A low temperature method of isolating normal human articular chondrocytes.
OBJECTIVE
Numerous methods for isolation of human chondrocytes are reported in the literature, most based on isolation from animal cartilage. Normal human articular cartilage (NHAC) poses particular problems for isolating chondrocytes when compared to animal or other types of human cartilage: a hardy matrix, combined with few and friable chondrocytes makes isolation difficult. Our objective was to develop an efficient method of isolating chondrocytes from NHAC without jeopardising the viability of these cells.


METHODS
In this study we demonstrate that lowering the enzymatic digestion temperature to 27 degrees C increases cell yield and chondrocyte viability. We then optimised this low temperature isolation of chondrocytes from NHAC by comparing the relative efficacies of trypsin and protease and hyaluronidase in combination with different types of collagenase (I, II and XI) at releasing chondrocytes from their surrounding cartilaginous matrix. Enzymes were tested at different concentrations and for differing times. Outcome measures included determining the amount of cartilage digested, the number of viable chondrocytes isolated per gram of cartilage and cell adherence rates.


CONCLUSIONS
From these set of experiments, the method that maximised cell yield without jeopardising cell viability proved to be a two stage process: pre-digestion step using trypsin for 15 min; followed by overnight digestion with a combination of two types of collagenase (types I and II) and at a lower temperature of 27 degrees C. This has resulted in an efficient and robust method of releasing chondrocytes from cartilage, without jeopardising the viability of these cells.
Hidvegi
N C
NC
Department of Plastic Surgery, Royal Free Hospital School of Medicine, London, UK. nhidvegi@hotmail.com
Sales
K M
KM
Izadi
D
D
Ong
J
J
Kellam
P
P
Eastwood
D
D
Butler
P E M
PE
D000368:Aged; D000369:Aged, 80 and over; D002358:Cartilage, Articular / Q000166:cytology* / Q000201:enzymology; D002448:Cell Adhesion / Q000502:physiology; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000201:enzymology; D003080:Cold Temperature; D017364:Collagenases / Q000378:metabolism; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D006821:Hyaluronoglucosaminidase; D010447:Peptide Hydrolases; D014357:Trypsin; D017364:Collagenases