We successfully developed a human vagina chip that supports spontaneous differentiation of squamous stratified vaginal epithelium. It forms a strong barrier response to hormones and generating microbiome supporting oxygen gradients. 2D and organoid cultures are currently used to study vaginal dysbiosis, but they aren't able to mimic the physiological complexities of the vaginal microenvironment due to their static nature.
Animal models too can't model the same hormonal changes present in the human menstrual cycle, and the vaginal microbiome across animal species and humans differ. We set out to explore whether organ chip technology can be used to develop a preclinical model of human vagina microbiome interactions, which could potentially be used for discovery and assessment of potential microbiome-based therapeutics. The vagina chip allows for dynamic fluid flow across the vaginal epithelium and stroma, which prolongs its co-culturing with vaginal microbial consortia, and maintains a physiologically relevant microenvironment.
The human vagina on a chip is an exciting tool that can be used to study varying diseases of the female reproductive tract and validate new biotherapeutics. To begin, take a 70 to 90%confluent culture of human uterine fibroblasts. Aspirate the medium from the flasks.
Wash the cells with five milliliters of warm calcium and magnesium free-DPBS. After aspirating the DPBS, pipette four milliliters of warm trypsin EDTA into each flask. Incubate the cells at 37 degrees Celsius for three to five minutes until the cells detach.
Next, add six milliliters of drips and neutralizing solution into each flask. Then transfer the cell suspension to a 15-milliliter conical tube. Use a pipette to mix the suspension well, and take a 10 microliter aliquot for cell counting.
Mix 10 microliters of the cell suspension with 10 microliters trypan blue. Transfer the stained cell suspension to a hemocytometer for cell counting. Next, centrifuge the cell suspension at 300 G for five minutes at room temperature.
After aspirating the supernatant, re-suspend the pellet in warm fibroblast medium. Now wash the basal channel of a microfluidic organ chip with 200 microliters of fibroblast medium. Then wash the apical channel with 200 microliters of warmed vaginal epithelial medium.
Add 200 microliters of complete vaginal epithelial medium to the apical channel inlet while plugging the outlet with the pipette tip. Dispense the medium until inlet and outlet tip volumes are equal. Then release the pipette tip from the pipette, leaving the tip in the inlet.
Slowly pipette 50 microliters of the human uterine fibroblast cell suspension into the basal channel inlet while simultaneously aspirating from the outlet. Remove the pipette tip from the inlet when approximately two microliters of the cell suspension remain in the pipette tip. Flip the plug chips upside down on a 15-milliliter tube rack.
Check the chips after incubation for cell attachment. Next, plug the outlet of the basal channel with a pipette tip. Then add 200 microliters of fibroblast medium to the basal channel inlet without pushing down on the pipette plunger.
Release the tip from the pipette to allow the medium to flow freely through the channel to the outlet pipette tip by gravitational flow. Incubate the fibroblast seeded chips overnight at 37 degrees Celsius with 5%carbon dioxide supplementation. To begin, take 50 milliliters each of fibroblast medium and vaginal epithelial medium into separate 50-milliliter conical tubes.
Degas the warmed media under a sterile vacuum for five minutes. Next, disinfecting clean trays with CaviCide and ethanol for the dynamic flow module. Remove the pods from the packaging and place them in the dynamic flow module trays.
Pipette two milliliters of degassed vaginal epithelial medium into the apical inlet reservoir. And add three milliliters of degassed fibroblast medium to the basal inlet reservoir. Pipette 500 microliters of degassed vaginal epithelial medium to the apical outlet reservoir, and 500 microliters of degassed fibroblast medium to the basal outlet reservoir.
Slide the trays containing pods into dynamic flow module. Now run the prime cycle two times. If a droplet does not form after four prime cycles, make direct contact with the port inside the outlet reservoir of the pod.
Pipette 200 microliters of the respective medium to allow the medium to flow between the reservoir in the channel to hand prime the pods. Next, remove pipette tips from human uterine fibroblast seeded organ chips. Place a droplet of respective medium over all the ports of each chip.
Slide the chips into the pods, and place the pods onto the trays. Aspirate any media on the surface of the chips. Then slide each tray into a dynamic flow module.
Next on the module, set the top and bottom as liquid, the apical flow to 15 microliters per hour, the basal flow to 30 microliters per hour, the stretch to 0%and the frequency to 0 hertz. Run the regulate cycle on the module, and allow flow overnight. A continuous sheet of vaginal epithelial cells that progressively formed multiple cell layers after differentiation was observed on the organ-chip device.
To begin, mix the calculated amount of each bacterial strain to total up to approximately five million colony-forming units per milliliter. Centrifuge the mix at 7, 000 G for seven minutes at four degrees Celsius. Next, carefully pipette out the supernatant.
Re-suspend the pellet in Hanks'Balanced Salt Solution with low buffering capacity and glucose. Now to touch the organ-chips containing the differentiated human vaginal epithelial cells from pods. Plug the outlet of the chip's basal channel with a pipette tip.
Add 200 microliters of antibiotic-free differentiation medium to the basal channel inlet without pushing down on the pipette plunger. Release the pipette tip from the pipette, allowing the medium to flow freely through the channel. Next, pipette 37 microliters of the bacterial inoculum to the chip's apical channel inlet while aspirating from the outlet.
Pull the tip, then plug both the apical channel inlet and outlet with pipette tips. For the control chips, pipette 37 microliters of Hank's Balanced Salt Solution with low buffering capacity and glucose into the apical channel inlet. After aspirating any media on the surface of the chip, place the chips in a 150 millimeter Petri dish.
Colonies of L.crispatus and G.vaginalis were detected within 48 hours of plating, confirming that both healthy and dysbiotic bacteria engrafted in the vagina chips. The pH of the vagina chips inoculated with L.crispatus was similar to that of uninoculated control chips, and when co-cultured with G.vaginalis, they experienced significantly increased pH.
