Tissue culture flask

All cell culture reagents were purchased from Sigma-Aldrich (Dorset, England) unless otherwise specified. Pancreatic insulinoma RINm5F β-cells (ATCC, CRL11605, Rockville, MD, USA) from Rattus norvegicus were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (32404, Gibco, Loughborough, England) supplemented with 10% (v/v) fetal bovine serum (F9665), 1 mM sodium pyruvate (11360070, Gibco, Loughborough, England), 4500 mg/L glucose (G8644), 2 mM Glutamax supplement (35050061, Gibco, Loughborough, England), 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and maintained at 37 °C, 5% CO₂, and 95% relative humidity in tissue culture flasks (Greiner Bio-One, Kremsmünster, Austria). Cultures were routinely split (1:2) at ~80% confluency by rinsing the cells with Dulbecco’s phosphate buffered saline (DPBS, 10 mm) without calcium or magnesium and released for sub-cultivation using 0.25% (v/v) trypsin-EDTA.

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and trypsin EDTA were purchased from Gibco (Gibco, Germany). Tissue culture flasks and disposable plastic ware purchased from Greiner Bio-One (Frickenhausen, Germany). N-Acetyl-L-cysteine and secondary antibodies were purchased from Sigma Aldrich (Taufkirchen, Germany). All primary antibodies were purchased from Abcam (Cambridge, UK). Polyvinylidene difluoride (PVDF) membrane was from Millipore (Schwal-bach, Germany). ECL reagents were from Amersham Pharmacia Corp. (Piscataway, NJ, USA). DHE and MitoSOX red were from Invitrogen (San Diego, CA, USA).

Soya phosphatidylcholine (SPC) was obtained from the Lipoid (Steinhausen, Switzerland), under the brand name of Phospholipon 90. The suppliers of other reagents were: PG (Chempure, Singapore), dialysis tubing (Thermo Scientific, Waltham, MA, USA), ibuprofen and ibuprofen sodium (Sigma, Singapore), tofacitinib citrate (Sigma, Singapore), rhodamine B (Sigma, Singapore), calcein (Sigma, Singapore), lidocaine (Tokyo Kasei, Tokyo, Japan), phosphate buffered saline (PBS) (Vivantis, Singapore). Polyethylene glycol-4-arm, succinimidyl glutarate, bovine fibrinogen, bovine thrombin and calcium chloride were purchased from Sigma-Aldrich, Singapore. Growth medium for culturing keratinocytes, fibroblasts, pericytes and human dermal microvascular cells were purchased from PromoCell, Heidelberg, Germany. ThinCert culture ware and tissue culture flasks were purchased from Greiner Bio-One, Kremsmünster, Austria. Water was purified by using Milli-Q system (Millipore, Billerica, MA, USA).

Human primary foreskin fibroblasts were obtained from American Type Culture Collection (ATCC nr. CRL 2429, LGC GmbH, Wesel, Germany). Cells were grown in growth medium (GM) consisting of Dulbecco’s modified Eagle Medium (DMEM GlutaMAX, Gibco; ThermoFisher Scientific, Roskilde, Denmark) supplemented with 10% fetal calf serum (FCS), 100 IU/mL penicillin, and 0.1 mg/mL streptomycin (all from ThermoFisher Scientific, Roskilde, Denmark). The cells were maintained in tissue culture flasks (Greiner Bio-one, Frickenhausen, Germany) in a standard humid incubator at 37 °C and 5% CO₂. The cells were passaged using TrypLE select (Gibco, ThermoFisher Scientific) when they reached 80–90% confluency. All experiments were conducted using cells between passages 3 and 5.

The IPL-LD 65Y cell line (Lymantria dispar) was obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany, No. ACC 181) and was maintained for routine culture in TC-100 medium (Lonza, Basel, Switzerland) with 11% fetal calf serum (FCS, PromoCell, Heidelberg, Germany). The cells were seeded with an initial concentration of 2E+05 cells per ml in tissue culture flasks (Greiner bio-one, Frickenhausen, Germany) and incubated at 27°C in a cooling incubator (Thermo Fisher Scientific, Schwerte, Germany). Cells were routinely passaged every seventh day.
Several commercially available substances were used in the screening assay. Fumagillin, surfactin, paromomycin, and metronidazole were obtained from Sigma-Aldrich (Taufkirchen, Germany); quinine, ornidazole, albendazole, tinidazole, clioquinol, and dimethylsulfoxid (DMSO) were obtained from VWR International (Darmstadt, Germany).

Fibroblasts were cultured in MEM (Gibco) supplemented with 10% (v/v) FBS (Gibco) 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco), 1 × MEM Vitamin Solution (Gibco), 1 mM sodium pyruvate (Gibco), 1 × NEAA (Gibco), 2 mM L-glutamine (Gibco) and 50 μg/ml uridine nanopure (Sigma). Cells were grown in tissue culture flasks (Greiner) at 37 °C in a humidified cell culture incubator containing 5% CO₂. For both patients and controls, we used cells at similar passage numbers (below 5). To investigate the effect of VPA on control and POLG-affected fibroblasts, cells were cultured in media supplemented with different VPA (Sigma) concentrations (0, 2, 5, 10 and 30 mM VPA) and various time points (3, 6, 8 and 10 days). All the cells died within few days of exposure to 30 mM VPA. At the same time, no obvious morphological changes were observed with 5 mM VPA after 10 days; hence, we decided on 10 mM to be the final VPA concentration in our model system. Cell viability was assessed by daily observations of morphology and density of fibroblasts using an inverted phase contrast microscope (Leica). The cellular pellets were obtained from (i) untreated fibroblast lines and (ii) cells cultured for 10 days in 10 mM VPA-supplemented growth media.