Effects of cryopreservation on human mesenchymal stem cells attached to different substrates.

There is a need to preserve cell-seeded scaffolds or cell-matrix constructs for tissue-engineering and other applications. Cryopreservation is likely to be the most practical method. The aim of this study was to investigate how cryopreservation affects cells attached to different substrates and how they respond differently from those in suspension. Human mesenchymal stem cells (hMSCs) were studied for their close relevance to tissue-engineering and stem cell therapy applications, in particular how cryopreservation affects cell adherence, cell growth and the viability of hMSCs attached to different substrates, including glass, gelatin, matrigel and a matrigel sandwich. The effects of cryopreservation on F-actin organization, intracellular pH and mitochondrial localization of the adherent hMSCs were further investigated. It was found that cells attached to a glass surface could hardly survive the common cryopreservation protocol using 10% DMSO and a 1°C/min cooling rate. By contrast, cells attached to gelatin and matrigel could survive to a greater extent. Furthermore, cryopreservation affected the potential of cell attachment and proliferation, resulted in distortion of F-actin, led to alteration of intracellular pH of the hMSCs for all tested substrates and caused a change in the mitochondrial localization of hMSCs on a matrigel substrate and in a matrigel sandwich. Our results showed that cell attachment and cell viability could be improved by changing the interaction between cell and substrate through modification of the substrate properties, which has implications for scaffold design if cell-seeded scaffolds or engineered tissues need to be cryopreserved.

Chitosan/gelatin porous scaffolds containing hyaluronic acid and heparan sulfate for neural tissue engineering.

The novel chitosan (Cs)/gelatin (Gel) porous scaffolds containing hyaluronic acid (HA) and heparan sulfate (HS) were fabricated via freeze-drying technique, and their physicochemical characteristics including pore size, porosity, water absorption, and in vitro degradation and biocompatibility were investigated. It was demonstrated that the Cs/Gel/HA/HS composite scaffolds had highly homogeneous and interconnected pores with porosity above 96% and average pore size ranging from 90 to 140 µm and a controllable degradation rate. The scanning electron microscopic images, cell viability assay, and fluorescence microscopy observation revealed that the presence of HA and HS in the scaffolds significantly promoted initial neural stem and progenitor cells (NS/PCs) adhesion and supported long-time growth in three-dimensional environment. Moreover, NS/PCs also maintained mutilineage differentiation potentials with enhanced neuronal differentiation upon induction in the Cs/Gel/HA/HS composite scaffolds in relation to Cs/Gel scaffolds. These results indicated that the Cs/Gel/HA/HS composite scaffolds were suitable for neural cells' adhesion, survival, and growth and could offer new and important options for neural tissue engineering applications.

Protocatechuic acid promotes the neuronal differentiation and facilitates survival of phenotypes differentiated from cultured neural stem and progenitor cells.

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia (A.) oxyphylla, plays crucial roles in the proliferation and neuroprotection of cultured neural stem and progenitor cells (NS/PCs) in our previous study. However, whether PCA modulates the differentiation of NS/PCs has remained to be elucidated. In this study, we show that PCA can promote the neuronal differentiation combined with fetal bovine serum (FBS) in vitro, although it cannot initiate the differentiation of NS/PCs by itself. Moreover, PCA is able to induce neuronal maturation and efficiently promote neurite outgrowth. On the other hand, PCA facilitates survival of phenotypes differentiated from cultured NS/PCs, which was associated with an increased percentage of the cellular viability and a decreased percentage of cells undergoing apoptosis under differentiation conditions. In addition, PCA-induced survival is also mediated with the activating of endogenous antioxidant enzymes. These results suggest that PCA may serve as a useful reference for future studies in designing stem cell strategies to promote brain recovery and repair in neurodegenerative diseases.

Slow-freezing cryopreservation of neural stem cell spheres with different diameters.

Neural stem cells (NSCs) are of great value for clinical application and scientific research. The development of efficient cryopreservation protocols could significantly facilitate the storage and transportation for clinic applications. The objective of the present study is to improve the survival rate and viability of NSCs. Neural stem cells with three states of single-cell suspension, NSC spheres with diameters of 30-50 microm and 80-100 microm, were cryopreserved by slow-freezing method with the cryoprotective agent (CPA) of dimethyl sulfoxide (Me(2)SO), respectively. Then the post-thawing NSCs were tested for the survival rate and the differentiation ability. As a result, NSC spheres with diameter of 80-100 microm and Me(2)SO concentration of 8% achieve the survival rate of 82.9%, and the NSCs still sustain the multi-differentiation potentiality. These results indicated that both the subtle interaction among NSCs and sphere diameters may affect the survival rate together.

Effect of protocatechuic acid from Alpinia oxyphylla on proliferation of human adipose tissue-derived stromal cells in vitro.

The effect of protocatechuic acid (PCA) from Alpinia oxyphylla and catapol from Rehmannia on the proliferation capacity of human adipose tissue-derived stromal cells (hADSCs) was investigated in vitro. Cell counts showed that treatment of hADSCs with PCA for 48 h increased the cell number in a dose-dependent manner, while no obvious effect of catapol on the proliferation of hADSCs was observed. In addition, the cell number of hADSCs treated by 1.5 mM PCA increased in a time-dependent manner. The flow cytometric analysis of DNA content demonstrated the cell cycle progress from the G0/G1 phase to the S phase. Western blot analysis revealed the elevated expression of cyclin D1 in hADSCs induced by PCA treatment. Cyclin D1-siRNA transfection significantly inhibit the promotion of cell proliferation by PCA. Furthermore, the flow cytometric analysis of the cell surface antigens and the multidifferential potential tests of PCA-treated hADSCs showed that the cells retained their functional characteristics of multipotential mesenchymal progenitors. It is concluded that PCA can effectively up-regulate the proliferation of hADSCs.

Cryoprotectants for the vitrification of corneal endothelial cells.

The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7kDa), dextran (MW 7kDa), chondroitin sulfate (CS, MW 18-30kDa), bovine serum albumin (MW 68kDa) and polyethylene glycol (MW 6kDa, 10kDa and 20kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4+/-2.1% (mean+/-SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.

Protocatechuic acid promotes cell proliferation and reduces basal apoptosis in cultured neural stem cells.

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, showed anti-oxidant neuroprotective property in our previous study. However, it is still unknown whether PCA have effects on the cultured neural stem cells (NSCs). In this study, we investigated the roles of PCA in the survival and apoptosis of rat NSCs under normal conditions. NSCs obtained from 13.5-day-old rat embryos were propagated as neurospheres and cultured under normal conditions with or without PCA for 4 and 7 days. The cell viability was determined by the cell counting kit-8 (CCK-8) test, while cell proliferation was assayed by bromodeoxyuridine (BrdU) labeling. PCA increased the cellular viability of NSCs and stimulated cell proliferation in a dose- and time-dependent manner. Apoptotic cells were detected after 4 days by observing the nuclear morphological changes and flow cytometric analysis. Compared with the control on both culture days, treatment with PCA effectively reduced the levels of apoptosis of NSCs. At the same time, the reactive oxygen species (ROS) level in NSCs was depressed. In addition, PCA also significantly decreased the activity of elevated caspase-3, indicating that PCA may inhibit apoptosis of NSCs via suppression of the caspase cascade. These results suggest that PCA may be a potential growth inducer and apoptosis inhibitor for NSCs.

Protocatechuic acid from Alpinia oxyphylla promotes migration of human adipose tissue-derived stromal cells in vitro.

Human adipose tissue-derived stromal cells (hADSCs) demonstrate promising potential in various clinical applications, including the transplantation to regenerate injured or degenerative tissues. The migration of engrafted hADSCs to the correct site of injure is essential for the curative effect of stem cell therapy. We found that protocatechuic acid (PCA) from Alpinia (A.) oxyphylla could promote the migration capacity of hADSCs through transwell coated with gelatin in vitro. PCA enhanced the cell migration rate in a dose-dependent and time-dependent manner. Meanwhile, RT-PCR and quantitative RT-PCR analysis revealed the elevation of membrane-type matrix metalloproteinase-1 (MT1-MMP) mRNA expression in 1.5 mM PCA-treated hADSCs. In the supernatants of these cells, the active matrix metalloproteinase-2 (MMP-2) increased compared with control cells with zymography. Moreover, the promotion of cell migration by PCA could be effectively and obviously inhibited by anti-MT1-MMP or anti-MMP-2 antibodies. Furthermore, flow cytometric analysis of the cell surface antigens, osteogenic induction, adipogenic induction and cardiomyocyte-like cell induction demonstrated that hADSCs retained their functional characteristics of multipotential mesenchymal progenitors after PCA treatment. These results suggest that PCA from A. oxyphylla promote the migration of hADSCs in vitro, which is partially due to the increased expression of MT1-MMP and the promotion of MMP-2 zymogen activation.

Enhanced L-(+)-lactic acid production by an adapted strain of Rhizopus oryzae using corncob hydrolysate.

Corncob is an economic feedstock and more than 20 million tons of corncobs are produced annually in China. Abundant xylose can be potentially converted from the large amount of hemicellulosic materials in corncobs, which makes the crop residue an attractive alternative substrate for a value-added production of a variety of bioproducts. Lactic acid can be used as a precursor for poly-lactic acid production. Although current industrial lactic acid is produced by lactic acid bacteria using enriched medium, production by Rhizopus oryzae is preferred due to its exclusive formation of the L-isomer and a simple nutrition requirement by the fungus. Production of L-(+)-lactic acid by R. oryzae using xylose has been reported; however, its yield and conversion rate are poor compared with that of using glucose. In this study, we report an adapted R. oryzae strain HZS6 that significantly improved efficiency of substrate utilization and enhanced production of L-(+)-lactic acid from corncob hydrolysate. It increased L-(+)-lactic acid final concentration, yield, and volumetric productivity more than twofold compared with its parental strain. The optimized growth and fermentation conditions for Strain HZS6 were defined.

Vitrification of corneal endothelial cells in a monolayer.

An in vitro model was developed for bovine natural corneal endothelia. The cells were cultured to a confluent monolayer and vitrified using 25% (w/w) 1,2-propanediol-35% (w/w) trehalose as cryoprotective agents. Approximately, 61.3% of the cells were viable using the protocol.

Three-dimensional expansion: in suspension culture of SD rat's osteoblasts in a rotating wall vessel bioreactor.

OBJECTIVE: To study large-scale expansion of SD (Sprague-Dawley) rat's osteoblasts in suspension culture in a rotating wall vessel bioreactor (RWVB). METHODS: The bioreactor rotation speeds were adjusted in the range of 0 to 20 rpm, which could provide low shear on the microcarriers around 1 dyn/cm2. The cells were isolated via sequential digestions of neonatal (less than 3 days old) SD rat calvaria. After the primary culture and several passages, the cells were seeded onto the microcarriers and cultivated in T-flask, spinner flask and RWVB respectively. During the culture period, the cells were counted and observed under the inverted microscope for morphology every 12 h. After 7 days, the cells were evaluated with scanning electron microscope (SEM) for histological examination of the aggregates. Also, the hematoxylin-eosin (HE) staining and alkaline phosphatase (ALP) staining were performed. Moreover, von-Kossa staining and Alizarin Red S staining were carried out for mineralized nodule formation. RESULTS: The results showed that in RWVB, the cells could be expanded by more than ten times and they presented better morphology and vitality and stronger ability to form bones. CONCLUSIONS: The developed RWVB can provide the culture environment with a relatively low shear force and necessary three-dimensional (3D) interactions among cells and is suitable for osteopath expansion in vitro.

Effect of extracellular ph on matrix synthesis by chondrocytes in 3D agarose gel.

In cartilage tissue engineering, the determination of the most appropriate cell/tissue culture conditions to maximize extracellular matrix synthesis is of major importance. The extracellular pH plays an important role in affecting energy metabolism and matrix synthesis by chondrocytes. In this study, chondrocytes were isolated from bovine articular cartilage, embedded in agarose gel, and cultured at varied pH levels (7.3-6.6). Rate of lactate production, total glycosaminoglycan (GAG) and collagen synthesis, as well as total cell numbers and cell viability were evaluated after culturing for up to 7 days. The results showed the rate of lactic acid production over the 7-day culture was significantly affected by extracellular pH; acidic pH markedly inhibited the production of lactate. Also, a biphasic response to extracellular pH in regard to total GAG synthesis was observed; the maximum synthesis was seen at pH 7.2. However, the collagen synthesis was not pH-dependent within the pH range explored. In addition, within the conditions studied, total cell numbers and cell viability were not significantly affected by extracellular pH. In conclusion, even minor changes in extracellular pH could markedly affect the metabolic activities and biosynthetic ability of chondrocytes. Consequently, the control of extracellular pH condition is crucially important for successful cartilage tissue engineering and for the study of chondrocyte physiology and functions.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: The model works well in describing the trajectory of a heavier microcarrier particle in rotating wall vessel.

Development of PDMS microbioreactor with well-defined and homogenous culture environment for chondrocyte 3-D culture.

Perfusion cell culture is believed to provide a stable culture environment due to the continuous supply of nutrients and removal of waste. However, the culture scales used in most cases were large, where the culture conditions can not be regarded as homogenous because of chemical gradients. To improve this, the concept of miniaturization is applied to 3-D cell culture. In this study, a simple perfusion microbioreactor was developed based on mass transport simulation to find out the reasonable culture scales with relatively lower chemical gradients. Besides, PDMS surface was treated with surfactant solution to reduce non-specific serum protein adsorption, which keeps the culture conditions steady. Chondrocyte 3-D culture using the proposed microbioreactors was compared with similar perfusion culture with a larger culture scale. Results showed that surfactant-treated PDMS surface could reduce serum protein adsorption by 85% over the native one. Also, microbioreactors were proved to provide a stable culture environment (e.g. pH) over the culture period. Cell culture scale of 200 microm thick culture construct was justified to have relatively lower chemical gradients than the larger scale perfusion culture. As a whole, the proposed culture system is capable of providing a well-defined and homogenous culture environment.

Comparison of different culture modes for long-term expansion of neural stem cells.

Neural stem cells (NSCs) can be cultured in two modes of suspension and monolayer in vitro. The cultured cells are different in both the ability to proliferate and heterogeneity. In order to find the appropriate methods for large-scale expansion of NSCs, we systematically compared the NSCs cultured in suspension with those cultured in monolayer. The forebrain tissue was removed from embryonic day 14 (E14) mice, then the tissue was dissociated into single-cell suspension by Accutase and mechanical trituration. The cells were cultured in both suspension and monolayer. The NSCs cultured in suspension and in monolayer were compared on viability, ability to proliferate and heterogeneity by fluorescent dyes, immunofluorescence and flow cytometry on DIV21 (21 days in vitro), DIV56 and DIV112, respectively. The results indicated that the NSCs cultured in both suspension and monolayer represented good viability in long-term cultures. But they displayed a distinct ability to proliferate in long-term cultures. The NSCs cultured in monolayer preceded those cultured in suspension on the ability to proliferate on DIV21 and DIV56, but no obvious difference on DIV112. The NSCs population cultured in suspension displayed more nestin-positive cells than those in monolayer during the whole process of culture. The NSCs population cultured in monolayer, however, displayed more betaIII tubulin-positive cells than those in suspension in the same period. The suspension culture mode excels the monolayer culture mode for large-scale expansion of NSCs.

A SU-8/PDMS hybrid microfluidic device with integrated optical fibers for online monitoring of lactate.

A microfluidic device with integrated optical fibres was developed for online monitoring of lactate. The device consists of a SU-8 waveguide, microfluidic channels and grooves for the insertion of optic fibres. It was fabricated by one-step photolithography of SU-8 polymer resist. Different channel widths (50-300 microm) were tested in terms of detection sensitivity. A wide range of flow rates were applied to investigate the influence of flow rate on signal fluctuations. The separation between optical fibre sensor and microfluidic channel and the width of fluidic channel have been optimized to maximize the detection sensitivity. It was revealed that 250 microm of channel width is the optimum light path length for a compromise between detection sensitivity and interference of ambient light. The independence of detection signals on flow rates was demonstrated within the range of flow rate (0.5-5 ml/hr) tested. Compared with conventional lactate detection, the device is proved to have high accuracy, relatively low limit of detection (50 mg/L) and reasonably fast response time (100 sec). The fabrication of device is simple and low cost. The present work has provided some fundamental data for further system optimization to meet specific detection requirements.