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.

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.

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%.

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.

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.

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.

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 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).

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 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.

Noninvasive discrimination between human normal and cancer cells by analysis of intracellular distribution of phase-shift data.

Aiming to establish a method for the noninvasive discrimination of cancer cells from normal cells in adherent culture, we investigated to employ all phase shift data for all pixels inside a cell. The bird's-eye views of phase shifts of human prostate epithelial cells (PRECs) and human prostatic carcinoma epithelial cell (PC-3) lines acquired by phase-shifting laser microscopy showed tableland and cone shapes, respectively, while treatment of PRECs with cytochalasin D resulted in the cone shape. So, the profile of phase shift in both sections towards the x- and y-axes of the views through the peaks of the phase shifts in PRECs and PC-3 cells were trapezoid-like and triangle-like, respectively. Typical profiles of phase shifts in a section in PRECs or PC-3 cells were calculated by averaging from 10 cells and smoothing. Cancer index is defined as the deduction of sums of the squared difference between a real cell and the typical profiles for a PREC and a PC-3 cell. The cancer indices for PC-3 and hepatocellular carcinoma cell lines were positive, while those for PRECs and human normal cryopreserved hepatocytes were negative. Cancer indices along the major axis of fibroblast-like cells of normal mesenchymal stem cells and the osteosarcoma cell line were negative and positive, respectively. Consequently, several cancer cells could be noninvasively discriminated from normal cells by calculating the cancer index employing phase shift for all pixels inside the cells.

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

Xeno-free and shrinkage-free preparation of scaffold-free cartilage-like disc-shaped cell sheet using human bone marrow mesenchymal stem cells.

Aiming for the clinical application of cartilage regeneration, the xeno-free cultivation method to obtain a scaffold-free cartilage-like disc-shaped cell sheet using mesenchymal stem cells (MSCs) derived from human bone marrow without the shrinkage of the sheet was investigated. MSCs were inoculated into Cell Culture Insert (0.3 cm(2), pore size; 0.4 µm, pore density; 1.0 × 10(8)/cm(2)) using serum-free chondrogenic differentiation medium containing TGF-ß3, IGF-1 and dexamethasone or other modified media, and cultured at 37 °C in 5% CO2 for 3 weeks. Sheet thickness, cartilage specific genes expression, ECM accumulation were determined, and the sections of sheets were stained with alcian blue. A novel mixed medium consisting of a growth medium (10% FCS) with a serum-free chondrogenic differentiation medium could prevent the shrinkage of the sheet and produced a disc-shaped cell sheet. The depth of the sheet was approximately 0.7 mm and the gene expression levels were higher than those in cells in normal human cartilage. The use of human serum instead of FCS did not cause shrinkage and did not decrease the accumulation levels of sGAG and type 2 collagen in the sheet. The cultivation of MSCs grown with completely xeno-free materials using the mixed medium containing human serum in a cell culture insert showed a sheet depth of 1.0 mm and gene expression levels higher than those in normal cartilage. The scaffold-free and xeno-free cartilage-like cell sheet was successfully formed without shrinkage using human bone marrow MSCs and the chondrogenic differentiation medium containing human serum.

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.

Correlation between actin content and laser phase shift of adhesive normal and malignant prostate epithelial cells.

Aiming to establish the noninvasive discrimination of cancer cells from normal cells in adherent culture, we examined in this study the reason why the laser phase shift, which is the product of refractive index and height of malignant cancer cells, was markedly smaller than that of normal cells. Both of the phase shift measured by phase-shifting laser microscopy (PLM) and the relative actin content of cells of the adhesive human prostatic carcinoma epithelial cell line (PC-3) were markedly lower than those of adhesive human prostate epithelial cells (PRECs), while there was almost no difference in morphology observed under a conventional inverted microscope, between them. The decrease in relative actin content by the addition of cytochalasin D resulted in the decrease in phase shift in both cell lines, and these cell lines showed a marked positive correlation between phase shift and relative actin content (r = 0.84). The height and refractive index of adhesive cells were determined using PLM and the height of PC-3 cells were apparently lower than those of PRECs, while there was no difference in refractive indices between PC-3 cells and PRECs. Consequently, the smaller height of PC-3 cells caused by lower actin content than of PRECs might be the reason for the lower phase shift of PC-3 cells.

Time-lapse analysis of laser phase shift for noninvasive discrimination of human normal cells and malignant tumor cells.

Time-lapse analysis of phase shift using phase-shifting laser microscopy (PLM) revealed that the laser phase shifts of primary normal human prostate epithelial cells (PRECs) and human prostatic carcinoma epithelial cell line (PC-3) cells during the mitotic phase were markedly higher than those in the interphase. The phase shift of PC-3 cells during the interphase was markedly lower than that of PRECs throughout the cell cycle. In conclusion, it was proposed that adherent PC-3 cancer cells could be noninvasively discriminated from normal adherent PRECs by the periodical measurement of phase shift using PLM during culture.

Effect of the distance between adherent mesenchymal stem cell and the focus of irradiation of femtosecond laser on cell replication capacity.

The effect of femtosecond laser irradiation on adherent mesenchymal stem cell was investigated with the aim to develop a novel noninvasive cell purification system. A single mesenchymal stem cell was irradiated with a femtosecond laser on the center of the cell using several energy values and the cell lost its replication capacity with one time irradiation with an energy of 2.0 µJ. Besides at a neighbor point in the major axis, when irradiated in the minor axis at a distance shorter than 10 µm, the cell stopped its replication capacity. The accumulation effect of cell damage caused by multiple laser shots at a neighbor point in the minor axis was correlated with the critical distance at which the cell lost its replication capacity. Finally, a novel equation of laser cell damage as a function of laser pulse energy and laser shot number is proposed.