# Isolate Circulating Mesenchymal Stromal Cells Without Growth Factor Administration and Using Density Gradient

## TL;DR

This paper introduces a new method to isolate mesenchymal stromal cells from blood without using growth factors, showing they can be cultured and used for medical purposes.

## Contribution

A novel protocol for isolating mesenchymal stromal cells from peripheral blood without growth factors, using density gradient and autologous serum.

## Key findings

- Isolated PB-MSCs showed increased CD34−CD45− cell populations compared to Ficoll gradient methods.
- PB-MSCs demonstrated differentiation into adipocytes, osteocytes, and chondrocytes.
- PB-MSCs exhibited immunomodulatory functions and suppressed T cell activation.

## Abstract

Mesenchymal stromal cells (MSCs) are recognized for their differentiation and immune regulation capabilities, which enhance their potential for treating various diseases. MSCs can be sourced from diverse tissues, with peripheral blood (PB) serving as a viable alternative to bone marrow. We now present an alternative strategy that eliminates the need for preadministering growth factors, utilizing density gradient methods, and culturing target cells in medium supplemented with autologous serum. PB was collected through venipuncture and then coincubated with glycerin. After incubation, a thin layer of cells above the red blood cells (RBCs) was isolated, showing an increased population of CD34−CD45− cells compared to PB mononuclear cell (PBMC) isolation using Ficoll gradient. After culture, adherent spindle-shaped cells were identified and collected to assess MSC surface markers, demonstrating their differentiation potential into adipocytes, osteocytes, and chondrocytes, thus, fulfilling the criteria for MSCs. The population doubling time (PDT) of isolated PB-MSCs was approximately 30–40 h in early passages. These PB-MSCs also exhibited immunomodulatory functions and are capable of suppressing T cell activation. We believe this protocol supports PB as a convenient alternative source for MSC isolation and offers new strategies for acquiring and maintaining PB-MSCs.