The work is funded by the National Eye Institute Grant EY019470 (D.A.D).

All human tissue was donated to the eye bank with prior informed consent and authorization of the donor for use in scientific research. Use of the human conjunctival tissue was reviewed by the Massachusetts Eye and Ear Human Studies Committee and determined to be exempt and not meeting the definition of research with human subjects.
1. Primary human goblet cell culture
<ol>
	<li>From the eye bank, obtain human conjunctival tissue16.</li>
	<li>Prepare the culture medium, RPMI-1640 culture medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 2 mM non-essential amino acids (NEAA), 2 mM sodium pyruvate, 100 &#181;g/mL penicillin-streptomycin, and 2 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES).</li>
	<li>Carry out primary cell culture as described below.
	<ol>
		<li>Mince the tissue into 1 mm3 pieces with a sterile scalpel and seed onto culture plates in a culture hood. Seed four pieces in each well of a 6-well plate, in 1 mL of complete RPMI media. Although the orientation of the tissue piece does not impact cell growth, ensure the pieces adhere to the culture plate to ensure outgrowth using the scalpel blade.</li>
		<li>Place the tissue pieces into the incubator containing 95% air and 5% CO2 at 37 &#176;C. Refresh the same culture medium every second day until the goblet cells reach 70%-80% confluency in approximately 14 days. Remove the tissue plug approximately 72 h after seeding. 
		&#8203;NOTE: Human conjunctival epithelium mainly contains stratified squamous cells and goblet cells. RPMI is a selective media for goblet cells. Rarely in human CGC culture, fibroblasts may grow around day 7. The method to purify the culture was described in a previous publication 22.</li>
		<li>Verify the purity of the GC culture using immunofluorescence microscopy (IFM) targeting cytokeratin (CK)7 and Helix pomatia lectin-1 (HPA-1)22 following the standard IFM method described in19; see representative results.</li>
	</ol>
	</li>
</ol>
2. Human conjunctival goblet cell passage and experimental preparation
<ol>
	<li>Rinse the cells with PBS (pH = 7.4) and detach the cells with trypsinization using 0.05% trypsin in 1x EDTA. Observe the cells every minute under a microscope. Once the cells detach from the bottom, deactivate the trypsin using complete RPMI media.</li>
	<li>Centrifuge the cells at 150 &#215; g for 5 min at room temperature. Resuspend the cell pellet in the complete RPMI culture medium and reseed onto glass-bottomed culture dishes for [Ca2+]i measurement. The total volume of the medium is 300 &#181;L per dish. Keep the media in the center of the glass part of the dish.</li>
	<li>Elimination of hormones in culture media: The culture medium may contain sex hormones, especially the FBS. As the phenol red contained in RPMI-1640 has estrogenic activity 23,24, replace the RPMI medium with phenol red-free RPMI medium and adjust the pH to 7.45 using pH strips after preparing the complete medium. The HEPES contained in the complete medium maintains the pH to a relatively small range.</li>
</ol>
3. Fura-2/acetoxymethyl (AM) assay for [Ca2+]i measurement
<ol>
	<li>Perform Fura-2/AM loading as described below.
	<ol>
		<li>Incubate the cells in glass bottom dishes for 1 h at 37 &#176;C, ambient atmosphere, and protected from light with Krebs-Ringer bicarbonate buffer (KRB; containing 119 mM NaCl, 4.8 mM KCl, 1.0 mM CaCl2, 1.2 mM MgSO4, and 25 mM NaHCO3) with 0.5% HEPES (Table 1), plus 0.5% BSA, 0.5 &#181;M fura-2/AM, 8 &#181;M pluronic acid F127, and 250 &#181;M sulfinpyrazone.</li>
		<li>Adjust the pH to 7.45 using a pH meter before use. After loading with fura-2/AM, wash the cells with KRB containing 250 &#181;M sulfinpyrazone immediately before [Ca2+]i measurement.</li>
	</ol>
	</li>
	<li>Perform [Ca2+]i measurement as described below.
	<ol>
		<li>Place the dishes containing the cells loaded with fura-2 under the microscope, locate a representative field containing between 20 cells and 50 cells at 200x magnification, and use the Freehand function to draw an outline around each cell to indicate to the software what is and what is not background fluorescence.</li>
		<li>Wait for the software to automatically subtract background fluorescence from the measurement and click the Start Experiment button.</li>
		<li>Then, wait between 8 s and 15 s to establish the basal calcium levels in the cells before carefully pipetting in the agonist of interest. Continue measuring for at least 120 s after adding the agonist or until calcium levels have returned to baseline.</li>
	</ol>
	</li>
	<li>Perform data analysis as described below.
	<ol>
		<li>Use the change in peak [Ca2+]i to represent the actions of the stimuli. Calculate the mean basal calcium level in each cell from the first 8-15 s of measurements. If that number is equal to or above 500 nM, remove that cell from the dataset as it is undergoing apoptosis or necrosis.</li>
		<li>Once the basal level of each cell has been calculated, subtract that amount from the maximum measured [Ca2+]i for the same cell. Average the change in peak [Ca2+]I for all of the cells in a given dish.</li>
		<li>To ensure consistency, record the responses of each stimulus in duplicate, and calculate the average value of the changes in peak [Ca2+]i obtained from the duplicates as one data point from the individual human sample. Compare the data from different groups using an appropriate data analysis method based on the study design.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest.

Investigating the sex-based differences in ocular tissues helps understand the processes of diseases, especially dry eye and allergic conjunctivitis, which disproportionately affect one sex4,5,6. Even though animal models can be used for these studies, data obtained directly from human tissue are essential due to the highest similarity to the human cells in vivo. The conjunctival tissues used in the current experimental setting are from deceased donors with an age range of 70-85 years (postmenopausal), making it easier to control the hormonal effect ante mortem. Here, we presented a cost-efficient method to study sex-based differences in human CGCs.
The critical step for acquiring consistent data is the removal of FBS and phenol red for 48 h before experiments to eliminate the effects of sex hormones. The actions of sex hormones in cells can be categorized into non-transcriptional and transcriptional effects. The non-transcriptional effects are rapidly mediated through G-proteins in a few seconds to minutes, and the transcriptional effect in which certain genes get activated takes 30 min to several hours27; the half-life of estrogen-induced estrogen receptor alpha (ER&#945;) is 2-6 h28,29. Even though the turnover rates of other types of sex hormone-induced proteins are rarely reported, results from the existing studies indicate that sex hormone-induced proteins are synthesized and degraded within hours. Therefore, 48 h is enough time to wash out the potential effect of hormones contained in the FBS while maintaining normal cellular function. Using the current method, we successfully reported sex-based differences in the expression of sex hormone receptors and cellular functions in CGCs30.
FBS is an essential factor for goblet cell cultures to remain healthy and viable. Replacing the normal heat-inactivated FBS with charcoal-stripped FBS is another possible way to eliminate sex hormones in the culture system. The limitation of using charcoal stripped FBS is the cost and the complex composition of the FBS. Completely removing FBS also eliminates the potential inconsistency from the undefined medium components of FBS.
The critical step for [Ca2+]i measurement is to stabilize the pH and temperature. Despite the HEPES supplement, we have noticed a shift of pH in the KRB buffer over time when stored at room temperature. Thus, a readjustment of the pH of the buffer is recommended for a long experiment, approximately every 3 h. The same [Ca2+]i method also applies to other types of cells such as stratified squamous cells of the conjunctiva, vascular endothelium cells, microglial cells, and neurons to study the action of different stimuli.
Binding of Fura-2 to free calcium in the cell causes a change in its peak absorption wavelength from 380 nm (unbound) to 340 nm (bound). However, the emission wavelength remains at 510 nm and these emissions are captured by the camera as black and white images. The ratio of emission intensity excited by 340 nm and 380 nm light is recorded. Besides comparing the 340/380 ratio to a stand curve to calculate a [Ca2+]i concentration, one may also report the fold change of the ratio without having to create a standard curve, therefore, without an actual concentration. Investigators can choose either way based on their equipment setup and the experimental design.
In conclusion, cultured primary conjunctival goblet cells can be used as a model to study the sex-based differences of ocular surface diseases. Incubation for 48 h in phenol red-free RPMI medium with 1% BSA before performing any experiments is a consistent and cost-efficient method to eliminate the potential effects of hormones in culture media and can be used as a basal medium to control hormone levels.

Sex-based differences affect multiple processes of the ocular surface1,2,3. The clinical manifestation of these sex-based differences is the difference in the prevalence of many ocular surface diseases between men and women, such as dry eye and conjunctivitis4,5,6. Evidence suggests that sex-based differences arise from multiple biological levels, including the different profiles of genes on X and Y chromosomes7 and the effects of hormones8. Studying the molecular basis of sex-based differences can provide a better understanding of disease and, eventually, improve personalized medicine.
The ocular surface comprises the overlying tear film, cornea, and conjunctiva. Sex-based differences are observed in multiple components of the ocular surface, including the tear film9,10, cornea11, the lacrimal gland12,13, and meibomian glands that also secrete tears12. Numerous mechanistic studies have investigated the effect of sex hormones on the cornea and its associated components14,15; however, little is known about the sex-based differences in the conjunctiva and its goblet cells. The conjunctiva is a mucous membrane that covers the sclera and the inner surface of the eyelid. The epithelium of the conjunctiva is comprised of nonkeratinizing, multi-layered, stratified squamous cells16.
Among the stratified squamous cells of the conjunctiva, there are goblet cells (CGCs) interspersed at the apical surface of the epithelium. These goblet cells are characterized by the large number of secretory granules located at the apical pole17. CGCs synthesize and secrete the gel-forming mucin MUC5AC to moisturize the ocular surface and lubricate it during blinking17. Mucin secretion is tightly regulated by the intracellular [Ca2+] ([Ca2+]i) and the activation of the Ras-dependent extracellular signal-regulated kinase (ERK1/2)18. Inability to secrete mucin results in dryness of the ocular surface and sequelae of pathological abnormalities. On an inflamed ocular surface, however, extensive mucin secretion stimulated by inflammatory mediators leads to a perception of stickiness and itchiness of the eye19. These conditions with disturbed mucin secretion will eventually lead to deterioration of the ocular surface.
The role of goblet cells as the major source of ocular mucin has long been recognized20, however, the sex-based differences in mucin regulation in both physiological and pathological states remain undiscovered. An in vitro system would be useful to monitor the function of goblet cells without the hormonal effect or with a precisely controlled level of sex hormones. Even though a conjunctival epithelial cell line has developed21, there is no goblet cell line with functional mucin secretion available. Therefore, we modified our developed primary human CGC culture to establish a method to analyze the sex-based difference in vitro16, and present it as below.

<table><tbody><tr><td>0.05% trypsin with 1x EDTA</td><td>Gibco (Grand Island, NY)</td><td>25300-054</td><td></td></tr><tr><td>4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid</td><td>Fisher Bioreagent (Pittsburgh, PA)</td><td>BP310-500</td><td></td></tr><tr><td>Advanced RPMI media</td><td>Gibco (Grand Island, NY)</td><td>12633020</td><td></td></tr><tr><td>carbachol</td><td>Cayman Chemical (Ann Arbor, MI)</td><td>144.86</td><td></td></tr><tr><td>Fetal Bovin Serum</td><td>R&amp;amp;D (Minneapolis, MN)</td><td>S11150H</td><td></td></tr><tr><td>Fura-2- acetoxymethyl ester&amp;nbsp;</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>F1221</td><td></td></tr><tr><td>Human conjunctival tissue</td><td>Eversight Eye Bank (Ann Arbor, MI)</td><td>N/A</td><td></td></tr><tr><td>inorganic salt for KRB buffer</td><td>Sigma-Aldrich (St. Louis, MO)</td><td></td><td>Any brand will work</td></tr><tr><td>L-glutamine&amp;nbsp;</td><td>Lonza Group (Basel, Switzerland)</td><td>17-605F</td><td></td></tr><tr><td>non-essential amino acids</td><td>Gibco (Grand Island, NY)</td><td>11140-050</td><td></td></tr><tr><td>penicillin/streptomycin</td><td>Gibco (Grand Island, NY)</td><td>15140-122</td><td></td></tr><tr><td>phenol red-free RPMI media&amp;nbsp;</td><td>Gibco (Grand Island, NY)</td><td>11835055</td><td></td></tr><tr><td>Pluronic acid F127</td><td>MilliporeSigma (Burlington, MA, USA)</td><td>P2443-250G</td><td></td></tr><tr><td>RPMI-1640 culture medium</td><td>Gibco (Grand Island, NY)</td><td>21875034</td><td></td></tr><tr><td>scalpel</td><td>Thermo Fisher Scientific (Waltham, MA, USA)</td><td>12460451</td><td>Any sterile surgical scalpel can work</td></tr><tr><td>sodium pyruvate</td><td>Gibco (Grand Island, NY)</td><td>11360-070</td><td></td></tr><tr><td>sulfinpyrazone</td><td>MilliporeSigma (Burlington, MA, USA)</td><td>S9509-5G</td><td></td></tr></tbody></table>

in vitro method to study sex-based differences in conjunctival goblet cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin that is secreted by conjunctival goblet cells (CGCs) onto the ocular surface contributes to multiple ocular surface diseases. The elimination of exogenous sex hormones is essential to obtain consistent results during in vitro study of sex-based differences in CGCs. This paper describes a method to minimize the presence of exogenous hormones in the study of sex-based differences in CGCs while maintaining their physiological function. CGCs from postmortem human donors of both sexes were cultured from pieces of the conjunctiva in RPMI medium with 10% fetal bovine serum (FBS) (referred to as the complete medium) until confluency. Nearly 48 h before the start of the experiments, CGCs were transferred to RPMI medium without phenol red or FBS but with 1% BSA (referred to as phenol-red-free medium). The normal cellular function was studied by measuring the increase in intracellular [Ca2+] ([Ca2+]i) after carbachol (Cch, 1 x 10-4 M) stimulation using fura 2/acetoxymethyl (AM) microscopy. The result shows that CGCs maintained normal function in the phenol-red-free media after 48 h. No significant difference in [Ca2+]i response was observed between phenol red-free RPMI medium and complete medium upon Cch stimulation. Therefore, we recommend using the phenol-red free RPMI medium with 1% BSA to eliminate exogenous hormones without altering the normal function of CGCs in the study of sex-based differences.

Human CGCs in primary culture grow to 80% confluency in approximately 14 days. The cell type was confirmed by immunofluorescence staining with antibodies to the goblet cell markers CK7 and HPA-125 (Figure 1). Even though the removal of FBS from the medium can eliminate the sex hormones, the lack of FBS could potentially affect the cellular response. To verify the hormone elimination method, a cholinergic agonist (carbachol, Cch 1 &#215; 10-4 M) was used as the stimulus to mimic the physiological goblet cell secretion mediated by different nerve endings surrounding the goblet cells26. No difference in the magnitude of the change in calcium was observed from the control complete RPMI medium, indicating that this method can be applied to study the sex-based difference in cell response.
The Fura-2/AM assay described in the protocol section was performed on dishes treated with complete RPMI + 10% FBS medium, advanced RPMI medium (RPMI supplied a proprietary concentration of insulin) supplemented with 1% BSA, and phenol red-free RPMI with 1% BSA. The results are obtained from four individuals. No significant difference was found when comparing the cells incubated in phenol red-free RPMI with 1% BSA and complete RPMI medium. However, a significantly increased [Ca2+]i response to Cch 1 &#215; 10-4 M was observed when comparing the advanced RPMI medium group to the complete RPMI medium group (Figure 2). In summary, phenol red-free RPMI with 1% BSA does not affect the cellular response of goblet cells, even outperforming the advanced RPMI medium in the current research setting.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64456/64456fig01.jpg" /> 
Figure 1: Human conjunctival goblet cell primary culture. The conjunctival goblet cells were passaged onto coverslips after 14 days in culture, and immunofluorescence staining using primary antibody against CK7 (red), and fluorescein-conjugated lectin HPA-1 (green) was performed. (A) Images were taken under a 200x magnification, and (B) images were taken under a 630x magnification. CK7 presents as a perinuclear fluorescent signal (left panel); HPA-1 presents a punctuate pattern perinuclearly (middle panel). Positive immunofluorescence signal of both CK7 and HPA-1 indicates a goblet cell. Negative control is incubation in the absence of the primary antibody (C). Scale bars = 50 &#181;m (A) and 8 &#181;m (B). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64456/64456fig01large.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/64456/64456fig02.jpg" /> 
Figure 2: Effects of different media on Cch-induced [Ca2+]i response. Cells were incubated with phenol red-free RPMI with 1% BSA, RPMI media with 10% FBS, or advanced RPMI with 1% BSA for 48 h incubation before the addition of 1 &#215; 10-4 M Cch. [Ca2+]i was measured by Fura2 assay and the change in peak [Ca2+]i was calculated and shown in the graph. The black bar indicates complete medium with phenol red and 10% FBS. The orange bar indicates advanced medium with 1% BSA, and the grey bar indicates RPMI without phenol red and with 1% BSA. Data are mean &#177; SEM, n = 4. One-way ANOVA with Dunnett correction was used in multiple group comparisons, p &#60; 0.05 was set as statistically significant. * indicates significant difference between the groups. Abbreviations: Cch = carbachol; [Ca2+]i = intracellular Ca2+ concentration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64456/64456fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Stock Component</td>
			<td>Amount for 250 mL of KRB</td>
			<td>Final Concentration</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>0.25 g</td>
			<td>0.1% w/v</td>
		</tr>
		<tr>
			<td>1 M NaCl</td>
			<td>30 mL</td>
			<td>119 mM</td>
		</tr>
		<tr>
			<td>0.5 M NaHCO3</td>
			<td>12.5 mL</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>1 M 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES)</td>
			<td>2.5 mL</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>1 M KCl</td>
			<td>1.2 mL</td>
			<td>4.8 mM</td>
		</tr>
		<tr>
			<td>1 M KH2PO4</td>
			<td>300 &#181;L</td>
			<td>1.2 mM</td>
		</tr>
		<tr>
			<td>1 M MgSO4</td>
			<td>300 &#181;L</td>
			<td>1.2 mM</td>
		</tr>
		<tr>
			<td>1 M CaCl2</td>
			<td>250 &#181;L</td>
			<td>1 mM</td>
		</tr>
	</tbody>
</table>
Table 1: Material and formula for KRB. Abbreviation:&#160;w/v, weight/volume.

Watch this Scientific Journal Video about In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells at JoVE.com

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Phenol red-free/fetal bovine serum-free medium is a better option than advanced RPMI to eliminate exogenous hormones without altering the normal function of conjunctival goblet cells in the study of sex-based differences.

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

Dry eye is a multi-factorial disease affecting ocular surface health, with a profoundly higher prevalence in women. Disruption of the gel-forming mucin ...

in-vitro-method-to-study-sex-based-differences-conjunctival-goblet

Nanyang Technological University

Research

JoVE Journal

Biology

434 Views.  Harvard Medical School. Door de functie van de bekercel in de primaire cultuur te bestuderen, krijgen we waardevolle inzichten in de mechanismen achter de op geslacht gebaseerde verschillen in de gezondheid van het oogoppervlak. We hebben de reeds gevestigde conjunctivale bekercel primaire kweekmethode aangepast door hormonen en oestrogene stoffen uit het systeem te verwijderen, waardoor een nauwkeurigere controle van de geslachtshormoonspiegels mogelijk wordt. Deze wijziging verbetert de nauwkeurigheid en betrouwba...

Video: In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

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 ...

Developmental Biology

Microdissection and Dissociation of the Murine Oviduct: Individual Segment Identification and Single Cell Isolation

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental treatments. However, because of their small body size, some structures, such as the oviduct with a diameter of 200-400 &#956;m, have proven to be relatively difficult to study except by immunohistochemistry. Recently, immunohistochemical studies have uncovered more complex differences in oviduct segments than were previously recognized; thus, the oviduct is divided into four functional segments with different ratios of seven distinct epithelial cell types. The different embryological origins and ratios of the epithelial cell types likely make the four functional regions differentially susceptible to disease. For example, precursor lesions to serous intraepithelial carcinomas arise from the infundibulum in mouse models and from the corresponding fimbrial region in the human fallopian tube. The protocol described here details a method for microdissection to subdivide the oviduct in such a way to yield a sufficient amount and purity of RNA necessary for downstream analysis such as reverse transcription-quantitative PCR (RT-qPCR) and RNA sequencing (RNAseq). Also described is a mostly non-enzymatic tissue dissociation method appropriate for flow cytometry or single cell RNAseq analysis of fully differentiated oviductal cells. The methods described will facilitate further research utilizing the murine oviduct in the field of reproduction, fertility, cancer, and immunology.

A method for microdissection of the mouse oviduct that allows collection of the individual segments while maintaining RNA integrity is presented. In addition, non-enzymatic oviductal cell dissociation procedure is described. The methods are appropriate for subsequent gene and protein analysis of the functionally different oviductal segments and dissociated oviductal cells.

Mouse model systems are unmatched for the analysis of disease processes because of their genetic manipulability and the low cost of experimental ...

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

Immunology and Infection

Impression Cytology of the Lid Wiper Area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

Medicine

Conjunctival Commensal Isolation and Identification in Mice

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. Identification and monitoring of bacterial species at the ocular mucosa have been challenging due to their low abundance and limited availability of appropriate methodology for commensal growth and identification. There are two standard approaches: culture based or DNA sequencing methods. The first method is problematic due to the limited recoverable bacteria and the second approach identifies both live and dead bacteria leading to an aberrant representation of the ocular space. We developed a robust and sensitive method for bacterial isolation by building upon standard microbiological culturing techniques. This is a swab-based technique, utilizing an &#8220;in-lab&#8221; made thin swab that targets the lower conjunctiva, followed by an amplification step for aerobic and facultative anaerobic genera. This protocol has allowed us to isolate and identify conjunctival species such as Corynebacterium spp., Coagulase Negative Staphylococcus spp., Streptococcus spp., etc. The approach is suitable to define commensal diversity in mice under different disease conditions.

Presented here is a protocol for the isolation and amplification of aerobic and facultative anaerobic mouse conjunctival commensal bacteria using a unique eye swab and culture-based enrichment step with subsequent identification by microbiological based methods and MALDI-TOF mass spectrometry.

The ocular surface was once considered immune privileged and abiotic, but recently it appears that there is a small, but persistent commensal presence. ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

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 ...

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). In experimental animal models with certain genetic modifications or artificial operations, it is useful to examine all parts of the OS epithelial sheet to evaluate relative pathogenetic changes of each part in parallel. However, dissection of OS tissue as a whole without distortion or damage has been challenging, primarily due to the softness and thinness of the OS affixed to physically separate yet movable eyelids and eyeball. Additionally, the deep eye socket formed by the hard skull/orbital bones is fully occupied by the eyeball leaving limited space for operating dissections. As a result, direct dissection of the eyeball with associated OS tissues from the facial side would often lead to tissue damages, especially the palpebral and bulbar conjunctiva. In this protocol, we described a method to remove the skull and orbital bones sequentially from a bisected mouse head, leaving eyelids, ocular surface, lens and retina altogether in one piece. The integrity of the OS sheet was well preserved and could be examined by histology or immunostaining in a single section.

This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its ...

Swab-based Conjunctival Commensal Bacteria Isolation: A Technique to Isolate Commensal Bacteria from Conjunctiva of Murine Model

Source: Smith-Page, K. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/61672" target="_blank">Conjunctival Commensal Isolation and Identification in Mice</a>. J. Vis. Exp. (2021)
This video describes a swab-based technique to isolate viable commensal bacteria from the ocular conjunctiva. The technique can be used to profile the commensal bacterial microbiome of the conjunctiva, which plays a defensive role against pathogens.

Source: Smith-Page, K. et al. Conjunctival Commensal Isolation and Identification in Mice. J. Vis. Exp. (2021)
This video describes a swab-based technique ...

In Vitro Method to Study Sex-Based Differences in Conjunctival Goblet Cells

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