Serum starvation as the most suitable method for inducing G₀/G₁ phase cell cycle arrest in six-banded armadillo (Euphractus sexcinctus) skin-derived fibroblast lines

Background: Several studies have evaluated different cell cycle synchronization methods to improve reprogramming efficiency aimed at wild species conservation. The six-banded armadillo is one of the wild mammals with significant ecological and biomedical interests but has not yet been evaluated for reprogramming purposes.

Objective: We investigated the effects in a time-dependent manner of serum starvation (SS; 0.5 % serum), contact inhibition (CI), and roscovitine (RSV 15 and 30 µM) on the cycle synchronization at G₀/G₁ phase of six-banded armadillo fibroblasts by flow cytometric analysis.

Methods: The morphology, viability, and apoptosis levels were determined by microscopic analysis.

Results: The results showed that SS significantly increased the percentage of cells arrested in the G₀/G₁ phase in a time-dependent manner, where SS for 72 h (90.5 % ± 0.8) and 120 h (90.1 % ± 0.9) showed the highest value compared to SS for 24 h (86.1 % ± 0.9) and control group (69.7 % ± 0.8; P<0.05). However, neither CI nor RSV exposure significantly increased the arrest of fibroblasts at the G₀/G₁ phase under any of the evaluated conditions (P>0.05). In contrast to SS and RSV, CI significantly induced necrosis, as evidenced by an increase in dead cells (P < 0.05) over the evaluated periods, thereby disrupting the synchronization efforts. Moreover, all methods demonstrated similar morphology and cell viability rates over the evaluated times, as assessed by the trypan blue exclusion assay.

Conclusion: These results indicated that SS was the most suitable method for cell cycle modulation in G₀/G₁ of six-banded armadillo fibroblasts, especially after 72-120 h of treatment. This finding has significant implications for future reprogramming efforts and conservation strategies for this unique species, as effective cell cycle synchronization can enhance the success of techniques, such as cloning and generation of induced cells to pluripotency.