Comparative study of fresh and cryopreserved rat mesenchymal stem cells: cell viability, immunological gene expression, and cytokine production

Abstract

Cryopreservation is fundamental for mesenchymal stem cell (MSC) banking, but the freeze–thaw cycle induces sub-cellular stress, potentially modifying the post-thaw genomic and paracrine identity of the cellular product. This study aimed to compare the biological, molecular, and functional stability of fresh and cryopreserved rat bone marrow MSCs (Rattus norvegicus BM-MSCs). Rat BM-MSCs were allocated into non-cryopreserved control cells (Fresh-MSCs) and cells frozen for 4 weeks in liquid nitrogen using 10 % DMSO (Cryo-MSCs). Cell survival was determined using the MTT assay. Immunoregulatory (Il10, Tgfβ1, Ido1) and apoptotic (Bax, Casp3, Bcl2) transcripts were evaluated via RT-qPCR. Paracrine protein concentrations of IL-10, TGF-β, TNF-α, IL-6, VEGF, and HGF were quantified via ELISA. Cryogenic processing significantly reduced post-thaw cell survival relative to fresh controls, though cellular viability safely remained high (87.32±2.91 % vs. 100.00±3.67 %; P<0.001). At the transcriptomic tier, Cryo-MSCs displayed a profound downregulation of all key immunomodulatory transcripts (P<0.05), with Il10, Tgfβ1, and Ido1 levels dropping to 0.68±0.07, 0.77±0.06, and 0.61±0.06, respectively. This matched a 21.58 % and 21.87 % drop in secreted IL-10 and TGF-β protein concentrations (P≤0.05), running parallel to a 15 % to 20 % loss in the paracrine release of VEGF and HGF (P≤0.01). Conversely, cryogenic shock triggered an acute stress response that severely inverted the mitochondrial apoptosis ratio, inducing a significant 1.61-fold upsurge in pro-apoptotic Bax and a 1.49-fold increase in executioner Casp3 transcripts, coupled with a 30.0 % reduction in survival-associated Bcl2 mRNA (P≤0.05). This transcriptomic disruption reprogrammed the post-thaw secretome toward an inflammatory phenotype, characterized by a dramatic 2.12-fold and 1.59-fold escalation in secreted TNF-α and IL-6 protein levels, respectively (P≤0.05). Standard cryopreservation effectively preserves cell viability but causes a temporary operational impairment encompassing transcriptomic suppression of immunomodulatory networks, downregulatory trophic trends, and accelerated apoptotic signaling. These findings reveal compromised functional quality post-thaw, highlighting the clinical necessity for an optimized post-thaw acclimation incubation period prior to cell administration.