Yuxin Yan1, Rao Fu1, Chuanqi Liu1,2, Jing Yang1, Qingfeng Li1, Ru-Lin Huang1. 1. Department of Plastic and Reconstructive Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China. 2. Department of Plastic and Burn Surgery, West China Hospital, Sichuan University, Shanghai, China.
Abstract
OBJECTIVE: The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. DESIGN: Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity. RESULTS: Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. CONCLUSIONS: The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.
OBJECTIVE: The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. DESIGN: Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity. RESULTS: Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. CONCLUSIONS: The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.
Authors: Yaima Campos; Amisel Almirall; Gastón Fuentes; Hans L Bloem; Eric L Kaijzel; Luis J Cruz Journal: Tissue Eng Part B Rev Date: 2019-08 Impact factor: 6.389
Authors: Heenam Kwon; Wendy E Brown; Cassandra A Lee; Dean Wang; Nikolaos Paschos; Jerry C Hu; Kyriacos A Athanasiou Journal: Nat Rev Rheumatol Date: 2019-07-11 Impact factor: 20.543