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. 2010 Jun 28;15(4):517–529. doi: 10.2478/s11658-010-0026-0

The proliferation and differentiation of osteoblasts in co-culture with human umbilical vein endothelial cells: An improved analysis using fluorescence-activated cell sorting

Yu Zhang 1,2,3, Andreas Schedle 3, Michael Matejka 1, Xiaohui Rausch-Fan 1, Oleh Andrukhov 1,✉
PMCID: PMC6275724  PMID: 20585887

Abstract

The interaction of osteoblasts and endothelial cells plays a pivotal role in osteogenesis. This interaction has been extensively studied using their direct co-culture in vitro. However, co-culture experiments require clear discrimination between the two different cell types in the mixture, but this was rarely achieved. This study is the first to use fluorescence-activated cell sorting (FACS) for the separation and quantitative analysis of the proliferation and differentiation of MG-63 cells grown in direct co-culture with human umbilical vein endothelial cells (HUVECs). The cells of the MG-63 cell line have properties consistent with the characteristics of normal osteoblasts. We labeled HUVECs with fluorescent antibody against CD31 and used FACS to measure the proportions of each cell type and to separate them based on their different fluorescence intensities. The rate of proliferation of the MG-63 cells was estimated based on a count of the total viable cells and the proportion of MG-63 cells in the mixture. The mRNA expression levels of the osteoblast differentiation markers alkaline phosphatase (ALP), collagen type 1 (Coll-1) and osteocalcin (OC) in the MG-63 cells were measured via real-time PCR after the separation via FACS. We found that HUVECs stimulated the proliferation of the MG-63 cells after 72 h of co-culture, and inhibited it after 120 h of co-culture. The mRNA expression levels of ALP and Coll-1 significantly increased, whereas that of OC significantly decreased in MG-63 after co-culture with HUVECs. Using FACS for the quantitative analysis of the proliferation and differentiation of osteoblasts directly interacting with endothelial cells could have merit for further co-culture research.

Key words: Osteoblasts, Endothelial cells, Co-culture, Proliferation, Differentiation

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Abbreviations used

ALP

alkaline phosphatise

BSA

bovine serum albumin

Coll-1

collagen type 1

ECs

endothelial cells

FACS

fluorescence-activated sell sorting

FC

flow cytometry

FCS

fetal calf serum

FITC

fluorescein isothiocyanate

HUVECs

human umbilical vein endothelial cells

mRNA

messenger ribonucleic acid

OBs

osteoblasts

OC

osteocalcin

VEGF

vascular endothelial growth factor

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