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.
目的: 优化半月板细胞提取的方法，缩短细胞提取时间，稳定地获取细胞数量多和细胞活率高的细胞悬液，使获得的细胞悬液悬液适用于单细胞RNA测序。
方法: 比较细胞悬液的细胞数量及细胞活率评价不同细胞提取方法的有效性，细胞提取方法包括传统半月板细胞提取法（短时间消化和长时间消化）和优化提取法；通过分析单细胞RNA测序数据集评价优化提取法获取的细胞悬液的稳定性；通过比较优化提取法获得的单细胞RNA测序数据集与公开的半月板单细胞RNA测序数据集，分析优化提取法的可靠性。
结果: 在3次重复实验中，优化提取法获得细胞数量最多的细胞悬液，细胞数量超过105，且细胞活率最高，平均达80%。通过分析单细胞RNA测序数据发现每个样本中80%以上细胞的线粒体基因含量少于20%。CD34+，MCAM+细胞以及COL1A1+胞均存在每个单细胞RNA测序数据集中。在比较基于优化提取法单细胞RNA测序数据集与公开的半月板单细胞RNA测序数据集时发现，优化提取法也能捕获COL3A1+细胞，COL1A1+细胞，MYLK+细胞，BMP2+细胞，CD93+细胞和CDK1+细胞。
结论: 优化细胞提取方法能稳定地获得适用于10×Genomics平台的单细胞RNA测序技术的单细胞悬液。
Keywords: cell extraction; meniscus; single-cell RNA sequencing; single-cell suspension.