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. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta3+regulates+assembly+of+a+non-fibrotic+matrix+in+a+3D+corneal+model.">Transforming growth factor-beta3 regulates assembly of a non-fibrotic matrix in a 3D corneal model.</a> J Tissue Eng Regen Med. 5 (8), e228-e238 (2011).</li><li>Karamichos, D., Lakshman, N., Petroll, W. 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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&#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...

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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 ...

corneal-tissue-engineering-an-vitro-model-stromal-nerve-interactions

Research

JoVE Journal

Developmental Biology

8.7K Views.  University of Oklahoma Health Sciences Center. 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.

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and the neurosciences. Traditional in vitro experimental models used in neurobiology include primary neuronal cultures from rats and mice, neuroblastoma cell lines including rat B35 and mouse Neuro-2A cells, rat PC12 cells, and short-term slice cultures. While many researchers rely on these models, they lack a human component and observed experimental effects could be exclusive to the respective species and may not occur identically in humans. Additionally, although these cells are neurons, they may have unstable karyotypes, making their use problematic for studies of gene expression and reproducible studies of cell signaling. It is therefore important to develop more consistent models of human neurological disease. 
The following procedure describes an easy-to-follow, reproducible method to obtain homogenous and viable human neuronal cultures, by differentiating the chromosomally stable human neuroblastoma cell line, SH-SY5Y. This method integrates several previously described methods1-4 and is based on sequential removal of serum from media. The timeline includes gradual serum-starvation, with introduction of extracellular matrix proteins and neurotrophic factors. This allows neurons to differentiate, while epithelial cells are selected against, resulting in a homogeneous neuronal culture. Representative results demonstrate the successful differentiation of SH-SY5Y neuroblastoma cells from an initial epithelial-like cell phenotype into a more expansive and branched neuronal phenotype. This protocol offers a reliable way to generate homogeneous populations of neuronal cultures that can be used for subsequent biochemical and molecular analyses, which provides researchers with a more accurate translational model of human infection and disease.

It is critical in neurobiology and neurovirology to have a reliable, replicable in vitro system that serves as a translational model for what occurs in vivo in human neurons. This protocol describes how to culture and differentiate SH-SY5Y human neuroblastoma cells into viable neurons for use in in vitro applications.

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing various types of stem cells to derive cell types of interest is often costly, time consuming and highly inefficient. Furthermore, undesired cell types must be removed prior to using this cell source, which requires another step in the process. In order to obtain enough esophageal epithelial cells to engineer the lumen of an esophageal construct or to screen therapeutic approaches for treating esophageal disease, native esophageal epithelial cells must be expanded without altering their gene expression or phenotype. Conditional reprogramming of esophageal epithelial tissue offers a promising approach to expanding patient-specific esophageal epithelial cells. Furthermore, these cells do not need to be sorted or purified and will return to a mature epithelial state after removing them from conditional reprogramming culture. This technique has been described in many cancer screening studies and allows for indefinite expansion of these cells over multiple passages. The ability to perform esophageal screening assays would help revolutionize the treatment of pediatric esophageal diseases like eosinophilic esophagitis by identifying the trigger mechanism causing the patient's symptoms. For those patients who suffer from congenital defect, disease or injury of the esophagus, this cell source could be used as a means to seed a synthetic construct for implantation to repair or replace the affected region.

Expansion of human pediatric esophageal epithelial cells utilizing conditional reprogramming provides investigators with a patient-specific population of cells that can be utilized for engineering esophageal constructs for autologous implantation to treat defects or injury and serve as a reservoir for therapeutic screening assays.

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...