All the culture cell materials from study subjects were collected with informed consent of the parents or the patient, following the recommendation from the Helsinki University Hospital ethical review board. The control cell lines originate from subjects eventually deemed not to manifest a mitochondrial disease.
The fibroblast cultures, previously immortalized by retroviral transduction of E6/E7 proteins of human papilloma virus, were cultivated at 37°C with 5% CO2 in DMEM (Dulbecco’s Modified Eagle’s Medium; Cat. #BE12-614F; Lonza) with 10% fetal bovine serum albumin (Cat. #11550356; Gibco), 1 × GlutaMAX Supplement (Cat. #35050061; Gibco) 50 mg/l uridine (Cat. #6680; Calbiochem), and 50 U/ml penicillin/streptomycin antibody (Cat. #15070063; Gibco) with media change in 3-d intervals. Cells were passaged after reaching 80% confluency by washing with PBS (Dulbecco’s Phosphate Buffered Saline; Cat. #D8537-6X500 ML; Sigma-Aldrich) and incubating in 1 × trypsin-EDTA (Cat. #15400-054; Gibco) in 37°C for 3 min prior to re-plating on two fresh 10-cm dishes. Cell lines were used no more than 30 passages.
The fibroblast cultures, previously immortalized by retroviral transduction of E6/E7 proteins of human papilloma virus, were cultivated at 37°C with 5% CO2 in DMEM (Dulbecco’s Modified Eagle’s Medium; Cat. #BE12-614F; Lonza) with 10% fetal bovine serum albumin (Cat. #11550356; Gibco), 1 × GlutaMAX Supplement (Cat. #35050061; Gibco) 50 mg/l uridine (Cat. #6680; Calbiochem), and 50 U/ml penicillin/streptomycin antibody (Cat. #15070063; Gibco) with media change in 3-d intervals. Cells were passaged after reaching 80% confluency by washing with PBS (Dulbecco’s Phosphate Buffered Saline; Cat. #D8537-6X500 ML; Sigma-Aldrich) and incubating in 1 × trypsin-EDTA (Cat. #15400-054; Gibco) in 37°C for 3 min prior to re-plating on two fresh 10-cm dishes. Cell lines were used no more than 30 passages.