The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Summary

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Abstract

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.

Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and "translatable" pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.

We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

Introduction

The complex process of angiogenesis improves and maintains tissue perfusion by promoting new blood vessel development from pre-existing vasculature¹. It is a tightly regulated, balanced process by pro-angiogenic and anti-angiogenic factors. Any deficiency in this system may lead to insufficient vessel maintenance or growth, causing severe ischemic diseases including myocardial disease, stroke, and neurodegenerative disorders. However, exaggerated vascular development is characteristic for conditions including cancer and inflammatory disorders².

Developing therapies that aim to control angiogen....

Protocol

All studies involving animals were conducted and reported according to ARRIVE guidelines²⁹. All studies were performed with institutional research ethics board approval (REB number 4276). All animal procedures were approved by the Animal Care Committee of the University Health Network (Toronto, Canada), and all animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, 8^th edition (National institutes of Health 2011).

1. Aortic Ring Assay Setup

1. Isolate the rat aorta.
   1. Euthanize 8 - 10-week-old Sprague-Dawley rats using CO₂

Results

The schematic work-flow for establishing the aortic ring/MSC co-culture assay is demonstrated in Figure 1. The main steps include: rat aorta isolation, sectioning and embedding of the aortic rings, monitoring the endothelial sprouting and network development, and finally labelling and administering the MSCs. The timeline of the endothelial network analysis outlines the window for the analyses feasible for each period of co-culturing: day 1, 5 & 7. Additio.......

Discussion

There are several critical stages in setting up a successful aortic ring assay MSC co-culture experiment. First, the most important steps when isolating and sectioning aorta are: 1) obtaining exclusively the thoracic segment of aorta; 2) carefully removing branching blood vessels, connective and adipose tissue and; 3) cutting even sections of the aorta (~1 mm) to limit variability between each assay. Second, the successful embedding of aortic rings into BME is critical for this assay. If the BME is not completely polymer.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alpha-MEM	Gibco	12571071	For FTM HUCPVC and BMSC culture media.
APC-conjugated anti human TRA-1-85	R&D Systems	FAB3195A	Human-specific cell marker for flow cytometry and cell sorting
Basal membrane extract (BME) (Matrigel)	Corning	354234	For aorta embedding
Bullet-kit	Lonza	CC-3162	Includes: Gentamicin/Amphotericin-B (GA)human Epidermal Growth Factor (hEGF); Vascular Endothelial Growth Factor (VEGF); R3- Insulin-like Growth Factor-1 (R3-IGF-1); Ascorbic Acid; Hydrocortisone; human Fibroblast Growth Factor-Beta (hFGF-β); Heparin; Fetal Bovine Serum (FBS). Required to prepare EGM
CellTracker Green CMFDA Dye	Thermo-fisher	C2925	For staining MSCs, green is picked up optimally by MSCs
CKX53 Culture Microscope	Olympus		For bright-field imaging of endothelial network development
Countess automated cell counter	Invitrogen	C10227	Cell counting for MSC culture, flow cytometry and qPCR
Dispase	StemCell technologies	7923	For dissociating aortic ring-MSC co-cultures (pre-warm at 37 °C)
Disposable sterile scalpels	VWR	21909-654	For sectioning aorta
Dulbecco's phosphate buffered saline	Sigma-Aldrich	D8537	PBS. 1X, Without calcium chloride and magnesium chloride
Endothelial basal media (EBM)	Lonza	CC-3156	Basal media required for culturing aortic ring assay-MSC co-cultures (warm at 37 °C before use). Required for EGM and EBM-FBS
Ethanol, 70%, Biotechnology Grade	VWR	97064-768	To sterilize surfaces
EVOS	Life Technologies		In-house fluorescent microscope to track MSC migration and integration
Fetal bovine serum (FBS) (Hyclone)	GE Healthcare	SH3039603	Serum component of cell culture medium
FITC-conjugated anti-CD31 antibody	BD	558068	Human endothelial marker for flow cytometry
FITC-conjugated anti-CD146antibody	BD	560846	Human pericyte marker for flow cytometry
Forceps	Almedic	7727-A10-704	For handing rat tissue. Can use any similar forceps
Hank's Balanced Salt Solution (HBSS)	Life Technologies	14175-094	1X Without calcium chloride and magnesium chloride
HERAcell 150i CO2 Incubator	Thermo Fisher Scientific	51026410	For incubating cells
Human Angiogenesis RT2 profiler PCR array	Qiagen	PAHS-024Z	Human specific and includes primers for 84 genes involved in angiogenesis. Each well is 1 primer reaction
ImageJ			Open source image processing software. Require Angiogenesis analyzer plugin
LSR II	BD		UHN SickKids FC Facility. For flow cytometry.
MoFlo Astrios	Beckman Coulter		UHN SickKids FC Facility. For cell sorting.
Penicillin/streptomycin	Gibco	15140122	Antibiotic component to buffers and cell culture medium
RNeasy Mini Kit	Qiagen	74104	For RNA purification. Includes cell lysis buffer
RT2Easy First Strand Kit	Qiagen	330421	For preparation of cDNA for qPCR
RT2PreAMP cDNA Synthesis Kit	Qiagen	330451	Pre-amplification of cDNA if low-yield RNA
Surgical scissors	Fine Science Tools	14059-11	For cutting skin, muscle and aorta
Sterile gauze	VWR	3084	To dampen and sterilize chest fur
TrypleE	Thermo Fisher Scientific	12605036	MSC dissociation enzyme pre-warm at 37 °C
0.2 μm pore filtration unit	Thermo Fisher Scientific	566-0020	To sterilize tissue culture media
0.25% Trypsin/EDTA	Gibco	25200056	For cell dissociation, pre-warm at 37 °C
10 cm tissue culture dishes	Corning	25382-428	For cleaning and sectioning aorta and MSC cell culture
12 well-cell culture plates	Corning-Sigma Aldrich	CLS3513	For setting up aortic ring assay-MSC co-cultures
15 mL tube	BD Falcon	352096	For general tissue culture procedures
70 μm cell strainer	Fisherbrand	22363548	To ensure a single cell suspension before flow cytometry or sorting