| Abstract |
Introduction: Transplantation of hematopoietic stem cells is often the only treatment option for many malignant and non-malignant diseases. A useful alternative source of HSCs that shows several advantages over the others is Umbilical Cord Blood, the presence of primordial hematopoietic cells in which has been demonstrated since the end of the 20th century. Cryopreservation is so far the only method for long-term storage of umbilical cord blood cells while ensuring the unit's quality is maintained. However, the cell viability of a cord blood unit is affected by its collection method, cryopreservation time and temperature, transport conditions, processing, and cryopreservation and thawing techniques. However, the effect of long-term cryopreservation on the viability of long-term cryopreserved cells has not yet been clarified.
Aim: The aim of this work is to evaluate the effect of cryopreservation on the viability and clonogenic capacity of umbilical cord blood primitive hematopoietic cells. The samples studied stored in the Transplantation Unit of the Children's Hematology-Oncology Clinic of Heraklion University Hospital at a temperature of -196°C for over 12 years.
Methods: For the present study, 14 UCB samples were used, which were stored for 12 to 22 years in the Transplantation Unit of the Children's Hematology-Oncology Clinic of the University General Hospital of Heraklion. These samples were cryopreserved in liquid nitrogen conditions at -196°C. The samples which studied was independent from primary grafts, they had the same cryopreservation procedure as the primary grafts but were stored separately.
Samples were rapidly thawed in a water bath at 37°C. Next, the viability of the samples was calculated after staining the cells with trypan blue. Using a Beckman Coulter flow cytometer, the percentage of primary hematopoietic cells expressing CD34 and CD133 surface antigens and the percentage of live, apoptotic and dead CD34+ cells were determined using the 7-AAD marker. The percentage of CD34+ cells was also determined without the use of 7-AAD and a comparison was made of the percentages before cryopreservation, a measurement in which no 7-AAD was used, and after the samples were thawed. Finally, the evaluation of the clonogenic capacity of the cells was performed by assessing colony formation on methylcellulose cultures.
At the same time, a bibliographic review was carried out regarding the evolution of UCB preservation techniques and the current status of umbilical cord blood transplantation and its modern applications. Results: Leukocyte viability rates determined using trypan blue were quite high. The highest viability rate was 99.79%, the lowest 94.97%, while the average was 98.2%. CD34+ cells were detected in 0.22% of the samples, the highest percentage detected in a sample was 0.43%, while the lowest was 0.10%. CD34+CD133+ cells were also detected at an average of 0.14%. The highest percentage of these cells was 0.27%, while the lowest was 0.02%. In several samples the percentage of CD34+ cells after cryopreservation both with and without the use of the apoptotic marker 7-AAD was found to be higher than before cryopreservation. The clonogenic capacity of the samples did not seem to be affected by their long-term cryopreservation, since each sample formed CFU-GM and BFU-E colonies, while a few CGU-GEMM colonies were also found in 3 samples, after culturing the cells in methylcellulose medium.
Conclusion: It appeared that long-term cryopreservation of umbilical cord blood progenitor cells in liquid nitrogen (-196°C) up to 22 years does not affect the quality of the units. Viability rates remained high regardless of the cryopreservation interval. However, with the existence of even low percentages of CD34+, CD133+ and CD34+CD133+ cells, the existence of progenitor hematopoietic cells was confirmed even after long-term cryopreservation. In addition, multi-year cryopreservation did not affect the ability of these cells to proliferate, differentiate, and form colonies.
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