058 Cryopreservation of non-manipulated stem cell products; clinical implication

Abstract

Cryopreservation is of critical importance to the successful clinical use of stem cell products since, except for freshly harvested marrow, most products are cryopreserved months to years before infusion into patients. Despite the importance, data regarding the optimization of cryopreservation of stem cells remains rather limited. Questions remaining that are of interest to the field include defining a shelf life for stem cell products, and determining how to assess successful freezing and thawing procedures. We recently performed two studies to help address these questions. In the first, we assessed the viability of peripheral blood stem cells (PBSC) cryopreserved for 16–18 years (average 17.5 years). Post-thaw cell recovery, viability and colony forming ability were assessed in vitro. To evaluate engraftment capabilities in vivo, four units were thawed, CD34+ cells isolated, and 2 ×105 CD34+ cells were infused into sublethally irradiated NOD/SCID/IL-2R γ null (NSG) mice. Average % cell recovery after thawing the seven units was 92% ± 11.4 with average % post-thaw viability of 72% ± 19. Average colony counts from the seven thawed units were 3.4 ± 3 CFU-GM and 3.1 ± 3 BFU-E. Significantly, at twelve weeks, NSG mice transplanted with two of the units showed evidence of long-term engraftment with the presence of human CD45+ cells by flow cytometry (hCD45+ range 26-81%). Mice transplanted with the other two units are in progress. This study demonstrates PBSC can be cryopreserved for over 16 years and retain colony forming ability, engraft into NSG mice and likely lead to successful clinical transplantation. In the second study, we examined the recovery of nucleated cells from cord blood units and correlated recovery with engraftment kinetics. Retrospective analysis was performed on 59 cord blood units transplanted into 52 patients at Indiana University (average age 23.6 years) with malignant and non-malignant diseases from January 2010 to October 2012. Cord blood units were obtained from 15 different cord blood banks and thawed using the Rubenstein thaw-wash method. Pre-freeze and post-thaw cell dose and viability with trypan blue were analyzed. The corrected recovery was calculated by (pre-freeze – post-thaw cell count) ×viability. This was correlated with neutrophil and platelet engraftment data. Average cell recovery was 81 ± 9.8% with post-thaw viability of 82 ± 6.4% for a corrected recovery of 66 ± 9.9%. Time to engraftment significantly correlated with infused cell dose/kg corrected for viability ( p = 0.0056). Platelet engraftment was significantly predicted by both corrected and uncorrected cell dose/kg ( p < 0.0005). In this study, infused cell dose/kg corrected for post-thaw viability was a stronger predictor of delayed neutrophil engraftment than cell dose/kg alone. In our patient cohort, we were surprised to find that this population averaged a corrected recovery of 66%, suggesting that it is essential to consider a loss of up to a third of the reported cell dose in cord blood unit selection practices. Along with HLA match, optimizing infused cell dose by accounting for additional cell losses using corrected recovery could impact cord selection and patient outcomes. These studies illustrate that while cryopreservation of hematopoietic stem cell products has been successful clinically for many years, work remains to optimize the freezing and thawing process to improve clinical results. Moreover, development of cryopreservation techniques for manipulated stem cell populations such as MSC are in their infancy, and established techniques used for hematopoietic stem cells may not necessarily be optimal for MSC. Some data from the studies in this abstract were presented at the Tandem ASPHO/PBMTC meeting in Miami FL April 2013, and accepted for a poster presentation at the annual AABB meeting in Denver CO, October 2013. Source of funding: None declared. Conflict of interest: None declared. sgoebel2@iupui.edu