Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. May 26, 2020; 12(5): 359-367
Published online May 26, 2020. 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
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
John Underwood, Departments of Internal Medicine and Pediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, United States
Mahvish Rahim, Karen E Pollok, Kent A Robertson, W Scott Goebel, Kerry M Hege, Department of Pediatrics, Division of Pediatric Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN 46202, United States
Carijo West, Rebecca Britton, Elaine Skipworth, Vicki Graves, Steven Sexton, Hillary Harris, Dave Schwering, Cellular Therapy Laboratory, Indiana University Health, Indianapolis, IN 46202, United States
Anthony Sinn, Karen E Pollok, 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
Author contributions: Skipworth E, Pollok KE, Robertson KA, Goebel WS and Hege KM designed and coordinated the study; West C, Britton R, Graves V, Sexton S, Harris H, Schwering D, Sinn A, Goebel WS and Hege KM performed the experiments, acquired and analyzed data; Underwood J, Rahim M, West C, Britton R, Graves V, Sexton S, Harris H, Schwering D, Pollok KE, Robertson KA, Goebel WS and Hege KM interpreted the data; Underwood J, Rahim M, Goebel WS and Hege KM wrote the manuscript; all authors approved the final version of the manuscript.
Supported by a pilot grant from the Indiana University Center of Excellence in Molecular Hematology, NIDDK, No. P30DK090948 (to Hege KM and Goebel WS); the NIH/NCI Cancer Center, No. P30CA082709 awarded to the Indiana University Simon Comprehensive Cancer Center (to Sinn A and Pollok KE).
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of Indiana University School of Medicine.
Institutional animal care and use committee statement: All animal experiments were performed under supervision of the In Vivo Therapeutics Core using procedures approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee (protocol 0000002985).
Conflict-of-interest statement: Goebel WS receives fees as a consulting medical director for Cook Regentec, LLC, and serves as medical director for Ossium Health, Inc. All other authors report no potential conflicts of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Corresponding author: W Scott Goebel, MD, PhD, Medical Director of the Cellular Therapy Laboratory, Associate Professor of Clinical Pediatrics, Division of Pediatric Hematology/Oncology, Indiana University School of Medicine, Room 2626, 705 Riley Hospital Drive, Indianapolis, IN 46202, United States. sgoebel2@iu.edu
Received: February 28, 2020
Peer-review started: February 28, 2020
First decision: April 2, 2020
Revised: April 14, 2020
Accepted: April 28, 2020
Article in press: April 28, 2020
Published online: May 26, 2020
Processing time: 88 Days and 8.6 Hours
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-2Rγnull (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 cells; Hematopoietic stem cell transplantation; In vitro techniques; Peripheral blood stem cell; Viability; Transplant; Long-term storage

Core tip: 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. 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.