Storage Effect on Cell Cycle Analysis

To determine the effect of storage conditions on cell cycle analysis in both immortalized cell lines and primary samples from patients. Cell lines were kept in sealed tubes for 1, 3, and 6 hours at either 37 degrees, room temperature, or 4 degrees. Following an hour rest in a 37 degree humidified incubator the cells were stained with IdU then smart tube fixed. A separate experiment tested the short term effect of storage by storing cells for 15, 30, and 45 minutes with no additional rest period before IdU staining and fixation. Finally an experiment was performed where cells were cryogenically frozen then thawed after a week in storage and were collected at serial time points for IdU stain and fixation. Bone marrow samples collected from patients was used to test the effects of short term and cryogenic storage on primary samples. At sample collection IdU was added to an aliquot of bone marrow
Conclusion:
Long term storage can have detrimental effects on IdU incorporation in cell type that may be exacerbated by a resting period. Cryopreservation and thawing causes significant disruption in IdU incorporation and cell cycling markers (Ki67 and pRb). After 48 hrs cells begin to show some normalization. Short time storage was not associated with significant IdU disruption or markers of cycling cells. In marrow studies certain populations showed IdU incorporation changes depending on storage length but overall 30 and 45 minutes storage were not associated with major changes to cycling markers. Additionally the effect of T cell and granulocyte activation during storage has been noted by other researchers and researchers performing cell cycle analysis should be aware of the possibility. A control sample in vented flasks was run, except for the short time points, with every cell type tested. For the cryogenic studies cells were continuously cultured for 2 weeks and a baseline sample was collected before freezing. For the human marrow studies an aliquot was stained with IdU immediately upon collection from the medical team drawing the bone marrow to serve as a control for subsequent experiments. The samples were also barcoded to ensure equivalent staining during data collection. Additionally for the remission marrow samples a normal marrow was included in the barcoding to serve as a template for lineage gating.