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.
