Isolation and ex vivo expansion of allo-WJ-MSCs
hepatitis B, syphilis
HEPATITIS B, VIRUS, SYPHILIS, HEPATITIS C, MYCOPLASMA
The umbilical cord was obtained after informed consent from healthy donors, and the acceptance of screening for pathogenic microorganisms (syphilis, hepatitis B virus, hepatitis C virus, human immunodeficiency virus type 1 and 2, human T-lymphotropic virus type 1 and 2, cytomegalovirus, and Toxoplasma Gondii).The umbilical cord was aseptically stored in sterile HBS. Allo-WJ-MSCs were isolated by enzymatic method. In brief, blood, vessels, and arteries were removed from the umbilical cord. Then, the remaining tissue was cut into smaller pieces which were placed in a collagenase enzymatic solution at 1 mg/mL for four hours, followed by incubation in trypsin-ethylene diamine-tetra-acetic-acid (EDTA) (Gibco, Paisley, UK) solution at 2.5 mg/mL, during 15 min in a humidified environment 37 °C containing 5% carbon dioxide. After incubation, the digested suspension was collected by gravity in a 50 mL conical tube, diluted, and centrifuged at 2000 revolutions per minute for 40 min at 18 ºC. The mononuclear cells were suspended in Minimum Essential Medium Eagle (MEM) (Gibco, Paisley, UK) supplemented with 10% human derivatives from platelet-rich plasma (hD-PRP) obtained from donors with type AB blood, 1% gentamicin, and 2 mM L-glutamine (Gibco, Grand Island, NY, U.S.), and seeded at a concentration of 0.5 × 10Finally, the cells were expanded in vitro to manufacture the required number of cells for the treatment. In the process, a donor master cell bank and working cell bank were maintained as a source of MSCs for future manufacturing purposes.The working cell bank was upscaled further to produce the IMP at passage 3, which was used for the pilot study. Once the desired number of cells was produced, aliquots of samples were provided for quality control testing. These include complete characterization according to the International Society for Cellular Therapy Guidelines [At passage 3, allo-WJ-MSCs were harvested and tested to confirm that were devoid of any microbial contaminants (mycoplasma, endotoxin (≤ 0.5 EU/mL), and aerobic anaerobic and fungi culture).