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

Summary

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.

Abstract

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.

Introduction

Sex-based differences affect multiple processes of the ocular surface^1,2,3. 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 conjunctivitis^4,5,6. 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 film^9,10, cornea¹¹, the lacrimal gland^12,13, and meibomian glands that also secrete tears¹². Numerous mechanistic studies have investigated the effect of sex hormones on the cornea and its associated components^14,15; 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.

Protocol

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

1. From the eye bank, obtain human conjunctival tissue¹⁶.
2. 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 µg/mL penicillin-streptomycin, and 2 mM 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES).
3. Carry out primary cell culture as described below.
   1. 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.
   2. Place the tissue pieces into the incubator containing 95% air and 5% CO₂ at 37 °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.
      ​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.
   3. 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.

2. Human conjunctival goblet cell passage and experimental preparation

1. 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.
2. Centrifuge the cells at 150 × 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 µL per dish. Keep the media in the center of the glass part of the dish.
3. 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.

3. Fura-2/acetoxymethyl (AM) assay for [Ca²⁺]_i measurement

1. Perform Fura-2/AM loading as described below.
   1. Incubate the cells in glass bottom dishes for 1 h at 37 °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 MgSO_4, and 25 mM NaHCO₃) with 0.5% HEPES (Table 1), plus 0.5% BSA, 0.5 µM fura-2/AM, 8 µM pluronic acid F127, and 250 µM sulfinpyrazone.
   2. 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 µM sulfinpyrazone immediately before [Ca²⁺]_i measurement.
2. Perform [Ca²⁺]_i measurement as described below.
   1. 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.
   2. Wait for the software to automatically subtract background fluorescence from the measurement and click the Start Experiment button.
   3. 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.
3. Perform data analysis as described below.
   1. 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.
   2. 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.
   3. 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.

Results

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 × 10^-4 M) was used as ...

Discussion

Investigating the sex-based differences in ocular tissues helps understand the processes of diseases, especially dry eye and allergic conjunctivitis, which disproportionately affect one sex^4,5,6. 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...

Materials

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