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 ...

<ol>
	<li>Gao, Y., 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=Female-specific+downregulation+of+tissue+polymorphonuclear+neutrophils+drives+impaired+regulatory+T+cell+and+amplified+effector+T+cell+responses+in+autoimmune+dry+eye+disease.">Female-specific downregulation of tissue polymorphonuclear neutrophils drives impaired regulatory T cell and amplified effector T cell responses in autoimmune dry eye disease.</a> Journal of Immunology. 195, 3086-3099 (2015).</li><li>Wang, S. B., 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=Estrogen+negatively+regulates+epithelial+wound+healing+and+protective+lipid+mediator+circuits+in+the+cornea.">Estrogen negatively regulates epithelial wound healing and protective lipid mediator circuits in the cornea.</a> FASEB Journal. 26, 1506-1516 (2012).</li><li>Sullivan, D. A., Block, L., Pena, 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=Influence+of+androgens+and+pituitary+hormones+on+the+structural+profile+and+secretory+activity+of+the+lacrimal+gland.">Influence of androgens and pituitary hormones on the structural profile and secretory activity of the lacrimal gland.</a> Acta Ophthalmologica Scandinavica. 74, 421-435 (1996).</li><li>Schaumberg, D. A., Dana, R., Buring, J. E., Sullivan, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+dry+eye+disease+among+US+men:+estimates+from+the+Physicians'+Health+Studies.">Prevalence of dry eye disease among US men: estimates from the Physicians' Health Studies.</a> Archives of Ophthalmology. 127, 763-768 (2009).</li><li>Tellefsen N&#248;land, 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=Sex+and+age+differences+in+symptoms+and+signs+of+dry+eye+disease+in+a+Norwegian+cohort+of+patients.">Sex and age differences in symptoms and signs of dry eye disease in a Norwegian cohort of patients.</a> The Ocular Surface. 19, 68-73 (2021).</li><li>Sullivan, D. A., 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=TFOS+DEWS+II+Sex,+gender,+and+hormones+report.">TFOS DEWS II Sex, gender, and hormones report.</a> The Ocular Surface. 15, 284-333 (2017).</li><li>Meester, I., 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=SeXY+chromosomes+and+the+immune+system:+reflections+after+a+comparative+study.">SeXY chromosomes and the immune system: reflections after a comparative study.</a> Biology of Sex Differences. 11, 3 (2020).</li><li>Yang, J. -. H., 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=Hormone+replacement+therapy+reverses+the+decrease+in+natural+killer+cytotoxicity+but+does+not+reverse+the+decreases+in+the+T-cell+subpopulation+or+interferon-gamma+production+in+postmenopausal+women.">Hormone replacement therapy reverses the decrease in natural killer cytotoxicity but does not reverse the decreases in the T-cell subpopulation or interferon-gamma production in postmenopausal women.</a> Fertility and Sterility. 74, 261-267 (2000).</li><li>Orucoglu, F., Akman, M., Onal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+age,+refractive+error+and+gender+related+changes+of+the+cornea+and+the+anterior+segment+of+the+eye+with+Scheimpflug+imaging.">Analysis of age, refractive error and gender related changes of the cornea and the anterior segment of the eye with Scheimpflug imaging.</a> Contact Lens &amp; Anterior Eye. 38, 345-350 (2015).</li><li>Strobbe, E., Cellini, M., Barbaresi, U., Campos, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+age+and+gender+on+corneal+biomechanical+properties+in+a+healthy+Italian+population.">Influence of age and gender on corneal biomechanical properties in a healthy Italian population.</a> Cornea. 33, 968-972 (2014).</li><li>Sullivan, D. A., Jensen, R. V., Suzuki, T., Richards, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+sex+steroids+exert+sex-specific+and/or+opposite+effects+on+gene+expression+in+lacrimal+and+meibomian+glands.">Do sex steroids exert sex-specific and/or opposite effects on gene expression in lacrimal and meibomian glands.</a> Molecular Vision. 15, 1553-1572 (2009).</li><li>Bukhari, A. A., Basheer, N. A., Joharjy, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age,+gender,+and+interracial+variability+of+normal+lacrimal+gland+volume+using+MRI.">Age, gender, and interracial variability of normal lacrimal gland volume using MRI.</a> Ophthalmic Plastic and Reconstructive Surgery. 30, 388-391 (2014).</li><li>Sullivan, B. D., Evans, J. E., Dana, M. R., Sullivan, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+aging+on+the+polar+and+neutral+lipid+profiles+in+human+meibomian+gland+secretions.">Influence of aging on the polar and neutral lipid profiles in human meibomian gland secretions.</a> Archives of Ophthalmology. 124, 1286-1292 (2006).</li><li>Ebeigbe, J. A., Ebeigbe, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+sex+hormone+levels+on+tear+production+in+postmenopausal+Nigerian+women.">The influence of sex hormone levels on tear production in postmenopausal Nigerian women.</a> African Journal of Medicine and Medical Sciences. 43, 205-211 (2014).</li><li>Suzuki, T., 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=Estrogen's+and+progesterone's+impact+on+gene+expression+in+the+mouse+lacrimal+gland.">Estrogen's and progesterone's impact on gene expression in the mouse lacrimal gland.</a> Investigative Ophthalmology &amp; Visual Science. 47, 158-168 (2006).</li><li>Shatos, M. A., 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=Isolation+and+characterization+of+cultured+human+conjunctival+goblet+cells.">Isolation and characterization of cultured human conjunctival goblet cells.</a> Investigative Ophthalmology &amp; Visual Science. 44, 2477-2486 (2003).</li><li>Huang, A. J., Tseng, S. C., Kenyon, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphogenesis+of+rat+conjunctival+goblet+cells.">Morphogenesis of rat conjunctival goblet cells.</a> Investigative Ophthalmology &amp; Visual Science. 29, 969-975 (1988).</li><li>Li, D., 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=Resolvin+D1+and+aspirin-triggered+resolvin+D1+regulate+histamine-stimulated+conjunctival+goblet+cell+secretion.">Resolvin D1 and aspirin-triggered resolvin D1 regulate histamine-stimulated conjunctival goblet cell secretion.</a> Mucosal Immunology. 6, 1119-1130 (2013).</li><li>Dartt, D. A., Masli, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjunctival+epithelial+and+goblet+cell+function+in+chronic+inflammation+and+ocular+allergic+inflammation.">Conjunctival epithelial and goblet cell function in chronic inflammation and ocular allergic inflammation.</a> Current Opinion in Allergy and Clinical Immunology. 14, 464-470 (2014).</li><li>Mantelli, F., Arg&#252;eso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+ocular+surface+mucins+in+health+and+disease.">Functions of ocular surface mucins in health and disease.</a> Current Opinion in Allergy and Clinical Immunology. 8, 477-483 (2008).</li><li>Garc&#237;a-Posadas, L., 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=Characterization+and+functional+performance+of+a+commercial+human+conjunctival+epithelial+cell+line.">Characterization and functional performance of a commercial human conjunctival epithelial cell line.</a> Experimental Eye Research. 223, 109220 (2022).</li><li>Shatos, M. A., 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=Isolation,+characterization,+and+propagation+of+rat+conjunctival+goblet+cells+in+vitro.">Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro.</a> Investigative Ophthalmology &amp; Visual Science. 42, 1455-1464 (2001).</li><li>Welshons, W. V., Wolf, M. F., Murphy, C. S., Jordan, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogenic+activity+of+phenol+red.">Estrogenic activity of phenol red.</a> Molecular and Cellular Endocrinology. 57, 169-178 (1988).</li><li>Berthois, Y., Katzenellenbogen, J. A., Katzenellenbogen, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenol+red+in+tissue+culture+media+is+a+weak+estrogen:+implications+concerning+the+study+of+estrogen-responsive+cells+in+culture.">Phenol red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 83, 2496-2500 (1986).</li><li>Garc&#237;a-Posadas, L., 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=Interaction+of+IFN-&#947;+with+cholinergic+agonists+to+modulate+rat+and+human+goblet+cell+function.">Interaction of IFN-&#947; with cholinergic agonists to modulate rat and human goblet cell function.</a> Mucosal Immunology. 9, 206-217 (2016).</li><li>Li, D., Jiao, J., Shatos, M. A., Hodges, R. R., Dartt, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+VIP+on+intracellular+[Ca2+],+extracellular+regulated+kinase+1/2,+and+secretion+in+cultured+rat+conjunctival+goblet+cells.">Effect of VIP on intracellular [Ca2 ], extracellular regulated kinase 1/2, and secretion in cultured rat conjunctival goblet cells.</a> Investigative Opthalmology &amp; Visual Science. 54, 2872-2884 (2013).</li><li>Contr&#242;, V., 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=Sex+steroid+hormone+receptors,+their+ligands,+and+nuclear+and+non-nuclear+pathways.">Sex steroid hormone receptors, their ligands, and nuclear and non-nuclear pathways.</a> AIMS Molecular Science. 2, 294-310 (2015).</li><li>Valley, C. C., Solodin, N. M., Powers, G. L., Ellison, S. J., Alarid, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variation+in+estrogen+receptor-alpha+protein+turnover+in+the+presence+of+estrogen.">Temporal variation in estrogen receptor-alpha protein turnover in the presence of estrogen.</a> Journal of Molecular Endocrinology. 40, 23-34 (2008).</li><li>Campen, C. A., Gorski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anomalous+behavior+of+protein+synthesis+inhibitors+on+the+turnover+of+the+estrogen+receptor+as+measured+by+density+labeling.">Anomalous behavior of protein synthesis inhibitors on the turnover of the estrogen receptor as measured by density labeling.</a> Endocrinology. 119, 1454-1461 (1986).</li><li>Yang, 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=Sex-based+differences+in+conjunctival+goblet+cell+responses+to+pro-inflammatory+and+pro-resolving+mediators.">Sex-based differences in conjunctival goblet cell responses to pro-inflammatory and pro-resolving mediators.</a> Scientific Reports. 12, 16305 (2022).</li></ol>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#38;D (Minneapolis, MN)</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Fura-2- acetoxymethyl ester&#160;</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&#160;</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&#160;</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 ...

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 ...

By studying the goblet cell function in primary culture, we gain valuable insights into the mechanisms behind the sex-based differences in ocular surface health. We have modified the already-established conjunctival goblet cell primary culture method by eliminating hormones and estrogenic substances from the system, enabling more precise control of sex hormone levels. This modification enhances the accuracy and reliability of our findings.The intracellular calcium measurement is a method to study the goblet cell function under various stimuli and the intracellular signaling pathways triggered by each stimulus. The technique can be extended to drug development for ocular surface diseases like ocular allergy, dry eye disease, or conjunctivitis. This technique facilitates the screening of various molecule's effect on goblet cells under specific hormone levels.People new to this technique often struggle with separating conjunctival tissue from connective tissue. Our advice is to pay close attention to the texture and material properties of the tissue. Conjunctiva should be elastic and semi-transparent while the connective tissue is usually lighter in color and cotton fiber-like.To begin, using a sterile scalpel, mince the human conjunctival tissue into one millimeter cubed pieces. In a six-well plate, seed four pieces in each well containing one millimeter of complete RPMI medium. Make sure to anchor the pieces to the plate.Incubate the tissue pieces at 37 degrees Celsius, 5%carbon dioxide, and 95%air. Refresh the culture medium every second day. After 72 hours from seeding, remove the tissue plug and continue incubation until goblet cells reach 70 to 80%confluency.Next, rinse the cells with PBS and add 0.05%Trypsin EDTA to detach the cells from the bottom of the plate. Observe the cell detachment under a microscope. And once the cells detach, add a complete RPMI medium to deactivate the trypsin.Centrifuge the cell suspension at 150 G for five minutes and resuspend the pellet in phenol red-free RPMI medium. Then reseed the cell suspension onto a glass bottom culture dish for intracellular calcium concentration measurement. For Fura-2 AM loading, add Krebs-Ringer bicarbonate buffer containing 0.5%HEPES, 0.5%BSA, and 0.5 micromolar Fura-2 AM, eight micromolar pluronic acid F127, and 250 micromolar sulfinpyrazone into the glass bottom dish containing goblet cells.Incubate the cells for one hour at 37 degrees Celsius, protecting them from light. After incubation, wash the cells with Krebs-Ringer bicarbonate buffer containing 250 micromolar sulfinpyrazone. For intracellular calcium concentration measurement, place the dish containing cells loaded with Fura-2 under the microscope.Then add 200 times magnification. Locate a representative field containing 20 to 50 cells. Using the freehand function, draw an outline around each cell to distinguish it from the background fluorescence and wait for the software to automatically subtract background fluorescence from the measurement.Click the Start Experiment button and wait for eight to 15 seconds to determine the baseline calcium levels in the cells. Then carefully add the cholinergic agonist carbachol and continue measuring for at least 120 seconds or until calcium levels have returned to baseline. Using the change in peak intracellular calcium concentration, calculate the mean basal calcium level in each cell from the first eight to 15 seconds of measurements.Remove cells having a mean basal calcium level equal to or above 500 nanomolar from the dataset. Next, calculate the maximum intracellular calcium concentration for each cell. Then subtract the basal calcium level value from the maximum measured intracellular concentration for the same cell.Finally, average the change in peak intracellular calcium concentration for all the cells in a dish. The purity of human conjunctival goblet cells was confirmed via immunofluorescent staining, utilizing antibodies specific to the goblet cell markers, cytokeratin and Helix pomatia lectin 1. The results of the Fura-2 AM assay showed no significant differences between cells incubated in phenol red-free RPMI with 1%BSA and complete RPMI medium.However, a significant increase intracellular calcium response to carbachol was observed when comparing the advanced RPMI medium group to the complete RPMI medium group. The most important thing is to obtain primary human goblet cell culture of sufficient cell density. During growth, ensure that the tissue pieces stick to the culture plate for effective outgrowth.The researchers could explore the involvement of specific pathways by applying various inhibitors, targeting receptors or signaling molecules to the cells before the stimulation. The other parameters indicative of goblet cell function, like mucin secretion and deactivation of mitogen-activated protein kinases can also be assessed. The complimentary measurements provide a comprehensive understanding of the goblet cells'response to different stimuli.

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

Research

JoVE Journal

Biology

376 Görüntüleme.  Harvard Medical School. Birincil kültürde goblet hücresi fonksiyonunu inceleyerek, oküler yüzey sağlığındaki cinsiyete dayalı farklılıkların arkasındaki mekanizmalar hakkında değerli bilgiler ediniyoruz. Hormonları ve östrojenik maddeleri sistemden çıkararak, seks hormonu seviyelerinin daha hassas kontrolünü sağlayarak, halihazırda kurulmuş olan konjonktival goblet hücre birincil kültür yöntemini değiştirdik. Bu değişiklik, bulgularımızın doğruluğunu ve güvenilirliğini artır?...