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

Pia Todtenhaupt; Laura A Franken; Sophie G Groene; Marcella van Hoolwerff; Lotte E van der Meeren; Jeanine M M van Klink; Arno A W Roest; Christiaan de Bruin; Yolande F M Ramos; Monique C Haak; Enrico Lopriore; Bastiaan T Heijmans; Melissa van Pel

doi:10.1016/j.jcyt.2023.07.004

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.

Affiliations

¹ Molecular Epidemiology, Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands; Neonatology, Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands.
² Molecular Epidemiology, Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands.
³ Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands; Department of Pathology, Erasmus Medical Center, Leiden, The Netherlands.
⁴ Neonatology, Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands.
⁵ Pediatric Cardiology, Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands.
⁶ Pediatric Endocrinology, Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands.
⁷ Fetal Medicine, Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands.
⁸ NecstGen, Leiden, The Netherlands; Department of Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: m.van_pel@necstgen.com.

PMID: 37516948
DOI: 10.1016/j.jcyt.2023.07.004

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