Viability of Stem Cells without Cryopreservation: How Good and How Long?

Although cryopreservation of stem cells is a standard practice for autologous haematopoietic progenitor cells transplantation to treat haematologic malignancies, it has many limitations including toxicities of cryo-protectant, requirement for cryopreservation facilities as well as trained personnel which causes barriers for transplant programs in resource limited countries. To overcome these limitations, a newly established first transplant centre at the North Okkalapa General and Teaching Hospital of Myanmar has been using ordinary refrigerator at 4 degree Celsius to store autologous peripheral blood stem cells collected by apheresis during the interval between collection and transplantation for patients with multiple myeloma who underwent high dose melphalan therapy and autologous stem cell rescue. During early days of the centre, in addition to microbiological testing, to ensure the quality of stem cells, serial measurement for stem cell count and viability were made on daily basis till the day of transplant and continued testing if adequate samples of aliquots were available for analysis. Viable CD34⁺ cells were analyzed by BD FACS Canto II flow cytometer using BD stem cell enumeration kits that incorporate 7-Amino Actinomycin D viability dyes. The results of the viable CD34+counts of 13 myeloma patients, 5 males and 8 females, median age- 57 years, who underwent autologous peripheral blood stem cell transplantation during early periods of the transplant center from May 2014 to January, 2019, were retrospectively reviewed for the quality of stem cells from the non-cryopreserved methods. Mean CD34 + cells enumerated at the time of reinfusion were 3.45 x 10⁶ cells/kg (range 1.45 - 4.87 x 10⁶ cells/kg). The mean viability of stem cells on day of collection (day 0) were 96.13% (range 83.33% -100%) which was almost unchanged one day after collection (day 1) at 95.93% (85.70% -99.24%). On second and third day after collection, mean viability of the collected cells although fallen below 90% were reasonably preserved at 88.7% (81.74-95.61) on day 2 and 88.08% (87.07-89.09) on day 3, and 85.37% on day 4. Median neutrophil engraftment time was 11 days (range 9-12) and that for platelet engraftment was 16 days (range 9-44). There were no graft failure or mortality during first 100 days. It was encouraging to observe that even at the refrigerator temperature at 4degree Celsius, without any additives or manipulation, stem cell viability could be maintained above 85% minimum of four days with good transplant outcome. Further studies are needed to investigate maximum storage period which could allow reasonable viability and function of the stem cells.