Purpose

Single-cell sequencing has potential to provide unprecedented insight into cell-to-cell communication and complex cross-talk between joint tissues. However, difficulty remains in preparing entire joints for comprehensive scRNA-seq analysis due to the great diversity of tissues. Joints tissues like bone and cartilage consist of calcified weight-bearing matrix sparsely populated by cells, whereas synovium and fat pads are highly cellular tissues, and immune cells may have little or no surrounding matrix. To capture this broad range of cell types we developed a two-stage digestion protocol.

Methods and Materials

Mouse knees from femoral and tibial growth plates were isolated, minced and digested in two stages. To protect softer cells from over-digestion a gentler 1% collagenase-IV (30 mins) digestion was used first. An aggressive digest, 2% collagenase-II (90 mins) followed to free cells within cartilaginous and bony matrix.

After each digest, released cells were strained, and RBCs lysed. 10X Chromium Single Cell Gene Expression platform and Illumina HiSeq 4000 were used. Cell Ranger and Seurat created PCA clustering.

Results

This sequential protocol demonstrated consistency of gene expression between contralateral knees by similar cell-types (Fig. 1a). Both digests are required for comprehensive cell-type identification and inclusion (Fig. 1b).

A diverse collection of cells was captured by this protocol, with many cell-types from hard and soft tissues shown here by selected signature genes (Fig. 2).

Conclusion

To understand the joint environment, we must understand the roles of all cells present in-vivo. This complex problem is currently best studied using scRNA-seq to assess what is happening at the cellular level. This protocol will be valuable for understanding the in-vivo conditions in which cartilage resides and provide unique insight for the study of joint preservation with age or injury.