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

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

To assemble 3D constructs, first, passage and count human corneal fibroblasts. Then, seed approximately 1 times 10 to the 6 HCFs onto a polycarbonate membrane insert with 0.4-micron pores. Add 1.5 milliliters of fresh medium to the top of the construct. Then, pipette another 1.5 milliliters of the same media to the bottom of each construct.
After 24 hours, add 1.5 milliliters of medium supplemented with 0.5 millimolar vitamin C to the top and bottom of each construct. Incubate at 37 degrees Celsius in 5% carbon dioxide for three weeks, adding fresh media containing vitamin C every other day. This will stimulate the cells to secrete and assemble their own extracellular matrix. After three weeks, add 8 times 10 to the 3 SH-SY5Y human neuroblastoma cells on the top of the previously assembled 3D constructs.
It is critical to calculate exact cell number in this step and to allow 24 hours before initiating cell differentiation.
After 24 hours of incubation, initiate SH-SY5Y cell differentiation by stimulating the cells with 1.5 milliliters of EMEM with 10% FBS and 10 micromolar retinoic acid. Then, add 1.5 milliliters of the medium containing vitamin C to the bottom of the construct. Retinoic acid is light-sensitive so this step should be performed in the dark.
After five days, when the cells reached the differentiation phase, add 1.5 milliliters of serum-free medium containing 2 nanomolar BDNF and 1% antibiotic-antimycotic to the top of the construct. Add fresh vitamin C-supplemented medium to the bottom of the construct. Culture the cells for two additional days before processing for further analysis.

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JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Co-culture and Interaction Studies

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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>

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.

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.

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视频: 生成人角膜成纤维细胞和神经元的 3D 共培养模型 - 实验

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

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

建立猪外维沃角膜模型研究药物治疗细菌性角膜炎

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

科研

JoVE Journal

免疫与感染

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.

眼毛刺角膜上皮磨损与角膜创面愈合的模型研究

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 ...

医学

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

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

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).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

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

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