[An optimized protocol of meniscus cell extraction for single-cell RNA sequencing].

Area of Science:

• Biotechnology
• Molecular Biology
• Regenerative Medicine

Background:

• Meniscus cell extraction is crucial for understanding knee joint health and disease.
• Current protocols may limit cell yield and viability, impacting downstream single-cell RNA sequencing (scRNA-seq).
• Optimizing cell suspension preparation is essential for accurate scRNA-seq analysis.

Purpose of the Study:

• To develop and validate an optimized protocol for meniscus cell extraction.
• To enhance cell yield and viability for scRNA-seq applications.
• To ensure the stability and reliability of meniscus-derived single-cell suspensions.

Main Methods:

• Compared routine (short-time and long-time digestion) and optimized cell extraction protocols.
• Evaluated cell number and viability post-extraction.
• Analyzed scRNA-seq data to assess cell suspension stability and cell type identification.
• Validated the protocol against published meniscus scRNA-seq datasets.

Main Results:

• The optimized protocol yielded over 1x10^5 cells, significantly more than routine methods.
• Achieved cell viability exceeding 80%, the highest among tested protocols.
• scRNA-seq data showed <20% mitochondrial gene content in >80% of cells, indicating high quality.
• Identified key cell populations including CD34+, MCAM+, COL1A1+, COL3A1+, MYLK+, BMP2+, CD93+, and CDK1+ cells.

Conclusions:

• The optimized protocol provides a stable method for preparing meniscus single-cell suspensions.
• This protocol is suitable for high-throughput scRNA-seq using platforms like 10x Genomics.
• The optimized method enhances the quality and quantity of cells for molecular analysis.