Alphamem culture medium

Metaphases were obtained from fibroblast cell cultures from a male sample of S. apella and C. capucinus (Cebidae), from Catoctin Zoo, Thurmont MD, USA and the Laboratory of Genomic Diversity of the National Cancer Institute, Frederick, MD, USA. Fibroblast cells were grown for 72 h in alphaMEM culture medium (Gibco, Waltham, MA, USA), 5% Antibiotics Penicillin/Streptomicin, 15% FBS, 10% amniomax (Gibco).
Lymphoblasts of a male sample of H. sapiens were grown in RPMI culture medium, following standardised protocols to obtain metaphases.
Cells harvesting was performed after 3 h incubation of colcemid 10 μL (10 μg/mL Gibco) followed by hypotonic treatments 0.075 M KCl for 20 min at 37 °C following a protocol from Small et al. [33 (link)].

Oral mucosa-derived mesenchymal stromal cells were isolated from a two square centimeter tissue biopsy obtained from the oral mucosa of a donor horse. Small cut pieces of the biopsies were digested by a solution containing 15 mL dispase II 240UI (Roche), 300 μL clinical grade MTF collagenase II (Invitrogen) complemented with amphotericin (2.5 μg/ml), penicillin (100 IU/ml), and gentamicin (50 μg/ml). The sample was digested at 37°C for 2 h30. After digestion, the suspension was sieved and the filtrate was centrifuged at room temperature at 400 g for 10 min. The resulting cell pellet was taken up in an alpha MEM culture medium (Gibco) at 2000 cells/cm². This medium was changed after 48 h with a low concentration of amphotericin (1 μg/ml) and then changed every 48 h until 80% confluence. MSCs obtained from the first passage were used for in vivo experiments. Trilineage differentiation of OM-MSC was verified according to the R&D Systems kit recommendations (R&D Systems).