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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 cm2, pore size; 0.4 μm, pore density; 1.0 × 108/cm2) using serum-free chondrogenic differentiation medium containing TGF-β3, 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. [ABSTRACT FROM AUTHOR]

Abstract: The noninvasive discrimination of cancer cells from normal cells in adherent culture by the measuring of the phase shift using phase-shifting laser microscopy (PLM) was investigated with the aim of noninvasive quality control of cell processing for transplantation. A human prostatic carcinoma epithelial cell line (PC-3) and human hepatocellular carcinoma cell lines (Hep3B, PLC, HLF, and Huh7) showed markedly lower phase shifts as measured by PLM than those of human prostate epithelial cells (PREC) and cryopreserved human hepatocytes, respectively, although there was no apparent difference in cell morphology between these sets of cells. Mixed cultures of PC-3 and PREC, in which the percentages of PC-3 cells were determined to be 43.3%, 14.0%, and 10.6% by immunofluorescence staining, were prepared, and the percentages were calculated to be 50.0%, 8.0%, and 2.5% using the distribution of the phase shift data of the cells, respectively. In conclusion, these adherent cancer cells can be noninvasively discriminated from normal adherent cells by phase shift measurement using PLM, and the sensitivity of the method of detecting cancer cell contamination reached 10%. [Copyright &y& Elsevier]

Background: Aiming to construct an osteochondral-like structure, the combination of a β-tricalcium phosphate (βTCP) 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 TGFβ3 for 3 weeks during which a cylindrical βTCP 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 βTCP 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 βTCP block, the expression levels of the aggrecan gene increased monotonically. The increase in the inoculum cell number from 1.86 to 3.72 × 10 cells resulted in marked increases in the thickness of cell sheet parts in the βTCP block and expression levels of the aggrecan and type II collagen genes, while the thickness of cell sheet parts on the βTCP 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 βTCP block, while the thickness of that in the βTCP block did not increase. Conclusion: A combined osteochondral-like structure was produced by putting a βTCP block on the sheet of MSC. The thickness of the cell sheet parts in and on the βTCP block could be increased by the increase in inoculum cell number and by providing an additional cell sheet, respectively. [ABSTRACT FROM AUTHOR]

Abstract: The effect of the addition of monosaccharides composing glycosaminoglycans (GAGs) on the accumulation of type 2 collagen (COL(II)) in a three-dimensional (3D) culture of porcine chondrocyte cells was investigated for possible application to cartilage regenerative medicine. Primary chondrocytes from porcine cartilage were cultivated in three-dimension employing atelo collagen gel for 3 weeks with the addition of several saccharides. The addition of d-glucuronic acid (d-GlcA), N-acetyl-d-galactosamine (d-GalNAc), chondroitin sulfate C (CSC), d-galactose, N-acetyl-d-glucosamine, and l-iduronic acid increased markedly not aggrecan but COL(II) accumulation although the addition of d-fructose and d-mannose not composing GAGs did not show such an effect. The addition of d-GlcA and d-GalNAc had no synergistic effect. The addition of CSC, d-GlcA, and d-GalNAc also increased COL(II) mRNA expression while aggrecan mRNA expression was not increased by these compounds. In conclusion, the addition of monosaccharides composing GAGs might be valuable for increasing COL(II) accumulation in the 3D culture of chondrocytes. [Copyright &y& Elsevier]

Collagen gel and a copolymer mesh of polylactate and polyglucuronic acid (PLGA) were combined for a three-dimensional (3D) culture of chondrocyte cells having both uniform cell distribution and mechanical strength. Although the 3D culture in 96-multi-wells caused decreases in the glucose consumption rate and cell density in the latter stages of cultivation, transfer of the culture gel from a 96-multi-well plate to a 24-multi-well plate and an increase in medium volume effectively increased the glucose consumption rate and the accumulation of glycosaminoglycan (GAG) in the gel. The reason for the decrease in glucose consumption rate in a 96-multi-well plate was not the depletion of glucose or the accumulation of lactate in the gel, but the accumulation of degradation products of PLGA. [Copyright &y& Elsevier]

Background: The combination of a β-tricalcium phosphate (βTCP) 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) 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 βTCP 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 cells tended to result in a curved cell sheet. Culture involving 18.6 × 10 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 βTCP block to the cell sheet (1-7 days) markedly affected the balance between the thickness of cell sheet parts on and in the βTCP block. Conclusion: Centrifugation and subsequent cultivation of chondrocytes (18.6 × 10 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 βTCP block on the cell sheet. The thickness of the cell sheet on the βTCP block and the binding strength between the cell sheet and the βTCP block could be optimized by adjusting the inoculum cell number and timing of βTCP block addition. [ABSTRACT FROM AUTHOR]

Abstract: Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6×105 cells/well) for 3weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0×105 MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6×105 MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet. [Copyright &y& Elsevier]

Abstract: The cell-specific production rate of melanoma inhibitory activity was quantitatively proportional to the aggrecan gene expression level in the early and middle phase during not only the redifferentiation culture of human cartilage chondrocytes but also the differentiation culture of human bone marrow mesenchymal stem cells to chondrocytes. [Copyright &y& Elsevier]