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

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 ....

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