establishing a co-culture of interneurons with periventricular endothelial cells

Establishing a Co-Culture of Interneurons with Periventricular Endothelial Cells

To perform the co-culture assay, suspend 30,000 GABAergic interneurons and 30,000 human periventricular endothelial cells in 70 microliters of co-culture medium. Then, seed this cell solution inside a one-well insert compartment. After 48 hours, remove the insert. Incubate the co-culture at 37 degrees Celsius and 5% carbon dioxide for five days.

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1. Culture and Storage of Human Periventricular Endothelial Cells
<ol>
	<li>Maintain human periventricular endothelial cells on basement membrane matrix-coated (see Table of Materials) 6-well plates in periventricular endothelial cell medium (E6 medium containing 50 ng/mL vascular endothelial growth factor A [VEGF-A], 100 ng/mL fibroblast growth factor 2 [FGF2] and 5 &#181;M gamma-aminobutyric acid [GABA]) at 37 &#176;C and 5% CO2. Change medium every alternate day.</li>
	<li>Thaw basement membrane matrix in 4 &#176;C, and make a 1:100 solution by diluting it in cold Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) medium. Coat each well of a 6-well plate with 1 mL of matrix solution. Incubate plates at 37 &#176;C for at least 1 h before use.</li>
	<li>Allow human periventricular endothelial cells to reach a confluency of 80%-90%. Aspirate medium from the well. Wash the wells once with 1 mL of sterile 1x phosphate-buffered saline (PBS) per well.</li>
	<li>Detach cells by adding 1 mL of cell dissociation solution (see Table of Materials) per well. Incubate at 37 &#176;C for 5 min. After 5 min, add 1 mL of periventricular endothelial cell medium. Transfer the cell solution into a 15 mL conical tube. 
	NOTE: We use Accutase for cell dissociation here, as opposed to TrypLE in sections 3 and 4.</li>
	<li>Centrifuge cells at 500 x g for 5 min at room temperature, aspirate the supernatant and resuspend the cell pellet in 1 mL of periventricular endothelial cell medium.</li>
	<li>Count live cells using the trypan blue exclusion method. Seed cells in fresh matrix-coated plates at a density of 1.2 x 105 cells/cm2. Incubate at 37 &#176;C and 5% CO2.</li>
	<li>Store human periventricular endothelial cells by cryopreserving in freezing medium (90% periventricular endothelial cell medium and 10% dimethyl sulfoxide [DMSO]).
	<ol>
		<li>Dissociate and collect cells following steps 1.3 and 1.4 above. Count cells in the solution by the trypan blue exclusion method.</li>
		<li>Centrifuge cells at 500 x g for 5 min at room temperature. Aspirate the supernatant and resuspend the cell pellet at 5 x 106 cells/ mL of freezing medium.</li>
		<li>Dispense 1 mL of freezing medium plus cells per cryovial. Place the vials in isopropanol-filled chamber and cool overnight in -80 &#176;C at 1 &#176;C/min. Transfer vials to a liquid nitrogen tank on the next day for long term storage.</li>
	</ol>
	</li>
</ol>
2. Preparation of Human Periventricular Endothelial Cells for Assay
<ol>
	<li>Allow human periventricular endothelial cells to reach 70%-80% confluency.</li>
	<li>Dissociate cells following steps 1.3 to 1.5 as described above. Count cells using the trypan blue exclusion method.</li>
</ol>
3. Preparation of Human GABAergic Interneurons for Assay
NOTE: Human induced pluripotent stem cell (iPSC)-derived GABAergic interneurons and the neuronal medium were commercially purchased (see Table of Materials). The neurons are generated by differentiating a human fibroblast-derived iPSC line following a protocol developed by the manufacturer. The cells were thawed and cultured according to manufacturer&#39;s protocol.
<ol>
	<li>Thaw human GABAergic interneurons and culture them in 12-well plate for two weeks to a confluency of 70%-80%.</li>
	<li>On the day of the assay, warm cell dissociation solution (see Table of Materials) and an aliquot of neuronal medium at 37 &#176;C for 10 min before use.</li>
	<li>Aspirate medium from each well containing the cells. Wash cells with 1 mL of sterile 1x PBS per well.</li>
	<li>Detach cells by adding 0.5 mL of pre-warmed dissociation solution per well and incubate at 37 &#176;C for 5 min. Add 1 mL of neuronal medium per well. Transfer cell solution into a 15 mL conical tube. Gently triturate to dissociate cell clumps.</li>
	<li>Centrifuge cells at 380 x g for 5 min at room temperature, aspirate the supernatant and resuspend the cell pellet in 1 mL of neuronal medium. Count live cells using the trypan blue exclusion method.</li>
</ol>
4. Preparation of Control Human Endothelial Cells for Assay
NOTE: Control human iPSC-derived endothelial cells and endothelial cell medium were commercially purchased (Table of Materials). These endothelial cells are generated by differentiating a human fibroblast-derived iPSC line to endothelial fate following a protocol developed by the manufacturer. The cells were thawed and cultured on Fibronectin substrate according to manufacturer&#39;s protocol. Fibronectin-coated plates were prepared following manufacturer&#39;s protocol.
<ol>
	<li>Thaw control human endothelial cells and culture them in 6-well plate to a confluency of 80%-90%.</li>
	<li>On the day of the assay, warm cell dissociation solution (see Table of Materials) and an aliquot of endothelial medium at 37 &#176;C for 10 min before use.</li>
	<li>Aspirate the medium from each well containing the cells. Wash cells with 1 mL of sterile 1x PBS per well.</li>
	<li>Detach cells by adding 0.5 mL of pre-warmed dissociation solution per well. Incubate at room temperature for 5 min. Add 1 mL of endothelial cell medium per well to neutralize the dissociation solution. Transfer cell solution into a 15 mL conical tube.</li>
	<li>Centrifuge cells at 200 x g for 5 min at room temperature. Aspirate supernatant and resuspend the cell pellet in 1 mL of endothelial cell medium. Count live cells using the trypan blue exclusion method.</li>
</ol>
5. Preparation of One-well Culture Inserts
<ol>
	<li>Thaw 1 mg/mL laminin solution at room temperature or overnight at 4 &#176;C.</li>
	<li>Coat an appropriate number of 35 mm dishes with 0.01% poly-L-ornithine solution (1 mL per dish). Incubate the dishes in room temperature for at least 1 h.</li>
	<li>Dilute 1 mg/mL laminin solution 1:300 in sterile water to a final concentration of 3.3 &#181;g/mL immediately before use.</li>
	<li>Completely aspirate poly-L-ornithine from each dish. Rinse each dish thoroughly 3x with sterile water and aspirate completely to avoid poly-L-ornithine-induced cell toxicity.</li>
	<li>Add 1 mL of 3.3 &#181;g/mL laminin solution to each dish and incubate at 37 &#176;C overnight or at least 1 h. Remove laminin solution from the dish immediately before use. 
	NOTE: Alternatively, store the laminin-containing dishes in 4 &#176;C. Equilibrate the dishes in a 37 &#176;C cell culture incubator before use.</li>
	<li>Cut three sides of one well of a two-well silicone culture insert (Figure 1A) using a sterile blade to generate a one-well insert (Figure 1B). 
	NOTE: Keep the two-well insert firmly attached to the surface of the original packaging while cutting to ensure a smooth cut and protect the adhesiveness of the insert.</li>
	<li>Aspirate laminin solution from the dishes. 
	NOTE: Do not wash the dishes with sterile PBS or water after laminin incubation. Wet surfaces will prevent tight adhesion of the culture insert.</li>
	<li>Remove one-well insert with sterile tweezers and place it in the center of the poly-L-ornithine/laminin coated dish. Press along the edges of the insert to fix it to the surface of the dish.</li>
	<li>Carefully turn the dish upside down to verify that the insert is firmly adhered.</li>
	<li>Keep the dish upside down and mark the boundary of the insert compartment using a permanent black marker with an ultra-fine tip (Figure 1C).</li>
</ol>
6. Co-culture Migration Assay
<ol>
	<li>Co-suspend 3 x 104 GABAergic interneurons and 3 x 104 human periventricular endothelial cells in 70 &#181;L of co-culture medium (50% periventricular maintenance medium without GABA and 50% neuronal medium). Seed this cell solution inside the one-well insert compartment. Prepare an appropriate number of such assay dishes. 
	NOTE: GABA was not added in the co-culture medium to exclude the effect of exogenous GABA on migration.</li>
	<li>Check under a microscope to verify that cells are not leaking from the insert compartment.</li>
	<li>Slowly add 1 mL of co-culture medium along the side of the dish to prevent the coating from drying. 
	NOTE: Add medium slowly along the edge of the dish so that the insert is not disturbed.</li>
	<li>As a first control, seed 3 x 104 human GABAergic interneurons only in 70 &#181;L of co-culture medium per one-well insert. Prepare an appropriate number of such dishes.</li>
	<li>As a second control, co-seed 3 x 104 GABAergic human interneurons with 3 x 104 control human endothelial cells in 70 &#181;L of co-culture medium per one-well insert. Prepare an appropriate number of dishes.</li>
	<li>Incubate the dishes for 24 h at 37 &#176;C and 5% CO2. After 24 h incubation, check under a microscope to verify that cells have attached properly and there is no leak.</li>
	<li>After 48 h of seeding, gently remove the insert using a sterile tweezer. Check under the microscope to verify that the cell layer is not disturbed (day 0).</li>
	<li>Remove medium and add 1 mL of fresh co-culture medium. 
	NOTE: Set aside an appropriate number of dishes for acquiring day 0 images.</li>
	<li>Incubate cells for 5 days at 37 &#176;C and 5% CO2.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase dissociation solution</td>
			<td>Millipore Sigma</td>
			<td>SCR005</td>
			<td>Cell dissociation solution (for periventricular endothelial cells, step 1.4)</td>
		</tr>
		<tr>
			<td>Control human endothelial cells</td>
			<td>Cellular Dynamics</td>
			<td>R1022</td>
			<td></td>
		</tr>
		<tr>
			<td>Control endothelial Cells Medium Supplement</td>
			<td>Cellular Dynamics</td>
			<td>M1019</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogenic vials</td>
			<td>Fisher Scientific</td>
			<td>03-337-7Y</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEMF/12 medium</td>
			<td>Thermofisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>E6 medium</td>
			<td>Thermofisher Scientific</td>
			<td>A1516401</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>Thermofisher Scientific</td>
			<td>PHG0261</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermofisher Scientific</td>
			<td>33016-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing Container</td>
			<td>Thermofisher Scientific</td>
			<td>5100</td>
			<td></td>
		</tr>
		<tr>
			<td>GABA</td>
			<td>Sigma-Aldrich</td>
			<td>A2129</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GABAergic neurons</td>
			<td>Cellular Dynamics</td>
			<td>R1013</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GABAergic neurons base medium</td>
			<td>Cellular Dynamics</td>
			<td>M1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Human GABAergic neuron Neural supplement</td>
			<td>Cellular Dynamics</td>
			<td>M1032</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector laboratories</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-L-ornithin</td>
			<td>Sigma</td>
			<td>p4957</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Thermofisher Scientific</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue</td>
			<td>Thermofisher Scientific</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE</td>
			<td>Thermofisher Scientific</td>
			<td>12563011</td>
			<td>Cell dissociation solution (for GABAergic interneurons and endothelial cells, sections 3 and 4)</td>
		</tr>
		<tr>
			<td>VEGF-A</td>
			<td>Peprotech</td>
			<td>100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF Medium Complete Kit</td>
			<td>Lifeline Cell Technologies</td>
			<td>LL-0003</td>
			<td>Component of control human endothelial cell medium</td>
		</tr>
		<tr>
			<td>2-well silicone culture-Insert</td>
			<td>ibidi</td>
			<td>80209</td>
			<td></td>
		</tr>
		<tr>
			<td>3-well silicone culture-Insert</td>
			<td>ibidi</td>
			<td>80369</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm dish</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml conical tube</td>
			<td>Fisher Scientific</td>
			<td>07-200-886</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA solution</td>
			<td>Fisher Scientific</td>
			<td>AAJ19943K2</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>14-832-11</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Datta, D., et al. <a href="https://app.jove.com/v/60295">Migration, Chemo-Attraction, and Co-Culture Assays for Human Stem Cell-Derived Endothelial Cells and GABAergic Neurons.</a> J. Vis. Exp. (2020)&#160;
This video demonstrates a technique for establishing a co-culture of interneurons with perivascular endothelial cells. The cells are added to a one-well insert placed inside a culture dish. After the cells adhere to the dish, the insert is removed, allowing the cells to proliferate and interact and form a co-culture.

<img alt="Figure 1" src="/files/ftp_upload/22383/22383_Figure1.jpg" /> 
Figure 1: Preparation of the culture insert. (A) A two-well culture insert. (B) A one-well insert fixed in the center of a 35 mm dish. (C) The outline of the rectangular patch as observed after removing the insert.

1. Culture and Storage of Human Periventricular Endothelial Cells

	Maintain human periventricular endothelial cells on basement membrane matrix-coated ...

Take a culture dish coated with a cell culture substrate to facilitate cell adhesion.
The dish contains a one-well culture insert without a base attached to its center, creating a defined compartment. The outline of this compartment is marked on the dish to observe cell migration.
Introduce a cell suspension into the compartment that contains periventricular endothelial cells or PVECs, cells lining the blood vessels in the brain, and interneurons that migrate along the PVECs during brain development.
Add a co-culture medium to the dish to maintain moisture, and incubate to facilitate the cells adhering to the substrate.
Remove the insert to eliminate the physical barrier; refresh the medium, and incubate again.
The PVECs proliferate, expanding the endothelial cell network beyond the insert boundary.
The PVECs secrete factors that act as guidance cues, directing the interneurons to migrate along the PVEC network.
The co-culture exhibiting interneuron migration is ready for analysis.

18 次观看. 建立中间神经元与脑室周围内皮细胞的共培养

视频: 建立中间神经元与脑室周围内皮细胞的共培养 - 实验

Using the Dot Assay to Analyze Migration of Cell Sheets

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move within tissues in a tightly orchestrated manner. During tumor development, this equilibrium is disturbed, and tumor cells leave the epithelium of origin to invade the local microenvironment, to travel to distant sites, and to ultimately form metastatic tumors at distant sites. The dot assay is a simple, two-dimensional unconstrained migration assay, to assess the net movement of cell sheets into a cell-free area, and to analyze parameters of cell migration using time-lapse imaging. Here, the dot assay is demonstrated using a human invasive, lung colony forming breast cancer cell line, MCF10CA1a, to analyze the cells' migratory response to epidermal growth factor (EGF), which is known to increase malignant potential of breast cancer cells and to alter the migratory phenotype of cells.

Cell migration is essential for development, tissue maintenance and repair, and tumorigenesis, and is regulated by growth factors, chemokines, and cytokines. This protocol describes the dot assay, a two-dimensional, unconstrained migration assay to assess the migratory phenotype of attached, cohesive cell sheets in response to microenvironmental cues.

用点法分析细胞板的迁移

Although complex organisms appear static, their tissues are under a continuous turnover. As cells age, die, and are replaced by new cells, cells move ...

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Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

A Cost Effective and Adaptable Scratch Migration Assay

Cell migration is a key component in both physiological and pathological events. Normal cell migration is required for essential functions such as development and mounting an immune response. When a defect or alteration occurs with the cell migration process, it can have detrimental outcomes (i.e., cancer metastasis, wound healing, and scar formation). Due to the importance of cell migration, it is necessary to have access to a cell migration assay that is affordable, adaptable, and repeatable. Utilizing the common scratch migration assay, we have developed a new approach to analyzing cell migration that uses general laboratory equipment. The method described uses visual markers that allow for recapturing specific areas of interest without the use of time-lapse microscopy. In addition, it provides flexibility in the experimental design, ranging from altering the migration matrix substrate to the addition of pharmacological modifiers. Furthermore, this protocol outlines a way to account for the area of cell migration, which is not considered by several methods when examining cell migration. This new approach offers a scratch migration assay to a larger audience and will provide greater opportunity for researchers to examine the physiological and pathophysiological impact of cell migration.

We present a cost-effective method to the scratch migration assay that provides a new approach for determining cell migration without the use of equipment-intensive methods. While fibroblasts were used in this protocol, it can be adapted and utilized to study additional cell types and influences on cell migration.

Establishing a Co-Culture of Interneurons with Periventricular Endothelial Cells

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Establishing a Co-Culture of Interneurons with Periventricular Endothelial Cells