A bioinspired and chemically defined alternative to dimethyl sulfoxide for the cryopreservation of human hematopoietic stem cells

Abstract

The cryopreservation of hematopoietic cells using dimethyl sulfoxide (DMSO) and serum is a common procedure used in transplantation. However, DMSO has clinical and biological side effects due to its toxicity, and serum introduces variation and safety risks. Inspired by natural antifreeze proteins, a novel class of ice-interactive cryoprotectants was developed. The corresponding DMSO-, protein-, and serum-free cryopreservation media candidates were screened through a series of biological assays using human cell lines, peripheral blood cells, and bone marrow cells. XT-Thrive-A and XT-Thrive-B were identified as lead candidates to rival cryopreservation with 10% DMSO in serum based on post-thaw cell survival and short-term proliferation assays. The effectiveness of the novel cryopreservation media in freezing hematopoietic stem cells from human whole bone marrow was assessed by extreme limiting dilution analysis in immunodeficient mice. Stem cell frequencies were measured 12 weeks after transplant based on bone marrow engraftment of erythroid, myeloid, B-lymphoid, and CD34+ progenitors measured by flow cytometry. The recovered numbers of cryopreserved stem cells were similar among XT-Thrive A, XT-Thrive B, and DMSO with serum groups. These findings show that cryoprotectants developed through biomimicry of natural antifreeze proteins offers a substitute for DMSO-based media for the cryopreservation of hematopoietic stem cells.

Assessment of the Impact of Post-Thaw Stress Pathway Modulation on Cell Recovery following Cryopreservation in a Hematopoietic Progenitor Cell Model

“…Specifically, it was found that an apparent >90% viable population 1 h post-thaw decreased to less than half that (between 20–40%) by 24 h post-thaw following hHPC cryopreservation using traditional culture media with 5 or 10% DMSO ( Figure 1 ). This finding was consistent with previous reports that describe HPC and/or HSC post-thaw viability to range between 40 and 60% of pre-freeze controls with varying cryopreservation media formulations and cryoprotective agents, including 10% DMSO [ 64 , 88 , 95 , 96 , 97 , 98 , 99 , 100 ]. When samples were cryopreserved using Unisol™, overall hHPC viability at 24 h post-thaw increased to 60–65% depending on the DMSO concentration ( Figure 2 ).…”

“…Furthermore, recognition of the critical role cryopreservation plays in the success of the field of regenerative medicine remains a hurdle. In an effort to address these challenges and improve the outcome, today’s researchers are focused on developing new CPAs [ 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 ], targeted apoptotic control [ 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ] during cryopreservation and new devices [ 69 , 70 , 71 ]. On the discovery science side, groups continue to investigate the pathways activated during cryopreservation, as well as during the recovery phase [ 72 , 73 , 74 , 75 , 76 , 77 ].…”

Natural Cryoprotective and Cytoprotective Agents in Cryopreservation: A Focus on Melatonin

“…These properties of natural AFPs have recently inspired the development of new ice-interactive cryoprotectants that have been used as an alternative to DMSO in the cryopreservation of human hematopoietic stem cells [ 52 ].…”

Cryopreservation of undifferentiated and differentiated human neuronal cells

“…However, ice crystal formation occurs throughout the cells during the freezing process if the cells are not sufficiently dehydrated. It is necessary to investigate vitrification and slow freezing with chemically defined freezing solutions (such as ethylene glycol, polyethylene glycol) without DMSO [ 27 , 31 ]. Damage-sensitive cells could be recovered by devising a suitable freezing strategy and optimizing the composition of the freezing solution.…”

“…A chemically defined solution without DMSO using vitrification was effective for the cryopreservation of human embryonic stem cells (ESCs) [ 27 ]. The use of a chemically defined solution using slow freezing for the cryopreservation of pluripotent stem cells and tissue stem cells have been widely reported [ 30 , 31 ]. Cell therapy with cellular and tissue-based products, such as Kymriah (Tisagenlecleucel) and TEMCELL HS Inj.…”