Optimization of human umbilical cord blood-derived mesenchymal stem cell isolation and culture methods in serum- and xeno-free conditions.

BACKGROUND: Although umbilical cord blood (UCB) is identified as a source of mesenchymal stem cells (MSCs) with various advantages, the success in cell isolation is volatile. Therefore, it is necessary to optimize methods of cord blood-derived MSC (UCB-MSC) isolation and culture. In this study, we evaluated the efficiency of UCB-MSC isolation and expansion using different commercially available serum- and xeno-free media and investigated the capacity of autologous serum and plasma as a supplement to support cell proliferation. Additionally, we defined the presence of multilineage-differentiating stress-enduring (Muse) cells in the UCB-MSC population. Functions of UCB-MSC in in vitro angiogenesis processes and anti-cancer were also verified. METHODS: Mononuclear cells were isolated using density gradient separation and cultured in four commercial media kits, as well as four surface coating solutions. UCB-MSCs were characterized and tested on tube formation assay, and co-cultured with SK-MEL cells in a transwell system. RESULTS: The results showed that only StemMACS™ MSC Expansion Media is more appropriate to isolate and culture UCB-MSCs. The cells exhibited a high cell proliferation rate, CFU forming capability, MSC surface marker expression, trilineage differentiate potential, and chromosome stability. In addition, the culture conditions with autologous serum coating and autologous plasma supplement enhanced cell growth and colony forming. This cell population contained Muse cells at rate of 0.3%. Moreover, UCB-MSCs could induce the tube formation of human umbilical vein endothelial cells and inhibit more than 50% of SK-MEL cell growth. CONCLUSIONS: UCB-MSCs could be high-yield isolated and expanded under serum- and xeno-free conditions by using the StemMACS™ MSC Expansion Media kit. Autologous serum coating and plasma supplement enhanced cell proliferation. These UCB-MSCs had effected the tube formation process and an anti-cancer impact.

Comparative Bioactivity Analysis for Off-the-Shelf and Culture-Rescued Umbilical Cord-Derived Mesenchymal Stem/Stromal Cells in a Xeno- and Serum-Free Culture System.

We recently reported a standardized xeno- and serum-free culture platform to isolate and expand umbilical cord-derived mesenchymal stem/stromal cells (UC-MSCs). Comparing populations from the same passage, cells that were cryopreserved and culture-rescued exhibited characteristics similar to those of their fresh counterparts, continuously cultured cells without interim cryopreservation. The culture rescue after thawing allowed for the cells to be fully recovered. However, since it would be more cost-effective and timesaving if cryopreserved cells can be used as an off-the-shelf product, we set out to compare the bioactivity of freshly thawed UC-MSCs versus culture-rescued UC-MSCs of the same batch that were recultured for an additional passage under our xeno- and serum-free protocol. UC-MSCs showed high viability in both the freshly thawed and the re-cultured group. Both populations displayed a similar proliferation capacity which is indicated by a comparable population doubling time and colony-forming ability. Both freshly thawed and culture-rescued UC-MSCs expressed the characteristic immunophenotype and were capable of differentiating into osteocytes, chondrocytes, and adipocytes. On the other hand, culture-rescued cells appeared to be more potent in immunosuppression than freshly thawed cells. In conclusion, freshly thawed and culture-rescued cell products share comparable bioactivity in cell growth and proliferation, immunophenotype, and differentiation potential. However, the culture-rescued cells that were allowed to grow for an additional passage appear to display a more favorable immunomodulatory potential when compared to their freshly thawed parent cells.