Analysis of the effect of cryoprotectant medium composition to viability of autologous hematopoietic cells collected by leukapheresis.
OBJECTIVE
Cryopreservation of hematopoietic stem cells intended for autologous transplantation is a crucial element of the banking process. Although cryopreservation techniques are well known, improvement is needed. This study was designed to optimize cryopreservation to improve the quantitative and qualitative parameters of hematopoietic stem cells in the material intended for transplantation. We used available opportunities to provide the best quantitative and qualitative parameters of hematopoietic stem cell transplants processed in a closed system.


METHODS
Two hundred forty-eight products of hematopoietic stem cells collected by leukapheresis from patients with lymphoproliferative disorders create the basis of this report. The material was frozen in a controlled-rate freezer and stored in containers in the vapor phase of LN2 (-160°C). The composition of a cryoprotectant medium was modified. For freezing, 192 probes were used with a cryoprotective medium containing 20% dimethyl sulfoxide (DMSO) and enriched RPMI 1640. For 56 samples, we used 20% DMSO in autologous plasma harvested during leukapheresis. Products of hematopoietic stem cells and cryoprotectant medium were combined in a 1:1 ratio. The final number of nuclear cells did not exceed 2 × 10(8)/mL. Analysis was performed after thawing the probes. Viability of nuclear cells has been assessed using the microscopic technique after incubation in Trypan blue and the CD34+ cells by flow cytometry using the 7-aminoactynomycin D. A statistical analysis has been conducted using the Statistica program (StatSoft, Cracow, Poland).


CONCLUSIONS
The results show that the application of autologous plasma is linked with higher viability of nuclear cells and CD34+ cells. Moreover, statistical analysis of the nuclear cells and CD34+ cells viability differs significantly between groups frozen using RPMI 1640 and autologous plasma (P < .05). To assess the viability of CD34+, cells frozen using RPMI 1640 results showed a large span of at 16.4% to 99.1% living cells.
Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan, Poland. Electronic address: maria.kozlowska-skrzypczak@skpp.edu.pl.
M
M
Kozlowska-Skrzypczak
Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan, Poland.
A
A
Kubiak
Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan, Poland.
E
E
Bembnista
Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan, Poland.
P
P
Matuszak
Department of Hematology and Bone Marrow Transplantation, Poznan University of Medical Sciences, Poznan, Poland.
M
M
Komarnicki
D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D003470:Culture Media / Q000494:pharmacology*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D005434:Flow Cytometry; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000187:drug effects*; D006801:Humans; D007937:Leukapheresis / Q000379:methods; D010949:Plasma; D011044:Poland; D014182:Transplantation, Autologous
D003451:Cryoprotective Agents; D003470:Culture Media; D004121:Dimethyl Sulfoxide