A robust and standardized method to isolate and expand mesenchymal stromal cells from human umbilical cord

Background aims: Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) are increasingly used in research and therapy. To obtain hUC-MSCs, a diversity of isolation and expansion methods are applied. Here, we report on a robust and standardized method for hUC-MSC isolation and expansion.Methods: Using 90 hUC donors, we compared and optimized critical variables during each phase of the multi-step procedure involving UC collection, processing, MSC isolation, expansion and characterization. Further-more, we assessed the effect of donor-to-donor variability regarding UC morphology and donor attributes on hUC-MSC characteristics.Results: We demonstrated robustness of our method across 90 UC donors at each step of the procedure. With our method, UCs can be collected up to 6 h after birth, and UC-processing can be initiated up to 48 h after col-lection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium a supplemented with human platelet lysate increased reproducibility of the expansion rate and MSC characteristics as compared with Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum. The isolated hUC-MSCs showed a purity of >> 98.9%, a viability of >97% and a high proliferative capacity. Trilineage differentiation capacity of hUC-MSCs was reduced as compared with bone marrow-derived MSCs. Functional assays indicated that the hUC-MSCs were able to inhibit T-cell proliferation demonstrating their immune-modulatory capacity.Conclusions: We present a robust and standardized method to isolate and expand hUC-MSCs, minimizing technical variability and thereby lay a foundation to advance reliability and comparability of results obtained from different donors and different studies.(c) 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)