Cryopreservation and storage effects on cell numbers and DNA damage in human lymphocytes

The comet assay measures DNA damage in individual cells (usually lymphocytes) and is widely used in biomonitoring studies. Lymphocytes are harvested and are usually cryopreserved for batch testing. We investigated cell loss during harvesting, cryopreservation, thawing, and washing of human peripheral lymphocytes and compared DNA damage, using the Fpg-assisted comet assay for oxidation-induced DNA lesions, in freshly harvested cells and cells that were thawed and tested after cryopreservation of 2-3 days and 4 weeks. Lymphocyte numbers were measured in fresh venous blood and after the steps of harvesting, cryopreservation, and washing. Results showed that >50% of lymphocytes in whole blood were harvested, but ∼60% were lost during washing. Loss during washing was not different (P>0.05) between fresh cells and cells thawed and washed after 2-3 days or 4 weeks cryopreservation. No change in DNA damage was seen after cryopreservation and thawing: mean (SD) % DNA in comet tail was 11.2 (1.53) in freshly harvested cells, 12.9 (1.39) in 2-3 days cryopreserved cells, and 12.9 (2.0) in cells tested after 4 weeks cryopreservation (P>0.05). Results indicate that there is no predominant loss of more highly damaged cells during cryopreservation and thawing and there is no induction of oxidation-induced DNA lesions in cryopreserved cells stored for up to 4 weeks.