Abstract
Cryopreservation is an important procedure in maintenance and clinical applications of mesenchymal stem/stromal cells (MSCs). Although the methods of cell freezing using various cryoprotectants are well developed and allow preserving structurally intact living cells, the freezing process can be considered as a severe cellular stress associated with ice formation, osmotic damage, cryoprotectants migration/cytotoxicity or rapid cell shrinkage. The cellular response to freezing stress is aimed at the restoring of homeostasis and repair of cell damage and is crucial for cell viability. In this study we evaluated the changes arising in the pig mesenchymal stromal cell transcriptome following cryopreservation and showed the vast alterations in cell transcriptional activity (5,575 genes with altered expression) suggesting the engagement in post-thawing cell recovery of processes connected with cell membrane tension regulation, membrane damage repair, cell shape maintenance, mitochondria-connected energy homeostasis and apoptosis mediation. We also evaluated the effect of known gene expression stimulator—Trichostain A (TSA) on the frozen/thawed cells transcriptome and showed that TSA is able to counteract to a certain extent transcriptome alterations, however, its specificity and advantages for cell recovery after cryopreservation require further studies.
Highlights
Mesenchymal stem cells (MSCs) are highly attractive for tissue engineering and clinical applications because of their inherent regenerative capacity, high proliferation potential, immunomodulatory activity, ability to differentiate into different cell lineages and low immunogenicity [1]
Mononuclear cells were separated by gradient centrifugation at 400 x g for 20 min over a layer of Ficoll-Paque Plus (Stem Cell Technologies; SCT, Canada) and cell suspensions were seeded at a concentration of 1x 105 cells/cm2 per 75 cm2 culture flask (Corning, USA) containing 17 ml medium comprised of low-glucose Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, Germany) supplemented with 10% foetal bovine serum (FBS; SigmaAldrich, Germany) and 1% antibiotic-antimycotic solution (AAS; Sigma-Aldrich, Germany)
No significant differences were noted in CD73, CD90 and CD105 proteins expression in MSCs from three treatment groups implying that Trichostain A (TSA) has neither positive nor negative impact on these markers’ expression (Figs 1 and 2)
Summary
Mesenchymal stem cells (MSCs) are highly attractive for tissue engineering and clinical applications because of their inherent regenerative capacity, high proliferation potential, immunomodulatory activity, ability to differentiate into different cell lineages and low immunogenicity [1]. Changes in the pig MSCs transcriptome following cryopreservation and TSA treatment procedures in regenerative medicine [3, 4], they are cost and time-consuming. This makes in vitro expansion of undifferentiated MSCs an indispensable procedure for both scientific and application purposes. Cryopreservation plays an important role in obtaining off-the-shelf availability for cells. It separates cell culture from application and prepares cells for long distance transportation and long term storage
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