Trypsin edta solution 1

The MNs were 3D-printed using microfluidic resin, MiiCraft BV-007A (Young Optics Inc., Hsinchu, Taiwan). Human dermal fibroblasts (HDFs), human dermal fibroblast growth medium and trypsin EDTA solution (1×) were purchased from Sigma Aldrich (Sigma Aldrich, Rochester, UK). The ReadyProbes^® Cell Viability Imaging Kit (Blue/Green) was purchased from Fisher Scientific™ (Fisher Scientific, Loughborough UK). The porcine skin was purchased freshly from a local slaughterhouse (Cheale Meats Ltd., Essex, UK).

NHLF were obtained from kidney donors with brain death upon previous consent of the family, clearly stating that the lung tissue will be used to extract cells for research purposes and data will remain anonymous. The protocol was accepted by the ethical boards of the Hospital General de Puebla and the Benemérita Universidad Autónoma de Puebla College of Medicine. After clamping the aorta, a lung sample was obtained from the left lower lobe and placed in HAM F-12 medium. The tissue was processed with Trypsin-EDTA solution 1× (Sigma, St. Louis, MO, USA) and serum-free F-12 medium. The filtrate was centrifuged at 200 g for 10 min and the pelleted cells were re-suspended in F-12 (10% fetal bovine serum) and layered in T-25 flasks. Cells were grown to a 75% confluence in F-12 medium, at 37 °C on an atmosphere of 95% of air and 5% of CO₂. For the experiments, cells from passages 5 to 10 were placed in 6 well plates and when confluence was reached, culture medium was substituted with serum-free F-12 for 24 h. DPPC, PG, PE (all from Sigma-Aldrich) or Beractant (Abbot) were used at different concentrations. Three cell lines were used for all the experiments, obtained from a thirty-year-old male, a sixty-one-year-old male and a fifteen-year-old female.

Two human glioblastoma cell lines (U118 MG, U251 MG) were used as an in vitro model. The U-118 MG was obtained from the ATCC^® (American Type Culture Collection, Virginia, No. HTB-15™) and was continuously cultured in Dulbecco’s modified Eagle’s medium (DMEM, ATCC^®, No. 30-2002). The U251 MG (No. 09063001) human glioblastoma astrocytoma cell line was purchased from PHE (Public Health England, Salisbury, UK) culture collections and cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, GlutaMAX™, Gibco^®, No. 31966021, Thermo Fisher Scientific, Waltham, MA, USA). The cells were maintained in 25 cm² tissue culture flasks (TPP^®, No. 90026, ) and the cultures were supplemented with 10% fetal bovine serum (FBS, Gibco^®, No. 10270106), in a humidified incubator at 37 °C with 5% CO₂. When 70% confluent cell monolayer was reached, the cells were detached with Trypsin/EDTA solution 1× (Sigma-Aldrich. No. T4049, Darmstadt, Germany) until complete cell detachment. The cell suspension with trypsin inactivated by a 5-fold dilution with fresh growth medium was spun down (3 min, 700 rpm) in 15 mL conical tubes, and the cell pellet was then resuspended in a fresh growth medium for cells scoring.