Conditioned medium: a new alternative for cryopreservation of equine umbilical cord mesenchymal stem cells

Abstract

Cryopreservation is a feasible alternative to maintaining several cell lines, particularly for immediate therapeutic use, transportation of samples, and implementation of new in vitro studies. This work parts from the hypothesis that the medium of cryopreservation composed by 90% of conditioned medium (CM) supports cryopreservation of equine umbilical cord intervascular matrix mesenchymal stem cells (UCIM-MSCs), allowing the maintenance of the biological properties for the establishment of cell banks intended for therapeutic use and in vitro studies. Thus, we evaluated the viability, apoptosis/necrosis rates, immunophenotypic profile (IP), chromosomal stability, clonicity, and differentiation potential of UCIM-MSCs cryopreserved with four different mediums (with FBS: M1, M3, M4 and without FBS: M2). After 3 months of cryopreservation, samples were thawed and analyzed. The potential of differentiation in the mesodermal lineages, clonicity, and the chromosomal stability were maintained after cryopreservation of UCIM-MSCs with medium containing FBS. Changes (P < 0.05) at IP for some markers were observed at cells cryopreserved with medium M1-M3. Only the UCIM-MSCs cryopreserved with the CM (M4) had similar viability post-thaw (P = 0.23) when compared with fresh cells. We proved the hypothesis that the medium of cryopreservation containing CM supports the cryopreservation of UCIM-MSCs, at the experimental conditions, being the medium that better maintains the biological characteristics observed at fresh cells. Thus, future studies of UCIM-MSCs secretome should be conducted to better understand the beneficial and protective effects of the CM during the freezing process.

Conditioned media and DMSO enhance the cryopreservation of bovine adipose tissue-derived mesenchymal stem cells

“…Using conditioned media is also more cost-effective than FBS because it reuses the previously used medium. Furthermore, the conditioned media produce soluble factors that help control the immune system and promote cell growth, communication, repair, and regeneration (Maia et al, 2017). Our findings are comparable with those of Maia et al (2017), who reported that conditioned media improves viability and proliferation rate during cryopreservation.…”

“…Furthermore, the conditioned media produce soluble factors that help control the immune system and promote cell growth, communication, repair, and regeneration (Maia et al, 2017). Our findings are comparable with those of Maia et al (2017), who reported that conditioned media improves viability and proliferation rate during cryopreservation. According to the data in Table 1, it is evident that M3 exhibits a higher proliferation rate compared to other media due to the presence of growth factors in the conditioned medium.…”

“…Reusing culture media as conditioned media can also replace FBS and make cryopreservation of MSCs more efficient. According to Maia et al (2017), conditioned media supported cryopreservation and better protected the biological characteristics of fresh cells. The factors present in an MSC-conditioned medium preserves cell viability and functionality during the freezing and thawing process.…”

Addition of synthetic polymer in the freezing solution of mesenchymal stem cells from equine adipose tissue as a future perspective for reducing of DMSO concentration

“…The results demonstrated that the FS containing DMSO, DMSO+X-1000, or DMSO+Trehalose+X-1000, did not show differences in the potential to form colonies with the control. These findings agreed to a study performed with MSCs in an umbilical cord in equine, which showed that the freezing exerted few or no effect on the capacity of these cells in adhering and form colonies (Maia et al, 2017).…”

“…These findings are under previous reports in equines, which showed that the utilization of DMSO in the FS of AT-MSCs had an elevation in the cell viability rate (84 ± 3.9%) post-thawing (Renzi et al, 2012). The DMSO has a more widely used penetrating cryoprotectant of MSCs from the different tissues, such as equine umbilical cord (Maia et al, 2017), human bone medulla (Kaplan et al, 2017), and adipose tissue of sheep and mouse (Renzi et al, 2012). This cryoprotectant shows a low molecular weight (78.13 g/mol), that is why it can quickly pass through the plasmatic membrane, blinds to water molecules in solution, and then, blocks the hydric efflux of the cytoplasm during the freezing process.…”