Small-molecule ice recrystallization inhibitors improve post-thaw recovery of mesenchymal stromal cells

Abstract

Background & Aim Mesenchymal stromal cells (MSCs) have therapeutic potential for a variety of diseases; however, preclinical effectiveness has not consistently been replicated in human trials. Rapidly thawed cryopreserved MSCs are often used in clinical trials as such products are more feasible from a logistic and cost perspective. However, recent reports suggest compromised MSC function in freshly thawed cells and this may result in diminished efficacy. To improve MSC function post-thaw, we tested a new class of cryoprotectants that can inhibit ice recrystallization and protect MSCs against cryoinjury. More specifically, we document the effect of two small-molecule ice recrystallization inhibitors (IRIs) N-(2-fluorophenyl)-D-gluconamide (2FA) and p-bromophenyl-β-D-glucopyranoside (pBrPh-Glc). Methods, Results & Conclusion Donor MSCs previously cryopreserved in passage 1 in Master cell banks were thawed and cultured in xeno- and serum-free media for 4 days. After 4 days in culture, MSCs were lifted, counted and cryopreserved at 2 × 106 cells/mL in Standard Freezing Media (SFM, 5% Human Albumin and 10% DMSO in PlasmaLyte A) with or without IRIs (15mM 2FA or 30mM pBrPh-Glc) using an optimized step-down program. Cryovials were subsequently thawed and cells were assessed for viability, identity and recovery post-thaw. Additionally, cell proliferation and interferon-gamma induced IDO expression were measured as surrogate markers of recovery and potency post-thaw. MSCs cryopreserved in SFM had a recovery of 80% and viabilities of 92.5% and 82.3% as measured by Trypan Blue exclusion and Annexin V and PI respectively. In comparison, MSCs cryopreserved in SFM containing 2FA or pBrPh-Glc had recoveries of 91.3 ± 0.7% or 73.3 ± 6.7% respectively. Cell viability as measured by Trypan blue exclusion was 90.9 ± 0.9% in the presence of 2FA versus 87.6 ± 0.8% for pBrPh-Glc. Significantly higher proliferation rates were observed 24h post-thaw in SFM containing 2FA (23.6% ± 1.9% cells in S-phase) than pBrPh-Glc (5.6% ± 0.5%) or SFM alone (6.88%). Additionally, MSCs cultured in XF-and SF-media had lower doubling times (27.4h ± 3.6h versus 59.7h ± 5.3h) and underwent more population doublings (2.71 ± 0.32 versus 1.22 ± 0.10) when cryopreserved in SFM containing 2FA. This effect was enhanced when cells were seeded at very low densities of 100 and 250 cells/cm2. In conclusion, we demonstrate marked improvement in post-thaw MSC recovery, viability and function when cryopreserved with the ice recrystallization inhibitor 2FA.