title: Xeno-free and shrinkage-free preparation of scaffold-free cartilage-like disc-shaped cell sheet using human bone marrow mesenchymal stem cells.
abstract: 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-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.
authors.affiliation: Division of Biotechnology and Macromolecular Chemistry, Graduate School of Engineering, Hokkaido University, Kita-ku, N13W8, Sapporo 060-8628, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Sato
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Wakitani
authors.forename: Mutsumi
authors.initials: M
authors.lastname: Takagi
mesh_terms: D053674:Aggrecans / Q000378:metabolism; D001798:Blood Proteins / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology*; D002356:Cartilage / Q000166:cytology*; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D019902:Chondrocytes / Q000166:cytology; D024043:Collagen Type II / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D008297:Male; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D023822:Tissue Engineering / Q000379:methods*; D055815:Young Adult
chemical_list: D053674:Aggrecans; D001798:Blood Proteins; D024043:Collagen Type II; D003470:Culture Media; D006025:Glycosaminoglycans; C013786:A73025
keywords: Cartilage regeneration; Chondrogenic differentiation; Mesenchymal stem cells; Scaffold-free; Xeno-free culture