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.
Highlights
Hematopoietic stem cell (HSC) transplantation is often a last resort for patients battling various inherited or acquired conditions [1, 2]
To prevent cell death cryoprotective agents (CPAs) are used that interfere with water crystallization, with 10% dimethyl sulfoxide (DMSO) in a solution with a high content of serum being standard for cryopreservation [7, 8]
peripheral blood mononuclear cells (PBMCs) were stained for monocytes (CD3-CD56-CD14+), B-cells (CD19+), and T-cells (CD3+) 24 h after culture
Summary
Hematopoietic stem cell (HSC) transplantation is often a last resort for patients battling various inherited or acquired conditions [1, 2]. To prevent cell death cryoprotective agents (CPAs) are used that interfere with water crystallization, with 10% dimethyl sulfoxide (DMSO) in a solution with a high content of serum being standard for cryopreservation [7, 8]. An effective CPA, DMSO is toxic to cells and to patients receiving cell products containing DMSO [9, 10]. 100% of bone marrow transplant recipients receiving DMSO-cryopreserved cells suffer side effects or serious complications during infusion [20]. Removal of DMSO before transplantation can lead to cell loss [22] and is time-consuming, cumbersome, and does not fall in line with minimal manipulation guidelines set by the FDA. As a strong solvent, DMSO leaches and etches plastic transfusion tubing as well as other containers, which increases risk for cGMP processes [23]
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