Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution.
OBJECTIVE
Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate-freezing and liquid nitrogen storage have become 'routine' practice in many cell-processing facilities, there is clearly room for improvement as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of dimethylsulfoxide (DMSO) that can contribute to the morbidity and mortality of the transplant therapy.


METHODS
This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor (BioLife Solutions Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 x 10(7) cells/1 mL sample vial were prepared for cryopreservation in the following solutions: (a) FHCRC standard, Normosol-R, 5% human serum albumin (HAS) and 10% DMSO; and (b) CryoStor CS10 (final diluted concentration of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of 1 week. Vials were then thawed and evaluated for total nucleated cell count (TNC), viability, CD34 and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose.


RESULTS
The PBSC samples frozen in CryoStor CS10 yielded significantly improved post-thaw recoveries for total viable CD34(+), colony-forming units (CFU) and granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8-fold increased recovery of viable CD34(+) cells (P=0.005), a 1.5-fold increase in CFU-granulocyte-macrophage (GM) numbers (P=0.030) and a 2.3-fold increase in granulocyte recovery (P=0.045).


CONCLUSIONS
This study indicates that use of CryoStor for cryopreservation can yield significantly improved recovery and in vitro functionality of stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary; however, these findings imply use of CryoStor for cryopreservation could result in improved engraftment for those patients with a lower content of CD34(+) cells in their PBSC collections, along with reducing the requirement for additional apheresis collections and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.
Clarke
Dominic M
DM
BioLife Solutions Inc., Bothell, Seattle, Washington, USA. Dclarke@BioLifeSolutions.com
Yadock
David J
DJ
Nicoud
Ian B
IB
Mathew
Aby J
AJ
Heimfeld
Shelly
S
D018952:Antigens, CD34; D003114:Colony-Forming Units Assay; D015925:Cryopreservation / Q000379:methods*; D005615:Freezing; D006098:Granulocytes / Q000166:cytology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry*; D012015:Reference Standards; D012996:Solutions / Q000737:chemistry*
D018952:Antigens, CD34; D012996:Solutions