Impact of freeze‐thaw on isolation of viable CD34+ cells from human cadaveric bone marrow
Authors: Bender Shannon , Gu Dongsheng , LaFontaine Michael , Johnstone Brian , Woods Erik 
Published: April 2020

Introduction

The CD34 surface protein is a marker used to distinguish hematopoietic stem and progenitor cells (HSPCs). CD34+ cells are currently used in stem cell transplantation to treat hematological malignancies following myeloablation of bone marrow (BM). Ossium Health is creating a large and genetically diverse bank of cryopreserved deceased donor BM for medical purposes such as hematopoietic stem cell transplantation (HSCT) as well as biomedical research. Given the growing diversity of the bank, it would be advantageous to cryogenically store whole BM for future isolation of CD34+ cells possessing desirable genetic and demographic attributes. While current protocols are designed to select cells from fresh BM aspirated from live donors, there are no reports of selecting these cells from either previously frozen or cadaveric BM. Therefore, the purpose of this study was to determine (1) whether cadaveric BM is an acceptable source of CD34+ cells, (2) whether functional CD34 cells could be selected from previously frozen BM, and (3) the integrity of CD34+ cells following multiple freeze‐thaw cycles.

Methods

Whole BM was obtained from vertebral bodies recovered from deceased donors consented for research use. CD34+ cells were selected from fresh and thawed BM by Ficoll separation followed by immunomagnetic bead isolation (EasySep, Stem Cell Technologies). Selected cells were subjected to further freeze‐thaw cycles and analyzed by flow cytometry and colony forming unit (CFU) assays to enumerate and evaluate CD34+ cell functionality, viability, purity, and population dynamics.

Results and discussion

Results thus far indicate no difference in CFU counts between fresh and thawed BM. Furthermore, 7‐AAD staining indicated a CD34+ average cell viability of 99+0% from fresh and 98+0% from thawed BM. Finally, long‐term hematopoietic stem cells (LT‐HSC) as defined by the cell surface protein markers CD34+ CD38‐CD90+CD45RA‐CD49f+, comprised 1.94% and 2.18% of CD34+ cells selected from fresh and previously frozen BM, respectively.

Conclusions and future directions

Together, these results indicate the suitability of deceased donor BM and the comparability of CD34+ cells selected from fresh and previously frozen BM. Ongoing experiments include evaluation of BM from additional donors as well as xenotranplantation studies in an irradiated immunocompromised mouse model. Validation of previously frozen cadaveric BM as an acceptable substrate for viable CD34+ cell isolation will enable a large and genetically diverse source of HSPC for both medical and research uses. Furthermore, due to the large volumes obtainable, BM from a single donor yields >100 million CD34+ cells, which is vastly greater than available from living donors, thus, providing a consistent source for study and to be used in therapeutic development.

Support or Funding Information

Thank you to the Marian University College of Osteopathic Medicine for their financial support of this project.