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 sterile Dulbecco&#39;s phosphate-buffered saline (DPBS) (1x).</li>
	<li>Scrape and remove the corneal epithelium by positioning the cornea dome face-up. To ensure complete removal, scrape off the epithelial layer thoroughly using a razor blade at a 45&#176; angle. Next, flip the cornea dome face-down and thoroughly scrape off the endothelium from the stroma with a razor blade at a 45&#176; angle to ensure complete removal. Wash the stromal tissue with DPBS.</li>
	<li>Cut the stromal tissues into small pieces, using a number 10 surgical blade, keeping the stromal pieces wet at all time using DPBS. Pick stromal pieces up with sterile forceps and place into a T25 culture flask without media (4 or 5 pieces of 2 x 2 mm tissue per flask).</li>
	<li>Allow the explants to adhere to the bottom of the flask at 37 &#176;C for about 30 - 40 min. Once adhered, slowly add Eagle&#39;s minimum essential medium (EMEM) containing 10% fetal bovine serum (FBS) and 1% Antibiotic/Antimycotic to the flask without disturbing the explants.</li>
	<li>Gently place the flask in the incubator at 37 &#176;C, 5% CO2. Leave the explants undisturbed until the cells begin isolating and migrating in the flask. After approximately 2 - 4 weeks, cells should reach 100% confluence. Upon 100% confluence, passage the cells by removing the tissue culture media and wash the cells with DPBS.</li>
	<li>Add trypsin to the cells and then incubate them at 37&#176;C. After approximately 5 min, view the cells under light microscopy to confirm that they have detached, and then halt the proteolysis by adding fresh media.</li>
	<li>Transfer the cell suspension into a 15 mL conical tube for centrifugation at 1,000 x g. Remove the supernatant carefully without disturbing the pellet and suspend the cells in fresh EMEM 10% FBS 1% Antibiotic. Count the cells and seed about 1 x 106 cells in T75 culture flask for further expansion with fresh EMEM 1% FBS 1% Antibiotic.</li>
</ol>
2. Primary HCFs 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>
3. 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.&#160;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.&#160;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 as indicated in Table 1.</li>
</ol>

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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. 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M., DeSimone, E., Chwalek, K., Kaplan, D. 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The authors declare no competing financial interests.

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&amp;rsquo;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&amp;rsquo;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-&amp;mu;m pores</td><td>Corning Costar</td><td>3412</td><td></td></tr><tr><td>2-O-&amp;alpha;-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...

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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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JoVE Journal

Developmental Biology

8.8K Views.  University of Oklahoma Health Sciences Center. Dit protocol beschrijft een nieuwe driedimensionale in vitro model, waar hoornvlies stromale cellen en gedifferentieerde neuronale cellen samen worden gekweekt om te helpen bij het onderzoek en het begrijpen van de interacties van de twee celtypes.

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

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioengineering

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Medicine

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

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).

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

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Corneal Sensory Fiber Damage Protocols for Axon Regeneration Studies

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

Author Spotlight: Advancing Corneal Innervation Research Through Innovative Models 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

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

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