Name: Video: Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons - Concept
Uploaded: 2024-09-27T15:58:18.000+00:00
Duration: 1 min 27 s
Description: 48 Views. Source: Sharif, R., et al. Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea. J. Vis. Exp. (2018) This video demonstrates a technique to generate a three-dimensional co-culture model system of primary human corneal fibroblasts and differentiated neuronal cells. This co-culture system can be used to study the stromal-nerve interactions.

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1. Primary HCFs (human corneal fibroblasts) Culture and 3D Constructs Assembly
<ol>
	<li>To assemble 3D constructs from explants previously grown in EMEM 10% FBS 1% Antibiotic/Antimycotic, passage and count HCFs.</li>
	<li>Seed approximately 1 x 106 cells/well of primary HCFs on polycarbonate membrane inserts with 0.4-&#181;m pores from the 3D constructs, along with 1.5 mL of fresh EMEM 10% FBS 1% Antibiotic/Antimycotic to the top of the construct. Add another 1.5 mL of the same media to the bottom of each construct.</li>
	<li>After 24 h of cell seeding, culture the cells with EMEM 10% FBS 1% Antibiotic/Antimycotic, stimulated with 0.5 mM 2-O-&#945;-D-glucopyranosyl-L-ascorbic acid (Vitamin C) by adding 1.5 mL on the top and 1.5 mL on the bottom of each construct.</li>
	<li>Maintain the cultures in an incubator at 37 &#176;C, 5 % CO2 for 3 weeks and supply fresh media every other day during the entire study period. No further passage is needed.</li>
</ol>
2. Cell Differentiation and Co-cultures
<ol>
	<li>After 3 weeks, add 8 x 103 cells/well of SH-SY5Y human neuroblastoma cells on the top of the previously assembled 3D constructs containing 1.5 mL of the EMEM containing 10% FBS, 1% Antibiotic/Antimycotic, and 0.5 mM 2-O-&#945;-D-glucopyranosyl-L-ascorbic acid (Vitamin C).
	<ol>
		<li>Allow the SH-SY5Y neuroblastoma cells to grow on top of the cornea stromal cells for at least 24 h before initiating cell differentiation.</li>
	</ol>
	</li>
	<li>Initiate SH-SY5Y cell differentiation by stimulating the cells with EMEM containing 1% FBS and 1.5 mL of 10 &#181;M Retinoic Acid on top of the construct. Add 1.5 mL of the EMEM 10% FBS 1% Antibiotic/Antimycotic 0.5 mM 2-O-&#945;-D-glucopyranosyl-L-ascorbic acid (Vitamin C) to the bottom of the construct. Note that the Retinoic Acid step should be performed in the dark.
	<ol>
		<li>Continue to culture the cells in this differentiation media for 5 days.</li>
	</ol>
	</li>
	<li>When the cells reach the differentiation phase, switch the cells to 1.5 mL of serum-free media containing 2 nM of Brain-derived neurotrophic factor (BDNF) and 1% Antibiotic/Antimycotic added to the EMEM, by adding to the top of the construct. Add 1.5 mL of the EMEM 10% FBS 1% Antibiotic/Antimycotic 0.5 mM 2-O-&#945;-D-glucopyranosyl-L-ascorbic acid (Vitamin C) to the bottom of the construct. Note that the BDNF step should be performed in the dark.</li>
	<li>Culture the cells for 2 additional days before processing for any further analysis.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Solution (1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile forceps</td>
			<td>Fischer Scientific</td>
			<td>13-812-42</td>
			<td>Fisherbrand Dissecting Extra-Fine-Pointed Splinter Forceps</td>
		</tr>
		<tr>
			<td>Single edge razor blades</td>
			<td>Personna</td>
			<td>270100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical scalpel blades No.10</td>
			<td>Feather Surgical Blade</td>
			<td>2976#10</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle&#39;s Minimum Essential Medium</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td>10% FBS is required for media preparation</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100X)</td>
			<td>Gibco by Life Technologies</td>
			<td>15240-062</td>
			<td>1% Antibiotic-Antimycotic is required for media preparation</td>
		</tr>
		<tr>
			<td>0.05% Trypsin EDTA(1X)</td>
			<td>Gibco by Life Technologies</td>
			<td>25300062</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane inserts with 0.4-&#956;m pores</td>
			<td>Corning Costar</td>
			<td>3412</td>
			<td></td>
		</tr>
		<tr>
			<td>2-O-&#945;-Dglucopyranosyl-L-ascorbic acid (Vitamin C)</td>
			<td>Sigma-Aldrich</td>
			<td>SMB00390-14</td>
			<td>A concentration of 0.5 mM should be used for the study</td>
		</tr>
		<tr>
			<td>SH-SY5Y Neuroblastoma cells</td>
			<td>ATCC</td>
			<td>SHSY5YATCC CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Retinoic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>SRP3014-10UG</td>
			<td>Final concentration of 10uM needs to be used</td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Sigma-Aldrich</td>
			<td>R2625-100MG</td>
			<td>Final concentration of 2nM needs to be used</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide(DMSO)</td>
			<td>VWR-Alfa Aesar</td>
			<td>67-68-5</td>
			<td>Ultra Pure Grade-Sterile DMSO to be used</td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T25)</td>
			<td>Fisher Scientific</td>
			<td>12-565-351</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T75)</td>
			<td>Fisher Scientific</td>
			<td>12-565-349</td>
			<td></td>
		</tr>
	</tbody>
</table>

generating a 3d co-culture model of human corneal fibroblasts and neurons

Source: Sharif, R., et al. <a href="https://app.jove.com/v/56308">Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea.</a> J. Vis. Exp. (2018)
This video demonstrates a technique to generate a three-dimensional co-culture model system of primary human corneal fibroblasts and differentiated neuronal cells. This co-culture system can be used to study the stromal-nerve interactions.

Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

1. Primary HCFs (human corneal fibroblasts) Culture and 3D Constructs Assembly

	To assemble 3D constructs from explants previously grown in EMEM 10% FBS ...

Take human corneal fibroblasts.
Transfer them to a membrane insert within a multi-well plate containing media.
Add media to the membrane insert. Incubate for fibroblast adherence to the insert.
Next, replace the media with media containing vitamin C in the membrane insert and the reservoir.
Incubate. Vitamin C stimulates the fibroblasts to secrete and assemble a 3D extracellular matrix.
Add a human neuroblastoma cell suspension over the fibroblasts.
Incubate, allowing neuroblastoma cell adhesion.
Now, add media containing retinoic acid over the cells and supplement the reservoir with fresh media.&#160;
Incubate. Retinoic acid triggers neuroblastoma cell differentiation into neural progenitor cells.
Next, remove the media. Introduce fresh media to the reservoir. Add media containing a neurotrophic factor to the membrane insert and incubate.
Neurotrophic factors trigger progenitor cell differentiation into neurons, generating a 3D co-culture model of human corneal fibroblasts and neurons.

generating-3d-co-culture-model-human-corneal-fibroblasts

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

48 Views. Source: Sharif, R., et al. Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea. J. Vis. Exp. (2018) This video demonstrates a technique to generate a three-dimensional co-culture model system of primary human corneal fibroblasts and differentiated neuronal cells. This co-culture system can be used to study the stromal-nerve interactions. 

Video: Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons - Concept

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JoVE Journal

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Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

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Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

Transcript

Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss