PMID- 37516948 OWN - NLM STAT- MEDLINE DCOM- 20230926 LR - 20231002 IS - 1477-2566 (Electronic) IS - 1465-3249 (Linking) VI - 25 IP - 10 DP - 2023 Oct TI - A robust and standardized method to isolate and expand mesenchymal stromal cells from human umbilical cord. PG - 1057-1068 LID - S1465-3249(23)01007-1 [pii] LID - 10.1016/j.jcyt.2023.07.004 [doi] AB - 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 alpha 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 approximately 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. CI - Copyright (c) 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved. FAU - Todtenhaupt, Pia AU - Todtenhaupt P AD - 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. FAU - Franken, Laura A AU - Franken LA AD - Molecular Epidemiology, Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands. FAU - Groene, Sophie G AU - Groene SG AD - 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. FAU - van Hoolwerff, Marcella AU - van Hoolwerff M AD - Molecular Epidemiology, Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands. FAU - van der Meeren, Lotte E AU - van der Meeren LE AD - Department of Pathology, Leiden University Medical Center, Leiden, The Netherlands; Department of Pathology, Erasmus Medical Center, Leiden, The Netherlands. FAU - van Klink, Jeanine M M AU - van Klink JMM AD - Neonatology, Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands. FAU - Roest, Arno A W AU - Roest AAW AD - Pediatric Cardiology, Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands. FAU - de Bruin, Christiaan AU - de Bruin C AD - Pediatric Endocrinology, Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands. FAU - Ramos, Yolande F M AU - Ramos YFM AD - Molecular Epidemiology, Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands. FAU - Haak, Monique C AU - Haak MC AD - Fetal Medicine, Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands. FAU - Lopriore, Enrico AU - Lopriore E AD - Neonatology, Willem-Alexander Children's Hospital, Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands. FAU - Heijmans, Bastiaan T AU - Heijmans BT AD - Molecular Epidemiology, Department of Biomedical Data Sciences, Leiden University Medical Center, Leiden, The Netherlands. FAU - van Pel, Melissa AU - van Pel M AD - NecstGen, Leiden, The Netherlands; Department of Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands. Electronic address: m.van_pel@necstgen.com. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20230728 PL - England TA - Cytotherapy JT - Cytotherapy JID - 100895309 SB - IM MH - Humans MH - Reproducibility of Results MH - Umbilical Cord MH - Cell Differentiation MH - Cell Proliferation MH - *Mesenchymal Stem Cells MH - *Mesenchymal Stem Cell Transplantation OTO - NOTNLM OT - differentiation OT - mesenchymal stromal cells OT - platelet lysate OT - protocol standardization OT - umbilical cord COIS- Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article. EDAT- 2023/07/30 13:09 MHDA- 2023/09/26 13:43 CRDT- 2023/07/30 10:29 PHST- 2023/01/20 00:00 [received] PHST- 2023/06/22 00:00 [revised] PHST- 2023/07/14 00:00 [accepted] PHST- 2023/09/26 13:43 [medline] PHST- 2023/07/30 13:09 [pubmed] PHST- 2023/07/30 10:29 [entrez] AID - S1465-3249(23)01007-1 [pii] AID - 10.1016/j.jcyt.2023.07.004 [doi] PST - ppublish SO - Cytotherapy. 2023 Oct;25(10):1057-1068. doi: 10.1016/j.jcyt.2023.07.004. Epub 2023 Jul 28.