Trypsin ethylene diamine tetra acetic acid edta

The whole procedure of isolation and characterization was performed at the Center of Excellence for Research in Regenerative Medicine and its Applications (CERRMA), Alexandria Faculty of Medicine.
On day 14 of pregnancy, the female rats were euthanized by an overdose of the inhalation anesthesia. The uterus was dissected, and each gestational sac was opened from the anti-mesenteric side to avoid bleeding from the placental site. Needles of 30-gauge calibration were used to aspirate AF from each sac separately [10 ]. The AF was then spun at 1400 × g for 5 min to pellet the cells. Pelleted cells were cultured in 60 mm culture dish where 5 ml of low glucose Dulbecco's Minimum Essential Medium (DMEM, Lonza) supplemented with 10% foetal bovine serum (FBS, Sigma Aldrich) and 1% penicillin/streptomycin (Lonza) were added. Cells were incubated at 37 °C and 5% CO₂ in a humidified incubator and observed daily under a phase contrast inverted microscope. The media was changed every 2–3 days. Cells were passaged after reaching 80% confluence using 0.25% (w/v) trypsin/ethylene diamine tetra acetic acid (EDTA) (Lonza), then cultured in T75 cm² flasks [11 ]. Cell viability was assessed using the trypan blue (Gibco) exclusion test, and cells were counted using a Neubauer hemocytometer [12 (link), 13 (link)].

Tetraethyl orthosilicate (TEOS, Si(OCH₂CH₃)₄), Triton X-100 (TX100), cetyl trimethylammonium bromide (CTAB, 99%), polyethylene glycol₂₀₀₀ (PEG₂₀₀₀), chitosan (75–85% deacetylated), sodium tripolyphosphate (TPP), Tweens 20, ammonia solution (28–30%), sulfuric acid, sodium carbonate (Na₂CO₃), 5-fluorouracil (5-FU), and deuterium oxide were all purchased from Sigma Aldrich (St Louis, MO, USA). Eagle’s minimum essential medium (EMEM), fetal bovine serum (FBS), penicillin/streptomycin solution (10,000 U/mL), and trypsin−ethylene diamine tetraacetic acid (EDTA) (0.25% trypsin, 0.1% EDTA) were obtained from Lonza (Viviers, Belgium). Phosphate-buffered saline (PBS) tablets were purchased from Calbiochem, San Diego, CA, USA. The MTT salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and trichloroacetic acid (TCA) were purchased from Merck, Darmstadt, Germany. The HeLa, Caco-2, and MCF-7 cells were originally purchased from the ATCC (Manassas, VA, USA), while the HEK293 cells were donated by the Anti-Viral Gene Therapy Unit, University of the Witwatersrand, Johannesburg, South Africa. All sterile plasticware for tissue cultures were obtained from Corning Inc., Corning, NY, USA. All other reagents were of analytical grade.

All chemicals were purchased from Sigma Aldrich (Madrid, Spain) unless otherwise stated. For the cell culture, L-Glutamine (200 mM), penicillin and streptomycin (P/S) (10,000 U/mL each), trypsin-ethylenediamine tetraacetic acid (EDTA) (200 mg/mL), a non-essential amino acid (NEAA) solution, and Leibovitz’s (L-15) cell culture medium were purchased from Lonza (Barcelona, Spain). Phenol red-free serum-free Minimum Essential Medium (MEM) was supplied by Gibco (Life Technologies, Madrid, Spain). Among the reactants used for determining cytotoxicity, resazurin (AlamarBlue^®, AB) and 5-carboxyfluorescein diacetate acetoxy methyl ester (CFDA-AM) were purchased from Invitrogen (Madrid, Spain). For the electron microscopy analyses, paraformaldehyde (16%) and glutaraldehyde (25%) were supplied by Electron Microscopy Sciences (Hatfiled, UK), and Spurr´s resin was provided by TAAB Laboratories Equipment Ltd. (Aldermaston, UK). High-grade purity water (>18 MΩ cm⁻¹) was obtained from a Milli-Q ElementA10 Century (Millipore Iberia, Madrid, Spain).