Abstract
Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16 h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5 h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.
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Acknowledgements
We thank staff of Medical Research Facilities at Jiangnan University Wuxi College of Medicine for providing us technical assistances.
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This work was supported by the Postgraduate Research & Practice Innovation Program of Jiangsu Province grant (KYCX22_2437) awarded to Jiamin Mao and by the Jiangsu Provincial Natural Science Foundation of China grant awarded to Lei Ding (BK20171143).
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Mao, J., Huang, L., Ding, Y. et al. Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement. Cell Tissue Bank 24, 759–768 (2023). https://doi.org/10.1007/s10561-023-10094-0
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DOI: https://doi.org/10.1007/s10561-023-10094-0