title: A theoretically optimized method for cord blood stem cell cryopreservation.
abstract: The objective of this study was to develop an optimal cryopreservation method for human umbilical cord blood hematopoietic progenitor cells as evidenced by improved retention of in vivo engraftment ability and multilineage differentiation. An extended understanding of the osmometric/permeability characteristics of cord blood stem cells was accomplished by measuring permeability of the cryoprotectant dimethyl sulfoxide (DMSO) at below-ambient temperatures (10 degrees and 3 degrees C). These data were combined with previously published osmotic and permeability data and the water-NaCl-DMSO phase diagram in conjunction with a mathematical model to determine an optimal initial DMSO concentration, cooling rate, and liquid nitrogen plunging temperature. Cells cryopreserved with the theoretically optimized procedure were then compared with cells frozen using standard methods for the ability to engraft in irradiated NOD/SCID mice. The optimal procedure was determined to include a 0.7 molal (approximately 5%) DMSO concentration at a cooling rate of 4 degrees C/min, and a plunging temperature of -44 degrees C. The optimized protocol resulted in significantly higher engraftment of human CD45(+) cells (17.2 +/- 1.6% vs. 8.4 +/- 1.6%), CD19(+) B lymphocytes (11.3 +/- 1.2% vs. 5.8 +/- 1.2%), and CD34(+) cells (1.9 +/- 0.09% vs. 0.6 +/- 0.09%) compared to cells frozen using a standard method. Engraftment of CD33(+) cells was not significantly different (4.0 +/- 0.3 vs. 3.2 +/- 0.6, respectively). This study demonstrated that the use of a theoretically determined optimal cryopreservation method is superior to standard methods for maintaining UCB PCBs with multilineage repopulation potential in NOD/SCID mice.
authors.affiliation: Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA. Erik@gnrlbiotech.com
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Woods
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Karen
authors.initials: K
authors.lastname: Pollok
authors.forename: Jennifer
authors.initials: J
authors.lastname: Hartwell
authors.forename: Franklin O
authors.initials: FO
authors.lastname: Smith
authors.forename: David A
authors.initials: DA
authors.lastname: Williams
authors.forename: Mervin C
authors.initials: MC
authors.lastname: Yoder
authors.forename: John K
authors.initials: JK
authors.lastname: Critser
mesh_terms: D015703:Antigens, CD / Q000097:blood; D018952:Antigens, CD34 / Q000097:blood; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D005312:Fetal Blood / Q000166:cytology*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D007231:Infant, Newborn; D008962:Models, Theoretical; D015203:Reproducibility of Results
chemical_list: D015703:Antigens, CD; D018952:Antigens, CD34; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide