (HP) is emerging as arguably one of the most important mechanical stimuli for cartilage, although no optimal treatment has been established across all culture systems. Therefore, this review evaluates prior studies on articular cartilage involving the use of HP, with a particular emphasis on the treatments that appear promising for use in future studies. Additionally, this review addresses HP bioreactor design, chondroprotective effects of HP, the use of HP for chondrogenic differentiation, the effects of high pressures, and HP mechanotransduction.

In Vitro Endothelialization of Biodegradable Vascular Grafts Via Endothelial Progenitor Cell Seeding and Maturation in a Tubular Perfusion System Bioreactor.

PubMed

Melchiorri, Anthony J; Bracaglia, Laura G; Kimerer, Lucas K; Hibino, Narutoshi; Fisher, John P

2016-07-01

A critical challenge to the success of biodegradable vascular grafts is the establishment of a healthy endothelium. To establish this monolayer of endothelial cells (ECs), a variety of techniques have been developed, including cell seeding. Vascular grafts may be seeded with relevant cell types and allowed to mature before implantation. Due to the low proliferative ability of adult ECs and issues with donor site morbidity, there has been increasing interest in using endothelial progenitor cells (EPCs) for vascular healing procedures. In this work, we combined the proliferative and differentiation capabilities of a commercial cell line of early EPCs with an established bioreactor system to support the maturation of cell-seeded vascular grafts. All components of the vascular graft and bioreactor setup are commercially available and allow for complete customization of the scaffold and culturing system. This bioreactor setup enables the control of flow through the graft, imparting fluid shear stress on EPCs and affecting cellular proliferation and differentiation. Grafts cultured with EPCs in the bioreactor system demonstrated greatly increased cell populations and neotissue formation compared with grafts seeded and cultured in a static system. Increased expression of markers for mature endothelial tissues were also observed in bioreactor-cultured EPC-seeded grafts. These findings suggest the distinct advantages of a customizable bioreactor setup for the proliferation and maturation of EPCs. Such a strategy may be beneficial for utilizing EPCs in vascular tissue engineering applications.

Effects of a Pseudophysiological Environment on the Elastic and Viscoelastic Properties of Collagen Gels

PubMed Central

Meghezi, Sébastien; Couet, Frédéric; Chevallier, Pascale; Mantovani, Diego

2012-01-01

Vascular tissue engineering focuses on the replacement of diseased small-diameter blood vessels with a diameter less than 6 mm for which adequate substitutes still do not exist. One approach to vascular tissue engineering is to culture vascular cells on a scaffold in a bioreactor. The bioreactor establishes pseudophysiological conditions for culture (medium culture, 37°C, mechanical stimulation). Collagen gels are widely used as scaffolds for tissue regeneration due to their biological properties; however, they exhibit low mechanical properties. Mechanical characterization of these scaffolds requires establishing the conditions of testing in regard to the conditions set in the bioreactor. The effects of different parameters used during mechanical testing on the collagen gels were evaluated in terms of mechanical and viscoelastic properties. Thus, a factorial experiment was adopted, and three relevant factors were considered: temperature (23°C or 37°C), hydration (aqueous saline solution or air), and mechanical preconditioning (with or without). Statistical analyses showed significant effects of these factors on the mechanical properties which were assessed by tensile tests as well as stress relaxation tests. The last tests provide a more consistent understanding of the gels' viscoelastic properties. Therefore, performing mechanical analyses on hydrogels requires setting an adequate environment in terms of temperature and aqueous saline solution as well as choosing the adequate test. PMID:22844285

Novel Bioreactor Platform for Scalable Cardiomyogenic Differentiation from Pluripotent Stem Cell-Derived Embryoid Bodies.

PubMed

Rungarunlert, Sasitorn; Ferreira, Joao N; Dinnyes, Andras

2016-01-01

Generation of cardiomyocytes from pluripotent stem cells (PSCs) is a common and valuable approach to produce large amount of cells for various applications, including assays and models for drug development, cell-based therapies, and tissue engineering. All these applications would benefit from a reliable bioreactor-based methodology to consistently generate homogenous PSC-derived embryoid bodies (EBs) at a large scale, which can further undergo cardiomyogenic differentiation. The goal of this chapter is to describe a scalable method to consistently generate large amount of homogeneous and synchronized EBs from PSCs. This method utilizes a slow-turning lateral vessel bioreactor to direct the EB formation and their subsequent cardiomyogenic lineage differentiation.

Tissue Culture in Microgravity

NASA Technical Reports Server (NTRS)

Pellis, Neal R.; Duray, Paul H.; Hatfill, Steven J.

1997-01-01

Attempts to simulate normal tissue micro-environments in vitro have been thwarted by the complexity and plasticity of the extracellular matrix, which is important in regulating cytoskeletal and nuclear matrix proteins. Gravity is one of the problems, tending to separate components that should be kept together. For space shuttle experiments, NASA engineers devised a double-walled rotating bioreactor, which is proving to be a useful tissue culture device on earth as well as in space.

Successful chondrogenesis within scaffolds, using magnetic stem cell confinement and bioreactor maturation.

PubMed

Luciani, Nathalie; Du, Vicard; Gazeau, Florence; Richert, Alain; Letourneur, Didier; Le Visage, Catherine; Wilhelm, Claire

2016-06-01

Tissue engineering strategies, such as cellularized scaffolds approaches, have been explored for cartilage replacement. The challenge, however, remains to produce a cartilaginous tissue incorporating functional chondrocytes and being large and thick enough to be compatible with the replacement of articular defects. Here, we achieved unprecedented cartilage tissue production into a porous polysaccharide scaffold by combining of efficient magnetic condensation of mesenchymal stem cells, and dynamic maturation in a bioreactor. In optimal conditions, all the hallmarks of chondrogenesis were enhanced with a 50-fold increase in collagen II expression compared to negative control, an overexpression of aggrecan and collagen XI, and a very low expression of collagen I and RUNX2. Histological staining showed a large number of cellular aggregates, as well as an increased proteoglycan synthesis by chondrocytes. Interestingly, electron microscopy showed larger chondrocytes and a more abundant extracellular matrix. In addition, the periodicity of the neosynthesized collagen fibers matched that of collagen II. These results represent a major step forward in replacement tissue for cartilage defects. A combination of several innovative technologies (magnetic cell seeding, polysaccharide porous scaffolds, and dynamic maturation in bioreactor) enabled unprecedented successful chondrogenesis within scaffolds. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Application of Disposable Bag Bioreactors in Tissue Engineering and for the Production of Therapeutic Agents

NASA Astrophysics Data System (ADS)

Eibl, R.; Eibl, D.

In order to increase process efficiency, many pharmaceutical and biotechnology companies have introduced disposable bag technology over the last 10 years. Because this technology also greatly reduces the risk of cross-contamination, disposable bags are preferred in applications in which an absolute or improved process safety is a necessity, namely the production of functional tissue for implantation (tissue engineering), the production of human cells for the treatment of cancer and immune system diseases (cellular therapy), the production of viruses for gene therapies, the production of therapeutic proteins, and veterinary as well as human vaccines.

Design of Electrical Stimulation Bioreactors for Cardiac Tissue Engineering

PubMed Central

Tandon, N.; Marsano, A.; Cannizzaro, C.; Voldman, J.; Vunjak-Novakovic, G.

2009-01-01

Electrical stimulation has been shown to improve functional assembly of cardiomyocytes in vitro for cardiac tissue engineering. Carbon electrodes were found in past studies to have the best current injection characteristics. The goal of this study was to develop rational experimental design principles for the electrodes and stimulation regime, in particular electrode configuration, electrode ageing, and stimulation amplitude. Carbon rod electrodes were compared via electrochemical impedance spectroscopy (EIS) and we identified a safety range of 0 to 8 V/cm by comparing excitation thresholds and maximum capture rates for neonatal rat cardiomyocytes cultured with electrical stimulation. We conclude with recommendations for studies involving carbon electrodes for cardiac tissue engineering. PMID:19163486

Biomedical engineering for health research and development.

PubMed

Zhang, X-Y

2015-01-01

Biomedical engineering is a new area of research in medicine and biology, providing new concepts and designs for the diagnosis, treatment and prevention of various diseases. There are several types of biomedical engineering, such as tissue, genetic, neural and stem cells, as well as chemical and clinical engineering for health care. Many electronic and magnetic methods and equipments are used for the biomedical engineering such as Computed Tomography (CT) scans, Magnetic Resonance Imaging (MRI) scans, Electroencephalography (EEG), Ultrasound and regenerative medicine and stem cell cultures, preparations of artificial cells and organs, such as pancreas, urinary bladders, liver cells, and fibroblasts cells of foreskin and others. The principle of tissue engineering is described with various types of cells used for tissue engineering purposes. The use of several medical devices and bionics are mentioned with scaffold, cells and tissue cultures and various materials are used for biomedical engineering. The use of biomedical engineering methods is very important for the human health, and research and development of diseases. The bioreactors and preparations of artificial cells or tissues and organs are described here.

Dynamic Single-Use Bioreactors Used in Modern Liter- and m(3)- Scale Biotechnological Processes: Engineering Characteristics and Scaling Up.

PubMed

Löffelholz, Christian; Kaiser, Stephan C; Kraume, Matthias; Eibl, Regine; Eibl, Dieter

2014-01-01

During the past 10 years, single-use bioreactors have been well accepted in modern biopharmaceutical production processes targeting high-value products. Up to now, such processes have mainly been small- or medium-scale mammalian cell culture-based seed inoculum, vaccine or antibody productions. However, recently first attempts have been made to modify existing single-use bioreactors for the cultivation of plant cells and tissue cultures, and microorganisms. This has even led to the development of new single-use bioreactor types. Moreover, due to safety issues it has become clear that single-use bioreactors are the "must have" for expanding human stem cells delivering cell therapeutics, the biopharmaceuticals of the next generation. So it comes as no surprise that numerous different dynamic single-use bioreactor types, which are suitable for a wide range of applications, already dominate the market today. Bioreactor working principles, main applications, and bioengineering data are presented in this review, based on a current overview of greater than milliliter-scale, commercially available, dynamic single-use bioreactors. The focus is on stirred versions, which are omnipresent in R&D and manufacturing, and in particular Sartorius Stedim's BIOSTAT family. Finally, we examine development trends for single-use bioreactors, after discussing proven approaches for fast scaling-up processes.

Development of Three-Dimensional Multicellular Tissue-Like Constructs for Mutational Analysis Using Macroporous Microcarriers

NASA Technical Reports Server (NTRS)

Jordan, Jacqueline A.; Fraga, Denise N.; Gonda, Steve R.

2002-01-01

A three-dimensional (3-D), tissue-like model was developed for the genotoxic assessment of space environment. In previous experiments, we found that culturing mammalian cells in a NASA-designed bioreactor, using Cytodex-3 beads as a scaffold, generated 3-D multicellular spheroids. In an effort to generate scaffold-free spheroids, we developed a new 3-D tissue-like model by coculturing fibroblast and epithelial cell in a NASA bioreactor using macroporous Cultispher-S(TradeMark) microcarriers. Big Blue(Registered Trademark) Rat 2(Lambda) fibroblasts, genetically engineered to contain multiple copies (>60 copies/cell) of the Lac I target gene, were cocultured with radio-sensitive human epithelial cells, H184F5. Over an 8-day period, samples were periodically examined by microscopy and histology to confirm cell attachment, growth, and viability. Immunohistochemistry and western analysis were used to evaluate the expression of specific cytoskeletal and adhesion proteins. Key cell culture parameters (glucose, pH, and lactate concentrations) were monitored daily. Controls were two-dimensional mono layers of fibroblast or epithelial cells cultured in T-flasks. Analysis of 3-D spheroids from the bioreactor suggests fibroblast cells attached to and completely covered the bead surface and inner channels by day 3 in the bioreactor. Treatment of the 3-day spheroids with dispase II dissolved the Cultisphers(TradeMark) and produced multicellular, bead-less constructs. Immunohistochemistry confirmed the presence of vi.mentin, cytokeratin and E-cadherin in treated spheroids. Examination of the dispase II treated spheroids with transmission electron microscopy (TEM) also showed the presence of desmosomes. These results suggest that the controlled enzymatic degradation of an artificial matrix in the low shear environment of the NASA-designed bioreactor can produce 3-D tissue-like spheroids. 2

Mathematical modeling and experimental testing of three bioreactor configurations based on windkessel models

PubMed Central

Ruel, Jean; Lachance, Geneviève

2010-01-01

This paper presents an experimental study of three bioreactor configurations. The bioreactor is intended to be used for the development of tissue-engineered heart valve substitutes. Therefore it must be able to reproduce physiological flow and pressure waveforms accurately. A detailed analysis of three bioreactor arrangements is presented using mathematical models based on the windkessel (WK) approach. First, a review of the many applications of this approach in medical studies enhances its fundamental nature and its usefulness. Then the models are developed with reference to the actual components of the bioreactor. This study emphasizes different conflicting issues arising in the design process of a bioreactor for biomedical purposes, where an optimization process is essential to reach a compromise satisfying all conditions. Two important aspects are the need for a simple system providing ease of use and long-term sterility, opposed to the need for an advanced (thus more complex) architecture capable of a more accurate reproduction of the physiological environment. Three classic WK architectures are analyzed, and experimental results enhance the advantages and limitations of each one. PMID:21977286

MOLD-SHAPED, NANOFIBER SCAFFOLD-BASED CARTILAGE ENGINEERING USING HUMAN MESENCHYMAL STEM CELLS AND BIOREACTOR

PubMed Central

Janjanin, Sasa; Li, Wan-Ju; Morgan, Meredith T.; Shanti, Rabie M.; Tuan, Rocky S.

2008-01-01

Background Mesenchymal stem cell (MSC)-based tissue engineering is a promising future alternative to autologous cartilage grafting. This study evaluates the potential of using MSCs, seeded into electrospun, biodegradable polymeric nanofibrous scaffolds, to engineer cartilage with defined dimensions and shape, similar to grafts used for subcutaneous implantation in plastic and reconstructive surgery. Materials and methods Human bone marrow derived MSCs seeded onto nanofibrous scaffolds and placed in custom-designed molds were cultured for up to 42 days in bioreactors. Chondrogenesis was induced with either transforming growth factor-β1 (TGF-β1) alone or in combination with insulin-like growth factor-I (IGF-I). Results Constructs exhibited hyaline cartilage histology with desired thickness and shape as well as favorable tissue integrity and shape retention, suggesting the presence of elastic tissue. Time-dependent increase in cartilage matrix gene expression was seen in both types of culture; at Day 42, TGF-β1/IGF-I treated cultures showed higher collagen type II and aggrecan expression. Both culture conditions showed significant time-dependent increase in sulfated glycosaminoglycan and hydroxyproline contents. TGF-β1/IGF-I treated samples were significantly stiffer; with equilibrium compressive Young’s modulus values reaching 17 kPa by Day 42. Conclusions The successful ex vivo development of geometrically defined cartilaginous construct using customized molding suggests the potential of cell-based cartilage tissue for reconstructive surgery. PMID:18316094

Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

PubMed Central

Rosa, Renata G.; Joazeiro, Paulo P.; Bianco, Juares; Kunz, Manuela; Weber, Joanna F.; Waldman, Stephen D.

2014-01-01

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation. PMID:25126941

Three-Dimensional Transgenic Cell Models to Quantify Space Genotoxic Effects

NASA Technical Reports Server (NTRS)

Gonda, S.; Wu, H.; Pingerelli, P.; Glickman, B.

2000-01-01

In this paper we describe a three-dimensional, multicellular tissue-equivalent model, produced in NASA-designed, rotating wall bioreactors using mammalian cells engineered for genomic containment of mUltiple copies of defined target genes for genotoxic assessment. The Rat 2(lambda) fibroblasts (Stratagene, Inc.) were genetically engineered to contain high-density target genes for mutagenesis. Stable three-dimensional, multicellular spheroids were formed when human mammary epithelial cells and Rat 2(lambda) fibroblasts were cocultured on Cytodex 3 Beads in a rotating wall bioreactor. The utility of this spheroidal model for genotoxic assessment was indicated by a linear dose response curve and by results of gene sequence analysis of mutant clones from 400micron diameter spheroids following low-dose, high-energy, neon radiation exposure

Biotechnology

NASA Image and Video Library

2003-01-22

Salmonella typhimurium appears green in on human intestinal tissue (stained red) cultured in a NASA rotating wall bioreactor. Dr. Cheryl Nickerson of Tulane University is studying the effects of simulated low-g on a well-known pathogen, Salmonella typhimurium, a bacterium that causes two to four million cases of gastrointestinal illness in the United States each year. While most healthy people recover readily, S. typhimurium can kill people with weakened immune systems. Thus, a simple case of food poisoning could disrupt a space mission. Using the NASA rotating-wall bioreactor, Nickerson cultured S. typhimurium in modeled microgravity. Mice infected with the bacterium died an average of three days faster than the control mice, indicating that S. typhimurium's virulence was enhanced by the bioreactor. Earlier research showed that 3 percent of the genes were altered by exposure to the bioreactor. Nickerson's work earned her a 2001 Presidential Early Career Award for Scientists and Engineers.

Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids

NASA Technical Reports Server (NTRS)

Chromiak, J. A.; Shansky, J.; Perrone, C.; Vandenburgh, H. H.

1998-01-01

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P < 0.05) over a 13-d period. Total protein synthesis rates could be determined accurately in the bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions necessary for space flight studies were validated as part of the studies. For example, the anabolic autocrine/paracrine skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.

Validation of Bioreactor and Human-on-a-Chip Devices for Chemical Safety Assessment.

PubMed

Rebelo, Sofia P; Dehne, Eva-Maria; Brito, Catarina; Horland, Reyk; Alves, Paula M; Marx, Uwe

2016-01-01

Equipment and device qualification and test assay validation in the field of tissue engineered human organs for substance assessment remain formidable tasks with only a few successful examples so far. The hurdles seem to increase with the growing complexity of the biological systems, emulated by the respective models. Controlled single tissue or organ culture in bioreactors improves the organ-specific functions and maintains their phenotypic stability for longer periods of time. The reproducibility attained with bioreactor operations is, per se, an advantage for the validation of safety assessment. Regulatory agencies have gradually altered the validation concept from exhaustive "product" to rigorous and detailed process characterization, valuing reproducibility as a standard for validation. "Human-on-a-chip" technologies applying micro-physiological systems to the in vitro combination of miniaturized human organ equivalents into functional human micro-organisms are nowadays thought to be the most elaborate solution created to date. They target the replacement of the current most complex models-laboratory animals. Therefore, we provide here a road map towards the validation of such "human-on-a-chip" models and qualification of their respective bioreactor and microchip equipment along a path currently used for the respective animal models.

Mesenchymal Stem Cells for Osteochondral Tissue Engineering

PubMed Central

Ng, Johnathan; Bernhard, Jonathan; Vunjak-Novakovic, Gordana

2017-01-01

Summary Mesenchymal stem cells (MSC) are of major interest to regenerative medicine, because of the ease of harvesting from a variety of sources (including bone marrow and fat aspirates) and ability to form a range of mesenchymal tissues, in vitro and in vivo. We focus here on the use of MSCs for engineering of cartilage, bone, and complex osteochondral tissue constructs, using protocols that replicate some aspects of the natural mesodermal development. For engineering of human bone, we discuss some of the current advances, and highlight the use of perfusion bioreactors for supporting anatomically exact human bone grafts. For engineering of human cartilage, we discuss limitations of current approaches, and highlight engineering of stratified, mechanically functional human cartilage interfaced with bone by mesenchymal condensation of MSCs. Taken together, the current advances enable engineering physiologically relevant bone, cartilage and osteochondral composites, and physiologically relevant studies of osteochondral development and disease. PMID:27236665

Creating homogenous strain distribution within 3D cell-encapsulated constructs using a simple and cost-effective uniaxial tensile bioreactor: Design and validation study.

PubMed

Subramanian, Gayathri; Elsaadany, Mostafa; Bialorucki, Callan; Yildirim-Ayan, Eda

2017-08-01

Mechanical loading bioreactors capable of applying uniaxial tensile strains are emerging to be a valuable tool to investigate physiologically relevant cellular signaling pathways and biochemical expression. In this study, we have introduced a simple and cost-effective uniaxial tensile strain bioreactor for the application of precise and homogenous uniaxial strains to 3D cell-encapsulated collagen constructs at physiological loading strains (0-12%) and frequencies (0.01-1 Hz). The bioreactor employs silicone-based loading chambers specifically designed to stretch constructs without direct gripping to minimize stress concentration at the ends of the construct and preserve its integrity. The loading chambers are driven by a versatile stepper motor ball-screw actuation system to produce stretching of the constructs. Mechanical characterization of the bioreactor performed through Finite Element Analysis demonstrated that the constructs experienced predominantly uniaxial tensile strain in the longitudinal direction. The strains produced were found to be homogenous over a 15 × 4 × 2 mm region of the construct equivalent to around 60% of the effective region of characterization. The strain values were also shown to be consistent and reproducible during cyclic loading regimes. Biological characterization confirmed the ability of the bioreactor to promote cell viability, proliferation, and matrix organization of cell-encapsulated collagen constructs. This easy-to-use uniaxial tensile strain bioreactor can be employed for studying morphological, structural, and functional responses of cell-embedded matrix systems in response to physiological loading of musculoskeletal tissues. It also holds promise for tissue-engineered strategies that involve delivery of mechanically stimulated cells at the site of injury through a biological carrier to develop a clinically useful therapy for tissue healing. Biotechnol. Bioeng. 2017;114: 1878-1887. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration.

PubMed

Van de Walle, Aurore; Wilhelm, Claire; Luciani, Nathalie

2017-04-27

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Latest status of the clinical and industrial applications of cell sheet engineering and regenerative medicine.

PubMed

Egami, Mime; Haraguchi, Yuji; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2014-01-01

Cell sheet engineering, which allows tissue engineering to be realized without the use of biodegradable scaffolds as an original approach, using a temperature-responsive intelligent surface, has been applied in regenerative medicine for various tissues, and a number of clinical studies have been already performed for life-threatening diseases. By using the results and findings obtained from the initial clinical studies, additional investigative clinical studies in several tissues with cell sheet engineering are currently in preparation stage. For treating many patients effectively by cell sheet engineering, an automated system integrating cell culture, cell-sheet fabrication, and layering is essential, and the system should include an advanced three-dimensional suspension cell culture system and an in vitro bioreactor system to scale up the production of cultured cells and fabricate thicker vascularized tissues. In this paper, cell sheet engineering, its clinical application, and further the authors' challenge to develop innovative cell culture systems under newly legislated regulatory platform in Japan are summarized and discussed.

Regeneration of the anterior cruciate ligament: Current strategies in tissue engineering

PubMed Central

Nau, Thomas; Teuschl, Andreas

2015-01-01

Recent advancements in the field of musculoskeletal tissue engineering have raised an increasing interest in the regeneration of the anterior cruciate ligament (ACL). It is the aim of this article to review the current research efforts and highlight promising tissue engineering strategies. The four main components of tissue engineering also apply in several ACL regeneration research efforts. Scaffolds from biological materials, biodegradable polymers and composite materials are used. The main cell sources are mesenchymal stem cells and ACL fibroblasts. In addition, growth factors and mechanical stimuli are applied. So far, the regenerated ACL constructs have been tested in a few animal studies and the results are encouraging. The different strategies, from in vitro ACL regeneration in bioreactor systems to bio-enhanced repair and regeneration, are under constant development. We expect considerable progress in the near future that will result in a realistic option for ACL surgery soon. PMID:25621217

Mechanical stimulation enhances integration in an in vitro model of cartilage repair.

PubMed

Theodoropoulos, John S; DeCroos, Amritha J N; Petrera, Massimo; Park, Sam; Kandel, Rita A

2016-06-01

(1) To characterize the effects of mechanical stimulation on the integration of a tissue-engineered construct in terms of histology, biochemistry and biomechanical properties; (2) to identify whether cells of the implant or host tissue were critical to implant integration; and (3) to study cells believed to be involved in lateral integration of tissue-engineered cartilage to host cartilage. We hypothesized that mechanical stimulation would enhance the integration of the repair implant with host cartilage in an in vitro integration model. Articular cartilage was harvested from 6- to 9-month-old bovine metacarpal-phalangeal joints. Constructs composed of tissue-engineered cartilage implanted into host cartilage were placed in spinner bioreactors and maintained on a magnetic stir plate at either 0 (static control) or 90 (experimental) rotations per minute (RPM). The constructs from both the static and spinner bioreactors were harvested after either 2 or 4 weeks of culture and evaluated histologically, biochemically, biomechanically and for gene expression. The extent and strength of integration between tissue-engineered cartilage and native cartilage improved significantly with both time and mechanical stimulation. Integration did not occur if the implant was not viable. The presence of stimulation led to a significant increase in collagen content in the integration zone between host and implant at 2 weeks. The gene profile of cells in the integration zone differs from host cartilage demonstrating an increase in the expression of membrane type 1 matrix metalloproteinase (MT1-MMP), aggrecan and type II collagen. This study shows that the integration of in vitro tissue-engineered implants with host tissue improves with mechanical stimulation. The findings of this study suggests that consideration should be given to implementing early loading (mechanical stimulation) into future in vivo studies investigating the long-term viability and integration of tissue-engineered cartilage for the treatment of cartilage injuries. This could simply be done through the use of continuous passive motion (CPM) in the post-operative period or through a more complex and structured rehabilitation program with a gradual increase in forces across the joint over time.

Alternating air-medium exposure in rotating bioreactors optimizes cell metabolism in 3D novel tubular scaffold polyurethane foams.

PubMed

Tresoldi, Claudia; Stefani, Ilaria; Ferracci, Gaia; Bertoldi, Serena; Pellegata, Alessandro F; Farè, Silvia; Mantero, Sara

2017-04-26

In vitro dynamic culture conditions play a pivotal role in developing engineered tissue grafts, where the supply of oxygen and nutrients, and waste removal must be permitted within construct thickness. For tubular scaffolds, mass transfer is enhanced by introducing a convective flow through rotating bioreactors with positive effects on cell proliferation, scaffold colonization and extracellular matrix deposition. We characterized a novel polyurethane-based tubular scaffold and investigated the impact of 3 different culture configurations over cell behavior: dynamic (i) single-phase (medium) rotation and (ii) double-phase exposure (medium-air) rotation; static (iii) single-phase static culture as control. A new mixture of polyol was tested to create polyurethane foams (PUFs) as 3D scaffold for tissue engineering. The structure obtained was morphologically and mechanically analyzed tested. Murine fibroblasts were externally seeded on the novel porous PUF scaffold, and cultured under different dynamic conditions. Viability assay, DNA quantification, SEM and histological analyses were performed at different time points. The PUF scaffold presented interesting mechanical properties and morphology adequate to promote cell adhesion, highlighting its potential for tissue engineering purposes. Results showed that constructs under dynamic conditions contain enhanced viability and cell number, exponentially increased for double-phase rotation; under this last configuration, cells uniformly covered both the external surface and the lumen. The developed 3D structure combined with the alternated exposure to air and medium provided the optimal in vitro biochemical conditioning with adequate nutrient supply for cells. The results highlight a valuable combination of material and dynamic culture for tissue engineering applications.

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration.

PubMed

Meghezi, Sébastien; Seifu, Dawit G; Bono, Nina; Unsworth, Larry; Mequanint, Kibret; Mantovani, Diego

2015-06-16

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled. The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The "static bioreactor" provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

Adipose-derived stem cells cultivated on electrospun l-lactide/glycolide copolymer fleece and gelatin hydrogels under flow conditions - aiming physiological reality in hypodermis tissue engineering.

PubMed

Gugerell, Alfred; Neumann, Anne; Kober, Johanna; Tammaro, Loredana; Hoch, Eva; Schnabelrauch, Matthias; Kamolz, Lars; Kasper, Cornelia; Keck, Maike

2015-02-01

Generation of adipose tissue for burn patients that suffer from an irreversible loss of the hypodermis is still one of the most complex challenges in tissue engineering. Electrospun materials with their micro- and nanostructures are already well established for their use as extracellular matrix substitutes. Gelatin is widely used in tissue engineering to gain thickness and volume. Under conventional static cultivation methods the supply of nutrients and transport of toxic metabolites is controlled by diffusion and therefore highly dependent on size and porosity of the biomaterial. A widely used method in order to overcome these limitations is the medium perfusion of 3D biomaterial-cell-constructs. In this study we combined perfusion bioreactor cultivation techniques with electrospun poly(l-lactide-co-glycolide) (P(LLG)) and gelatin hydrogels together with adipose-derived stem cells (ASCs) for a new approach in soft tissue engineering. ASCs were seeded on P(LLG) scaffolds and in gelatin hydrogels and cultivated for 24 hours under static conditions. Thereafter, biomaterials were cultivated under static conditions or in a bioreactor system for three, nine or twelve days with a medium flow of 0.3ml/min. Viability, morphology and differentiation of cells was monitored. ASCs seeded on P(LLG) scaffolds had a physiological morphology and good viability and were able to migrate from one electrospun scaffold to another under flow conditions but not migrate through the mesh. Differentiated ASCs showed lipid droplet formations after 21 days. Cells in hydrogels were viable but showed rounded morphology. Under flow conditions, morphology of cells was more diffuse. ASCs could be cultivated on P(LLG) scaffolds and in gelatin hydrogels under flow conditions and showed good cell viability as well as the potential to differentiate. These results should be a next step to a physiological three-dimensional construct for soft tissue engineering and regeneration. Copyright © 2014 Elsevier Ltd and ISBI. All rights reserved.

Flow dynamics in bioreactors containing tissue engineering scaffolds.

PubMed

Lawrence, Benjamin J; Devarapalli, Mamatha; Madihally, Sundararajan V

2009-02-15

Bioreactors are widely used in tissue engineering as a way to distribute nutrients within porous materials and provide physical stimulus required by many tissues. However, the fluid dynamics within the large porous structure are not well understood. In this study, we explored the effect of reactor geometry by using rectangular and circular reactors with three different inlet and outlet patterns. Geometries were simulated with and without the porous structure using the computational fluid dynamics software Comsol Multiphysics 3.4 and/or ANSYS CFX 11 respectively. Residence time distribution analysis using a step change of a tracer within the reactor revealed non-ideal fluid distribution characteristics within the reactors. The Brinkman equation was used to model the permeability characteristics with in the chitosan porous structure. Pore size was varied from 10 to 200 microm and the number of pores per unit area was varied from 15 to 1,500 pores/mm(2). Effect of cellular growth and tissue remodeling on flow distribution was also assessed by changing the pore size (85-10 microm) while keeping the number of pores per unit area constant. These results showed significant increase in pressure with reduction in pore size, which could limit the fluid flow and nutrient transport. However, measured pressure drop was marginally higher than the simulation results. Maximum shear stress was similar in both reactors and ranged approximately 0.2-0.3 dynes/cm(2). The simulations were validated experimentally using both a rectangular and circular bioreactor, constructed in-house. Porous structures for the experiments were formed using 0.5% chitosan solution freeze-dried at -80 degrees C, and the pressure drop across the reactor was monitored.

Biomimetic and synthetic esophageal tissue engineering.

PubMed

Jensen, Todd; Blanchette, Alex; Vadasz, Stephanie; Dave, Apeksha; Canfarotta, Michael; Sayej, Wael N; Finck, Christine

2015-07-01

A tissue-engineered esophagus offers an alternative for the treatment of pediatric patients suffering from severe esophageal malformations, caustic injury, and cancer. Additionally, adult patients suffering from carcinoma or trauma would benefit. Donor rat esophageal tissue was physically and enzymatically digested to isolate epithelial and smooth muscle cells, which were cultured in epithelial cell medium or smooth muscle cell medium and characterized by immunofluorescence. Isolated cells were also seeded onto electrospun synthetic PLGA and PCL/PLGA scaffolds in a physiologic hollow organ bioreactor. After 2 weeks of in vitro culture, tissue-engineered constructs were orthotopically transplanted. Isolated cells were shown to give rise to epithelial, smooth muscle, and glial cell types. After 14 days in culture, scaffolds supported epithelial, smooth muscle and glial cell phenotypes. Transplanted constructs integrated into the host's native tissue and recipients of the engineered tissue demonstrated normal feeding habits. Characterization after 14 days of implantation revealed that all three cellular phenotypes were present in varying degrees in seeded and unseeded scaffolds. We demonstrate that isolated cells from native esophagus can be cultured and seeded onto electrospun scaffolds to create esophageal constructs. These constructs have potential translatable application for tissue engineering of human esophageal tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization and Application of a Disposable Rotating Bed Bioreactor for Mesenchymal Stem Cell Expansion.

PubMed

Neumann, Anne; Lavrentieva, Antonina; Heilkenbrinker, Alexandra; Loenne, Maren; Kasper, Cornelia

2014-11-27

Recruitment of mesenchymal stromal cells (MSC) into the field of tissue engineering is a promising development since these cells can be expanded vivo to clinically relevant numbers and, after expansion, retain their ability to differentiate into various cell lineages. Safety requirements and the necessity to obtain high cell numbers without frequent subcultivation of cells raised the question of the possibility of expanding MSC in one-way (single-use) disposable bioreactors. In this study, umbilical cord-derived MSC (UC-MSC) were expanded in a disposable Z 2000 H bioreactor under dynamic conditions. Z was characterized regarding residence time and mixing in order to evaluate the optimal bioreactor settings, enabling optimal mass transfer in the absence of shear stress, allowing an reproducible expansion of MSC, while maintaining their stemness properties. Culture of the UC-MSC in disposable Z 2000 H bioreactor resulted in a reproducible 8-fold increase of cell numbers after 5 days. Cells were shown to maintain specific MSC surface marker expression as well as trilineage differentiation potential and lack stress-induced premature senescence.

A Review of Cellularization Strategies for Tissue Engineering of Whole Organs

PubMed Central

Scarritt, Michelle E.; Pashos, Nicholas C.; Bunnell, Bruce A.

2015-01-01

With the advent of whole organ decellularization, extracellular matrix scaffolds suitable for organ engineering were generated from numerous tissues, including the heart, lung, liver, kidney, and pancreas, for use as alternatives to traditional organ transplantation. Biomedical researchers now face the challenge of adequately and efficiently recellularizing these organ scaffolds. Herein, an overview of whole organ decellularization and a thorough review of the current literature for whole organ recellularization are presented. The cell types, delivery methods, and bioreactors employed for recellularization are discussed along with commercial and clinical considerations, such as immunogenicity, biocompatibility, and Food and Drug Administartion regulation. PMID:25870857

NASA-approved rotary bioreactor enhances proliferation of human epidermal stem cells and supports formation of 3D epidermis-like structure.

PubMed

Lei, Xiao-hua; Ning, Li-na; Cao, Yu-jing; Liu, Shuang; Zhang, Shou-bing; Qiu, Zhi-fang; Hu, Hui-min; Zhang, Hui-shan; Liu, Shu; Duan, En-kui

2011-01-01

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a "stimulatory" environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues currently being cultured in rotating bioreactors by investigators

Rotating Bioreactor

NASA Technical Reports Server (NTRS)

1988-01-01

The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues currently being cultured in rotating bioreactors by investigators.

Establishing Early Functional Perfusion and Structure in Tissue Engineered Cardiac Constructs

PubMed Central

Wang, Bo; Patnaik, Sourav S.; Brazile, Bryn; Butler, J. Ryan; Claude, Andrew; Zhang, Ge; Guan, Jianjun; Hong, Yi; Liao, Jun

2016-01-01

Myocardial infarction (MI) causes massive heart muscle death and remains a leading cause of death in the world. Cardiac tissue engineering aims to replace the infarcted tissues with functional engineered heart muscles or revitalize the infarcted heart by delivering cells, bioactive factors, and/or biomaterials. One major challenge of cardiac tissue engineering and regeneration is the establishment of functional perfusion and structure to achieve timely angiogenesis and effective vascularization, which are essential to the survival of thick implants and the integration of repaired tissue with host heart. In this paper, we review four major approaches to promoting angiogenesis and vascularization in cardiac tissue engineering and regeneration: delivery of pro-angiogenic factors/molecules, direct cell implantation/cell sheet grafting, fabrication of prevascularized cardiac constructs, and the use of bioreactors to promote angiogenesis and vascularization. We further provide a detailed review and discussion on the early perfusion design in nature-derived biomaterials, synthetic biodegradable polymers, tissue-derived acellular scaffolds/whole hearts, and hydrogel derived from extracellular matrix. A better understanding of the current approaches and their advantages, limitations, and hurdles could be useful for developing better materials for future clinical applications. PMID:27480586

Establishing Early Functional Perfusion and Structure in Tissue Engineered Cardiac Constructs.

PubMed

Wang, Bo; Patnaik, Sourav S; Brazile, Bryn; Butler, J Ryan; Claude, Andrew; Zhang, Ge; Guan, Jianjun; Hong, Yi; Liao, Jun

2015-01-01

Myocardial infarction (MI) causes massive heart muscle death and remains a leading cause of death in the world. Cardiac tissue engineering aims to replace the infarcted tissues with functional engineered heart muscles or revitalize the infarcted heart by delivering cells, bioactive factors, and/or biomaterials. One major challenge of cardiac tissue engineering and regeneration is the establishment of functional perfusion and structure to achieve timely angiogenesis and effective vascularization, which are essential to the survival of thick implants and the integration of repaired tissue with host heart. In this paper, we review four major approaches to promoting angiogenesis and vascularization in cardiac tissue engineering and regeneration: delivery of pro-angiogenic factors/molecules, direct cell implantation/cell sheet grafting, fabrication of prevascularized cardiac constructs, and the use of bioreactors to promote angiogenesis and vascularization. We further provide a detailed review and discussion on the early perfusion design in nature-derived biomaterials, synthetic biodegradable polymers, tissue-derived acellular scaffolds/whole hearts, and hydrogel derived from extracellular matrix. A better understanding of the current approaches and their advantages, limitations, and hurdles could be useful for developing better materials for future clinical applications.

Preparation of acellular myocardial scaffolds with well-preserved cardiomyocyte lacunae, and method for applying mechanical and electrical simulation to tissue construct.

PubMed

Wang, Bo; Williams, Lakiesha N; de Jongh Curry, Amy L; Liao, Jun

2014-01-01

Cardiac tissue engineering/regeneration using decellularized myocardium has attracted great research attention due to its potential benefit for myocardial infarction (MI) treatment. Here we describe an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulation for facilitating cardiac construct development.

Experimental orthotopic transplantation of a tissue-engineered oesophagus in rats

PubMed Central

Sjöqvist, Sebastian; Jungebluth, Philipp; Ling Lim, Mei; Haag, Johannes C.; Gustafsson, Ylva; Lemon, Greg; Baiguera, Silvia; Angel Burguillos, Miguel; Del Gaudio, Costantino; Rodríguez, Antonio Beltrán; Sotnichenko, Alexander; Kublickiene, Karolina; Ullman, Henrik; Kielstein, Heike; Damberg, Peter; Bianco, Alessandra; Heuchel, Rainer; Zhao, Ying; Ribatti, Domenico; Ibarra, Cristián; Joseph, Bertrand; Taylor, Doris A.; Macchiarini, Paolo

2014-01-01

A tissue-engineered oesophageal scaffold could be very useful for the treatment of pediatric and adult patients with benign or malignant diseases such as carcinomas, trauma or congenital malformations. Here we decellularize rat oesophagi inside a perfusion bioreactor to create biocompatible biological rat scaffolds that mimic native architecture, resist mechanical stress and induce angiogenesis. Seeded allogeneic mesenchymal stromal cells spontaneously differentiate (proven by gene-, protein and functional evaluations) into epithelial- and muscle-like cells. The reseeded scaffolds are used to orthotopically replace the entire cervical oesophagus in immunocompetent rats. All animals survive the 14-day study period, with patent and functional grafts, and gain significantly more weight than sham-operated animals. Explanted grafts show regeneration of all the major cell and tissue components of the oesophagus including functional epithelium, muscle fibres, nerves and vasculature. We consider the presented tissue-engineered oesophageal scaffolds a significant step towards the clinical application of bioengineered oesophagi. PMID:24736316

A double chamber rotating bioreactor for enhanced tubular tissue generation from human mesenchymal stem cells: a promising tool for vascular tissue regeneration.

PubMed

Stefani, I; Asnaghi, M A; Cooper-White, J J; Mantero, S

2018-01-01

Cardiovascular diseases represent a major global health burden, with high rates of mortality and morbidity. Autologous grafts are commonly used to replace damaged or failing blood vessels; however, such approaches are hampered by the scarcity of suitable graft tissue, donor site morbidity and poor long-term stability. Tissue engineering has been investigated as a means by which exogenous vessel grafts can be produced, with varying levels of success to date, a result of mismatched mechanical properties of these vessel substitutes and inadequate ex vivo vessel tissue genesis. In this work, we describe the development of a novel multifunctional dual-phase (air/aqueous) bioreactor, designed to both rotate and perfuse small-diameter tubular scaffolds and encourage enhanced tissue genesis throughout such scaffolds. Within this novel dynamic culture system, an elastomeric nanofibrous, microporous composite tubular scaffold, composed of poly(caprolactone) and acrylated poly(lactide-co-trimethylene-carbonate) and with mechanical properties approaching those of native vessels, was seeded with human mesenchymal stem cells (hMSCs) and cultured for up to 14 days in inductive (smooth muscle) media. This scaffold/bioreactor combination provided a dynamic culture environment that enhanced (compared with static controls) scaffold colonization, cell growth, extracellular matrix deposition and in situ differentiation of the hMSCs into mature smooth muscle cells, representing a concrete step towards our goal of creating a mature ex vivo vascular tissue for implantation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Adipogenesis of human adipose-derived stem cells within three-dimensional hollow fiber-based bioreactors.

PubMed

Gerlach, Jörg C; Lin, Yen-Chih; Brayfield, Candace A; Minteer, Danielle M; Li, Han; Rubin, J Peter; Marra, Kacey G

2012-01-01

To further differentiate adipose-derived stem cells (ASCs) into mature adipocytes and create three-dimensional (3D) adipose tissue in vitro, we applied multicompartment hollow fiber-based bioreactor technology with decentral mass exchange for more physiological substrate gradients and integral oxygenation. We hypothesize that a dynamic 3D perfusion in such a bioreactor will result in longer-term culture of human adipocytes in vitro, thus providing metabolically active tissue serving as a diagnostic model for screening drugs to treat diabetes. ASCs were isolated from discarded human abdominal subcutaneous adipose tissue and then inoculated into dynamic 3D culture bioreactors to undergo adipogenic differentiation. Insulin-stimulated glucose uptake from the medium was assessed with and without TNF-alpha. 3D adipose tissue was generated in the 3D-bioreactors. Immunohistochemical staining indicated that 3D-bioreactor culture displayed multiple mature adipocyte markers with more unilocular morphologies as compared with two-dimensional (2D) cultures. Results of real-time polymerase chain reaction showed 3D-bioreactor treatment had more efficient differentiation in fatty acid-binding protein 4 expression. Repeated insulin stimulation resulted in increased glucose uptake, with a return to baseline between testing. Importantly, TNF-alpha inhibited glucose uptake, an indication of the metabolic activity of the tissue. 3D bioreactors allow more mature adipocyte differentiation of ASCs compared with traditional 2D culture and generate adipose tissue in vitro for up to 2 months. Reproducible metabolic activity of the adipose tissue in the bioreactor was demonstrated, which is potentially useful for drug discovery. We present here, to the best of our knowledge for the first time, the development of a coherent 3D high density fat-like tissue consisting of unilocular structure from primary adipose stem cells in vitro.

Adipogenesis of Human Adipose-Derived Stem Cells Within Three-Dimensional Hollow Fiber-Based Bioreactors

PubMed Central

Gerlach, Jörg C.; Lin, Yen-Chih; Brayfield, Candace A.; Minteer, Danielle M.; Li, Han; Rubin, J. Peter

2012-01-01

To further differentiate adipose-derived stem cells (ASCs) into mature adipocytes and create three-dimensional (3D) adipose tissue in vitro, we applied multicompartment hollow fiber-based bioreactor technology with decentral mass exchange for more physiological substrate gradients and integral oxygenation. We hypothesize that a dynamic 3D perfusion in such a bioreactor will result in longer-term culture of human adipocytes in vitro, thus providing metabolically active tissue serving as a diagnostic model for screening drugs to treat diabetes. ASCs were isolated from discarded human abdominal subcutaneous adipose tissue and then inoculated into dynamic 3D culture bioreactors to undergo adipogenic differentiation. Insulin-stimulated glucose uptake from the medium was assessed with and without TNF-alpha. 3D adipose tissue was generated in the 3D-bioreactors. Immunohistochemical staining indicated that 3D-bioreactor culture displayed multiple mature adipocyte markers with more unilocular morphologies as compared with two-dimensional (2D) cultures. Results of real-time polymerase chain reaction showed 3D-bioreactor treatment had more efficient differentiation in fatty acid-binding protein 4 expression. Repeated insulin stimulation resulted in increased glucose uptake, with a return to baseline between testing. Importantly, TNF-alpha inhibited glucose uptake, an indication of the metabolic activity of the tissue. 3D bioreactors allow more mature adipocyte differentiation of ASCs compared with traditional 2D culture and generate adipose tissue in vitro for up to 2 months. Reproducible metabolic activity of the adipose tissue in the bioreactor was demonstrated, which is potentially useful for drug discovery. We present here, to the best of our knowledge for the first time, the development of a coherent 3D high density fat-like tissue consisting of unilocular structure from primary adipose stem cells in vitro. PMID:21902468

A bioreactor for the dynamic mechanical stimulation of vocal-fold fibroblasts based on vibro-acoustography

NASA Astrophysics Data System (ADS)

Chan, Roger W.; Rodriguez, Maritza

2005-09-01

During voice production, the vocal folds undergo airflow-induced self-sustained oscillation at a fundamental frequency of around 100-1000 Hz, with an amplitude of around 1-3 mm. The vocal-fold extracellular matrix (ECM), with appropriate tissue viscoelastic properties, is optimally tuned for such vibration. Vocal-fold fibroblasts regulate the gene expressions for key ECM proteins (e.g., collagen, fibronectin, fibromodulin, and hyaluronic acid), and these expressions are affected by the stress fields experi- enced by the fibroblasts. This study attempts to develop a bioreactor for cultivating cells under a micromechanical environment similar to that in vivo, based on the principle of vibro-acoustography. Vocal-fold fibroblasts from primary culture were grown in 3D, biodegradable scaffolds, and were excited dynamically by the radiation force generated by amplitude modulation of two confocal ultrasound beams of slightly different frequencies. Low-frequency acoustic radiation force was applied to the scaffold surface, and its vibratory response was imaged by videostroboscopy. A phantom tissue (standard viscoelastic material) with known elastic modulus was also excited and its vibratory frequency and amplitude were measured by videostroboscopy. Results showed that the bioreactor was capable of delivering mechanical stimuli to the tissue constructs in a physiological frequency range (100-1000 Hz), supporting its potential for vocal-fold tissue engineering applications. [Work supported by NIH Grant R01 DC006101.

Developing a Customized Perfusion Bioreactor Prototype with Controlled Positional Variability in Oxygen Partial Pressure for Bone and Cartilage Tissue Engineering.

PubMed

Lee, Poh Soo; Eckert, Hagen; Hess, Ricarda; Gelinsky, Michael; Rancourt, Derrick; Krawetz, Roman; Cuniberti, Gianaurelio; Scharnweber, Dieter

2017-05-01

Skeletal development is a multistep process that involves the complex interplay of multiple cell types at different stages of development. Besides biochemical and physical cues, oxygen tension also plays a pivotal role in influencing cell fate during skeletal development. At physiological conditions, bone cells generally reside in a relatively oxygenated environment whereas chondrocytes reside in a hypoxic environment. However, it is technically challenging to achieve such defined, yet diverse oxygen distribution on traditional in vitro cultivation platforms. Instead, engineered osteochondral constructs are commonly cultivated in a homogeneous, stable environment. In this study, we describe a customized perfusion bioreactor having stable positional variability in oxygen tension at defined regions. Further, engineered collagen constructs were coaxed into adopting the shape and dimensions of defined cultivation platforms that were precasted in 1.5% agarose bedding. After cultivating murine embryonic stem cells that were embedded in collagen constructs for 50 days, mineralized constructs of specific dimensions and a stable structural integrity were achieved. The end-products, specifically constructs cultivated without chondroitin sulfate A (CSA), showed a significant increase in mechanical stiffness compared with their initial gel-like constructs. More importantly, the localization of osteochondral cell types was specific and corresponded to the oxygen tension gradient generated in the bioreactor. In addition, CSA in complementary with low oxygen tension was also found to be a potent inducer of chondrogenesis in this system. In summary, we have demonstrated a customized perfusion bioreactor prototype that is capable of generating a more dynamic, yet specific cultivation environment that could support propagation of multiple osteochondral lineages within a single engineered construct in vitro. Our system opens up new possibilities for in vitro research on human skeletal development.

Myocardial scaffold-based cardiac tissue engineering: application of coordinated mechanical and electrical stimulations.

PubMed

Wang, Bo; Wang, Guangjun; To, Filip; Butler, J Ryan; Claude, Andrew; McLaughlin, Ronald M; Williams, Lakiesha N; de Jongh Curry, Amy L; Liao, Jun

2013-09-03

Recently, we developed an optimal decellularization protocol to generate 3D porcine myocardial scaffolds, which preserve the natural extracellular matrix structure, mechanical anisotropy, and vasculature templates and also show good cell recellularization and differentiation potential. In this study, a multistimulation bioreactor was built to provide coordinated mechanical and electrical stimulation for facilitating stem cell differentiation and cardiac construct development. The acellular myocardial scaffolds were seeded with mesenchymal stem cells (10(6) cells/mL) by needle injection and subjected to 5-azacytidine treatment (3 μmol/L, 24 h) and various bioreactor conditioning protocols. We found that after 2 days of culturing with mechanical (20% strain) and electrical stimulation (5 V, 1 Hz), high cell density and good cell viability were observed in the reseeded scaffold. Immunofluorescence staining demonstrated that the differentiated cells showed a cardiomyocyte-like phenotype by expressing sarcomeric α-actinin, myosin heavy chain, cardiac troponin T, connexin-43, and N-cadherin. Biaxial mechanical testing demonstrated that positive tissue remodeling took place after 2 days of bioreactor conditioning (20% strain + 5 V, 1 Hz); passive mechanical properties of the 2 day and 4 day tissue constructs were comparable to those of the tissue constructs produced by stirring reseeding followed by 2 weeks of static culturing, implying the effectiveness and efficiency of the coordinated simulations in promoting tissue remodeling. In short, the synergistic stimulations might be beneficial not only for the quality of cardiac construct development but also for patients by reducing the waiting time in future clinical scenarios.

Myocardial Scaffold-based Cardiac Tissue Engineering: Application of Coordinated Mechanical and Electrical Stimulations

PubMed Central

Wang, Bo; Wang, Guangjun; To, Filip; Butler, J. Ryan; Claude, Andrew; McLaughlin, Ronald M.; Williams, Lakiesha N.; de Jongh Curry, Amy L.; Liao, Jun

2013-01-01

Recently, we have developed an optimal decellularization protocol to generate 3D porcine myocardial scaffolds, which preserved natural extracellular matrix structure, mechanical anisotropy, and vasculature templates, and also showed good cell recellularization and differentiation potential. In this study, a multi-stimulation bioreactor was built to provide coordinated mechanical and electrical stimulations for facilitating stem cell differentiation and cardiac construct development. The acellular myocardial scaffolds were seeded with mesenchymal stem cells (106 cells/ml) by needle injection and subjected to 5-azacytidine treatment (3 μmol/L, 24 h) and various bioreactor conditioning protocols. We found that, after 2-day culture with mechanical (20% strain) and electrical stimulation (5 V, 1 Hz), high cell density and good cell viability were observed in the reseeded scaffold. Immunofluorescence staining demonstrated that the differentiated cells showed cardiomyocyte-like phenotype, by expressing sarcomeric α-actinin, myosin heavy chain, cardiac troponin T, connexin-43, and N-cadherin. Biaxial mechanical testing demonstrated that positive tissue remodeling took place after 2-day bioreactor conditioning (20% strain + 5 V, 1 Hz); passive mechanical properties of the 2-day and 4-day tissue constructs were comparable to the tissue constructs produced by stirring reseeding followed by 2-week static culture, implying the effectiveness and efficiency of the coordinated simulations in promoting tissue remodeling. In short, the synergistic stimulations might be beneficial not only for the quality of cardiac construct development, but also for patients by reducing the waiting time in future clinical scenarios. PMID:23923967

Bioreactor principles

NASA Technical Reports Server (NTRS)

2001-01-01

Cells cultured on Earth (left) typically settle quickly on the bottom of culture vessels due to gravity. In microgravity (right), cells remain suspended and aggregate to form three-dimensional tissue. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Electronics control module for the NASA Bioreactor. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Interior view of the gas supply for the NASA Bioreactor. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2001-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Final samples from Mir and Earth appeared histologically cartilaginous throughout their entire cross sections (5-8 mm thick), with the exception of fibrous outer capsules. Constructs grown on Earth (A) appeared to have a more organized extracellular matrix with more uniform collagen orientation as compared with constructs grown on Mir (B), but the average collagen fiber diameter was similar in the two groups (22 +- 2 nm) and comparable to that previously reported for developing articular cartilage. Randomly oriented collagen in Mir samples would be consistent with previous reports that microgravity disrupts fibrillogenesis. These are transmission electron micrographs of constructs from Mir (A) and Earth (B) groups at magnifications of x3,500 and x120,000 (Inset). The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Credit: Proceedings of the National Academy of Sciences.

Commercial considerations in tissue engineering

PubMed Central

Mansbridge, Jonathan

2006-01-01

Tissue engineering is a field with immense promise. Using the example of an early tissue-engineered skin implant, Dermagraft, factors involved in the successful commercial development of devices of this type are explored. Tissue engineering has to strike a balance between tissue culture, which is a resource-intensive activity, and business considerations that are concerned with minimizing cost and maximizing customer convenience. Bioreactor design takes place in a highly regulated environment, so factors to be incorporated into the concept include not only tissue culture considerations but also matters related to asepsis, scaleup, automation and ease of use by the final customer. Dermagraft is an allogeneic tissue. Stasis preservation, in this case cryopreservation, is essential in allogeneic tissue engineering, allowing sterility testing, inventory control and, in the case of Dermagraft, a cellular stress that may be important for hormesis following implantation. Although the use of allogeneic cells provides advantages in manufacturing under suitable conditions, it raises the spectre of immunological rejection. Such rejection has not been experienced with Dermagraft. Possible reasons for this and the vision of further application of allogeneic tissues are important considerations in future tissue-engineered cellular devices. This review illustrates approaches that indicate some of the criteria that may provide a basis for further developments. Marketing is a further requirement for success, which entails understanding of the mechanism of action of the procedure, and is illustrated for Dermagraft. The success of a tissue-engineered product is dependent on many interacting operations, some discussed here, each of which must be performed simultaneously and well. PMID:17005024

Commercial considerations in tissue engineering.

PubMed

Mansbridge, Jonathan

2006-10-01

Tissue engineering is a field with immense promise. Using the example of an early tissue-engineered skin implant, Dermagraft, factors involved in the successful commercial development of devices of this type are explored. Tissue engineering has to strike a balance between tissue culture, which is a resource-intensive activity, and business considerations that are concerned with minimizing cost and maximizing customer convenience. Bioreactor design takes place in a highly regulated environment, so factors to be incorporated into the concept include not only tissue culture considerations but also matters related to asepsis, scaleup, automation and ease of use by the final customer. Dermagraft is an allogeneic tissue. Stasis preservation, in this case cryopreservation, is essential in allogeneic tissue engineering, allowing sterility testing, inventory control and, in the case of Dermagraft, a cellular stress that may be important for hormesis following implantation. Although the use of allogeneic cells provides advantages in manufacturing under suitable conditions, it raises the spectre of immunological rejection. Such rejection has not been experienced with Dermagraft. Possible reasons for this and the vision of further application of allogeneic tissues are important considerations in future tissue-engineered cellular devices. This review illustrates approaches that indicate some of the criteria that may provide a basis for further developments. Marketing is a further requirement for success, which entails understanding of the mechanism of action of the procedure, and is illustrated for Dermagraft. The success of a tissue-engineered product is dependent on many interacting operations, some discussed here, each of which must be performed simultaneously and well.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Laptop computer sits atop the Experiment Control Computer for a NASA Bioreactor. The flight crew can change operating conditions in the Bioreactor by using the graphical interface on the laptop. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Production of oncolytic adenovirus and human mesenchymal stem cells in a single-use, Vertical-Wheel bioreactor system: Impact of bioreactor design on performance of microcarrier-based cell culture processes.

PubMed

Sousa, Marcos F Q; Silva, Marta M; Giroux, Daniel; Hashimura, Yas; Wesselschmidt, Robin; Lee, Brian; Roldão, António; Carrondo, Manuel J T; Alves, Paula M; Serra, Margarida

2015-01-01

Anchorage-dependent cell cultures are used for the production of viruses, viral vectors, and vaccines, as well as for various cell therapies and tissue engineering applications. Most of these applications currently rely on planar technologies for the generation of biological products. However, as new cell therapy product candidates move from clinical trials towards potential commercialization, planar platforms have proven to be inadequate to meet large-scale manufacturing demand. Therefore, a new scalable platform for culturing anchorage-dependent cells at high cell volumetric concentrations is urgently needed. One promising solution is to grow cells on microcarriers suspended in single-use bioreactors. Toward this goal, a novel bioreactor system utilizing an innovative Vertical-Wheel™ technology was evaluated for its potential to support scalable cell culture process development. Two anchorage-dependent human cell types were used: human lung carcinoma cells (A549 cell line) and human bone marrow-derived mesenchymal stem cells (hMSC). Key hydrodynamic parameters such as power input, mixing time, Kolmogorov length scale, and shear stress were estimated. The performance of Vertical-Wheel bioreactors (PBS-VW) was then evaluated for A549 cell growth and oncolytic adenovirus type 5 production as well as for hMSC expansion. Regarding the first cell model, higher cell growth and number of infectious viruses per cell were achieved when compared with stirred tank (ST) bioreactors. For the hMSC model, although higher percentages of proliferative cells could be reached in the PBS-VW compared with ST bioreactors, no significant differences in the cell volumetric concentration and expansion factor were observed. Noteworthy, the hMSC population generated in the PBS-VW showed a significantly lower percentage of apoptotic cells as well as reduced levels of HLA-DR positive cells. Overall, these results showed that process transfer from ST bioreactor to PBS-VW, and scale-up was successfully carried out for two different microcarrier-based cell cultures. Ultimately, the data herein generated demonstrate the potential of Vertical-Wheel bioreactors as a new scalable biomanufacturing platform for microcarrier-based cell cultures of complex biopharmaceuticals. © 2015 American Institute of Chemical Engineers.

Modeling of flow-induced shear stress applied on 3D cellular scaffolds: Implications for vascular tissue engineering.

PubMed

Lesman, Ayelet; Blinder, Yaron; Levenberg, Shulamit

2010-02-15

Novel tissue-culture bioreactors employ flow-induced shear stress as a means of mechanical stimulation of cells. We developed a computational fluid dynamics model of the complex three-dimensional (3D) microstructure of a porous scaffold incubated in a direct perfusion bioreactor. Our model was designed to predict high shear-stress values within the physiological range of those naturally sensed by vascular cells (1-10 dyne/cm(2)), and will thereby provide suitable conditions for vascular tissue-engineering experiments. The model also accounts for cellular growth, which was designed as an added cell layer grown on all scaffold walls. Five model variants were designed, with geometric differences corresponding to cell-layer thicknesses of 0, 50, 75, 100, and 125 microm. Four inlet velocities (0.5, 1, 1.5, and 2 cm/s) were applied to each model. Wall shear-stress distribution and overall pressure drop calculations were then used to characterize the relation between flow rate, shear stress, cell-layer thickness, and pressure drop. The simulations showed that cellular growth within 3D scaffolds exposes cells to elevated shear stress, with considerably increasing average values in correlation to cell growth and inflow velocity. Our results provide in-depth analysis of the microdynamic environment of cells cultured within 3D environments, and thus provide advanced control over tissue development in vitro. 2009 Wiley Periodicals, Inc.

Coculture strategies in bone tissue engineering: the impact of culture conditions on pluripotent stem cell populations.

PubMed

Janardhanan, Sathyanarayana; Wang, Martha O; Fisher, John P

2012-08-01

The use of pluripotent stem cell populations for bone tissue regeneration provides many opportunities and challenges within the bone tissue engineering field. For example, coculture strategies have been utilized to mimic embryological development of bone tissue, and particularly the critical intercellular signaling pathways. While research in bone biology over the last 20 years has expanded our understanding of these intercellular signaling pathways, we still do not fully understand the impact of the system's physical characteristics (orientation, geometry, and morphology). This review of coculture literature delineates the various forms of coculture systems and their respective outcomes when applied to bone tissue engineering. To understand fully the key differences between the different coculture methods, we must appreciate the underlying paradigms of physiological interactions. Recent advances have enabled us to extrapolate these techniques to larger dimensions and higher geometric resolutions. Finally, the contributions of bioreactors, micropatterned biomaterials, and biomaterial interaction platforms are evaluated to give a sense of the sophistication established by a combination of these concepts with coculture systems.

Aortic valve cell seeding into decellularized animal pericardium by perfusion-assisted bioreactor.

PubMed

Amadeo, Francesco; Boschetti, Federica; Polvani, Gianluca; Banfi, Cristina; Pesce, Maurizio; Santoro, Rosaria

2018-04-27

Animal-derived pericardium is the elective tissue employed in manufacturing heart valve prostheses. The preparation of this tissue for biological valve production consists of fixation with aldehydes, which reduces, but not eliminates, the xenoantigens and the donor cellular material. As a consequence, especially in patients below 65-70 years of age, the employment of valve substitutes contaning pericardium is not indicated due to progressive calcification that causes tissue degeneration and recurrence of valve insufficiency. Decellularization with ionic or nonionic detergents has been proposed as an alternative procedure to prepare aldehyde- or xenoantigen-free pericardium for biological valve manufacturing. In the present contribution, we optimized a decellularization procedure that is permissive for seeding and culturing valve competent cells able to colonize and reconstitute a valve-like tissue. A high-efficiency cellularization was achieved by forcing cell penetration inside the pericardium matrix using a perfusion bioreactor. Because the decellularization procedure was found not to alter the collagen composition of the pericardial matrix and cells seeded in the tissue constructs consistently grew and acquired the phenotype of "quiescent" valve interstitial cells, our investigation sets a novel standard in pericardium application for tissue engineering of "living" valve implants. Copyright © 2018 John Wiley & Sons, Ltd.

Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces

NASA Astrophysics Data System (ADS)

Luo, Wei; Pulsipher, Abigail; Dutta, Debjit; Lamb, Brian M.; Yousaf, Muhammad N.

2014-09-01

We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies.

Deformation simulation of cells seeded on a collagen-GAG scaffold in a flow perfusion bioreactor using a sequential 3D CFD-elastostatics model.

PubMed

Jungreuthmayer, C; Jaasma, M J; Al-Munajjed, A A; Zanghellini, J; Kelly, D J; O'Brien, F J

2009-05-01

Tissue-engineered bone shows promise in meeting the huge demand for bone grafts caused by up to 4 million bone replacement procedures per year, worldwide. State-of-the-art bone tissue engineering strategies use flow perfusion bioreactors to apply biophysical stimuli to cells seeded on scaffolds and to grow tissue suitable for implantation into the patient's body. The aim of this study was to quantify the deformation of cells seeded on a collagen-GAG scaffold which was perfused by culture medium inside a flow perfusion bioreactor. Using a microCT scan of an unseeded collagen-GAG scaffold, a sequential 3D CFD-deformation model was developed. The wall shear stress and the hydrostatic wall pressure acting on the cells were computed through the use of a CFD simulation and fed into a linear elastostatics model in order to calculate the deformation of the cells. The model used numerically seeded cells of two common morphologies where cells are either attached flatly on the scaffold wall or bridging two struts of the scaffold. Our study showed that the displacement of the cells is primarily determined by the cell morphology. Although cells of both attachment profiles were subjected to the same mechanical load, cells bridging two struts experienced a deformation up to 500 times higher than cells only attached to one strut. As the scaffold's pore size determines both the mechanical load and the type of attachment, the design of an optimal scaffold must take into account the interplay of these two features and requires a design process that optimizes both parameters at the same time.

A three-dimensional computational fluid dynamics model of shear stress distribution during neotissue growth in a perfusion bioreactor.

PubMed

Guyot, Y; Luyten, F P; Schrooten, J; Papantoniou, I; Geris, L

2015-12-01

Bone tissue engineering strategies use flow through perfusion bioreactors to apply mechanical stimuli to cells seeded on porous scaffolds. Cells grow on the scaffold surface but also by bridging the scaffold pores leading a fully filled scaffold following the scaffold's geometric characteristics. Current computational fluid dynamic approaches for tissue engineering bioreactor systems have been mostly carried out for empty scaffolds. The effect of 3D cell growth and extracellular matrix formation (termed in this study as neotissue growth), on its surrounding fluid flow field is a challenge yet to be tackled. In this work a combined approach was followed linking curvature driven cell growth to fluid dynamics modeling. The level-set method (LSM) was employed to capture neotissue growth driven by curvature, while the Stokes and Darcy equations, combined in the Brinkman equation, provided information regarding the distribution of the shear stress profile at the neotissue/medium interface and within the neotissue itself during growth. The neotissue was assumed to be micro-porous allowing flow through its structure while at the same time allowing the simulation of complete scaffold filling without numerical convergence issues. The results show a significant difference in the amplitude of shear stress for cells located within the micro-porous neo-tissue or at the neotissue/medium interface, demonstrating the importance of taking along the neotissue in the calculation of the mechanical stimulation of cells during culture.The presented computational framework is used on different scaffold pore geometries demonstrating its potential to be used a design as tool for scaffold architecture taking into account the growing neotissue. Biotechnol. Bioeng. 2015;112: 2591-2600. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Effects of Hydrostatic Loading on a Self-Aggregating, Suspension Culture–Derived Cartilage Tissue Analog

PubMed Central

Kraft, Jeffrey J.; Jeong, Changhoon; Novotny, John E.; Seacrist, Thomas; Chan, Gilbert; Domzalski, Marcin; Turka, Christina M.; Richardson, Dean W.; Dodge, George R.

2011-01-01

Objective: Many approaches are being taken to generate cartilage replacement materials. The goal of this study was to use a self-aggregating suspension culture model of chondrocytes with mechanical preconditioning. Design: Our model differs from others in that it is based on a scaffold-less, self-aggregating culture model that produces a cartilage tissue analog that has been shown to share many similarities with the natural cartilage phenotype. Owing to the known loaded environment under which chondrocytes function in vivo, we hypothesized that applying force to the suspension culture–derived chondrocyte biomass would improve its cartilage-like characteristics and provide a new model for engineering cartilage tissue analogs. Results: In this study, we used a specialized hydrostatic pressure bioreactor system to apply mechanical forces during the growth phase to improve biochemical and biophysical properties of the biomaterial formed. We demonstrated that using this high-density suspension culture, a biomaterial more consistent with the hyaline cartilage phenotype was produced without any foreign material added. Unpassaged chondrocytes responded to a physiologically relevant hydrostatic load by significantly increasing gene expression of critical cartilage molecule collagen and aggrecan along with other cartilage relevant genes, CD44, perlecan, decorin, COMP, and iNOS. Conclusions: This study describes a self-aggregating bioreactor model without foreign material or scaffold in which chondrocytes form a cartilage tissue analog with many features similar to native cartilage. This study represents a promising scaffold-less, methodological advancement in cartilage tissue engineering with potential translational applications to cartilage repair. PMID:26069584

Effects of Hydrostatic Loading on a Self-Aggregating, Suspension Culture-Derived Cartilage Tissue Analog.

PubMed

Kraft, Jeffrey J; Jeong, Changhoon; Novotny, John E; Seacrist, Thomas; Chan, Gilbert; Domzalski, Marcin; Turka, Christina M; Richardson, Dean W; Dodge, George R

2011-07-01

Many approaches are being taken to generate cartilage replacement materials. The goal of this study was to use a self-aggregating suspension culture model of chondrocytes with mechanical preconditioning. Our model differs from others in that it is based on a scaffold-less, self-aggregating culture model that produces a cartilage tissue analog that has been shown to share many similarities with the natural cartilage phenotype. Owing to the known loaded environment under which chondrocytes function in vivo, we hypothesized that applying force to the suspension culture-derived chondrocyte biomass would improve its cartilage-like characteristics and provide a new model for engineering cartilage tissue analogs. In this study, we used a specialized hydrostatic pressure bioreactor system to apply mechanical forces during the growth phase to improve biochemical and biophysical properties of the biomaterial formed. We demonstrated that using this high-density suspension culture, a biomaterial more consistent with the hyaline cartilage phenotype was produced without any foreign material added. Unpassaged chondrocytes responded to a physiologically relevant hydrostatic load by significantly increasing gene expression of critical cartilage molecule collagen and aggrecan along with other cartilage relevant genes, CD44, perlecan, decorin, COMP, and iNOS. This study describes a self-aggregating bioreactor model without foreign material or scaffold in which chondrocytes form a cartilage tissue analog with many features similar to native cartilage. This study represents a promising scaffold-less, methodological advancement in cartilage tissue engineering with potential translational applications to cartilage repair.

Microgravity

NASA Image and Video Library

1996-06-01

The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues currently being cultured in rotating bioreactors by investigators

Microgravity

NASA Image and Video Library

1988-07-14

The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues currently being cultured in rotating bioreactors by investigators.

Use of NASA Bioreactor in Engineering Tissue for Bone Repair

NASA Technical Reports Server (NTRS)

Duke, Pauline

1998-01-01

This study was proposed in search for a new alternative for bone replacement or repair. Because the systems commonly used in repair of bony defects form bone by going through a cartilaginous phase, implantation of a piece of cartilage could enhance the healing process by having a more advanced starting point. However, cartilage has seldom been used to replace bone due, in part, to the limitations in conventional culture systems that did not allow production of enough tissue for implants. The NASA-developed bioreactors known as STLV (Slow Turning Lateral Vessel) provide homogeneous distribution of cells, nutrients, and waste products, with less damaging turbulence and shear forces than conventional systems. Cultures under these conditions have higher growth rates, viability, and longevity, allowing larger "tissue-like" aggregates to form, thus opening the possibilities of producing enough tissue for implantation, along with the inherent advantages of in vitro manipulations. To assure large numbers of cells and to eliminate the use of timed embryos, we proposed to use an immortalized mouse limb bud cell line as the source of cells.

Microgravity

NASA Image and Video Library

1996-01-01

Electronics control module for the NASA Bioreactor. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Microgravity

NASA Image and Video Library

1996-01-01

Interior view of the gas supply for the NASA Bioreactor. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Cell sheet-based tissue engineering for fabricating 3-dimensional heart tissues.

PubMed

Shimizu, Tatsuya

2014-01-01

In addition to stem cell biology, tissue engineering is an essential research field for regenerative medicine. In contrast to cell injection, bioengineered tissue transplantation minimizes cell loss and has the potential to repair tissue defects. A popular approach is scaffold-based tissue engineering, which utilizes a biodegradable polymer scaffold for seeding cells; however, new techniques of cell sheet-based tissue engineering have been developed. Cell sheets are harvested from temperature-responsive culture dishes by simply lowering the temperature. Monolayer or stacked cell sheets are transplantable directly onto damaged tissues and cell sheet transplantation has already been clinically applied. Cardiac cell sheet stacking produces pulsatile heart tissue; however, lack of vasculature limits the viable tissue thickness to 3 layers. Multistep transplantation of triple-layer cardiac cell sheets cocultured with endothelial cells has been used to form thick vascularized cardiac tissue in vivo. Furthermore, in vitro functional blood vessel formation within 3-dimensional (3D) tissues has been realized by successfully imitating in vivo conditions. Triple-layer cardiac cell sheets containing endothelial cells were layered on vascular beds and the constructs were media-perfused using novel bioreactor systems. Interestingly, cocultured endothelial cells migrate into the vascular beds and form perfusable blood vessels. An in vitro multistep procedure has also enabled the fabrication of thick, vascularized heart tissues. Cell sheet-based tissue engineering has revealed great potential to fabricate 3D cardiac tissues and should contribute to future treatment of severe heart diseases and human tissue model production.

Microgravity

NASA Image and Video Library

1996-01-01

Laptop computer sits atop the Experiment Control Computer for a NASA Bioreactor. The flight crew can change operating conditions in the Bioreactor by using the graphical interface on the laptop. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Interior of a Biotechnology Refrigerator that preserves samples for use in (or after culturing in) the NASA Bioreactor. The unit is shown extracted from a middeck locker shell. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Biotechnology Refrigerator that preserves samples for use in (or after culturing in) the NASA Bioreactor. The unit is shown extracted from a middeck locker shell. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Biotechnology Refrigerator that preserves samples for use in (or after culturing in) the NASA Bioreactor. The unit is shown extracted from a middeck locker shell and with thermal blankets partially removed. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

A novel flow-perfusion bioreactor supports 3D dynamic cell culture.

PubMed

Sailon, Alexander M; Allori, Alexander C; Davidson, Edward H; Reformat, Derek D; Allen, Robert J; Warren, Stephen M

2009-01-01

Bone engineering requires thicker three-dimensional constructs than the maximum thickness supported by standard cell-culture techniques (2 mm). A flow-perfusion bioreactor was developed to provide chemotransportation to thick (6 mm) scaffolds. Polyurethane scaffolds, seeded with murine preosteoblasts, were loaded into a novel bioreactor. Control scaffolds remained in static culture. Samples were harvested at days 2, 4, 6, and 8 and analyzed for cellular distribution, viability, metabolic activity, and density at the periphery and core. By day 8, static scaffolds had a periphery cell density of 67% +/- 5.0%, while in the core it was 0.3% +/- 0.3%. Flow-perfused scaffolds demonstrated peripheral cell density of 94% +/- 8.3% and core density of 76% +/- 3.1% at day 8. Flow perfusion provides chemotransportation to thick scaffolds. This system may permit high throughput study of 3D tissues in vitro and enable prefabrication of biological constructs large enough to solve clinical problems.

Microgravity

NASA Image and Video Library

2001-06-01

Cells cultured on Earth (left) typically settle quickly on the bottom of culture vessels due to gravity. In microgravity (right), cells remain suspended and aggregate to form three-dimensional tissue. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Novel anisotropic engineered cardiac tissues: studies of electrical propagation.

PubMed

Bursac, Nenad; Loo, Yihua; Leong, Kam; Tung, Leslie

2007-10-05

The goal of this study was to engineer cardiac tissue constructs with uniformly anisotropic architecture, and to evaluate their electrical function using multi-site optical mapping of cell membrane potentials. Anisotropic polymer scaffolds made by leaching of aligned sucrose templates were seeded with neonatal rat cardiac cells and cultured in rotating bioreactors for 6-14 days. Cells aligned and interconnected inside the scaffolds and when stimulated by a point electrode, supported macroscopically continuous, anisotropic impulse propagation. By culture day 14, the ratio of conduction velocities along vs. across cardiac fibers reached a value of 2, similar to that in native neonatal ventricles, while action potential duration and maximum capture rate, respectively, decreased to 120ms and increased to approximately 5Hz. The shorter culture time and larger scaffold thickness were associated with increased incidence of sustained reentrant arrhythmias. In summary, this study is the first successful attempt to engineer a cm(2)-size, functional anisotropic cardiac tissue patch.

Man as a living bioreactor: Long-term histological aspects of a mandibular replacement engineered in the patient's own body.

PubMed

Naujokat, H; Açil, Y; Gülses, A; Birkenfeld, F; Wiltfang, J

2018-05-26

In 2016, we reported the world's first reconstruction of a mandibular discontinuity defect using a custom-made bone transplant that had been prefabricated in the gastrocolic omentum using tissue engineering strategies. However, the tissue of an engineered human neomandible has not been evaluated histologically until now. The current study assessed the long-term histological characteristics of biopsies of the neomandible 9months after transplantation. Histological analysis showed an increased amount of vital mineralized bone tissue after 10months, in comparison to biopsies obtained earlier. The engineered bone covered the surface of the bone substitute material but also grew out typical structures of cancellous bone tissue without a core of BioOss. The amount of induced bone tissue was 32% in the biopsy. In addition, the soft tissue showed an alignment of the connective tissue fibres parallel to the trabecular bone. Increasing time and mechanical forces at the mandible led to an increased amount of mineralized tissue and remodelling of the connective tissue fibres after transplantation. Further research should focus on developing advanced scaffold materials, as the outer titanium mesh cage leads to complications. Copyright © 2018 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Bioreactors Drive Advances in Tissue Engineering

NASA Technical Reports Server (NTRS)

2012-01-01

It was an unlikely moment for inspiration. Engineers David Wolf and Ray Schwarz stopped by their lab around midday. Wolf, of Johnson Space Center, and Schwarz, with NASA contractor Krug Life Sciences (now Wyle Laboratories Inc.), were part of a team tasked with developing a unique technology with the potential to enhance medical research. But that wasn t the focus at the moment: The pair was rounding up colleagues interested in grabbing some lunch. One of the lab s other Krug engineers, Tinh Trinh, was doing something that made Wolf forget about food. Trinh was toying with an electric drill. He had stuck the barrel of a syringe on the bit; it spun with a high-pitched whirr when he squeezed the drill s trigger. At the time, a multidisciplinary team of engineers and biologists including Wolf, Schwarz, Trinh, and project manager Charles D. Anderson, who formerly led the recovery of the Apollo capsules after splashdown and now worked for Krug was pursuing the development of a technology called a bioreactor, a cylindrical device used to culture human cells. The team s immediate goal was to grow human kidney cells to produce erythropoietin, a hormone that regulates red blood cell production and can be used to treat anemia. But there was a major barrier to the technology s success: Moving the liquid growth media to keep it from stagnating resulted in turbulent conditions that damaged the delicate cells, causing them to quickly die. The team was looking forward to testing the bioreactor in space, hoping the device would perform more effectively in microgravity. But on January 28, 1986, the Space Shuttle Challenger broke apart shortly after launch, killing its seven crewmembers. The subsequent grounding of the shuttle fleet had left researchers with no access to space, and thus no way to study the effects of microgravity on human cells. As Wolf looked from Trinh s syringe-capped drill to where the bioreactor sat on a workbench, he suddenly saw a possible solution to both problems. It dawned on me that rotating the wall of the reactor would solve one of our fundamental fluid mechanical problems, specifically by removing the velocity gradient of the tissue culture fluid media near the reactor s walls, says Wolf. It looked as though it would allow us to suspend the growing cells within the reactor without introducing turbulent fluid mechanical conditions.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

For 5 days on the STS-70 mission, a bioreactor cultivated human colon cancer cells, such as the culture section shown here, which grew to 30 times the volume of control specimens grown on Earth. This significant result was reproduced on STS-85 which grew mature structures that more closely match what are found in tumors in humans. The two white circles within the tumor are part of a plastic lattice that helped the cells associate. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Tissue grown in space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens of cartilage tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Constructs grown on Mir (A) tended to become more spherical, whereas those grown on Earth (B) maintained their initial disc shape. These findings might be related to differences in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1g (Earth's gravity). In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion, increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. A and B are full cross sections of constructs from Mir and Earth groups shown at 10-power. C and D are representative areas at the construct surfaces enlarged to 200-power. They are stained red with safranin-O. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Photo credit: Proceedings of the National Academy of Sciences.

Microgravity

NASA Image and Video Library

1996-01-01

Interior of a Biotechnology Refrigerator that preserves samples for use in (or after culturing in) the NASA Bioreactor. The unit is shown extracted from a middeck locker shell. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Microgravity

NASA Image and Video Library

1996-01-01

Biotechnology Refrigerator that preserves samples for use in (or after culturing in) the NASA Bioreactor. The unit is shown extracted from a middeck locker shell and with thermal blankets partially removed. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Microgravity

NASA Image and Video Library

1996-01-01

Close-up view of the interior of a NASA Bioreactor shows the plastic plumbing and valves (cylinders at right center) to control fluid flow. The rotating wall vessel is at top center. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Microgravity

NASA Image and Video Library

1996-01-01

Biotechnology Refrigerator that preserves samples for use in (or after culturing in) the NASA Bioreactor. The unit is shown extracted from a middeck locker shell. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Bioreactor rotating wall vessel

NASA Technical Reports Server (NTRS)

2001-01-01

The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Cell constructs grown in a rotating bioreactor on Earth (left) eventually become too large to stay suspended in the nutrient media. In the microgravity of orbit, the cells stay suspended. Rotation then is needed for gentle stirring to replenish the media around the cells.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Close-up view of the interior of a NASA Bioreactor shows the plastic plumbing and valves (cylinders at right center) to control fluid flow. The rotating wall vessel is at top center. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Discovery of Hyperpolarized Molecular Imaging Biomarkers in a Novel Prostate Tissue Slice Culture Model

DTIC Science & Technology

2013-06-01

start of tissue 2D culture , and 3 and 20 hours post culturing of the TSC’s in the 3D bioreactor (left) and corresponding representative tissue... culture tissue slice 13P spectrum obtained after 20 hours in the 5mm 3D MR compatible tissue culture bioreactor . 5 TSC’s with ≈ 90% viability...hours (≈98%) of being in 3D culture within the MR compatible bioreactor . • We demonstrated that the increased flux from pyruvate to lactate in the

Effects of scaffold architecture on mechanical characteristics and osteoblast response to static and perfusion bioreactor cultures.

PubMed

Bartnikowski, Michal; Klein, Travis J; Melchels, Ferry P W; Woodruff, Maria A

2014-07-01

Tissue engineering focuses on the repair and regeneration of tissues through the use of biodegradable scaffold systems that structurally support regions of injury while recruiting and/or stimulating cell populations to rebuild the target tissue. Within bone tissue engineering, the effects of scaffold architecture on cellular response have not been conclusively characterized in a controlled-density environment. We present a theoretical and practical assessment of the effects of polycaprolactone (PCL) scaffold architectural modifications on mechanical and flow characteristics as well as MC3T3-E1 preosteoblast cellular response in an in vitro static plate and custom-designed perfusion bioreactor model. Four scaffold architectures were contrasted, which varied in inter-layer lay-down angle and offset between layers, while maintaining a structural porosity of 60 ± 5%. We established that as layer angle was decreased (90° vs. 60°) and offset was introduced (0 vs. 0.5 between layers), structural stiffness, yield stress, strength, pore size, and permeability decreased, while computational fluid dynamics-modeled wall shear stress was increased. Most significant effects were noted with layer offset. Seeding efficiencies in static culture were also dramatically increased due to offset (∼ 45% to ∼ 86%), with static culture exhibiting a much higher seeding efficiency than perfusion culture. Scaffold architecture had minimal effect on cell response in static culture. However, architecture influenced osteogenic differentiation in perfusion culture, likely by modifying the microfluidic environment. © 2014 Wiley Periodicals, Inc.

Reduced-Gravity Experiments Conducted to Help Bioreactor Development

NASA Technical Reports Server (NTRS)

Niederhaus, Charles E.; Nahra, Henry K.; Kizito, John P.

2004-01-01

The NASA Glenn Research Center and the NASA Johnson Space Center are collaborating on fluid dynamic investigations for a future cell science bioreactor to fly on the International Space Station (ISS). Project Manager Steven Gonda from the Cellular Biotechnology Program at Johnson is leading the development of the Hydrodynamic Focusing Bioreactor--Space (HFB-S) for use on the ISS to study tissue growth in microgravity. Glenn is providing microgravity fluid physics expertise to help with the design and evaluation of the HFB-S. These bioreactors are used for three-dimensional tissue culture, which cannot be done in ground-based labs in normal gravity. The bioreactors provide a continual supply of oxygen for cell growth, as well as periodic replacement of cell culture media with nutrients. The bioreactor must provide a uniform distribution of oxygen and nutrients while minimizing the shear stresses on the tissue culture.

Tissue Engineering Under Microgravity Conditions-Use of Stem Cells and Specialized Cells.

PubMed

Grimm, Daniela; Egli, Marcel; Krüger, Marcus; Riwaldt, Stefan; Corydon, Thomas J; Kopp, Sascha; Wehland, Markus; Wise, Petra; Infanger, Manfred; Mann, Vivek; Sundaresan, Alamelu

2018-03-29

Experimental cell research studying three-dimensional (3D) tissues in space and on Earth using new techniques to simulate microgravity is currently a hot topic in Gravitational Biology and Biomedicine. This review will focus on the current knowledge of the use of stem cells and specialized cells for tissue engineering under simulated microgravity conditions. We will report on recent advancements in the ability to construct 3D aggregates from various cell types using devices originally created to prepare for spaceflights such as the random positioning machine (RPM), the clinostat, or the NASA-developed rotating wall vessel (RWV) bioreactor, to engineer various tissues such as preliminary vessels, eye tissue, bone, cartilage, multicellular cancer spheroids, and others from different cells. In addition, stem cells had been investigated under microgravity for the purpose to engineer adipose tissue, cartilage, or bone. Recent publications have discussed different changes of stem cells when exposed to microgravity and the relevant pathways involved in these biological processes. Tissue engineering in microgravity is a new technique to produce organoids, spheroids, or tissues with and without scaffolds. These 3D aggregates can be used for drug testing studies or for coculture models. Multicellular tumor spheroids may be interesting for radiation experiments in the future and to reduce the need for in vivo experiments. Current achievements using cells from patients engineered on the RWV or on the RPM represent an important step in the advancement of techniques that may be applied in translational Regenerative Medicine.

Microgravity-Enhanced Stem Cell Selection

NASA Technical Reports Server (NTRS)

Claudio, Pier Paolo; Valluri, Jagan

2011-01-01

Stem cells, both embryonic and adult, promise to revolutionize the practice of medicine in the future. In order to realize this potential, a number of hurdles must be overcome. Most importantly, the signaling mechanisms necessary to control the differentiation of stem cells into tissues of interest remain to be elucidated, and much of the present research on stem cells is focused on this goal. Nevertheless, it will also be essential to achieve large-scale expansion and, in many cases, assemble cells in 3D as transplantable tissues. To this end, microgravity analog bioreactors can play a significant role. Microgravity bioreactors were originally conceived as a tool to study the cellular responses to microgravity. However, the technology can address some of the shortcomings of conventional cell culture systems; namely, the deficiency of mass transport in static culture and high mechanical shear forces in stirred systems. Unexpectedly, the conditions created in the vessel were ideal for 3D cell culture. Recently, investigators have demonstrated the capability of the microgravity bioreactors to expand hematopoietic stem cells compared to static culture, and facilitate the differentiation of umbilical cord stem cells into 3D liver aggregates. Stem cells are capable of differentiating into functional cells. However, there are no reliable methods to induce the stem cells to form specific cells or to gain enough cells for transplantation, which limits their application in clinical therapy. The aim of this study is to select the best experimental setup to reach high proliferation levels by culturing these cells in a microgravity-based bioreactor. In typical cell culture, the cells sediment to the bottom surface of their container and propagate as a one-cell-layer sheet. Prevention of such sedimentation affords the freedom for self-assembly and the propagation of 3D tissue arrays. Suspension of cells is easily achievable using stirred technologies. Unfortunately, in conventional bioreactors, stirring invokes deleterious forces that disrupt cell aggregation and results in cell death. First-generation rotating bioreactors provided rotation on the horizontal axis, which resulted in the suspension of cells without stirring, thus providing a suitable environment to propagate cells without sedimentation to a surface. The rotating wall bioreactors did not provide a way to remove air bubbles that were causing shear and disrupting 3D cultures. Johnson Space Center successfully engineered the hydrofocusing bioreactor (HFB) that resolved the problem of removing the air bubbles from the fluid medium of NASA's rotating-wall space bioreactors. The HFB uses the principle of hydrodynamic focusing that simultaneously produces a low-shear fluid culture environment and a variable hydrofocusing force that can control the movement, location, and removal of suspended cells, tissues, and air bubbles from the bioreactor. The HFB is a rotating, domeshaped cell culture vessel with a centrally located sampling port and an internal viscous spinner. The vessel and spinner can rotate at different speeds either in the same or opposite directions. Rotation of the vessel and viscous interaction at the spinner generate a hydrofocusing force. Adjusting the differential rotation rate between vessel and spinner controls the magnitude of the force.

Process quality engineering for bioreactor-driven manufacturing of tissue-engineered constructs for bone regeneration.

PubMed

Papantoniou Ir, Ioannis; Chai, Yoke Chin; Luyten, Frank P; Schrooten Ir, Jan

2013-08-01

The incorporation of Quality-by-Design (QbD) principles in tissue-engineering bioprocess development toward clinical use will ensure that manufactured constructs possess prerequisite quality characteristics addressing emerging regulatory requirements and ensuring the functional in vivo behavior. In this work, the QbD principles were applied on a manufacturing process step for the in vitro production of osteogenic three-dimensional (3D) hybrid scaffolds that involves cell matrix deposition on a 3D titanium (Ti) alloy scaffold. An osteogenic cell source (human periosteum-derived cells) cultured in a bioinstructive medium was used to functionalize regular Ti scaffolds in a perfusion bioreactor, resulting in an osteogenic hybrid carrier. A two-level three-factor fractional factorial design of experiments was employed to explore a range of production-relevant process conditions by simultaneously changing value levels of the following parameters: flow rate (0.5-2 mL/min), cell culture duration (7-21 days), and cell-seeding density (1.5×10(3)-3×10(3) cells/cm(2)). This approach allowed to evaluate the individual impact of the aforementioned process parameters upon key quality attributes of the produced hybrids, such as collagen production, mineralization level, and cell number. The use of a fractional factorial design approach helped create a design space in which hybrid scaffolds of predefined quality attributes may be robustly manufactured while minimizing the number of required experiments.

Growing Tissues in Real and Simulated Microgravity: New Methods for Tissue Engineering

PubMed Central

Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Ulbrich, Claudia; Magnusson, Nils E.; Infanger, Manfred; Bauer, Johann

2014-01-01

Tissue engineering in simulated (s-) and real microgravity (r-μg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-μg in Space or to s-μg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals. PMID:24597549

Growing tissues in real and simulated microgravity: new methods for tissue engineering.

PubMed

Grimm, Daniela; Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Ulbrich, Claudia; Magnusson, Nils E; Infanger, Manfred; Bauer, Johann

2014-12-01

Tissue engineering in simulated (s-) and real microgravity (r-μg) is currently a topic in Space medicine contributing to biomedical sciences and their applications on Earth. The principal aim of this review is to highlight the advances and accomplishments in the field of tissue engineering that could be achieved by culturing cells in Space or by devices created to simulate microgravity on Earth. Understanding the biology of three-dimensional (3D) multicellular structures is very important for a more complete appreciation of in vivo tissue function and advancing in vitro tissue engineering efforts. Various cells exposed to r-μg in Space or to s-μg created by a random positioning machine, a 2D-clinostat, or a rotating wall vessel bioreactor grew in the form of 3D tissues. Hence, these methods represent a new strategy for tissue engineering of a variety of tissues, such as regenerated cartilage, artificial vessel constructs, and other organ tissues as well as multicellular cancer spheroids. These aggregates are used to study molecular mechanisms involved in angiogenesis, cancer development, and biology and for pharmacological testing of, for example, chemotherapeutic drugs or inhibitors of neoangiogenesis. Moreover, they are useful for studying multicellular responses in toxicology and radiation biology, or for performing coculture experiments. The future will show whether these tissue-engineered constructs can be used for medical transplantations. Unveiling the mechanisms of microgravity-dependent molecular and cellular changes is an up-to-date requirement for improving Space medicine and developing new treatment strategies that can be translated to in vivo models while reducing the use of laboratory animals.

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds.

PubMed

Bhaskar, Birru; Owen, Robert; Bahmaee, Hossein; Rao, Parcha Sreenivasa; Reilly, Gwendolen C

2018-06-01

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30 mm diameter × 5 mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds were either maintained in static conditions or transferred to the bioreactor 3 days post-seeding and a continuous flow rate of 3.47 mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10 days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.

[Design and verification of Luo-Ye pump-based stress formation for cultivation of tissue-engineered blood vessel].

PubMed

Liao, Wen-Jun; Chen, Wan-Wen; Wen, Zhang; Wu, Yue-Heng; Li, Dong-Feng; Zhou, Jia-Hui; Zheng, Jian-Yi; Lin, Zhan-Yi

2016-06-20

To improve Luo-Ye pump-based stress-forming system and optimize the stimulating effect on smooth muscle cells during cultivation of tissue-engineered blood vessels (TEBV). A new Luo-Ye pump-based TEBV 3D culture system was developed by adding an air pump to the output of the bioreactor. A pressure guide wire was used to measure the stress at different points of the silicone tube inside the TEBV bio-reactor, and fitting curves of the stress changes over time was created using Origin 8.0 software. The TEBVs were constructed by seeding vascular smooth muscle cells (VSMCs) isolated from human umbilical artery on polyglycolic acid (PGA) and cultured under dynamic conditions with 40 mmHg resistance (improved group), dynamic conditions without resistance (control group) or static condition (static group) for 4 weeks. The harvested TEBVs were then examined with HE staining, masson staining, α-SMA immunohistochemical staining, and scanning and transmission electron microscopy with semi-quantitative analysis of collagen content and α-SMA expression. The measured stress values and the fitting curves showed that the stress stimuli from the Luo-Ye pump were enhanced by adding an air pump to the output of the bioreactor. Histological analysis revealed improved VSMC density, collagen content and α-SMA expression in the TEBVs constructed with the improved method as compared with those in the control and static groups. Adding an air pump to the Luo-Ye pump significantly enhances the stress stimulation in the TEBV 3-D culture system to promote the secretion function of VSMCs.

Engineering tissue constructs to mimic native aortic and pulmonary valve leaflets' structures and mechanics

NASA Astrophysics Data System (ADS)

Masoumi, Nafiseh

There are several disadvantages correlated with current heart valve replacement, including anticoagulation therapy for patients with mechanical valves and the low durability of bioprosthetic valves. The non-viable nature of such devices is a critical drawback especially for pediatric cases due to the inability of the graft to grow in vivo with the patients. A tissue engineered heart valve (TEHV) with remodeling and growth ability, is conceptually appealing to use in the surgical repair and could serve as a permanent replacements when operating for pediatric valvular lesions. It is critical that scaffolds for functional heart valve tissue engineering, be capable of mimicking the native leaflet's structure and mechanical properties at the time of implantation. Meanwhile, the scaffolds should be able to support cellular proliferation and native-like tissue formation as the TEHV remodels toward a scaffold-free state. Our overall hypothesis is that an "ideal" engineered construct, designed based on native leaflet's structure and mechanics, will complement a native heart valve leaflet in providing benchmarks for use in the design of clinically-applicable TEHV. This hypothesis was addressed through several experiments conducted in the present study. To establish a functional biomimetic TEHV, we developed scaffolds capable of matching the anisotropic stiffness of native leaflet while promoting native-like cell and collagen content and supporting the ECM generation. Scaffolds with various polymer contents (e.g., poly (glycerol sebacate) (PGS) and poly (epsilon-caprolactone) (PCL)) and structural designs (e.g., microfabricated and microfibrous scaffolds), were fabricated based on native leaflet's structure and mechanics. It was found that the tri-layered scaffold, designed with assembly of microfabricated PGS and microfibrous PGS/PCL was a functional leaflet capable of promoting tissue formation. Furthermore, to investigate the effect of cyclic stress and flexure individually on the TEHV development, we designed a simple and novel stretch-flexure bioreactor in which samples were subjected to well-defined stimulations with a controlled strain-rate. The stretch and flexure was found to accelerate and increase tissue formation on the microfabricated PGS scaffolds cultivated in the bioreactors.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

The heart of the bioreactor is the rotating wall vessel, shown without its support equipment. Volume is about 125 mL. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Bioreactor culture duration of engineered constructs influences bone formation by mesenchymal stem cells.

PubMed

Mitra, Debika; Whitehead, Jacklyn; Yasui, Osamu W; Leach, J Kent

2017-11-01

Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs in vitro to boost their bone forming capacity in vivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks in vivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days in vitro improves the quantity and quality of bone formation in vivo. This study highlights the need for optimizing in vitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

A kinetic modeling of chondrocyte culture for manufacture of tissue-engineered cartilage.

PubMed

Kino-Oka, Masahiro; Maeda, Yoshikatsu; Yamamoto, Takeyuki; Sugawara, Katsura; Taya, Masahito

2005-03-01

For repairing articular cartilage defects, innovative techniques based on tissue engineering have been developed and are now entering into the practical stage of clinical application by means of grafting in vitro cultured products. A variety of natural and artificial materials available for scaffolds, which permit chondrocyte cells to aggregate, have been designed for their ability to promote cell growth and differentiation. From the viewpoint of the manufacturing process for tissue-engineered cartilage, the diverse nature of raw materials (seeding cells) and end products (cultured cartilage) oblige us to design a tailor-made process with less reproducibility, which is an obstacle to establishing a production doctrine based on bioengineering knowledge concerning growth kinetics and modeling as well as designs of bioreactors and culture operations for certification of high product quality. In this article, we review the recent advances in the manufacturing of tissue-engineered cartilage. After outlining the manufacturing processes for tissue-engineered cartilage in the first section, the second and third sections, respectively, describe the three-dimensional culture of chondrocytes with Aterocollagen gel and kinetic model consideration as a tool for evaluating this culture process. In the final section, culture strategy is discussed in terms of the combined processes of monolayer growth (ex vivo chondrocyte cell expansion) and three-dimensional growth (construction of cultured cartilage in the gel).

NASA Bioreactor Demonstration System

NASA Technical Reports Server (NTRS)

2002-01-01

Leland W. K. Chung (left), Director, Molecular Urology Therapeutics Program at the Winship Cancer Institute at Emory University, is principal investigator for the NASA bioreactor demonstration system (BDS-05). With him is Dr. Jun Shu, an assistant professor of Orthopedics Surgery from Kuming Medical University China. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: Emory University.

Prostate tumor grown in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2001-01-01

This prostate cancer construct was grown during NASA-sponsored bioreactor studies on Earth. Cells are attached to a biodegradable plastic lattice that gives them a head start in growth. Prostate tumor cells are to be grown in a NASA-sponsored Bioreactor experiment aboard the STS-107 Research-1 mission in 2002. Dr. Leland Chung of the University of Virginia is the principal investigator. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: NASA and the University of Virginia.

Microgravity

NASA Image and Video Library

2001-05-31

The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Cell constructs grown in a rotating bioreactor on Earth (left) eventually become too large to stay suspended in the nutrient media. In the microgravity of orbit, the cells stay suspended. Rotation then is needed for gentle stirring to replenish the media around the cells.

A Flow Perfusion Bioreactor System for Vocal Fold Tissue Engineering Applications

PubMed Central

Heris, Hossein K.; Thomson, Scott L.; Taher, Rani; Kazemirad, Siavash; Sheibani, Sara; Li-Jessen, Nicole Y.K.; Vali, Hojatollah; Mongeau, Luc

2016-01-01

The human vocal folds (VFs) undergo complex biomechanical stimulation during phonation. The aim of the present study was to develop and validate a phono-mimetic VF flow perfusion bioreactor, which mimics the mechanical microenvironment of the human VFs in vitro. The bioreactor uses airflow-induced self-oscillations, which have been shown to produce mechanical loading and contact forces that are representative of human phonation. The bioreactor consisted of two synthetic VF replicas within a silicone body. A cell-scaffold mixture (CSM) consisting of human VF fibroblasts, hyaluronic acid, gelatin, and a polyethylene glycol cross-linker was injected into cavities within the replicas. Cell culture medium (CCM) was perfused through the scaffold by using a customized secondary flow loop. After the injection, the bioreactor was operated with no stimulation over a 3-day period to allow for cell adaptation. Phonation was subsequently induced by using a variable speed centrifugal blower for 2 h each day over a period of 4 days. A similar bioreactor without biomechanical stimulation was used as the nonphonatory control. The CSM was harvested from both VF replicas 7 days after the injection. The results confirmed that the phono-mimetic bioreactor supports cell viability and extracellular matrix proteins synthesis, as expected. Many scaffold materials were found to degrade because of challenges from phonation-induced biomechanical stimulation as well as due to biochemical reactions with the CCM. The bioreactor concept enables future investigations of the effects of different phonatory characteristics, that is, voice regimes, on the behavior of the human VF cells. It will also help study the long-term functional outcomes of the VF-specific biomaterials before animal and clinical studies. PMID:27537192

A Flow Perfusion Bioreactor System for Vocal Fold Tissue Engineering Applications.

PubMed

Latifi, Neda; Heris, Hossein K; Thomson, Scott L; Taher, Rani; Kazemirad, Siavash; Sheibani, Sara; Li-Jessen, Nicole Y K; Vali, Hojatollah; Mongeau, Luc

2016-09-01

The human vocal folds (VFs) undergo complex biomechanical stimulation during phonation. The aim of the present study was to develop and validate a phono-mimetic VF flow perfusion bioreactor, which mimics the mechanical microenvironment of the human VFs in vitro. The bioreactor uses airflow-induced self-oscillations, which have been shown to produce mechanical loading and contact forces that are representative of human phonation. The bioreactor consisted of two synthetic VF replicas within a silicone body. A cell-scaffold mixture (CSM) consisting of human VF fibroblasts, hyaluronic acid, gelatin, and a polyethylene glycol cross-linker was injected into cavities within the replicas. Cell culture medium (CCM) was perfused through the scaffold by using a customized secondary flow loop. After the injection, the bioreactor was operated with no stimulation over a 3-day period to allow for cell adaptation. Phonation was subsequently induced by using a variable speed centrifugal blower for 2 h each day over a period of 4 days. A similar bioreactor without biomechanical stimulation was used as the nonphonatory control. The CSM was harvested from both VF replicas 7 days after the injection. The results confirmed that the phono-mimetic bioreactor supports cell viability and extracellular matrix proteins synthesis, as expected. Many scaffold materials were found to degrade because of challenges from phonation-induced biomechanical stimulation as well as due to biochemical reactions with the CCM. The bioreactor concept enables future investigations of the effects of different phonatory characteristics, that is, voice regimes, on the behavior of the human VF cells. It will also help study the long-term functional outcomes of the VF-specific biomaterials before animal and clinical studies.

Micro and nanotechnologies in heart valve tissue engineering.

PubMed

Hasan, Anwarul; Saliba, John; Pezeshgi Modarres, Hassan; Bakhaty, Ahmed; Nasajpour, Amir; Mofrad, Mohammad R K; Sanati-Nezhad, Amir

2016-10-01

Due to the increased morbidity and mortality resulting from heart valve diseases, there is a growing demand for off-the-shelf implantable tissue engineered heart valves (TEHVs). Despite the significant progress in recent years in improving the design and performance of TEHV constructs, viable and functional human implantable TEHV constructs have remained elusive. The recent advances in micro and nanoscale technologies including the microfabrication, nano-microfiber based scaffolds preparation, 3D cell encapsulated hydrogels preparation, microfluidic, micro-bioreactors, nano-microscale biosensors as well as the computational methods and models for simulation of biological tissues have increased the potential for realizing viable, functional and implantable TEHV constructs. In this review, we aim to present an overview of the importance and recent advances in micro and nano-scale technologies for the development of TEHV constructs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dynamic Mechanical Compression of Chondrocytes for Tissue Engineering: A Critical Review.

PubMed

Anderson, Devon E; Johnstone, Brian

2017-01-01

Articular cartilage functions to transmit and translate loads. In a classical structure-function relationship, the tissue resides in a dynamic mechanical environment that drives the formation of a highly organized tissue architecture suited to its biomechanical role. The dynamic mechanical environment includes multiaxial compressive and shear strains as well as hydrostatic and osmotic pressures. As the mechanical environment is known to modulate cell fate and influence tissue development toward a defined architecture in situ , dynamic mechanical loading has been hypothesized to induce the structure-function relationship during attempts at in vitro regeneration of articular cartilage. Researchers have designed increasingly sophisticated bioreactors with dynamic mechanical regimes, but the response of chondrocytes to dynamic compression and shear loading remains poorly characterized due to wide variation in study design, system variables, and outcome measurements. We assessed the literature pertaining to the use of dynamic compressive bioreactors for in vitro generation of cartilaginous tissue from primary and expanded chondrocytes. We used specific search terms to identify relevant publications from the PubMed database and manually sorted the data. It was very challenging to find consensus between studies because of species, age, cell source, and culture differences, coupled with the many loading regimes and the types of analyses used. Early studies that evaluated the response of primary bovine chondrocytes within hydrogels, and that employed dynamic single-axis compression with physiologic loading parameters, reported consistently favorable responses at the tissue level, with upregulation of biochemical synthesis and biomechanical properties. However, they rarely assessed the cellular response with gene expression or mechanotransduction pathway analyses. Later studies that employed increasingly sophisticated biomaterial-based systems, cells derived from different species, and complex loading regimes, did not necessarily corroborate prior positive results. These studies report positive results with respect to very specific conditions for cellular responses to dynamic load but fail to consistently achieve significant positive changes in relevant tissue engineering parameters, particularly collagen content and stiffness. There is a need for standardized methods and analyses of dynamic mechanical loading systems to guide the field of tissue engineering toward building cartilaginous implants that meet the goal of regenerating articular cartilage.

Live imaging flow bioreactor for the simulation of articular cartilage regeneration after treatment with bioactive hydrogel.

PubMed

Bar, Assaf; Ruvinov, Emil; Cohen, Smadar

2018-06-05

Osteochondral defects (OCDs) are conditions affecting both cartilage and the underlying bone. Since cartilage is not spontaneously regenerated, our group has recently developed a strategy of injecting bioactive alginate hydrogel into the defect for promoting endogenous regeneration of cartilage via presentation of affinity-bound transforming growth factor β1 (TGF-β1). As in vivo model systems often provide only limited insights as for the mechanism behind regeneration processes, here we describe a novel flow bioreactor for the in vitro modeling of the OCD microenvironment, designed to promote cell recruitment from the simulated bone marrow compartment into the hydrogel, under physiological flow conditions. Computational fluid dynamics modeling confirmed that the bioreactor operates in a relevant slow-flowing regime. Using a chemotaxis assay, it was shown that TGF-β1 does not affect human mesenchymal stem cell (hMSC) chemotaxis in 2D culture. Accessible through live imaging, the bioreactor enabled monitoring and discrimination between erosion rates and profiles of different alginate hydrogel compositions, using green fluorescent protein-expressing cells. Mathematical modeling of the erosion front progress kinetics predicted the erosion rate in the bioreactor up to 7 days postoperation. Using quantitative real-time polymerase chain reaction of early chondrogenic markers, the onset of chondrogenic differentiation in hMSCs was detected after 7 days in the bioreactor. In conclusion, the designed bioreactor presents multiple attributes, making it an optimal device for mechanistical studies, serving as an investigational tool for the screening of other biomaterial-based, tissue engineering strategies. © 2018 Wiley Periodicals, Inc.

Pulsatile perfusion bioreactor for cardiac tissue engineering.

PubMed

Brown, Melissa A; Iyer, Rohin K; Radisic, Milica

2008-01-01

Cardiovascular disease is the number one cause of mortality in North America. Cardiac tissue engineering aims to engineer a contractile patch of physiological thickness to use in surgical repair of diseased heart tissue. We previously reported that perfusion of engineered cardiac constructs resulted in improved tissue assembly. Because heart tissues respond to mechanical stimuli in vitro and experience rhythmic mechanical forces during contraction in vivo, we hypothesized that provision of pulsatile interstitial medium flow to an engineered cardiac patch would result in enhanced tissue assembly by way of mechanical conditioning and improved mass transport. Thus, we constructed a novel perfusion bioreactor capable of providing pulsatile fluid flow at physiologically relevant shear stresses and flow rates. Pulsatile perfusion (PP) was achieved by incorporation of a normally closed solenoid pinch valve into the perfusion loop and was carried out at a frequency of 1 Hz and a flow rate of 1.50 mL/min (PP) or 0.32 mL/min (PP-LF). Nonpulsatile flow at 1.50 mL/min (NP) or 0.32 mL/min (NP-LF) served as controls. Static controls were cultivated in well plates. The main experimental groups were seeded with cells enriched for cardiomyocytes by one preplating step (64% cardiac Troponin I+, 34% prolyl-4-hydroxylase+), whereas pure cardiac fibroblasts and cells enriched for cardiomyocytes by two preplating steps (81% cardiac Troponin I+, 16% prolyl-4-hydroxylase+) served as controls. Cultivation under pulsatile flow had beneficial effects on contractile properties. Specifically, the excitation threshold was significantly lower in the PP condition (pulsatile perfusion at 1.50 mL/min) than in the Static control, and the contraction amplitude was the highest; whereas high maximum capture rate was observed for the PP-LF conditions (pulsatile perfusion at 0.32 mL/min). The enhanced hypertrophy index observed for the PP-LF group was consistent with the highest cellular length and diameter in this group. Within the same cultivation groups (Static, NP-LF, PP-LF, PP, and NP) there were no significant differences in the diameter between fibroblasts and cardiomyocytes, although cardiomyocytes were significantly more elongated than fibroblasts under PP-LF conditions. Cultivation of control cell populations resulted in noncontractile constructs when cardiac fibroblasts were used (as expected) and no overall improvement in functional properties when two steps of preplating were used to enrich for cardiomyocytes in comparison with only one step of preplating.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Astronaut John Blaha replaces an exhausted media bag and filled waste bag with fresh bags to continue a bioreactor experiment aboard space station Mir in 1996. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. This image is from a video downlink. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC).

Coupling curvature-dependent and shear stress-stimulated neotissue growth in dynamic bioreactor cultures: a 3D computational model of a complete scaffold.

PubMed

Guyot, Y; Papantoniou, I; Luyten, F P; Geris, L

2016-02-01

The main challenge in tissue engineering consists in understanding and controlling the growth process of in vitro cultured neotissues toward obtaining functional tissues. Computational models can provide crucial information on appropriate bioreactor and scaffold design but also on the bioprocess environment and culture conditions. In this study, the development of a 3D model using the level set method to capture the growth of a microporous neotissue domain in a dynamic culture environment (perfusion bioreactor) was pursued. In our model, neotissue growth velocity was influenced by scaffold geometry as well as by flow- induced shear stresses. The neotissue was modeled as a homogenous porous medium with a given permeability, and the Brinkman equation was used to calculate the flow profile in both neotissue and void space. Neotissue growth was modeled until the scaffold void volume was filled, thus capturing already established experimental observations, in particular the differences between scaffold filling under different flow regimes. This tool is envisaged as a scaffold shape and bioprocess optimization tool with predictive capacities. It will allow controlling fluid flow during long-term culture, whereby neotissue growth alters flow patterns, in order to provide shear stress profiles and magnitudes across the whole scaffold volume influencing, in turn, the neotissue growth.

Rotating microgravity-bioreactor cultivation enhances the hepatic differentiation of mouse embryonic stem cells on biodegradable polymer scaffolds.

PubMed

Wang, Yingjie; Zhang, Yunping; Zhang, Shichang; Peng, Guangyong; Liu, Tao; Li, Yangxin; Xiang, Dedong; Wassler, Michael J; Shelat, Harnath S; Geng, Yongjian

2012-11-01

Embryonic stem (ES) cells are pluripotent cells that are capable of differentiating all the somatic cell lineages, including those in the liver tissue. We describe the generation of functional hepatic-like cells from mouse ES (mES) cells using a biodegradable polymer scaffold and a rotating bioreactor that allows simulated microgravity. Cells derived from ES cells cultured in the three-dimensional (3D) culture system with exogenous growth factors and hormones can differentiate into hepatic-like cells with morphologic characteristics of typical mature hepatocytes. Reverse-transcription polymerase chain-reaction testing, Western blot testing, immunostaining, and flow cytometric analysis show that these cells express hepatic-specific genes and proteins during differentiation. Differentiated cells on scaffolds further exhibit morphologic traits and biomarkers characteristic of liver cells, including albumin production, cytochrome P450 activity, and low-density lipoprotein uptake. When these stem cell-bearing scaffolds are transplanted into severe combined immunodeficient mice, the 3D constructs remained viable, undergoing further differentiation and maturation of hepatic-like cells in vivo. In conclusion, the growth and differentiation of ES cells in a biodegradable polymer scaffold and a rotating microgravity bioreactor can yield functional and organizational hepatocytes useful for research involving bioartificial liver and engineered liver tissue.

Mechanical control of tissue-engineered bone.

PubMed

Hung, Ben P; Hutton, Daphne L; Grayson, Warren L

2013-01-31

Bone is a load-bearing tissue and physical forces play key roles in the development and maintenance of its structure. Mechanical cues can stimulate the expression of an osteogenic phenotype, enhance matrix and mineral deposition, and influence tissue organization to improve the functional outcome of engineered bone grafts. In recent years, a number of studies have investigated the effects of biophysical forces on the bone formation properties of osteoprogenitor cells. The application of physiologically relevant stimuli to tissue-engineered bone may be determined through observation and understanding of forces to which osteoblasts, osteoclasts, and osteocytes are exposed in native bone. Subsequently, these cues may be parameterized and their effects studied in well-defined in vitro systems. The osteo-inductive effects of three specific mechanical cues - shear stress, substrate rigidity, and nanotopography - on cells cultured in monolayer or in three-dimensional biomaterial scaffolds in vitro are reviewed. Additionally, we address the time-dependent effects of mechanical cues on vascular infiltration and de novo bone formation in acellular scaffolds implanted into load-bearing sites in vivo. Recent studies employing cutting-edge advances in biomaterial fabrication and bioreactor design have provided key insights into the role of mechanical cues on cellular fate and tissue properties of engineered bone grafts. By providing mechanistic understanding, future studies may go beyond empirical approaches to rational design of engineering systems to control tissue development.

In vitro culture of large bone substitutes in a new bioreactor: importance of the flow direction.

PubMed

Olivier, V; Hivart, Ph; Descamps, M; Hardouin, P

2007-09-01

New biomaterials combined with osteogenic cells are now being developed as an alternative to autogeneous bone grafts when the skeletal defect reaches a critical size. Yet, the size issue appears to be a key obstacle in the development of bone tissue engineering. Bioreactors are needed to allow the in vitro expansion of cells inside large bulk materials under appropriate conditions. However, no bioreactor has yet been designed for large-scale 3D structures and custom-made scaffolds. In this study, we evaluate the efficiency of a new bioreactor for the in vitro development of large bone substitutes, ensuring the perfusion of large ceramic scaffolds by the nutritive medium. The survival and proliferation of cells inside the scaffolds after 7 and 28 days in this dynamic culture system and the impact of the direction of the flow circulation are evaluated. The follow-up of glucose consumption, DNA quantification and microscopic evaluation all confirmed cell survival and proliferation for a sample under dynamic culture conditions, whereas static culture leads to the death of cells inside the scaffolds. Two directions of flow perfusion were assayed; the convergent direction leads to enhanced results compared to divergent flow.

Biotechnology

NASA Image and Video Library

2001-05-15

This prostate cancer construct was grown during NASA-sponsored bioreactor studies on Earth. Cells are attached to a biodegradable plastic lattice that gives them a head start in growth. Prostate tumor cells are to be grown in a NASA-sponsored Bioreactor experiment aboard the STS-107 Research-1 mission in 2002. Dr. Leland Chung of the University of Virginia is the principal investigator. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: NASA and the University of Virginia.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1996-01-01

Close-up view of the interior of a NASA Bioreactor shows the plastic plumbing and valves (cylinders at center) to control fluid flow. A fresh nutrient bag is installed at top; a flattened waste bag behind it will fill as the nutrients are consumed during the course of operation. The drive chain and gears for the rotating wall vessel are visible at bottom center center. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Engineered skeletal muscle tissue for soft robotics: fabrication strategies, current applications, and future challenges.

PubMed

Duffy, Rebecca M; Feinberg, Adam W

2014-01-01

Skeletal muscle is a scalable actuator system used throughout nature from the millimeter to meter length scales and over a wide range of frequencies and force regimes. This adaptability has spurred interest in using engineered skeletal muscle to power soft robotics devices and in biotechnology and medical applications. However, the challenges to doing this are similar to those facing the tissue engineering and regenerative medicine fields; specifically, how do we translate our understanding of myogenesis in vivo to the engineering of muscle constructs in vitro to achieve functional integration with devices. To do this researchers are developing a number of ways to engineer the cellular microenvironment to guide skeletal muscle tissue formation. This includes understanding the role of substrate stiffness and the mechanical environment, engineering the spatial organization of biochemical and physical cues to guide muscle alignment, and developing bioreactors for mechanical and electrical conditioning. Examples of engineered skeletal muscle that can potentially be used in soft robotics include 2D cantilever-based skeletal muscle actuators and 3D skeletal muscle tissues engineered using scaffolds or directed self-organization. Integration into devices has led to basic muscle-powered devices such as grippers and pumps as well as more sophisticated muscle-powered soft robots that walk and swim. Looking forward, current, and future challenges include identifying the best source of muscle precursor cells to expand and differentiate into myotubes, replacing cardiomyocytes with skeletal muscle tissue as the bio-actuator of choice for soft robots, and vascularization and innervation to enable control and nourishment of larger muscle tissue constructs. © 2013 Wiley Periodicals, Inc.

Biotechnology

NASA Image and Video Library

2002-07-02

Leland W. K. Chung (left), Director, Molecular Urology Therapeutics Program at the Winship Cancer Institute at Emory University, is principal investigator for the NASA bioreactor demonstration system (BDS-05). With him is Dr. Jun Shu, an assistant professor of Orthopedics Surgery from Kuming Medical University China. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: Emory University.

Biotechnology

NASA Image and Video Library

2002-07-02

Diagram depicts the importance of cell-cell communication as central to the understanding of cancer growth and progression, the focus of the NASA bioreactor demonstration system (BDS-05) investigation. Microgravity studies will allow us to unravel the signaling and communication between these cells with the host and potential development of therapies for the treatment of cancer metastasis. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: Emory University.

Colon tumor cells grown in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2001-01-01

These photos compare the results of colon carcinoma cells grown in a NASA Bioreactor flown on the STS-70 Space Shuttle in 1995 flight and ground control experiments. The cells grown in microgravity (left) have aggregated to form masses that are larger and more similar to tissue found in the body than the cells cultured on the ground (right). The principal investigator is Milburn Jessup of the University of Texas M. D. Anderson Cancer Center. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Cell constructs grown in a rotating bioreactor on Earth (left) eventually become too large to stay suspended in the nutrient media. In the microgravity of orbit, the cells stay suspended. Rotation then is needed for gentle stirring to replenish the media around the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Credit: NASA and University of Texas M. D. Anderson Cancer Center.

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures.

PubMed

Chouinard, Julie A; Gagnon, Serge; Couture, Marc G; Lévesque, Alain; Vermette, Patrick

2009-12-15

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real-time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48-h culture period. Cells were uniformly dispersed within the 14.40 mm x 17.46 mm x 6.35 mm chamber. A larger fraction of the cells suspended in 6.35-mm thick gels and cultured in a traditional CO(2) incubator were found to be round and dead [corrected]. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. 2009 Wiley Periodicals, Inc.

Arterial specification of endothelial cells derived from human induced pluripotent stem cells in a biomimetic flow bioreactor.

PubMed

Sivarapatna, Amogh; Ghaedi, Mahboobe; Le, Andrew V; Mendez, Julio J; Qyang, Yibing; Niklason, Laura E

2015-01-01

Endothelial cells (ECs) exist in different microenvironments in vivo, including under different levels of shear stress in arteries versus veins. Standard stem cell differentiation protocols to derive ECs and EC-subtypes from human induced pluripotent stem cells (hiPSCs) generally use growth factors or other soluble factors in an effort to specify cell fate. In this study, a biomimetic flow bioreactor was used to subject hiPSC-derived ECs (hiPSC-ECs) to shear stress to determine the impacts on phenotype and upregulation of markers associated with an anti-thrombotic, anti-inflammatory, arterial-like phenotype. The in vitro bioreactor system was able to efficiently mature hiPSC-ECs into arterial-like cells in 24 h, as demonstrated by qRT-PCR for arterial markers EphrinB2, CXCR4, Conexin40 and Notch1, as well protein-level expression of Notch1 intracellular domain (NICD). Furthermore, the exogenous addition of soluble factors was not able to fully recapitulate this phenotype that was imparted by shear stress exposure. The induction of these phenotypic changes was biomechanically mediated in the shear stress bioreactor. This biomimetic flow bioreactor is an effective means for the differentiation of hiPSC-ECs toward an arterial-like phenotype, and is amenable to scale-up for culturing large quantities of cells for tissue engineering applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Microgravity

NASA Image and Video Library

1998-01-01

The heart of the bioreactor is the rotating wall vessel, shown without its support equipment. Volume is about 125 mL. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Synergistic effect between bioactive glass foam and a perfusion bioreactor on osteogenic differentiation of human adipose stem cells.

PubMed

Silva, A R P; Paula, A C C; Martins, T M M; Goes, A M; Pereria, M M

2014-03-01

Tissue engineering is a multidisciplinary science that combines a structural scaffold and cells to form a construct able to promote regeneration of injured tissue. Bioactive glass foam produced by sol-gel is an osteoinductive material with a network of interconnected macropores necessary for cell colonization. The use of human adipose-derived stem cell (hASC) presents advantages as the potential for a large number of cells, rapid expansion in vitro and the capability of differentiating into osteoblasts. The use of a bioreactor in three-dimensional cell culture enables greater efficiency for cell nutrition and application of mechanical forces, important modulators of bone physiology. The hASC seeded in a bioactive glass scaffold and cultured in osteogenic Leibovitz L-15 medium in a bioreactor with a flow rate of 0.1 mL min(-1) demonstrated a significant increase in cell proliferation and viability and alkaline phosphatase (ALP) activity peak after 14 days. The immunofluorescence assay revealed an expression of osteopontin, osteocalcin and type I collagen from 7 to 21 days after culture. The cells changed from a spindle shape to a cuboidal morphology characteristic of osteoblasts. The polymerase chain reaction assay confirmed that osteopontin, osteocalcin, and ALP genes were expressed. These results indicate that hASCs differentiated into an osteogenic phenotype when cultured in bioactive glass scaffold, osteogenic Leibovitz L-15 medium and a perfusion bioreactor. Therefore, these results highlight the synergism between a bioactive glass scaffold and the effect of perfusion on cells and indicate the differentiation into an osteogenic phenotype. Copyright © 2013 Wiley Periodicals, Inc.

Bioprocess Forces and Their Impact on Cell Behavior: Implications for Bone Regeneration Therapy

PubMed Central

Brindley, David; Moorthy, Kishaani; Lee, Jae-Ho; Mason, Chris; Kim, Hae-Won; Wall, Ivan

2011-01-01

Bioprocess forces such as shear stress experienced during routine cell culture are considered to be harmful to cells. However, the impact of physical forces on cell behavior is an area of growing interest within the tissue engineering community, and it is widely acknowledged that mechanical stimulation including shear stress can enhance osteogenic differentiation. This paper considers the effects of bioprocess shear stress on cell responses such as survival and proliferation in several contexts, including suspension-adapted cells used for recombinant protein and monoclonal antibody manufacture, adherent cells for therapy in suspension, and adherent cells attached to their growth substrates. The enhanced osteogenic differentiation that fluid flow shear stress is widely found to induce is discussed, along with the tissue engineering of mineralized tissue using perfusion bioreactors. Recent evidence that bioprocess forces produced during capillary transfer or pipetting of cell suspensions can enhance osteogenic responses is also discussed. PMID:21904661

The effect of hydrodynamic shear on 3D engineered chondrocyte systems subject to direct perfusion.

PubMed

Raimondi, Manuela T; Moretti, Matteo; Cioffi, Margherita; Giordano, Carmen; Boschetti, Federica; Laganà, Katia; Pietrabissa, Riccardo

Bioreactors allowing direct-perfusion of culture medium through tissue-engineered constructs may overcome diffusion limitations associated with static culturing, and may provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed on cells within scaffolds is directly dependent on scaffold microstructure and on bioreactor configuration. Aim of this study is to investigate optimal shear stress ranges and to quantitatively predict the levels of hydrodynamic shear imposed to cells during the experiments. Bovine articular chondrocytes were seeded on polyestherurethane foams and cultured for 2 weeks in a direct perfusion bioreactor designed to impose 4 different values of shear level at a single flow rate (0.5 ml/min). Computational fluid dynamics (CFD) simulations were carried out on reconstructions of the scaffold obtained from micro-computed tomography images. Biochemistry analyses for DNA and sGAG were performed, along with electron microscopy. The hydrodynamic shear induced on cells within constructs, as estimated by CFD simulations, ranged from 4.6 to 56 mPa. This 12-fold increase in the level of applied shear stress determined a 1.7-fold increase in the mean content in DNA and a 2.9-fold increase in the mean content in sGAG. In contrast, the mean sGAG/DNA ratio showed a tendency to decrease for increasing shear levels. Our results suggest that the optimal condition to favour sGAG synthesis in engineered constructs, at least at the beginning of culture, is direct perfusion at the lowest level of hydrodynamic shear. In conclusion, the presented results represent a first attempt to quantitatively correlate the imposed hydrodynamic shear level and the invoked biosynthetic response in 3D engineered chondrocyte systems.

Stem Cell-based Tissue Engineering Approaches for Musculoskeletal Regeneration

PubMed Central

Brown, Patrick T.; Handorf, Andrew M.; Jeon, Won Bae; Li, Wan-Ju

2014-01-01

The field of regenerative medicine and tissue engineering is an ever evolving field that holds promise in treating numerous musculoskeletal diseases and injuries. An important impetus in the development of the field was the discovery and implementation of stem cells. The utilization of mesenchymal stem cells, and later embryonic and induced pluripotent stem cells, opens new arenas for tissue engineering and presents the potential of developing stem cell-based therapies for disease treatment. Multipotent and pluripotent stem cells can produce various lineage tissues, and allow for derivation of a tissue that may be comprised of multiple cell types. As the field grows, the combination of biomaterial scaffolds and bioreactors provides methods to create an environment for stem cells that better represent their microenvironment for new tissue formation. As technologies for the fabrication of biomaterial scaffolds advance, the ability of scaffolds to modulate stem cell behavior advances as well. The composition of scaffolds could be of natural or synthetic materials and could be tailored to enhance cell self-renewal and/or direct cell fates. In addition to biomaterial scaffolds, studies of tissue development and cellular microenvironments have determined other factors, such as growth factors and oxygen tension, that are crucial to the regulation of stem cell activity. The overarching goal of stem cell-based tissue engineering research is to precisely control differentiation of stem cells in culture. In this article, we review current developments in tissue engineering, focusing on several stem cell sources, induction factors including growth factors, oxygen tension, biomaterials, and mechanical stimulation, and the internal and external regulatory mechanisms that govern proliferation and differentiation. PMID:23432679

Whole-organ re-engineering: a regenerative medicine approach to digestive organ replacement.

PubMed

Yagi, Hiroshi; Soto-Gutierrez, Alejandro; Kitagawa, Yuko

2013-06-01

Recovery from end-stage organ failure presents a challenge for the medical community, considering the limitations of extracorporeal assist devices and the shortage of donors when organ replacement is needed. There is a need for new methods to promote recovery from organ failure and regenerative medicine is an option that should be considered. Recent progress in the field of tissue engineering has opened avenues for potential clinical applications, including the use of microfluidic devices for diagnostic purposes, and bioreactors or cell/tissue-based therapies for transplantation. Early attempts to engineer tissues produced thin, planar constructs; however, recent approaches using synthetic scaffolds and decellularized tissue have achieved a more complex level of tissue organization in organs such as the urinary bladder and trachea, with some success in clinical trials. In this context, the concept of decellularization technology has been applied to produce whole organ-derived scaffolds by removing cellular content while retaining all the necessary vascular and structural cues of the native organ. In this review, we focus on organ decellularization as a new regenerative medicine approach for whole organs, which may be applied in the field of digestive surgery.

Cells growing in NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

For 5 days on the STS-70 mission, a bioreactor cultivated human colon cancer cells, which grew to 30 times the volume of control specimens grown on Earth. This significant result was reproduced on STS-85 which grew mature structures that more closely match what are found in tumors in humans. Shown here, clusters of cells slowly spin inside a bioreactor. On Earth, the cells continually fall through the buffer medium and never hit bottom. In space, they are naturally suspended. Rotation ensures gentle stirring so waste is removed and fresh nutrient and oxygen are supplied. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Microgravity

NASA Image and Video Library

1998-01-01

For 5 days on the STS-70 mission, a bioreactor cultivated human colon cancer cells, which grew to 30 times the volume of control specimens grown on Earth. This significant result was reproduced on STS-85 which grew mature structures that more closely match what are found in tumors in humans. Shown here, clusters of cells slowly spin inside a bioreactor. On Earth, the cells continually fall through the buffer medium and never hit bottom. In space, they are naturally suspended. Rotation ensures gentle stirring so waste is removed and fresh nutrient and oxygen are supplied. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Diagram of Cell to Cell Communication

NASA Technical Reports Server (NTRS)

2002-01-01

Diagram depicts the importance of cell-cell communication as central to the understanding of cancer growth and progression, the focus of the NASA bioreactor demonstration system (BDS-05) investigation. Microgravity studies will allow us to unravel the signaling and communication between these cells with the host and potential development of therapies for the treatment of cancer metastasis. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. Credit: Emory University.

Microgravity

NASA Image and Video Library

1996-01-01

Close-up view of the interior of a NASA Bioreactor shows the plastic plumbing and valves (cylinders at center) to control fluid flow. A fresh nutrient bag is installed at top; a flattened waste bag behind it will fill as the nutrients are consumed during the course of operation. The drive chain and gears for the rotating wall vessel are visible at bottom center center. The NASA Bioreactor provides a low turbulence culture environment which promotes the formation of large, three-dimensional cell clusters. The Bioreactor is rotated to provide gentle mixing of fresh and spent nutrient without inducing shear forces that would damage the cells. Due to their high level of cellular organization and specialization, samples constructed in the bioreactor more closely resemble the original tumor or tissue found in the body. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

PubMed

Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

2015-01-01

Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

Microgravity

NASA Image and Video Library

2004-04-15

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

Microgravity

NASA Image and Video Library

2004-04-15

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

Current concepts: tissue engineering and regenerative medicine applications in the ankle joint

PubMed Central

Correia, S. I.; Pereira, H.; Silva-Correia, J.; Van Dijk, C. N.; Espregueira-Mendes, J.; Oliveira, J. M.; Reis, R. L.

2014-01-01

Tissue engineering and regenerative medicine (TERM) has caused a revolution in present and future trends of medicine and surgery. In different tissues, advanced TERM approaches bring new therapeutic possibilities in general population as well as in young patients and high-level athletes, improving restoration of biological functions and rehabilitation. The mainstream components required to obtain a functional regeneration of tissues may include biodegradable scaffolds, drugs or growth factors and different cell types (either autologous or heterologous) that can be cultured in bioreactor systems (in vitro) prior to implantation into the patient. Particularly in the ankle, which is subject to many different injuries (e.g. acute, chronic, traumatic and degenerative), there is still no definitive and feasible answer to ‘conventional’ methods. This review aims to provide current concepts of TERM applications to ankle injuries under preclinical and/or clinical research applied to skin, tendon, bone and cartilage problems. A particular attention has been given to biomaterial design and scaffold processing with potential use in osteochondral ankle lesions. PMID:24352667

Current concepts: tissue engineering and regenerative medicine applications in the ankle joint.

PubMed

Correia, S I; Pereira, H; Silva-Correia, J; Van Dijk, C N; Espregueira-Mendes, J; Oliveira, J M; Reis, R L

2014-03-06

Tissue engineering and regenerative medicine (TERM) has caused a revolution in present and future trends of medicine and surgery. In different tissues, advanced TERM approaches bring new therapeutic possibilities in general population as well as in young patients and high-level athletes, improving restoration of biological functions and rehabilitation. The mainstream components required to obtain a functional regeneration of tissues may include biodegradable scaffolds, drugs or growth factors and different cell types (either autologous or heterologous) that can be cultured in bioreactor systems (in vitro) prior to implantation into the patient. Particularly in the ankle, which is subject to many different injuries (e.g. acute, chronic, traumatic and degenerative), there is still no definitive and feasible answer to 'conventional' methods. This review aims to provide current concepts of TERM applications to ankle injuries under preclinical and/or clinical research applied to skin, tendon, bone and cartilage problems. A particular attention has been given to biomaterial design and scaffold processing with potential use in osteochondral ankle lesions.

Microgravity

NASA Image and Video Library

1998-01-01

Astronaut John Blaha replaces an exhausted media bag and filled waste bag with fresh bags to continue a bioreactor experiment aboard space station Mir in 1996. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. This image is from a video downlink. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC).

Spaceflight bioreactor studies of cells and tissues.

PubMed

Freed, Lisa E; Vunjak-Novakovic, Gordana

2002-01-01

Studies of the fundamental role of gravity in the development and function of biological organisms are a central component of the human exploration of space. Microgravity affects numerous physical phenomena relevant to biological research, including the hydrostatic pressure in fluid filled vesicles, sedimentation of organelles, and buoyancy-driven convection of flow and heat. These physical phenomena can in turn directly and indirectly affect cellular morphology, metabolism, locomotion, secretion of extracellular matrix and soluble signals, and assembly into functional tissues. Studies aimed at distinguishing specific effects of gravity on biological systems require the ability to: (i) control and systematically vary gravity, e.g. by utilizing the microgravity environment of space in conjunction with an in-flight centrifuge; and (ii) maintain constant all other factors in the immediate environment, including in particular concentrations and exchange rates of biochemical species and hydrodynamic shear. The latter criteria imply the need for gravity-independent mechanisms to provide for mass transport between the cells and their environment. Available flight hardware has largely determined the experimental design and scientific objectives of spaceflight cell and tissue culture studies carried out to date. Simple culture vessels have yielded important quantitative data, and helped establish in vitro models of cell locomotion, growth and differentiation in various mammalian cell types including embryonic lung cells [6], lymphocytes [2,8], and renal cells [7,31]. Studies done using bacterial cells established the first correlations between gravity-dependent factors such as cell settling velocity and diffusional distance and the respective cell responses [12]. The development of advanced bioreactors for microgravity cell and tissue culture and for tissue engineering has benefited both research areas and provided relevant in vitro model systems for studies of astronaut well-being (loss of muscle and skeletal tissues [15-17]) and gene- and cell-level responses to the mechanical environment [13,14,18]. All five of the spaceflight bioreactor studies described above utilized three-dimensional cell culture systems in which the cells were associated with biodegradable polymer scaffolds [17], collagen gel [16], or microcarrier beads [13-15,18] in order to promote the expression of differentiated cell function. In four of the five spaceflight bioreactor studies [15-18], cells were cultured in perfused vessels (cartridges or rotating bioreactors) within recirculating loops designed to maintain medium composition within target ranges by a combination of gas exchange and fresh medium supply. Future spaceflight studies of cells and tissues are likely to involve a three-dimensional culture system, to promote cellular differentiation, and perfusion with or without rotation, to provide a gravity-independent mechanism for fluid mixing and mass transport. Previous spaceflight studies have guided the ongoing development of NASA flight hardware for the ISS (e.g. the EDU-2 and the CCU). This next generation of hardware will have extended operational capabilities including on-line microscopy, in-line sensors for the monitoring and control of metabolic parameters, modular design for replicate cultures, and, perhaps most importantly of all, compatibility with the ISS centrifuge. The latter will permit in-flight, 1 g control cultures, and thereby allow the experimental variable to be gravity itself rather than the more general "spaceflight environment". Technical limitations of spaceflight studies (e.g. allowable size, mass, and power) continue to motivate a creative approach to system design and to result in "spin-off" technologies (e.g. the STLV) for ground-based cell and tissue culture research. The increasing scientific and medical relevance of this work is evidenced by the growing number of publications in which advanced bioreactors are used for in vitro studies in physiologically relevant cell and tissue models.

Fluid mechanics of spinner-flask bioreactors

NASA Astrophysics Data System (ADS)

Sucosky, Philippe; Neitzel, G. Paul

2000-11-01

The dynamic environment within bioreactors used for in vitro tissue growth has been observed to affect the development of mammalian cells. Many studies have shown that moderate mechanical stress enhances growth of some tissues whereas high shear levels and turbulence seem to damage cells. In order to optimize the design and the operating conditions of bioreactors, it is important to understand the fluid-dynamic characteristics and to control the stress levels within these devices. The present research focuses on the characterization of the flow field within a spinner-flask bioreactor. The dynamic properties of the flow are investigated experimentally using particle-image velocimetry with a refractive-index-matched model. Phase-locked ensemble-averaging is employed to provide some information on the turbulence characteristics of the model culture medium in the vicinity of a model tissue construct.

Hyperpolarized 13C MR Markers of Renal Tumor Aggressiveness

DTIC Science & Technology

2014-10-01

as a biomarker of tumor aggressiveness in a MR compatible 3D cell and tissue culture bioreactor ” to be presented at the ISMRM Workshop on Magnetic... Cell Carcinoma, Hyperpolarized 13C MR, Sub-renal capsule, patient derived tissue slice cultures , bioreactor 3. OVERALL PROJECT SUMMARY: Aim...grade from high grade RCCs using human TSCs cultured in a bioreactor . Aim 2:Identify HP 13C metabolic markers that discriminate low grade from

The productivity limit of manufacturing blood cell therapy in scalable stirred bioreactors

PubMed Central

Bayley, Rachel; Ahmed, Forhad; Glen, Katie; McCall, Mark; Stacey, Adrian

2017-01-01

Abstract Manufacture of red blood cells (RBCs) from progenitors has been proposed as a method to reduce reliance on donors. Such a process would need to be extremely efficient for economic viability given a relatively low value product and high (2 × 1012) cell dose. Therefore, the aim of these studies was to define the productivity of an industry standard stirred‐tank bioreactor and determine engineering limitations of commercial red blood cells production. Cord blood derived CD34+ cells were cultured under erythroid differentiation conditions in a stirred micro‐bioreactor (Ambr™). Enucleated cells of 80% purity could be created under optimal physical conditions: pH 7.5, 50% oxygen, without gas‐sparging (which damaged cells) and with mechanical agitation (which directly increased enucleation). O2 consumption was low (~5 × 10–8 μg/cell.h) theoretically enabling erythroblast densities in excess of 5 × 108/ml in commercial bioreactors and sub‐10 l/unit production volumes. The bioreactor process achieved a 24% and 42% reduction in media volume and culture time, respectively, relative to unoptimized flask processing. However, media exchange limited productivity to 1 unit of erythroblasts per 500 l of media. Systematic replacement of media constituents, as well as screening for inhibitory levels of ammonia, lactate and key cytokines did not identify a reason for this limitation. We conclude that the properties of erythroblasts are such that the conventional constraints on cell manufacturing efficiency, such as mass transfer and metabolic demand, should not prevent high intensity production; furthermore, this could be achieved in industry standard equipment. However, identification and removal of an inhibitory mediator is required to enable these economies to be realized. Copyright © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd. PMID:27696710

Effects of combined mechanical stimulation on the proliferation and differentiation of pre-osteoblasts

PubMed Central

Kang, Kyung Shin; Lee, Seung-Jae; Lee, Haksue; Moon, Wonkyu

2011-01-01

We observed how combined mechanical stimuli affect the proliferation and differentiation of pre-osteoblasts. For this research, a bioreactor system was developed that can simultaneously stimulate cells with cyclic strain and ultrasound, each of which is known to effectively stimulate bone tissue regeneration. MC3T3-E1 pre-osteoblasts were chosen for bone tissue engineering due to their osteoblast-like characteristics. 3-D scaffolds were fabricated with polycaprolactone and poly-L-lactic acid using the salt leaching method. The cells were stimulated by the bioreactor with cyclic strain and ultrasound. The bioreactor was set at a frequency of 1.0 Hz and 10% strain for cyclic strain and 1.0 MHz and 30 mW/cm2 for ultrasound. Three experimental groups (ultrasound, cyclic strain, and combined stimulation) and a control group were examined. Each group was stimulated for 20 min/day. Mechanical stimuli did not affect MC3T3-E1 cell proliferation significantly up to 10 days when measured with the cell counting kit-8. However, gene expression analysis of collagen type-I, osteocalcin, RUNX2, and osterix revealed that the combined mechanical stimulation accelerated the matrix maturation of MC3T3-E1 cells. These results indicate that the combined mechanical stimulation can enhance the differentiation of pre-osteoblasts more efficiently than simple stimuli, in spite of no effect on cell proliferation. PMID:21532314

Multi-cellular, three-dimensional living mammalian tissue

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J. (Inventor); Wolf, David A. (Inventor)

1994-01-01

The present invention relates to a multicellular, three-dimensional, living mammalian tissue. The tissue is produced by a co-culture process wherein two distinct types of mammalian cells are co-cultured in a rotating bioreactor which is completely filled with culture media and cell attachment substrates. As the size of the tissue assemblies formed on the attachment substrates changes, the rotation of the bioreactor is adjusted accordingly.

Estimation of fundamental kinetic parameters of polyhydroxybutyrate fermentation process of Azohydromonas australica using statistical approach of media optimization.

PubMed

Gahlawat, Geeta; Srivastava, Ashok K

2012-11-01

Polyhydroxybutyrate or PHB is a biodegradable and biocompatible thermoplastic with many interesting applications in medicine, food packaging, and tissue engineering materials. The present study deals with the enhanced production of PHB by Azohydromonas australica using sucrose and the estimation of fundamental kinetic parameters of PHB fermentation process. The preliminary culture growth inhibition studies were followed by statistical optimization of medium recipe using response surface methodology to increase the PHB production. Later on batch cultivation in a 7-L bioreactor was attempted using optimum concentration of medium components (process variables) obtained from statistical design to identify the batch growth and product kinetics parameters of PHB fermentation. A. australica exhibited a maximum biomass and PHB concentration of 8.71 and 6.24 g/L, respectively in bioreactor with an overall PHB production rate of 0.75 g/h. Bioreactor cultivation studies demonstrated that the specific biomass and PHB yield on sucrose was 0.37 and 0.29 g/g, respectively. The kinetic parameters obtained in the present investigation would be used in the development of a batch kinetic mathematical model for PHB production which will serve as launching pad for further process optimization studies, e.g., design of several bioreactor cultivation strategies to further enhance the biopolymer production.

Biophysical Stimulation for Engineering Functional Skeletal Muscle.

PubMed

Somers, Sarah M; Spector, Alexander A; DiGirolamo, Douglas J; Grayson, Warren L

2017-08-01

Tissue engineering is a promising therapeutic strategy to regenerate skeletal muscle. However, ex vivo cultivation methods typically result in a low differentiation efficiency of stem cells as well as grafts that resemble the native tissues morphologically, but lack contractile function. The application of biomimetic tensile strain provides a potent stimulus for enhancing myogenic differentiation and engineering functional skeletal muscle grafts. We reviewed integrin-dependent mechanisms that potentially link mechanotransduction pathways to the upregulation of myogenic genes. Yet, gaps in our understanding make it challenging to use these pathways to theoretically determine optimal ex vivo strain regimens. A multitude of strain protocols have been applied to in vitro cultures for the cultivation of myogenic progenitors (adipose- and bone marrow-derived stem cells and satellite cells) and transformed murine myoblasts, C2C12s. Strain regimens are characterized by orientation, amplitude, and time-dependent factors (effective frequency, duration, and the rest period between successive strain cycles). Analysis of published data has identified possible minimum/maximum values for these parameters and suggests that uniaxial strains may be more potent than biaxial strains, possibly because they more closely mimic physiologic strain profiles. The application of these biophysical stimuli for engineering 3D skeletal muscle grafts is nontrivial and typically requires custom-designed bioreactors used in combination with biomaterial scaffolds. Consideration of the physical properties of these scaffolds is critical for effective transmission of the applied strains to encapsulated cells. Taken together, these studies demonstrate that biomimetic tensile strain generally results in improved myogenic outcomes in myogenic progenitors and differentiated myoblasts. However, for 3D systems, the optimization of the strain regimen may require the entire system including cells, biomaterials, and bioreactor, to be considered in tandem.

Mechanobiologic Research in a Microgravity Environment Bioreactor

NASA Astrophysics Data System (ADS)

Guidi, A.; Dubini, G.; Tominetti, F.; Raimondi, M.

A current problem in tissue culturing technology is the unavailability of an effective Bioreactor for the in vitro cultivation of cells and explants. It has, in fact, proved extremely difficult to promote the high-density three-dimensional in vitro growth of human tissues that have been removed from the body and deprived of their normal in vivo vascular sources of nutrients and gas exchange. A variety of tissue explants can be maintained for a short period of time on a supportive collagen matrix surrounded by culture medium. But this system provides only limited mass transfer of nutrients and wastes through the tissue, and gravity-induced sedimentation prevents complete three- dimensional cell-cell and cell-matrix interactions. Several devices presently on the market have been used with only limited success since each has limitations, which restrict usefulness and versatility. Further, no Bioreactor or culture vessel is known that will allow for unimpeded growth of three dimensional cellular aggregates or tissue. Extensive research on the effect of mechanical stimuli on cell metabolism suggests that tissues may respond to mechanical stimulation via loading-induced flow of the interstitial fluids. During the culture, cells are subject to a flow of culture medium. Flow properties such as flow field, flow regime (e.g. turbulent or laminar), flow pattern (e.g. circular), entity and distribution of the shear stress acting on the cells greatly influence fundamental aspects of cell function, such as regulation and gene expression. This has been demonstrated for endothelial cells and significant research efforts are underway to elucidate these mechanisms in various other biological systems. Local fluid dynamics is also responsible of the mass transfer of nutrients and catabolites as well as oxygenation through the tissue. Most of the attempts to culture tissue-engineered constructs in vitro have utilized either stationary cultures or systems generating relatively small mechanical forces. For example, cartilage constructs have been cultured in spinner flasks under mixed or unmixed conditions, in simulated and in real microgravity. In these mixing studies, however, it is difficult to definitively quantify the effects of mixing-induced mechanical forces from those of convection-enhanced transport of nutrients to and of catabolites away from the cells. At the state of the art, the presence of a more controlled mechanical environment may be the condition required in order to study the biochemical and mechanical response of these biological systems. Such a controlled environment could lead to an advanced fluid dynamic design of the culture chamber that could both enhance the local mass transfer phenomena and match the needs of specific macroscopic mechanical effects in tissue development. The bioreactor is an excellent example of how the skills and resources of two distinctly different fields can complement each other. Microgravity can be used to enhance the formation of tissue like aggregates in specially designed bioreactors. Theoretical and experimental projects are under way to improve cell culture techniques using microgravity conditions experienced during space flights. Bioreactors usable under space flight conditions impose constructional principles which are different from those intended solely for ground applications. The Columbus Laboratory as part of the International Space Station (ISS) will be an evolving facility in low Earth orbit. Its mission is to support scientific, technological, and commercial activities in space. A goal of this research is to design a unique bioreactor for use sequentially from ground research to space research. One of the particularities of the simulated microgravity obtained through time averaging of the weight vector is that by varying the rotational velocity the same results can be obtained with a different value of g. One of the first applications of this technique in space biology was in fact the Rotating Wall Vessel developed by NASA, and originally designed to protect cell culture from the high shear forces generated during the launch and the landing of the Space Shuttle. A Bioreactor that is used both for ground and flight experiments provides the additional benefit of isolating dependent variable of gravity. This continuity will provide a means to compare results to a control experiment.

The BioStent: novel concept for a viable stent structure.

PubMed

Weinandy, Stefan; Rongen, Lisanne; Schreiber, Fabian; Cornelissen, Christian; Flanagan, Thomas Cormac; Mahnken, Andreas; Gries, Thomas; Schmitz-Rode, Thomas; Jockenhoevel, Stefan

2012-09-01

Percutaneous stenting of occluded peripheral vessels is a well-established technique in clinical practice. Unfortunately, the patency rates of small-caliber vessels after stenting remain unsatisfactory. The aim of the BioStent concept is to overcome in-stent restenosis by excluding the diseased vessel segment entirely from the blood stream, in addition to providing an intact endothelial cell layer. The concept combines the principles of vascular tissue engineering with a self-expanding stent: casting of the stent within a cellularized fibrin gel structure, followed by bioreactor conditioning, allows complete integration of the stent within engineered tissue. Small-caliber BioStents (Ø=6 mm; n=4) were produced by casting a nitinol stent within a thin fibrin/vascular smooth muscle cell (vSMC) mixture, followed by luminal endothelial cell seeding, and conditioning of the BioStent within a bioreactor system. The potential remodeling of the fibrin component into tissue was analyzed using routine histological methods. Scanning electron microscopy was used to assess the luminal endothelial cell coverage following the conditioning phase and crimping of the stent. The BioStent was shown to be noncytotoxic, with no significant effect on cell proliferation. Gross and microscopic analysis revealed complete integration of the nitinol component within a viable tissue structure. Hematoxylin and eosin staining revealed a homogenous distribution of vSMCs throughout the thickness of the BioStent, while a smooth, confluent luminal endothelial cell lining was evident and not significantly affected by the crimping/release process. The BioStent concept is a platform technology offering a novel opportunity to generate a viable, self-expanding stent structure with a functional endothelial cell lining. This platform technology can be transferred to different applications depending on the luminal cell lining required.

Development of Scaffold-Free Elastic Cartilaginous Constructs with Structural Similarities to Auricular Cartilage

PubMed Central

Giardini-Rosa, Renata; Joazeiro, Paulo P.; Thomas, Kathryn; Collavino, Kristina; Weber, Joanna

2014-01-01

External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm2) directly from a small population of donor cells (20,000–40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model. PMID:24124666

Development of scaffold-free elastic cartilaginous constructs with structural similarities to auricular cartilage.

PubMed

Giardini-Rosa, Renata; Joazeiro, Paulo P; Thomas, Kathryn; Collavino, Kristina; Weber, Joanna; Waldman, Stephen D

2014-03-01

External ear reconstruction with autologous cartilage still remains one of the most difficult problems in the fields of plastic and reconstructive surgery. As the absence of tissue vascularization limits the ability to stimulate new tissue growth, relatively few surgical approaches are currently available (alloplastic implants or sculpted autologous cartilage grafts) to repair or reconstruct the auricle (or pinna) as a result of traumatic loss or congenital absence (e.g., microtia). Alternatively, tissue engineering can offer the potential to grow autogenous cartilage suitable for implantation. While tissue-engineered auricle cartilage constructs can be created, a substantial number of cells are required to generate sufficient quantities of tissue for reconstruction. Similarly, as routine cell expansion can elicit negative effects on chondrocyte function, we have developed an approach to generate large-sized engineered auricle constructs (≥3 cm(2)) directly from a small population of donor cells (20,000-40,000 cells/construct). Using rabbit donor cells, the developed bioreactor-cultivated constructs adopted structural-like characteristics similar to native auricular cartilage, including the development of distinct cartilaginous and perichondrium-like regions. Both alterations in media composition and seeding density had profound effects on the formation of engineered elastic tissue constructs in terms of cellularity, extracellular matrix accumulation, and tissue structure. Higher seeding densities and media containing sodium bicarbonate produced tissue constructs that were closer to the native tissue in terms of structure and composition. Future studies will be aimed at improving the accumulation of specific tissue constituents and determining the clinical effectiveness of this approach using a reconstructive animal model.

The impact of left ventricular stretching in model cultivations with neonatal cardiomyocytes in a whole-heart bioreactor.

PubMed

Hülsmann, Jörn; Aubin, Hug; Wehrmann, Alexander; Lichtenberg, Artur; Akhyari, Payam

2017-05-01

Here, we investigate the impact of integrated three-dimensional (3D) left ventricular (LV) stretching on myocardial maturation in a whole-heart bioreactor setting. Therefore, decellularized rat hearts were selectively repopulated with rodent neonatal cardiomyocytes (5 · 10 6 cells per heart) and cultured over 5 days. Continuous medium perfusion was maintained through the coronary artery system in a customized whole-heart bioreactor system with or without integrated biomechanical stimulation of LV. 3D repopulation effectiveness and cellular vitality were evaluated by repetitive metabolic WST-1 assays and 3D confocal microscopy analysis through fluorescent staining, also assessing cellular organization. Moreover, specific myocardial vitality was verified by detecting spontaneous electrophysiological activity using a multielectrode assay. Western blot analysis of cardiac myosin heavychain (MHC) and quantitative RT-PCR for Connexin 43 was used to analyze cardiomyocyte maturation. Decellularized whole-heart constructs repopulated with neonatal cardiomyocytes (repopWHC) showed vital 3D cell populations throughout the repopulation sites within the LV with a significant increase in metabolic activity (326 ± 113% for stimulated constructs vs. 162 ± 32% for non-stimulated controls after 96 h of continuous cultivation as compared to their state 24 h after injection, directly prior to bioreactor cultivation). Further, bioreactor cultivation under integrated mechanical LV stimulation not only led to a higher degree of cellular organization and an increased MHC content, but also to a significant increase of Cx43 gene expression resulting in a regain of 60 ± 19% of native neonatal hearts expression level in contrast to 20 ± 9% for non-stimulated controls (P = 0.03). Therefore, our study suggests that the integration of LV stretching into whole-heart bioreactor cultivation may enhance cardiac maturation not only by promoting cellular organization but also through adaptive protein and gene expression with particular implications for the formation of the conductive apparatus. Further, this study emphasizes the importance of suitable bioprocessing strategies within sophisticated bioreactor systems as tools for customized stimulation and cultivation of tissue engineered tissues and organs. Biotechnol. Bioeng. 2017;114: 1107-1117. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Bioreactor strategy in bone tissue engineering: pre-culture and osteogenic differentiation under two flow configurations.

PubMed

Kim, Junho; Ma, Teng

2012-11-01

Since robust osteogenic differentiation and mineralization are integral to the engineering of bone constructs, understanding the impact of the cellular microenvironments on human mesenchymal stem cell (hMSCs) osteogenic differentiation is crucial to optimize bioreactor strategy. Two perfusion flow conditions were utilized in order to understand the impact of the flow configuration on hMSC construct development during both pre-culture (PC) in growth media and its subsequent osteogenic induction (OI). The media in the in-house perfusion bioreactor was controlled to perfuse either around (termed parallel flow [PF]) the construct surfaces or penetrate through the construct (termed transverse flow [TF]) for 7 days of the PC followed by 7 days of the OI. The flow configuration during the PC not only changed growth kinetics but also influenced cell distribution and potency of osteogenic differentiation and mineralization during the subsequent OI. While shear stress resulted from the TF stimulated cell proliferation during PC, the convective removal of de novo extracellular matrix (ECM) proteins and growth factors (GFs) reduced cell proliferation on OI. In contrast, the effective retention of de novo ECM proteins and GFs in the PC constructs under the PF maintained cell proliferation under the OI but resulted in localized cell aggregations, which influenced their osteogenic differentiation. The results revealed the contrasting roles of the convective flow as a mechanical stimulus, the redistribution of the cells and macromolecules in 3D constructs, and their divergent impacts on cellular events, leading to bone construct formation. The results suggest that the modulation of the flow configuration in the perfusion bioreactor is an effective strategy that regulates the construct properties and maximizes the functional outcome.

Development and Translation of a Tissue-Engineered Disc in a Preclinical Rodent Model

DTIC Science & Technology

2012-10-01

DAPS AF constructs, fabricated complete DAPS comprised of a PCL nanofiber AF and a hyaluronic acid hydrogel NP, and designed and commencement of...of a PCL nanofiber AF and a hyaluronic acid hydrogel NP, and design and commencement of construction of a novel multi-axis bioreactor that will be... nanofiber AF and a hyaluronic acid hydrogel NP We have commenced fabrication and in vitro pre-culture of composite DAPS constructs. An AF region

Monitoring tissue formation and organization of engineered tendon by optical coherence tomography

NASA Astrophysics Data System (ADS)

Bagnaninchi, P. O.; Yang, Y.; Maffulli, N.; Wang, R. K.; El Haj, A.

2006-02-01

The uniaxial orientation and bundle formation of collagen fibres determine the mechanical properties of tendons. Thus the particular challenge of tendon tissue engineering is to build the tissue with a highly organized structure of collagen fibres. Ultimately the engineered construct will be used as autologous grafts in tendon surgery, withstanding physiological loading. We grew pig tenocytes in porous chitosan scaffolds with multiple microchannels of 250-500 μm. The cell proliferation and production of extra-cellular matrix (ECM) within the scaffolds have been successfully monitored by Optical Coherence Tomography (OCT), a bench-top OCT system equipped with a broadband light source centred at 1300 nm. Under sterile condition, the measurements were performed on-line and in a non-destructive manner. In addition, a novel method based on OCT imaging, which calculates the occupation ratio of the microchannel derived from the scattered intensity has been developed. It is confirmed that the occupation ratio is correlated to cell proliferation and ECM production in the scaffolds. Thus this method has been utilised to assess the effect of different culture conditions on the tissue formation. The use of a perfusion bioreactor has resulted in a significantly (p<1e -3) higher cell proliferation and matrix production.

The effect of mechanical extension stimulation combined with epithelial cell sorting on outcomes of implanted tissue-engineered muscular urethras.

PubMed

Fu, Qiang; Deng, Chen-Liang; Zhao, Ren-Yan; Wang, Ying; Cao, Yilin

2014-01-01

Urethral defects are common and frequent disorders and are difficult to treat. Simple natural or synthetic materials do not provide a satisfactory curative solution for long urethral defects, and urethroplasty with large areas of autologous tissues is limited and might interfere with wound healing. In this study, adipose-derived stem cells were used. These cells can be derived from a wide range of sources, have extensive expansion capability, and were combined with oral mucosal epithelial cells to solve the problem of finding seeding cell sources for producing the tissue-engineered urethras. We also used the synthetic biodegradable polymer poly-glycolic acid (PGA) as a scaffold material to overcome issues such as potential pathogen infections derived from natural materials (such as de-vascular stents or animal-derived collagen) and differing diameters. Furthermore, we used a bioreactor to construct a tissue-engineered epithelial-muscular lumen with a double-layer structure (the epithelial lining and the muscle layer). Through these steps, we used an epithelial-muscular lumen built in vitro to repair defects in a canine urethral defect model (1 cm). Canine urethral reconstruction was successfully achieved based on image analysis and histological techniques at different time points. This study provides a basis for the clinical application of tissue engineering of an epithelial-muscular lumen. Copyright © 2013 Elsevier Ltd. All rights reserved.

Microgravity

NASA Image and Video Library

1998-10-10

Time-lapse exposure depicts Bioreactor rotation. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunourous tissues.

Growing Three-Dimensional Corneal Tissue in a Bioreactor

NASA Technical Reports Server (NTRS)

Spaulding, Glen F.; Goodwin, Thomas J.; Aten, Laurie; Prewett, Tacey; Fitzgerald, Wendy S.; OConnor, Kim; Caldwell, Delmar; Francis, Karen M.

2003-01-01

Spheroids of corneal tissue about 5 mm in diameter have been grown in a bioreactor from an in vitro culture of primary rabbit corneal cells to illustrate the production of optic cells from aggregates and tissue. In comparison with corneal tissues previously grown in vitro by other techniques, this tissue approximates intact corneal tissue more closely in both size and structure. This novel three-dimensional tissue can be used to model cell structures and functions in normal and abnormal corneas. Efforts continue to refine the present in vitro method into one for producing human corneal tissue to overcome the chronic shortage of donors for corneal transplants: The method would be used to prepare corneal tissues, either from in vitro cultures of a patient s own cells or from a well-defined culture from another human donor known to be healthy. As explained in several articles in prior issues of NASA Tech Briefs, generally cylindrical horizontal rotating bioreactors have been developed to provide nutrient-solution environments conducive to the 30 NASA Tech Briefs, October 2003 growth of delicate animal cells, with gentle, low-shear flow conditions that keep the cells in suspension without damaging them. The horizontal rotating bioreactor used in this method, denoted by the acronym "HARV," was described in "High-Aspect-Ratio Rotating Cell-Culture Vessel" (MSC-21662), NASA Tech Briefs, Vol. 16, No. 5 (May, 1992), page 150.

Numerical simulation of microcarrier motion in a rotating wall vessel bioreactor.

PubMed

Ju, Zhi-Hao; Liu, Tian-Qing; Ma, Xue-Hu; Cui, Zhan-Feng

2006-06-01

To analyze the forces of rotational wall vessel (RWV) bioreactor on small tissue pieces or microcarrier particles and to determine the tracks of microcarrier particles in RWV bioreactor. The motion of the microcarrier in the rotating wall vessel (RWV) bioreactor with both the inner and outer cylinders rotating was modeled by numerical simulation. The continuous trajectory of microcarrier particles, including the possible collision with the wall was obtained. An expression between the minimum rotational speed difference of the inner and outer cylinders and the microcarrier particle or aggregate radius could avoid collisions with either wall. The range of microcarrier radius or tissue size, which could be safely cultured in the RWV bioreactor, in terms of shear stress level, was determined. The model works well in describing the trajectory of a heavier microcarrier particle in rotating wall vessel.

The impact of simulated and real microgravity on bone cells and mesenchymal stem cells.

PubMed

Ulbrich, Claudia; Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Magnusson, Nils; Infanger, Manfred; Grosse, Jirka; Eilles, Christoph; Sundaresan, Alamelu; Grimm, Daniela

2014-01-01

How microgravity affects the biology of human cells and the formation of 3D cell cultures in real and simulated microgravity (r- and s-µg) is currently a hot topic in biomedicine. In r- and s-µg, various cell types were found to form 3D structures. This review will focus on the current knowledge of tissue engineering in space and on Earth using systems such as the random positioning machine (RPM), the 2D-clinostat, or the NASA-developed rotating wall vessel bioreactor (RWV) to create tissue from bone, tumor, and mesenchymal stem cells. To understand the development of 3D structures, in vitro experiments using s-µg devices can provide valuable information about modulations in signal-transduction, cell adhesion, or extracellular matrix induced by altered gravity conditions. These systems also facilitate the analysis of the impact of growth factors, hormones, or drugs on these tissue-like constructs. Progress has been made in bone tissue engineering using the RWV, and multicellular tumor spheroids (MCTS), formed in both r- and s-µg, have been reported and were analyzed in depth. Currently, these MCTS are available for drug testing and proteomic investigations. This review provides an overview of the influence of µg on the aforementioned cells and an outlook for future perspectives in tissue engineering.

Assembly of tissue engineered blood vessels with spatially-controlled heterogeneities.

PubMed

Strobel, Hannah A; Hookway, Tracy; Piola, Marco; Fiore, Gianfranco Beniamino; Soncini, Monica; Alsberg, Eben; Rolle, Marsha

2018-05-04

Tissue-engineered human blood vessels may enable in vitro disease modeling and drug screening to accelerate advances in vascular medicine. Existing methods for tissue engineered blood vessel (TEBV) fabrication create homogenous tubes not conducive to modeling the focal pathologies characteristic of vascular disease. We developed a system for generating self-assembled human smooth muscle cell ring-units, which were fused together into TEBVs. The goal of this study was to assess the feasibility of modular assembly and fusion of ring building units to fabricate spatially-controlled, heterogeneous tissue tubes. We first aimed to enhance fusion and reduce total culture time, and determined that reducing ring pre-culture duration improved tube fusion. Next, we incorporated electrospun polymer ring units onto tube ends as reinforced extensions, which allowed us to cannulate tubes after only 7 days of fusion, and culture tubes with luminal flow in a custom bioreactor. To create focal heterogeneities, we incorporated gelatin microspheres into select ring units during self-assembly, and fused these rings between ring units without microspheres. Cells within rings maintained their spatial position within tissue tubes after fusion. This work describes a platform approach for creating modular TEBVs with spatially-defined structural heterogeneities, which may ultimately be applied to mimic focal diseases such as intimal hyperplasia or aneurysm.

The Impact of Simulated and Real Microgravity on Bone Cells and Mesenchymal Stem Cells

PubMed Central

Wehland, Markus; Pietsch, Jessica; Aleshcheva, Ganna; Wise, Petra; van Loon, Jack; Magnusson, Nils; Infanger, Manfred; Grosse, Jirka; Eilles, Christoph

2014-01-01

How microgravity affects the biology of human cells and the formation of 3D cell cultures in real and simulated microgravity (r- and s-µg) is currently a hot topic in biomedicine. In r- and s-µg, various cell types were found to form 3D structures. This review will focus on the current knowledge of tissue engineering in space and on Earth using systems such as the random positioning machine (RPM), the 2D-clinostat, or the NASA-developed rotating wall vessel bioreactor (RWV) to create tissue from bone, tumor, and mesenchymal stem cells. To understand the development of 3D structures, in vitro experiments using s-µg devices can provide valuable information about modulations in signal-transduction, cell adhesion, or extracellular matrix induced by altered gravity conditions. These systems also facilitate the analysis of the impact of growth factors, hormones, or drugs on these tissue-like constructs. Progress has been made in bone tissue engineering using the RWV, and multicellular tumor spheroids (MCTS), formed in both r- and s-µg, have been reported and were analyzed in depth. Currently, these MCTS are available for drug testing and proteomic investigations. This review provides an overview of the influence of µg on the aforementioned cells and an outlook for future perspectives in tissue engineering. PMID:25110709

Three-Dimensional Cell Culture Models for Infectious Disease and Drug Development

NASA Technical Reports Server (NTRS)

Nickerson, Cheryl A.; Honer zu Bentrup, Kerstin; Ott, C. Mark

2005-01-01

Three-dimensional (3-D) cell cultures hold enormous potential to advance our understanding of infectious disease and to effectively translate basic cellular research into clinical applications. Using novel NASA bioreactor technology, the rotating wall vessel (RWV), we have engineered physiologically relevant 3-D human tissue culture models for infectious disease studies. The design of the RWV is based on the understanding that organs and tissues function in a 3-D environment, and that this 3-D architecture is critical for the differentiated form and function of tissues in vivo. The RWV provides large numbers of cells which are amenable to a wide variety of experimental manipulations and provides an easy, reproducible, and cost-effective approach to enhance differentiated features of cell culture models.

NASA Bioreactor Schematic

NASA Technical Reports Server (NTRS)

2001-01-01

The schematic depicts the major elements and flow patterns inside the NASA Bioreactor system. Waste and fresh medium are contained in plastic bags placed side-by-side so the waste bag fills as the fresh medium bag is depleted. The compliance vessel contains a bladder to accommodate pressure transients that might damage the system. A peristolic pump moves fluid by squeezing the plastic tubing, thus avoiding potential contamination. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Tissue engineering as innovative chance for organ replacement in radical tumor surgery.

PubMed

Alberti, C

2013-03-01

Different pathological conditions such as congenital organ absence, severe organ injuries, end-stage organ failure and malignancy-related organ removal, have few effective therapeutic options a part from a whole organ transplant, that, however, often meets with a serious shortage of suitable donor organs. The purpose of this paper consists in highlighting what the novel tissue engineering approaches might help to solve such problems. EMERGING CONCEPTS: A recent approach in tissue/organ engineering, particularly to build bioartificial airways, is the procedure of decellularizing a whole donor organ to obtain a complex 3D-biomatrix-scaffold maintaining the intrinsic vascular network, that is subsequently recellularized with recipient's autologous organ-specific differentiated cells or/and stem cells, to build a potentially functional biological substitute. Such strategy has been clinically used to replace organ in trachea/broncus tumor patients. In another approach, mainly used to construct a bioartificial urinary bladder tissue, different types of either biodegradable synthetic polymers or naturally-derived matrices or even polymer/biomatrix-composite materials are used as scaffold for either cell-free or autologous cell-seeded tissue engineering procedures. So far, such technique has been mainly used to make an augmentation cystoplasty in patients with end-stage poorly compliant neuropathic bladder or in exstrophic bladder subjects. Intriguing developments in biomaterial science, nanotechnologies, stem cell biology, and further improvements in bioreactor manufacturing will allow to generate, in the near future, tissue engineered organs that, as for structure/function so the native one-like, might represent the optimum solution to replace organs in tumor surgery.

Bone mechanobiology, gravity and tissue engineering: effects and insights.

PubMed

Ruggiu, Alessandra; Cancedda, Ranieri

2015-12-01

Bone homeostasis strongly depends on fine tuned mechanosensitive regulation signals from environmental forces into biochemical responses. Similar to the ageing process, during spaceflights an altered mechanotransduction occurs as a result of the effects of bone unloading, eventually leading to loss of functional tissue. Although spaceflights represent the best environment to investigate near-zero gravity effects, there are major limitations for setting up experimental analysis. A more feasible approach to analyse the effects of reduced mechanostimulation on the bone is represented by the 'simulated microgravity' experiments based on: (1) in vitro studies, involving cell cultures studies and the use of bioreactors with tissue engineering approaches; (2) in vivo studies, based on animal models; and (3) direct analysis on human beings, as in the case of the bed rest tests. At present, advanced tissue engineering methods allow investigators to recreate bone microenvironment in vitro for mechanobiology studies. This group and others have generated tissue 'organoids' to mimic in vitro the in vivo bone environment and to study the alteration cells can go through when subjected to unloading. Understanding the molecular mechanisms underlying the bone tissue response to mechanostimuli will help developing new strategies to prevent loss of tissue caused by altered mechanotransduction, as well as identifying new approaches for the treatment of diseases via drug testing. This review focuses on the effects of reduced gravity on bone mechanobiology by providing the up-to-date and state of the art on the available data by drawing a parallel with the suitable tissue engineering systems. Copyright © 2014 John Wiley & Sons, Ltd.

The Potential for Microalgae as Bioreactors to Produce Pharmaceuticals

PubMed Central

Yan, Na; Fan, Chengming; Chen, Yuhong; Hu, Zanmin

2016-01-01

As photosynthetic organisms, microalgae can efficiently convert solar energy into biomass. Microalgae are currently used as an important source of valuable natural biologically active molecules, such as carotenoids, chlorophyll, long-chain polyunsaturated fatty acids, phycobiliproteins, carotenoids and enzymes. Significant advances have been achieved in microalgae biotechnology over the last decade, and the use of microalgae as bioreactors for expressing recombinant proteins is receiving increased interest. Compared with the bioreactor systems that are currently in use, microalgae may be an attractive alternative for the production of pharmaceuticals, recombinant proteins and other valuable products. Products synthesized via the genetic engineering of microalgae include vaccines, antibodies, enzymes, blood-clotting factors, immune regulators, growth factors, hormones, and other valuable products, such as the anticancer agent Taxol. In this paper, we briefly compare the currently used bioreactor systems, summarize the progress in genetic engineering of microalgae, and discuss the potential for microalgae as bioreactors to produce pharmaceuticals. PMID:27322258

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

Time-lapse exposure depicts Bioreactor rotation. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunourous tissues.

Organ engineering--combining stem cells, biomaterials, and bioreactors to produce bioengineered organs for transplantation.

PubMed

Murphy, Sean Vincent; Atala, Anthony

2013-03-01

Often the only treatment available for patients suffering from diseased and injured organs is whole organ transplant. However, there is a severe shortage of donor organs for transplantation. The goal of organ engineering is to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Recent progress in stem cell biology, biomaterials, and processes such as organ decellularization and electrospinning has resulted in the generation of bioengineered blood vessels, heart valves, livers, kidneys, bladders, and airways. Future advances that may have a significant impact for the field include safe methods to reprogram a patient's own cells to directly differentiate into functional replacement cell types. The subsequent combination of these cells with natural, synthetic and/or decellularized organ materials to generate functional tissue substitutes is a real possibility. This essay reviews the current progress, developments, and challenges facing researchers in their goal to create replacement tissues and organs for patients. Copyright © 2013 WILEY Periodicals, Inc.

Microgravity

NASA Image and Video Library

2001-10-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Final samples from Mir and Earth appeared histologically cartilaginous throughout their entire cross sections (5-8 mm thick), with the exception of fibrous outer capsules. Constructs grown on Earth (A) appeared to have a more organized extracellular matrix with more uniform collagen orientation as compared with constructs grown on Mir (B), but the average collagen fiber diameter was similar in the two groups (22 +- 2 nm) and comparable to that previously reported for developing articular cartilage. Randomly oriented collagen in Mir samples would be consistent with previous reports that microgravity disrupts fibrillogenesis. These are transmission electron micrographs of constructs from Mir (A) and Earth (B) groups at magnifications of x3,500 and x120,000 (Inset). The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Credit: Proceedings of the National Academy of Sciences.

Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs.

PubMed

Sonnaert, Maarten; Kerckhofs, Greet; Papantoniou, Ioannis; Van Vlierberghe, Sandra; Boterberg, Veerle; Dubruel, Peter; Luyten, Frank P; Schrooten, Jan; Geris, Liesbet

2015-01-01

To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial 'design of experiments' approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.

Visualizing medium and biodistribution in complex cell culture bioreactors using in vivo imaging.

PubMed

Ratcliffe, E; Thomas, R J; Stacey, A J

2014-01-01

There is a dearth of technology and methods to aid process characterization, control and scale-up of complex culture platforms that provide niche micro-environments for some stem cell-based products. We have demonstrated a novel use of 3d in vivo imaging systems to visualize medium flow and cell distribution within a complex culture platform (hollow fiber bioreactor) to aid characterization of potential spatial heterogeneity and identify potential routes of bioreactor failure or sources of variability. This can then aid process characterization and control of such systems with a view to scale-up. Two potential sources of variation were observed with multiple bioreactors repeatedly imaged using two different imaging systems: shortcutting of medium between adjacent inlet and outlet ports with the potential to create medium gradients within the bioreactor, and localization of bioluminescent murine 4T1-luc2 cells upon inoculation with the potential to create variable seeding densities at different points within the cell growth chamber. The ability of the imaging technique to identify these key operational bioreactor characteristics demonstrates an emerging technique in troubleshooting and engineering optimization of bioreactor performance. © 2013 American Institute of Chemical Engineers.

Microgravity

NASA Image and Video Library

2001-06-01

The schematic depicts the major elements and flow patterns inside the NASA Bioreactor system. Waste and fresh medium are contained in plastic bags placed side-by-side so the waste bag fills as the fresh medium bag is depleted. The compliance vessel contains a bladder to accommodate pressure transients that might damage the system. A peristolic pump moves fluid by squeezing the plastic tubing, thus avoiding potential contamination. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Use of bioreactors for culturing human retinal organoids improves photoreceptor yields.

PubMed

Ovando-Roche, Patrick; West, Emma L; Branch, Matthew J; Sampson, Robert D; Fernando, Milan; Munro, Peter; Georgiadis, Anastasios; Rizzi, Matteo; Kloc, Magdalena; Naeem, Arifa; Ribeiro, Joana; Smith, Alexander J; Gonzalez-Cordero, Anai; Ali, Robin R

2018-06-13

The use of human pluripotent stem cell-derived retinal cells for cell therapy strategies and disease modelling relies on the ability to obtain healthy and organised retinal tissue in sufficient quantities. Generating such tissue is a lengthy process, often taking over 6 months of cell culture, and current approaches do not always generate large quantities of the major retinal cell types required. We adapted our previously described differentiation protocol to investigate the use of stirred-tank bioreactors. We used immunohistochemistry, flow cytometry and electron microscopy to characterise retinal organoids grown in standard and bioreactor culture conditions. Our analysis revealed that the use of bioreactors results in improved laminar stratification as well as an increase in the yield of photoreceptor cells bearing cilia and nascent outer-segment-like structures. Bioreactors represent a promising platform for scaling up the manufacture of retinal cells for use in disease modelling, drug screening and cell transplantation studies.

Optimal 3D culture of primary articular chondrocytes for use in the rotating wall vessel bioreactor.

PubMed

Mellor, Liliana F; Baker, Travis L; Brown, Raquel J; Catlin, Lindsey W; Oxford, Julia Thom

2014-08-01

Reliable culturing methods for primary articular chondrocytes are essential to study the effects of loading and unloading on joint tissue at the cellular level. Due to the limited proliferation capacity of primary chondrocytes and their tendency to dedifferentiate in conventional culture conditions, long-term culturing conditions of primary chondrocytes can be challenging. The goal of this study was to develop a suspension culturing technique that not only would retain the cellular morphology, but also maintain the gene expression characteristics of primary articular chondrocytes. Three-dimensional culturing methods were compared and optimized for primary articular chondrocytes in the rotating wall vessel bioreactor, which changes the mechanical culture conditions to provide a form of suspension culture optimized for low shear and turbulence. We performed gene expression analysis and morphological characterization of cells cultured in alginate beads, Cytopore-2 microcarriers, primary monolayer culture, and passaged monolayer cultures using reverse transcription-PCR and laser scanning confocal microscopy. Primary chondrocytes grown on Cytopore-2 microcarriers maintained the phenotypical morphology and gene expression pattern observed in primary bovine articular chondrocytes, and retained these characteristics for up to 9 d. Our results provide a novel and alternative culturing technique for primary chondrocytes suitable for studies that require suspension such as those using the rotating wall vessel bioreactor. In addition, we provide an alternative culturing technique for primary chondrocytes that can impact future mechanistic studies of osteoarthritis progression, treatments for cartilage damage and repair, and cartilage tissue engineering.

Cell Culturing of Cytoskeleton

NASA Technical Reports Server (NTRS)

2004-01-01

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc., has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc., is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

Cell Culturing of Cytoskeleton

NASA Technical Reports Server (NTRS)

2004-01-01

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Cell culturing, such as this bone cell culture, is an important part of biomedical research. The BioDyn payload includes a tissue engineering investigation. The commercial affiliate, Millenium Biologix, Inc. has been conducting bone implant experiments to better understand how synthetic bone can be used to treat bone-related illnesses and bone damaged in accidents. On STS-95, the BioDyn payload will include a bone cell culture aimed to help develop this commercial synthetic bone product. Millenium Biologix, Inc. is exploring the potential for making human bone implantable materials by seeding its proprietary artificial scaffold material with human bone cells. The product of this tissue engineering experiment using the Bioprocessing Modules (BPMs) on STS-95 is space-grown bone implants, which could have potential for dental implants, long bone grafts, and coating for orthopedic implants such as hip replacements.

High-Throughput Nuclear Magnetic Resonance Metabolomic Footprinting for Tissue Engineering

PubMed Central

Seagle, Christopher; Christie, Megan A.; Winnike, Jason H.; McClelland, Randall E.; Ludlow, John W.; O'Connell, Thomas M.; Gamcsik, Michael P.

2008-01-01

Abstract We report a high-throughput (HTP) nuclear magnetic resonance (NMR) method for analysis of media components and a metabolic schematic to help easily interpret the data. Spin-lattice relaxation values and concentrations were measured for 19 components and 2 internal referencing agents in pure and 2-day conditioned, hormonally defined media from a 3-dimensional (3D) multicoaxial human bioartificial liver (BAL). The 1H NMR spectral signal-to-noise ratio is 21 for 0.16 mM alanine in medium and is obtained in 12 min using a 400 MHz NMR spectrometer. For comparison, 2D gel cultures and 3D multicoaxial BALs were batch cultured, with medium changed every day for 15 days after inoculation with human liver cells in Matrigel–collagen type 1 gels. Glutamine consumption was higher by day 8 in the BAL than in 2D culture; lactate production was lower through the 15-day culture period. Alanine was the primary amino acid produced and tracked with lactate or urea production. Glucose and pyruvate consumption were similar in the BAL and 2D cultures. NMR analysis permits quality assurance of the bioreactor by identifying contaminants. Ethanol was observed because of a bioreactor membrane “wetting” procedure. A biochemical scheme is presented illustrating bioreactor metabolomic footprint results and demonstrating how this can be translated to modify bioreactor operational parameters or quality assurance issues. PMID:18544027

Engineering of oriented myocardium on three-dimensional micropatterned collagen-chitosan hydrogel.

PubMed

Chiu, Loraine L Y; Janic, Katarina; Radisic, Milica

2012-04-30

Surface topography and electrical field stimulation are important guidance cues that aid the organization and contractility of cardiomyocytes in vivo. We report here on the use of these biomimetic cues in vitro to engineer an implantable contractile cardiac tissue. Photocrosslinkable collagen-chitosan hydrogels with microgrooves of 10 µm, 20 µm and 100 µm in width were fabricated using polydimethylsiloxane (PDMS) molds. The hydrogels were seeded with cardiomyocytes, placed into a bioreactor array with the microgrooves aligned with the electrical field lines, and stimulated with biphasic square pulses at 1 Hz and 2.5 V/cm. At Day 6, cardiomyocytes were aligned in the direction of the microgrooves. When cultivated without electrical stimulation, the excitation threshold of engineered cardiac tissues using micropatterned hydrogels was significantly lower than using smooth hydrogels, thus showing the importance of cell alignment to cardiac function. The success rate of achieving beating constructs was higher with the application of electrical stimulation. In addition, formation of dense contractile cardiac organoids was observed in groups with both biomimetic cues. The cultivation of cardiomyocytes on hydrogels with 10 µm grooves yielded 100% beating tissues with or without electrical stimulation, thus suggesting a smaller groove width is necessary for cells to communicate and form proper gap junctions. However, electrical field stimulation further increased cell density and enhanced tissue morphology which may be essential for the integration of the tissue construct to the native heart tissue upon implantation. The biodegradability of the hydrogel substrate allows for the rapid translation of the engineered, oriented cardiac tissue to clinical applications.

Modulating the Behaviors of Mesenchymal Stem Cells Via the Combination of High-Frequency Vibratory Stimulations and Fibrous Scaffolds

PubMed Central

Tong, Zhixiang; Duncan, Randall L.

2013-01-01

We are interested in the in vitro engineering of artificial vocal fold tissues via the strategic combination of multipotent mesenchymal stem cells (MSCs), physiologically relevant mechanical stimulations, and biomimetic artificial matrices. We have constructed a vocal fold bioreactor that is capable of imposing vibratory stimulations on the cultured cells at human phonation frequencies. Separately, fibrous poly (ɛ-caprolactone) (PCL) scaffolds emulating the ligamentous structure of the vocal fold were prepared by electrospinning, were incorporated in the vocal fold bioreactor, and were driven into a wave-like motion in an axisymmetrical fashion by the oscillating air. MSC-laden PCL scaffolds were subjected to vibrations at 200 Hz with a normal center displacement of ∼40 μm for a total of 7 days. A continuous (CT) or a 1 h-on-1 h-off (OF) regime with a total dynamic culture time of 12 h per day was applied. The dynamic loading did not cause any physiological trauma to the cells. Immunohistotochemical staining revealed the reinforcement of the actin filament and the enhancement of α5β1 integrin expression under selected dynamic culture conditions. Cellular expression of essential vocal fold extracellular matrix components, such as elastin, hyaluronic acid, and matrix metalloproteinase-1, was significantly elevated as compared with the static controls, and the OF regime is more conducive to matrix production than the CT vibration mode. Analyses of genes of typical fibroblast hallmarks (tenascin-C, collagen III, and procollagen I) as well as markers for MSC differentiation into nonfibroblastic lineages confirmed MSCs' adaptation of fibroblastic behaviors. Overall, the high-frequency vibratory stimulation, when combined with a synthetic fibrous scaffold, serves as a potent modulator of MSC functions. The novel bioreactor system presented here, as a versatile, yet well-controlled model, offers an in vitro platform for understanding vibration-induced mechanotransduction and for engineering of functional vocal fold tissues. PMID:23516973

New advances in MR-compatible bioartificial liver

PubMed Central

Jeffries, Rex E.; Macdonald, Jeffrey M.

2015-01-01

MR-compatible bioartificial liver (BAL) studies have been performed for 30 years and are reviewed. There are two types of study: (i) metabolism and drug studies using multinuclear MRS; primarily short-term (< 8 h) studies; (ii) the use of multinuclear MRS and MRI to noninvasively define the features and functions of BAL systems for long-term liver tissue engineering. In the latter, these systems often undergo not only modification of the perfusion system, but also the construction of MR radiofrequency probes around the bioreactor. We present novel MR-compatible BALs and the use of multinuclear MRS (13C, 19F, 31P) for the noninvasive monitoring of their growth, metabolism and viability, as well as 1H MRI methods for the determination of flow profiles, diffusion, cell distribution, quality assurance and bioreactor integrity. Finally, a simple flexible coil design and circuit, and life support system, are described that can make almost any BAL MR-compatible. PMID:22351642

A study of the Coriolis effect on the fluid flow profile in a centrifugal bioreactor.

PubMed

Detzel, Christopher J; Thorson, Michael R; Van Wie, Bernard J; Ivory, Cornelius F

2009-01-01

Increasing demand for tissues, proteins, and antibodies derived from cell culture is necessitating the development and implementation of high cell density bioreactors. A system for studying high density culture is the centrifugal bioreactor (CCBR), which retains cells by increasing settling velocities through system rotation, thereby eliminating diffusional limitations associated with mechanical cell retention devices. This article focuses on the fluid mechanics of the CCBR system by considering Coriolis effects. Such considerations for centrifugal bioprocessing have heretofore been ignored; therefore, a simpler analysis of an empty chamber will be performed. Comparisons are made between numerical simulations and bromophenol blue dye injection experiments. For the non-rotating bioreactor with an inlet velocity of 4.3 cm/s, both the numerical and experimental results show the formation of a teardrop shaped plume of dye following streamlines through the reactor. However, as the reactor is rotated, the simulation predicts the development of vortices and a flow profile dominated by Coriolis forces resulting in the majority of flow up the leading wall of the reactor as dye initially enters the chamber, results are confirmed by experimental observations. As the reactor continues to fill with dye, the simulation predicts dye movement up both walls while experimental observations show the reactor fills with dye from the exit to the inlet. Differences between the simulation and experimental observations can be explained by excessive diffusion required for simulation convergence, and a slight density difference between dyed and un-dyed solutions. Implications of the results on practical bioreactor use are also discussed. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009.

Bioinspired methodology for preparing magnetic responsive chitosan beads to be integrated in a tubular bioreactor for biomedical applications.

PubMed

Song, Wenlong; Oliveira, Mariana B; Sher, Praveen; Gil, Sara; Nóbrega, J Miguel; Mano, João F

2013-08-01

Magnetic responsive chitosan beads were prepared using a methodology inspired by the rolling of water droplets over lotus leaves. Liquid precursors containing chitosan and magnetic microparticles were dispensed in the form of spherical droplets and crosslinked with genipin over synthetic superhydrophobic surfaces. Scanning electronic microscopy, histology and micro-computed tomography were employed to characterize the structure of the prepared composite beads and the inner distribution of the magnetic particles. Cellular metabolic activity tests showed that fibroblasts-like (L929 cell line) can adhere and proliferate on the prepared chitosan beads. We hypothesize that such spherical biomaterials could be integrated in a new concept of tubular bioreactor. The magnetic beads can be immobilized by an external magnetic field at specific positions and may be transported along the bioreactor by the drag of the culture medium flow. The system behavior was also studied through numerical modeling, which allowed to identify the relative importance of the main parameters, and to conclude that the distance between carrier beads plays a major role on their interaction with the culture medium and, consequently, on the overall system performance. In an up-scaled version of this bioreactor, the herein presented system may comprise different chambers in serial or parallel configurations. This constitutes a simple way of preparing magnetic responsive beads combined with a new design of bioreactor, which may find application in biomedicine and biotechnology, including in cell expansion for tissue engineering or for the production of therapeutic proteins to be used in cell therapies.

Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors.

PubMed

Gareau, Tia; Lara, Giovanna G; Shepherd, Robert D; Krawetz, Roman; Rancourt, Derrick E; Rinker, Kristina D; Kallos, Michael S

2014-04-01

Pluripotent embryonic stem cells (ESCs) have been used increasingly in research as primary material for various tissue-engineering applications. Pluripotency, or the ability to give rise to all cells of the body, is an important characteristic of ESCs. Traditional methods use leukaemia inhibitory factor (LIF) to maintain murine embryonic stem cell (mESC) pluripotency in static and bioreactor cultures. When LIF is removed from mESCs in static cultures, pluripotency genes are downregulated and the cultures will spontaneously differentiate. Recently we have shown the maintenance of pluripotency gene expression of mESCs in stirred suspension bioreactors during differentiation experiments in the absence of LIF. This is undesired in a differentiation experiment, where the goal is downregulation of pluripotency gene expression and upregulation of gene expression characteristic to the differentiation. Thus, the objective of this study was to examine how effectively different levels of shear stress [100 rpm (6 dyne/cm(2) ), 60 rpm (3 dyne/cm(2) )] maintained and influenced pluripotency in suspension bioreactors. The pluripotency markers Oct-4, Nanog, Sox-2 and Rex-1 were assessed using gene expression profiles and flow-cytometry analysis and showed that shear stress does maintain and influence the gene expression of certain pluripotency markers. Some significant differences between the two levels of shear stress were seen and the combination of shear stress and LIF was observed to synergistically increase the expression of certain pluripotency markers. Overall, this study provides a better understanding of the environmental conditions within suspension bioreactors and how these conditions affect the pluripotency of mESCs. Copyright © 2012 John Wiley & Sons, Ltd.

Tissue engineering of cartilage using a mechanobioreactor exerting simultaneous mechanical shear and compression to simulate the rolling action of articular joints.

PubMed

Shahin, Kifah; Doran, Pauline M

2012-04-01

The effect of dynamic mechanical shear and compression on the synthesis of human tissue-engineered cartilage was investigated using a mechanobioreactor capable of simulating the rolling action of articular joints in a mixed fluid environment. Human chondrocytes seeded into polyglycolic acid (PGA) mesh or PGA-alginate scaffolds were precultured in shaking T-flasks or recirculation perfusion bioreactors for 2.5 or 4 weeks prior to mechanical stimulation in the mechanobioreactor. Constructs were subjected to intermittent unconfined shear and compressive loading at a frequency of 0.05 Hz using a peak-to-peak compressive strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5%. The mechanical treatment was carried out for up to 2.5 weeks using a loading regime of 10 min duration each day with the direction of the shear forces reversed after 5 min and release of all loading at the end of the daily treatment period. Compared with shaking T-flasks and mechanobioreactor control cultures without loading, mechanical treatment improved the amount and quality of cartilage produced. On a per cell basis, synthesis of both major structural components of cartilage, glycosaminoglycan (GAG) and collagen type II, was enhanced substantially by up to 5.3- and 10-fold, respectively, depending on the scaffold type and seeding cell density. Levels of collagen type II as a percentage of total collagen were also increased after mechanical treatment by up to 3.4-fold in PGA constructs. Mechanical treatment had a less pronounced effect on the composition of constructs precultured in perfusion bioreactors compared with perfusion culture controls. This work demonstrates that the quality of tissue-engineered cartilage can be enhanced significantly by application of simultaneous dynamic mechanical shear and compression, with the greatest benefits evident for synthesis of collagen type II. Copyright © 2011 Wiley Periodicals, Inc.

A Novel Bioreactor System for the Assessment of Endothelialization on Deformable Surfaces

PubMed Central

Bachmann, Björn J.; Bernardi, Laura; Loosli, Christian; Marschewski, Julian; Perrini, Michela; Ehrbar, Martin; Ermanni, Paolo; Poulikakos, Dimos; Ferrari, Aldo; Mazza, Edoardo

2016-01-01

The generation of a living protective layer at the luminal surface of cardiovascular devices, composed of an autologous functional endothelium, represents the ideal solution to life-threatening, implant-related complications in cardiovascular patients. The initial evaluation of engineering strategies fostering endothelial cell adhesion and proliferation as well as the long-term tissue homeostasis requires in vitro testing in environmental model systems able to recapitulate the hemodynamic conditions experienced at the blood-to-device interface of implants as well as the substrate deformation. Here, we introduce the design and validation of a novel bioreactor system which enables the long-term conditioning of human endothelial cells interacting with artificial materials under dynamic combinations of flow-generated wall shear stress and wall deformation. The wall shear stress and wall deformation values obtained encompass both the physiological and supraphysiological range. They are determined through separate actuation systems which are controlled based on validated computational models. In addition, we demonstrate the good optical conductivity of the system permitting online monitoring of cell activities through live-cell imaging as well as standard biochemical post-processing. Altogether, the bioreactor system defines an unprecedented testing hub for potential strategies toward the endothelialization or re-endothelialization of target substrates. PMID:27941901

Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue

NASA Technical Reports Server (NTRS)

Klement, B. J.; Spooner, B. S.

1993-01-01

Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment. These vessels will also provide an advantageous, low maintenance culture system on space station Freedom. Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue. We have characterized an organ culture system utilizing embryonic mouse pre-metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells. Further development to form bone tissue is achieved by supplementation of the culture medium. Research using pre-metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity. Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth.

Microgravity

NASA Image and Video Library

1998-10-10

Dr. Harry Mahtani analyzes the gas content of nutrient media from Bioreactor used in research on human breast cancer. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunourous tissues.

Bioreactor-engineered cancer tissue-like structures mimic phenotypes, gene expression profiles and drug resistance patterns observed "in vivo".

PubMed

Hirt, Christian; Papadimitropoulos, Adam; Muraro, Manuele G; Mele, Valentina; Panopoulos, Evangelos; Cremonesi, Eleonora; Ivanek, Robert; Schultz-Thater, Elke; Droeser, Raoul A; Mengus, Chantal; Heberer, Michael; Oertli, Daniel; Iezzi, Giandomenica; Zajac, Paul; Eppenberger-Castori, Serenella; Tornillo, Luigi; Terracciano, Luigi; Martin, Ivan; Spagnoli, Giulio C

2015-09-01

Anticancer compound screening on 2D cell cultures poorly predicts "in vivo" performance, while conventional 3D culture systems are usually characterized by limited cell proliferation, failing to produce tissue-like-structures (TLS) suitable for drug testing. We addressed engineering of TLS by culturing cancer cells in porous scaffolds under perfusion flow. Colorectal cancer (CRC) HT-29 cells were cultured in 2D, on collagen sponges in static conditions or in perfused bioreactors, or injected subcutaneously in immunodeficient mice. Perfused 3D (p3D) cultures resulted in significantly higher (p < 0.0001) cell proliferation than static 3D (s3D) cultures and yielded more homogeneous TLS, with morphology and phenotypes similar to xenografts. Transcriptome analysis revealed a high correlation between xenografts and p3D cultures, particularly for gene clusters regulating apoptotic processes and response to hypoxia. Treatment with 5-Fluorouracil (5-FU), a frequently used but often clinically ineffective chemotherapy drug, induced apoptosis, down-regulation of anti-apoptotic genes (BCL-2, TRAF1, and c-FLIP) and decreased cell numbers in 2D, but only "nucleolar stress" in p3D and xenografts. Conversely, BCL-2 inhibitor ABT-199 induced cytotoxic effects in p3D but not in 2D cultures. Our findings advocate the importance of perfusion flow in 3D cultures of tumor cells to efficiently mimic functional features observed "in vivo" and to test anticancer compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

Beta-tricalcium phosphate granules improve osteogenesis in vitro and establish innovative osteo-regenerators for bone tissue engineering in vivo.

PubMed

Gao, Peng; Zhang, Haoqiang; Liu, Yun; Fan, Bo; Li, Xiaokang; Xiao, Xin; Lan, Pingheng; Li, Minghui; Geng, Lei; Liu, Dong; Yuan, Yulin; Lian, Qin; Lu, Jianxi; Guo, Zheng; Wang, Zhen

2016-03-22

The drawbacks of traditional bone-defect treatments have prompted the exploration of bone tissue engineering. This study aimed to explore suitable β-tricalcium phosphate (β-TCP) granules for bone regeneration and identify an efficient method to establish β-TCP-based osteo-regenerators. β-TCP granules with diameters of 1 mm and 1-2.5 mm were evaluated in vitro. The β-TCP granules with superior osteogenic properties were used to establish in vivo bioreactors, referred to as osteo-regenerators, which were fabricated using two different methods. Improved proliferation of bone mesenchymal stem cells (BMSCs), glucose consumption and ALP activity were observed for 1-2.5 mm β-TCP compared with 1-mm granules (P < 0.05). In addition, BMSCs incubated with 1-2.5 mm β-TCP expressed significantly higher levels of the genes for runt-related transcription factor-2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type-1 and the osteogenesis-related proteins alkaline phosphatase, collagen type-1 and runt-related transcription factor-2 compared with BMSCs incubated with 1 mm β-TCP (P < 0.05). Fluorochrome labelling, micro-computed tomography and histological staining analyses indicated that the osteo-regenerator with two holes perforating the femur promoted significantly greater bone regeneration compared with the osteo-regenerator with a periosteum incision (P < 0.05). This study provides an alternative to biofunctionalized bioreactors that exhibits improved osteogenesis.

Beta-tricalcium phosphate granules improve osteogenesis in vitro and establish innovative osteo-regenerators for bone tissue engineering in vivo

PubMed Central

Gao, Peng; Zhang, Haoqiang; Liu, Yun; Fan, Bo; Li, Xiaokang; Xiao, Xin; Lan, Pingheng; Li, Minghui; Geng, Lei; Liu, Dong; Yuan, Yulin; Lian, Qin; Lu, Jianxi; Guo, Zheng; Wang, Zhen

2016-01-01

The drawbacks of traditional bone-defect treatments have prompted the exploration of bone tissue engineering. This study aimed to explore suitable β-tricalcium phosphate (β-TCP) granules for bone regeneration and identify an efficient method to establish β-TCP-based osteo-regenerators. β-TCP granules with diameters of 1 mm and 1–2.5 mm were evaluated in vitro. The β-TCP granules with superior osteogenic properties were used to establish in vivo bioreactors, referred to as osteo-regenerators, which were fabricated using two different methods. Improved proliferation of bone mesenchymal stem cells (BMSCs), glucose consumption and ALP activity were observed for 1–2.5 mm β-TCP compared with 1-mm granules (P < 0.05). In addition, BMSCs incubated with 1–2.5 mm β-TCP expressed significantly higher levels of the genes for runt-related transcription factor-2, alkaline phosphatase, osteocalcin, osteopontin, and collagen type-1 and the osteogenesis-related proteins alkaline phosphatase, collagen type-1 and runt-related transcription factor-2 compared with BMSCs incubated with 1 mm β-TCP (P < 0.05). Fluorochrome labelling, micro-computed tomography and histological staining analyses indicated that the osteo-regenerator with two holes perforating the femur promoted significantly greater bone regeneration compared with the osteo-regenerator with a periosteum incision (P < 0.05). This study provides an alternative to biofunctionalized bioreactors that exhibits improved osteogenesis. PMID:27000963

Ultrasound Elastography for Estimation of Regional Strain of Multilayered Hydrogels and Tissue-Engineered Cartilage

PubMed Central

Chung, Chen-Yuan; Heebner, Joseph; Baskaran, Harihara; Welter, Jean F.; Mansour, Joseph M.

2015-01-01

Tissue-engineered (TE) cartilage constructs tend to develop inhomogeneously, thus, to predict the mechanical performance of the tissue, conventional biomechanical testing, which yields average material properties, is of limited value. Rather, techniques for evaluating regional and depth-dependent properties of TE cartilage, preferably non-destructively, are required. The purpose of this study was to build upon our previous results and to investigate the feasibility of using ultrasound elastography to non-destructively assess the depth-dependent biomechanical characteristics of TE cartilage while in a sterile bioreactor. As a proof-of-concept, and to standardize an assessment protocol, a well-characterized three-layered hydrogel construct was used as a surrogate for TE cartilage, and was studied under controlled incremental compressions. The strain field of the construct predicted by elastography was then validated by comparison with a poroelastic finite-element analysis (FEA). On average, the differences between the strains predicted by elastography and the FEA were within 10%. Subsequently engineered cartilage tissue was evaluated in the same test fixture. Results from these examinations showed internal regions where the local strain was 1–2 orders of magnitude greater than that near the surface. These studies document the feasibility of using ultrasound to evaluate the mechanical behaviors of maturing TE constructs in a sterile environment. PMID:26077987

Influence of the mechanical environment on the engineering of mineralised tissues using human dental pulp stem cells and silk fibroin scaffolds.

PubMed

Woloszyk, Anna; Holsten Dircksen, Sabrina; Bostanci, Nagihan; Müller, Ralph; Hofmann, Sandra; Mitsiadis, Thimios A

2014-01-01

Teeth constitute a promising source of stem cells that can be used for tissue engineering and regenerative medicine purposes. Bone loss in the craniofacial complex due to pathological conditions and severe injuries could be treated with new materials combined with human dental pulp stem cells (hDPSCs) that have the same embryonic origin as craniofacial bones. Optimising combinations of scaffolds, cells, growth factors and culture conditions still remains a great challenge. In the present study, we evaluate the mineralisation potential of hDPSCs seeded on porous silk fibroin scaffolds in a mechanically dynamic environment provided by spinner flask bioreactors. Cell-seeded scaffolds were cultured in either standard or osteogenic media in both static and dynamic conditions for 47 days. Histological analysis and micro-computed tomography of the samples showed low levels of mineralisation when samples were cultured in static conditions (0.16±0.1 BV/TV%), while their culture in a dynamic environment with osteogenic medium and weekly µCT scans (4.9±1.6 BV/TV%) significantly increased the formation of homogeneously mineralised structures, which was also confirmed by the elevated calcium levels (4.5±1.0 vs. 8.8±1.7 mg/mL). Molecular analysis of the samples showed that the expression of tooth correlated genes such as Dentin Sialophosphoprotein and Nestin were downregulated by a factor of 6.7 and 7.4, respectively, in hDPSCs when cultured in presence of osteogenic medium. This finding indicates that hDPSCs are able to adopt a non-dental identity by changing the culture conditions only. Also an increased expression of Osteocalcin (1.4x) and Collagen type I (1.7x) was found after culture under mechanically dynamic conditions in control medium. In conclusion, the combination of hDPSCs and silk scaffolds cultured under mechanical loading in spinner flask bioreactors could offer a novel and promising approach for bone tissue engineering where appropriate and rapid bone regeneration in mechanically loaded tissues is required.

Bioreactors for Tissue Engineering of Cartilage

NASA Astrophysics Data System (ADS)

Concaro, S.; Gustavson, F.; Gatenholm, P.

The cartilage regenerative medicine field has evolved during the last decades. The first-generation technology, autologous chondrocyte transplantation (ACT) involved the transplantation of in vitro expanded chondrocytes to cartilage defects. The second generation involves the seeding of chondrocytes in a three-dimensional scaffold. The technique has several potential advantages such as the ability of arthroscopic implantation, in vitro pre-differentiation of cells and implant stability among others (Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L, N Engl J Med 331(14):889-895, 1994; Henderson I, Francisco R, Oakes B, Cameron J, Knee 12(3):209-216, 2005; Peterson L, Minas T, Brittberg M, Nilsson A, Sjogren-Jansson E, Lindahl A, Clin Orthop (374):212-234, 2000; Nagel-Heyer S, Goepfert C, Feyerabend F, Petersen JP, Adamietz P, Meenen NM, et al. Bioprocess Biosyst Eng 27(4):273-280, 2005; Portner R, Nagel-Heyer S, Goepfert C, Adamietz P, Meenen NM, J Biosci Bioeng 100(3):235-245, 2005; Nagel-Heyer S, Goepfert C, Adamietz P, Meenen NM, Portner R, J Biotechnol 121(4):486-497, 2006; Heyland J, Wiegandt K, Goepfert C, Nagel-Heyer S, Ilinich E, Schumacher U, et al. Biotechnol Lett 28(20):1641-1648, 2006). The nutritional requirements of cells that are synthesizing extra-cellular matrix increase along the differentiation process. The mass transfer must be increased according to the tissue properties. Bioreactors represent an attractive tool to accelerate the biochemical and mechanical properties of the engineered tissues providing adequate mass transfer and physical stimuli. Different reactor systems have been [5] developed during the last decades based on different physical stimulation concepts. Static and dynamic compression, confined and nonconfined compression-based reactors have been described in this review. Perfusion systems represent an attractive way of culturing constructs under dynamic conditions. Several groups showed increased matrix production using confined and unconfined systems. Development of automatic culture systems and noninvasive monitoring of matrix production will take place during the next few years in order to improve the cost affectivity of tissue-engineered products.

Regeneration of Tissues and Organs Using Autologous Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anthony Atala, M D

2012-10-11

The proposed work aims to address three major challenges to the field of regenerative medicine: 1) the growth and expansion of regenerative cells outside the body in controlled in vitro environments, 2) supportive vascular supply for large tissue engineered constructs, and 3) interactive biomaterials that can orchestrate tissue development in vivo. Toward this goal, we have engaged a team of scientists with expertise in cell and molecular biology, physiology, biomaterials, controlled release, nanomaterials, tissue engineering, bioengineering, and clinical medicine to address all three challenges. This combination of resources, combined with the vast infrastructure of the WFIRM, have brought to bearmore » on projects to discover and test new sources of autologous cells that can be used therapeutically, novel methods to improve vascular support for engineered tissues in vivo, and to develop intelligent biomaterials and bioreactor systems that interact favorably with stem and progenitor cells to drive tissue maturation. The Institute's ongoing programs are aimed at developing regenerative medicine technologies that employ a patient's own cells to help restore or replace tissue and organ function. This DOE program has provided a means to solve some of the vexing problems that are germane to many tissue engineering applications, regardless of tissue type or target disease. By providing new methods that are the underpinning of tissue engineering, this program facilitated advances that can be applied to conditions including heart disease, diabetes, renal failure, nerve damage, vascular disease, and cancer, to name a few. These types of conditions affect millions of Americans at a cost of more than $400 billion annually. Regenerative medicine holds the promise of harnessing the body's own power to heal itself. By addressing the fundamental challenges of this field in a comprehensive and focused fashion, this DOE program has opened new opportunities to treat conditions where other approaches have failed.« less

Advances in vascular tissue engineering.

PubMed

Thomas, Anita C; Campbell, Gordon R; Campbell, Julie H

2003-01-01

Coronary and peripheral artery bypass grafting is commonly used to relieve the symptoms of vascular deficiencies, but the supply of autologous artery or vein may not be sufficient or suitable for multiple bypass or repeat procedures, necessitating the use of other materials. Synthetic materials are suitable for large bore arteries but often thrombose when used in smaller arteries. Suitable replacement grafts must have appropriate characteristics, including resistance to infection, low immunogenicity and good biocompatability and thromboresistance, with appropriate mechanical and physiological properties and cheap and fast manufacture. Current avenues of graft development include coating synthetic grafts with either biological chemicals or cells with anticoagulatory properties. Matrix templates or acellular tubes of extracellular matrix (such as collagen) may be coated or infiltrated with cultured cells. Once placed into the artery, these grafts may become colonised by host cells and gain many of the properties of normal artery. "Tissue-engineered blood vessels" may also be formed from layers of human vascular cells grown in culture. These engineered vessels have many of the characteristics of arteries formed in vivo. "Artificial arteries" may be also be derived from peritoneal granulation tissue in body "bioreactors" by adapting the body's natural wound healing response to produce a hollow tube.

Predicting cell viability within tissue scaffolds under equiaxial strain: multi-scale finite element model of collagen-cardiomyocytes constructs.

PubMed

Elsaadany, Mostafa; Yan, Karen Chang; Yildirim-Ayan, Eda

2017-06-01

Successful tissue engineering and regenerative therapy necessitate having extensive knowledge about mechanical milieu in engineered tissues and the resident cells. In this study, we have merged two powerful analysis tools, namely finite element analysis and stochastic analysis, to understand the mechanical strain within the tissue scaffold and residing cells and to predict the cell viability upon applying mechanical strains. A continuum-based multi-length scale finite element model (FEM) was created to simulate the physiologically relevant equiaxial strain exposure on cell-embedded tissue scaffold and to calculate strain transferred to the tissue scaffold (macro-scale) and residing cells (micro-scale) upon various equiaxial strains. The data from FEM were used to predict cell viability under various equiaxial strain magnitudes using stochastic damage criterion analysis. The model validation was conducted through mechanically straining the cardiomyocyte-encapsulated collagen constructs using a custom-built mechanical loading platform (EQUicycler). FEM quantified the strain gradients over the radial and longitudinal direction of the scaffolds and the cells residing in different areas of interest. With the use of the experimental viability data, stochastic damage criterion, and the average cellular strains obtained from multi-length scale models, cellular viability was predicted and successfully validated. This methodology can provide a great tool to characterize the mechanical stimulation of bioreactors used in tissue engineering applications in providing quantification of mechanical strain and predicting cellular viability variations due to applied mechanical strain.

Engineered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors.

PubMed

Schmidt, Dörthe; Asmis, Lars M; Odermatt, Bernhard; Kelm, Jens; Breymann, Christian; Gössi, Matthias; Genoni, Michele; Zund, Gregor; Hoerstrup, Simon P

2006-10-01

Tissue-engineered living blood vessels (TEBV) with growth capacity represent a promising new option for the repair of congenital malformations. We investigate the functionality of TEBV with endothelia generated from human umbilical cord blood-derived endothelial progenitor cells. Tissue-engineered living blood vessels were generated from human umbilical cord-derived myofibroblasts seeded on biodegradable vascular scaffolds, followed by endothelialization with differentiated cord blood-derived endothelial progenitor cells. During in vitro maturation the TEBV were exposed to physiologic conditioning in a flow bioreactor. For functional assessment, a subgroup of TEBV was stimulated with tumor necrosis factor-alpha. Control vessels endothelialized with standard vascular endothelial cells were treated in parallel. Analysis of the TEBV included histology, immunohistochemistry, biochemistry (extracellular matrix analysis, DNA), and biomechanical testing. Endothelia were analyzed by flow cytometry and immunohistochemistry (CD31, von Willebrand factor, thrombomodulin, tissue factor, endothelial nitric oxide synthase). Histologically, a three-layered tissue organization of the TEBV analogous to native vessels was observed, and biochemistry revealed the major matrix constituents (collagen, proteoglycans) of blood vessels. Biomechanical properties (Young's modulus, 2.03 +/- 0.65 MPa) showed profiles resembling those of native tissue. Endothelial progenitor cells expressed typical endothelial cell markers CD31, von Willebrand factor, and endothelial nitric oxide synthase comparable to standard vascular endothelial cells. Stimulation with tumor necrosis factor-alpha resulted in physiologic upregulation of tissue factor and downregulation of thrombomodulin expression. These results indicate that TEBV with tissue architecture and functional endothelia similar to native blood vessels can be successfully generated from human umbilical cord progenitor cells. Thus, blood-derived progenitor cells obtained before or at birth may enable the clinical realization of tissue engineering constructs for pediatric applications.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Biotechnology Specimen Temperature Controller (BSTC) will cultivate cells until their turn in the bioreactor; it can also be used in culturing experiments that do not require the bioreactor. The BSTC comprises four incubation/refrigeration chambers individually set at 4 to 50 deg. C (near-freezing to above body temperature). Each chamber holds three rugged tissue chamber modules (12 total), clear Teflon bags holding 30 ml of growth media, all positioned by a metal frame. Every 7 to 21 days (depending on growth rates), an astronaut uses a shrouded syringe and the bags' needleless injection ports to transfer a few cells to a fresh media bag, and to introduce a fixative so that the cells may be studied after flight. The design also lets the crew sample the media to measure glucose, gas, and pH levels, and to inspect cells with a microscope. The controller is monitored by the flight crew through a 23-cm (9-inch) color computer display on the face of the BSTC. This view shows the BTSC with the front panel open. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Biotechnology Specimen Temperature Controller (BSTC) will cultivate cells until their turn in the bioreactor; it can also be used in culturing experiments that do not require the bioreactor. The BSTC comprises four incubation/refrigeration chambers individually set at 4 to 50 degreesC (near-freezing to above body temperature). Each chamber holds three rugged tissue chamber modules (12 total), clear Teflon bags holding 30 ml of growth media, all positioned by a metal frame. Every 7 to 21 days (depending on growth rates), an astronaut uses a shrouded syringe and the bags' needleless injection ports to transfer a few cells to a fresh media bag, and to introduce a fixative so that the cells may be studied after flight. The design also lets the crew sample the media to measure glucose, gas, and pH levels, and to inspect cells with a microscope. The controller is monitored by the flight crew through a 23-cm (9-inch) color computer display on the face of the BSTC. This view shows the BTSC with the front panel open. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Tissue-engineered vascular grafts composed of marine collagen and PLGA fibers using pulsatile perfusion bioreactors.

PubMed

Jeong, Sung In; Kim, So Yeon; Cho, Seong Kwan; Chong, Moo Sang; Kim, Kyung Soo; Kim, Hyuck; Lee, Sang Bong; Lee, Young Moo

2007-02-01

Novel tubular scaffolds of marine source collagen and PLGA fibers were fabricated by freeze drying and electrospinning processes for vascular grafts. The hybrid scaffolds, composed of a porous collagen matrix and a fibrous PLGA layer, had an average pore size of 150+/-50 microm. The electrospun fibrous PLGA layer on the surface of a porous tubular collagen scaffold improved the mechanical strength of the collagen scaffolds in both the dry and wet states. Smooth muscle cells (SMCs)- and endothelial cells (ECs)-cultured collagen/PLGA scaffolds exhibited mechanical properties similar to collagen/PLGA scaffolds unseeded with cells, even after culturing for 23 days. The effect of a mechanical stimulation on the proliferation and phenotype of SMCs and ECs, cultured on collagen/PLGA scaffolds, was evaluated. The pulsatile perfusion system enhanced the SMCs and ECs proliferation. In addition, a significant cell alignment in a direction radial to the distending direction was observed in tissues exposed to radial distention, which is similar to the phenomenon of native vessel tissues in vivo. On the other hand, cells in tissues engineered in the static condition were randomly aligned. Immunochemical analyses showed that the expressions of SM alpha-actin, SM myosin heavy chain, EC von Willebrand factor, and EC nitric oxide were upregulated in tissues engineered under a mechano-active condition, compared to vessel tissues engineered in the static condition. These results indicated that the co-culturing of SMCs and ECs, using collagen/PLGA hybrid scaffolds under a pulsatile perfusion system, leads to the enhancement of vascular EC development, as well as the retention of the differentiated cell phenotype.

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

Dr. Harry Mahtani analyzes the gas content of nutrient media from Bioreactor used in research on human breast cancer. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunourous tissues.

Video of Tissue Grown in Space in NASA Bioreactor

NASA Technical Reports Server (NTRS)

2003-01-01

Principal investigator Leland Chung grew prostate cancer and bone stromal cells aboard the Space Shuttle Columbia during the STS-107 mission. Although the experiment samples were lost along with the ill-fated spacecraft and crew, he did obtain downlinked video of the experiment that indicates the enormous potential of growing tissues in microgravity. Cells grown aboard Columbia had grown far larger tissue aggregates at day 5 than did the cells grown in a NASA bioreactor on the ground.

Scaling down of a clinical three-dimensional perfusion multicompartment hollow fiber liver bioreactor developed for extracorporeal liver support to an analytical scale device useful for hepatic pharmacological in vitro studies.

PubMed

Zeilinger, Katrin; Schreiter, Thomas; Darnell, Malin; Söderdahl, Therese; Lübberstedt, Marc; Dillner, Birgitta; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B

2011-05-01

Within the scope of developing an in vitro culture model for pharmacological research on human liver functions, a three-dimensional multicompartment hollow fiber bioreactor proven to function as a clinical extracorporeal liver support system was scaled down in two steps from 800 mL to 8 mL and 2 mL bioreactors. Primary human liver cells cultured over 14 days in 800, 8, or 2 mL bioreactors exhibited comparable time-course profiles for most of the metabolic parameters in the different bioreactor size variants. Major drug-metabolizing cytochrome P450 activities analyzed in the 2 mL bioreactor were preserved over up to 23 days. Immunohistochemical studies revealed tissue-like structures of parenchymal and nonparenchymal cells in the miniaturized bioreactor, indicating physiological reorganization of the cells. Moreover, the canalicular transporters multidrug-resistance-associated protein 2, multidrug-resistance protein 1 (P-glycoprotein), and breast cancer resistance protein showed a similar distribution pattern to that found in human liver tissue. In conclusion, the down-scaled multicompartment hollow fiber technology allows stable maintenance of primary human liver cells and provides an innovative tool for pharmacological and kinetic studies of hepatic functions with small cell numbers.

Long term organ culture of human prostate tissue in a NASA-designed rotating wall bioreactor

NASA Technical Reports Server (NTRS)

Margolis, L.; Hatfill, S.; Chuaqui, R.; Vocke, C.; Emmert-Buck, M.; Linehan, W. M.; Duray, P. H.

1999-01-01

PURPOSE: To maintain ex vivo integral prostatic tissue including intact stromal and ductal elements using the NASA-designed Rotating Wall Vessel (RWV) which maintains colocalized cells in an environment that promotes both three-dimensional cellular interactions together with the uniform mass transfer of nutrients and metabolic wastes. MATERIALS AND METHODS: Samples of normal prostate were obtained as a byproduct of transurethral prostatectomy or needle biopsy. Prostatic tissue dissected into small 1 x 1 mm. blocks was cultured in the Rotating Wall Vessel (RWV) Bioreactor for various time periods and analyzed using histological, immunochemical, and total cell RNA assays. RESULTS: We report the long term maintenance of benign explanted human prostate tissue grown in simple culture medium, under the simulated microgravity conditions afforded by the RWV bioreactor. Mesenchymal stromal elements including blood vessels and architecturally preserved tubuloglandular acini were maintained for a minimum of 28 days. Cytokeratins, vimentin and TGF-beta2 receptor and ligand were preserved through the entire culture period as revealed by immunocytochemistry. Prostatic acid phosphatase (PAP) was continuously expressed during the culture period, although somewhat decreased. Prostatic specific antigen (PSA) and its transcript were down regulated over time of culture. Prostatic carcinoma cells from the TSU cell line were able to invade RWV-cultured benign prostate tissue explants. CONCLUSIONS: The RWV bioreactor represents an additional new technology for culturing prostate tissue for further investigations concerning the basic physiology and pathobiology of this clinically important tissue.

Tissue Engineering-based Therapeutic Strategies for Vocal Fold Repair and Regeneration

PubMed Central

Li, Linqing; Stiadle, Jeanna M.; Lau, Hang K.; Zerdoum, Aidan B.; Jia, Xinqiao; L.Thibeault, Susan; Kiick, Kristi L.

2016-01-01

Vocal folds are soft laryngeal connective tissues with distinct layered structures and complex multicomponent matrix compositions that endow phonatory and respiratory functions. This delicate tissue is easily damaged by various environmental factors and pathological conditions, altering vocal biomechanics and causing debilitating vocal disorders that detrimentally affect the daily lives of suffering individuals. Modern techniques and advanced knowledge of regenerative medicine have led to a deeper understanding of the microstructure, microphysiology, and micropathophysiology of vocal fold tissues. State-of-the-art materials ranging from extracecullar-matrix (ECM)-derived biomaterials to synthetic polymer scaffolds have been proposed for the prevention and treatment of voice disorders including vocal fold scarring and fibrosis. This review intends to provide a thorough overview of current achievements in the field of vocal fold tissue engineering, including the fabrication of injectable biomaterials to mimic in vitro cell microenvironments, novel designs of bioreactors that capture in vivo tissue biomechanics, and establishment of various animal models to characterize the in vivo biocompatibility of these materials. The combination of polymeric scaffolds, cell transplantation, biomechanical stimulation, and delivery of antifibrotic growth factors will lead to successful restoration of functional vocal folds and improved vocal recovery in animal models, facilitating the application of these materials and related methodologies in clinical practice. PMID:27619243

Effects of flow configuration on bone tissue engineering using human mesenchymal stem cells in 3D chitosan composite scaffolds.

PubMed

Sellgren, Katelyn L; Ma, Teng

2015-08-01

Perfusion bioreactor plays important role in supporting 3D bone construct development. Scaffolds of chitosan composites have been studied to support bone tissue regeneration from osteogenic progenitor cells including human mesenchymal stem cells (hMSC). In this study, porous scaffolds of hydroxyapatite (H), chitosan (C), and gelatin (G) were fabricated by phase-separation and press-fitted in the perfusion bioreactor system where media flow is configured either parallel or transverse with respect to the scaffolds to investigate the impact of flow configuration on hMSC proliferation and osteogenic differentiation. The in vitro results showed that the interstitial flow in the transverse flow (TF) constructs reduced cell growth during the first week of culture but improved spatial cell distribution and early onset of osteogenic differentiation measured by alkaline phosphatase and expression of osteogenic genes. After 14 days of bioreactor culture, the TF constructs have comparable cell number but higher expression of bone markers genes and proteins compared to the parallel flow constructs. To evaluate ectopic bone formation, the HCG constructs seeded with hMSCs pre-cultured under two flow configurations for 7 days were implanted in CD-1 nude mice. While Masson's Trichrom staining revealed bone formation in both constructs, the TF constructs have improved spatial cell and osteoid distribution throughout the 2.0 mm constructs. The results highlight the divergent effects of media flow over the course of construct development and suggest that the flow configuration is an important parameter regulating the cellular events leading to bone construct formation in the HCG scaffolds. © 2014 Wiley Periodicals, Inc.

Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI-TOF-MS.

PubMed

Karst, Daniel J; Steinhoff, Robert F; Kopp, Marie R G; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

2017-11-01

The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13 C 6 glucose containing feed media during constant operation at 20 × 10 6 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13 C 6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13 C 6 glucose in the bioreactor and thus the intracellular 13 C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017. © 2017 American Institute of Chemical Engineers.

Microgravity

NASA Image and Video Library

1998-10-10

Human primary breast tumor cells after 49 days of growth in a NASA Bioreactor. Tumor cells aggregate on microcarrier beads (indicated by arrow). NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida

Channeled Scaffolds for Engineering Myocardium with Mechanical Stimulation

PubMed Central

Zhang, Ting; Wan, Leo Q.; Xiong, Zhuo; Marsano, Anna; Maidhof, Robert; Park, Miri; Yan, Yongnian; Vunjak-Novakovic, Gordana

2011-01-01

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) are critically important for engineering functional myocardial tissue. We report the development of chitosan-collagen scaffolds with micro-pores and an array of parallel channels (~200 μm in diameter) that were specifically designed for cardiac tissue engineering with mechanical stimulation. The scaffolds were designed to have the structural and mechanical properties similar to those of the native human heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom-designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1–2 mm thick) consisted of metabolically active cells and started to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stresses promoted cell alignment, elongation, and the expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs. PMID:22081518

Channelled scaffolds for engineering myocardium with mechanical stimulation.

PubMed

Zhang, Ting; Wan, Leo Q; Xiong, Zhuo; Marsano, Anna; Maidhof, Robert; Park, Miri; Yan, Yongnian; Vunjak-Novakovic, Gordana

2012-10-01

The characteristics of the matrix (composition, structure, mechanical properties) and external culture environment (pulsatile perfusion, physical stimulation) of the heart are important characteristics in the engineering of functional myocardial tissue. This study reports on the development of chitosan-collagen scaffolds with micropores and an array of parallel channels (~ 200 µm in diameter) that were specifically designed for cardiac tissue engineering using mechanical stimulation. The scaffolds were designed to have similar structural and mechanical properties of those of native heart matrix. Scaffolds were seeded with neonatal rat heart cells and subjected to dynamic tensile stretch using a custom designed bioreactor. The channels enhanced oxygen transport and facilitated the establishment of cell connections within the construct. The myocardial patches (14 mm in diameter, 1-2 mm thick) consisted of metabolically active cells that began to contract synchronously after 3 days of culture. Mechanical stimulation with high tensile stress promoted cell alignment, elongation, and expression of connexin-43 (Cx-43). This study confirms the importance of scaffold design and mechanical stimulation for the formation of contractile cardiac constructs. Copyright © 2011 John Wiley & Sons, Ltd.

Isolation and Expansion of Hepatic Stem-like Cells from a Healthy Rat Liver and their Efficient Hepatic Differentiation of under Well-defined Vivo Hepatic like Microenvironment in a Multiwell Bioreactor

PubMed Central

Giri, Shibashish; Acikgöz, Ali; Bader, Augustinus

2015-01-01

Background Currently, undifferentiated cells are found in all tissue and term as local stem cells which are quiescent in nature and less in number under normal healthy conditions but activate upon injury and repair the tissue or organs via automated activating mechanism. Due to very scanty presence of local resident somatic local stem cells in healthy organs, isolation and expansion of these adult stems is an immense challenge for medical research and cell based therapy. Particularly organ like liver, there is an ongoing controversy about existence of liver stem cells. Methods Herein, Hepatic stem cells population was identified during culture of primary hepatocyte cells upon immediate isolation of primary hepatocyte cells. These liver stem cells has been expanded extensively and differentiated into primary hepatocytes under defined culture conditions in a nanostructured self assembling peptides modular bioreactor that mimic the state of art of liver microenvironment and compared with Matrigel as a positive control. Nanostructured self assembling peptides were used a defined extracellular matrix and Matrigel was used for undefined extracellular matrix. Proliferation of hepatic stem cells was investigated by two strategies. First strategy is to provide high concentration of hepatocyte growth factor (HGF) and second strategy is to evaluate the role of recombinant human erythropoietin (rHuEPO) in presence of trauma/ischemia cytokines (IL-6, TNF-α). Expansion to hepatic differentiation is observed by morphological analysis and was evaluated for the expression of hepatocyte-specific genes using RT-PCR and biochemical methods. Results Hepatocyte-specific genes are well expressed at final stage (day 21) of differentiation period. The differentiated hepatocytes exhibited functional hepatic characteristics such as albumin secretion, urea secretion and cytochrome P450 expression. Additionally, immunofluorescence analysis revealed that hepatic stem cells derived hepatocytes exhibited mature hepatocyte markers (albumin, CK-19, CPY3A1, alpha 1-antitrypsin). Expansion and hepatic differentiation was efficiently in nanostructured self assembling peptides without such batch to batch variation while there was much variation in Matrigel coated bioreactor. In conclusion, the results of the study suggest that the nanostructured self assembling peptides coated bioreactor supports expansion as well as hepatic differentiation of liver stem cells which is superior than Matrigel. Conclusion This defined microenvironment conditions in bioreactor module can be useful for research involving bioartificial liver system, stem cell research and engineered liver tissue which could contribute to regenerative cell therapies or drug discovery and development. PMID:26155038

Integration of technologies for hepatic tissue engineering.

PubMed

Nahmias, Yaakov; Berthiaume, Francois; Yarmush, Martin L

2007-01-01

The liver is the largest internal organ in the body, responsible for over 500 metabolic, regulatory, and immune functions. Loss of liver function leads to liver failure which causes over 25,000 deaths/year in the United States. Efforts in the field of hepatic tissue engineering include the design of bioartificial liver systems to prolong patient's lives during liver failure, for drug toxicity screening and for the study of liver regeneration, ischemia/reperfusion injury, fibrosis, viral infection, and inflammation. This chapter will overview the current state-of-the-art in hepatology including isolated perfused liver, culture of liver slices and tissue explants, hepatocyte culture on collagen "sandwich" and spheroids, coculture of hepatocytes with non-parenchymal cells, and the integration of these culture techniques with microfluidics and reactor design. This work will discuss the role of oxygen and medium composition in hepatocyte culture and present promising new technologies for hepatocyte proliferation and function. We will also discuss liver development, architecture, and function as they relate to these culture techniques. Finally, we will review current opportunities and major challenges in integrating cell culture, bioreactor design, and microtechnology to develop new systems for novel applications.

A comparison of orbitally-shaken and stirred-tank bioreactors: pH modulation and bioreactor type affect CHO cell growth and protein glycosylation.

PubMed

Monteil, Dominique T; Juvet, Valentin; Paz, Jonathan; Moniatte, Marc; Baldi, Lucia; Hacker, David L; Wurm, Florian M

2016-09-01

Orbitally shaken bioreactors (OSRs) support the suspension cultivation of animal cells at volumetric scales up to 200 L and are a potential alternative to stirred-tank bioreactors (STRs) due to their rapid and homogeneous mixing and high oxygen transfer rate. In this study, a Chinese hamster ovary cell line producing a recombinant antibody was cultivated in a 5 L OSR and a 3 L STR, both operated with or without pH control. Effects of bioreactor type and pH control on cell growth and metabolism and on recombinant protein production and glycosylation were determined. In pH-controlled bioreactors, the glucose consumption and lactate production rates were higher relative to cultures grown in bioreactors without pH control. The cell density and viability were higher in the OSRs than in the STRs, either with or without pH control. Volumetric recombinant antibody yields were not affected by the process conditions, and a glycan analysis of the antibody by mass spectrometry did not reveal major process-dependent differences in the galactosylation index. The results demonstrated that OSRs are suitable for recombinant protein production from suspension-adapted animal cells. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1174-1180, 2016. © 2016 American Institute of Chemical Engineers.

Bioethanol production from uncooked raw starch by immobilized surface-engineered yeast cells.

PubMed

Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

2008-03-01

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis alpha-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.

Bioethanol Production from Uncooked Raw Starch by Immobilized Surface-engineered Yeast Cells

NASA Astrophysics Data System (ADS)

Chen, Jyh-Ping; Wu, Kuo-Wei; Fukuda, Hideki

Surface-engineered yeast Saccharomyces cerevisiae codisplaying Rhizopus oryzae glucoamylase and Streptococcus bovis α-amylase on the cell surface was used for direct production of ethanol from uncooked raw starch. By using 50 g/L cells during batch fermentation, ethanol concentration could reach 53 g/L in 7 days. During repeated batch fermentation, the production of ethanol could be maintained for seven consecutive cycles. For cells immobilized in loofa sponge, the concentration of ethanol could reach 42 g/L in 3 days in a circulating packed-bed bioreactor. However, the production of ethanol stopped thereafter because of limited contact between cells and starch. The bioreactor could be operated for repeated batch production of ethanol, but ethanol concentration dropped to 55% of its initial value after five cycles because of a decrease in cell mass and cell viability in the bioreactor. Adding cells to the bioreactor could partially restore ethanol production to 75% of its initial value.

Dynamic 3D culture promotes spontaneous embryonic stem cell differentiation in vitro.

PubMed

Gerlach, Jörg C; Hout, Mariah; Edsbagge, Josefina; Björquist, Petter; Lübberstedt, Marc; Miki, Toshio; Stachelscheid, Harald; Schmelzer, Eva; Schatten, Gerald; Zeilinger, Katrin

2010-02-01

Spontaneous in vitro differentiation of mouse embryonic stem cells (mESC) is promoted by a dynamic, three-dimensional (3D), tissue-density perfusion technique with continuous medium perfusion and exchange in a novel four-compartment, interwoven capillary bioreactor. We compared ectodermal, endodermal, and mesodermal immunoreactive tissue structures formed by mESC at culture day 10 with mouse fetal tissue development at gestational day E9.5. The results show that the bioreactor cultures more closely resemble mouse fetal tissue development at gestational day E9.5 than control mESC cultured in Petri dishes.

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells.

PubMed

Freeman, Cassandra A; Samuel, Premsingh S D; Kompala, Dhinakar S

2017-07-01

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent-pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month-long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed-batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913-922, 2017. © 2017 American Institute of Chemical Engineers.

CASKAD. Manual Mixing in Bioreactor

NASA Image and Video Library

2013-10-05

ISS037-E-005692 (5 Oct. 2013) --- Russian cosmonaut Sergey Ryazanskiy, Expedition 37 flight engineer, prepares to manually mix samples in a Bioreactor for the CASKAD experiment in the Poisk Mini-Research Module 2 (MRM2) of the International Space Station.

CASKAD. Manual Mixing in Bioreactor

NASA Image and Video Library

2013-10-05

ISS037-E-005694 (5 Oct. 2013) --- Russian cosmonaut Sergey Ryazanskiy, Expedition 37 flight engineer, prepares to manually mix samples in a Bioreactor for the CASKAD experiment in the Poisk Mini-Research Module 2 (MRM2) of the International Space Station.

Tissue grown in NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Cells from kidneys lose some of their special features in conventional culture but form spheres replete with specialized cell microvilli (hair) and synthesize hormones that may be clinically useful. Ground-based research studies have demonstrated that both normal and neoplastic cells and tissues recreate many of the characteristics in the NASA bioreactor that they display in vivo. Proximal kidney tubule cells that normally have rich apically oriented microvilli with intercellular clefts in the kidney do not form any of these structures in conventional two-dimensional monolayer culture. However, when normal proximal renal tubule cells are cultured in three-dimensions in the bioreactor, both the microvilli and the intercellular clefts form. This is important because, when the morphology is recreated, the function is more likely also to be rejuvenated. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC).

TexMi: Development of Tissue-Engineered Textile-Reinforced Mitral Valve Prosthesis

PubMed Central

Moreira, Ricardo; Gesche, Valentine N.; Hurtado-Aguilar, Luis G.; Schmitz-Rode, Thomas; Frese, Julia

2014-01-01

Mitral valve regurgitation together with aortic stenosis is the most common valvular heart disease in Europe and North America. Mechanical and biological prostheses available for mitral valve replacement have significant limitations such as the need of a long-term anticoagulation therapy and failure by calcifications. Both types are unable to remodel, self-repair, and adapt to the changing hemodynamic conditions. Moreover, they are mostly designed for the aortic position and do not reproduce the native annular-ventricular continuity, resulting in suboptimal hemodynamics, limited durability, and gradually decreasing ventricular pumping efficiency. A tissue-engineered heart valve specifically designed for the mitral position has the potential to overcome the limitations of the commercially available substitutes. For this purpose, we developed the TexMi, a living textile-reinforced mitral valve, which recapitulates the key elements of the native one: annulus, asymmetric leaflets (anterior and posterior), and chordae tendineae to maintain the native annular-ventricular continuity. The tissue-engineered valve is based on a composite scaffold consisting of the fibrin gel as a cell carrier and a textile tubular structure with the twofold task of defining the gross three-dimensional (3D) geometry of the valve and conferring mechanical stability. The TexMi valves were molded with ovine umbilical vein cells and stimulated under dynamic conditions for 21 days in a custom-made bioreactor. Histological and immunohistological stainings showed remarkable tissue development with abundant aligned collagen fibers and elastin deposition. No cell-mediated tissue contraction occurred. This study presents the proof-of-principle for the realization of a tissue-engineered mitral valve with a simple and reliable injection molding process readily adaptable to the patient's anatomy and pathological situation by producing a patient-specific rapid prototyped mold. PMID:24665896

Quantitative Validation of the Presto Blue Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System.

PubMed

Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P; Schrooten, Jan Ir

2015-06-01

As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required.

Quantitative Validation of the Presto Blue™ Metabolic Assay for Online Monitoring of Cell Proliferation in a 3D Perfusion Bioreactor System

PubMed Central

Sonnaert, Maarten; Papantoniou, Ioannis; Luyten, Frank P.

2015-01-01

As the fields of tissue engineering and regenerative medicine mature toward clinical applications, the need for online monitoring both for quantitative and qualitative use becomes essential. Resazurin-based metabolic assays are frequently applied for determining cytotoxicity and have shown great potential for monitoring 3D bioreactor-facilitated cell culture. However, no quantitative correlation between the metabolic conversion rate of resazurin and cell number has been defined yet. In this work, we determined conversion rates of Presto Blue™, a resazurin-based metabolic assay, for human periosteal cells during 2D and 3D static and 3D perfusion cultures. Our results showed that for the evaluated culture systems there is a quantitative correlation between the Presto Blue conversion rate and the cell number during the expansion phase with no influence of the perfusion-related parameters, that is, flow rate and shear stress. The correlation between the cell number and Presto Blue conversion subsequently enabled the definition of operating windows for optimal signal readouts. In conclusion, our data showed that the conversion of the resazurin-based Presto Blue metabolic assay can be used as a quantitative readout for online monitoring of cell proliferation in a 3D perfusion bioreactor system, although a system-specific validation is required. PMID:25336207

The Application of Ultrasound in 3D Bio-Printing.

PubMed

Zhou, Yufeng

2016-05-05

Three-dimensional (3D) bioprinting is an emerging and promising technology in tissue engineering to construct tissues and organs for implantation. Alignment of self-assembly cell spheroids that are used as bioink could be very accurate after droplet ejection from bioprinter. Complex and heterogeneous tissue structures could be built using rapid additive manufacture technology and multiple cell lines. Effective vascularization in the engineered tissue samples is critical in any clinical application. In this review paper, the current technologies and processing steps (such as printing, preparation of bioink, cross-linking, tissue fusion and maturation) in 3D bio-printing are introduced, and their specifications are compared with each other. In addition, the application of ultrasound in this novel field is also introduced. Cells experience acoustic radiation force in ultrasound standing wave field (USWF) and then accumulate at the pressure node at low acoustic pressure. Formation of cell spheroids by this method is within minutes with uniform size and homogeneous cell distribution. Neovessel formation from USWF-induced endothelial cell spheroids is significant. Low-intensity ultrasound could enhance the proliferation and differentiation of stem cells. Its use is at low cost and compatible with current bioreactor. In summary, ultrasound application in 3D bio-printing may solve some challenges and enhance the outcomes.

Microgravity

NASA Image and Video Library

1998-01-01

Biotechnology Specimen Temperature Controller (BSTC) will cultivate cells until their turn in the bioreactor; it can also be used in culturing experiments that do not require the bioreactor. The BSTC comprises four incubation/refrigeration chambers individually set at 4 to 50 deg. C (near-freezing to above body temperature). Each chamber holds three rugged tissue chamber modules (12 total), clear Teflon bags holding 30 ml of growth media, all positioned by a metal frame. Every 7 to 21 days (depending on growth rates), an astronaut uses a shrouded syringe and the bags' needleless injection ports to transfer a few cells to a fresh media bag, and to introduce a fixative so that the cells may be studied after flight. The design also lets the crew sample the media to measure glucose, gas, and pH levels, and to inspect cells with a microscope. The controller is monitored by the flight crew through a 23-cm (9-inch) color computer display on the face of the BSTC. This view shows the BTSC with the front panel open. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Microgravity

NASA Image and Video Library

1998-01-01

Biotechnology Specimen Temperature Controller (BSTC) will cultivate cells until their turn in the bioreactor; it can also be used in culturing experiments that do not require the bioreactor. The BSTC comprises four incubation/refrigeration chambers individually set at 4 to 50 degreesC (near-freezing to above body temperature). Each chamber holds three rugged tissue chamber modules (12 total), clear Teflon bags holding 30 ml of growth media, all positioned by a metal frame. Every 7 to 21 days (depending on growth rates), an astronaut uses a shrouded syringe and the bags' needleless injection ports to transfer a few cells to a fresh media bag, and to introduce a fixative so that the cells may be studied after flight. The design also lets the crew sample the media to measure glucose, gas, and pH levels, and to inspect cells with a microscope. The controller is monitored by the flight crew through a 23-cm (9-inch) color computer display on the face of the BSTC. This view shows the BTSC with the front panel open. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators.

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

Human primary breast tumor cells after 49 days of growth in a NASA Bioreactor. Tumor cells aggregate on microcarrier beads (indicated by arrow). NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida

Additively Manufactured Device for Dynamic Culture of Large Arrays of 3D Tissue Engineered Constructs.

PubMed

Costa, Pedro F; Hutmacher, Dietmar W; Theodoropoulos, Christina; Gomes, Manuela E; Reis, Rui L; Vaquette, Cédryck

2015-04-22

The ability to test large arrays of cell and biomaterial combinations in 3D environments is still rather limited in the context of tissue engineering and regenerative medicine. This limitation can be generally addressed by employing highly automated and reproducible methodologies. This study reports on the development of a highly versatile and upscalable method based on additive manufacturing for the fabrication of arrays of scaffolds, which are enclosed into individualized perfusion chambers. Devices containing eight scaffolds and their corresponding bioreactor chambers are simultaneously fabricated utilizing a dual extrusion additive manufacturing system. To demonstrate the versatility of the concept, the scaffolds, while enclosed into the device, are subsequently surface-coated with a biomimetic calcium phosphate layer by perfusion with simulated body fluid solution. 96 scaffolds are simultaneously seeded and cultured with human osteoblasts under highly controlled bidirectional perfusion dynamic conditions over 4 weeks. Both coated and noncoated resulting scaffolds show homogeneous cell distribution and high cell viability throughout the 4 weeks culture period and CaP-coated scaffolds result in a significantly increased cell number. The methodology developed in this work exemplifies the applicability of additive manufacturing as a tool for further automation of studies in the field of tissue engineering and regenerative medicine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Continuous Digital Light Processing (cDLP): Highly Accurate Additive Manufacturing of Tissue Engineered Bone Scaffolds.

PubMed

Dean, David; Jonathan, Wallace; Siblani, Ali; Wang, Martha O; Kim, Kyobum; Mikos, Antonios G; Fisher, John P

2012-03-01

Highly accurate rendering of the external and internal geometry of bone tissue engineering scaffolds effects fit at the defect site, loading of internal pore spaces with cells, bioreactor-delivered nutrient and growth factor circulation, and scaffold resorption. It may be necessary to render resorbable polymer scaffolds with 50 μm or less accuracy to achieve these goals. This level of accuracy is available using Continuous Digital Light processing (cDLP) which utilizes a DLP(®) (Texas Instruments, Dallas, TX) chip. One such additive manufacturing device is the envisionTEC (Ferndale, MI) Perfactory(®). To use cDLP we integrate a photo-crosslinkable polymer, a photo-initiator, and a biocompatible dye. The dye attenuates light, thereby limiting the depth of polymerization. In this study we fabricated scaffolds using the well-studied resorbable polymer, poly(propylene fumarate) (PPF), titanium dioxide (TiO(2)) as a dye, Irgacure(®) 819 (BASF [Ciba], Florham Park, NJ) as an initiator, and diethyl fumarate as a solvent to control viscosity.

Sensing in tissue bioreactors

NASA Astrophysics Data System (ADS)

Rolfe, P.

2006-03-01

Specialized sensing and measurement instruments are under development to aid the controlled culture of cells in bioreactors for the fabrication of biological tissues. Precisely defined physical and chemical conditions are needed for the correct culture of the many cell-tissue types now being studied, including chondrocytes (cartilage), vascular endothelial cells and smooth muscle cells (blood vessels), fibroblasts, hepatocytes (liver) and receptor neurones. Cell and tissue culture processes are dynamic and therefore, optimal control requires monitoring of the key process variables. Chemical and physical sensing is approached in this paper with the aim of enabling automatic optimal control, based on classical cell growth models, to be achieved. Non-invasive sensing is performed via the bioreactor wall, invasive sensing with probes placed inside the cell culture chamber and indirect monitoring using analysis within a shunt or a sampling chamber. Electroanalytical and photonics-based systems are described. Chemical sensing for gases, ions, metabolites, certain hormones and proteins, is under development. Spectroscopic analysis of the culture medium is used for measurement of glucose and for proteins that are markers of cell biosynthetic behaviour. Optical interrogation of cells and tissues is also investigated for structural analysis based on scatter.

Tenogenesis of bone marrow-, adipose-, and tendon-derived stem cells in a dynamic bioreactor.

PubMed

Youngstrom, Daniel W; LaDow, Jade E; Barrett, Jennifer G

2016-11-01

Tendons are frequently damaged and fail to regenerate, leading to pain, loss of function, and reduced quality of life. Mesenchymal stem cells (MSCs) possess clinically useful tissue-regenerative properties and have been exploited for use in tendon tissue engineering and cell therapy. However, MSCs exhibit phenotypic heterogeneity based on the donor tissue used, and the efficacy of cell-based treatment modalities may be improved by optimizing cell source based on relative differentiation capacity. Equine MSCs were isolated from bone marrow (BM), adipose (AD), and tendon (TN), expanded in monolayer prior to seeding on decellularized tendon scaffolds (DTS), and cell-laden constructs were placed in a bioreactor designed to mimic the biophysical environment of the tendon. It was hypothesized that TN MSCs would differentiate toward a tendon cell phenotype better than BM and AD MSCs in response to a conditioning period involving cyclic mechanical stimulation for 1 hour per day at 3% strain and 0.33 Hz. All cell types integrated into DTS adopted an elongated morphology similar to tenocytes, expressed tendon marker genes, and improved tissue mechanical properties after 11 days. TN MSCs expressed the greatest levels of scleraxis, collagen type-I, and cartilage oligomeric matrix protein. Major histocompatibility class-II protein mRNA expression was not detected in any of the MSC types, suggesting low immunogenicity for allogeneic transplantation. The results suggest that TN MSCs are the ideal cell type for regenerative medicine therapies for tendinopathies, exhibiting the most mature tendon-like phenotype in vitro. When TN MSCs are unavailable, BM or AD MSCs may serve as robust alternatives.

Three Dimensional Primary Hepatocyte Culture

NASA Technical Reports Server (NTRS)

Yoffe, Boris

1998-01-01

Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

Towards the feasibility of using ultrasound to determine mechanical properties of tissues in a bioreactor.

PubMed

Mansour, Joseph M; Gu, Di-Win Marine; Chung, Chen-Yuan; Heebner, Joseph; Althans, Jake; Abdalian, Sarah; Schluchter, Mark D; Liu, Yiying; Welter, Jean F

2014-10-01

Our ultimate goal is to non-destructively evaluate mechanical properties of tissue-engineered (TE) cartilage using ultrasound (US). We used agarose gels as surrogates for TE cartilage. Previously, we showed that mechanical properties measured using conventional methods were related to those measured using US, which suggested a way to non-destructively predict mechanical properties of samples with known volume fractions. In this study, we sought to determine whether the mechanical properties of samples, with unknown volume fractions could be predicted by US. Aggregate moduli were calculated for hydrogels as a function of SOS, based on concentration and density using a poroelastic model. The data were used to train a statistical model, which we then used to predict volume fractions and mechanical properties of unknown samples. Young's and storage moduli were measured mechanically. The statistical model generally predicted the Young's moduli in compression to within <10% of their mechanically measured value. We defined positive linear correlations between the aggregate modulus predicted from US and both the storage and Young's moduli determined from mechanical tests. Mechanical properties of hydrogels with unknown volume fractions can be predicted successfully from US measurements. This method has the potential to predict mechanical properties of TE cartilage non-destructively in a bioreactor.

Towards the feasibility of using ultrasound to determine mechanical properties of tissues in a bioreactor

PubMed Central

Mansour, Joseph M.; Gu, Di-Win Marine; Chung, Chen-Yuan; Heebner, Joseph; Althans, Jake; Abdalian, Sarah; Schluchter, Mark D.; Liu, Yiying; Welter, Jean F.

2016-01-01

Introduction Our ultimate goal is to non-destructively evaluate mechanical properties of tissue-engineered (TE) cartilage using ultrasound (US). We used agarose gels as surrogates for TE cartilage. Previously, we showed that mechanical properties measured using conventional methods were related to those measured using US, which suggested a way to non-destructively predict mechanical properties of samples with known volume fractions. In this study, we sought to determine whether the mechanical properties of samples, with unknown volume fractions could be predicted by US. Methods Aggregate moduli were calculated for hydrogels as a function of SOS, based on concentration and density using a poroelastic model. The data were used to train a statistical model, which we then used to predict volume fractions and mechanical properties of unknown samples. Young's and storage moduli were measured mechanically. Results The statistical model generally predicted the Young's moduli in compression to within < 10% of their mechanically measured value. We defined positive linear correlations between the aggregate modulus predicted from US and both the storage and Young's moduli determined from mechanical tests. Conclusions Mechanical properties of hydrogels with unknown volume fractions can be predicted successfully from US measurements. This method has the potential to predict mechanical properties of TE cartilage non-destructively in a bioreactor. PMID:25092421

MODULAR FIELD-BIOREACTOR FOR ACID MINE DRAINAGE TREATMENT

EPA Science Inventory

The presentation focuses on the improvements to engineered features of a passive technology that has been used for remediation of acid rock drainage (ARD). This passive remedial technology, a sulfate-reducing bacteria (SRB) bioreactor, takes advantage of the ability of SRB that,...

Microgravity

NASA Image and Video Library

1998-01-01

Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues have reported that initially disc-like specimens of cartilage tend to become spherical in space, demonstrating that tissues can grow and differentiate into distinct structures in microgravity. The Mir Increment 3 (Sept. 16, 1996 - Jan. 22, 1997) samples were smaller, more spherical, and mechanically weaker than Earth-grown control samples. These results demonstrate the feasibility of microgravity tissue engineering and may have implications for long human space voyages and for treating musculoskeletal disorders on earth. Constructs grown on Mir (A) tended to become more spherical, whereas those grown on Earth (B) maintained their initial disc shape. These findings might be related to differences in cultivation conditions, i.e., videotapes showed that constructs floated freely in microgravity but settled and collided with the rotating vessel wall at 1g (Earth's gravity). In particular, on Mir the constructs were exposed to uniform shear and mass transfer at all surfaces such that the tissue grew equally in all directions, whereas on Earth the settling of discoid constructs tended to align their flat circular areas perpendicular to the direction of motion, increasing shear and mass transfer circumferentially such that the tissue grew preferentially in the radial direction. A and B are full cross sections of constructs from Mir and Earth groups shown at 10-power. C and D are representative areas at the construct surfaces enlarged to 200-power. They are stained red with safranin-O. NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). Photo credit: Proceedings of the National Academy of Sciences.

Microgravity

NASA Image and Video Library

2004-04-15

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Anchorage dependent cells on STS-95 will be grown on beads similar to these cells produced during previous investigations. Recombinant proteins may offer the possibility of reducing or eliminating transplant rejections. Research by Synthecon, Inc. using the BioDyn Bioreactor will focus on the preliminary process for growing a proprietary recombinant protein that can decrease rejection of transplanted tissue. The cells producing this protein are anchorage dependent, meaning that they must attach to something to grow. These cells will be cultured in the bioreactor in a medium containing polymer microbeads. Synthecon hopes that the data from this mission will lead to the development of a commercial protein that will aid in prevention of transplant rejection.

Microgravity

NASA Image and Video Library

2004-04-15

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Anchorage dependent cells on STS-95 will be grown on beads, similar to these cells produced during previous investigations. Recombinant proteins may offer the possibility of reducing or eliminating transplant rejections. Research by Synthecon, Inc. using the BioDyn Bioreactor will focus on the preliminary process for growing a proprietary recombinant protein that can decrease rejection of transplanted tissue. The cells producing this protein are anchorage dependent, meaning that they must attach to something to grow. These cells will be cultured in the bioreactor in a medium containing polymer microbeads. Synthecon hopes that the data from this mission will lead to the development of a commercial protein that will aid in prevention of transplant rejection.

Anchorage Dependent Cells Attached to a Polymer

NASA Technical Reports Server (NTRS)

2004-01-01

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Anchorage dependent cells on STS-95 will be grown on beads similar to these cells produced during previous investigations. Recombinant proteins may offer the possibility of reducing or eliminating transplant rejections. Research by Synthecon, Inc. using the BioDyn Bioreactor will focus on the preliminary process for growing a proprietary recombinant protein that can decrease rejection of transplanted tissue. The cells producing this protein are anchorage dependent, meaning that they must attach to something to grow. These cells will be cultured in the bioreactor in a medium containing polymer microbeads. Synthecon hopes that the data from this mission will lead to the development of a commercial protein that will aid in prevention of transplant rejection.

Anchorage Dependent Cells Attached to a Polymer

NASA Technical Reports Server (NTRS)

2004-01-01

Biomedical research offers hope for a variety of medical problems, from diabetes to the replacement of damaged bone and tissues. Bioreactors, which are used to grow cells and tissue cultures, play a major role in such research and production efforts. Anchorage dependent cells on STS-95 will be grown on beads, similar to these cells produced during previous investigations. Recombinant proteins may offer the possibility of reducing or eliminating transplant rejections. Research by Synthecon, Inc. using the BioDyn Bioreactor will focus on the preliminary process for growing a proprietary recombinant protein that can decrease rejection of transplanted tissue. The cells producing this protein are anchorage dependent, meaning that they must attach to something to grow. These cells will be cultured in the bioreactor in a medium containing polymer microbeads. Synthecon hopes that the data from this mission will lead to the development of a commercial protein that will aid in prevention of transplant rejection.

Osteogenic Performance of Donor-Matched Human Adipose and Bone Marrow Mesenchymal Cells Under Dynamic Culture

PubMed Central

Wu, Wei; Le, Andrew V.; Mendez, Julio J.; Chang, Julie; Niklason, Laura E.

2015-01-01

Adipose-derived mesenchymal cells (ACs) and bone marrow-derived mesenchymal cells (BMCs) have been widely used for bone regeneration and can be seeded on a variety of rigid scaffolds. However, to date, a direct comparison of mesenchymal cells (MC) harvested from different tissues from the same donor and cultured in identical osteogenic conditions has not been investigated. Indeed, it is unclear whether marrow-derived or fat-derived MC possess intrinsic differences in bone-forming capabilities, since within-patient comparisons have not been previously done. This study aims at comparing ACs and BMCs from three donors ranging in age from neonatal to adult. Matched cells from each donor were studied in three distinct bioreactor settings, to determine the best method to create a viable osseous engineered construct. Human ACs and BMCs were isolated from each donor, cultured, and seeded on decellularized porcine bone (DCB) constructs. The constructs were then subjected to either static or dynamic (stirring or perfusion) bioreactor culture conditions for 7–21 days. Afterward, the constructs were analyzed for cell adhesion and distribution and osteogenic differentiation. ACs demonstrated higher seeding efficiency than BMCs. However, static and dynamic culture significantly increased BMCs proliferation more than ACs. In all conditions, BMCs demonstrated stronger osteogenic activity as compared with ACs, through higher alkaline phosphatase activity and gene expression for various bony markers. Conversely, ACs expressed more collagen I, which is a nonspecific matrix molecule in most connective tissues. Overall, dynamic bioreactor culture conditions enhanced osteogenic gene expression in both ACs and BMCs. Scaffolds seeded with BMCs in dynamic stirring culture conditions exhibit the greatest osteogenic proliferation and function in vitro, proving that marrow-derived MC have superior bone-forming potential as compared with adipose-derived cells. PMID:25668104

Engineering-derived approaches for iPSC preparation, expansion, differentiation and applications.

PubMed

Li, Yang; Li, Ling; Chen, Zhi-Nan; Gao, Ge; Yao, Rui; Sun, Wei

2017-07-31

Remarkable achievements have been made since induced pluripotent stem cells (iPSCs) were first introduced in 2006. Compared with non-pluripotent stem cells, iPSC research faces several additional complexities, such as the choice of extracellular matrix proteins, growth and differentiation factors, as well as technical challenges related to self-renewal and directed differentiation. Overcoming these challenges requires the integration of knowledge and technologies from multiple fields including cell biology, biomaterial science, engineering, physics and medicine. Here, engineering-derived iPSC approaches are reviewed according to three aspects of iPSC studies: preparation, expansion, differentiation and applications. Engineering strategies, such as 3D systems establishment, cell-matrix mechanics and the regulation of biophysical and biochemical cues, together with engineering techniques, such as 3D scaffolds, cell microspheres and bioreactors, have been applied to iPSC studies and have generated insightful results and even mini-organs such as retinas, livers and intestines. Specific results are given to demonstrate how these approaches impact iPSC behavior, and related mechanisms are discussed. In addition, cell printing technologies are presented as an advanced engineering-derived approach since they have been applied in both iPSC studies and the construction of diverse tissues and organs. Further development and possible innovations of cell printing technologies are presented in terms of creating complex and functional iPSC-derived living tissues and organs.

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective

PubMed Central

Sonnaert, Maarten; Luyten, Frank P.; Papantoniou, Ioannis

2015-01-01

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion. PMID:26313143

Bioreactor-Based Online Recovery of Human Progenitor Cells with Uncompromised Regenerative Potential: A Bone Tissue Engineering Perspective.

PubMed

Sonnaert, Maarten; Luyten, Frank P; Schrooten, Jan; Papantoniou, Ioannis

2015-01-01

The use of a 3D perfusion culture environment for stem cell expansion has been shown to be beneficial for maintenance of the original cell functionality but due to several system inherent characteristics such as the presence of extracellular matrix, the continued development and implementation of 3D perfusion bioreactor technologies is hampered. Therefore, this study developed a methodology for harvesting a progenitor cell population from a 3D open porous culture surface after expansion in a perfusion bioreactor and performed a functional characterization of the expanded cells. An initial screening showed collagenase to be the most interesting reagent to release the cells from the 3D culture surface as it resulted in high yields without compromising cell viability. Subsequently a Design of Experiment approach was used to obtain optimized 3D harvest conditions by assessing the interplay of flow rate, collagenase concentration and incubation time on the harvest efficiency, viability and single cell fraction. Cells that were recovered with the optimized harvest protocol, by perfusing a 880 U/ml collagenase solution for 7 hours at a flow rate of 4 ml/min, were thereafter functionally analyzed for their characteristics as expanded progenitor cell population. As both the in vitro tri-lineage differentiation capacity and the in vivo bone forming potential were maintained after 3D perfusion bioreactor expansion we concluded that the developed seeding, culture and harvest processes did not significantly compromise the viability and potency of the cells and can contribute to the future development of integrated bioprocesses for stem cell expansion.

A Cost-Effective Culture System for the In Vitro Assembly, Maturation, and Stimulation of Advanced Multilayered Multiculture Tubular Tissue Models.

PubMed

Loy, Caroline; Pezzoli, Daniele; Candiani, Gabriele; Mantovani, Diego

2018-01-01

The development of tubular engineered tissues is a challenging research area aiming to provide tissue substitutes but also in vitro models to test drugs, medical devices, and even to study physiological and pathological processes. In this work, the design, fabrication, and validation of an original cost-effective tubular multilayered-tissue culture system (TMCS) are reported. By exploiting cellularized collagen gel as scaffold, a simple moulding technique and an endothelialization step on a rotating system, TMCS allowed to easily prepare in 48 h, trilayered arterial wall models with finely organized cellular composition and to mature them for 2 weeks without any need of manipulation. Multilayered constructs incorporating different combinations of vascular cells are compared in terms of cell organization and viscoelastic mechanical properties demonstrating that cells always progressively aligned parallel to the longitudinal direction. Also, fibroblast compacted less the collagen matrix and appeared crucial in term of maturation/deposition of elastic extracellular matrix. Preliminary studies under shear stress stimulation upon connection with a flow bioreactor are successfully conducted without damaging the endothelial monolayer. Altogether, the TMCS herein developed, thanks to its versatility and multiple functionalities, holds great promise for vascular tissue engineering applications, but also for other tubular tissues such as trachea or oesophagus. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Three dimensional optic tissue culture and process

NASA Technical Reports Server (NTRS)

Spaulding, Glenn F. (Inventor); Prewett, Tacey L. (Inventor); Goodwin, Thomas J. (Inventor); Francis, Karen M. (Inventor); Cardwell, Delmar R. (Inventor); Oconnor, Kim (Inventor); Fitzgerald, Wendy S. (Inventor); Aten, Laurie A. (Inventor)

1994-01-01

A process for artificially producing three-dimensional optic tissue has been developed. The optic cells are cultured in a bioreactor at low shear conditions. The tissue forms normal, functional tissue organization and extracellular matrix.

Apparatus and methods for manipulation and optimization of biological systems

NASA Technical Reports Server (NTRS)

Sun, Ren (Inventor); Ho, Chih-Ming (Inventor); Wong, Pak Kin (Inventor); Yu, Fuqu (Inventor)

2012-01-01

The invention provides systems and methods for manipulating, e.g., optimizing and controlling, biological systems, e.g., for eliciting a more desired biological response of biological sample, such as a tissue, organ, and/or a cell. In one aspect, systems and methods of the invention operate by efficiently searching through a large parametric space of stimuli and system parameters to manipulate, control, and optimize the response of biological samples sustained in the system, e.g., a bioreactor. In alternative aspects, systems include a device for sustaining cells or tissue samples, one or more actuators for stimulating the samples via biochemical, electromagnetic, thermal, mechanical, and/or optical stimulation, one or more sensors for measuring a biological response signal of the samples resulting from the stimulation of the sample. In one aspect, the systems and methods of the invention use at least one optimization algorithm to modify the actuator's control inputs for stimulation, responsive to the sensor's output of response signals. The compositions and methods of the invention can be used, e.g., to for systems optimization of any biological manufacturing or experimental system, e.g., bioreactors for proteins, e.g., therapeutic proteins, polypeptides or peptides for vaccines, and the like, small molecules (e.g., antibiotics), polysaccharides, lipids, and the like. Another use of the apparatus and methods includes combination drug therapy, e.g. optimal drug cocktail, directed cell proliferations and differentiations, e.g. in tissue engineering, e.g. neural progenitor cells differentiation, and discovery of key parameters in complex biological systems.

A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies.

PubMed

Christoffersson, Jonas; Bergström, Gunnar; Schwanke, Kristin; Kempf, Henning; Zweigerdt, Robert; Mandenius, Carl-Fredrik

2016-01-01

Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here, we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging.

Microgravity

NASA Image and Video Library

1998-10-10

High magnification of view of tumor cells aggregate on microcarrier beads, illustrting breast cells with intercellular boundaires on bead surface and aggregates of cells achieving 3-deminstional growth outward from bead after 56 days of culture in a NASA Bioreactor. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida.

Microgravity

NASA Image and Video Library

1998-10-10

High magnification view of human primary breast tumor cells after 56 days of culture in a NASA Bioreactor. The arrow points to bead surface indicating breast cancer cells (as noted by the staining of tumor cell intermediate filaments). NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida

A Guide for Using Mechanical Stimulation to Enhance Tissue-Engineered Articular Cartilage Properties.

PubMed

Salinas, Evelia Y; Hu, Jerry C; Athanasiou, Kyriacos

2018-04-26

The use of tissue-engineered articular cartilage (TEAC) constructs has the potential to become a powerful treatment option for cartilage lesions resulting from trauma or early stages of pathology. Although fundamental tissue-engineering strategies based on the use of scaffolds, cells, and signals have been developed, techniques that lead to biomimetic AC constructs that can be translated to in vivo use are yet to be fully confirmed. Mechanical stimulation during tissue culture can be an effective strategy to enhance the mechanical, structural, and cellular properties of tissue-engineered constructs toward mimicking those of native AC. This review focuses on the use of mechanical stimulation to attain and enhance the properties of AC constructs needed to translate these implants to the clinic. In vivo, mechanical loading at maximal and supramaximal physiological levels has been shown to be detrimental to AC through the development of degenerative changes. In contrast, multiple studies have revealed that during culture, mechanical stimulation within narrow ranges of magnitude and duration can produce anisotropic, mechanically robust AC constructs with high cellular viability. Significant progress has been made in evaluating a variety of mechanical stimulation techniques on TEAC, either alone or in combination with other stimuli. These advancements include determining and optimizing efficacious loading parameters (e.g., duration and frequency) to yield improvements in construct design criteria, such as collagen II content, compressive stiffness, cell viability, and fiber organization. With the advancement of mechanical stimulation as a potent strategy in AC tissue engineering, a compendium detailing the results achievable by various stimulus regimens would be of great use for researchers in academia and industry. The objective is to list the qualitative and quantitative effects that can be attained when direct compression, hydrostatic pressure, shear, and tensile loading are used to tissue-engineer AC. Our goal is to provide a practical guide to their use and optimization of loading parameters. For each loading condition, we will also present and discuss benefits and limitations of bioreactor configurations that have been used. The intent is for this review to serve as a reference for including mechanical stimulation strategies as part of AC construct culture regimens.

CONSTRUCTION OF MODULAR FIELD-BIOREACTOR FOR ACID MINE DRAINAGE TREATMENT

EPA Science Inventory

The paper focuses on the improvements to engineered features of a passive technology that has been used for remediation of acid rock drainage (ARD). This passive remedial technology, a sulfate-reducing bacteria (SRB) bioreactor, takes advantage of the ability of SRB that, if sup...

Denitrifying woodchip bioreactor and phosphorus filter pairing to minimize pollution swapping

USDA-ARS?s Scientific Manuscript database

Pairing denitrifying woodchip bioreactors and phosphorus-sorbing filters provides a unique, engineered approach for dual nutrient removal from waters impaired with both nitrogen (N) and phosphorus (P). This column study aimed to test placement of two P-filter media (acid mine drainage treatment resi...

The Flow in a Model Rotating-Wall Bioreactor.

NASA Astrophysics Data System (ADS)

Smith, Marc K.; Neitzel, G. Paul

1997-11-01

Aggregates of mammalian cells can be grown on artificial polymer constructs in a reactor vessel in order to produce high-quality tissue for medical applications. The growth and differentiation of these cells is greatly affected by the fluid flow and mass transfer within the bioreactor. The surface shear stress on the constructs is an especially important quantity of interest. Here, we consider a bioreactor in the form of two concentric, independently-rotating cylinders with the axis of rotation in a horizontal plane. We shall examine the flow around a model tissue construct in the form of a disk fixed in the flow produced by the rotating walls of the bioreactor. Using CFD techniques, we shall determine the flow field and the surface shear stress distribution on the construct as a function of the wall velocities, the Reynolds number of the flow, and the construct size and position. The results will be compared to the PIV measurements of this system reported by Brown & Neitzel(1997 Meeting of the APS/DFD.).

Tissue Engineered Skeletal Myofibers can Directly "Sense" Gravitational Force Changes

NASA Technical Reports Server (NTRS)

Vandenburgh, Herman H.; Shansky, J.; DelTatto, M.; Lee, Peter; Meir, J.

1999-01-01

Long-term manned space flight requires a better understanding of skeletal muscle atrophy resulting from microgravity. Atrophy most likely results from changes at both the systemic level (e.g. decreased circulating growth hormone, increased circulating glucocorticoids) and locally (e.g. decreased myofiber resting tension). Differentiated skeletal myofibers in tissue culture have provided a model system over the last decade for gaining a better understanding of the interactions of exogenous growth factors, endogenous growth factors, and muscle fiber tension in regulating protein turnover rates and muscle cell growth. Tissue engineering these cells into three dimensional bioartificial muscle (BAM) constructs has allowed us to extend their use to Space flight studies for the potential future development of countermeasures. Embryonic avian muscle cells were isolated and BAMs tissue engineered as described previously. The myoblasts proliferate and fuse into aligned postmitotic myofibers after ten to fourteen days in vitro. A cylindrical muscle-like structure containing several thousand myofibers is formed which is approximately 30 mm in length, 2-3 mm in diameter, and attached at each end. For the Space Shuttle experiments, the BAMs were transferred to 55 mL bioreactor cartridges (6 BAMs/cartridge). At Kennedy Space Center, the cartridges were mounted in two Space Tissue Loss (STL) Modules (three to four cartridges per Module) and either maintained as ground controls or loaded in a Mid-Deck locker of the Space Shuttle. The BAM cartridges were continuously perfused during the experiment at 1.5 mL/ min with tissue culture medium. Eighteen BAMs were flown for nine days on Mission STS66 while eighteen BAMs served as ground controls. The complete experiment was repeated on Mission STS77 with twenty four BAMs in each group. BAMs could be maintained in a healthy state for at least 30 days in the perfusion bioreactor cartridges. The BAM muscle fibers directly detected both the loss of gravity and the reloading effects of 1 x g. While total cellular metabolism and total protein degradation rates were not altered during 9 to 10 days in Space, protein synthesis rates were significantly reduced and resulted in significant myofiber atrophy compared to ground controls. One g reloading of the flight muscle cells post-flight significantly increased protein synthesis rates and the synthesis rates of myosin heavy chain, fibronectin, and collagen. Tissue cultured muscle cells can directly "sense" changes in gravity and provide a valid model to begin the study of countermeasures. Based on our ground based experiments, and the experiments of others, growth hormone and/or insulin-like growth factors are attractive protein therapeutics which may assisting attenuating skeletal muscle wasting in space. Our laboratory is developing a new cell-based delivery system for this and other potential therapeutic factors for attenuating muscle and bone wasting.

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunourous tissues. Here, two High-Aspect Ratio Vessels turn at about 12 rmp to keep breast tissue constructs suspended inside the culture media. Syringes allow scientists to pull for analysis during growth sequences. The tube in the center is a water bubbler that dehumidifies the air to prevent evaporation of the media and thus the appearance of destructive bubbles in the bioreactor.

Microgravity

NASA Image and Video Library

1998-10-10

NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cells (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunourous tissues. Here, two High-Aspect Ratio Vessels turn at about 12 rmp to keep breast tissue constructs suspended inside the culture media. Syringes allow scientists to pull for analysis during growth sequences. The tube in the center is a water bubbler that dehumidifies the air to prevent evaporation of the media and thus the appearance of destructive bubbles in the bioreactor.

Fibre-reinforced hydrogels for tissue engineering

NASA Astrophysics Data System (ADS)

Waters, Sarah; Byrne, Helen; Chen, Mike; Dias Castilho, Miguel; Kimpton, Laura; Please, Colin; Whiteley, Jonathan

2017-11-01

Tissue engineers aim to grow replacement tissues in vitro to replace those in the body that have been damaged through age, trauma or disease. One approach is to seed cells within a scaffold consisting of an interconnected 3D-printed lattice of polymer fibres, cast in a hydrogel, and subject the construct (cell-seeded scaffold) to an applied load in a bioreactor. A key question is to understand how this applied load is distributed throughout the construct to the mechanosensitive cells. To address this, we exploit the disparate length scales (small inter-fibre spacing compared with construct dimensions). The fibres are treated as a linear elastic material and the hydrogel as a poroelastic material. We employ homogenisation theory to derive equations governing the material properties of a periodic, elastic-poroelastic composite. To validate the mobel, model solutions are compared to experimental data describing the unconfined compression of the fibre-reinforced hydrogels. The model is used to derive the bulk mechanical properties of a cylindrical construct of the composite material for a range of fibre spacings, and the local mechanical environment experienced by cells embedded within the construct is determined. Funded by the European Union Seventh Framework Programme (FP7/2007-2013).

Finite element study of scaffold architecture design and culture conditions for tissue engineering.

PubMed

Olivares, Andy L; Marsal, Elia; Planell, Josep A; Lacroix, Damien

2009-10-01

Tissue engineering scaffolds provide temporary mechanical support for tissue regeneration and transfer global mechanical load to mechanical stimuli to cells through its architecture. In this study the interactions between scaffold pore morphology, mechanical stimuli developed at the cell microscopic level, and culture conditions applied at the macroscopic scale are studied on two regular scaffold structures. Gyroid and hexagonal scaffolds of 55% and 70% porosity were modeled in a finite element analysis and were submitted to an inlet fluid flow or compressive strain. A mechanoregulation theory based on scaffold shear strain and fluid shear stress was applied for determining the influence of each structures on the mechanical stimuli on initial conditions. Results indicate that the distribution of shear stress induced by fluid perfusion is very dependent on pore distribution within the scaffold. Gyroid architectures provide a better accessibility of the fluid than hexagonal structures. Based on the mechanoregulation theory, the differentiation process in these structures was more sensitive to inlet fluid flow than axial strain of the scaffold. This study provides a computational approach to determine the mechanical stimuli at the cellular level when cells are cultured in a bioreactor and to relate mechanical stimuli with cell differentiation.

Trickle-bed root culture bioreactor design and scale-up: growth, fluid-dynamics, and oxygen mass transfer.

PubMed

Ramakrishnan, Divakar; Curtis, Wayne R

2004-10-20

Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L. Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)). These results demonstrate that trickle-bed reactor systems can sustain tissue concentrations, growth rates and volumetric biomass productivities substantially higher than other reported bioreactor configurations. Mass transfer and fluid dynamics are characterized in trickle-bed root reactors to identify appropriate operating conditions and scale-up criteria. Root tissue respiration goes through a minimum with increasing liquid flow, which is qualitatively consistent with traditional trickle-bed performance. However, liquid hold-up is much higher than traditional trickle-beds and alternative correlations based on liquid hold-up per unit tissue mass are required to account for large changes in biomass volume fraction. Bioreactor characterization is sufficient to carry out preliminary design calculations that indicate scale-up feasibility to at least 10,000 liters.

Development of an Autonomous, Dual Chamber Bioreactor for the Growth of 3-Dimensional Epithelial-Stromal Tissues in Microgravity

NASA Technical Reports Server (NTRS)

Patel, Zarana S.; Wettergreen, Matthew A.; Huff, Janice L.

2014-01-01

We are developing a novel, autonomous bioreactor that can provide for the growth and maintenance in microgravity of 3-D organotypic epithelial-stromal cultures that require an air-liquid interface. These complex 3-D tissue models accurately represent the morphological features, differentiation markers, and growth characteristics observed in normal human epithelial tissues, including the skin, esophagus, lung, breast, pancreas, and colon. However, because of their precise and complex culture requirements, including that of an air-liquid interface, these 3-D models have yet to be utilized for life sciences research aboard the International Space Station. The development of a bioreactor for these cultures will provide the capability to perform biological research on the ISS using these realistic, tissue-like human epithelial-stromal cell models and will contribute significantly to advances in fundamental space biology research on questions regarding microgravity effects on normal tissue development, aging, cancer, and other disease processes. It will also allow for the study of how combined stressors, such as microgravity with radiation and nutritional deficiencies, affect multiple biological processes and will provide a platform for conducting countermeasure investigations on the ISS without the use of animal models. The technology will be autonomous and consist of a cell culture chamber that provides for air-liquid, liquid-liquid, and liquid-air exchanges within the chambers while maintaining the growth and development of the biological samples. The bioreactor will support multiple tissue types and its modular design will provide for incorporation of add-on capabilities such as microfluidics drug delivery, media sampling, and in situ biomarker analysis. Preliminary flight testing of the hardware will be conducted on a parabolic platform through NASA's Flight Opportunities Program.

Perfusion-decellularization of human ear grafts enables ECM-based scaffolds for auricular vascularized composite tissue engineering.

PubMed

Duisit, Jérôme; Amiel, Hadrien; Wüthrich, Tsering; Taddeo, Adriano; Dedriche, Adeline; Destoop, Vincent; Pardoen, Thomas; Bouzin, Caroline; Joris, Virginie; Magee, Derek; Vögelin, Esther; Harriman, David; Dessy, Chantal; Orlando, Giuseppe; Behets, Catherine; Rieben, Robert; Gianello, Pierre; Lengelé, Benoît

2018-06-01

Human ear reconstruction is recognized as the emblematic enterprise in tissue engineering. Up to now, it has failed to reach human applications requiring appropriate tissue complexity along with an accessible vascular tree. We hereby propose a new method to process human auricles in order to provide a poorly immunogenic, complex and vascularized ear graft scaffold. 12 human ears with their vascular pedicles were procured. Perfusion-decellularization was applied using a SDS/polar solvent protocol. Cell and antigen removal was examined by histology and DNA was quantified. Preservation of the extracellular matrix (ECM) was assessed by conventional and 3D-histology, proteins and cytokines quantifications. Biocompatibility was assessed by implantation in rats for up to 60 days. Adipose-derived stem cells seeding was conducted on scaffold samples and with human aortic endothelial cells whole graft seeding in a perfusion-bioreactor. Histology confirmed cell and antigen clearance. DNA reduction was 97.3%. ECM structure and composition were preserved. Implanted scaffolds were tolerated in vivo, with acceptable inflammation, remodeling, and anti-donor antibody formation. Seeding experiments demonstrated cell engraftment and viability. Vascularized and complex auricular scaffolds can be obtained from human source to provide a platform for further functional auricular tissue engineered constructs, hence providing an ideal road to the vascularized composite tissue engineering approach. The ear is emblematic in the biofabrication of tissues and organs. Current regenerative medicine strategies, with matrix from donor tissues or 3D-printed, didn't reach any application for reconstruction, because critically missing a vascular tree for perfusion and transplantation. We previously described the production of vascularized and cell-compatible scaffolds, from porcine ear grafts. In this study, we ---- applied findings directly to human auricles harvested from postmortem donors, providing a perfusable matrix that retains the ear's original complexity and hosts new viable cells after seeding. This approach unlocks the ability to achieve an auricular tissue engineering approach, associated with possible clinical translation. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Three-Dimensional Rotating Wall Vessel-Derived Cell Culture Models for Studying Virus-Host Interactions

PubMed Central

Gardner, Jameson K.; Herbst-Kralovetz, Melissa M.

2016-01-01

The key to better understanding complex virus-host interactions is the utilization of robust three-dimensional (3D) human cell cultures that effectively recapitulate native tissue architecture and model the microenvironment. A lack of physiologically-relevant animal models for many viruses has limited the elucidation of factors that influence viral pathogenesis and of complex host immune mechanisms. Conventional monolayer cell cultures may support viral infection, but are unable to form the tissue structures and complex microenvironments that mimic host physiology and, therefore, limiting their translational utility. The rotating wall vessel (RWV) bioreactor was designed by the National Aeronautics and Space Administration (NASA) to model microgravity and was later found to more accurately reproduce features of human tissue in vivo. Cells grown in RWV bioreactors develop in a low fluid-shear environment, which enables cells to form complex 3D tissue-like aggregates. A wide variety of human tissues (from neuronal to vaginal tissue) have been grown in RWV bioreactors and have been shown to support productive viral infection and physiological meaningful host responses. The in vivo-like characteristics and cellular features of the human 3D RWV-derived aggregates make them ideal model systems to effectively recapitulate pathophysiology and host responses necessary to conduct rigorous basic science, preclinical and translational studies. PMID:27834891

The Effect of Simulated Microgravity Environment of RWV Bioreactors on Surface Reactions and Adsorption of Serum Proteins on Bone-bioactive Microcarriers

NASA Technical Reports Server (NTRS)

Radin, Shula; Ducheyne, P.; Ayyaswamy, P. S.

2003-01-01

Biomimetically modified bioactive materials with bone-like surface properties are attractive candidates for use as microcarriers for 3-D bone-like tissue engineering under simulated microgravity conditions of NASA designed rotating wall vessel (RWV) bioreactors. The simulated microgravity environment is attainable under suitable parametric conditions of the RWV bioreactors. Ca-P containing bioactive glass (BG), whose stimulatory effect on bone cell function had been previously demonstrated, was used in the present study. BG surface modification via reactions in solution, resulting formation of bone-like minerals at the surface and adsorption of serum proteins is critical for obtaining the stimulatory effect. In this paper, we report on the major effects of simulated microgravity conditions of the RWV on the BG reactions surface reactions and protein adsorption in physiological solutions. Control tests at normal gravity were conducted at static and dynamic conditions. The study revealed that simulated microgravity remarkably enhanced reactions involved in the BG surface modification, including BG dissolution, formation of bone-like minerals at the surface and adsorption of serum proteins. Simultaneously, numerical models were developed to simulate the mass transport of chemical species to and from the BG surface under normal gravity and simulated microgravity conditions. The numerical results showed an excellent agreement with the experimental data at both testing conditions.

Improvement of In Vitro Three‐Dimensional Cartilage Regeneration by a Novel Hydrostatic Pressure Bioreactor

PubMed Central

Chen, Jie; Yuan, Zhaoyuan; Liu, Yu; Zheng, Rui; Dai, Yao; Tao, Ran; Xia, Huitang; Liu, Hairong; Zhang, Zhiyong; Zhang, Wenjie; Liu, Wei; Cao, Yilin

2016-01-01

Abstract In vitro three‐dimensional (3D) cartilage regeneration is a promising strategy for repair of cartilage defects. However, inferior mechanical strength and tissue homogeneity greatly restricted its clinical translation. Simulation of mechanical stress through a bioreactor is an important approach for improving in vitro cartilage regeneration. The current study developed a hydrostatic pressure (HP) bioreactor based on a novel pressure‐transmitting mode achieved by slight deformation of a flexible membrane in a completely sealed stainless steel device. The newly developed bioreactor efficiently avoided the potential risks of previously reported pressure‐transmitting modes and simultaneously addressed a series of important issues, such as pressure scopes, culture chamber sizes, sealability, contamination control, and CO2 balance. The whole bioreactor system realized stable long‐term (8 weeks) culture under high HP (5–10 MPa) without the problems of medium leakage and contamination. Furthermore, the results of in vitro 3D tissue culture based on a cartilage regeneration model revealed that HP provided by the newly developed bioreactor efficiently promoted in vitro 3D cartilage formation by improving its mechanical strength, thickness, and homogeneity. Detailed analysis in cell proliferation, cartilage matrix production, and cross‐linking level of collagen macromolecules, as well as density and alignment of collagen fibers, further revealed the possible mechanisms that HP regulated in vitro cartilage regeneration. The current study provided a highly efficient and stable bioreactor system for improving in vitro 3D cartilage regeneration and thus will help to accelerate its clinical translation. Stem Cells Translational Medicine 2017;6:982–991 PMID:28297584

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Bioreactor Demonstration System (BDS) comprises an electronics module, a gas supply module, and the incubator module housing the rotating wall vessel and its support systems. Nutrient media are pumped through an oxygenator and the culture vessel. The shell rotates at 0.5 rpm while the irner filter typically rotates at 11.5 rpm to produce a gentle flow that ensures removal of waste products as fresh media are infused. Periodically, some spent media are pumped into a waste bag and replaced by fresh media. When the waste bag is filled, an astronaut drains the waste bag and refills the supply bag through ports on the face of the incubator. Pinch valves and a perfusion pump ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system through a laptop computer displaying graphics designed for easy crew training and operation. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. See No. 0101825 for a version with major elements labeled, and No. 0103180 for an operational schematic. 0101816

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Bioreactor Demonstration System (BDS) comprises an electronics module, a gas supply module, and the incubator module housing the rotating wall vessel and its support systems. Nutrient media are pumped through an oxygenator and the culture vessel. The shell rotates at 0.5 rpm while the irner filter typically rotates at 11.5 rpm to produce a gentle flow that ensures removal of waste products as fresh media are infused. Periodically, some spent media are pumped into a waste bag and replaced by fresh media. When the waste bag is filled, an astronaut drains the waste bag and refills the supply bag through ports on the face of the incubator. Pinch valves and a perfusion pump ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system through a laptop computer displaying graphics designed for easy crew training and operation. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. See No. 0101816 for a version without labels, and No. 0103180 for an operational schematic.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Bioreactor Demonstration System (BDS) comprises an electronics module, a gas supply module, and the incubator module housing the rotating wall vessel and its support systems. Nutrient media are pumped through an oxygenator and the culture vessel. The shell rotates at 0.5 rpm while the irner filter typically rotates at 11.5 rpm to produce a gentle flow that ensures removal of waste products as fresh media are infused. Periodically, some spent media are pumped into a waste bag and replaced by fresh media. When the waste bag is filled, an astronaut drains the waste bag and refills the supply bag through ports on the face of the incubator. Pinch valves and a perfusion pump ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system through a laptop computer displaying graphics designed for easy crew training and operation. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. See No. 0101823 for a version without labels, and No. 0103180 for an operational schematic.

NASA Bioreactor

NASA Technical Reports Server (NTRS)

1998-01-01

Bioreactor Demonstration System (BDS) comprises an electronics module, a gas supply module, and the incubator module housing the rotating wall vessel and its support systems. Nutrient media are pumped through an oxygenator and the culture vessel. The shell rotates at 0.5 rpm while the irner filter typically rotates at 11.5 rpm to produce a gentle flow that ensures removal of waste products as fresh media are infused. Periodically, some spent media are pumped into a waste bag and replaced by fresh media. When the waste bag is filled, an astronaut drains the waste bag and refills the supply bag through ports on the face of the incubator. Pinch valves and a perfusion pump ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system through a laptop computer displaying graphics designed for easy crew training and operation. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. See No. 0101824 for a version with labels, and No. 0103180 for an operational schematic.

Method and Apparatus for a Miniature Bioreactor System for Long-Term Cell Culture

NASA Technical Reports Server (NTRS)

Kleis, Stanley J. (Inventor); Geffert, Sandra K. (Inventor); Gonda, Steve R. (Inventor)

2015-01-01

A bioreactor and method that permits continuous and simultaneous short, moderate, or long term cell culturing of one or more cell types or tissue in a laminar flow configuration is disclosed, where the bioreactor supports at least two laminar flow zones, which are isolated by laminar flow without the need for physical barriers between the zones. The bioreactors of this invention are ideally suited for studying short, moderate and long term studies of cell cultures and the response of cell cultures to one or more stressors such as pharmaceuticals, hypoxia, pathogens, or any other stressor. The bioreactors of this invention are also ideally suited for short, moderate or long term cell culturing with periodic cell harvesting and/or medium processing for secreted cellular components.

Growing Three-Dimensional Cartilage-Cell Cultures

NASA Technical Reports Server (NTRS)

Spaulding, Glenn F.; Prewett, Tacey L.; Goodwin, Thomas J.

1995-01-01

Process for growing three-dimensional cultures of mammalian cartilage from normal mammalian cells devised. Effected using horizontal rotating bioreactor described in companion article, "Simplified Bioreactor for Growing Mammalian Cells" (MSC-22060). Bioreactor provides quiescent environment with generous supplies of nutrient and oxygen. Initiated with noncartilage cells. Artificially grown tissue resembles that in mammalian cartilage. Potential use in developing therapies for damage to cartilage by joint and back injuries and by such inflammatory diseases as arthritis and temporal-mandibular joint disease. Also used to test nonsteroid anti-inflammation medicines.

Culturing and applications of rotating wall vessel bioreactor derived 3D epithelial cell models.

PubMed

Radtke, Andrea L; Herbst-Kralovetz, Melissa M

2012-04-03

Cells and tissues in the body experience environmental conditions that influence their architecture, intercellular communications, and overall functions. For in vitro cell culture models to accurately mimic the tissue of interest, the growth environment of the culture is a critical aspect to consider. Commonly used conventional cell culture systems propagate epithelial cells on flat two-dimensional (2-D) impermeable surfaces. Although much has been learned from conventional cell culture systems, many findings are not reproducible in human clinical trials or tissue explants, potentially as a result of the lack of a physiologically relevant microenvironment. Here, we describe a culture system that overcomes many of the culture condition boundaries of 2-D cell cultures, by using the innovative rotating wall vessel (RWV) bioreactor technology. We and others have shown that organotypic RWV-derived models can recapitulate structure, function, and authentic human responses to external stimuli similarly to human explant tissues (1-6). The RWV bioreactor is a suspension culture system that allows for the growth of epithelial cells under low physiological fluid shear conditions. The bioreactors come in two different formats, a high-aspect rotating vessel (HARV) or a slow-turning lateral vessel (STLV), in which they differ by their aeration source. Epithelial cells are added to the bioreactor of choice in combination with porous, collagen-coated microcarrier beads (Figure 1A). The cells utilize the beads as a growth scaffold during the constant free fall in the bioreactor (Figure 1B). The microenvironment provided by the bioreactor allows the cells to form three-dimensional (3-D) aggregates displaying in vivo-like characteristics often not observed under standard 2-D culture conditions (Figure 1D). These characteristics include tight junctions, mucus production, apical/basal orientation, in vivo protein localization, and additional epithelial cell-type specific properties. The progression from a monolayer of epithelial cells to a fully differentiated 3-D aggregate varies based on cell type(1, 7-13). Periodic sampling from the bioreactor allows for monitoring of epithelial aggregate formation, cellular differentiation markers and viability (Figure 1D). Once cellular differentiation and aggregate formation is established, the cells are harvested from the bioreactor, and similar assays performed on 2-D cells can be applied to the 3-D aggregates with a few considerations (Figure 1E-G). In this work, we describe detailed steps of how to culture 3-D epithelial cell aggregates in the RWV bioreactor system and a variety of potential assays and analyses that can be executed with the 3-D aggregates. These analyses include, but are not limited to, structural/morphological analysis (confocal, scanning and transmission electron microscopy), cytokine/chemokine secretion and cell signaling (cytometric bead array and Western blot analysis), gene expression analysis (real-time PCR), toxicological/drug analysis and host-pathogen interactions. The utilization of these assays set the foundation for more in-depth and expansive studies such as metabolomics, transcriptomics, proteomics and other array-based applications. Our goal is to present a non-conventional means of culturing human epithelial cells to produce organotypic 3-D models that recapitulate the human in vivo tissue, in a facile and robust system to be used by researchers with diverse scientific interests.

The chorioallantoic membrane (CAM) assay for the study of human bone regeneration: a refinement animal model for tissue engineering

NASA Astrophysics Data System (ADS)

Moreno-Jiménez, Inés; Hulsart-Billstrom, Gry; Lanham, Stuart A.; Janeczek, Agnieszka A.; Kontouli, Nasia; Kanczler, Janos M.; Evans, Nicholas D.; Oreffo, Richard Oc

2016-08-01

Biomaterial development for tissue engineering applications is rapidly increasing but necessitates efficacy and safety testing prior to clinical application. Current in vitro and in vivo models hold a number of limitations, including expense, lack of correlation between animal models and human outcomes and the need to perform invasive procedures on animals; hence requiring new predictive screening methods. In the present study we tested the hypothesis that the chick embryo chorioallantoic membrane (CAM) can be used as a bioreactor to culture and study the regeneration of human living bone. We extracted bone cylinders from human femoral heads, simulated an injury using a drill-hole defect, and implanted the bone on CAM or in vitro control-culture. Micro-computed tomography (μCT) was used to quantify the magnitude and location of bone volume changes followed by histological analyses to assess bone repair. CAM blood vessels were observed to infiltrate the human bone cylinder and maintain human cell viability. Histological evaluation revealed extensive extracellular matrix deposition in proximity to endochondral condensations (Sox9+) on the CAM-implanted bone cylinders, correlating with a significant increase in bone volume by μCT analysis (p < 0.01). This human-avian system offers a simple refinement model for animal research and a step towards a humanized in vivo model for tissue engineering.

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

High magnification view of human primary breast tumor cells after 56 days of culture in a NASA Bioreactor. The arrow points to bead surface indicating breast cancer cells (as noted by the staining of tumor cell intermediate filaments). NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

High magnification of view of tumor cells aggregate on microcarrier beads, illustrting breast cells with intercellular boundaires on bead surface and aggregates of cells achieving 3-deminstional growth outward from bead after 56 days of culture in a NASA Bioreactor. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida.

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions.

PubMed

Rath, Subha N; Strobel, Leonie A; Arkudas, Andreas; Beier, Justus P; Maier, Anne-Kathrin; Greil, Peter; Horch, Raymund E; Kneser, Ulrich

2012-10-01

In many tissue engineering approaches, the basic difference between in vitro and in vivo conditions for cells within three-dimensional (3D) constructs is the nutrition flow dynamics. To achieve comparable results in vitro, bioreactors are advised for improved cell survival, as they are able to provide a controlled flow through the scaffold. We hypothesize that a bioreactor would enhance long-term differentiation conditions of osteogenic cells in 3D scaffolds. To achieve this either primary rat osteoblasts or bone marrow stromal cells (BMSC) were implanted on uniform-sized biphasic calcium phosphate (BCP) scaffolds produced by a 3D printing method. Three types of culture conditions were applied: static culture without osteoinduction (Group A); static culture with osteoinduction (Group B); dynamic culture with osteoinduction (Group C). After 3 and 6 weeks, the scaffolds were analysed by alkaline phosphatase (ALP), dsDNA amount, SEM, fluorescent labelled live-dead assay, and real-time RT-PCR in addition to weekly alamarBlue assays. With osteoinduction, increased ALP values and calcium deposition are observed; however, under static conditions, a significant decrease in the cell number on the biomaterial is observed. Interestingly, the bioreactor system not only reversed the decreased cell numbers but also increased their differentiation potential. We conclude from this study that a continuous flow bioreactor not only preserves the number of osteogenic cells but also keeps their differentiation ability in balance providing a suitable cell-seeded scaffold product for applications in regenerative medicine. © 2012 The Authors Journal of Cellular and Molecular Medicine © 2012 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

STEADY-STATE DESIGN OF VERTICAL WELLS FOR LIQUIDS ADDITION AT BIOREACTOR LANDFILLS

EPA Science Inventory

This paper presents design charts that a landfill engineer can use for the design of a vertical well system for liquids addition at bioreactor landfills. The flow rate and lateral and vertical zones of impact of a vertical well were estimated as a function of input variables su...

Bioreactor Engineering of Stem Cell Environments

PubMed Central

Tandon, Nina; Marolt, Darja; Cimetta, Elisa; Vunjak-Novakovic, Gordana

2013-01-01

Stem cells hold promise to revolutionize modern medicine by development of new therapies, disease models and drug screening systems. Standard cell culture systems have limited biological relevance because they do not recapitulate the complex 3-dimensional interactions and biophysical cues that characterize the in vivo environment. In this review, we discuss the current advances in engineering stem cell environments using novel biomaterials and bioreactor technologies. We also reflect on the challenges the field is currently facing with regard to translation of stem cell based therapies into the clinic. PMID:23531529

Characterization of TAP Ambr 250 disposable bioreactors, as a reliable scale-down model for biologics process development.

PubMed

Xu, Ping; Clark, Colleen; Ryder, Todd; Sparks, Colleen; Zhou, Jiping; Wang, Michelle; Russell, Reb; Scott, Charo

2017-03-01

Demands for development of biological therapies is rapidly increasing, as is the drive to reduce time to patient. In order to speed up development, the disposable Automated Microscale Bioreactor (Ambr 250) system is increasingly gaining interest due to its advantages, including highly automated control, high throughput capacity, and short turnaround time. Traditional early stage upstream process development conducted in 2 - 5 L bench-top bioreactors requires high foot-print, and running cost. The establishment of the Ambr 250 as a scale-down model leads to many benefits in process development. In this study, a comprehensive characterization of mass transfer coefficient (k L a) in the Ambr 250 was conducted to define optimal operational conditions. Scale-down approaches, including dimensionless volumetric flow rate (vvm), power per unit volume (P/V) and k L a have been evaluated using different cell lines. This study demonstrates that the Ambr 250 generated comparable profiles of cell growth and protein production, as seen at 5-L and 1000-L bioreactor scales, when using k L a as a scale-down parameter. In addition to mimicking processes at large scales, the suitability of the Ambr 250 as a tool for clone selection, which is traditionally conducted in bench-top bioreactors, was investigated. Data show that cell growth, productivity, metabolite profiles, and product qualities of material generated using the Ambr 250 were comparable to those from 5-L bioreactors. Therefore, Ambr 250 can be used for clone selection and process development as a replacement for traditional bench-top bioreactors minimizing resource utilization during the early stages of development in the biopharmaceutical industry. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:478-489, 2017. © 2017 American Institute of Chemical Engineers.

Design challenges for space bioreactors

NASA Technical Reports Server (NTRS)

Seshan, P. K.; Petersen, G. R.

1989-01-01

The design of bioreactors for operation under conditions of microgravity presents problems and challenges. Absence of a significant body force such as gravity can have profound consequences for interfacial phenomena. Marangoni convection can no longer be overlooked. Many speculations on the advantages and benefits of microgravity can be found in the literature. Initial bioreactor research considerations for space applications had little regard for the suitability of the designs for conditions of microgravity. Bioreactors can be classified in terms of their function and type of operation. The complex interaction of parameters leading to optimal design and operation of a bioreactor is illustrated by the JSC mammalian cell culture system. The design of a bioreactor is strongly dependent upon its intended use as a production unit for cell mass and/or biologicals or as a research reactor for the study of cell growth and function. Therefore a variety of bioreactor configurations are presented in rapid summary. Following this, a rationale is presented for not attempting to derive key design parameters such as the oxygen transfer coefficient from ground-based data. A set of themes/objectives for flight experiments to develop the expertise for design of space bioreactors is then proposed for discussion. These experiments, carried out systematically, will provide a database from which engineering tools for space bioreactor design will be derived.

Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model

NASA Technical Reports Server (NTRS)

Lewis, M. L.; Moriarity, D. M.; Campbell, P. S.

1993-01-01

During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research.

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system.

PubMed

Kang, Chang-Wei; Wang, Yan; Tania, Marshella; Zhou, Huancheng; Gao, Yi; Ba, Te; Tan, Guo-Dong Sean; Kim, Sangho; Leo, Hwa Liang

2013-01-01

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell-culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell-carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications. © 2013 American Institute of Chemical Engineers.

Tissue-Engineered Heart Valve with a Tubular Leaflet Design for Minimally Invasive Transcatheter Implantation

PubMed Central

Moreira, Ricardo; Velz, Thaddaeus; Alves, Nuno; Gesche, Valentine N.; Malischewski, Axel; Schmitz-Rode, Thomas; Frese, Julia

2015-01-01

Transcatheter aortic valve implantation of (nonviable) bioprosthetic valves has been proven a valid alternative to conventional surgical implantation in patients at high or prohibitive mortality risk. In this study we present the in vitro proof-of-principle of a newly developed tissue-engineered heart valve for minimally invasive implantation, with the ultimate aim of adding the unique advantages of a living tissue with regeneration capabilities to the continuously developing transcatheter technologies. The tube-in-stent is a fibrin-based tissue-engineered valve with a tubular leaflet design. It consists of a tubular construct sewn into a self-expandable nitinol stent at three commissural attachment points and along a circumferential line so that it forms three coaptating leaflets by collapsing under diastolic back pressure. The tubular constructs were molded with fibrin and human umbilical vein cells. After 3 weeks of conditioning in a bioreactor, the valves were fully functional with unobstructed opening (systolic phase) and complete closure (diastolic phase). Tissue analysis showed a homogeneous cell distribution throughout the valve's thickness and deposition of collagen types I and III oriented along the longitudinal direction. Immunohistochemical staining against CD31 and scanning electron microscopy revealed a confluent endothelial cell layer on the surface of the valves. After harvesting, the valves underwent crimping for 20 min to simulate the catheter-based delivery. This procedure did not affect the valvular functionality in terms of orifice area during systole and complete closure during diastole. No influence on the extracellular matrix organization, as assessed by immunohistochemistry, nor on the mechanical properties was observed. These results show the potential of combining tissue engineering and minimally invasive implantation technology to obtain a living heart valve with a simple and robust tubular design for transcatheter delivery. The effect of the in vivo remodeling on the functionality of the tube-in-stent valve remains to be tested. PMID:25380414

An Optimization of Pulsed ElectroMagnetic Fields Study

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J.

2006-01-01

To date, in our research we have focused on the use of normal human neuronal progenitor (NHNP) cells because of their importance in human nervous system regeneration, development and maintenance, but we have developed 2-D and 3-D bioreactors that can accommodate any cell line. In this Project, we will include the use of tissues important for physiological regeneration: Human osteoblasts or chondrocytes, and vascular cells. Our initial results with the NHNP cells were quite startling using extremely low-level electromagnetic fields (5 microtesla at 10Hz; 6mA). The low-amplitude, rapidly time-varying electromagnetic fields exert a very potent effect on the proliferation, morphology, and gene expression of the cells in culture, both in standard 2-dimensional culture plates as well as cells organized into 3-dimensional tissue-like assemblies (TLAs) in a 3D bioreactor. We have replicated our preliminary results many, many times, have analyzed the gene expression using gene arrays (followed by Luminex analysis for protein production), and have monitored cell proliferation, orientation, morphology, and glucose metabolism, and we are confident that we have a stable and reliable model to study the control of high-level cellular processes by application of low-amplitude, time varying electromagnetic fields (TVEMF) (1, 2). In additional studies at the University of Michigan, we have been able to generate functional in vitro engineered mammalian skeletal muscle, and have employed nerve-muscle co-culture techniques to promote axonal sprouting. We believe that nearly all tissues, in particular, neural, are susceptible to the influences of low-level TVEMF.

Upcyte® Microvascular Endothelial Cells Repopulate Decellularized Scaffold

PubMed Central

Dally, Iris; Hartmann, Nadja; Münst, Bernhard; Braspenning, Joris; Walles, Heike

2013-01-01

A general problem in tissue engineering is the poor and insufficient blood supply to guarantee tissue cell survival as well as physiological tissue function. To address this limitation, we have developed an in vitro vascularization model in which a decellularized porcine small bowl segment, representing a capillary network within a collagen matrix (biological vascularized scaffold [BioVaSc]), is reseeded with microvascular endothelial cells (mvECs). However, since the supply of mvECs is limited, in general, and as these cells rapidly dedifferentiate, we have applied a novel technology, which allows the generation of large batches of quasi-primary cells with the ability to proliferate, whilst maintaining their differentiated functionality. These so called upcyte mvECs grew for an additional 15 population doublings (PDs) compared to primary cells. Upcyte mvECs retained endothelial characteristics, such as von Willebrandt Factor (vWF), CD31 and endothelial nitric oxide synthase (eNOS) expression, as well as positive Ulex europaeus agglutinin I staining. Upcyte mvECs also retained biological functionality such as tube formation, cell migration, and low density lipoprotein (LDL) uptake, which were still evident after PD27. Initial experiments using MTT and Live/Dead staining indicate that upcyte mvECs repopulate the BioVaSc Scaffold. As with conventional cultures, these cells also express key endothelial molecules (vWF, CD31, and eNOS) in a custom-made bioreactor system even after a prolonged period of 14 days. The combination of upcyte mvECs and the BioVaSc represents a novel and promising approach toward vascularizing bioreactor models which can better reflect organs, such as the liver. PMID:22799502

A multiphase model for tissue construct growth in a perfusion bioreactor.

PubMed

O'Dea, R D; Waters, S L; Byrne, H M

2010-06-01

The growth of a cell population within a rigid porous scaffold in a perfusion bioreactor is studied, using a three-phase continuum model of the type presented by Lemon et al. (2006, Multiphase modelling of tissue growth using the theory of mixtures. J. Math. Biol., 52, 571-594) to represent the cell population (and attendant extracellular matrix), culture medium and porous scaffold. The bioreactor system is modelled as a 2D channel containing the cell-seeded rigid porous scaffold (tissue construct) which is perfused with culture medium. The study concentrates on (i) the cell-cell and cell-scaffold interactions and (ii) the impact of mechanotransduction mechanisms on construct composition. A numerical and analytical analysis of the model equations is presented and, depending upon the relative importance of cell aggregation and repulsion, markedly different cell movement is revealed. Additionally, mechanotransduction effects due to cell density, pressure and shear stress-mediated tissue growth are shown to generate qualitative differences in the composition of the resulting construct. The results of our simulations indicate that this model formulation (in conjunction with appropriate experimental data) has the potential to provide a means of identifying the dominant regulatory stimuli in a cell population.

Platelet bioreactor: accelerated evolution of design and manufacture.

PubMed

Thon, Jonathan N; Dykstra, Brad J; Beaulieu, Lea M

2017-07-01

Platelets, responsible for clot formation and blood vessel repair, are produced by megakaryocytes in the bone marrow. Platelets are critical for hemostasis and wound healing, and are often provided following surgery, chemotherapy, and major trauma. Despite their importance, platelets today are derived exclusively from human volunteer donors. They have a shelf life of just five days, making platelet shortages common during long weekends, civic holidays, bad weather, and during major emergencies when platelets are needed most. Megakaryocytes in the bone marrow generate platelets by extruding long cytoplasmic extensions called proplatelets through gaps/fenestrations in blood vessels. Proplatelets serve as assembly lines for platelet production by sequentially releasing platelets and large discoid-shaped platelet intermediates called preplatelets into the circulation. Recent advances in platelet bioreactor development have aimed to mimic the key physiological characteristics of bone marrow, including extracellular matrix composition/stiffness, blood vessel architecture comprising tissue-specific microvascular endothelium, and shear stress. Nevertheless, how complex interactions within three-dimensional (3D) microenvironments regulate thrombopoiesis remains poorly understood, and the technical challenges associated with designing and manufacturing biomimetic microfluidic devices are often under-appreciated and under-reported. We have previously reviewed the major cell culture, platelet quality assessment, and regulatory roadblocks that must be overcome to make human platelet production possible for clinical use [1]. This review builds on our previous manuscript by: (1) detailing the historical evolution of platelet bioreactor design to recapitulate native platelet production ex vivo, and (2) identifying the associated challenges that still need to be addressed to further scale and validate these devices for commercial application. While platelets are among the first cells whose ex vivo production is spearheading major engineering advancements in microfluidic design, the resulting discoveries will undoubtedly extend to the production of other human tissues. This work is critical to identify the physiological characteristics of relevant 3D tissue-specific microenvironments that drive cell differentiation and elaborate upon how these are disrupted in disease. This is a burgeoning field whose future will define not only the ex vivo production of platelets and development of targeted therapies for thrombocytopenia, but the promise of regenerative medicine for the next century.

Breast Cancer Research at NASA

NASA Technical Reports Server (NTRS)

1998-01-01

Human primary breast tumor cells after 56 days of culture in a NASA Bioreactor. A cross-section of a construct, grown from surgical specimens of brease cancer, stained for microscopic examination, reveals areas of tumor cells dispersed throughout the non-epithelial cell background. The arrow denotes the foci of breast cancer cells. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida

Microgravity

NASA Image and Video Library

1998-10-10

Human primary breast tumor cells after 56 days of culture in a NASA Bioreactor. A cross-section of a construct, grown from surgical specimens of brease cancer, stained for microscopic examination, reveals areas of tumor cells dispersed throughout the non-epithelial cell background. The arrow denotes the foci of breast cancer cells. NASA's Marshall Space Flight Center (MSFC) is sponsoring research with Bioreactors, rotating wall vessels designed to grow tissue samples in space, to understand how breast cancer works. This ground-based work studies the growth and assembly of human mammary epithelial cell (HMEC) from breast cancer susceptible tissue. Radiation can make the cells cancerous, thus allowing better comparisons of healthy vs. tunorous tissue. Credit: Dr. Jearne Becker, University of South Florida

A bioreactor test system to mimic the biological and mechanical environment of oral soft tissues and to evaluate substitutes for connective tissue grafts.

PubMed

Mathes, Stephanie H; Wohlwend, Lorenz; Uebersax, Lorenz; von Mentlen, Roger; Thoma, Daniel S; Jung, Ronald E; Görlach, Christoph; Graf-Hausner, Ursula

2010-12-15

Gingival cells of the oral connective tissue are exposed to complex mechanical forces during mastication, speech, tooth movement and orthodontic treatments. Especially during wound healing following surgical procedures, internal and external forces may occur, creating pressure upon the newly formed tissue. This clinical situation has to be considered when developing biomaterials to augment soft tissue in the oral cavity. In order to pre-evaluate a collagen sponge intended to serve as a substitute for autogenous connective tissue grafts (CTGs), a dynamic bioreactor system was developed. Pressure and shear forces can be applied in this bioreactor in addition to a constant medium perfusion to cell-material constructs. Three-dimensional volume changes and stiffness of the matrices were analyzed. In addition, cell responses such as cell vitality and extracellular matrix (ECM) production were investigated. The number of metabolic active cells constantly increased under fully dynamic culture conditions. The sponges remained elastic even after mechanical forces were applied for 14 days. Analysis of collagen type I and fibronectin revealed a statistically significant accumulation of these ECM molecules (P < 0.05-0.001) when compared to static cultures. An increased expression of tenascin-c, indicating tissue remodeling processes, was observed under dynamic conditions only. The results indicate that the tested in vitro cell culture system was able to mimic both the biological and mechanical environments of the clinical situation in a healing wound. © 2010 Wiley Periodicals, Inc.

Mixtures of tense and relaxed state polymerized human hemoglobin regulate oxygen affinity and tissue construct oxygenation

PubMed Central

Belcher, Donald Andrew; Banerjee, Uddyalok; Baehr, Christopher Michael; Richardson, Kristopher Emil; Cabrales, Pedro; Berthiaume, François

2017-01-01

Pure tense (T) and relaxed (R) quaternary state polymerized human hemoglobins (PolyhHbs) were synthesized and their biophysical properties characterized, along with mixtures of T- and R-state PolyhHbs. It was observed that the oxygen affinity of PolyhHb mixtures varied linearly with T-state mole fraction. Computational analysis of PolyhHb facilitated oxygenation of a single fiber in a hepatic hollow fiber (HF) bioreactor was performed to evaluate the oxygenation potential of T- and R-state PolyhHb mixtures. PolyhHb mixtures with T-state mole fractions greater than 50% resulted in hypoxic and hyperoxic zones occupying less than 5% of the total extra capillary space (ECS). Under these conditions, the ratio of the pericentral volume to the perivenous volume in the ECS doubled as the T-state mole fraction increased from 50 to 100%. These results show the effect of varying the T/R-state PolyhHb mole fraction on oxygenation of tissue-engineered constructs and their potential to oxygenate tissues. PMID:29020036

Multiple-Step Injection Molding for Fibrin-Based Tissue-Engineered Heart Valves

PubMed Central

Weber, Miriam; Gonzalez de Torre, Israel; Moreira, Ricardo; Frese, Julia; Oedekoven, Caroline; Alonso, Matilde; Rodriguez Cabello, Carlos J.

2015-01-01

Heart valves are elaborate and highly heterogeneous structures of the circulatory system. Despite the well accepted relationship between the structural and mechanical anisotropy and the optimal function of the valves, most approaches to create tissue-engineered heart valves (TEHVs) do not try to mimic this complexity and rely on one homogenous combination of cells and materials for the whole construct. The aim of this study was to establish an easy and versatile method to introduce spatial diversity into a heart valve fibrin scaffold. We developed a multiple-step injection molding process that enables the fabrication of TEHVs with heterogeneous composition (cell/scaffold material) of wall and leaflets without the need of gluing or suturing components together, with the leaflets firmly connected to the wall. The integrity of the valves and their functionality was proved by either opening/closing cycles in a bioreactor (proof of principle without cells) or with continuous stimulation over 2 weeks. We demonstrated the potential of the method by the two-step molding of the wall and the leaflets containing different cell lines. Immunohistology after stimulation confirmed tissue formation and demonstrated the localization of the different cell types. Furthermore, we showed the proof of principle fabrication of valves using different materials for wall (fibrin) and leaflets (hybrid gel of fibrin/elastin-like recombinamer) and with layered leaflets. The method is easy to implement, does not require special facilities, and can be reproduced in any tissue-engineering lab. While it has been demonstrated here with fibrin, it can easily be extended to other hydrogels. PMID:25654448

Multiple-Step Injection Molding for Fibrin-Based Tissue-Engineered Heart Valves.

PubMed

Weber, Miriam; Gonzalez de Torre, Israel; Moreira, Ricardo; Frese, Julia; Oedekoven, Caroline; Alonso, Matilde; Rodriguez Cabello, Carlos J; Jockenhoevel, Stefan; Mela, Petra

2015-08-01

Heart valves are elaborate and highly heterogeneous structures of the circulatory system. Despite the well accepted relationship between the structural and mechanical anisotropy and the optimal function of the valves, most approaches to create tissue-engineered heart valves (TEHVs) do not try to mimic this complexity and rely on one homogenous combination of cells and materials for the whole construct. The aim of this study was to establish an easy and versatile method to introduce spatial diversity into a heart valve fibrin scaffold. We developed a multiple-step injection molding process that enables the fabrication of TEHVs with heterogeneous composition (cell/scaffold material) of wall and leaflets without the need of gluing or suturing components together, with the leaflets firmly connected to the wall. The integrity of the valves and their functionality was proved by either opening/closing cycles in a bioreactor (proof of principle without cells) or with continuous stimulation over 2 weeks. We demonstrated the potential of the method by the two-step molding of the wall and the leaflets containing different cell lines. Immunohistology after stimulation confirmed tissue formation and demonstrated the localization of the different cell types. Furthermore, we showed the proof of principle fabrication of valves using different materials for wall (fibrin) and leaflets (hybrid gel of fibrin/elastin-like recombinamer) and with layered leaflets. The method is easy to implement, does not require special facilities, and can be reproduced in any tissue-engineering lab. While it has been demonstrated here with fibrin, it can easily be extended to other hydrogels.

Characterization of local fluid flow in 3D porous construct characterized by Fourier domain Doppler optical coherence tomography

NASA Astrophysics Data System (ADS)

Bagnaninchi, P. O.; Yang, Y.; El Haj, A.; Hinds, M. T.; Wang, R. K.

2007-02-01

In order to achieve functional tissue with the correct biomechanical properties it is critical to stimulate mechanically the cells. Perfusion bioreactor induces fluid shear stress that has been well characterized for two-dimensional culture where both simulation and experimental data are available. However these results can't be directly translated to tissue engineering that makes use of complex three-dimensional porous scaffold. Moreover, stimulated cells produce extensive extra-cellular matrix (ECM) that alter dramatically the micro-architecture of the constructs, changing the local flow dynamic. In this study a Fourier domain Doppler optical coherent tomography (FD-DOCT) system working at 1300nm with a bandwidth of 50nm has been used to determine the local flow rate inside different types of porous scaffolds used in tissue engineering. Local flow rates can then be linearly related, for Newtonian fluid, to the fluid shear stress occurring on the pores wall. Porous chitosan scaffolds (\\fgr 1.5mm x 3mm) with and without a central 250 μm microchannel have been produced by a freeze-drying technique. This techniques allow us to determine the actual shear stress applied to the cells and to optimise the input flow rate consequently, but also to relate the change of the flow distribution to the amount of ECM production allowing the monitoring of tissue formation.

Microgravity

NASA Image and Video Library

1998-01-01

Biotechnology Refrigerator (BTR) holds fixed tissue culture bags at 4 degrees C to preserve them for return to Earth and postflight analysis. The cultures are used in research with the NASA Bioreactor cell science program. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC).

Unique cell culture systems for ground based research

NASA Technical Reports Server (NTRS)

Lewis, Marian L.

1990-01-01

The horizontally rotating fluid-filled, membrane oxygenated bioreactors developed at NASA Johnson for spacecraft applications provide a powerful tool for ground-based research. Three-dimensional aggregates formed by cells cultured on microcarrier beads are useful for study of cell-cell interactions and tissue development. By comparing electron micrographs of plant seedlings germinated during Shuttle flight 61-C and in an earth-based rotating bioreactor it is shown that some effects of microgravity are mimicked. Bioreactors used in the UAH Bioreactor Laboratory will make it possible to determine some of the effects of altered gravity at the cellular level. Bioreactors can be valuable for performing critical, preliminary-to-spaceflight experiments as well as medical investigations such as in vitro tumor cell growth and chemotherapeutic drug response; the enrichment of stem cells from bone marrow; and the effect of altered gravity on bone and muscle cell growth and function and immune response depression.

High-throughput miniaturized bioreactors for cell culture process development: reproducibility, scalability, and control.

PubMed

Rameez, Shahid; Mostafa, Sigma S; Miller, Christopher; Shukla, Abhinav A

2014-01-01

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr™) is an automated micro-bioreactor system with miniature single-use bioreactors with a 10-15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in-line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr™ resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr™ was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr™ system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers.

Bioreactor engineering of stem cell environments.

PubMed

Tandon, Nina; Marolt, Darja; Cimetta, Elisa; Vunjak-Novakovic, Gordana

2013-11-15

Stem cells hold promise to revolutionize modern medicine by the development of new therapies, disease models and drug screening systems. Standard cell culture systems have limited biological relevance because they do not recapitulate the complex 3-dimensional interactions and biophysical cues that characterize the in vivo environment. In this review, we discuss the current advances in engineering stem cell environments using novel biomaterials and bioreactor technologies. We also reflect on the challenges the field is currently facing with regard to the translation of stem cell based therapies into the clinic. Copyright © 2013 Elsevier Inc. All rights reserved.

An overview of cartilage tissue engineering.

PubMed

Kim, H W; Han, C D

2000-12-01

Articular cartilage regeneration refers to the formation of new tissue that is indistinguishable from the native articular cartilage with respect to zonal organization, biochemical composition, and mechanical properties. Due to a limited capacity to repair cartilage, scar tissue frequently has a poorly organized structure and lacks the functional characteristics of normal cartilage. The degree of success to date achieved using a purely cell- or biological-based approach has been modest. Potentially the development of a hybrid strategy, whereby, chondrocytes or chondrogenic stem cells are combined with a matrix, making cartilage in vitro, which is then subsequently transplanted, offers a route towards a new successful treatment modality. The success of this approach depends upon the material being biocompatible, processable into a suitable three-dimensional structure and eventually biodegradable without harmful effects. In addition, the material should have a sufficient porosity to facilitate high cell loading and tissue ingrowth, and it should be able to support cell proliferation, differentiation, and function. The cell-polymer-bioreactor system provides a basis for studying the structural and functional properties of the cartilaginous matrix during its development, because tissue concentrations of glycosaminoglycan and collagen can be modulated by altering the conditions of tissue cultivation.

Bridging the gap between traditional cell cultures and bioreactors applied in regenerative medicine: practical experiences with the MINUSHEET perfusion culture system.

PubMed

Minuth, Will W; Denk, Lucia

2016-03-01

To meet specific requirements of developing tissues urgently needed in tissue engineering, biomaterial research and drug toxicity testing, a versatile perfusion culture system was developed. First an individual biomaterial is selected and then mounted in a MINUSHEET(®) tissue carrier. After sterilization the assembly is transferred by fine forceps to a 24 well culture plate for seeding cells or mounting tissue on it. To support spatial (3D) development a carrier can be placed in various types of perfusion culture containers. In the basic version a constant flow of culture medium provides contained tissue with always fresh nutrition and respiratory gas. For example, epithelia can be transferred to a gradient container, where they are exposed to different fluids at the luminal and basal side. To observe development of tissue under the microscope, in a different type of container a transparent lid and base are integrated. Finally, stem/progenitor cells are incubated in a container filled by an artificial interstitium to support spatial development. In the past years the described system was applied in numerous own and external investigations. To present an actual overview of resulting experimental data, the present paper was written.

Stem cell engineered bone with calcium-phosphate coated porous titanium scaffold or silicon hydroxyapatite granules for revision total joint arthroplasty.

PubMed

García-Gareta, Elena; Hua, Jia; Rayan, Faizal; Blunn, Gordon W

2014-06-01

Aseptic loosening in total joint replacements (TJRs) is mainly caused by osteolysis which leads to a reduction of the bone stock necessary for implant fixation in revision TJRs. Our aim was to develop bone tissue-engineered constructs based on scaffolds of clinical relevance in revision TJRs to reconstitute the bone stock at revision operations by using a perfusion bioreactor system (PBRS). The hypothesis was that a PBRS will enhance mesenchymal stem cells (MSCs) proliferation and osteogenic differentiation and will provide an even distribution of MSCs throughout the scaffolds when compared to static cultures. A PBRS was designed and implemented. Scaffolds, silicon substituted hydroxyapatite granules and calcium-phosphate coated porous TiAl6V4 cylinders, were seeded with MSCs and cultured either in static conditions or in the PBRS at 0.75 mL/min. Statistically significant increased cell proliferation and alkaline phosphatase activity was found in samples cultured in the PBRS. Histology revealed a more even cell distribution in the perfused constructs. SEM showed that cells arranged in sheets. Long cytoplasmic processes attached the cells to the scaffolds. We conclude that a novel tissue engineering approach to address the issue of poor bone stock at revision operations is feasible by using a PBRS.

Teaching bioprocess engineering to undergraduates: Multidisciplinary hands-on training in a one-week practical course.

PubMed

Henkel, Marius; Zwick, Michaela; Beuker, Janina; Willenbacher, Judit; Baumann, Sandra; Oswald, Florian; Neumann, Anke; Siemann-Herzberg, Martin; Syldatk, Christoph; Hausmann, Rudolf

2015-01-01

Bioprocess engineering is a highly interdisciplinary field of study which is strongly benefited by practical courses where students can actively experience the interconnection between biology, engineering, and physical sciences. This work describes a lab course developed for 2nd year undergraduate students of bioprocess engineering and related disciplines, where students are challenged with a real-life bioprocess-engineering application, the production of recombinant protein in a fed-batch process. The lab course was designed to introduce students to the subject of operating and supervising an experiment in a bioreactor, along with the analysis of collected data and a final critical evaluation of the experiment. To provide visual feedback of the experimental outcome, the organism used during class was Escherichia coli which carried a plasmid to recombinantly produce enhanced green fluorescent protein (eGFP) upon induction. This can easily be visualized in both the bioreactor and samples by using ultraviolet light. The lab course is performed with bioreactors of the simplest design, and is therefore highly flexible, robust and easy to reproduce. As part of this work the implementation and framework, the results, the evaluation and assessment of student learning combined with opinion surveys are presented, which provides a basis for instructors intending to implement a similar lab course at their respective institution. © 2015 by the International Union of Biochemistry and Molecular Biology.

Microgravity

NASA Image and Video Library

1998-01-01

Bioreactor Demonstration System (BDS) comprises an electronics module, a gas supply module, and the incubator module housing the rotating wall vessel and its support systems. Nutrient media are pumped through an oxygenator and the culture vessel. The shell rotates at 0.5 rpm while the irner filter typically rotates at 11.5 rpm to produce a gentle flow that ensures removal of waste products as fresh media are infused. Periodically, some spent media are pumped into a waste bag and replaced by fresh media. When the waste bag is filled, an astronaut drains the waste bag and refills the supply bag through ports on the face of the incubator. Pinch valves and a perfusion pump ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system through a laptop computer displaying graphics designed for easy crew training and operation. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. See No. 0101823 for a version without labels, and No. 0103180 for an operational schematic.

Microgravity

NASA Image and Video Library

1998-01-01

Bioreactor Demonstration System (BDS) comprises an electronics module, a gas supply module, and the incubator module housing the rotating wall vessel and its support systems. Nutrient media are pumped through an oxygenator and the culture vessel. The shell rotates at 0.5 rpm while the irner filter typically rotates at 11.5 rpm to produce a gentle flow that ensures removal of waste products as fresh media are infused. Periodically, some spent media are pumped into a waste bag and replaced by fresh media. When the waste bag is filled, an astronaut drains the waste bag and refills the supply bag through ports on the face of the incubator. Pinch valves and a perfusion pump ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system through a laptop computer displaying graphics designed for easy crew training and operation. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. See No. 0101816 for a version without labels, and No. 0103180 for an operational schematic.

Microgravity

NASA Image and Video Library

1998-01-01

Bioreactor Demonstration System (BDS) comprises an electronics module, a gas supply module, and the incubator module housing the rotating wall vessel and its support systems. Nutrient media are pumped through an oxygenator and the culture vessel. The shell rotates at 0.5 rpm while the irner filter typically rotates at 11.5 rpm to produce a gentle flow that ensures removal of waste products as fresh media are infused. Periodically, some spent media are pumped into a waste bag and replaced by fresh media. When the waste bag is filled, an astronaut drains the waste bag and refills the supply bag through ports on the face of the incubator. Pinch valves and a perfusion pump ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system through a laptop computer displaying graphics designed for easy crew training and operation. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. See No. 0101825 for a version with major elements labeled, and No. 0103180 for an operational schematic. 0101816

Microgravity

NASA Image and Video Library

1998-01-01

Bioreactor Demonstration System (BDS) comprises an electronics module, a gas supply module, and the incubator module housing the rotating wall vessel and its support systems. Nutrient media are pumped through an oxygenator and the culture vessel. The shell rotates at 0.5 rpm while the irner filter typically rotates at 11.5 rpm to produce a gentle flow that ensures removal of waste products as fresh media are infused. Periodically, some spent media are pumped into a waste bag and replaced by fresh media. When the waste bag is filled, an astronaut drains the waste bag and refills the supply bag through ports on the face of the incubator. Pinch valves and a perfusion pump ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system through a laptop computer displaying graphics designed for easy crew training and operation. The work is sponsored by NASA's Office of Biological and Physical Research. The bioreactor is managed by the Biotechnology Cell Science Program at NASA's Johnson Space Center (JSC). NASA-sponsored bioreactor research has been instrumental in helping scientists to better understand normal and cancerous tissue development. In cooperation with the medical community, the bioreactor design is being used to prepare better models of human colon, prostate, breast and ovarian tumors. Cartilage, bone marrow, heart muscle, skeletal muscle, pancreatic islet cells, liver and kidney are just a few of the normal tissues being cultured in rotating bioreactors by investigators. See No. 0101824 for a version with labels, and No. 0103180 for an operational schematic.

Engineering three dimensional micro nerve tissue using postnatal stem cells from human dental apical papilla.

PubMed

Kim, Byung-Chul; Jun, Sung-Min; Kim, So Yeon; Kwon, Yong-Dae; Choe, Sung Chul; Kim, Eun-Chul; Lee, Jae-Hyung; Kim, Jinseok; Suh, Jun-Kyo Francis; Hwang, Yu-Shik

2017-04-01

The in vitro generation of cell-based three dimensional (3D) nerve tissue is an attractive subject to improve graft survival and integration into host tissue for neural tissue regeneration or to model biological events in stem cell differentiation. Although 3D organotypic culture strategies are well established for 3D nerve tissue formation of pluripotent stem cells to study underlying biology in nerve development, cell-based nerve tissues have not been developed using human postnatal stem cells with therapeutic potential. Here, we established a culture strategy for the generation of in vitro cell-based 3D nerve tissue from postnatal stem cells from apical papilla (SCAPs) of teeth, which originate from neural crest-derived ectomesenchyme cells. A stem cell population capable of differentiating into neural cell lineages was generated during the ex vivo expansion of SCAPs in the presence of EGF and bFGF, and SCAPs differentiated into neural cells, showing neural cell lineage-related molecular and gene expression profiles, morphological changes and electrophysical property under neural-inductive culture conditions. Moreover, we showed the first evidence that 3D cell-based nerve-like tissue with axons and myelin structures could be generated from SCAPs via 3D organotypic culture using an integrated bioprocess composed of polyethylene glycol (PEG) microwell-mediated cell spheroid formation and subsequent dynamic culture in a high aspect ratio vessel (HARV) bioreactor. In conclusion, the culture strategy in our study provides a novel approach to develop in vitro engineered nerve tissue using SCAPs and a foundation to study biological events in the neural differentiation of postnatal stem cells. Biotechnol. Bioeng. 2017;114: 903-914. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Bioreactor design for successive culture of anchorage-dependent cells operated in an automated manner.

PubMed

Kino-Oka, Masahiro; Ogawa, Natsuki; Umegaki, Ryota; Taya, Masahito

2005-01-01

A novel bioreactor system was designed to perform a series of batchwise cultures of anchorage-dependent cells by means of automated operations of medium change and passage for cell transfer. The experimental data on contamination frequency ensured the biological cleanliness in the bioreactor system, which facilitated the operations in a closed environment, as compared with that in flask culture system with manual handlings. In addition, the tools for growth prediction (based on growth kinetics) and real-time growth monitoring by measurement of medium components (based on small-volume analyzing machinery) were installed into the bioreactor system to schedule the operations of medium change and passage and to confirm that culture proceeds as scheduled, respectively. The successive culture of anchorage-dependent cells was conducted with the bioreactor running in an automated way. The automated bioreactor gave a successful culture performance with fair accordance to preset scheduling based on the information in the latest subculture, realizing 79- fold cell expansion for 169 h. In addition, the correlation factor between experimental data and scheduled values through the bioreactor performance was 0.998. It was concluded that the proposed bioreactor with the integration of the prediction and monitoring tools could offer a feasible system for the manufacturing process of cultured tissue products.

In vitro biomimetic platforms featuring a perfusion system and 3D spheroid culture promote the construction of tissue-engineered corneal endothelial layers.

PubMed

Li, Shanyi; Han, Yuting; Lei, Hao; Zeng, Yingxin; Cui, Zekai; Zeng, Qiaolang; Zhu, Deliang; Lian, Ruiling; Zhang, Jun; Chen, Zhe; Chen, Jiansu

2017-04-10

Corneal endothelial cells (CECs) are very important for the maintenance of corneal transparency. However, in vitro, CECs display limited proliferation and loss of phenotype via endothelial to mesenchymal transformation (EMT) and cellular senescence. In this study, we demonstrate that continuous supplementary nutrition using a perfusion culture bioreactor and three-dimensional (3D) spheroid culture can be used to improve CEC expansion in culture and to construct a tissue-engineered CEC layer. Compared with static culture, perfusion-derived CECs exhibited an increased proliferative ability as well as formed close cell-cell contact junctions and numerous surface microvilli. We also demonstrated that the CEC spheroid culture significantly down-regulated gene expression of the proliferation marker Ki67 and EMT-related markers Vimentin and α-SMA, whereas the gene expression level of the CEC marker ATP1A1 was significantly up-regulated. Furthermore, use of the perfusion system in conjunction with a spheroid culture on decellularized corneal scaffolds and collagen sheets promoted the generation of CEC monolayers as well as neo-synthesized ECM formation. This study also confirmed that a CEC spheroid culture on a curved collagen sheet with controlled physiological intraocular pressure could generate a CEC monolayer. Thus, our results show that the use of a perfusion system and 3D spheroid culture can promote CEC expansion and the construction of tissue-engineered corneal endothelial layers in vitro.

Aligned poly(L-lactic-co-e-caprolactone) electrospun microfibers and knitted structure: a novel composite scaffold for ligament tissue engineering.

PubMed

Vaquette, Cédryck; Kahn, Cyril; Frochot, Céline; Nouvel, Cécile; Six, Jean-Luc; De Isla, Natalia; Luo, Li-Hua; Cooper-White, Justin; Rahouadj, Rachid; Wang, Xiong

2010-09-15

We developed a novel technique involving knitting and electrospinning to fabricate a composite scaffold for ligament tissue engineering. Knitted structures were coated with poly(L-lactic-co-e-caprolactone) (PLCL) and then placed onto a rotating cylinder and a PLCL solution was electrospun onto the structure. Highly aligned 2-microm-diameter microfibers covered the space between the stitches and adhered to the knitted scaffolds. The stress-strain tensile curves exhibited an initial toe region similar to the tensile behavior of ligaments. Composite scaffolds had an elastic modulus (150 +/- 14 MPa) similar to the modulus of human ligaments. Biological evaluation showed that cells proliferated on the composite scaffolds and they spontaneously orientated along the direction of microfiber alignment. The microfiber architecture also induced a high level of extracellular matrix secretion, which was characterized by immunostaining. We found that cells produced collagen type I and type III, two main components found in ligaments. After 14 days of culture, collagen type III started to form a fibrous network. We fabricated a composite scaffold having the mechanical properties of the knitted structure and the morphological properties of the aligned microfibers. It is difficult to seed a highly macroporous structure with cells, however the technique we developed enabled an easy cell seeding due to presence of the microfiber layer. Therefore, these scaffolds presented attractive properties for a future use in bioreactors for ligament tissue engineering. (c) 2010 Wiley Periodicals, Inc.

The effects of dynamic compression on the development of cartilage grafts engineered using bone marrow and infrapatellar fat pad derived stem cells.

PubMed

Luo, Lu; Thorpe, Stephen D; Buckley, Conor T; Kelly, Daniel J

2015-09-21

Bioreactors that subject cell seeded scaffolds or hydrogels to biophysical stimulation have been used to improve the functionality of tissue engineered cartilage and to explore how such constructs might respond to the application of joint specific mechanical loading. Whether a particular cell type responds appropriately to physiological levels of biophysical stimulation could be considered a key determinant of its suitability for cartilage tissue engineering applications. The objective of this study was to determine the effects of dynamic compression on chondrogenesis of stem cells isolated from different tissue sources. Porcine bone marrow (BM) and infrapatellar fat pad (FP) derived stem cells were encapsulated in agarose hydrogels and cultured in a chondrogenic medium in free swelling (FS) conditions for 21 d, after which samples were subjected to dynamic compression (DC) of 10% strain (1 Hz, 1 h d(-1)) for a further 21 d. Both BM derived stem cells (BMSCs) and FP derived stem cells (FPSCs) were capable of generating cartilaginous tissues with near native levels of sulfated glycosaminoglycan (sGAG) content, although the spatial development of the engineered grafts strongly depended on the stem cell source. The mechanical properties of cartilage grafts generated from both stem cell sources also approached that observed in skeletally immature animals. Depending on the stem cell source and the donor, the application of DC either enhanced or had no significant effect on the functional development of cartilaginous grafts engineered using either BMSCs or FPSCs. BMSC seeded constructs subjected to DC stained less intensely for collagen type I. Furthermore, histological and micro-computed tomography analysis showed mineral deposition within BMSC seeded constructs was suppressed by the application of DC. Therefore, while the application of DC in vitro may only lead to modest improvements in the mechanical functionality of cartilaginous grafts, it may play an important role in the development of phenotypically stable constructs.

Generation of Neural Progenitor Spheres from Human Pluripotent Stem Cells in a Suspension Bioreactor.

PubMed

Yan, Yuanwei; Song, Liqing; Tsai, Ang-Chen; Ma, Teng; Li, Yan

2016-01-01

Conventional two-dimensional (2-D) culture systems cannot provide large numbers of human pluripotent stem cells (hPSCs) and their derivatives that are demanded for commercial and clinical applications in in vitro drug screening, disease modeling, and potentially cell therapy. The technologies that support three-dimensional (3-D) suspension culture, such as a stirred bioreactor, are generally considered as promising approaches to produce the required cells. Recently, suspension bioreactors have also been used to generate mini-brain-like structure from hPSCs for disease modeling, showing the important role of bioreactor in stem cell culture. This chapter describes a detailed culture protocol for neural commitment of hPSCs into neural progenitor cell (NPC) spheres using a spinner bioreactor. The basic steps to prepare hPSCs for bioreactor inoculation are illustrated from cell thawing to cell propagation. The method for generating NPCs from hPSCs in the spinner bioreactor along with the static control is then described. The protocol in this study can be applied to the generation of NPCs from hPSCs for further neural subtype specification, 3-D neural tissue development, or potential preclinical studies or clinical applications in neurological diseases.

Mechanical regulation of chondrogenesis

PubMed Central

2013-01-01

Mechanical factors play a crucial role in the development of articular cartilage in vivo. In this regard, tissue engineers have sought to leverage native mechanotransduction pathways to enhance in vitro stem cell-based cartilage repair strategies. However, a thorough understanding of how individual mechanical factors influence stem cell fate is needed to predictably and effectively utilize this strategy of mechanically-induced chondrogenesis. This article summarizes some of the latest findings on mechanically stimulated chondrogenesis, highlighting several new areas of interest, such as the effects of mechanical stimulation on matrix maintenance and terminal differentiation, as well as the use of multifactorial bioreactors. Additionally, the roles of individual biophysical factors, such as hydrostatic or osmotic pressure, are examined in light of their potential to induce mesenchymal stem cell chondrogenesis. An improved understanding of biomechanically-driven tissue development and maturation of stem cell-based cartilage replacements will hopefully lead to the development of cell-based therapies for cartilage degeneration and disease. PMID:23809493

Greater scaffold permeability promotes growth of osteoblastic cells in a perfused bioreactor.

PubMed

Fan, Jie; Jia, Xiaoling; Huang, Yan; Fu, Bingmei M; Fan, Yubo

2015-12-01

Pore size and porosity have been widely acknowledged as important structural factors in tissue-engineered scaffolds. In fact, scaffolds with similar pore size and porosity can provide important and varied permeability due to different pore shape, interconnectivity and tortuosity. However, the effects of scaffold permeability on seeded cells remains largely unknown during tissue regeneration in vitro. In this study, we measured the Darcy permeability (K) of tri-calcium phosphate scaffolds by distributed them into three groups: Low, Medium and High. As a result, the effects of scaffold permeability on cell proliferation, cellular activity and growth in the inner pores were investigated in perfused and static cultures in vitro. Results demonstrated that higher permeable scaffolds exhibited superior performance during bone regeneration in vitro and the advantages of higher scaffold permeability were amplified in perfused culture. Based on these findings, scaffold permeability should be considered in future scaffold fabrications. Copyright © 2013 John Wiley & Sons, Ltd.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture.

PubMed

Storm, Michael P; Sorrell, Ian; Shipley, Rebecca; Regan, Sophie; Luetchford, Kim A; Sathish, Jean; Webb, Steven; Ellis, Marianne J

2016-05-26

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Large-scale culture of a megakaryocytic progenitor cell line with a single-use bioreactor system.

PubMed

Nurhayati, Retno Wahyu; Ojima, Yoshihiro; Dohda, Takeaki; Kino-Oka, Masahiro

2018-03-01

The increasing application of regenerative medicine has generated a growing demand for stem cells and their derivatives. Single-use bioreactors offer an attractive platform for stem cell expansion owing to their scalability for large-scale production and feasibility of meeting clinical-grade standards. The current work evaluated the capacity of a single-use bioreactor system (1 L working volume) for expanding Meg01 cells, a megakaryocytic (MK) progenitor cell line. Oxygen supply was provided by surface aeration to minimize foaming and orbital shaking was used to promote oxygen transfer. Oxygen transfer rates (k L a) of shaking speeds 50, 100, and 125 rpm were estimated to be 0.39, 1.12, and 10.45 h -1 , respectively. Shaking speed was a critical factor for optimizing cell growth. At 50 rpm, Meg01 cells exhibited restricted growth due to insufficient mixing. A negative effect occurred when the shaking speed was increased to 125 rpm, likely caused by high hydrodynamic shear stress. The bioreactor culture achieved the highest growth profile when shaken at 100 rpm, achieving a total expansion rate up to 5.7-fold with a total cell number of 1.2 ± 0.2 × 10 9 cells L -1 . In addition, cells expanded using the bioreactor system could maintain their potency to differentiate following the MK lineage, as analyzed from specific surface protein and morphological similarity with the cells grown in the conventional culturing system. Our study reports the impact of operational variables such as shaking speed for growth profile and MK differentiation potential of a progenitor cell line in a single-use bioreactor. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:362-369, 2018. © 2017 American Institute of Chemical Engineers.

A xenogeneic-free bioreactor system for the clinical-scale expansion of human mesenchymal stem/stromal cells.

PubMed

Dos Santos, Francisco; Campbell, Andrew; Fernandes-Platzgummer, Ana; Andrade, Pedro Z; Gimble, Jeffrey M; Wen, Yuan; Boucher, Shayne; Vemuri, Mohan C; da Silva, Cláudia L; Cabral, Joaquim M S

2014-06-01

The large cell doses (>1 × 10(6)  cells/kg) used in clinical trials with mesenchymal stem/stromal cells (MSC) will require an efficient production process. Moreover, monitoring and control of MSC ex-vivo expansion is critical to provide a safe and reliable cell product. Bioprocess engineering approaches, such as bioreactor technology, offer the adequate tools to develop and optimize a cost-effective culture system for the rapid expansion of human MSC for cellular therapy. Herein, a xenogeneic (xeno)-free microcarrier-based culture system was successfully established for bone marrow (BM) MSC and adipose tissue-derived stem/stromal cell (ASC) cultivation using a 1L-scale controlled stirred-tank bioreactor, allowing the production of (1.1 ± 0.1) × 10(8) and (4.5 ± 0.2) × 10(7) cells for BM MSC and ASC, respectively, after 7 days. Additionally, the effect of different percent air saturation values (%Airsat ) and feeding regime on the proliferation and metabolism of BM MSC was evaluated. No significant differences in cell growth and metabolic patterns were observed under 20% and 9%Airsat . Also, the three different feeding regimes studied-(i) 25% daily medium renewal, (ii) 25% medium renewal every 2 days, and (iii) fed-batch addition of concentrated nutrients and growth factors every 2 days-yielded similar cell numbers, and only slight metabolic differences were observed. Moreover, the immunophenotype (positive for CD73, CD90 and CD105 and negative for CD31, CD80 and HLA-DR) and multilineage differentiative potential of expanded cells were not affected upon bioreactor culture. These results demonstrated the feasibility of expanding human MSC from different sources in a clinically relevant expansion configuration in a controlled microcarrier-based stirred culture system under xeno-free conditions. The further optimization of this bioreactor culture system will represent a crucial step towards an efficient GMP-compliant clinical-scale MSC production system. © 2014 Wiley Periodicals, Inc.

Perfusion directed 3D mineral formation within cell-laden hydrogels.

PubMed

Sawyer, Stephen William; Shridhar, Shivkumar Vishnempet; Zhang, Kairui; Albrecht, Lucas; Filip, Alex; Horton, Jason; Soman, Pranav

2018-06-08

Despite the promise of stem cell engineering and the new advances in bioprinting technologies, one of the major challenges in the manufacturing of large scale bone tissue scaffolds is the inability to perfuse nutrients throughout thick constructs. Here, we report a scalable method to create thick, perfusable bone constructs using a combination of cell-laden hydrogels and a 3D printed sacrificial polymer. Osteoblast-like Saos-2 cells were encapsulated within a gelatin methacrylate (GelMA) hydrogel and 3D printed polyvinyl alcohol (PVA) pipes were used to create perfusable channels. A custom-built bioreactor was used to perfuse osteogenic media directly through the channels in order to induce mineral deposition which was subsequently quantified via microCT. Histological staining was used to verify mineral deposition around the perfused channels, while COMSOL modeling was used to simulate oxygen diffusion between adjacent channels. This information was used to design a scaled-up construct containing a 3D array of perfusable channels within cell-laden GelMA. Progressive matrix mineralization was observed by cells surrounding perfused channels as opposed to random mineral deposition in static constructs. MicroCT confirmed that there was a direct relationship between channel mineralization within perfused constructs and time within the bioreactor. Furthermore, the scalable method presented in this work serves as a model on how large-scale bone tissue replacement constructs could be made using commonly available 3D printers, sacrificial materials, and hydrogels. © 2018 IOP Publishing Ltd.

Three-dimensional co-culture of human hepatocytes and mesenchymal stem cells: improved functionality in long-term bioreactor cultures.

PubMed

Rebelo, Sofia P; Costa, Rita; Silva, Marta M; Marcelino, Paulo; Brito, Catarina; Alves, Paula M

2017-07-01

The development of human cell models that can efficiently restore hepatic functionality and cope with the reproducibility and scalability required for preclinical development poses a significant effort in tissue engineering and biotechnology. Primary cultures of human hepatocytes (HHs), the preferred model for in vitro toxicity testing, dedifferentiate and have short-term viability in two-dimensional (2D) cultures. In this study, hepatocytes isolated from human liver tissue were co-cultured with human bone marrow mesenchymal stem cells (BM-MSCs) as spheroids in automated, computer-controlled, stirred-tank bioreactors with perfusion operation mode. A dual-step inoculation strategy was used, resulting in an inner core of parenchymal liver tissue with an outer layer of stromal cells. Hepatocyte polarization and morphology as well as the mesenchymal phenotype of BM-MSCs were maintained throughout the culture period and the crosstalk between the two cell types was depicted. The viability, compact morphology and phenotypic stability of hepatocytes were enhanced in co-cultures in comparison to monocultures. Gene expression of phase I and II enzymes was higher and CYP3A4 and CYP1A2 activity was inducible until week 2 of culture, being applicable for repeated-dose toxicity testing. Moreover, the excretory activity was maintained in co-cultures and the biosynthetic hepatocellular functions (albumin and urea secretion) were not affected by the presence of BM-MSCs. This strategy might be extended to other hepatic cell sources and the characterization performed brings knowledge on the interplay between the two cell types, which may be relevant for therapeutic applications. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Introducing Molecular Biology to Environmental Engineers through Development of a New Course.

ERIC Educational Resources Information Center

Oerther, Daniel B.

2002-01-01

Introduces a molecular biology course designed for environmental engineering majors using 16S ribosomal ribonucleic acid-targeted technology that allows students to identify and study microorganisms in bioreactor environments. (Contains 17 references.) (YDS)

Use of Orbital Shaken Disposable Bioreactors for Mammalian Cell Cultures from the Milliliter-Scale to the 1,000-Liter Scale

NASA Astrophysics Data System (ADS)

Zhang, Xiaowei; Stettler, Matthieu; de Sanctis, Dario; Perrone, Marco; Parolini, Nicola; Discacciati, Marco; de Jesus, Maria; Hacker, David; Quarteroni, Alfio; Wurm, Florian

Driven by the commercial success of recombinant biopharmaceuticals, there is an increasing demand for novel mammalian cell culture bioreactor systems for the rapid production of biologicals that require mammalian protein processing. Recently, orbitally shaken bioreactors at scales from 50 mL to 1,000 L have been explored for the cultivation of mammalian cells and are considered to be attractive alternatives to conventional stirred-tank bioreactors because of increased flexibility and reduced costs. Adequate oxygen transfer capacity was maintained during the scale-up, and strategies to increase further oxygen transfer rates (OTR) were explored, while maintaining favorable mixing parameters and low-stress conditions for sensitive lipid membrane-enclosed cells. Investigations from process development to the engineering properties of shaken bioreactors are underway, but the feasibility of establishing a robust, standardized, and transferable technical platform for mammalian cell culture based on orbital shaking and disposable materials has been established with further optimizations and studies ongoing.

Use of orbital shaken disposable bioreactors for mammalian cell cultures from the milliliter-scale to the 1,000-liter scale.

PubMed

Zhang, Xiaowei; Stettler, Matthieu; De Sanctis, Dario; Perrone, Marco; Parolini, Nicola; Discacciati, Marco; De Jesus, Maria; Hacker, David; Quarteroni, Alfio; Wurm, Florian

2009-01-01

Driven by the commercial success of recombinant biopharmaceuticals, there is an increasing demand for novel mammalian cell culture bioreactor systems for the rapid production of biologicals that require mammalian protein processing. Recently, orbitally shaken bioreactors at scales from 50 mL to 1,000 L have been explored for the cultivation of mammalian cells and are considered to be attractive alternatives to conventional stirred-tank bioreactors because of increased flexibility and reduced costs. Adequate oxygen transfer capacity was maintained during the scale-up, and strategies to increase further oxygen transfer rates (OTR) were explored, while maintaining favorable mixing parameters and low-stress conditions for sensitive lipid membrane-enclosed cells. Investigations from process development to the engineering properties of shaken bioreactors are underway, but the feasibility of establishing a robust, standardized, and transferable technical platform for mammalian cell culture based on orbital shaking and disposable materials has been established with further optimizations and studies ongoing.

Human cell culture in a space bioreactor

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1988-01-01

Microgravity offers new ways of handling fluids, gases, and growing mammalian cells in efficient suspension cultures. In 1976 bioreactor engineers designed a system using a cylindrical reactor vessel in which the cells and medium are slowly mixed. The reaction chamber is interchangeable and can be used for several types of cell cultures. NASA has methodically developed unique suspension type cell and recovery apparatus culture systems for bioprocess technology experiments and production of biological products in microgravity. The first Space Bioreactor was designed for microprocessor control, no gaseous headspace, circulation and resupply of culture medium, and slow mixing in very low shear regimes. Various ground based bioreactors are being used to test reactor vessel design, on-line sensors, effects of shear, nutrient supply, and waste removal from continuous culture of human cells attached to microcarriers. The small Bioreactor is being constructed for flight experiments in the Shuttle Middeck to verify systems operation under microgravity conditions and to measure the efficiencies of mass transport, gas transfer, oxygen consumption and control of low shear stress on cells.

Effect of human patient plasma ex vivo treatment on gene expression and progenitor cell activation of primary human liver cells in multi-compartment 3D perfusion bioreactors for extra-corporeal liver support.

PubMed

Schmelzer, Eva; Mutig, Kerim; Schrade, Petra; Bachmann, Sebastian; Gerlach, Jörg C; Zeilinger, Katrin

2009-07-01

Cultivation of primary human liver cells in innovative 3D perfusion multi-compartment capillary membrane bioreactors using decentralized mass exchange and integral oxygenation provides in vitro conditions close to the physiologic environment in vivo. While a few scale-up bioreactors were used clinically, inoculated liver progenitors in these bioreactors were not investigated. Therefore, we characterized regenerative processes and expression patterns of auto- and paracrine mediators involved in liver regeneration in bioreactors after patient treatment. Primary human liver cells containing parenchymal and non-parenchymal cells co-cultivated in bioreactors were used for clinical extra-corporeal liver support to bridge to liver transplantation. 3D tissue re-structuring in bioreactors was studied; expression of proteins and genes related to regenerative processes and hepatic progenitors was analyzed. Formation of multiple bile ductular networks and colonies of putative progenitors were observed within parenchymal cell aggregates. HGF was detected in scattered cells located close to vascular-like structures, expression of HGFA and c-Met was assigned to biliary cells and hepatocytes. Increased expression of genes associated to hepatic progenitors was detected following clinical application. The results confirm auto- and paracrine interactions between co-cultured cells in the bioreactor. The 3D bioreactor provides a valuable tool to study mechanisms of progenitor activation and hepatic regeneration ex vivo under patient plasma treatment. (c) 2009 Wiley Periodicals, Inc.

Design concepts for bioreactors in space

NASA Technical Reports Server (NTRS)

Seshan, P. K.; Peterson, G. R.; Beard, B.; Dunlop, E. H.

1986-01-01

Microbial food sources are becoming viable and more efficient alternatives to conventional food sources especially in the context of Closed Ecological Life Support Systems (CELSS) in space habitats. Since bioreactor designs for terrestrial operation will not readily apply to conditions of microgravity, there is an urgent need to learn about the differences. These differences cannot be easily estimated due to the complex nature of the mass transport and mixing mechanisms in fermenters. Therefore, a systematic and expeditious experimental program must be undertaken to obtain the engineering data necessary to lay down the foundations of designing bioreactors for microgravity. Two bioreactor design concepts presented represent two dissimilar approaches to grappling with the absence of gravity in space habitats and deserve to be tested for adoption as important components of the life support function aboard spacecrafts, space stations and other extra-terrestrial habitats.

Scaffold-free, Human Mesenchymal Stem Cell-Based Tissue Engineered Blood Vessels.

PubMed

Jung, Youngmee; Ji, HaYeun; Chen, Zaozao; Fai Chan, Hon; Atchison, Leigh; Klitzman, Bruce; Truskey, George; Leong, Kam W

2015-10-12

Tissue-engineered blood vessels (TEBV) can serve as vascular grafts and may also play an important role in the development of organs-on-a-chip. Most TEBV construction involves scaffolding with biomaterials such as collagen gel or electrospun fibrous mesh. Hypothesizing that a scaffold-free TEBV may be advantageous, we constructed a tubular structure (1 mm i.d.) from aligned human mesenchymal cell sheets (hMSC) as the wall and human endothelial progenitor cell (hEPC) coating as the lumen. The burst pressure of the scaffold-free TEBV was above 200 mmHg after three weeks of sequential culture in a rotating wall bioreactor and perfusion at 6.8 dynes/cm(2). The interwoven organization of the cell layers and extensive extracellular matrix (ECM) formation of the hMSC-based TEBV resembled that of native blood vessels. The TEBV exhibited flow-mediated vasodilation, vasoconstriction after exposure to 1 μM phenylephrine and released nitric oxide in a manner similar to that of porcine femoral vein. HL-60 cells attached to the TEBV lumen after TNF-α activation to suggest a functional endothelium. This study demonstrates the potential of a hEPC endothelialized hMSC-based TEBV for drug screening.

Application of Hydrogels in Heart Valve Tissue Engineering

PubMed Central

Zhang, Xing; Xu, Bin; Puperi, Daniel S.; Wu, Yan; West, Jennifer L.; Grande-Allen, K. Jane

2015-01-01

With an increasing number of patients requiring valve replacement, there is heightened interest in advancing heart valve tissue engineering (HVTE) to provide solutions to the many limitations of current surgical treatments. A variety of materials have been developed as scaffolds for HVTE including natural polymers, synthetic polymers, and decellularized valvular matrices. Among them, biocompatible hydrogels are generating growing interest. Natural hydrogels, such as collagen and fibrin, generally show good bioactivity, but poor mechanical durability. Synthetic hydrogels, on the other hand, have tunable mechanical properties; however, appropriate cell-matrix interactions are difficult to obtain. Moreover, hydrogels can be used as cell carriers when the cellular component is seeded into the polymer meshes or decellularized valve scaffolds. In this review, we discuss current research strategies for HVTE with an emphasis on hydrogel applications. The physicochemical properties and fabrication methods of these hydrogels, as well as their mechanical properties and bioactivities are described. Performance of some hydrogels including in vitro evaluation using bioreactors and in vivo tests in different animal models are also discussed. For future HVTE, it will be compelling to examine how hydrogels can be constructed from composite materials to replicate mechanical properties and mimic biological functions of the native heart valve. PMID:25955010

Automated Decellularization of Intact, Human-Sized Lungs for Tissue Engineering

PubMed Central

Price, Andrew P.; Godin, Lindsay M.; Domek, Alex; Cotter, Trevor; D'Cunha, Jonathan; Taylor, Doris A.

2015-01-01

We developed an automated system that can be used to decellularize whole human-sized organs and have shown lung as an example. Lungs from 20 to 30 kg pigs were excised en bloc with the trachea and decellularized with our established protocol of deionized water, detergents, sodium chloride, and porcine pancreatic DNase. A software program was written to control a valve manifold assembly that we built for selection and timing of decellularization fluid perfusion through the airway and the vasculature. This system was interfaced with a prototypic bioreactor chamber that was connected to another program, from a commercial source, which controlled the volume and flow pressure of fluids. Lung matrix that was decellularized by the automated method was compared to a manual method previously used by us and others. Automation resulted in more consistent acellular matrix preparations as demonstrated by measuring levels of DNA, hydroxyproline (collagen), elastin, laminin, and glycosaminoglycans. It also proved highly beneficial in saving time as the decellularization procedure was reduced from days down to just 24 h. Developing a rapid, controllable, automated system for production of reproducible matrices in a closed system is a major step forward in whole-organ tissue engineering. PMID:24826875