Dulbecco s modified eagle s medium dmem ham s f12 medium 1 1

Lapine cruciate ligaments used for cell isolation were obtained from the abattoir. Lapine cruciate ligamentocytes were isolated from four healthy female and one male New Zealand Rabbits (mean age of 12 months). Surrounding connective tissue was removed and 1–2-mm sized pieces of the cruciate ligaments were prepared and transferred into a T-25 culture flask (Sarstedt AG & Co. KG, Nürnbrecht, Germany) with 2 mL culture medium (Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F12 medium [1:1] (Bio&SELL), containing 10% fetal bovine serum (FBS, Bio&SELL), 1% penicillin/streptomycin solution, 25 μg/mL ascorbic acid (Sigma-Aldrich, Munich, Germany), 2.5 μg/mL amphotericin B (Bio&SELL) and MEM amino acid solution (Sigma-Aldrich)). Culture medium was changed every 2–3 days. After around 1 week, cruciate ligamentocytes emigrated from the explant. Cells were detached after being 80% confluent using 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) solution (Bio&SELL). Cell numbers and viability were calculated with a hemocytometer using the trypan blue exclusion assay.

For isolation of lapine cruciate ligamentocytes anteroir (ACLs) and posterior cruciate ligaments (PCLs) of 10 healthy male and female New Zealand rabbits (between 4 and 24 months old) obtained from the abattoir were harvested. Slices of 1–2 mm of the ACLs and PCLs (Cls) were prepared and transferred into T25 culture flasks (Sarstedt AG & Co. KG, Nürnbrecht, Germany) with growth medium (Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F12 medium (1:1) (Bio&SELL, Feucht, Germany), containing 10% fetal bovine serum (FBS, Bio&SELL), 1% penicillin/streptomycin solution, 25 μg/mL ascorbic acid (Sigma-Aldrich, Munich, Germany), 2.5 μg/mL amphotericin B (Bio&SELL) and MEM amino acid solution (Sigma-Aldrich)).
Growth medium was changed 2–3 times a week. After 7–10 days, CL-derived ligamentocytes emigrated and were detached using 0.05% trypsin/ 0.02% ethylenediaminetetraacetic acid (EDTA) solution (Bio&SELL). The number of viable cells was determined using the trypan blue exclusion assay with a hemocytometer.