COMPARISON OF EXTRACTION METHODS AND CULTURE MEDIUM FOR UMBILICAL CORD LINING- AND WHARTON'S JELLY-DERIVED MESENCHYMAL STROMAL CELLS

Background & Aim The umbilical cord (UC) is a rich source of mesenchymal stromal cells (MSC). The function of UC-MSC extracted by enzymatic digestion and long-term culture in bovine serum-supplemented medium may be adversely affected. Using a higher proportion of animal-based supplements may introduce variability in manufactured MSC. We investigated if a cGMP-compliant method would overcome these challenges. This study compared the yield, purity, differentiation potential and chromosomal stability of MSC derived from cryopreserved UC lining and Wharton's Jelly, under the influence of different extraction methods (enzymatic digest versus explant) and culture media (PTT-6 versus DMEM:F12 with 10% FBS). Methods, Results & Conclusion Six donated UC samples were disinfected, cut into discs and frozen in cryopreservant containing 10% DMSO. Samples were thawed at 37°C, then incubated in either media for 3 - 4 days at 37°C in 5% CO 2 . MSC were extracted from the incubated tissues and differentially treated to generate every permutation of cell source, extraction method and culture media (total conditions: 8). At every passage, cell doubling rate was calculated and morphology was captured. At Passage 6 (P6), trilineage differentiation and karyotype were evaluated by StemPro® differentiation kits and G-banding. MSC extracted from all conditions displayed fibroblastic, spindle-shaped morphology and plastic-adherence. Only P6 MSC cultured in DMEM:F12 but not PPT-6 displayed enlarged and polygonal morphology. Overall doubling rate of PTT-6 cultured MSC was significantly faster than its DMEM:F12 counterpart (50.1 ± 9.7 h versus 98.4 ± 37.5 h, P Tissue source and extraction method did not influence trilineage differentiation. PTT-6 favoured adipogenesis while MSC from DMEM:F12 were inclined towards osteogenesis. Both media allowed for chondrogenesis. No gross chromosomal abnormalities were observed after at least 10 population doublings. The patented PTT-6 medium maintained MSC stemness and significantly hastened UC-MSC expansion without detriment to trilineage differentiation or chromosomal stability. Further work is warranted to determine whether replicative senescence was averted.