# Simultaneous isolation and culture of endothelial colony-forming cells, endothelial cells and vascular smooth muscle cells from human umbilical cords

## TL;DR

This paper introduces a new method to isolate and culture multiple cell types from human umbilical cords to better understand how the umbilical circulation is regulated.

## Contribution

A simplified, cost-effective protocol for simultaneous isolation of multiple umbilical vascular cell types.

## Key findings

- The protocol successfully isolates ECFCs, ECs, and SMCs from human umbilical cords.
- The method is simpler and faster compared to previously published techniques.

## Abstract

Regulation of the human umbilical circulation under physiological and pathological conditions remains poorly understood. We previously demonstrated that intrauterine growth restriction (IUGR) is associated with sex-specific alterations in the human umbilical circulation. Our data strongly suggest a differential contribution of subcellular compartmentation depending on fetal sex, vessel type and the presence of IUGR. We therefore developed a protocol to isolate and culture umbilical vascular cells to further investigate the relative contribution of each cell type and subcellular compartmentation to the human umbilical circulation regulation.

Human umbilical cords and cord blood were collected just after delivery. Mononuclear cells were recovered from cord blood using a Ficoll gradient and cultured to obtain endothelial colony-forming cells (ECFCs). Endothelial cells (ECs) were isolated from human umbilical vein (HUV) and arteries (HUAs) by collagenase/dispase digestion, and vascular smooth muscle cells (SMCs) by migration from vascular explants. All cell types were characterized by visualization, and by analysis of biomarkers using immunocytofluorescence and Western blot. ECFCs were also submitted to polychromatic flow cytometry analysis.

This protocol enables simultaneous isolation and culture of ECFCs, HUVECs, HUAECs, HUVSMCs and HUASMCs from the same umbilical cord. It is simpler, faster and more cost-effective than other previously published methods, with good success rates. This will be helpful to further investigate the regulatory mechanisms implicated in the human umbilical circulation under physiological and pathological conditions and to study the influence of fetal sex.

The online version contains supplementary material available at 10.1007/s11033-025-10418-1.