The authors thank Alcon for providing financial support for these studies.

1. Human corneal epithelial cell culture
<ol>
	<li>Grow immortalized HCEC19 in collagen-coated 75 cm2 flasks with 20 mL of Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 &#176;C with 5% CO2. 
	NOTE: Shaking is not required.</li>
	<li>Change the media every 2&#8722;3 days.</li>
</ol>
2. Preparation of cells for testing
<ol>
	<li>After the cells are almost confluent remove culture media.</li>
	<li>Add 4&#8722;6 mL of cell disassociation solution to each 75 cm2 flasks. Incubate the cells at 37 &#176;C until the cells detach (approximately 20&#8722;30 min), checking under the microscope periodically.</li>
	<li>Add 2&#8722;6 mL of DMEM/F12 containing 10% FBS to each 75 cm2 flask.</li>
	<li>Transfer the contents of the flask to a 50 mL centrifuge tube.</li>
	<li>Centrifuge the cells at 450&#8722;500 x g for 5 min. Pipet out the supernatant and resuspend the cells in pre-warmed media.</li>
	<li>Count the cells using a hemocytometer. Calculate the volume of media that contains a total of 1 x 105 cells.</li>
	<li>Add 1 x 105 cells to each well of a 48 well collagen-1 coated culture plate. Add enough media to the well so that the final volume of media in the well is 0.5 mL of culture DMEM/F12 with 10% FBS. 
	NOTE: An artificial tear formulation may cause cytotoxicity that can be measured by a decrease in the metabolic activity of HCEC. The consistency of the data is dependent on adding the same number of HCEC to each well when seeding. The culture concentration that is recommended for 48 well plates is 1 x 105. Because mammalian cells can settle in the centrifuge tube after resuspending, it is recommended to resuspend the cells by agitation frequently while seeding the plates with cells so that the cell suspension has the HCEC equally distributed.</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2 for 24 h.</li>
</ol>
3. No desiccation protocol
<ol>
	<li>Control procedure
	<ol>
		<li>After the 24 h incubation remove the culture media from the plate.</li>
		<li>Immediately treat with 150 &#181;L of a test formulation and media control solution.</li>
		<li>Incubate the cells at 37 &#176;C and 5% CO2 for 30 min.</li>
		<li>Remove the test solutions from the cells.</li>
		<li>Add 0.5 mL of 10% metabolic dye solution to test for metabolic activity. Incubate the cells for another 4 h at 37 &#176;C and 5% CO2.</li>
		<li>After the 4 h incubation period, remove 100 &#181;L of metabolic dye solution from each well and place each 100 &#181;L into a well of a 96 well plate.</li>
		<li>Measure the fluorescence of each well using a fluorescent plate reader (Table of Materials). Set the excitation wavelength at 540 nm and the emission wavelength at 590 nm.</li>
	</ol>
	</li>
	<li>No desiccation protocol (recovery procedure)
	<ol>
		<li>Repeat steps 3.1.1&#8722;3.1.4.</li>
		<li>Add 0.5 mL of DMEM/F12 media to each well.</li>
		<li>Incubate one set of cultures for 18 h at 37 &#176;C and 5% CO2.</li>
		<li>Remove the media and add 0.5 mL of 10% metabolic dye solution. Repeat steps 3.1.6 and 3.1.7.</li>
	</ol>
	</li>
</ol>
4. Desiccation protocol
<ol>
	<li>Control procedure
	<ol>
		<li>After the 24 h incubation (step 2.8), remove the culture media from the plate.</li>
		<li>Immediately treat with 150 &#181;L of a test formulation and media control solution.</li>
		<li>Incubate the cells at 37 &#176;C and 5% CO2 for 30 min.</li>
		<li>Remove the test solutions from the cells.</li>
		<li>Place the cells in a 37 &#176;C and 45% RH chamber for 5 min to desiccate the cells.</li>
		<li>Add 0.5 mL of 10% metabolic dye solution to test metabolic activity. Incubate the cells for another 4 h at 37 &#176;C and 5% CO2.</li>
		<li>After the 4 h incubation period, remove 100 &#181;L of metabolic dye solution from each well and place each 100 &#181;L into a well of a 96 well plate.</li>
		<li>Measure the fluorescence of each well using a fluorescent plate reader. Set the excitation wavelength at 540 nm and the emission wavelength at 590 nm.</li>
	</ol>
	</li>
	<li>Recovery procedure
	<ol>
		<li>Repeat steps 4.1.1-4.1.5.</li>
		<li>Add 0.5 mL of DMEM/F12 media to each well.</li>
		<li>Incubate one set of cultures for 18 h at 37 &#176;C and 5% CO2.</li>
		<li>Remove the media and add 0.5 mL of 10% metabolic dye solution. Repeat steps 4.1.7 and 4.1.8.</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Calculate the average fluorescence of the media control. Calculate the percent viability of the sample using the follow formula: 
	Percent viability of the sample = test sample fluorescence/media control fluorescence x 100 
	NOTE: The media control average is 100% viability. The metabolic dye by itself has a small amount of fluorescence associated with it. The fluorescence of the metabolic dye reagent can be subtracted from the control and test sample fluorescence prior to calculating % viability of the test samples to the media control.</li>
	<li>Perform a normality test and a test for equal variances on the control and test samples.</li>
	<li>If the normality test passes and the variances are equal then perform a one-way ANOVA. To identify significant differences between specific groups perform a pairwise comparisons post hoc test (i.e., Tukey or Dunnett&#39;s multiple comparisons tests).</li>
	<li>If the normality test passes and the variances are different then perform the Welch&#39;s ANOVA. To identify significant differences between specific groups perform a pairwise comparisons post hoc test (i.e., Dunnett&#39;s or Games-Howell multiple comparisons tests).</li>
	<li>If the normality test fails use a nonparametric test (i.e., Kruskal-Wallis test). To identify significant differences between specific groups perform a pairwise comparisons post hoc test (i.e., Dunn&#39;s test).</li>
</ol>

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Three authors (Rekha Rangarajan, Howard A. Ketelson, Lakshman Subbaraman) are employees of Alcon. Alcon is the sole source of funding and sponsor of this project. Lyndon W. Jones, over the past 3 years, has received research support or lectureship honoraria from the following companies: Alcon, Allergan, Contamac, CooperVision, GL Chemtec, Inflamax Research, J&#38;J Vision, Menicon, Nature&#39;s Way, Novartis, PS Therapy, Santen, Shire, SightGlass and Visioneering. Lyndon Jones is also a consultant and/or serves on an advisory board for Alcon, CooperVision, J&#38;J Vision, Novartis and Ophtecs. The following list of authors have nothing to disclose: Richard Do, David J. McCanna, Adeline Suko, Daryl Enstone, Jaya Dantam.

This protocol is designed to determine the protective effects of artificial tear solutions against cell desiccation. Initially, the cells are exposed to the dry eye drops to coat the cells. Next, the cells are dried, and the metabolic activity is monitored to assess whether the formulations can mitigate the detrimental effects of desiccation stress. These steps are critical for understanding the overall impact the dry eye formulations could have on the overall viability of corneal epithelial cells.
After most of the dry eye formulations are removed from the culture wells, the chemistry of the remaining molecules that are in contact with the cells will impact cell desiccation. Some products contain micro-emulsions of oils that minimize cell evaporation20. A study that evaluated friction analysis after rinsing showed maintenance of low friction for some dry eye formulations which may be an indicator of the enhanced residence time of the dry eye formulations21. One of the limitations of this assay is that although desiccation protection can be evaluated in comparison to other eye drop formulations the duration of this protection cannot be determined due to the short drying time assessed. Longer drying times in this in vitro assay may require the use of multilayer cultures that have overall more moisture content than a cell monolayer.
The specific incubation times for the formulations on the cells and the duration of drying time may need to be adjusted if temperature and humidity values are changed. Selecting a temperature, RH and air flow for simulating environmental stress conditions that can contribute to dry eye can be difficult as the seasonal and local environmental conditions are quite variable21. Modern-day environment chambers for human studies can vary the temperature between 5 &#176;C and 35 &#176;C, and RH between 5% and 95%22. Using an evaporimeter and tightly fitted goggles it was determined that at RH of 40&#8722;45% the evaporation rate from the cornea was 0.037 &#181;L/cm2/min and at RH of 20&#8722;25% the evaporation rate was 0.065 &#181;L/cm2/min23. If the in vitro model is looking at the protective effect of formulations under extremely low RH conditions such as in airplanes15, deserts, or dry indoor environments16, incubation times may need to be reduced to accommodate the enhanced evaporation rates from the cells. In the human tear fluid, a variety of lipids derived from meibomian and other glands are present that protect the tears from evaporation. These lipids have different melting points24. Temperature changes could impact the fluidity of the tear fluid so tests performed at ambient temperature could have significantly different evaporation rates than tests conducted at 37 &#176;C. Since the average ocular surface temperature range is from 32.9 to 36 &#176;C25, performing the test near this temperature range is recommended.
In addition to the metabolic dye (alamarBlue) used in this protocol, there are other chemical reagents that can be used to assess the effects of product formulations on cell metabolic activity. The tetrazolium salts (MTT, XTT, MTS, WST) are alternative chemicals that can be used. The difference in the tetrazolium salts is that MTT is a positively charged molecule so it can enter the cytoplasm of living cells26. MTT becomes insoluble after its reduction by NAD(P)H27. It must be solubilized prior to reading absorbance on a plate reader. The other tetrazolium salts are negatively charged28. They must be reduced by secondary molecules outside the living cell because they cannot enter the cell and interact directly with intracellular molecules29. For the XTT, MTS, and WST-1 assays, the addition of an electron coupling agent 1-methoxy phenazine methosulfate (PMS) can improve the performance of the assays30. In contrast to the metabolic assays that use tetrazolium salts, alamarBlue does not require an additional solubilizing or electron coupling agents. Also, unlike XTT, MTS or WST-1, alamarBlue penetrates cell membranes and can be reduced directly by cellular enzymes27,29. Direct comparison of alamarBlue to tetrazolium salts have shown that alamarBlue is more sensitive for evaluating the metabolic activity of various cell lines including HCEC3,27,31,32.
Other biochemical endpoints can also be considered for evaluating the protection of human corneal epithelial cells by desiccation. Fluorescent dyes can be used to detect cell membrane permeability and cell esterase activity1. Also, apoptosis can be detected by staining for apoptotic markers on the cell surface or by measuring for caspase activity1,33. Other assays have determined the effects of ophthalmic products on HCEC tight junctions34. These assays could be incorporated in future assessments utilizing the desiccation procedures described in this article.
There are many causes of dry eye. Dry eye can be caused by decreased secretion of tears, increased ocular surface inflammation or increased dehydration at the ocular surface. For the individuals that have evaporative dry eye use of a dry eye drop that prevents their cells from drying during desiccation conditions may alleviate the symptoms of dry eye. One of the problems in some dry eye formulations is the presence of preservatives that may harm the ocular surface. Injuring cells is not helpful to dry eye patients as it can cause more cells to die as injured cells are more susceptible to desiccation stress35.
A recent review article by Garrigue et al.36 provides an assessment of the current knowledge of how lipid-based products act to alleviate evaporative dry eye. Adding lipids to lipid deficient tears can prevent evaporation, thus protecting the cells from desiccation. In addition, humectants are molecules that attract and retain water at the ocular surface37. Both glycerin in solution #1 and propylene glycol in solution #3 are humectants. Solution #3 showed a greater protective effect of the HCEC that may have been due to the lipid and humectant properties of the formulation ingredients.
This protocol is designed to evaluate the metabolic activity of HCEC after exposure to the dry eye formulations and to desiccation stress. By using the methods described in this article, new nonirritating and protective eye drops can be developed for people that suffer from evaporative dry eye.

Many different ingredients in multiple-dose ophthalmic solutions are needed to maintain stability, pH, osmolarity, and efficacy. However, some chemicals such as preservatives or surfactants in ocular solutions can cause damage to the corneal epithelium1,2. Cell viability tests using human corneal epithelial cells (HCEC) can be performed to assess the potential damage caused by these various ingredients in ophthalmic formulations. One of the in vitro assays that is particularly useful for assessing cell damage is the alamarBlue assay3. In this assay, the metabolic dye (i.e., alamarBlue) contains resazurin, which functions as a cell viability indicator. During cellular respiration, resazurin is reduced to resorufin by accepting electrons4. The reduction of resorufin causes a change from a non-fluorescent molecule to a fluorescent molecule that can be detected using a spectrofluorometer5.
This investigation uses two different treatments of the corneal epithelial cells to determine how dry eye products may perform in an in vitro model. In the first evaluation, the cells are treated with these products for 30 min. After treatment, the cells are then evaluated for metabolic activity immediately after exposure to the dry eye products and after a recovery time interval. This assessment determines the effect of these products on cells prior to desiccation in a humidity chamber. Solution preservatives, surfactants or buffers in these solutions may have some cytotoxic effects on the cells that can be detected using the metabolic dye.
The second evaluation determines the metabolic activity of the cells after exposure to the dry eye products and desiccation. In this assessment, if the product treatment offers protection to the cells from desiccation damage, the metabolic activity of the cells will be maintained. Cell protection from the harmful effects of desiccation may occur if the dry eye product can coat the cells with lipids or the product may absorb liquid from the surroundings adding moisture to the cells.
This protocol is designed to simulate severe conditions of environmental stress on cells. Other studies have used various models to create this external stress. Some studies have dried the cells in laminar flow hoods6,7,8, whereas others have used room temperature and various relative humidity (RH) values9,10,11. A method of simulating dry eye conditions is to increase the osmolarity of the incubating solution12,13. Another in vitro method for simulating adverse environmental conditions is to add cytokines to the cell media to simulate the tear fluid of dry eye patients14. Although these tests are interesting models that can evaluate how chemicals reduce osmotic stress or stimulate the immune system, these models do not directly show how chemical formulations can protect cells from desiccation stress.
The desiccation model uses a temperature/humidity chamber 37 &#176;C and 45% RH. It simulates environmental conditions that can cause evaporation from the ocular surface. Simulations of other extreme environmental conditions such as the low humidity found in airplane cabins15 or in indoor environments during the winter months16 can also be performed using this method.
Cell culture modeling is used to simulate the effect of desiccation stress on the human cornea. In order to detect cell stress, cell viability testing is undertaken to determine the impact on cell health. Several different physiological tests can be performed on cells to determine if they are stressed17. The analysis performed in this test procedure uses the commercially available metabolic dye (Table of Materials). It has the advantage over many other cell stress tests in that it is non-toxic, soluble in growth media and can evaluate cells without cell fixation18. In this procedure the cells are tested for metabolic activity immediately after each treatment and a second set of cells are placed back into growth media and evaluated after 18 h recovery. The reason for testing immediately and then after a recovery period is to identify cell damage that causes immediate effects on metabolic activity and cell damage that causes delayed effects. Also, it provides an opportunity to evaluate the impact of these formulations on short-term versus long-term damage and the ability of the formulations to recover/not recover from the initial insult.
This investigation is used to compare various lipid-containing dry eye products for their effect on HCEC metabolic activity with and without desiccation. The ingredients in the dry eye products tested are described in Table of Materials. The ingredients in solution #1 are carboxymethylcellulose sodium (0.5%), glycerin (1%), and polysorbate 80 (0.5%), in solution #2 they are light mineral oil (1.0%) and mineral oil (4.5%), and in solution #3 the lipid is mineral oil and propylene glycol is the demulcent. These lipids are the formulation components that are expected to provide protection of the HCEC from desiccation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>75 cm2 Vented Flask</td>
			<td>Corning</td>
			<td>354485</td>
			<td>This is the BioCoat brand collagen coated</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>costar</td>
			<td>3370</td>
			<td></td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Fisher Scientific</td>
			<td>dal 1025</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 48 Well plates</td>
			<td>Corning</td>
			<td>354505</td>
			<td>This is the BioCoat brand collagen coated</td>
		</tr>
		<tr>
			<td>Cytation 5</td>
			<td>BioTek</td>
			<td>CYT5MPV</td>
			<td>Can read fluorescence from 280 - 700 nm (for assay 540/590)</td>
		</tr>
		<tr>
			<td>DMEM/F12 with L-glutamine and 15 mM HEPES</td>
			<td>Gibco</td>
			<td>11039-021</td>
			<td>There is No Phenol Red in this media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hycone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Human Corneal Epithelial Cells</td>
			<td>University of Ottawa</td>
			<td>N/A</td>
			<td>SV40-immortalized human corneal epithelial cells from Dr. M. Griffith (Ottawa Eye Research Institute, Ottawa, Canada) and have been characterized Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169&#8211;72.</td>
		</tr>
		<tr>
			<td>Humidity/Temperature Chamber</td>
			<td>Associated Environmental Systems</td>
			<td>LH-1.5</td>
			<td>Economy Line Humidity Chamber</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>100X concentration so add 1ml to each 99ml of media</td>
		</tr>
		<tr>
			<td>Refresh Optive Advanced (solution #1)</td>
			<td>Allergan, Inc</td>
			<td></td>
			<td>Carboxymethylcellulose Sodium (0.5%),Glycerin (1%),polysorbate 80 (0.5%),-Lubricants- Inactive ingredients-boric acid; carbomer copolymer type A; castor oil; erythritol; levocarnitine; purified water; Purite (stablized oxychloro complex)and may contain sodium hydroxide to adjust pH.</td>
		</tr>
		<tr>
			<td>Soothe XP (solution #2)</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>Light mineral oil (1.0%), Mineral Oil (4.5%)-emolients-Inactive ingredients-boric acid, edetate disodium, octoxynol-40, polyquaternium-1 (preservative), polysorbate 80, purified water, sodium borate decahydrate. May contain hydrochloric acid and/or sodium hydroxide (to adjust pH).</td>
		</tr>
		<tr>
			<td>Systane Complete (solution #3)</td>
			<td>Alcon Inc.</td>
			<td></td>
			<td>Propylene Glycol 0.6% Lubricant Inactive ingredients- boric acid, dimyristoyl phosphatidylglycerol, edatate disodium, hydroxypropyl guar, mineral oil, polyoxl 40 stearate, POLYQUAD&#174; (polyquaternium-1) 0.001% preservative, sorbitan tristearate, sorbitol and purified water. May contain hydrochloric acid and/or sodium hydroxide to adjust pH.</td>
		</tr>
		<tr>
			<td>TrypLE Express (cell disassociation solution)</td>
			<td>Fisher Scientific</td>
			<td><a href="https://www.thermofisher.com/order/catalog/product/12605036" target="_blank">12605036</a></td>
			<td></td>
		</tr>
	</tbody>
</table>

effect of artificial tear formulations on the metabolic activity of human corneal epithelial cells after exposure to desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

Results from a study comparing three dry eye formulations are shown in Figure 1. This figure shows that there are differences in the effect the three products, solution #1, solution #2 and solution #3 (Table of Materials) have on the viability of HCEC. Solution #1 and solution #2 had a significant effect on the metabolic activity of HCEC before desiccation. This means that there are formulation components in these products that can disrupt the metabolic activity of the HCEC. The additional drop in cell metabolic activity that occurred after 18 h recovery of cells exposed to solution #1 shows that HCEC were injured initially and after 18 h the injury was not repaired. In comparison, solution #3 had only a mild effect on the metabolic activity of the HCEC.
Figure 2 shows the effect of these lipid-containing dry eye formulations on cell protection. With a near 0% HCEC metabolic activity at the 18 h recovery interval, solution #1 and solution #2 did not protect cells from desiccation stress. Solution #3, however, offered some protection against desiccation stress.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60812/60812fig1.jpg" /> 
Figure 1: Effect of dry eye lipid enhanced products on cell viability. Relative viability measured by HCEC metabolic activity after treatment with lipid-containing dry eye products. * = statistical significance (p &#60; 0.05) relative to solution #3, &#916; = statistical significance (p &#60; 0.05) relative to the media control for the appropriate recovery period. The height of the bar represents the mean value, and the error bars are the SD. <a href="https://www.jove.com/files/ftp_upload/60812/60812fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60812/60812fig2.jpg" /> 
Figure 2: Effect of dry eye lipid enhanced products on desiccation protection. Effect of lipid-containing dry eye formulations on cell protection. Solution #1 and solution #2 did not protect cells from desiccation stress, and they did not recover from desiccation effects over time. In contrast, solution #3 offered some protection against desiccation stress. * = p &#60; 0.05 relative to solution #3, and &#916; = p &#60; 0.05 relative to untreated (control) media for the appropriate recovery time. The height of the bar represents the mean value, and the error bars are the SD. <a href="https://www.jove.com/files/ftp_upload/60812/60812fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation at JoVE.com

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

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3.7K Views.  Alcon Vision, LLC. The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Video: Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Systems Biology of Metabolic Regulation by Estrogen Receptor Signaling in Breast Cancer

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective mining of the information in the large-scale datasets that are obtained from gene expression microarrays, deep sequencing experiments or metabolic profiling is essential to uncover and then effectively target the critical regulators of diseased cell phenotypes. Estrogen Receptor &#945; (ER&#945;) is one of the master transcription factors regulating the gene programs that are important for estrogen responsive breast cancers. In order to understand to role of ER&#945; signaling in breast cancer metabolism we utilized transcriptomic, cistromic and metabolomic data from MCF-7 cells treated with estradiol. In this report we described generation of samples for RNA-Seq, ChIP-Seq and metabolomics experiments and the integrative computational analysis of the obtained data. This approach is useful in delineating novel molecular mechanisms and gene regulatory circuits that are regulated by a particular transcription factor which impacts metabolism of normal or diseased cells.

Integration of data from genome-wide sequencing experiments and metabolomics experiments is a challenge. In this paper we report, for the first time, generation, analysis and integration of transcriptome, cistrome and metabolome data from breast cancer cells treated with estradiol.

With the advent of the -omics approaches our understanding of the chronic diseases like cancer and metabolic syndrome has improved. However, effective ...

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.

We describe a straightforward method for the isolation of washed platelets from human blood followed by agonist-induced platelet aggregation measurements by turbidimetry. As an example we apply this method for studying the aggregation response of human platelets to collagen after a pre-incubation with the Pannexin1 channel inhibitor Brilliant Blue FCF.

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or ...

A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be attributed to, in part, the dysregulation of autophagy. Aberrant changes in the expression of autophagy regulators in the placentas from obese pregnancies may be regulated by inflammatory processes associated with both obesity and pregnancy. Described here is a protocol for sampling of villous tissue and isolation of villous cytotrophoblasts from the term human placenta for primary cell culture. This is followed by a method for simulating the inflammatory milieu in the obese intrauterine environment by treating primary trophoblasts from lean pregnancies with tumor necrosis factor alpha (TNF&#945;), a proinflammatory cytokine that is elevated in obesity and in pregnancy. Through the implementation of the protocol described here, it is found that exposure to exogenous TNF&#945; regulates the expression of Rubicon, a negative regulator of autophagy, in trophoblasts from lean pregnancies with female fetuses. While a variety of biological factors in the obese intrauterine environment maintain the potential to modulate critical pathways in trophoblasts, this ex vivo system is especially useful for determining if expression patterns observed in vivo in human placentas with maternal obesity are a direct result of TNF&#945; signaling. Ultimately, this approach affords the opportunity to parse out the regulatory and molecular implications of inflammation associated with maternal obesity on autophagy and other critical cellular pathways in trophoblasts that have the potential to impact placental function.

Presented here is a protocol for sampling of human placental villous tissue followed by isolation of cytotrophoblasts for primary cell culture. Treatment of trophoblasts with TNF&#945; recapitulates inflammation in the obese intrauterine environment and facilitates the discovery of molecular targets regulated by inflammation in placentas with maternal obesity.

Maternal obesity is associated with an increased risk of adverse perinatal outcomes that are likely mediated by compromised placental function that can be ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...

Advancing Cryostorage of Human Stem Cell-Derived Retinal Pigment Epithelial Cells

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of&#160;RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2&#8242;-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

Author Spotlight: Development of an Efficient and Feasible Method of Retinal Pigment Epithelial Cell Freezing

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...