期刊
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
卷 10, 期 -, 页码 -出版社
FRONTIERS MEDIA SA
DOI: 10.3389/fbioe.2022.1046127
关键词
chondrocyte isolation; human articular cartilage; small chondrocytes; large chondrocytes; transcriptome preservation; single-cell RNA sequencing
资金
- Willy Robert Pitzer Foundation (Pitzer Laboratory of Osteoarthritis Research)
- Rolf M. Schwiete Foundation (Osteoarthritis Research Program)
- Einstein Center for Regenerative Therapies
- German Research Foundation (DFG) [EZ-2016-289]
- German Federal Ministry of Education and Research (BMBF) [TP28, LO 1542/4-1, LO 1542/5-1, EFRE 1.8/11]
- National Natural Science Foundation of China [SFB650]
- European Regional Development Fund (ERDF) [01KC 2011C]
- state of Berlin [81671619]
This study optimized the isolation procedure of human chondrocytes from articular cartilage to preserve the in vivo transcriptome. By using appropriate concentrations of collagenase and digestion time, the maximum release and preservation of chondrocyte transcriptome were achieved.
The isolation of chondrocytes from human articular cartilage for single-cell RNA sequencing requires extensive and prolonged tissue digestion at 37 C. Modulations of the transcriptional activity likely take place during this period such that the transcriptomes of isolated human chondrocytes no longer match their original status in vivo. Here, we optimized the human chondrocyte isolation procedure to maximally preserve the in vivo transcriptome. Cartilage tissues were transferred into a hypoxia chamber (4% O-2) immediately after being removed from OA patients and minced finely. Collagenase II at concentrations of 0.02%, 0.1%, 0.25%, 0.5%, 1%, and 2% was applied for 0.5, 1, 2, 4, and 18 h to digest the minced tissue. Actinomycin D (ActD) was added to test its capacity in stabilizing the transcriptome. Cell yield, viability, cell size, and transcriptome were determined using counter chamber, flow cytometry, and RNA sequencing (RNA-seq). Collagenase II at 2% concentration released small chondrocytes from cartilage matrix during the first digestion hour and started to release large cells thereafter, reaching a complete release at 4 h. During 4-h digestions, collagenase II at 2% and 1% but not at lower concentrations yielded maximal release also of the large chondrocyte population. RNA-seq analysis revealed that a 4-h digestion period with 1% or 2% collagenase II plus Actinomycin D optimally preserved the transcriptome. Thus, this study provides an isolation protocol for single chondrocytes from human articular cartilage optimized for transcriptome preservation and RNA-seq analysis.
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