Cell Thawing Protocol

Background

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Thawing cells incorrectly can lead to cellular damage, loss of viability, and alterations in cellular characteristics, compromising the reliability and reproducibility of research results or therapeutic outcomes. By employing appropriate thawing methods, such as gradual warming and the use of protective agents, researchers can ensure the preservation of cell integrity, genetic stability, and functional properties, thus maximizing the efficiency and success of downstream applications. Additionally, adherence to standardized thawing protocols minimizes experimental variability, enhances reproducibility, and ultimately contributes to advancing scientific knowledge and the development of effective cell-based therapies and biologics.

Protocol

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Tips

• For best results, it is highly recommended to use validated and optimized media from BPS Bioscience. Other preparations or formulations of media may result in suboptimal performance.
• When receiving a new cell line, it is recommended to expand the cells and freeze at least 10 vials at an early passage for future use.
• Thaw media do not contain selective antibiotics.

Adherent Cell Thawing Protocol

This protocol is applicable to adherent cell lines, such as HEK293, CHO or HeLa cells.

1. Swirl the vial of frozen cells for approximately 60 seconds in a 37°C water bath. As soon as the cells are thawed (it may be slightly faster or slower than 60 seconds), quickly transfer the entire contents of the vial to a tube containing 10 ml of pre-warmed Thaw Medium not containing a selection antibiotic. Leaving the cells in the water bath at 37°C for too long will result in rapid loss of viability.
• An alternative to step 1, which may be less harsh to cells, would be to quickly transfer the entire content of the vial to an empty 50 ml conical tube immediately after thaw and then slowly add 10 ml of pre-warmed Thaw Medium dropwise while gently rocking the conical tube to permit gentle mixing and avoid osmotic shock.
2. Immediately spin down the cells at 300 x g for 5 minutes, remove the medium and resuspend the cells in 5 ml of pre-warmed thaw medium (no selection antibiotic).
3. Transfer the resuspended cells to a T25 flask or T75 flask and incubate at 37°C in a 5% CO₂.
4. After 24 hours of culture, check for cell attachment and viability. Change medium to fresh Thaw Medium and continue growing in a 5% CO₂ incubator at 37°C until the cells are ready to passage.
5. Cells should be passaged before they are fully confluent. At first passage and subsequent passages, use Growth Medium (containing appropriate selection antibiotics).

Suspension Cell Thawing Protocol

This protocol is applicable to suspension cell lines, such as Jurkat, THP-1, etc.

1. Swirl the vial of frozen cells for approximately 60 seconds in a 37°C water bath. As soon as the cells are thawed (it may be slightly faster or slower than 60 seconds), quickly transfer the entire contents of the vial to a tube containing 10 ml of pre-warmed Thaw Medium not containing a selection antibiotic. Leaving the cells in the water bath at 37°C for too long will result in rapid loss of viability.
• An alternative to step 1, which may be less harsh to cells, would be to quickly transfer the entire content of the vial to an empty 50 ml conical tube immediately after thaw and then slowly add 10 ml of pre-warmed Thaw Medium dropwise while gently rocking the conical tube to permit gentle mixing and avoid osmotic shock.
2. Immediately spin down the cells at 300 x g for 5 minutes, remove the medium and resuspend the cells in 5 ml of pre-warmed thaw medium (no selection antibiotic).
3. Transfer the resuspended cells to a T25 flask and incubate at 37°C in a 5% CO₂.
4. After 24 hours of culture, check for cell viability. For a T25 flask, add 3-4 ml of Thaw Medium (no selection antibiotic), and continue growing in a 5% CO₂ incubator at 37°C until the cells are ready to passage.
5. Cells should be passaged before they reach a density of approximately 2 x 10⁶ cells/ml. At first passage and subsequent passages, use Growth Medium (containing appropriate selection antibiotics).

PBMC Thawing Protocol

This protocol is applicable to cryopreserved PBMCs or purified T cells.

1. Swirl the vial of frozen cells in a 37°C water bath until 90% of the vial is thawed.
• Note: Leaving the cells in the water bath at 37°C for too long will result in rapid loss of viability.
2. Clean the outside of the vial with 70% ethanol.
3. As soon as the cells are thawed, quickly transfer the entire contents of the vial to a 15 ml tube.
4. Rinse the vial with Thaw Medium 10 (#79704) and add to the 15 ml tube.
5. Slowly add 10 ml of pre-warmed Thaw Medium 10.
6. Gently mix by inverting the tube.
7. Immediately spin down the cells at 200 x g for 10 minutes at Room Temperature (RT).
8. Carefully remove the supernatant with a pipette without disturbing the pellet.
9. Resuspend the pellet with 10 ml of pre-warmed Thaw Medium 10.
10. Gently mix by inverting the tube.
11. Spin down the cells at 200 x g for 10 minutes at RT.
12. Carefully remove the supernatant with a pipette without disturbing the pellet.
• Note: Up to 20% cell loss can be expected during washing steps.
13. Cells are now ready for downstream applications.