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 institutional review board of Duksung Women&#8217;s University (IRB No. 2017-002-01).
1. Cell Culture
<ol>
	<li>Preparing ECFCs and MSCs medium and coat plates
	<ol>
		<li>Prepare endothelial cell growth medium MV2 (ECGM-MV2, except hydrocortisone) supplemented with 10% fetal bovine serum (FBS) and 1% glutamine-penicillin-streptomycin (GPS).</li>
		<li>Prepare mesenchymal stem cell growth medium-2 (MSCGM-2) containing 10% FBS and 1% GPS.</li>
		<li>For ECFC culture, coat the cell culture plates with 1% gelatin solution. To prepare 1% gelatin solution, dissolve 1 g of gelation powder in 500 mL of PBS with the magnetic stirrer, and sterilize with autoclave. Plates can be coated with 3 mL/60 mm, 5 mL/100 mm, or 15 mL/150 mm of 1% gelatin solution. Incubate coated plates for 15 min in a cell culture incubator (37 &#176;C and 5% CO2). After then, remove the remaining 1% gelatin solution by aspiration and wash the coated plates one time with PBS.</li>
	</ol>
	</li>
	<li>Growing ECFCs and MSCs
	<ol>
		<li>Seed 1 x 106 ECFCs on 1% gelatin-coated 150 mm plates, and grow using ECGM-MV2 (10% FBS, 1% GPS) in a cell culture incubator (37&#176;C and 5% CO2) to 80-90% confluency. Use ECFCs at passage numbers 7-10 to obtain consistent results.</li>
		<li>Seed 1 x 106 MSCs on non-coated 150 mm plates, and grow using MSCGM-2 in a cell culture incubator (37&#176;C and 5% CO2) to 80-90% confluency. Use MSCs at passage numbers 7-10 to obtain consistent results.</li>
	</ol>
	</li>
</ol>
2. Preparation of 1.2% w/v Methylcellulose Solution
<ol>
	<li>Measure 6 g of methyl cellulose in a 500 mL glass bottle and sterilize in an autoclave.</li>
	<li>Heat ECGM-MV2 (without FBS and GPS) at 60 &#176;C for 20 min, and add 250 mL of heated ECGM-MV2 to sterilized methyl cellulose powder. To maintain sterile conditions, carry out the process inside the flow hood.</li>
	<li>Add a sterilized magnetic stir bar and mix the solution for 20 min on the magnetic stirrer until the methyl cellulose is thoroughly wetted and evenly dispersed with no lumps. Add 250 mL of cold (4 &#176;C) ECGM-MV2 (without FBS and GPS) under the sterile condition and mix for an additional 10 min on the magnetic stirrer. Then, chill the solution in a refrigerator (4 &#176;C) overnight. 
	NOTE: Methylcellulose is a carbohydrate polymer that dissolve well in cool temperatures due to swelling and subsequent hydration.</li>
	<li>The next day, aliquot the solution in a 50 mL tube and centrifuge at 5,000 x g for 3 h at 4 &#176;C. Take the clear viscous supernatant solution and store at 4 &#176;C until use for up to 3-6 months. 
	NOTE: The sediment may contain residual cellulose fiber, so gently take the supernatant solution leaving behind at least 5 mL of volume. This volume can be adjusted according to experimental conditions.</li>
</ol>
3. Generation and Embedding of ECFCs-only, MSCs-only, and ECFCs-MSCs Spheroids
Day 1 
<ol>
	<li>Preparing ECFCs and MSCs cell suspension
	<ol>
		<li>Wash the 80-90% confluent ECFCs and MSCs by adding phosphate buffered saline (PBS) without calcium and magnesium, and aspirate.</li>
		<li>To detach ECFCs and MSCs from the culture plate, incubate them with 0.05% trypsin-EDTA for 3 min and 5 min in a cell culture incubator (37&#176;C and 5% CO2), respectively, in a cell culture incubator.</li>
		<li>Inactivate trypsin by adding DMEM medium containing 10% FBS and 1% GPS. Pipette the cells up and down to create a single cell suspension.</li>
		<li>Sediment the cells by centrifuging at 282 x g for 5 min at RT.</li>
		<li>Remove the supernatant, and re-suspend the cells in the respective medium.</li>
	</ol>
	</li>
	<li>Labeling ECFCs and MSCs with fluorescent dye 
	NOTE: Perform cell membrane labelling of ECFCs with PKH67 (green) and MSCs with PKH26 (red) fluorescent dye following the manufacturer&#8217;s instructions with slightly modifications as follows.
	<ol>
		<li>Wash the cells two times with serum free medium to remove FBS. 
		NOTE: Proteins and lipids in FBS reduces the effective dye concentration for labelling cells by binding.</li>
		<li>Count ECFCs and MSCs using a hemocytometer under the microscope, and transfer 3 x 106 ECFCs and 2 x 106 MSCs into 2 mL micro-tubes. 
		NOTE: These are the optimum cell densities for labelling cells with dyes. Use of a large number of cells causes poor and heterogeneous staining, whereas use of too few cells yields poor cells recovery.</li>
		<li>Centrifuge the tubes at 100 x g for 5 min at RT&#160;to obtain a cell pellet. Carefully aspirate the supernatant leaving no more than 15-25 &#181;L of residual volume.</li>
		<li>Re-suspend ECFCs and MSCs in 250 &#181;L of Diluent C from the dye kit. Gently pipette the cell suspension to ensure complete dispersion. 
		NOTE: Diluent C is a physiologic salt, and may reduce the staining efficiency of dye by forming micelles. Therefore, do not let cells stand in diluent C for long, and do not vortex the tubes.</li>
		<li>Prepare the staining solution for ECFCs by adding 5 &#181;L of PKH67 to 250 &#181;L of Diluent C (final concentration of 20 &#181;M) and for MSCs by adding 3 &#181;L of PKH26 to 250 &#181;L of Diluent C (final concentration of 12 &#181;M ). 
		NOTE: The final dye concentrations are different from the manufacturer&#8217; recommendations. High concentration would lead to cell clumping and cell toxicity. Low concentration would not be sufficient for staining.</li>
		<li>Add 250 &#181;L of ECFCs cell suspension to 250 &#181;L of PKH67 dye solution, and 250 &#181;L of MSCs to 250 &#181;L of PKH26 dye solution. Immediately mix the cells with dyes by pipetting up and down and incubate for 5 min at room temperature. For better results, cover the tube with aluminum foil and place on a shaker to ensure sufficient mixing during staining.</li>
		<li>Stop the staining process by adding 0.5 mL of FBS to the stained cell suspension, and then incubate for 1 min to allow FBS to bind excess unbound dye. Sediment the stained cells by centrifuging at 100 x g for 5 min. 
		NOTE: ECFCs and MSCs pellets are light yellow and pink, respectively, after staining.</li>
		<li>Carefully remove the supernatant and wash the cells two times with complete medium to remove excess dye. Centrifuge the tubes at 100 x g for 5 min and re-suspend the pellet in 2 mL of each complete medium.</li>
	</ol>
	</li>
	<li>Generating ECFC, MSC, or ECFC+MSC spheroids 
	NOTE: Apply the hanging drop technique to generate spheroids as described previously28. The steps for the preparation of spheroids are given below.
	<ol>
		<li>Count the stained ECFCs and MSCs.</li>
		<li>Suspend stained ECFCs, MSCs or ECFCs+MSCs in ECGM-MV2 containing 20% Methylcellulose solution and 5% FBS. Prepare at a cell density of 4 x 104 cells/mL for ECFCs or MSCs (25 &#181;L of cell suspension contains 1,000 cells). For two cell suspension of ECFCs and MSCs, prepare at a cell density of 2 x 104 cells/mL ECFCs and 0.4 x 104 cell/mL MSCs (25 &#181;L contains 500 ECFCs and 100 MSCs). 
		NOTE: Ratio of ECFCs to MSCs should not exceed 5:1. A larger number of MSCs could lead to excessive migration and irregular sprout morphology. A smaller number of MSCs could cause poor interaction between cells, yielding poor sprouting.</li>
		<li>Prepare a hydration unit by adding 15 mL of PBS into the bottom of a 150 mm culture plate.</li>
		<li>Transfer the cell suspension into the sterilized polystyrene rectangular reservoir for the use of multichannel pipette. Disperse the cells suspension evenly by pipetting.</li>
		<li>Deposit 25 &#181;L drops of cell suspension onto the cover of a 150 mm culture plate using a multi-channel pipette (approximately 100 drops/cover of the 150 mm plate). Invert the cover over a PBS-filled hydration unit and incubate in a cell culture incubator for 24 h. 
		NOTE: Methylcellulose solution provides a proper viscosity to the suspension solution. When the hanging drop method was performed without methylcellulose solution, drops were easily slipped down when the cover was inverted (Supplemental Figure 1). Furthermore, after overnight incubation, the spheroids were not perfectly formed without methylcellulose solution. This result indicate that proper viscosity by methylcellulose solution is necessary to form round shape of the spheroids.</li>
	</ol>
	</li>
</ol>
Day 2
<ol start="4">
	<li>Embeding spheroids in neutralized collagen gel
	<ol>
		<li>Warm FBS and ECGM-MV2 (without FBS and GPS) in a water bath (37 &#176;C). Place cold methylcellulose solution on a work bench to bring to room temperature.</li>
		<li>Place a 24-well plate in a cell culture incubator (37 &#176;C) for warming.</li>
		<li>Cut the pointed 1 mL pipette tips 3-5 mm to aspirate spheroids and viscous suspensions, such as methylcellulose solution and type I collagen, comfortably and accurately.</li>
		<li>Prepare 3 mg/mL neutralized type I collagen gel on ice following the type I collagen gel manufacturer&#8217;s instruction. 
		NOTE: Neutralization should be performed on ice to avoid gelation of collagen at room temperature. For one well of a 24-well plate, 500 &#181;L of neutralized collagen is needed. Always prepare 1 mL extra volume of collagen. Collagen gel can be used up to 2-3 h after neutralization if kept on ice.</li>
		<li>Harvest the spheroids by rinsing the cover containing spheroids with 5 mL of PBS. Collect spheroid-suspended solution in a 50 mL conical tube. Re-rinse the cover with 5 mL of PBS to obtain the remaining spheroids. 
		NOTE: During harvest, closely observe to confirm the existence of spheroids. For more visual inspection, transfer about 50-100 &#181;L of spheroid-suspended solution on the slide glass, and check the round shape of the spheroids under the microscope.</li>
		<li>Sediment the spheroids by centrifuging at 282 x g for 5 min. Discard the supernatant carefully without disturbing the spheroids leaving behind not more than 100-200 &#181;L of residual volume.</li>
		<li>Gently tap on the wall of the tube so that spheroids are freely suspended in the remaining 100-200 &#181;L residual solution.</li>
		<li>Add ECGM-MV2 containing 5% FBS and 40% methylcellulose solution to the spheroid-containing tube. The volume added is determined based on approximately 100 spheroids/mL. Gently mix the spheroid suspension by pipetting with a blunt 1 mL pipette tip. 
		NOTE: Methylcellulose solution is widely used as a suspending agent that does not allow spheroids to sediment. FBS concentration should be 5%. Higher FBS concentration causes abnormal sprouting due to excessive growth factors. Lower FBS concentration cannot maintain healthy conditions for cells.</li>
		<li>Mix spheroid-suspension solution and neutralized-type I collagen gel (3 mg/mL) at a 1:1 ratio on ice. Use a blunt 1 mL pipette tip to avoid breaking of spheroids. 
		NOTE: 1 mL of the spheroids-suspending collagen gel solution is required for one well of a 24-well plate. Make an extra mixed suspension solution if possible.</li>
		<li>If the angiogenic properties of any agent(s) or chemical(s) need to be tested, transfer 1 mL of the spheroid-suspending collagen gel solution in a 1 mL micro-tube and add agent(s) or chemical(s) followed by gentle mixing with a blunt 1mL pipette tip. 
		NOTE:&#160;The volume of agent and chemical can sum to 200 &#956;L, which dilutes the type I collagen gel concentration from 1.5 mg/mL to 1.25 mg/mL, but does not affect type I collagen gel polymerization. Add same volume of vehicle to the control spheroids.</li>
		<li>Add the spheroid-suspending collagen gel solution into wells of a pre-warmed 24-well plate (0.9 mL/well) by pipetting, and then incubate for 30 min in a cell culture incubator for polymerization. 
		NOTE: During the spheroid embedding process, do not disturb the gel by agitating the plate.</li>
		<li>Cover the spheroid-suspending collagen gel by adding 100 &#181;L of ECGM-MV2 containing 2.5% FBS with/without VEGF. For ECFC-only spheroids, stimulate the cells with exogenous addition of VEGF (final concentration of 50 ng/mL) to form proper sprouts. ECFC+MSC spheroids do not require exogenous stimulation by any growth factors.</li>
		<li>Place the plate in a real-time cell recorder installed in a cell culture incubator (37 &#176;C and 5% CO2), and randomly focus on 5-10 spheroids (10x objective lens). Monitor the sprouting formation of each fluorescence-labeled spheroids every 1 h for 24 h without any disturbance.</li>
	</ol>
	</li>
</ol>
4. Quantitate Spheroid Sprouting
<ol>
	<li>Import the image files to ImageJ software to quantitate the number of sprouts and measure the length of each sprout. For co-culture spheroids, label ECFCs with green fluorescent dye before making spheroids. Then, measure the number and length of sprouts stained with green fluorescence (Supplemental Figure 2). Five randomly-selected spheroids were quantified per experimental group. 
	NOTE: Sprouts are collaboratively elongated structures formed by several ECFCs extending out of spheroids. Sprout length is measured as the length from the point of sprouts origin from the surface of the spheroids to the tip of sprouts.</li>
	<li>Express the values as means &#177; SEM of at least three independent experiments. Determine the statistically significant difference by one-way ANOVA for multiple comparisons or Student&#8217;s t-test for paired comparisons. 
	NOTE: P &#8804; 0.05 is considered statistically significant.</li>
</ol>

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Cell+Spheroid+Angiogenesis+Assay+System+Using+Both+Endothelial+Colony+Forming+Cells+and+Mesenchymal+Stem+Cells.">Two-Cell Spheroid Angiogenesis Assay System Using Both Endothelial Colony Forming Cells and Mesenchymal Stem Cells.</a> Biomolecules &amp; Therapeutics (Seoul). 26 (5), 474-480 (2018).</li></ol>

The authors have nothing to disclose.

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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	<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 ...

three-dimensional-angiogenesis-assay-system-using-co-culture

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9.7K Views.  Duksung Women's University. 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.

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

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 distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;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 different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based 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 gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

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 mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

This protocol describes methods for purifying, quantitating, and characterizing extracellular vesicles (EVs)/exosomes from non-adherent/mesenchymal mammary epithelial cells and for using them to transfer mammary gland-forming ability to luminal mammary epithelial cells. EVs/exosomes derived from stem-like mammary epithelial cells can transfer this cell property to cells that ingest the EVs/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

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 &#34;translatable&#34; 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.

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

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.

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 microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

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 disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

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, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

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

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

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

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

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

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...