Name: three-dimensional angiogenesis assay system using co-culture spheroids formed by endothelial colony forming cells and mesenchymal stem cells
Uploaded: 2019-09-18T12:30:00
Description: Watch this Scientific Journal Video about Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells at JoVE.com

This research was supported by a grant (17172MFDS215) from Ministry of Food and Drug Safety, the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIP) (2017R1A2B4005463), and the Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (2016R1A6A1A03007648).

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 institut...

<ol>
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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...

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 experiments2. Therefore, improved in vitro assay systems are needed to obtain more accurate and predictable data leading to the better decision-making before animal experiments. Current in vitro angiogenesis assays usually measure proliferation, invasion, migration, or tubular structure formation of endothelial cells (ECs) seeded in two-dimensional (2D) culture plates3. These 2D angiogenesis assays are quick, simple, quantitative, and cost-effective, and have significantly contributed to the discovery of angiogenesis-modulating drugs. However, several issues remain to be improved.
Such 2D in vitro assay systems cannot reflect complex multi-step events of angiogenesis that occurs in in vivo physiologic conditions, leading to inaccurate results that cause discrepancies between in vitro assay data and clinical trial outcomes4. 2D culture conditions also induce the change of cellular phenotypes. For example, after proliferation in 2D culture plates, ECs have a weak cellular phenotype as manifested by reduced expression of CD34 and several signals that govern cellular responses5,6. To overcome the limitations of 2D culture-based angiogenesis assay systems, three-dimensional (3D) spheroid angiogenesis assay systems have been developed. Sprouting followed by tubular structure formation from spheroids formed by ECs reflect in vivo neo-vascularization processes7,8. Thus, the 3D spheroid angiogenesis assay has been considered an effective assay system for screening potential pro- or anti-angiogenesis drugs.
Most 3D spheroid angiogenesis assays utilize only ECs, mainly human umbilical vein endothelial cells (HUVECs) or human dermal microvascular endothelial cells (HDMECs) to focus on the cellular response of ECs during angiogenesis. However, blood capillaries are composed of two cell types: ECs and pericytes. Elaborating bi-directional interaction between ECs and pericytes is critical for proper vascular integrity and function. Several diseases, such as hereditary stroke, diabetic retinopathy, and venous malformation, are associated with altered pericyte density or decreased pericyte attachment to the endothelium9. Pericytes are also known as a key element of the angiogenic process. Pericytes are recruited to stabilize newly-formed vessel structures by ECs. In this regard, mono-culture spheroid angiogenesis assay does not incorporate pericytes7,10. Therefore, co-culture spheroids formed by ECs and pericytes may provide a valuable approach to more closely mimic physiologic angiogenic events.
The present study aimed to develop a 3D co-culture spheroid angiogenesis assay with a combination of human endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) to more closely reflect in vivo angiogenesis. Co-culture spheroid system as an in vitro representation assembly of a normal blood vessel was first established by Korff et al. in 200111. They combined HUVECs and human umbilical artery smooth muscle cells (HUSMCs), and demonstrated that co-culture of two mature vascular cells decreased the sprouting potential. Mature ECs (HUVECs) are known to progressively lose their ability to proliferate and differentiate, which negatively affects their angiogenesis responses12,13. Mature perivascular cells (HUSMCs) can cause endothelial cell inactivation through the abrogation of the vascular endothelial growth factor (VEGF) responsiveness11. The main difference between Korff&#8217;s and our co-culture spheroid system is the cell types used. We applied two vascular precursors, ECFCs and MSCs, to establish a proper angiogenesis assay system to screen and investigate pro-or-anti-angiogenic agents. ECFCs are the precursor of ECs. ECFCs have robust proliferation capacity compared with mature ECs14, which enable to overcome the limitation of ECs. ECFCs contribute to new vessel formation in many post-natal pathophysiologic conditions15,16,17. MSCs are pluripotent stem cells that have the capacity to differentiate into pericytes, thereby contributing to angiogenesis18,19.
In previous reports, ECFCs and MSCs showed synergistic effects on in vitro tube formation20, in vivo neo-vascularization21,22, and improved reperfusion of ischemic tissues23,24. In the present study, ECFCs and MSCs were used to form co-culture spheroids and embedded in type I collagen gel to reflect an in vivo 3D environment. Collagen is considered as a major constituents of the extracellular matrix (ECM) surrounding ECs25. The ECM plays a critical role in regulating cell behavior26. The assay protocol proposed here can be easily and quickly carried out within two days using common laboratory techniques. For effective cell tracking during the sprouting process, each cell type can be fluorescently labeled and monitored using a real-time cell recorder. The newly-established 3D co-culture spheroid angiogenesis assay system is designed to increase sensitivity for evaluating potential angiogenic modulators and to provide predictable information in advance of in vivo study.

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		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">0.05 % Trypsin EDTA (1X)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">25300-054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Bevacizumab</td>
			<td style="width:123px;">Roche</td>
			<td style="width:161px;">NA</td>
			<td>Commercial name: Avastin</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Dulbecco Modified Eagle Medium</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">11885-084</td>
			<td>DMEM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Dulbeco&#39;s Phosphate buffered saline (10X)</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">21600-010</td>
			<td>PBS (10X)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Dulbeco&#39;s Phosphate buffered saline (1X)</td>
			<td style="width:123px;">Corning</td>
			<td style="width:161px;">21-031-CVR</td>
			<td>PBS (1X)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Endothelial cell Growth medium MV2 kit</td>
			<td style="width:123px;">Promocell</td>
			<td style="width:161px;">C-22121</td>
			<td>ECGM-MV2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:123px;">Atlas</td>
			<td style="width:161px;">FP-0500A</td>
			<td>FBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Gelatin</td>
			<td style="width:123px;">BD Sciences</td>
			<td style="width:161px;">214340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">L-Glutamine&#8211;Penicillin&#8211;Streptomycin</td>
			<td style="width:123px;">Gibco</td>
			<td style="width:161px;">10378-016</td>
			<td>GPS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:259px;">Mesenchymal stem cell growth medium-2</td>
			<td style="width:123px;">Promocell</td>
			<td style="width:161px;">C-28009</td>
			<td>MSCGM-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Methyl cellulose</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">M0512</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PKH26 Fluorescent Cell Linker Kits</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">MINI26</td>
			<td>PKH26</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">PKH67 Fluorescent Cell Linker Kits</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">MINI67</td>
			<td>PKH67</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Sodium Hydroxide</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:161px;">S5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Type I collagen gel</td>
			<td style="width:123px;">Corning</td>
			<td style="width:161px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Vascular endothelial growth factor A</td>
			<td style="width:123px;">R&#38;D</td>
			<td style="width:161px;">293-VE-010</td>
			<td>VEGF</td>
		</tr>
	</tbody>
</table>

three-dimensional angiogenesis assay system using co-culture spheroids formed by endothelial colony forming cells and mesenchymal stem cells

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.

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...

Watch this Scientific Journal Video about Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells at JoVE.com

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

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.

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 ...

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Developmental Biology

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal ...

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

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

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...