Alternative Methods of Cryopreservation of Human Peripheral Blood Stem Cells for Marrow Transplantation

Abstract

Background: Peripheral blood stem cells (PBSC) have become a widely applied therapeutic option inhematological, immunological diseses.Simplified methods for PBSC cryopreservation and storage are needed.Objectives: To select an alternative cryopreservation protocols of (PBSC) to reduce the cost, time and enhance thesafety and effectiveness of these cryopreservation methods.Subjects Methods: The current study was the first Iraqi study in this field, conducted at BMT (Bone Marrowtransplantation) center in Baghdad Iraq, on nine healthy male donors, median age 34.8 years (range 21-46years). The plan of the study was as follow:Mobilization of peripheral blood stem cells (PBSC) with G-CSF, Collection of PBSC (leukapheresis) was doneusing a blood cell separator (Haemonatics MCS +) Processing of PBSC& mixing with the following differentcryopreservative solutions:(Sol 1): 10% DMSO (dimathylsulfoxide) + autologous plasma(Sol 2): 10% DMSO + autologous plasma + Hank’s solution.(Sol3):5%DMSO+Autologousplasma+phosphate buffer saline.Then Cryopreservation in 3 different methods:(Freeze- A): controlled rate freezing (CRF) & stored in liquid nitrogen -196 ºC.(Freeze - B): liquid nitrogen (LN2) -196 ºC.(Freeze -C): blood bank (B.B) mechanical freezer at -60 ºC.All samples were stored for one month. Viability test for PBSC using Try pan blue stain was conducted on eachdifferent sample after thawing at day 1, 3, 7&30.Results: Comparing the results obtained from the three different types of solutions 1, 2, 3 &the three types freezingA, B, & C, the following points were observed:Using Sol 1, Sol 2& Sol 3 ,the survival rate of PBSC in Sol 1,2,3 all values showed statistically significantdifferences between days 1,3,7&30 with P value <0.05 in the three freezing methods .The best results were foundin CRF ,then LN2 & the lowest in blood bank freezer ,by using Sol 1&Sol 3,while using Sol 2,results of CRF wasequal to LN2&both were higher than blood bank freezer. All the three methods of freezing can be applied safely forcryopreservation of PBSC.Conclusion: In the cryopreservation method of blood bank mechanical freezer applying Sol 3 is an alternativemethod which has lower percent of DMSO =5% and therefore lower toxicity effects with effective resultsregarding viability of PBSC. We can use Sol 2 instead of Sol 1 to have the same effective results ofcryopreservation in liquid nitrogen equal to that of controlled rate freezing. So we can obtain high results ofviability in PBSC by simpler, cheaper and less time consuming method of cryopreservation.Key words: cryopreservation, peripheral blood stem cells.