Effects of oxygen transport on 3-d human mesenchymal stem cell metabolic activity in perfusion and static cultures: experiments and mathematical model

Human mesenchymal stem cells (hMSCs) have unique potential to develop into functional tissue constructs to replace a wide range of tissues damaged by disease or injury. While recent studies have highlighted the necessity for 3-D culture systems to facilitate the proper biological, physiological, and developmental processes of the cells, the effects of the physiological environment on the intrinsic tissue development characteristics in the 3-D scaffolds have not been fully investigated. In this study, experimental results from a 3-D perfusion bioreactor system and the static culture are combined with a mathematical model to assess the effects of oxygen transport on hMSC metabolism and proliferation in 3-D constructs grown in static and perfusion conditions. Cells grown in the perfusion culture had order of magnitude higher metabolic rates, and the perfusion culture supports higher cell density at the end of cultivation. The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 micromol/10(6) cells/h as cell density increases, suggesting intrinsic physiological change at high cell density. BrdU staining revealed the noneven spatial distribution of the proliferating cells in the constructs grown under static culture conditions compared to the cells that were grown in the perfusion system. The hypothesis that the constructs in static culture grow under oxygen limitation is supported by higher Y(L/G) in static culture. Modeling results show that the oxygen tension in the static culture is lower than that of the perfusion unit, where the cell density was 4 times higher. The experimental and modeling results show the dependence of cell metabolism and spatial growth patterns on the culture environment and highlight the need to optimize the culture parameters in hMSC tissue engineering.     

接种方式对细胞在三维材料中接种率和分布的影响

考察了静态和动态接种方式对成纤维细胞在胶原壳聚糖支架材料中接种率和分布的影响。将人成纤维细胞制成细胞悬液,分别采用静态接种、转瓶接种和灌注接种方式将细胞接入三维胶原壳聚糖海绵。通过MTT法和切片HE染色分别考察细胞接种率及细胞在三维材料中的分布。实验结果表明：在低的接种密度下静态接种有较高的接种率(889%),但随着接种密度的增加接种率下降显著,细胞结团且分布不均匀;转瓶接种的接种率约为60%,细胞分布也不均匀;灌注接种的接种率始终维持在77%以上,能得到高的起始细胞密度,且细胞分布均匀,是一种理想的接种方式。细胞接种方式的优化为改善工程化组织的结构和功能、缩短体外构建时间奠定了基础。     

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor

It has been widely demonstrated that perfusion bioreactors improve in vitro three‐dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C₂C₁₂ muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G₂/M) owing to medium perfusion in long‐term cultures. After 7–10 days, the percentage of proliferating cells was 8–12% for dynamic cultures and 3–5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Three-dimensional cell seeding and growth in radial-flow perfusion bioreactor for in vitro tissue reconstruction

Radial-flow perfusion bioreactor systems have been designed and evaluated to enable direct cell seeding into a three-dimensional (3-D) porous scaffold and subsequent cell culture for in vitro tissue reconstruction. However, one of the limitations of in vitro regeneration is the tissue necrosis that occurs at the central part of the 3-D scaffold. In the present study, tubular poly-L-lactic acid (PLLA) porous scaffolds with an optimized pore size and porosity were prepared by the lyophilization method, and the effect of different perfusion conditions on cell seeding and growth were compared with those of the conventional static culture. The medium flowed radially from the lumen toward the periphery of the tubular scaffolds. It was found that cell seeding under a radial-flow perfusion condition of 1.1 mL/cm2 x min was effective, and that the optimal flow rate for cell growth was 4.0 mL/cm2 x min. At this optimal rate, the increase in seeded cells in the perfusion culture over a period of 5 days was 7.3-fold greater than that by static culture over the same period. The perfusion cell seeding resulted in a uniform distribution of cells throughout the scaffold. Subsequently, the perfusion of medium and hence the provision of nutrients and oxygen permitted growth and maintenance of the tissue throughout the scaffold. The perfusion seeding/culture system was a much more effective strategy than the conventional system in which cells are seeded under a static condition and cultured in a bioreactor such as a spinner flask.     

Osteoinduction and survival of osteoblasts and bone-marrow stromal cells in 3D biphasic calcium phosphate scaffolds under static and dynamic culture conditions

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.     

Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices

The cell seeding density and spatial distribution in a 3-D scaffold are critical to the morphogenetic development of an engineered tissue. A dynamic depth-filtration seeding method was developed to improve the initial cell seeding density and spatial distribution in 3-D nonwoven fibrous matrices commonly used as tissue scaffolds. In this work, trophoblast-like ED27 cells were seeded in poly(ethylene terephthalate) (PET) matrices with various porosities (0.85-0.93). The effects of the initial concentration of cells in the suspension used to seed the PET matrix and the pore size of the matrix on the resulting seeding density and subsequent cell proliferation and tissue development were studied. Compared to the conventional static seeding method, the dynamic depth-filtration seeding method gave a significantly higher initial seeding density (2-4 x 10(7) vs 4 x 10(6) cells/cm3), more uniform cell distribution, and a higher final cell density in the tissue scaffold. The more uniform initial cell spatial distribution from the filtration seeding method also led to more cells in S phase and a prolonged proliferation period. However, both uniform spatial cell distribution and the pore size of the matrices are important to cell proliferation and morphological development in the seeded tissue scaffold. Large-pore matrices led to the formation of cell aggregates and thus might reduce cell proliferation. The dynamic depth-filtration seeding method is better in providing a higher initial seeding density and more uniform cell distribution and is easier to apply to large tissue scaffolds. A depth-filtration model was also developed and can be used to simulate the seeding process and to predict the maximum initial seeding densities in matrices with different porosities.     

Oncostatin M stimulates proliferation and functions of mouse fetal liver cells in three-dimensional cultures

In order to develop a tissue engineered bioartificial liver (BAL), long-term three-dimensional (3-D) culture of fetal liver cells (FLCs) utilizing porous polymer as a scaffold was performed for up to 1 month. The effects of the basal medium and supplementation with oncostatin M (OSM) on the proliferation and differentiation of mouse FLCs were examined in both 3-D culture and conventional monolayer dish culture. Compared with monolayer culture, cell numbers and hepatic function of FLCs were better maintained by 3-D culture. When two kinds of basal media were tested in this study, Williams' medium E (WE) was superior to minimum essential medium alpha (alphaMEM) in expressing hepatic function of FLCs in both 3-D and monolayer cultures, although higher cell densities were obtained with alphaMEM. OSM potently stimulated both cell growth and metabolic activity, especially in 3-D culture. When WE supplemented with OSM was used for 3-D culture, albumin secretion by FLCs increased dramatically after day 5, and a high level of secretion was maintained until the end of culture. During a period of over 1 month, no decrease of albumin secretion was observed. In conclusion, this 3-D culture method was expected to be one of the realistic attempts to develop a tissue engineered BAL.     

3-D computational modeling of media flow through scaffolds in a perfusion bioreactor

Media perfusion bioreactor systems have been developed to improve mass transport throughout three-dimensional (3-D) tissue-engineered constructs cultured in vitro. In addition to enhancing the exchange of nutrients and wastes, these systems simultaneously deliver flow-mediated shear stresses to cells seeded within the constructs. Local shear stresses are a function of media flow rate and dynamic viscosity, bioreactor configuration, and porous scaffold microarchitecture. We have used the Lattice-Boltzmann method to simulate the flow conditions within perfused cell-seeded cylindrical scaffolds. Microcomputed tomography imaging was used to define the scaffold microarchitecture for the simulations, which produce a 3-D fluid velocity field throughout the scaffold porosity. Shear stresses were estimated at various media flow rates by multiplying the symmetric part of the gradient of the velocity field by the dynamic viscosity of the cell culture media. The shear stress algorithm was validated by modeling flow between infinite parallel plates and comparing the calculated shear stress distribution to the analytical solution. Relating the simulation results to perfusion experiments, an average surface shear stress of 5x10(-5)Pa was found to correspond to increased cell proliferation, while higher shear stresses were associated with upregulation of bone marker genes. This modeling approach can be used to compare results obtained for different perfusion bioreactor systems or different scaffold microarchitectures and may allow specific shear stresses to be determined that optimize the amount, type, or distribution of in vitro tissue growth.     

Heterogenous morphogenesis of Caco-2 cells reveals that flow induces three-dimensional growth and maturation at high initial seeding cell densities

Here, we introduce a customized hanging insert fitting a six-well plate to culture Caco-2 cells on hydrogel membranes under flow conditions. The cells are cultured in the apical channel-like chamber, which provides about 1.3 dyn/cm² shear, while the basolateral chamber is mixed when the device is rocked. The device was tested by investigating the functional impact of the initial seeding density in combination with flow applied at confluency. The low seeding density cultures grew in two dimensional (2D) irrespective of the flow. Flow and higher seeding density resulted in a mixture of three dimensional (3D) structures and 2D layers. Static culture and high cell seeding density resulted in 2D layers. The flow increased the height and ZO-1 expression of cells in 2D layers, which correlated with an improved barrier function. Cultures with 3D structures had higher ZO-1 expression than 2D cultures, but this did not correlate with an increased barrier function. 2D monolayers in static and dynamic cultures had similar morphology and heterogeneity in the expression of Mucin-2 and Villin, while the 3D structures had generally higher expression of these markers. The result shows that the cell density and flow determine 3D growth and that the highest barrier function was obtained with low-density cultures and flow.     

A dynamic culture platform enhances the efficiency of the 3D HUVEC-based tube formation assay

Cell-based in vitro biological models traditionally use monolayer cell cultures grown over plastic surfaces bathing in static media. Higher fidelity to a natural biological tissue is expected to result from growing the cells in a three-dimensional (3D) matrix. However, due to the decreased rate of diffusion inherent to increased distances within a tridimensional space, proper fluidic conditions are needed in this setting to better approximate a physiological environment. To this aim, we here propose a prototypal dynamic cell culture platform for the automatic medium replacement, via periodic perfusion flow, in a human umbilical vein endothelial cell (HUVECs) culture seeded in a Geltrex™ matrix. A state-of-the-art angiogenesis assay performed in these dynamic conditions showed sizable effects with respect to conventional static control cultures, with significantly enhanced pro-(dual antiplatelet therapy [DAPT]) and anti-(EDTA) angiogenic compound activity. In particular, dynamic culture conditions (a) enhance the 3D-organization of HUVECs into microtubule structure; (b) accelerate and improve endothelial tube formation by HUVECs in the presence of DAPT; (c) are able to completely revert the blocking effects of EDTA. These evidence emphasize the need of setting proper fluidic conditions for a better approximation of a physiological environment as an appropriate evolution of current cell culture paradigms.     

Three-dimensional polymeric systems for cancer cell studies

Three-dimensional (3-D) culture of cancer cells and of normal mammalian cells in a polymeric matrix is generally a better alternate model for understanding the regulation of cancer cell proliferation and for evaluation of different anticancer drugs. A substantial amount of evidence demonstrates important differences in the behavior of cells grown in monolayer, i.e., two-dimensional (2-D), and in 3-D cultures. Cancer cells grown in 3-D culture are more resistant to cytotoxic agents than cells in 2-D culture; growth of cells in vitro in 3-D requires a suitable polymer that provides a structural scaffold for cell adhesion and growth. Many naturally derived polymers as well as synthetic polymers have been investigated as scaffolds. The aim of this review is to overview the polymeric materials of natural and synthetic origin that are of specific interest to 3-D cell cultures, and discuss the development of new polymers that should be specifically designed for 3-D culture applications.     

Various seeding methods for tissue development of human umbilical-cord-derived mesenchymal stem cells in 3-dimensional PET matrix

Mesenchymal stem cells derived from human umbilical cords (hUCMSCs) are attractive as a new cell source for tissue engineering. It is essential to investigate and optimize the seeding process of these cells for the success of cell culture and tissue regeneration in vitro. In this study, a static seeding method (SSM), a centrifugal seeding method (CSM), and a novel method-cycling filtration seeding method (CFSM) are evaluated in terms of seeding efficiency, cell damage, and distribution inside the scaffolds, cell proliferation, and osteogenic differentiation. Cells were seeded on three-dimensional (3-D) nonwoven PET discs at a density of 1×10⁴ cells/disc, followed by 21 days of cell culture and 20 days of osteogenic differentiation. Cells grown in 3-D conditions exhibited higher metabolic activity than those grown on a 2-D control surface. The CSM and CFSM groups showed higher seeding efficiency, proliferation capacity, and differentiation potential. H&E staining indicated a more uniform spatial distribution of cells in CFSM groups. LDH level measurements suggested that more cell damage was caused by the CFSM process. Above all, the results showed that the cells maintained their proliferation ability and differentiation potential ex vivo during approximately 7 weeks of culture. The CSM and CFSM are recommended for hUCMSC tissue engineering, although the seeding parameters still require further investigation and optimization.     

Effect of pulse frequency on the osteogenic differentiation of mesenchymal stem cells in a pulsatile perfusion bioreactor

Perfusion bioreactors are a promising in vitro strategy to engineer bone tissue because they supply needed oxygen and nutrients and apply an osteoinductive mechanical stimulus to osteoblasts within large porous three-dimensional scaffolds. Model two-dimensional studies have shown that dynamic flow conditions (e.g., pulsatile oscillatory waveforms) elicit an enhanced mechanotransductive response and elevated expression of osteoblastic proteins relative to steady flow. However, dynamic perfusion of three-dimensional scaffolds has been primarily examined in short term cultures to probe for early markers of mechanotransduction. Therefore, the objective of this study was to investigate the effect of extended dynamic perfusion culture on osteoblastic differentiation of primary mesenchymal stem cells (MSCs). To accomplish this, rat bone marrow-derived MSCs were seeded into porous foam scaffolds and cultured for 15 days in osteogenic medium under pulsatile regimens of 0.083, 0.050, and 0.017 Hz. Concurrently, MSCs seeded in scaffolds were also maintained under static conditions or cultured under steady perfusion. Analysis of the cells after 15 days of culture indicated that alkaline phosphatase (ALP) activity, mRNA expression of osteopontin (OPN), and accumulation of OPN and prostaglandin E(2) were enhanced for all four perfusion conditions relative to static culture. ALP activity, OPN and OC mRNA, and OPN protein accumulation were slightly higher for the intermediate frequency (0.05 Hz) as compared with the other flow conditions, but the differences were not statistically significant. Nevertheless, these results demonstrate that dynamic perfusion of MSCs may be a useful strategy for stimulating osteoblastic differentiation in vitro.     

Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development

Human mesenchymal stem cells (hMSCs) have great potential for therapeutic applications. A bioreactor system that supports long-term hMSCs growth and three-dimensional (3-D) tissue formation is an important technology for hMSC tissue engineering. A 3-D perfusion bioreactor system was designed using non-woven poly (ethylene terepthalate) (PET) fibrous matrices as scaffolds. The main features of the perfusion bioreactor system are its modular design and integrated seeding operation. Modular design of the bioreactor system allows the growth of multiple engineered tissue constructs and provides flexibility in harvesting the constructs at different time points. In this study, four chambers with three matrices in each were utilized for hMSC construct development. The dynamic depth filtration seeding operation is incorporated in the system by perfusing cell suspensions perpendicularly through the PET matrices, achieving a maximum seeding efficiency of 68%, and the operation effectively reduced the complexity of operation and the risk of contamination. Statistical analyses suggest that the cells are uniformly distributed in the matrices. After seeding, long-term construct cultivation was conducted by perfusing the media around the constructs from both sides of the matrices. Compared to the static cultures, a significantly higher cell density of 4.22 x 10(7) cell/mL was reached over a 40-day culture period. Cellular constructs at different positions in the flow chamber have statistically identical cell densities over the culture period. After expansion, the cells in the construct maintained the potential to differentiate into osteoblastic and adipogenic lineages at high cell density. The perfusion bioreactor system is amenable to multiple tissue engineered construct production, uniform tissue development, and yet is simple to operate and can be scaled up for potential clinical use. The results also demonstrate that the multi-lineage differentiation potential of hMSCs are preserved even after extensive expansion, thus indicating the potential of hMSCs for functional tissue construct development. The system has important applications in stem cell tissue engineering.     

Development of a bioreactor for evaluating novel nerve conduits

We describe an experimental closed bioreactor device for studying novel tissue engineered peripheral nerve conduits in vitro. The system integrates a closed loop system consisting of one, two, or three experimental nerve conduits connected in series or parallel, with the ability to study novel scaffolds within guidance conduits. The system was established using aligned synthetic microfiber scaffolds of viscose rayon and electrospun polystyrene. Schwann cells were seeded directly into conduits varying from 10 to 80 mm in length and allowed to adhere under 0 flow for 1 h, before being cultured for 4 days under static or continuous flow conditions. In situ viability measurements showed the distribution of live Schwann cells within each conduit and enabled quantification thereafter. Under static culture viable cells only existed in short conduit scaffolds (10 mm) or at the ends of longer conduits (20-80 mm) with a variation in viable cell distribution. Surface modification of scaffold fibers with type-1 collagen or acrylic acid increased cell number by 17% and 30%, respectively. However, a continuous medium flow of 0.8 mL/h was found to increase total cell number by 2.5-fold verses static culture. Importantly, under these conditions parallel viability measurements revealed a ninefold increase compared to static culture. Fluorescence microscopy of scaffolds showed cellular adhesion and alignment on the longitudinal axis. We suggest that such a system will enable a rigorous and controlled approach for evaluating novel conduits for peripheral nerve repair, in particular using hydrolysable materials for the parallel organization of nerve support cells, prior to in vivo study.     

Perfusion affects the tissue developmental patterns of human mesenchymal stem cells in 3D scaffolds

Human mesenchymal stem cells (hMSCs) developed in three‐dimensional (3D) scaffolds are significantly affected by culture conditions. We hypothesized that the hydrodynamic forces generated in perfusion bioreactors significantly affected hMSC functionality in 3D scaffolds by shaping the extracellular matrix (ECM) proteins. In this study, hMSCs were grown in 3D poly(ethylene terephthalate) (PET) scaffolds in static and a parallel perfusion system under similar initial conditions for up to 35 days. Results demonstrated that even at very low media velocities (O [10^?4 cm/sec]), perfusion cultures affected the ability of hMSCs to form an organized ECM network as illustrated by the immunostaining of collagen I and laminin fibrous structure. The change in the ECM microenvironment consequently influenced the nuclear shape. The hMSCs grown at the lower surface of static culture displayed a 15.2 times higher nuclear elongation than those at the upper surface, whereas cells grown in the perfusion bioreactor displayed uniform spherical nuclei on both surfaces. The difference in ECM organization and nuclear morphology associated with gene expression and differentiation characteristics of hMSCs. The cells exhibited lower CFU‐F colony forming ability and decreased expressions of stem‐cell genes of Rex‐1 and Oct‐4, implying a less primitive stem‐cell phenotype was maintained in the perfusion culture relative to the static culture conditions. The significantly higher expression level of osteonectin gene in the perfusion culture at day 28 indicated an upregulation of osteogenic ability of hMSCs. The study highlights the critical role of dynamic culture conditions on 3D hMSC construct development and properties. J. Cell. Physiol. 219: 421–429, 2009. © 2009 Wiley‐Liss, Inc.     

Engineering of osteoinductive grafts by isolation and expansion of ovine bone marrow stromal cells directly on 3D ceramic scaffolds

In this work, we investigated whether osteoinductive constructs can be generated by isolation and expansion of sheep bone marrow stromal cells (BMSC) directly within three-dimensional (3D) ceramic scaffolds, bypassing the typical phase of monolayer (2D) expansion prior to scaffold loading. Nucleated cells from sheep bone marrow aspirate were seeded into 3D ceramic scaffolds either by static loading or under perfusion flow and maintained in culture for up to 14 days. The resulting constructs were exposed to enzymatic treatment to assess the number and lineage of extracted cells, or implanted subcutaneously in nude mice to test their capacity to induce bone formation. As a control, BMSC expanded in monolayer for 14 days were also seeded into the scaffolds and implanted. BMSC could be isolated and expanded directly in the 3D ceramic scaffolds, although they proliferated slower than in 2D. Upon ectopic implantation, the resulting constructs formed a higher amount of bone tissue than constructs loaded with the same number of 2D-expanded cells. Constructs cultivated for 14 days generated significantly more bone tissue than those cultured for 3 days. No differences in bone formation were found between samples seeded by static loading or under perfusion. In conclusion, the culture of bone marrow nucleated cells directly on 3D ceramic scaffolds represents a promising approach to expand BMSC and streamline the engineering of osteoinductive grafts.     

3D culture of osteoblast‐like cells by unidirectional or oscillatory flow for bone tissue engineering

A medium perfusion system is expected to be beneficial for three‐dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast‐like MC 3T3‐E1 cells were 3D‐cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell‐seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering. Biotechnol. Bioeng. 2009;102: 1670–1678. © 2008 Wiley Periodicals, Inc.     

Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: An in vitro system with application to the bioartificial liver

A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.Abbreviations FDA fluorescein diacetate - EB ethidium bromide - MEGX monoethylglycinexylidide     

Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization.

Cardiac tissue engineering has been motivated by the need to create functional tissue equivalents for scientific studies and cardiac tissue repair. We previously demonstrated that contractile cardiac cell-polymer constructs can be cultivated using isolated cells, 3-dimensional scaffolds, and bioreactors. In the present work, we examined the effects of (1) cell source (neonatal rat or embryonic chick), (2) initial cell seeding density, (3) cell seeding vessel, and (4) tissue culture vessel on the structure and composition of engineered cardiac muscle. Constructs seeded under well-mixed conditions with rat heart cells at a high initial density ((6-8) x 10(6) cells/polymer scaffold) maintained structural integrity and contained macroscopic contractile areas (approximately 20 mm(2)). Seeding in rotating vessels (laminar flow) rather than mixed flasks (turbulent flow) resulted in 23% higher seeding efficiency and 20% less cell damage as assessed by medium lactate dehydrogenase levels (p < 0.05). Advantages of culturing constructs under mixed rather than static conditions included the maintenance of metabolic parameters in physiological ranges, 2-4 times higher construct cellularity (p &le 0.0001), more aerobic cell metabolism, and a more physiological, elongated cell shape. Cultivations in rotating bioreactors, in which flow patterns are laminar and dynamic, yielded constructs with a more active, aerobic metabolism as compared to constructs cultured in mixed or static flasks. After 1-2 weeks of cultivation, tissue constructs expressed cardiac specific proteins and ultrastructural features and had approximately 2-6 times lower cellularity (p < 0.05) but similar metabolic activity per unit cell when compared to native cardiac tissue.