A proteomics investigation of primary human articular chondrocyte isolation.

Objective: Human primary articular chondrocytes (hPACs) are routinely isolated from articular cartilage for pre-clinical OA research. Collagenase digest of tissue is an essential step, yet the impact of lengthy enzymatic incubation on the hPAC phenotype is unclear. We aimed to delineate this through proteomic analysis. Design: hPACs were isolated from human knee cartilage (n ​= ​4) from patients undergoing total knee replacement. Collagenase treatment was performed with or without prior fixation of the tissue. Proteomes were quantified using LC-MS/MS. The Proteomic Ruler was employed to estimate protein copy numbers and cell protein masses. Significant differences in protein intensities were determined using paired t-testing and Benjamini-Hochberg correction. Proteomic data were integrated with existing transcriptomes (GSE217871) of hPACs and ground cartilage (ex vivo) RNA. Results: Following collagenase treatment, we identified 498 differentially expressed proteins (DEP) in the unfixed cells. We observed depletion of FOXO signaling and enrichment of ribosomal RNA processing, indicative of increased cell cycle progression. This was supported by depletion of cell cycle inhibitors including CDKN1C. Transcriptomic analysis identified 3937 differentially expressed genes (DEG), and a 53 ​% overlap in DEP and DEG. Propidium iodide staining did not identify significant differences in cell cycle between fixed and unfixed hPACs. Conclusions: We identified shifts in the proteome and transcriptome of hPACs following collagenase digest, supporting the use of tissue fixation before extracting nucleic acids for analysis where possible. Despite widespread expression changes, hPACs largely retain their chondrocyte phenotype. These datasets and analyses will serve as a valuable resource for the OA and cartilage research community.