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

Cytotherapy. 2023 Oct;25(10):1057-1068. doi: 10.1016/j.jcyt.2023.07.004. Epub 2023 Jul 28.

Abstract

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. Furthermore, 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 collection without impacting on hUC-MSC characteristics. The removal of blood vessels before explant cultures improved hUC-MSC purity. Expansion in Minimum essential medium α 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.

Keywords: differentiation; mesenchymal stromal cells; platelet lysate; protocol standardization; umbilical cord.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Differentiation
  • Cell Proliferation
  • Humans
  • Mesenchymal Stem Cell Transplantation*
  • Mesenchymal Stem Cells*
  • Reproducibility of Results
  • Umbilical Cord