Human mesenchymal stem cells tissue development in 3D PET matrices

Human mesenchymal stem cells (hMSCs) are attractive cell sources for engineered tissue constructs with broad therapeutic potential. Three-dimensional (3D) hMSC tissue development in nonwoven poly(ethylene terephthalate) (PET) fibrous matrices was investigated. HMSCs were seeded onto 3D PET scaffolds and were cultured for over 1 month. Their proliferation rates were affected by seeding density but remained much lower than those of 2D controls. Compared to 2D surfaces, hMSCs grown in 3D scaffolds secreted and embedded themselves in an extensive ECM network composed of collagen I, collagen IV, fibronectin, and laminin. HMSCs were influenced by the orientation of adjacent PET fibers to organize the ECM proteins into highly aligned fibrils. We observed the increased expressions of alpha(2)beta(1) integrin but a slight decrease in the expression of alpha(5)beta(1) integrin in 3D compared to 2D culture and found that alpha(V)beta(3) was expressed only in 2D. Paxillin expression was down-regulated in 3D culture with a concomitant change in its localization patterns. We demonstrated the multi-lineage potentials of the 3D tissue constructs by differentiating the cells grown in the scaffolds into osteoblasts and adipocytes. Taken together, these results showed that hMSCs grown in 3D scaffolds display tissue development patterns distinct from their 2D counterparts and provide important clues for designing 3D scaffolds for developing tissue engineered constructs.     

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.     

Stem cell-derived extracellular matrix enables survival and multilineage differentiation within superporous hydrogels

Hydrophilic poly(ethylene glycol) diacrylate (PEGDA) hydrogel surfaces resist protein adsorption and are generally thought to be unsuitable for anchorage-dependent cells to adhere. Intriguingly, our previous findings revealed that PEGDA superporous hydrogel scaffolds (SPHs) allow anchorage of bone marrow derived human mesenchymal stem cells (hMSCs) and support their long-term survival. Therefore, we hypothesized that the physicochemical characteristics of the scaffold impart properties that could foster cellular responses. We examined if hMSCs alter their microenvironment to allow cell attachment by synthesizing their own extracellular matrix (ECM) proteins. Immunofluorescence staining revealed extensive expression of collagen type I, collagen type IV, laminin, and fibronectin within hMSC-seeded SPHs by the end of the third week. Whether cultured in serum-free or serum-supplemented medium, hMSC ECM protein gene expression patterns exhibited no substantial changes. The presence of serum proteins is required for initial anchorage of hMSCs within the SPHs but not for the hMSC survival after 24 h. In contrast to 2D expansion on tissue culture plastic (TCP), hMSCs cultured within SPHs proliferate similarly in the presence or absence of serum. To test whether hMSCs retain their undifferentiated state within the SPHs, cell-seeded constructs were cultured for 3 weeks in stem cell maintenance medium and the expression of hMSC-specific cell surface markers were evaluated by flow cytometry. CD105, CD90, CD73, and CD44 were present to a similar extent in the SPH and in 2D monolayer culture. We further demonstrated multilineage potential of hMSCs grown in the PEGDA SPHs, whereby differentiation into osteoblasts, chondrocytes, and adipocytes could be induced. The present study demonstrates the potential of hMSCs to alter the "blank" PEGDA environment to a milieu conducive to cell growth and multilineage differentiation by secreting adhesive ECM proteins within the porous network of the SPH scaffolds.     

Autocrine fibroblast growth factor 2‐mediated interactions between human mesenchymal stem cells and the extracellular matrix under varying oxygen tension

Human mesenchymal stromal or stem cells (hMSCs) are being investigated for cell therapy in a wide range of diseases. MSCs are a potent source of trophic factors and actively remodel their immediate microenvironment through the secretion of bioactive factors in response to external stimuli such as oxygen tension. In this study, we examined the hypothesis that hypoxia influences hMSC properties in part through the regulation of extracellular milieu characterized by the extracellular matrix (ECM) matrices and the associated fibroblast growth factor‐2 (FGF‐2). The decellularized ECM matrices derived from hMSC culture under both hypoxic (e.g., 2% O₂) and the standard culture (e.g., 20% O₂) conditions have different binding capacities to the cell‐secreted and exogenenous FGF‐2. The reduced hMSC proliferation in the presence of FGF‐2 inhibitor and the differential capacity of the decellularized ECM matrices in regulating hMSC osteogeneic and adipogenic differentiation suggest an important role of the endogenous FGF‐2 in sustaining hMSC proliferation and regulating hMSC fate. Additionally, the combination of the ECM adhesion and hypoxic culture preserved hMSC viability under serum withdrawal. Together, the results suggest the synergistic effect of hypoxia and the ECM matrices in sustaining hMSC ex vivo expansion and preserving their multi‐potentiality and viability under nutrient depletion. The results have important implication in optimizing hMSC expansion and delivery strategies to obtain hMSCs in sufficient quantity with required potency and to enhance survival and function upon transplantation. J. Cell. Biochem. 114: 716–727, 2013. © 2012 Wiley Periodicals, Inc.     

Endogenous extracellular matrices enhance human mesenchymal stem cell aggregate formation and survival

Human mesenchymal stem or stromal cell (hMSC) therapies have promise across a wide range of diseases. However, inefficient cell delivery and low cell survival at injury sites reduce efficacy and are the major barriers in hMSC‐based therapy. Formation of three‐dimensional (3D) hMSC aggregates has been found to activate hMSC functions from enhancing secretion of therapeutic factors for improving cell migration and survival. As the stromal cells in bone marrow, hMSCs are significant sources of extracellular matrix (ECM) proteins and growth factors, which form an interactive microenvironment to influence hMSC fate via paracrine and autocrine actions. To date, however, the impact of the extracellular microenvironment on hMSC properties in the aggregates remains unknown. In the present study, we investigated the role of endogenous ECM matrices on hMSC aggregate formation and survival under ischemic stress. The results demonstrated that the preservation of endogenous ECM in the aggregates formed by thermal lifting (termed TLAs) as opposed to the aggregates formed by enzymatically detached hMSCs (termed EDAs) enhanced cell proliferation, multilineage potential, and survival under ischemic stress. The improved cell proliferation and viability in the TLAs is attributed to the incorporation of endogenous ECM proteins in the TLAs and their promitotic and antioxidant properties. The results demonstrate a novel method for the formation of hMSC aggregates via thermal responsive surface and highlight the significant contribution of the ECM in preserving hMSC properties in the 3D aggregates. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 441–451, 2013     

Regulation of autocrine fibroblast growth factor‐2 signaling by perfusion flow in 3D human mesenchymal stem cell constructs

Perfusion bioreactor systems play a crucial role in mitigating nutrient limitation as well as providing biomechanical stimuli and redistributing regulatory macromolecules that influence human mesenchymal stem cells (hMSC) fate in three‐dimensional (3D) scaffolds. As fibroblast growth factor‐2 (FGF‐2) is known to regulate hMSC phenotype, understanding the role of autocrine FGF‐2 signaling in the 3D construct under the different perfusion flow provides important insight into an optimal bioreactor design. To investigate FGF‐2 signaling inhibition in hMSC cultured in the porous poly(ethylene terephthalate) (PET) scaffolds perfused under two flow configurations, PD173074, an FGFR1 inhibitor, was added in growth media after 7 day of pre‐culture and its impact on hMSC proliferation and clonogenicity during the subsequent 7 days of cultivation was analyzed. Compared with control constructs in growth media, the addition of PD173074 resulted in significant reduction in hMSC proliferation and colony formation in both constructs with a more dramatic reduction in the parallel flow constructs. The results demonstrate that autocrine FGF‐2 plays a significant role in 3D scaffold and suggest modulation of the perfusion flow in the bioreactor as a strategy to influence autocrine actions and cell fate in the 3D scaffold. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Perfusion regulation of hMSC microenvironment and osteogenic differentiation in 3D scaffold

The combination of hMSCs with 3D scaffolds has become an important approach to creating functional bone constructs. Bioreactors are important tools to mitigate mass transfer limitations and to provide controlled physiochemical and biomechanical environments for the 3D bone construct development. Media flow in the bioreactor systems is generally controlled either parallel or transverse with respect to the 3D construct, creating different cellular and biomechanical microenvironments in the 3D constructs. In this study, a custom designed modular perfusion bioreactor system was operated under either the parallel or transverse flow. The influence of the flow patterns on the characteristics of the hMSCs' cellular microenvironment and subsequent construct development was investigated. The parallel flow configuration retained ECM proteins and mitogenic growth factors within the scaffold, effectively preserving hMSC progenicity and proliferation potential (e.g., CFU-F, proliferation, and OCT-4), whereas the transverse flow induced hMSC osteogenic differentiation with higher ALP activity and calcium deposition and up-regulation of osteogenic bone markers (e.g., BMP-2, ALP, RUNX2, OSX, and OC). These results demonstrate the regulatory role of the macroscopic flow on the cellular microenvironment of the 3D hMSC construct, and suggest configuring media flow as a strategy for directing hMSC fate and 3D bone construct development in the perfusion bioreactor.     

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 decellularized amniotic membrane as a bioscaffold for bone marrow-derived mesenchymal stem cells: ultrastructural study

Developing effective stem cell-based therapies requires the design of complex in vitro culture systems for accurate representation of the physiological stem cell niche. Human amniotic membrane (hAM) has been successfully used in clinical grafting applications due to its unique biological and regenerative properties. Decellularized hAM (d-hAM) has been previously applied to the culture of human bone marrow mesenchymal stem cells (hMSCs), promoting their expansion and differentiation into adipogenic and osteogenic lineages. In the present study, hAM was decellularized by NaOH-treatment, to provide the three-dimensional (3D) bioscaffold for culturing hMSCs. The ultrastructural differences between intact hAM and decellularized hAM were characterized using the transmission electron microscope (TEM), as well as the 3D interaction between d-hAM and hMSCs cultured on the membrane. TEM examination of the intact hAM showed many microvilli on the epithelial layer cells, active Golgi apparatus, smooth endolplasmic reticulum and the characteristic pinocytic vesicles. The epithelial layer with its structures was absent in the d-hAM. However, no observable difference was detected in the ultrastructural characteristics of the compact stromal layer of d-hAM compared to intact hAM. Both contained bundles of extra cellular matrix (ECM) proteins, and scattered elastic fibres. Cultured human mesenchymal stem cells (hMSCs) examined by TEM appeared oval to spherical in shape and had a rough and non-uniform surface with distinct protrusions or irregular fillopodia. Their diameter ranged from 20.49 to 21.6 µm. Most of the cellular organelles were also noticed. SEM examination of the prepared samples revealed unique 3D interaction between the hMSC and d-hAM, where the latter seems to envelop the segments of the hMSCs lying on the surrounding membrane. This study shows that the decellularization process affected the epithelial layer only of hAM and had no effect on altering the presence of ECM components present in the stromal layer of the d-hAM. The interaction between hMSCs and d-hAM maybe mediated by hAM components other than human amniotic epithelial cells, such as ECM components or MSCs present in the deeper spongy layer of the membrane or/and the adhesive components of the basement membrane of the removed epithelial layer.     

Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells

Changes in oxygen concentrations affect many of the innate characteristics of stem and progenitor cells. Human mesenchymal stem cells (hMSCs) were maintained under hypoxic atmospheres (2% O(2)) for up to seven in vitro passages. This resulted in approximately 30-fold higher hMSC expansion over 6 weeks without loss of multi-lineage differentiation capabilities. Under hypoxia, hMSCs maintained their growth-rates even after reaching confluence, resulting in the formation of multiple cell layers. Hypoxic hMSCs also displayed differences in the cell and nuclear morphologies as well as enhanced ECM formation and organization. These changes in cellular characteristics were accompanied by higher mRNA levels of Oct-4 and HIF-2alpha, as well as increased expression levels of connexin-43, a protein used in gap junction formation. The results from this study demonstrated that oxygen concentrations affected many aspects of stem-cell physiology, including growth and in vitro development, and may be a critical parameter during expansion and differentiation.     

Human mesenchymal stem cell position within scaffolds influences cell fate during dynamic culture

Cell-based tissue engineering is limited by the size of cell-containing constructs that can be successfully cultured in vitro. This limit is largely a result of the slow diffusion of molecules such as oxygen into the interior of three-dimensional scaffolds in static culture. Bioreactor culture has been shown to overcome these limits. In this study we utilize a tubular perfusion system (TPS) bioreactor for the three-dimensional dynamic culture of human mesenchymal stem cells (hMSCs) in spherical alginate bead scaffolds. The goal of this study is to examine the effect of shear stress in the system and then quantify the proliferation and differentiation of hMSCs in different radial annuli of the scaffold. Shear stress was shown to have a temporal effect on hMSC osteoblastic differentiation with a strong correlation of shear stress, osteopontin, and bone morphogenic protein-2 occurring on day 21, and weaker correlation occurring at early timepoints. Further results revealed an approximate 2.5-fold increase in cell number in the inner annulus of TPS cultured constructs as compared to statically cultured constructs after 21 days. This result demonstrated a nutrient transfer limitation in static culture which can be mitigated by dynamic culture. A significant increase (P < 0.05) in mineralization in the inner and outer annuli of bioreactor cultured 4 mm scaffolds occurred on day 21 with 79 ± 29% and 53 ± 25% mineralization area, respectively, compared to 6 ± 4% and 19 ± 6% mineralization area, respectively, in inner and outer annuli of 4 mm statically cultured scaffolds. Surprising lower mineralization area was observed in 2 mm bioreactor cultured beads which had the highest levels of proliferation. These results may demonstrate a relationship between scaffold position and stem cell fate. In addition the decreased proliferation and matrix production in statically cultured scaffolds compared to bioreactor cultured constructs demonstrate the need for bioreactor systems and the effectiveness of the TPS bioreactor in promoting hMSC proliferation and differentiation in three-dimensional scaffolds.     

Aggregation of human mesenchymal stem cells enhances survival and efficacy in stroke treatment

Human mesenchymal stem cells (hMSCs) have been shown to enhance stroke lesion recovery by mediating inflammation and tissue repair through secretion of trophic factors. However, low cell survival and reduced primitive stem cell function of culture-expanded hMSCs are the major challenges limiting hMSC therapeutic efficacy in stroke treatment. In this study, we report the effects of short-term preconditioning of hMSCs via three-dimensional (3D) aggregation on stroke lesion recovery after intra-arterial (IA) transplantation of 3D aggregate-derived hMSCs (Agg-D hMSCs) in a transient middle cerebral artery occlusion (MCAO) stroke model. Compared with two-dimensional (2D) monolayer culture, Agg-D hMSCs exhibited increased resistance to ischemic stress, secretory function and therapeutic outcome. Short-term preconditioning via 3D aggregation reconfigured hMSC energy metabolism and altered redox cycle, which activated the PI3K/AKT pathway and enhanced resistance to in vitro oxidative stress. Analysis of transplanted hMSCs in MCAO rats using ultra-high-field magnetic resonance imaging at 21.1 T showed increased hMSC persistence and stroke lesion reduction by sodium (²³Na) imaging in the Agg-D hMSC group compared with 2D hMSC control. Behavioral analyses further revealed functional improvement in MCAO animal treated with Agg-D hMSCs compared with saline control. Together, the results demonstrated the improved outcome for Agg-D hMSCs in the MCAO model and suggest short-term 3D aggregation as an effective preconditioning strategy for hMSC functional enhancement in stroke treatment.     

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.     

N‐isopropylacrylamide‐based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion

Human mesenchymal stem cells (hMSCs) are colony‐forming unit fibroblasts (CFU‐F) derived from adult bone marrow and have significant potential for many cell‐based tissue‐engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N‐isopropylacrylamide (NIPAM)‐based thermoresponsive polyelectrolyte multilayer (N‐PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N‐PEMU films were made via layer‐by‐layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU‐F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer‐by‐layer approach is an efficient way to form PNIPAM‐based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU‐forming ability and their multi‐lineage differentiation potential. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Aggregation kinetics of human mesenchymal stem cells under wave motion

Human mesenchymal stem cells (hMSCs) are primary candidates in cell therapy and regenerative medicine but preserving their therapeutic potency following culture expansion is a significant challenge. hMSCs can spontaneously assemble into three‐dimensional (3D) aggregates that enhance their regenerative properties. The present study investigated the impact of hydrodynamics conditions on hMSC aggregation kinetics under controlled rocking motion. While various laboratory methods have been developed for hMSC aggregate production, the rocking platform provides gentle mixing and can be scaled up using large bags as in wave motion bioreactors. The results show that the hMSC aggregation is mediated by cell adhesion molecules and that aggregate size distribution is influenced by seeding density, culture time, and hydrodynamic conditions. The analysis of fluid shear stress by COMSOL indicated that aggregate size distribution is inversely correlated with shear stress and that the rocking angle had a more pronounced effect on aggregate size distribution than the rocking speed due to its impact on shear stress. hMSC aggregates obtained from the bioreactor exhibit increased stemness, migratory properties, and expression of angiogenic factors. The results demonstrate the potential of the rocking platform to produce hMSC aggregates with controlled size distribution for therapeutic application.     

Osteostimulation scaffolds of stem cells: BMP‐7‐derived peptide‐decorated alginate porous scaffolds promote the aggregation and osteo‐differentiation of human mesenchymal stem cells

The scaffolds for stem cell‐based bone tissue engineering should hold the ability to guide stem cells osteo‐differentiating. Otherwise, stem cells will differentiate into unwanted cell types or will form tumors in vivo. Alginate, a natural polysaccharide with great biocompatibility, was widely used in biomedical applications. However, the limited bioactivity and poor osteogenesis capability of pristine alginate hampered its further application in tissue engineering. In this work, a bone forming peptide‐1 (BFP‐1), derived from bone morphogenetic protein‐7, was grafted to alginate polymer chains to prepare peptide‐decorated alginate porous scaffolds (pep‐APS) for promoting osteo‐differentiation of human mesenchymal stem cells (hMSCs). SEM images of pep‐APS exhibited porous structure with about 90% porosity (pore size 100–300 μm), which was appropriate for hMSCs ingrowth. The adhesion, proliferation and aggregation of hMSCs grown on pep‐APS were enhanced in vitro. Moreover, pep‐APS promoted the alkaline phosphatase (ALP) activity of hMSCs, and the osteo‐related genes expression was obviously up‐regulated. The immunochemical staining and western blot analysis results showed high expression level of OCN and Col1a1 in the hMSCs grown on pep‐APS. This work provided a facile and valid strategy to endow the alginate polymers themselves with specific bioactivity and prepare osteopromoting scaffold with enhanced osteogenesis ability, possessing potential applications in stem cell therapy and regenerative medicine.     

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     

Effects of hypoxia on human mesenchymal stem cell expansion and plasticity in 3D constructs

Low oxygen tension is thought to be an integral component of the human mesenchymal stem cell (hMSC) native bone marrow microenvironment. HMSC were cultured under physiologically relevant oxygen environments (2% O2) in three-dimensional (3D) constructs for up to 1 month in order to investigate the combined effects of chronic hypoxia and 3D architecture on hMSC tissue-development patterns. Hypoxic hMSC exhibited an extended lag phase in order to acclimatize to culture conditions. However, they subsequently proliferated continuously throughout the culture period, while maintaining significantly higher colony-forming unit capabilities and expressing higher levels of stem cell genes than hMSC cultured at 20% O2 (normoxic) conditions. Upon induction, hypoxic hMSC also expressed higher levels of osteoblastic and adipocytic differentiation markers than normoxic controls. Hypoxia induced increased total protein levels in hMSC throughout the culture period, as well as significantly different fibronectin expression patterns suggesting that oxygen levels can significantly affect tissue-development patterns. Importantly, hMSC maintained the ability to thrive in prolonged hypoxic conditions suggesting that hypoxia may be an essential element of the in vivo hMSC niche. Further studies are required to determine how variations in cellular characteristics and ECM expression impact on the physiological properties of the engineered tissue, yet these results strongly indicate that oxygen tension is a key parameter that influences the in vitro characteristics of hMSC and their development into tissues.