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.

<table border="0" cellpadding="0" cellspacing="0" style="width:709px;" width="708">
	<colgroup>
		<col />
		<col />
		<col />
		<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...

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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 ...

This 3D co-culture spheroid system is designed to mimic physiologic angiogenesis. It will be effective for evaluating potential angiogenic modulators and provides predictable information in advance of in vivo studies. This system utilizes co-culture spheroids formed by two vascular progenitor cells and can be used to investigate cellular interactions, sprouting, and the tubular maturation of physiologic angiogenesis.If we can use patient cells to form co-culture spheroids, we can use this system to develop personalized treatments for abnormal angiogenesis-related diseases, such as cancer. This assay system can increase the sensitivity and efficiency of studies of angiogenesis and drug development. Begin by using 05%trypsin-EDTA to detach ECFCs and MSCs from 80 to 90%confluent cell cultures for three or five minutes in a cell culture incubator, respectively.Inactivate trypsin with fresh complete DMEM medium, and pipette the cells a few times to generate a single cell suspension. Collect the cells by centrifugation, followed by two washes with serum-free medium. After the second wash, dilute the ECFCs to a three times 10 to the six cells per milliliter of medium concentration and the MSCs to a two times 10 to the six cells per millimeter of medium concentration in individual two-milliliter microcentrifuge tubes.Pellet the cells by centrifugation, and carefully aspirate all but the last 15 to 25 microliters of supernatant from each cell sample. Next, resuspend each pellet in 250 microliters of diluent C from a fluorescent dye kit, and gently pipette the cell suspensions to ensure a complete dispersion. Add 250 microliters of freshly prepared 20-micromolar PKH67 to the tube of ECFCs.Add 250 microliters of 12-micromolar PKH26 to the tube of MSCs. Immediately mix both cell suspensions. After five minutes at room temperature with shaking, protected from light, stop the staining process with a one-minute incubation in 0.5 milliliters of FBS per tube.At the end of the incubation, collect the cells by centrifugation, and carefully remove the supernatant, followed by two washes in two milliliters of fresh complete medium per wash. Then suspend the cells in respective medium in 15-milliliter tubes. For spheroid generation, after the second wash, first dilute the cell suspensions in the appropriate concentration of endothelial cell growth medium MV2 supplemented with 20%methyl cellulose solution and 5%fetal bovine serum concentration.Next, add each cell suspension into a sterilized polystyrene rectangular reservoir. Use a multichannel pipette to add approximately 125-microliter droplets of cells onto the cover of a 150-millimeter culture plate. When all of the cells have been added, invert the cover over the bottom of a dish containing 15 milliliters of PBS, and place the plates in the cell culture incubator for 24 hours.The next day, rinse the dish covers with five milliliters of PBS into one 50-milliliter tube per dish. Rinse the covers with an additional five milliliters of PBS to collect any remaining spheroids, and pellet the spheroids by centrifugation. Carefully discard all but the last 100 to 200 microliters of supernatant, and gently tap on the wall of the tube so that the spheroids are freely suspended in the remaining supernatant.Add the appropriate volume of endothelial cell growth medium MV2 supplemented with 5%fetal bovine serum and 40%methyl cellulose solution to each tube to resuspend the spheroids at a 100 spheroids per milliliter of medium concentration. Use a one-milliliter pipette tip cut to a three-to five-millimeter diameter to gently mix the spheroid suspensions. Use a new wide-tip, one-milliliter pipette to mix the spheroid suspension solution with a neutralized type I collagen gel at a one-to-one ratio on ice.Add 900 microliters of each spheroid-suspending collagen gel solution into the wells of a pre-warmed 24-well plate. Allow the suspensions to polymerize for 30 minutes in the cell culture incubator, before covering the spheroids with 100 microliters of endothelial cell growth medium MV2 supplemented with 2.5%FBS and 50 nanograms per milliliter of VEGF to the ECFC-only spheroids and medium supplemented with FBS only to the MSC and ECFC-plus-MSC spheroid cultures. Then place each plate in a real-time cell recorder installed in a cell culture incubator, and randomly focus on five to 10 spheroids at a 10x magnification to monitor the sprouting formation of each fluorescence-labeled spheroid every hour for 24 hours.To quantify the spheroid sprouting, import the image files to ImageJ, and measure the number and length of sprouts expressing the appropriate fluorescent signal from five randomly selected spheroids per experimental group. For ECFC-plus-MSC spheroids, the number of sprouts and cumulative sprout length are significantly higher compared to those of ECFC-only spheroids at all time points. MSCs-only spheroids do not form sprouts but demonstrate an individual migration of the MSC outside of spheroids.The labeling of ECFC with green-fluorescent dye and MSC with red-fluorescent dye before combining to generate ECFC-plus-MSC spheroids demonstrates that ECFC-mediated sprout structures are covered with MSCs, suggesting that combined MSCs function as perivascular cells during sprout formation, enhancing sprout stability and durability by the tight association between two vascular cells. ECFC-plus-MSC spheroids pretreated with an angiogenesis inhibitor show a decreased sprout number and a cumulative sprout length in a dose-dependent manner compared to control ECFC-plus-MSC spheroids. In parallel experiments, ECFC-only spheroids pretreated with the inhibitor followed by stimulation with VEGF also demonstrate a decreased growth factor-induced sprout number and cumulative sprout length in a dose-dependent manner.Of note, a higher concentration of angiogenesis inhibitor is needed to inhibit ECFC-plus-MSC spheroids compared to ECFC-only spheroids. Tumor growth is significantly inhibited in high dose angiogenesis inhibitor-treated animals compared to both control-treated animals and low dose angiogenesis inhibitor-treated animals in a human xenograft tumor mouse model. The plasma concentration of bevacizumab in high dose-treated mice was 568 plus or minus 40.62 micrograms per milliliter, which was closer to the IC50 value of bevacizumab for inhibiting cumulative sprout length in ECFC-plus-MSC spheroids rather than ECFC-only spheroids.This suggests that the ECFC-plus-MSC co-culture spheroid system is suitable for predicting effective plasma concentrations in advance of an animal study. Using a wide-tip, one-mL pipette to gently mix the spheroids while they are in collagen gel on ice is important for protecting the integrity of the spheroids. We can modify this system to introduce other cell types such as immune cells and other extracellular matrices to investigate cell-matrix crosstalk during angiogenesis.

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JoVE Journal

Developmental Biology

9.6K 조회수.  Duksung Women's University. 이 3D 공동 배양 스페로이드 시스템은 생리성 혈관 신생을 모방하도록 설계되었습니다. 잠재적인 혈관 유발 변조기를 평가하는 데 효과적이며 생체 내 연구에 앞서 예측 가능한 정보를 제공합니다. 이 시스템은 두 개의 혈관 전구 세포에 의해 형성된 공동 배양 스페로이드를 이용하고 생리성 혈관신생의 세포 상호 작용, 발아 및 관 성숙을 조사하는 데 사용될 수 있습니다....