PB2175: TRYPAN BLUE AND FLOW CYTOMETRY CELL VIABILITY TESTING OF PACKED OR DILUTED CRYOPRESERVED AT -80ºC HEMATOPOIETIC STEM CELLS SHOW SIMILAR RESULTS

Abstract

Background: Trypan blue exclusion and flow cytometry were compared for testing of cell viability of hemopoietic stem cells (HSCs) cryopreserved at -80ºC. Stem cells were frozen and stored with 5% final DMSO concentration with different degree of cell packing. Aims: We wanted to evaluate the difference in viability results with folw cytometry and trypan blue exclusion with different degree of cell packing. Methods: We analyzed 47 samples stored at -80ºС in a cryoprotectant solution with final concentrations of 5% DMSO, 3.6% hydroxyethyl starch (HES 450 000 mw) and 3% of human serum albumin. The cell concentration in the frozen suspension ranged between 69х106/ml and 665х106/ml (median 310,25х106/ml). The cell viability was tested simultaneously by trypan blue exclusion test and by flow cytometry with 7 -aminoactinomycin D (7-AAD) without lysing solution. Results: Both viability tests gave similar results. When tested by trypan blue, the viability was 95,5% (80-99%), and by flow cytometry was 95,92% (89,1-98,4%). The additional statistical Spearman’s Rho analysis does not show statistically significant correlation between viability results, performed with either test, and the degree of cell packing. Summary/Conclusion: Flow cytometry and trypan blue tests show similar viability results. Since trypan blue testing is well established, has lower cost, and can be performed faster, we accepted it as a routine for our further analyses of cell viability of packed prior to freezing stem cell concentrates. HSC viability after storage at -80ºС with 5% final DMSO concentration is very good and is not affected by higher cell packing thus allowing the reduction of infused DMSO and the side effects (cardiac, neurologic, respiratory, etc.) during transplantation.