Question: Can cryopreserved cells be used for gene expression profiling?
Answer: We have successfully generated Single Cell libraries from cryopreserved human and mouse cell lines and PBMCs. We expect some decrease in viability and cell recovery after thawing, dependent on starting cell integrity.
We expect at least 50% cell loss to occur during cryopreservation and thawing steps (and greater loss may occur with low numbers of cells); therefore, the number of frozen cells should be higher than the targeted cell recovery rate to account for cell loss.
The Cell Thawing Guide (Cell Thawing Protocols for Single Cell Assays CG000447) outlines four different protocols to thaw cryopreserved cell suspensions for use with 10x Genomics Single Cell assays.
When reviving frozen cells for sample preparation, we recommend following these best practices for sample handling:
- Remove cell debris/dead cells with appropriate filters and/or other methods (e.g., FACS, Miltenyi Dead Cell Removal)
- Remove freezing media through washing steps to avoid carryover of contaminants that may inhibit downstream reactions
- Minimize time between cell thawing and chip loading to avoid premature cell lysis
- Resuspend cells in PBS containing 0.04% BSA or other tested media
- Re-quantify single cells and visually inspect cells before loading to confirm viability and cell concentration, avoid underloading/overloading, and reduce the risk of clogs.
Note: Depending on the cell type, sample preparation may need to be adjusted to preserve cell viability.
Products: Single Cell Gene Expression, Single Cell Immune Profiling, Fixed RNA Profiling Gene Expression