Ultraglutamine

Human embryonic kidney 293 cells were maintained at subconfluence in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose (Sigma‐Aldrich) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Sigma‐Aldrich), 2 mM Ultraglutamine (Lonza), 10 U/mL penicillin, and 100 U/mL streptomycin (Lonza). Cells were grown at 37°C in a humidified atmosphere with 5% CO₂. The myogenic C2C12 mouse cell line was maintained at subconfluence in DMEM containing 4.5 g/L glucose (Sigma‐Aldrich) supplemented with 20% heat inactivated FBS, 2 mM Ultraglutamine, 10 U/mL penicillin, and 100 U/mL streptomycin and grown at 37°C in a humidified atmosphere with 5% CO₂. C2C12 cells were differentiated at 95% confluence by reducing serum levels to 2% horse serum (Thermo Fisher Scientific).

Two gastric adenocarcinoma cell lines were used: the EPG85-257 P – sensitive and EPG85-257 RDB – daunorubicin-resistant cell line. Human gastric cancer cells were obtained as a kind gift from Prof. Herman Lage. The EPG85-257 RDB cell line reveals the overexpression of MDR1 and the resistance to daunorubicin [37 (link)]. Both cell lines were cultured in Leibovitz L-15 medium (Sigma-Aldrich, Poznan, Poland) supplemented by 10% fetal bovine serum (FBS, Lonza, Poland), 1% antibiotics (penicillin/streptomycin, Sigma-Aldrich, Poznan, Poland), 1 mM ultraglutamine (Sigma-Aldrich, Poznan, Poland), 6.25 mg/L fetuin (Sigma-Aldrich, Poznan, Poland), 2.5 mg/mL transferrin (Sigma-Aldrich, Poznan, Poland), 0.5 g/L glucose, 1.1 g/L NaHCO₃, and 1% minimal essential vitamins (Mem-Vit, Sigma-Aldrich, Poznan, Poland). Cell cultures were cultured as a monolayer on a 25 and 75 cm² plastic flask (Sarstedt, Germany) maintained in a humidified atmosphere at 37 °C and 5% CO₂, and detached for the experiments by trypsinization (trypsin 0.025% and EDTA 0.02% solution, Sigma-Aldrich, Poznan, Poland). Cells were passed every 2–3 days and a day before the experiment.

SkMCs were obtained from the patient from remaining muscle tissue after a cruciate ligament reconstruction procedure. Skeletal myogenic cells were isolated and enriched using a previously described methodology called “pre-plating” with gelatin coated flasks [16 (link)]. All procedures here employed were approved by the Local Bioethical Committee of Poznan Medical University (permission no. 818/13) and were conducted in accordance with the principles of Good Clinical Practice. Written consents from the patients were obtained. All the methods used in the study with human biological material adhered to the principles outlined in the Declaration of Helsinki. Cells were cultured in standard Dulbecco’s modified Eagle’s medium with 4.5 g/L of glucose, supplemented with 20% bovine fetal serum (Lonza Group, Basel, Switzerland), 1% antibiotics, 1% Ultraglutamine, and bFGF (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in vitro in standard cell culture conditions (95% humidity and 5% CO₂ at 37 °C). The medium was changed every 48 h, and to avoid spontaneous myotube formation the cells were passaged when they reached 70% confluence using 0.25% trypsin (Lonza Group, Basel, Switzerland). Cells were in vitro cultured until they reached appropriate distortion quantity and confluence, then the polymeric agent was applied for 24 h, either loaded with BCB or without cargo.