Serum starvation is as efficient as roscovitine on the cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.

Fibroblast cycle synchronization in G0/G1 is an essential step for nuclear reprogramming by cloning or induced cells to pluripotency. Considering the diversity among rodents and the ecological and scientific importance of these animals, we compared the contact inhibition, serum starvation, and 10 µM of roscovitine as methods of synchronization of red-rumped agouti fibroblasts. The effects of each protocol were evaluated on the percentage of cycle phase, morphology, viability, and apoptosis levels. The results showed that culturing the cells to serum starvation for 24 h (75.9%), 48 h (81.6%), 72 h (86.2%), 96 h (84.0%), and 120 h (83.7%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P < 0.05) phase than cells not subjected to any cell cycle synchronization method (31.4%). Also, this effect was not different between the times of 48 and 120 h (P > 0.05). A similar response was observed for cells cultured with roscovitine for 12 h (86.9%), 24 h (74.8%), and 48 h (81.7%), with a higher percentage of synchronized cells in G0/G1 compared to cells not submitted to any synchronization treatment (52.2%). Nevertheless, this effect was best evidenced at 12 h (P < 0.05). Also, the contact inhibition for 24-120 h could not synchronize cells in G0/G1, with values ranging from 70.9 to 77.9% (P > 0.05). Moreover, no difference was observed for morphology, viability, and apoptosis levels in any synchronization method (P > 0.05). Therefore, serum starvation is as efficient as roscovitine on cycle synchronization in G0/G1 of red-rumped agouti fibroblasts.

Long-term preservation of established fibroblast lines from six-banded armadillos (Euphractus sexcintus, Linnaeus, 1758) by extended passage and cryopreservation.

Establishing new somatic cell cultures has raised significant attention as an effective and convenient way to preserve genetic samples for different applications. Although many lines have been established in model animals, none derived from six-banded armadillo species is currently available. We report the successful isolation and characterization of fibroblasts from six-banded armadillos, evaluating the cell quality after extended culture and cryopreservation. Initially, we collected ear skin from five captive adult individuals and identified fibroblast lines by morphology, karyotyping, and immunophenotyping assays. The isolated fibroblasts were evaluated after several passages (fourth, seventh, and tenth passages) and cryopreservation by slow freezing. Cell morphology, viability, metabolism, proliferative activity, mitochondrial membrane potential, and apoptosis levels were analyzed. The skin explants had great adhesion, and cell outgrowth could be seen after 3-6 d. The cells were verified as fibroblasts at the fourth passage by vimentin expression and normal karyotype (2n = 58). The viability remained high (> 87%) and constant from the fourth to the tenth passage (p > 0.05). The passages did not change the cell morphology and metabolic and growth rates. Moreover, cryopreservation did not affect most evaluated parameters; post-thawed cells maintained their viability, growth, metabolism, and apoptosis levels. Nevertheless, cryopreservation increased mitochondrial membrane permeability and cell population doubling time compared to non-cryopreserved cells (p < 0.05). In summary, viable fibroblasts can be obtained from six-banded armadillo skin while conserving their quality as the number of passages increases and featuring few changes after cryopreservation.