Recovery of human mesenchymal stem cells grown on novel microcarrier coated with thermoresponsive polymer.

The cultivation of cells on microcarriers (MCs) in stirred suspension system is useful method for the large-scale culture of human mesenchymal stem cells (hMSCs) for allogenic transplantation. To harvest hMSCs from MCs without using proteolytic enzyme treatment but by lowering temperature, polystyrene MCs coated with a copolymer called CAT having zwitterionic and thermoresponsive characteristics, which has a lower critical solution temperature (LCST) in the range of 28-32 â„ƒ, were developed and compared with those coated with poly(N-isopropylacrylamide) (PNIPAM), which has an LCST almost the same as that of the CAT copolymer. A preliminary study using polystyrene dishes coated with the CAT copolymer at various densities showed superior adhesion efficiency and cell growth compared with those coated with PNIPAM; however, the rate of cell recovery by lowering the temperature to 24 â„ƒ was only about 80% in both cases. Although cells grew on polystyrene MCs coated with PNIPAM (0.64-16 µg/cm2) and on those coated with CAT (0.0050-1.0 µg/cm2), the cell recovery rate at 24 â„ƒ was lower than 20%. The decrease in recovery temperature from 24 to 4 â„ƒ resulted in about 50% cell recovery from CAT-coated (0.010-0.10 µg/cm2) MC, whereas the rate of cell recovery from PNIPAM-coated MC remained at about 20%. CAT (0.20 µg/cm2) coating after treatment of polystyrene MCs with oxygen plasma discharge increased the cell recovery rate to 72% at 4 â„ƒ. Consequently, the combination of oxygen plasma discharge treatment and CAT coating of polystyrene MCs might provide not only adhesion efficiency and growth of MSCs comparable to those on polystyrene MCs without any treatment but also a high cell recover rate of more than 70%.

Effect of epigallocatechin-3-gallate on the increase in type II collagen accumulation in cartilage-like MSC sheets.

With the aim to increase type II collagen content in the scaffold-free cartilage-like cell sheet using human bone marrow mesenchymal stem cells, we examined the effect of epigallocatechin-3-gallate (EGCG) addition to the chondrogenic medium for the cell sheet culture. The addition of EGCG (10 µM) increased the content of type II collagen 2-fold, while the addition did not markedly change the expression level of the genes encoding type II collagen and Sox 9. The reactive oxygen species level in the cells in cell sheets was thought to be too low to suppress the accumulation of type II collagen. On the other hand, the addition of EGCG markedly decreased both the matrix metalloproteinase-13 concentration in the supernatant of cell sheet culture and the type II collagen degradation activity in that supernatant. Taken together, EGCG may enhance the accumulation of type II collagen by suppressing type II collagen degradation.

Effect of epigallocatechin-3-o-gallate and quercetin on the cryopreservation of cartilage tissue.

The effects of epigallocatechin-3-o-gallate (EGCG) and quercetin on the contents of extracellular matrix (ECM) in porcine cartilage at 4 °C were investigated. The addition of quercetin at 0.01 mM for the incubation of porcine cartilage disks at 4 °C for 2 week could suppress the decrease in ECM and the compliance of the disks, markedly greater than those of EGCG (1.0 mM).

Construction of an osteochondral-like tissue graft combining ß-tricalcium phosphate block and scaffold-free mesenchymal stem cell sheet.

BACKGROUND: Aiming to construct an osteochondral-like structure, the combination of a ß-tricalcium phosphate (ßTCP) block with a scaffold-free sheet formed using mesenchymal stem cells (MSCs) was investigated. METHODS: Human bone marrow MSCs in a cell culture insert that was set in a 24-well plate were cultivated using a chondrogenic medium containing dexamethasone, IGF-1, and TGFß3 for 3 weeks during which a cylindrical ßTCP block was put on the sheet at day 1, and the cell sheet construct was harvested. In other experiments, at day 14, the construct was put on a cell sheet that was prepared the day before and cultivated for 3 weeks. RESULTS: The addition of a ßTCP block resulted in a combined osteochondral-like construct and comparable staining intensity by Alcian blue, while the expression levels of the aggrecan and type II collagen genes decreased a little. During the culture with the ßTCP block, the expression levels of the aggrecan gene increased monotonically. The increase in the inoculum cell number from 1.86 to 3.72 × 10(6) cells resulted in marked increases in the thickness of cell sheet parts in the ßTCP block and expression levels of the aggrecan and type II collagen genes, while the thickness of cell sheet parts on the ßTCP block scarcely changed. On the other hand, the addition of a cell sheet that was prepared a day before to the construct at day 14 resulted in the marked increase in thickness of the cell sheet part on the ßTCP block, while the thickness of that in the ßTCP block did not increase. CONCLUSION: A combined osteochondral-like structure was produced by putting a ßTCP block on the sheet of MSC. The thickness of the cell sheet parts in and on the ßTCP block could be increased by the increase in inoculum cell number and by providing an additional cell sheet, respectively.

Effect of combination of residual glucose concentration and subsequent increment by temporal glucose feeding on oscillation of clock gene Per2 expression.

With the aim of regulating clock gene expression to control cell activities in cell processing engineering, the effect of the combination of residual glucose concentration and subsequent increment by temporal glucose feeding on the oscillation of the expression of clock gene Per2 was investigated employing rat Mesenchymal stem cell (MSC)-like cells having Per2 promoter gene with a destabilized luciferase gene (Per2-dLuc). Two experiments with several initial glucose concentrations and different times of cultures (2 and 5 days) before temporal glucose feeding (0.9 g/L) were employed to realize various concentrations of residual glucose in the medium before the feeding. In these experiments, the lower residual glucose concentrations (0.002-0.02 g/L) before temporal glucose feeding tended to induce the larger amplitude of oscillation of Per2 expression than the higher ones (0.55-0.74 g/L). When the residual glucose concentration before glucose feeding was low (0.014-0.038 g/L), the higher temporal glucose concentration (0.23-0.9 g/L) feeding tended to induce the larger amplitude of oscillation of Per2 expression than the lower ones (0.012-0.023 g/L). Taken together, we found that the amplitude of oscillation of the expression of clock gene Per2 could be controlled by the combination of residual glucose concentration and glucose concentration of subsequent temporal feeding.

Construction of osteochondral-like tissue graft combining ß-tricalcium phosphate block and scaffold-free centrifuged chondrocyte cell sheet.

BACKGROUND: The combination of a ß-tricalcium phosphate (ßTCP) block with a scaffold-free chondrocyte sheet formed by the centrifugation of chondrocytes in a well was investigated with the aim of constructing an osteochondral-like structure. METHODS: Human and porcine articular cartilage chondrocytes were respectively centrifuged in a 96-well plate or cell culture insert (0.32 cm(2)) that was set in a 24-well plate, cultivated in the respective vessel for 3 weeks, and the cell sheets were harvested. In some cases, a cylindrical ßTCP block (diameter 5 mm, height 3 mm) was placed on the sheet on days 1-7. The sheet size, cell number, and sulfated glycosaminoglycan accumulation were determined. RESULTS: The use of a 96-well plate for not suspension but adhesion culture and the initial centrifugation of a well containing cells were crucial to obtaining a uniformly thick cell sheet. The glycosaminoglycan density of the harvested cell sheet was comparable to that of the pellet culture. An inoculum cell number of more than 31 × 10(5) cells tended to result in a curved cell sheet. Culture involving 18.6 × 10(5) cells and the 96-well plate for adhesion culture showed no curving of the cell sheet (thickness of 0.85 mm), and these were found to be the best of the culture conditions tested. The timing of the addition of a ßTCP block to the cell sheet (1-7 days) markedly affected the balance between the thickness of cell sheet parts on and in the ßTCP block. CONCLUSION: Centrifugation and subsequent cultivation of chondrocytes (18.6 × 10(5) cells) in a 96-well plate for adhesion culture led to the production of a scaffold-free cartilage-like cell sheet with a thickness of 0.85 mm. A combined osteochondral-like structure was produced by putting a ßTCP block on the cell sheet. The thickness of the cell sheet on the ßTCP block and the binding strength between the cell sheet and the ßTCP block could be optimized by adjusting the inoculum cell number and timing of ßTCP block addition.

Polymer fraction including exosomes derived from Chinese hamster ovary cells promoted their growth during serum-free repeated batch culture.

While continuous (perfusion) culture of mammalian cells might reduce the reactor size owing to the high cell density, there is the problem of higher medium cost; however, this problem is expected to be solved by the reuse of growth-promoting components in the culture supernatant. The polymer fraction (PF, 10 kDa-220 nm) collected from the supernatant of serum-free repeated-batch culture of Chinese hamster ovary (CHO) cells in not only adhesion but also suspension promoted the cell growth in respective serum-free cultures. PF contained CD81-positive exosomes and proteins, both of which were necessary for its growth-promoting activity. Consequently, the medium cost for the continuous (perfusion) serum-free suspension culture of CHO cells may be decreased by the repeated collection and addition of PF that contains exosomes and growth factor proteins.

Comparison of percentage of CD90-positive cells and osteogenic differentiation potential between mesenchymal stem cells grown on dish and nonwoven fabric.

Although nonwoven fabric (NWF) has been reported to be a candidate scaffold for the large-scale expansion of mesenchymal stem cells (MSCs), the quality of cells grown in NWF has not been well clarified. In this report, MSCs grown in an NWF disc for 3 weeks showed higher osteogenic differentiation potential and percentage of CD90 (+) cells than MSCs grown on the bottom surface of dish. The amount of the extracellular matrix (ECM) per unit surface area of fibers was larger than that on the bottom surface of the dish in the first 2 weeks of culture. The osteogenic differentiation potential of MSCs inoculated onto cell-free ECM increased with increasing amount of ECM. The higher percentage of CD90 (+) cells and osteogenic differentiation potential of cells grown in an NWF disc than of cells grown on a dish might, at least in part, be due to the higher amount of ECM.

Correlation between cell cycle phase of adherent Chinese hamster ovary cells and laser phase shift determined by phase-shifting laser microscopy.

The simultaneous determination of the cell cycle phase of individual adherent Chinese hamster ovary cells using a fluorescence microscope after staining with 4',6-diamidine-2'-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by a phase-shifting laser microscopy revealed that the laser phase shift of cells in the G2/M phase was markedly higher than that of cells in the G1 and S phases.

Protocol of mesenchymal stem cell inoculation to nonwoven fabric scaffold.

To obtain a large number of human mesenchymal stem cell (hMSCs) for allograft, nonwoven fabrics (NWF) were used as a cell culture scaffold. NWF are three-dimensional fiber aggregates formed by heat bonding and have a high surface area for cell adhesion and elongation. Inoculation hMSC was done to a center of NWF disc (diameter, 15.1 mm; depth, 0.1 mm). A cell suspension inoculum had a volume of 10 µL, which was close to the void volume of the disc, and resulted in a high initial (24 h) cell adhesion efficiency. Use of green fluorescent protein expressing rat MSCs and fluorescence microscopy revealed that adding an additional 10 µL of medium at 0-2 h after the cell inoculation made the initial horizontal distribution of cells in the NWF disc more uniform. Addition of 10 µL of the medium after 1 and 2 h of hMSC inoculation (0.15 × 103 cells/cm2 NWF-fiber) markedly increased the final cell density (21 days) from 2.48 to 7.45 × 103 cells/cm2 NWF-fiber and fold increase in cell density by 16-48-fold. In conclusion, the addition of an additional medium after inoculation made the initial cells distribution in NWF more uniform, which might result in higher final cell density.

Noninvasive measurement of three-dimensional morphology of adhered animal cells employing phase-shifting laser microscope.

Noninvasive measurement of 3-D morphology of adhered animal cells employing a phase-shifting laser microscope (PLM) is investigated, in which the phase shift for each pixel in the view field caused by cell height and the difference in refractive indices between the cells and the medium is determined. By employing saline with different refractive indices instead of a culture medium, the refractive index of the cells, which is necessary for the determination of cell height, is determined under PLM. The observed height of Chinese hamster ovary (CHO) cells cultivated under higher osmolarity is lower than that of the cells cultivated under physiological osmolarity, which is in agreement with previous data observed under an atomic force microscope (AFM). Maximum heights of human bone marrow mesenchymal stem cells and human umbilical cord vein endothelial cells measured under PLM and AFM agree well with each other. The maximum height of nonadherent spherical CHO cells observed under PLM is comparable to the cell diameter measured under a phase contrast inverted microscope. Laser irradiation, which is necessary for the observation under PLM, did not affect 3-D cell morphology. In conclusion, 3-D morphology of adhered animal cells can be noninvasively measured under PLM.

Effect of static pressure on intracellular pH of adhesive Chinese hamster ovary cells.

The effect of static pressure on the intracellular pH of the Chinese hamster ovary (CHO) cell line DR1000L4N was investigated. In cultivation of CHO cells at 0.9 MPa, two distinct populations were observed in the histogram of a flow cytometer, while single population was observed in cultivation at 0.1 MPa. The intracellular pH of the major population at 0.9 MPa was markedly lower than that of the single population at 0.1 MPa.

High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells.

The effects of the density of human mesenchymal stem cells (MSCs) on their differentiation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGFbeta3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05 x 10(4) to 0.9 x 10(4) cells/cm(2) accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2) accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2), whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05 x 10(4) cells/cm(2) did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3 x 10(4) cells/cm(2) showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05 x 10(4) cells/cm(2). Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes.

Effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of human mesenchymal stem cells.

With the aim to utilize human mesenchymal stem cells (hMSCs) grown in large scale for regenerative medicine, effects of agitation rate on aggregation during beads-to-beads subcultivation of microcarrier culture of hMSCs were studied. hMSCs could attach and grew on surface-type microcarriers of Cytodex 1, whereas almost no cell elongation and growth were observed on porous type microcarriers of Cytopores. The percentages of aggregated Cytodex 1 microcarriers at an agitation rate of 60 and 90 rpm were lower than that at 30 rpm, which was the lowest agitation rate necessary for the suspension of Cytodex 1 microcarriers, and the cells grew fastest at 60 rpm. hMSC could be subcultivated on Cytodex 1 by the beads-to-beads method at both 30 and 60 rpm without trypsinization. However, agitation at 60 rpm resulted in a markedly lower percentage of aggregated microcarriers not only before but also after subcultivation. The percentages of CD90- and CD166-positive cells among cells grown on Cytodex 1 at 60 rpm (91.5 and 87.6 %) were comparable to those of cells grown in the pre-culture on dishes. In conclusion, hMSCs could be subcultivated on Cytodex 1 by beads-to-beads method maintaining the expressions of the cell surface antigens CD90 and CD166, while adjusting agitation rate could decrease the microcarrier aggregation.

Synergistic effect of ascorbic acid and collagen addition on the increase in type 2 collagen accumulation in cartilage-like MSC sheet.

Aiming to increase the content of type 2 collagen in scaffold-free cartilage-like cell sheets prepared using human bone marrow mesenchymal stem cells, the effect of several kinds of additives in a chondrogenic medium was investigated. Addition of ascorbic acid 2 phosphate (VCP) at a high concentration (250 µg/ml) and type 1 atelocollagen (5 µg/ml) increased the accumulation of type 2 collagen by fourfold and twofold, respectively. On the other hand, an antioxidant, glutathione showed no such effect. The synergistic effect of VCP and type 1 atelocollagen resulted in an eightfold increase in the accumulation level of type 2 collagen. Furthermore, the gene expression level of type 2 collagen increased and that of matrix metalloproteinase-13 (MMP-13) decreased to approximately one-third of the control. The increase in type 2 collagen accumulation in the scaffold-free cartilage-like cell sheet might be due to not only the enhancement of the synthesis but also the suppression of the degradation of type 2 collagen by MMP-13.

Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells.

In order to get the high liquefaction yield from marine algae cell mass to fuel oil, the effect of salt stress on the accumulation of lipids and triacylglyceride in Dunaliella cells was investigated. Although initial NaCl concentration higher than 1.5 M markedly inhibited cell growth, increase of initial NaCl concentration from 0.5 (equal to sea water) to 1.0 M resulted in a higher intracellular lipid content (67%) in comparison with 60% for the salt concentration of 0.5 M. Addition of 0.5 or 1.0 M NaCl at mid-log phase or the end of log phase during cultivation with initial NaCl concentration of 1.0 M further increased the lipid content (70%).

Cell Processing Engineering for Regenerative Medicine : Noninvasive Cell Quality Estimation and Automatic Cell Processing.

The cell processing engineering including automatic cell processing and noninvasive cell quality estimation of adherent mammalian cells for regenerative medicine was reviewed. Automatic cell processing necessary for the industrialization of regenerative medicine was introduced. The cell quality such as cell heterogeneity should be noninvasively estimated before transplantation to patient, because cultured cells are usually not homogeneous but heterogeneous and most protocols of regenerative medicine are autologous system. The differentiation level could be estimated by two-dimensional cell morphology analysis using a conventional phase-contrast microscope. The phase-shifting laser microscope (PLM) could determine laser phase shift at all pixel in a view, which is caused by the transmitted laser through cell, and might be more noninvasive and more useful than the atomic force microscope and digital holographic microscope. The noninvasive determination of the laser phase shift of a cell using a PLM was carried out to determine the three-dimensional cell morphology and estimate the cell cycle phase of each adhesive cell and the mean proliferation activity of a cell population. The noninvasive discrimination of cancer cells from normal cells by measuring the phase shift was performed based on the difference in cytoskeleton density. Chemical analysis of the culture supernatant was also useful to estimate the differentiation level of a cell population. A probe beam, an infrared beam, and Raman spectroscopy are useful for diagnosing the viability, apoptosis, and differentiation of each adhesive cell.

Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair: A Preclinical Study.

OBJECTIVE: The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow-derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats. DESIGN: (Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery. RESULTS: The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks. CONCLUSIONS: These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.

Cell processing engineering for ex-vivo expansion of hematopoietic cells.

The cell processing engineering for ex vivo expansion of hematopoietic cells is reviewed. All hematopoietic cells of different lineages and/or at various stages of differentiation are derived from the same precursor, pluripotent hematopoietic stem cells. Bone marrow stromal cells promote and regulate the self-renewal, commitment, differentiation, and proliferation of stem cells and progenitors through their secreted extracellular matrices and cytokine environment in the hematopoietic microenvironment. Although stroma-mediated hematopoiesis has been studied in vitro using the Dexter culture system in tissue culture flasks, hematopoiesis in the Dexter culture system is almost limited to a granulocyte lineage and the system could not expand primitive cells. The addition of large amounts of cytokines to the culture of hematopoietic cells enabled their expansion, but is too expensive. Some clonal stromal cell lines have been established from the Dexter culture of murine bone marrow cells in order to simplify and stimulate the ex vivo expansion of hematopoietic cells. In order to solve the problem regarding the usage of exogeneic stromal cell lines, a novel membrane-separated coculture system, in which stromal cells adhere onto the lower surface of a porous membrane and hematopoietic cells are incubated on the upper surface of the membrane, was proposed. In order to mimic the contact between stromal and hematopoietic cells in vivo in the bone marrow, several types of three-dimensional (3-D) culture of hematopoietic cells were developed. The 3-D coculture of hematopoietic cells with spatial development of stromal cells in nonwoven fabrics enabled the expansion of progenitors without cytokine addition. Progenitors in cord blood mononucleated cells were also successfully expanded without the addition in the 3-D coculture with primary human bone marrow stromal cells in 3-D. Heparin addition to the 3-D coculture and coating the nonwoven fabrics with N-(O-beta-(6-O-sulfogalactopyranosyl)-6-oxyhexyl)-3,5-bis(dodecyloxy)-benzamide further increased the number of progenitors.

Effect of chondroitin sulfate and hyaluronic acid on gene expression in a three-dimensional culture of chondrocytes.

The effect of glycosaminoglycan addition on a three-dimensional (3D) culture of porcine chondrocyte cells was investigated with a view to use in cartilage regenerative medicine. Chondroitin sulfate C increased the mRNA expression of type 2 collagen, while chondroitin sulfate A did not. Hyaluronic acid of high molecular weight markedly decreased the mRNA expression of both aggrecan and type 2 collagen, although hyaluronic acid of low molecular weight showed no apparent effect.