Freezing and Cryostorage of Hematopoietic Progenitor Cells (HPC) Apheresis Using 5% Dimethyl Sulfoxide (DMSO) without Hydroxyethyl Starch (HES) in Cryocyte Freezing Bags and in Cryogenic Vials.
Authors: Chan Wai Shun , Pecora Andrew L. , Nadasi Suzi , Myers Lester , Munoz Margarita , Jashari Besa , Preti Robert A. 
Published: April 2020

Abstract
Cryoprotectant formulated with 5% DMSO, 6% HES, 0.2% dextrose and 3.75% human serum albumin (HSA) [DMSO/(+)HES], originally adapted by Stiff et al (Blood1987, 70:974–978) for cryopreservation of mononuclear cells from bone marrow in a −86°C mechanical freezer (MF) (dump freezing) without a controlled rate freezer (CRF), is commonly used for cryopreserving human HPC derived from apheresis (HPC-A) and bone marrow. However, use of HES for cryopreserving HPC in clinical setting requires IND approval in the US. As no formulated GMP-grade DMSO/(+)HES is currently commercially available, the preparation of such cryoprotectant requires in-house manufacture and release testing, which can be time consuming and costly.
In this study, we evaluated a cryoprotectant formulated in 5% DMSO and 2.25% HSA in saline without HES [DMSO/(−)HES] for cryopreservation of HPC-A using various freezing and cryostorage procedures. Non-mobilized HPC-A from normal donors were cryopreserved with an equal volume of DMSO/(−)HES and DMSO/(+)HES as controls to achieve 5% DMSO in cell suspensions loaded in Cryocyte freezing bags (Baxter R4R9953) and cryogenic vials. Samples were frozen either by dump freezing in −86°C MF or program freezing in CRF. Cryostorage of frozen HPC samples was accomplished in either the −86°C MF or the vapor phase (&lt; −135°C) of liquid nitrogen (VLN) for a duration of 2–7 days before thawing for quality assessments. Additionally, cells were frozen in both bags and retain vials in order to evalaute the validity of the use of such retains to represent the contents of the freezing containers.
While the viability of HPC-A cryopreservevd in DMSO/(−)HES in bags and vials using combinations of freezing and storage procedures was 92±6% as determined by both trypan blue (TB) and 7-AAD flow cytometry assays, the sample group that cryostored in MF versus that in VLN showed a slight reduction in viability. The recovery of both CD34+ cells (by flow cytometry) and CFU from the pre-frozen samples were also slightly reduced in bag samples cryostored in MF (CD34+ cell recovery of 87±25% and 79±11%; and CFU recovery of 72±8% and 63±7% for samples initially frozen in MF and CRF, respectively) than in VLN (CD34+ cell recovery of 102±44% and 96±35%; and CFU recovery of 104±44% and 116±97% for samples initially frozen in MF and CRF, respectively). Such differences were not observed within the cryogenic vial sample group as the cell recovery in these samples cryostored in VLN (CD34+ cell recovery of 79±11% and 76±38%; and CFU recovery of 79±43% and 56±44% for samples initially frozen in MF and CRF, respectively) was lower than those for the bag group. However, all these observed differences were not statistically significant (paired t-Test analyses).
TB viability, 7-AAD viability, CD34 recovery and CFU recovery of HPC-A cryopreserved in freezing bag using DMSO/(+)HES, frozen and cryostored in MF were 98±1%, 96±1%, 94±18% and 103±9%, respectively; in comparison, samples cryopreserved in cryogenic vials showed a slight, although not significant reduction, in CD34+ cell and CFU recovery.
In conclusion, 5% DMSO and 2.25% HSA in saline appears to be suitable as a cryoprotectant for preserving HPC-A for clinical use. Cell quality of HPC is likely better preserved in freezing bag containers and in VLN for longer term storage. Further studies to assess patient engraftment efficiency of the infused HPC-A cryopreserved with this cryoprotectant are in progress.