Effects of cryopreservation protocols on the viability of umbilical cord blood stem cells

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Effects of cryopreservation protocols on the viability of umbilical cord blood stem cells

 

Author: Chan, Lai-shan Judy
Title: Effects of cryopreservation protocols on the viability of umbilical cord blood stem cells
Degree: M.Sc.
Year: 2006
Subject: Hong Kong Polytechnic University -- Dissertations.
Fetal blood.
Cryopreservation of organs, tissues, etc.
Department: Dept. of Health Technology and Informatics
Pages: x, 153 leaves : col. ill. ; 30 cm.
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2174226
URI: http://theses.lib.polyu.edu.hk/handle/200/2979
Abstract: Hematopoietic umbilical cord blood (UCB) stem cells have been demonstrated as important resources for allogeneic transplantation over the last decade. Owing to the limited volume and low cell numbers available in UCB samples, an optimized cryopreservation protocol for preserving UCB is of utmost importance to enforce maximal cell recovery. An experimental design for testing the main and interactive effects of cryoprotectant and the freezing program of a cryopreservation protocol was introduced in the study. A total of seven cryoprotectant combinations and three freezing programs were investigated. Three variables, namely, viable nucleated cell recovery, viable CD34+ cell recovery and viable leukocyte recovery, were analyzed to compare the various cryopreservation combinations. Two detection methods were employed in the study: Trypan Blue exclusion test for the viable nucleated cell recovery and flow cytometry for the remaining two variables. The 5% DMSO was selected as the best performing cryoprotectant among the seven cryoprotectant combinations. By modifying the freezing program in a controlled-rate programmable freezer, an optimized cryopreservation protocol was finally established, and consisted of 5% DMSO with an optimized freezing program. The viable CD34+ cell recovery attained for the optimized protocol was found to be 66.4% with a standard deviation of 9.1%. The present study also suggested a practical process of manipulating the freezing program in a controlled-rate freezer for UCB preservation in the cryovial and cryogenic bag. In addition, the relationship between the cryovial and cryogenic bag was investigated by a simulation study using normal blood as a substitute for cord blood. The present data proved that the simulation was applicable for evaluation purpose. Importantly, to a certain extent, the constraint of resources in future studies can be alleviated by applying the simulation. Better understanding of the cryopreservation protocol, especially the freezing curve was also provided in this study which will definitely be useful in the future development of a cord blood bank service. In conclusion, the present study accentuates the various aspects of an optimized cryopreservation protocol that affect the cryopreservation of the umbilical cord blood stem cells.

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