How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years - implications for clinical transplantation and stability programs

John Underwood; Mahvish Rahim; Carijo West; Rebecca Britton; Elaine Skipworth; Vicki Graves; Steven Sexton; Hillary Harris; Dave Schwering; Anthony Sinn; Karen E Pollok; Kent A Robertson; W Scott Goebel; Kerry M Hege

doi:10.4252/wjsc.v12.i5.359

How old is too old? In vivo engraftment of human peripheral blood stem cells cryopreserved for up to 18 years - implications for clinical transplantation and stability programs

World J Stem Cells. 2020 May 26;12(5):359-367. doi: 10.4252/wjsc.v12.i5.359.

Authors

Affiliations

¹ Departments of Internal Medicine and Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, United States.
² Department of Pediatrics, Division of Pediatric Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN 46202, United States.
³ Cellular Therapy Laboratory, Indiana University Health, Indianapolis, IN 46202, United States.
⁴ Department of Pediatrics, Herman B Wells Center for Pediatric Research, In Vivo Therapeutics Core and Angio Biocore Shared Resource Facilities for the Indiana University Simon Cancer Center, Indiana University School of Medicine, Indianapolis, IN 46202, United States.
⁵ Department of Pediatrics, Division of Pediatric Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN 46202, United States. sgoebel2@iu.edu.

Abstract

Background: Peripheral blood stem cells (PBSC) are commonly cryopreserved awaiting clinical use for hematopoietic stem cell transplant. Long term cryopreservation is commonly defined as five years or longer, and limited data exists regarding how long PBSC can be cryopreserved and retain the ability to successfully engraft. Clinical programs, stem cell banks, and regulatory and accrediting agencies interested in product stability would benefit from such data. Thus, we assessed recovery and colony forming ability of PBSC following long-term cryopreservation as well as their ability to engraft in NOD/SCID/IL-2Rγ^null (NSG) mice.

Aim: To investigate the in vivo engraftment potential of long-term cryopreserved PBSC units.

Methods: PBSC units which were collected and frozen using validated clinical protocols were obtained for research use from the Cellular Therapy Laboratory at Indiana University Health. These units were thawed in the Cellular Therapy Laboratory using clinical standards of practice, and the pre-freeze and post-thaw characteristics of the units were compared. Progenitor function was assessed using standard colony-forming assays. CD34-selected cells were transplanted into immunodeficient mice to assess stem cell function.

Results: Ten PBSC units with mean of 17 years in cryopreservation (range 13.6-18.3 years) demonstrated a mean total cell recovery of 88% ± 12% (range 68%-110%) and post-thaw viability of 69% ± 17% (range 34%-86%). BFU-E growth was shown in 9 of 10 units and CFU-GM growth in 7 of 10 units post-thaw. Immunodeficient mice were transplanted with CD34-selected cells from four randomly chosen PBSC units. All mice demonstrated long-term engraftment at 12 wk with mean 34% ± 24% human CD45+ cells, and differentiation with presence of human CD19+, CD3+ and CD33+ cells. Harvested bone marrow from all mice demonstrated growth of erythroid and myeloid colonies.

Conclusion: We demonstrated engraftment of clinically-collected and thawed PBSC following cryopreservation up to 18 years in NSG mice, signifying likely successful clinical transplantation of PBSC following long-term cryopreservation.

Keywords: Colony-forming units assay; Cryopreservation; Hematopoietic stem cell transplantation; Hematopoietic stem cells; In vitro techniques; Long-term storage; Peripheral blood stem cell; Transplant; Viability.