Name: corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea
Uploaded: 2018-01-24T13:30:00
Description: Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

We would like to extend our sincere thanks to Dr. Ben Fowler for his technical help with the TEM experiments.

This protocol follows the guidelines of the University of Oklahoma Health Sciences Center/Institutional review board (IRB #4509). All parts of the protocol met the tenets of the declaration of Helsinki. Corneal samples were obtained from the National Development and Research Institute (NDRI) and the Oklahoma Lions Eye Bank.
1. Isolation of Primary Cells
<ol>
	<li>Upon receipt of the human cornea tissue samples, transfer the tissues into a 21.5 cm2 Petri dish containing 2 mL of ste...

<ol>
	<li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. 96 (5), 614-618 (2012).</li><li>R&#246;nkk&#246;, S., Vellonen, K. -. S., J&#228;rvinen, K., Toropainen, E., Urtti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+cell+culture+models+for+drug+toxicity+studies.">Human corneal cell culture models for drug toxicity studies.</a> Drug Delivery and Translational Research. 6 (6), 660-675 (2016).</li><li>Ghezzi, C. E., Rnjak-Kovacina, J., Kaplan, D. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+fibrosis:+an+in+vitro+model.">Human corneal fibrosis: an in vitro model.</a> Invest Ophthalmol Vis Sci. 51 (3), 1382-1388 (2010).</li><li>Chen, F. M., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+biomaterials+of+human+origin+for+tissue+engineering.">Advancing biomaterials of human origin for tissue engineering.</a> Prog Polym Sci. 53, 86-168 (2016).</li><li>Priyadarsini, S., Sarker-Nag, A., Rowsey, T. G., Ma, J. X., Karamichos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+3D+In+Vitro+Model+to+Accelerate+the+Development+of+Human+Therapies+against+Corneal+Diabetes.">Establishment of a 3D In Vitro Model to Accelerate the Development of Human Therapies against Corneal Diabetes.</a> PLoS One. 11 (12), e0168845 (2016).</li><li>Karamichos, D., Hjortdal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratoconus:+tissue+engineering+and+biomaterials.">Keratoconus: tissue engineering and biomaterials.</a> J Funct Biomater. 5 (3), 111-134 (2014).</li><li>Wilson, S. L., Yang, Y., El Haj, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Stromal+Cell+Plasticity:+In+Vitro+Regulation+of+Cell+Phenotype+Through+Cell-Cell+Interactions+in+a+Three-Dimensional+Model.">Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model.</a> Tissue Engineering Part A. 20 (1-2), 225-238 (2014).</li><li>Proulx, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+a+human+cornea+by+the+self-assembly+approach+of+tissue+engineering+using+the+three+native+cell+types.">Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.</a> Molecular Vision. 16 (234-236), 2192-2201 (2010).</li><li>Gonzalez-Andrades, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+in+vitro+model+of+stratified+epithelial+wound+healing+with+barrier+function.">Establishment of a novel in vitro model of stratified epithelial wound healing with barrier function.</a> Sci Rep. 6, 19395 (2016).</li><li>Hopkins, A. M., DeSimone, E., Chwalek, K., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+in+vitro+modeling+of+the+central+nervous+system.">3D in vitro modeling of the central nervous system.</a> Prog Neurobiol. 125, 1-25 (2015).</li><li>Schulz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+Corneal+Cell-Based+Microenvironment+as+Prerequisite+for+Balanced+3D+Corneal+Epithelial+Morphogenesis:+A+Promising+Animal+Experiment-Abandoning+Tool+in+Ophthalmology.">Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology.</a> Tissue Engineering Part C-Methods. 20 (4), 297-307 (2014).</li><li>Gao, J., Wang, Y., Zhao, X., Chen, P., Xie, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204-5p-Mediated+Regulation+of+SIRT1+Contributes+to+the+Delay+of+Epithelial+Cell+Cycle+Traversal+in+Diabetic+Corneas.">MicroRNA-204-5p-Mediated Regulation of SIRT1 Contributes to the Delay of Epithelial Cell Cycle Traversal in Diabetic Corneas.</a> Invest Ophthalmol Vis Sci. 56 (3), 1493-1504 (2015).</li><li>Koulikovska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+regeneration+of+corneal+tissue+via+a+bioengineered+collagen+construct+implanted+by+a+nondisruptive+surgical+technique.">Enhanced regeneration of corneal tissue via a bioengineered collagen construct implanted by a nondisruptive surgical technique.</a> Tissue Eng Part A. 21 (5-6), 1116-1130 (2015).</li><li>Ljubimov, A. V., Saghizadeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+corneal+wound+healing.">Progress in corneal wound healing.</a> Progress in retinal and eye research. 49, 17-45 (2015).</li><li>Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+and+wound+healing.">Extracellular matrix and wound healing.</a> Curr Opin Ophthalmol. 12 (4), 237-241 (2001).</li><li>Karamichos, D., Hutcheon, A. E., Zieske, J. 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Several studies have been focused on developing various animal models that can help develop a better understanding of corneal diseases as well as discover treatments. However, a significant value to humans from these studies has not been verified. To date, various in vitro models have been developed and widely investigated due to their remarkable clinical significance. Our previously established 3D in vitro model is a novel system that significantly contributes to medical science advancements in vision ...

In the human body, the cornea is the most densely innervated tissue. The nerves are responsible for various sensations like touch, pain, temperature, and also have an essential role in wound healing, blink reflexes, tear production, and secretion5,6,7. In the cornea, stromal nerve trunks arise from the limbal plexus and enter the peripheral corneal stroma radially. The stromal nerve organization is parallel to the collagen lamellae and they further branch into smaller fascicles as they proceed towards the superficial stroma5,8. The nerve fibers further penetrate the epithelial layer and thus, innervation is widely spread out across the corneal epithelium and stroma. Therefore, innervation has an essential role in the healthy and diseased state of the cornea. In this protocol, we reveal the advancement of a novel 3D in vitro model, which is the first of its kind to mimic the in vivo stromal-nerve interactions. The SH-SY5Y cell line was used for this study, as it is one of the most established, well characterized lines used to study neuronal growth. The SH-SY5Y cell line has been described to produce both substrate adherent (S-type) and neuroblastic (N-type) cells that can undergo transdifferentiation9. As a result, even though this cell line is derived from a triple successive subclone selection of N-type cells, it also contains a small number of S-type cells capable of undergoing differentiation into neurons through the use of retinoic acid and brain-derived neurotrophic factor9. This provides a tool that may lead to a better understanding of the corneal complications associated with diabetic retinopathy (DM) and other ocular diseases. Due to the difficulties associated with obtaining and culturing neurons from patients with ocular disease, this 3D in vitro model provides substantial implications in studying the neuronal interactions and signaling with the corneal stroma.
Diseased conditions often affect various tissues of the body at a very large scale, leading to a compromised quality of life. Ocular dystrophies are common complications often associated with systemic diseases and lead to loss of visual acuity or even permanent vision loss. Comprehensive studies are often essential for a better understanding of the disease condition as well as the effects at the basal cellular level. To study the effects of such diseases, various in vivo and in vitro models have been developed with the help of tissue engineering applications. Corneal tissue engineering applications have garnered great interest across various fields of science10,11,12,13,14, but there are still major limitations during actual application, including corneal graft rejections, infections, and scarring10,11,12,13,14. There are several studies that have successfully developed and established various in vitro models3,15,16,17,18,19,20,21,22,23,24,25,26. The 3D in vitro models are the most promising and of great scientific interest. 3D models are known to better mirror the in vivo cellular and physiological events that are critical during fibrosis and wound healing15,27,28,29. These in vitro models play an integral role in finding new therapeutic approaches for treating different disease conditions including corneal complications. Despite the critical role of innervation in corneal functions, little effort has been made to promote peripheral nerve proliferation within corneal tissue-engineered constructs2,3. However, the proposed 3D in vitro cell constructs mimic the target tissue in order to attain the desired tissue functionality.
While diabetic keratopathy is an obvious application for the model described here due to the neuronal defects, there are several other corneal diseases that can benefit from a human in vitro model including Keratoconus and Fuchs dystrophies. Our 3D model emerges from this prospect and proposes the development of an in vitro representation of corneal tissue to assess drug delivery and safety of new ocular medicines.

<table>
	<tbody>
		<tr>
			<td>Healthy corneal tissue</td>
			<td>NDRI</td>
			<td></td>
			<td>Samples from donors with no ocular trauma or systemic disease</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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&#8217;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>Wax block</td>
			<td>VWR</td>
			<td>50-949-027</td>
			<td></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>

corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Figure 1 is a step-by-step representative image of the working 3D in vitro model. At the first step, cells are isolated from human corneas. Then, they are grown on a polycarbonate membrane and stimulated with Vitamin C to assemble a self-secreted 3D matrix. This 3D construct system induces the synthesis of a multilayered cellular in vivo-like stromal matrix. Thereafter, neuroblastoma cells are seeded on top of the stromal cells followed by i...

Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

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