title: 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.
authors.affiliation: Post Graduate Program in Animal Biology, University of Brasília, Brasilia, Brazil; CAPES Support scholar ship, Brasilia, Brazil.
authors.forename: E R
authors.initials: ER
authors.lastname: da Cunha
authors.forename: C F
authors.initials: CF
authors.lastname: Martins
authors.forename: C G
authors.initials: CG
authors.lastname: Silva
authors.forename: H C
authors.initials: HC
authors.lastname: Bessler
authors.forename: S N
authors.initials: SN
authors.lastname: Báo
mesh_terms: D000653:Amniotic Fluid / Q000166:cytology*; D000818:Animals; D002417:Cattle / Q000502:physiology*; D018929:Cell Culture Techniques / Q000379:methods / Q000662:veterinary*; D002470:Cell Survival; D015925:Cryopreservation / Q000662:veterinary*; D003582:Cytogenetics; D053938:DNA Fragmentation; D013997:Time Factors; D014470:Umbilical Cord / Q000166:cytology*