Question: Are there gene expression differences between fresh and frozen-thawed cells?
Answer: We have compared fresh and frozen-thawed human PBMCs from the same donor using the Single Cell 3' assay. Global gene expression was highly correlated between fresh and cryopreserved PBMCs. Cell type distribution was consistent before and after cryopreservation across multiple donors except for Red Blood Cells (RBCs) which are known to be sensitive to freeze-thaw conditions.
Cryopreservation has also been demonstrated as a viable method of storage for other sample types (see citations below). However, the effect of freezing and thawing may vary as different cell types may have different sensitivities.
Best practices for cryopreserving cell suspensions:
- Start with high cell viability (>90%)
- Freeze >1Million cells/mL
- Freezing cells in a dilute form may cause cell stress leading to cell death.
- Some cell loss is expected during the thawing process.
- Use a cryoprotectant or specialized cryopreservation solution
- 10% DMSO in a cell culture media or serum is typical—media can be swapped out for the media of choice for your specific sample type
- It may be possible to use commercially available cryopreservation media (i.e. Cryostor). Consult with the manufacturer for applicability to your sample type and recommended protocols.
- Control the rate of freezing using a freezing container
- Specialized freezing containers (i.e. Mr. Frosty, CoolCell) control the rate of freezing to avoid shocking cells and inducing cell death. We recommend cooling the cell suspension to -80C in these freezing containers for four hours before transferring to liquid nitrogen
- After initial freezing, frozen cell suspensions should be stored in Liquid Nitrogen tanks long-term until usage
- Thawing protocols can affect cell viability post cryopreservation
- Cultured cell lines can be thawed using a quick-thaw protocol: Fresh Frozen Human-Mouse Cell Line Mixtures for Single Cell RNA Sequencing
- Gentle thawing is recommended for primary cell lines such as PBMCs: Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing
- If low cell viability is observed, Dead Cell Removal or other cleanup methods may be used to improve cell viability
Products: Single Cell Gene Expression