Gentamycin amphotericin

Primary human neonatal fibroblasts were isolated from circumcised foreskins. Informed consent was obtained prior to collection of human skin samples with approval from the Oxford Research Ethics Committee; reference 10 (link)/H0605/1. The tissue was cut into small pieces and digested overnight at 4 °C with 0.5 mg/ml Liberase DH in CnT-07 keratinocyte medium (CellnTech) supplemented with penicillin/streptomycin (Sigma) and gentamycin/amphotericin (Life Tech). Following digestion, the epidermis was peeled off from the tissue pieces. The de-epidermized tissue pieces were placed faced down on plastic cell culture plates and allowed to partially dry before addition of DMEM supplemented with 10% FBS and antibiotics. After several days incubation in a 37 °C, 5% CO₂ humidified environment, fibroblasts can be seen to migrate out from the tissue pieces and when their growth reached confluence, they were trypsinized, counted and seeded into fresh plates for experiments. Adult human coronary artery endothelial cells (HCAEC) were purchased from Cell Applications (USA) and cultured in MesoEndo Cell Growth Medium (Sigma) at 37 °C humidified incubator with 5% CO₂.

HEK293 expressing DsRed2-NES (10⁵ cells) were seeded on μ-dish (35 mm, high, ibidi) and incubated at 37 °C for 24 h in growth media (GM, DMEM (Sigma), to which were added 10% FBS (Gibco), 2 mM Glutamax (Life Technologies), 4 μl of gentamycin/amphotericin (Life Technologies)). DsRed2-NES was used in order to confirm nucleus position. The medium was exchanged to Opti-MEM (Life Technologies) before the insertion test. The MB-modified nanoneedle array (the method of modification is described prior section) was then approached to the cells using the manipulator until contact with surface of the dish was confirmed. Then, the array was retracted back several μm and was held still (no oscillation). Confocal fluorescence images of the nanoneedles and cells were acquired starting from the base of the culture dish up to a height of 30 μm, every 1 μm. Fluorescence intensity of the nanoneedles was analyzed taking into account the first 10-μm segment of the nanoneedle, from the tip towards the base, using ImageJ.

Primary human neonatal fibroblasts were isolated from circumcised foreskins. Informed consent was obtained prior to collection of human skin samples with approval from the Oxford Research Ethics Committee; reference 10 (link)/H0605/1. The tissue was cut into small pieces and digested overnight at 4 °C with 0.5 mg/ml Liberase DH in CnT-07 keratinocyte medium (CellnTech) supplemented with penicillin/streptomycin (Sigma) and gentamycin/amphotericin (Life Tech). Following digestion, the epidermis was peeled off from the tissue pieces. The de-epidermised tissue pieces were placed faced down on plastic cell culture plates and allowed partially dry before addition of DMEM supplemented with 10% FBS and antibiotics. After several days incubation in a 37 °C, 5% CO₂ humidified environment, fibroblasts can be seen to migrate out from the tissue pieces and when their growth reached confluence, they were trypsinised, counted and seeded into fresh plates for experiments. Adult human coronary artery endothelial cells (HCAEC) were purchased from Cell Applications (USA) and cultured in MesoEndo Cell Growth Medium (Sigma) at 37 °C humidified incubator with 5% CO₂.