title: The optimal conditions of chondrocyte isolation and its seeding in the preparation for cartilage tissue engineering.
abstract: To optimize the chondrocyte numbers obtained after collagenase digestion for cartilage tissue engineering, we examined the enzyme concentration and incubation time for collagenase digestion. The appropriate cell density in the chondrocyte primary culture was also verified. The collagenase digestion conditions that maximized the viable cell numbers were 24 h in 0.15% and 0.3% collagenase, 6 h in 0.6%, and 4 h in 1.2%, leading to ∼5 × 10⁵ cells from 0.05 g. When seeded at 10,000 cells/cm², all of these cells became almost confluent within 1 week. Cells digested in 0.3% for 24 h or 0.6% for 6 h and seeded at 3000 cells/cm² may also be acceptable, and similarly reached confluence within 1 week. Results regarding expression of the p53, tumor necrosis factor-α, and interleukin-1β genes, as well as apoptosis enzyme-linked immunosorbent assay results, show that excessive collagenase exposure may decrease chondrocyte viability or activity. We recommend a 24-h incubation in 0.3% collagenase or 6 h in 0.6% collagenase, and a cell-seeding density of 3000-10,000 cells/cm². These conditions maximize the harvest of isolated chondrocytes from a small amount of biopsied tissue and significantly aid in obtaining a large quantity of cultured cells in a short period.
authors.affiliation: Department of Cartilage and Bone Regeneration (Fujisoft), Graduate School of Medicine, University of Tokyo, Tokyo, Japan.
authors.forename: Kazumichi
authors.initials: K
authors.lastname: Yonenaga
authors.forename: Satoru
authors.initials: S
authors.lastname: Nishizawa
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fujihara
authors.forename: Yukiyo
authors.initials: Y
authors.lastname: Asawa
authors.forename: Kanazawa
authors.initials: K
authors.lastname: Sanshiro
authors.forename: Satoru
authors.initials: S
authors.lastname: Nagata
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Takato
authors.forename: Kazuto
authors.initials: K
authors.lastname: Hoshi
mesh_terms: D002138:Calibration; D002356:Cartilage / Q000166:cytology* / Q000254:growth & development* / Q000502:physiology; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods / Q000592:standards; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods / Q000592:standards; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000502:physiology; D006801:Humans; D023822:Tissue Engineering / Q000379:methods*