Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Summary

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Abstract

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC₅₀ value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Introduction

Approximately 500 million people worldwide are expected to benefit from angiogenesis-modulating therapy for vascular malformation-associated diseases such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis¹. Thus, the development of drugs that control angiogenesis has become an important research area in the pharmaceutical industry. During the drug development process, in vivo animal study is necessary to explore the effects of drug candidates on physiologic functions and systemic interactions between organs. However, ethical and cost issues have increased the concerns regarding animal experiments

Protocol

Human ECFCs were isolated from human peripheral blood as described in a previous report²⁷. Briefly, the mononuclear cell layer was separated from the whole blood using the Ficoll-Paque Plus, and cultured in the proper medium until the endothelial-like colonies were appeared. Colonies were collected and ECFCs were isolated using CD31-coated magnetic beads. MSCs were isolated from the adherent mononuclear cell (MNC) fraction of human adult bone marrow. The study protocol was approved by the institutional review board of Duksung Women’s University (IRB No. 2017-002-01).

1. Cell Culture

1. Preparin....

Results

Comparison experiments were performed using mono-culture spheroids (ECFCs-only) and co-culture spheroids (ECFCs+MSCs) to examine whether MSCs play a considerable role in ECFCs-mediated angiogenesis. Sprouting formation from each spheroid was monitored for 24 h by a real-time cell recorder that could capture the progression of angiogenic sprouting from spheroids. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from individual spheroids. Five ra.......

Discussion

The present study introduce an improved in vitro angiogenesis assay system utilizing co-culture spheroids formed by two human vascular cell progenitors, ECFCs and MSCs. Co-culture spheroid system can mimic in vivo vascular sprout formation, which is accomplished by interaction and incorporation between endothelial cells and pericytes. Compared to other in vitro angiogenesis assays that reflect only ECs-mediated angiogenesis, this co-culture assay system is more representative of the multistep cascade of physiologic angio.......

Materials

Name	Company	Catalog Number	Comments
0.05 % Trypsin EDTA (1X)	Gibco	25300-054
Bevacizumab	Roche	NA	Commercial name: Avastin
Dulbecco Modified Eagle Medium	Gibco	11885-084	DMEM
Dulbeco's Phosphate buffered saline (10X)	Gibco	21600-010	PBS (10X)
Dulbeco's Phosphate buffered saline (1X)	Corning	21-031-CVR	PBS (1X)
Endothelial cell Growth medium MV2 kit	Promocell	C-22121	ECGM-MV2
Fetal bovine serum (FBS)	Atlas	FP-0500A	FBS
Gelatin	BD Sciences	214340
L-Glutamine–Penicillin–Streptomycin	Gibco	10378-016	GPS
Mesenchymal stem cell growth medium-2	Promocell	C-28009	MSCGM-2
Methyl cellulose	Sigma-Aldrich	M0512
PKH26 Fluorescent Cell Linker Kits	Sigma-Aldrich	MINI26	PKH26
PKH67 Fluorescent Cell Linker Kits	Sigma-Aldrich	MINI67	PKH67
Sodium Hydroxide	Sigma-Aldrich	S5881
Type I collagen gel	Corning	354236
Vascular endothelial growth factor A	R&D	293-VE-010	VEGF