Automated dry thawing of cryopreserved haematopoietic cells is not adversely influenced by cryostorage time, patient age or gender

Abstract

Cell therapies are becoming increasingly widely used, and their production and cryopreservation should take place under tightly controlled GMP conditions, with minimal batch-to-batch variation. One potential source of variation is in the thawing of cryopreserved samples, typically carried out in water baths. This study looks at an alternative, dry thawing, to minimise variability in the thawing of a cryopreserved cell therapy, and compares the cellular outcome on thaw. Factors such as storage time, patient age, and gender are considered in terms of cryopreservation and thawing outcomes. Cryopreserved leukapheresis samples from 41 donors, frozen by the same protocol and stored for up to 17 years, have been thawed using automated, water-free equipment and by conventional wet thawing using a water bath. Post-thaw viability, assessed by both trypan blue and flow cytometry, showed no significant differences between the techniques. Similarly, there was no negative effect of the duration of frozen storage, donor age at sample collection or donor gender on post-thaw viability using either thawing method. The implication of these results is that the cryopreservation protocol chosen initially remains robust and appropriate for use with a wide range of donors. The positive response of the samples to water-free thawing offers potential benefits for clinical situations by removing the subjective element inherent in water bath thawing and eliminating possible contamination issues.

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 ).…”

“…To this end, initial observations of the CD34+ cells within the surviving HPC samples suggest a similar % fraction between samples cryopreserved in media or Unisol + DMSO and cultured in recovery media with or without n-acetyl cysteine supplementation. While preliminary, these initial findings are consistent with previous reports [ 51 , 88 , 95 , 96 , 97 , 98 , 99 , 100 , 101 ], and suggest that post-thaw conditioning may provide for increased recovery of total viable cells as well as maintenance of the CD34+ subpopulation, correlating to a higher number of viable CD34+ cells post-thaw. While encouraging, these observations are highly preliminary; therefore, further investigation must be completed prior to drawing any definitive conclusions.…”

“…Reports have shown that a 50 to 75% reduction in overall cell viability can result in the initial 24 h post-thaw period in numerous cell systems [ 22 , 94 ]. However, detailed reports on the extent of hHPC cell loss over this interval are limited [ 88 , 95 , 96 , 97 , 98 , 99 , 100 ]. Our data illustrated that there was a substantial level of hHPC loss over the initial 24 h recovery period ( Figure 1 ).…”

Walking on thin ice: controlled freezing & thawing of pharmaceutical active molecules

“…Otherwise, it could take up to three weeks [9]. It should be noted, however, that fast thawing is not necessarily associated with improved post-thaw recovery [13].…”