A method of isolating viable chondrocytes with proliferative capacity from cryopreserved human articular cartilage.
This study aimed to optimise methods of cryopreserving human articular cartilage (AC) tissue for the isolation of late chondrocytes. Human AC specimens from osteoarthritis patients who had undergone total knee replacement were used to optimise the chondrocyte isolation process and the choice of cryoprotective agent (CPA). For AC tissue cryopreservation, intact cored cartilage discs (5 mm diameter) and diced cartilage (0.2-1 mm cubes) from the same sized discs were step cooled and stored in liquid nitrogen for up to 48 h before chondrocyte isolation and in vitro assay of cell viability and proliferative potential. The results showed that 10 % dimethyl sulphoxide in 90 % foetal bovine serum was a successful CPA for chondrocyte cryopreservation. Compared with intact cored discs, dicing of AC tissue into 0.2-1 mm cubes significantly increased the viability and proliferative capacity of surviving chondrocytes after cryopreservation. In situ cross-section imaging using focused ion beam microscopy revealed that dicing of cored AC discs into small cubes reduced the cryo-damage to cartilage tissue matrix. In conclusion, modification of appropriate factors, such as the size of the tissue, cryoprotective agent, and isolation protocol, can allow successful isolation of viable chondrocytes with high proliferative capacity from cryopreserved human articular cartilage tissue. Further studies are required to determine whether these cells may retain cartilage differentiation capacity and provide sufficient chondrocytes for use as implants in clinical applications.
Nuffield Department of Orthopaedic Surgery, Botnar Research Centre, Oxford University Institute of Musculoskeletal Sciences, University of Oxford, Oxford, OX3 7LD, UK. zhidao.xia@gmail.com
Zhidao
Z
Xia
Xin
X
Duan
David
D
Murray
James T
JT
Triffitt
Andrew J
AJ
Price
D000368:Aged; D000369:Aged, 80 and over; D002358:Cartilage, Articular / Q000187:drug effects / Q000473:pathology* / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000473:pathology* / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D020370:Osteoarthritis, Knee / Q000473:pathology / Q000503:physiopathology; D013696:Temperature
D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide