Abstract: Objective To optimize the protocol of meniscus cell extraction to enhance the efficiency of cell suspension preparation and maintain a high cell viability for single-cell RNA sequencing. Methods We compared the efficiency of the routine cell extraction methods (short-time digestion and long-time digestion) and the optimized protocol for obtaining meniscus cell suspensions by evaluating the cell number obtained and the cell viability. Single-cell RNA sequencing datasets were analyzed to evaluate the stability of the cell suspension prepared using the optimized protocol. The reliability of the optimized protocol was assessed by comparing the single-cell RNA sequencing dataset obtained by the optimized protocol with published single-cell RNA sequencing datasets of the meniscus. Results The optimized protocol harvested a greater number of cells (over 1×105) than the routine protocols. The cell suspension prepared with the optimized protocol showed a cell viability higher than 80%, the highest among the 3 methods. Analysis of single-cell RNA sequencing datasets showed that the ratio of the mitochondrial genes was below 20% in over 80% of the cells. CD34+ cells, MCAM+ cells and COL1A1+ cells were identified in the datasets. Comparison with the publish datasets showed that the optimized protocol was capable of harvesting COL3A1+, COL1A1+, MYLK+, BMP2+, CD93+ and CDK1+ cells. Conclusion Single-cell suspension prepared from the meniscus can be stably obtained using the optimized protocol for single-cell RNA sequencing using the 10× Genomics platform.

Key words: meniscus; cell extraction; single-cell suspension; single-cell RNA sequencing