Effects of Prolonged in vitro Culture and Cryopreservation on Viability, DNA Fragmentation, Chromosome Stability and Ultrastructure of Bovine Cells from Amniotic Fluid and Umbilical Cord

Abstract

The objective of this work was to study cellular types that did not participated in the gastrulation process, amniotic fluid cells (AFCs) and umbilical cord cells (UCCs), in conditions of long-term culture and cryopreserved with different solutions. The AFCs and UCCs were used in a comparative study with ear fibroblast cells (EFCs) that were cultured in vitro until 20 cellular passages and cryopreserved in 10% dimethylsulphoxide (DMSO), 5% dimethyl formamide (DMF) and 7% glycerol (Gly) solutions. The cellular viability, ultrastructure, DNA fragmentation and chromosome stability were evaluated to determine the cellular type most resistant. In all cell types, it was possible to evaluate the AFCs until 15 passages and UCCs until 20 passages with different periods of cellular growth to reach the confluence phase. Solutions containing 10% DMSO ensured viability of 90.33 ± 5.58%, 90.56 ± 4.40% and 81.90 ± 3.31%, respectively for EFCs, AFCs and UCCs, being significantly more efficient and with less variation than other cryoprotectant solutions. The AFCs were more sensitive to cryopreservation and presented low viability rate at the passage 20 (17.2 ± 8.87%). There was no change in karyotype and nuclear fragmentation was low in all cellular passages studied. With the scanning electron analysis was possible the characterization of AFCs and UCCs in suspension. The three cellular types of cells presented different shapes and characteristics on the surface. The results demonstrate that bovine AFCs and UCCs can be isolated, cultured in vitro and cryopreserved in 10% DMSO, not causing damage to DNA and chromosomes. The UCCs were more resistant than AFCs in all aspects.

Isolation and characterization of mesenchymal stem cells derived from bovine Wharton's jelly and their potential for use in cloning by nuclear transfer

“…SEM images demonstrated that WJC assumed a spherical shape without villosities in the transition suspension-adherence phase (Figure 1). Our data corroborated the findings of CUNHA et al (2014).…”

“…Evaluation of cells by SEM was performed as described previously by CUNHA et al (2014). In short, WJC in eighth passage were washed in PBS and fixed with 2% glutaraldehyde and 2% paraformaldehyde in 0.1M sodium cacodylate buffer.…”

Long-term cryopreservation of human oocytes does not increase embryonic aneuploidy

“…Mounting data support the safety of OC, the maintenance of DNA integrity after prolonged cryopreservation of other cell types, as well as the safety of long-term embryo cryopreservation (29,30). However, to our knowledge, no clinical data have been reported on the risk of embryonic aneuploidy after prolonged periods of OC.…”

Bovine endometrial cells: a source of mesenchymal stem/progenitor cells

“…The cryopreservation using DMSO as a protective solution in combination with a progressive reduction of temperature is efficient in the preservation of good viability rates for bovine cells from amniotic fluid and umbilical cord, once enables the culture after thawed (Cunha et al, ). To our knowledge, this is the first report showing the possibility of cryopreservation of bovine eMSCs using two medium of cryopreservation at a controlled temperature rate, allowing the formation of cell biobank.…”