Modeling Healthy and Dysbiotic Vaginal Microenvironments in a Human Vagina-on-a-Chip

Summary

This article describes a protocol for creating a microfluidic vagina-on-a-chip (Vagina Chip) culture device that enables the study of human host interactions with a living vaginal microbiome under microaerophilic conditions. This chip can be used as a tool to investigate vaginal diseases as well as to develop and test potential therapeutic countermeasures.

Abstract

Women's health, and particularly diseases of the female reproductive tract (FRT), have not received the attention they deserve, even though an unhealthy reproductive system may lead to life-threatening diseases, infertility, or adverse outcomes during pregnancy. One barrier in the field is that there has been a dearth of preclinical, experimental models that faithfully mimic the physiology and pathophysiology of the FRT. Current in vitro and animal models do not fully recapitulate the hormonal changes, microaerobic conditions, and interactions with the vaginal microbiome. The advent of Organ-on-a-Chip (Organ Chip) microfluidic culture technology that can mimic tissue-tissue interfaces, vascular perfusion, interstitial fluid flows, and the physical microenvironment of a major subunit of human organs can potentially serve as a solution to this problem. Recently, a human Vagina Chip that supports co-culture of human vaginal microbial consortia with primary human vaginal epithelium that is also interfaced with vaginal stroma and experiences dynamic fluid flow has been developed. This chip replicates the physiological responses of the human vagina to healthy and dysbiotic microbiomes. A detailed protocol for creating human Vagina Chips has been described in this article.

Introduction

A vaginal microbiome dominated by Lactobacillus spp. that helps to maintain an acidic microenvironment plays an important role in maintaining female reproductive health¹. However, at times there can be a change in the composition of microbial communities that comprise the microbiome, which results in an increase in the diversity of vaginal bacteria. These dysbiotic changes, which often result in a switch from a Lactobacillus-dominated state to one dominated by more diverse anaerobic bacterial species (e.g., Gardnerella vaginalis), are associated with various diseases of the reproductive system, such as bacterial vaginosis, atrophic vaginitis, urinary tract infection, vulvovaginal candidiasis, urethritis, and chorioamnionitis^2,3,4,5. These diseases, in turn, increase a woman's chances of acquiring sexually transmitted diseases and pelvic inflammatory disease^6,7,8,9. They also pose a higher risk for pre-term birth and miscarriages in pregnant women^10,11,12 and have also been implicated in infertility^13,14,15,16.

Although efforts have been made to model vaginal dysbiosis using vaginal epithelial cells cultured in static, two-dimensional (2D) culture systems^17,18, they do not effectively mimic the physiology and complexity of the vaginal microenvironment¹⁹. Animal models also have been used to study vaginal dysbiosis; however, their menstrual phases and host-microbiome interactions differ greatly from that in humans, and thus, the physiological relevance of results from these studies remains unclear^19,20,21. To counteract these issues, organoids and Transwell insert models of human vaginal tissue also have been used to study host-pathogen interactions in the FRT^19,22,23,24. But because these are static cultures, they can only support co-culture of human cells with living microbes for a short period of time (<16-24 h), and they lack many other potentially important physical features of the human vaginal microenvironment, such as mucus production and fluid flow²².

Organ Chips are three-dimensional (3D) microfluidic culture systems that contain one or more parallel hollow microchannels lined by living cells cultured under dynamic fluid flow. The two-channel chips enable the recreation of organ-level tissue-tissue interfaces by culturing different cell types (e.g., epithelium and stromal fibroblasts or epithelium and vascular endothelium) on opposite sides of a porous membrane that separates the two parallel channels (Figure 1). Both tissues can be independently exposed to fluid flow, and they can also experience microaerobic conditions to enable co-culture with a complex microbiome^25,26,27,28. This approach was recently leveraged to develop a human Vagina Chip lined by hormone-sensitive, primary vaginal epithelium interfaced with underlying stromal fibroblasts, which sustains a low physiological oxygen concentration in the epithelial lumen and enables co-culture with healthy and dysbiotic microbiomes for at least 3 days in vitro²⁹. It was demonstrated that the Vagina Chip could be used to study colonization by optimal (healthy) L. crispatus consortia and detect inflammation and injury caused by non-optimal (non-healthy) G. vaginalis containing consortia. Here, we describe in detail the methods that are used to create the human Vagina Chip as well as to establish healthy and dysbiotic bacterial communities on-chip.

Protocol

This research was performed in compliance with institutional guidelines for the use of human cells. The cells were obtained commercially (see Table of Materials). All steps should be performed aseptically in a biosafety cabinet (BSC). Use only filter (or barrier) pipette tips for this protocol.

1. Culturing human vaginal epithelial cells

1. Warm 50 mL of vaginal epithelial medium (VEM, see Table of Materials) to 37 °C.
2. Aliquot 9 mL of VEM to a 15 mL tube. Then, thaw a vial of human vaginal epithelial cells (HVECs), and add it to the tube containing VEM.
3. Centrifuge the 15 mL tube at 300 x g for 5 min at room temperature (RT) and aspirate the supernatant, leaving the pellet behind.
4. Gently resuspend the pellet in 2 mL of VEM and add 1 mL to each of the two T75 flasks containing 14 mL of VEM.
5. Incubate the flasks at 37 °C with 5% CO_2. Change VEM every 2 days until HVECs are approximately 70% confluent (about 5 days).

2. Culturing human uterine fibroblast cells

1. Prepare 10 mL of 15 μg/mL Poly-L-lysine solution (PLL) in double distilled water (ddH₂O). Add 5 mL of PLL solution to each of the two T75 flasks and incubate at 37 °C for 1 h.
2. Warm 50 mL of fibroblast medium (FM, see Table of Materials) to 37 °C.
3. Aspirate the PLL solution and wash each flask with 5 mL of ddH₂O.
4. Aliquot 9 mL of FM to a 15 mL tube and thaw a vial of human uterine fibroblasts (HUFs).
5. Add the HUFs to the 15 mL tube containing FM.
6. Centrifuge the 15 mL tube at 300 x g for 5 min at RT and aspirate the supernatant, leaving the cell pellet behind.
7. Gently resuspend the pellet in 2 mL of FM and add 1 mL to each of the two T75 flasks containing 14 mL of FM.
8. Incubate flasks at 37 °C with 5% CO₂ until cells are approximately 70% confluent while changing the medium every 2 days.

3. Chip activation and channel coating

1. Degas the chips (obtained commercially, see Table of Materials) for 30 min in a vacuum desiccator.
2. Allow the Activation Reagent 1 (AR-1) and Activation Reagent 2 (AR-2) (see Table of Materials) to equilibrate to RT for 15 min without removing their packaging.
3. Wrap a 15 mL conical tube in foil to protect it from light. Slowly add 1 mL of AR-2 solution to the walls of the AR-1 vial and mix well. Transfer the mixture to the foil-wrapped 15 mL tube.
4. Repeatedly add AR-2 solution to the AR-1 vial in 1 mL increments until the AR-1 powder is fully washed from the vial.
5. Top up the AR-1 reconstituted solution to 10 mL with AR-2 solution.
6. Add 200 µL of this solution to the apical channel inlet of each chip while aspirating from the outlet (Figure 1A). Repeat for the basal channel. Keep the pipette perpendicular to the chip while adding the solution to maintain a tight seal with unobstructed flow.
7. Repeat step 3.6 for all chips.
8. Check all chips for bubbles. Remove any bubbles by adding more solution to the affected channel(s).
9. Aspirate all excess AR-1 solution from the chip surface while avoiding channel inlets and outlets.
10. Place chips in a 150 mm Petri dish and insert this uncovered dish into a UV light box.
11. Face the UV light box to the back of the BSC and leave the chips under constant UV light for 30 min. The color of the solution in the chips will change from dark pink to mahogany.
12. Wash each channel by adding 200 µL of AR-2 solution to the inlet while simultaneously aspirating from the outlet.
13. Wash each channel twice by adding 200 µL of cold DPBS (-/-) to the inlet while simultaneously aspirating from the outlet.
14. Prepare the apical channel coating (200 µg/mL Collagen I and 30 µg/mL Collagen IV mixture in DMEM) (see Table of Materials). Keep on ice.
15. Prepare the basal channel coating (15 µg/mL PLL and 200 µg/mL collagen I mixture in DMEM). Keep on ice.
16. Add 200 µL of basal channel coating to the basal channel inlet. Plug the outlet with a P200 tip when the coating solution appears at the outlet. Dispense the solution until inlet and outlet tip volumes are equal, and then release the pipette tip from the pipette, leaving the tip in the inlet.
17. Similarly, add 200 µL of apical channel coating to the apical channel.
18. Aspirate excess solution from the chip surface.
19. Check all chips for bubbles. Remove any bubbles by adding more channel coating to the affected channel(s).
20. Incubate the chips overnight in a 150 mm Petri dish at 37 °C with 5% CO₂.

4. Seeding chip basal channel with HUFs

1. View the growth of HUFs in the flask under a microscope daily.
2. Once HUF cultures are 70%-90% confluent (~3 days after plating), warm 25 mL of FM, 5 mL of Ca²⁺/Mg²⁺ free DPBS (DPBS (-/-), 10 mL of trypsin/EDTA, and 15 mL of trypsin neutralizing solution (TNS, see Table of Materials) to 37 °C.
3. Aspirate the medium from the flasks. Wash with 5 mL of DPBS (-/-), then aspirate again.
4. Add 4 mL of trypsin-EDTA to each flask and incubate at 37 °C for 3-5 min until cells detach.
5. Add 6 mL of TNS to each flask and transfer the cell suspension to a 15 mL conical tube.
6. Mix suspension well with a pipette and take a 10 µL aliquot for cell counting. Mix 10 µL of cell suspension with 10 µL trypan blue and count using a hemocytometer.
7. Centrifuge cell suspension at 300 x g for 5 min at RT. Aspirate the supernatant and resuspend the pellet in warm FM to a final concentration of 7.5 x 10⁵ cells/mL.
8. Wash the basal channel with 200 µL of FM.
9. Warm 15 mL of VEM to 37 °C. Wash the apical channel with 200 µL of VEM.
10. Add 200 µL of complete VEM to the apical channel inlet while plugging the outlet with a 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. Keep the top channel filled and plugged at both the inlet and outlet.
11. Slowly pipette 50 µL of HUF cell suspension into the basal channel inlet while simultaneously aspirating from the outlet. Remove the pipette tip from the inlet when ~2 µL of the cell suspension remains in the pipette tip without pressing on or releasing the pipette plunger to avoid bubble formation. Plug the inlet and outlet with pipette tips.
12. Check for bubbles under a microscope. If they are present, wash the basal channel with FM and repeat step 4.11.
13. Flip the plugged chips upside down on a 15 mL tube rack and incubate at 37 °C with 5% CO₂ for 1 h. Observe the chips after incubation and check for cell attachment.
14. Plug the outlet of the basal channel with a pipette tip. Add 200 µL of FM to the basal channel inlet without pushing down on the pipette plunger. Release the tip from the pipette and allow the medium to flow freely through the channel to the outlet pipette tip by gravitational flow.
15. Incubate HUF-seeded chips overnight at 37 °C with 5% CO₂.

5. Seeding chip apical channel with vaginal epithelial cells

1. Warm 50 mL of VEM to 37 °C.
2. Prepare apical channel coating (200 µg/mL Collagen I in DMEM). Keep on ice.
3. Plug the apical channel outlet with a pipette tip.
4. Add 200 µL of apical channel coating to the apical channel inlet. Dispense the apical coating solution until inlet and outlet tip volumes are equal, then release the pipette tip from the pipette, leaving the tip in the inlet.
5. Aspirate excess solution from the surface of the chip.
6. Incubate chips at 37 °C with 5% CO₂ for 1 h.
7. After 1 h, wash the apical channel coating by adding 200 µL of VEM to the apical channel inlet while aspirating from the outlet.
8. Check HVEC growth under a microscope for ~70%-90% confluency.
9. If cells are 70%-90% confluent, warm 6 mL of complete vaginal epithelial cell medium and 4 mL of trypsin/EDTA per flask, to 37 °C.
10. Aspirate medium from the HVEC flask and wash with 5 mL of DPBS (-/-), then aspirate.
11. Add 4 mL of trypsin to each flask and incubate at 37 °C with 5% CO₂ for 3-5 min until cells detach.
12. Add 6 mL of VEM to the flask to inactivate trypsin and transfer the cell suspension to a 15 mL conical tube.
13. Mix suspension well with a pipette and take a 10 µL aliquot for cell counting. Mix 10 µL of cell suspension with 10 µL trypan blue and count using a hemocytometer.
14. Centrifuge cell suspension at 300 x g for 5 min at RT. Aspirate the supernatant and resuspend the pellet in VEM to a final concentration of 3.5-4 million cells/mL.
15. Warm 25 mL of FM to 37 °C.
16. Plug the apical channel outlet with a pipette tip. Slowly pipette at least 40 µL of HVEC cell suspension into the apical channel inlet. Dispense the cell suspension until inlet and outlet tip volumes are equal, then release the pipette tip from the pipette, leaving the tip in the inlet. Keep the basal channel filled with FM and plugged at both the inlet and outlet.
17. Carefully aspirate excess medium on the surface of chips and check for bubbles under a microscope. If they are present, repeat step 5.16.
18. Place chips in a large Petri dish and incubate at 37 °C with 5% CO₂ overnight.
19. On the next day, observe chips under a microscope for cell attachment.
20. Remove the pipette tips from the inlets and outlets of both the apical and basal channels.
21. Plug the basal channel outlet with a pipette tip and add 200 µL of FM to the basal channel inlet without pushing down on the pipette plunger. Release the pipette tip from the pipette and allow the medium to flow freely through the channel to the outlet pipette tip by gravitational flow.
22. Repeat step 5.21 for the apical channel using VEM.
23. Incubate the dually seeded chips at 37 °C with 5% CO₂ for 24 h.

6. Connecting chips to pods and differentiating vaginal epithelial cells

1. Aliquot 50 mL of FM and VEM to separate 50 mL conical tubes and warm to 37 °C.
2. Degas the FM and VEM media warmed to 37 °C under a sterile vacuum for 5 min.
3. Disinfect and clean trays for the Dynamic Flow Module (DFM, see Table of Materials). Remove pods from packaging and place them in the trays.
4. Add 2 mL of degassed VEM to the apical inlet reservoir (top right reservoir; Figure 1B). Add medium along the reservoir walls to avoid bubble formation.
5. Add 3 mL of degassed FM to the basal inlet reservoir (top left reservoir, Figure 1B). Add medium along the reservoir walls to avoid bubble formation.
6. Add 500 µL of degassed VEM to the apical outlet reservoir (bottom right reservoir, Figure 1B). Tilt the pod so that the medium covers the entire bottom surface of the reservoir.
7. Add 500 µL of degassed FM to the basal outlet reservoir (bottom left reservoir, Figure 1B). Tilt the pod so that the medium covers the entire bottom surface of the reservoir.
8. Slide trays containing pods into DFM and run the Prime cycle twice. Check for droplets coming out of the ports on the underside of each pod.
9. If a droplet does not form after 4 "Prime" cycles, make direct contact with the port inside the outlet reservoir of the pod (Figure 1B) and pipette 200 µL of the respective medium to allow the medium to flow between the reservoir and the channel. This is called "Hand-priming".
10. Remove pipette tips from chips and place a droplet of respective medium over all the ports for each chip.
11. Slide chips into pods and place pods onto trays.
12. Aspirate any media on the surface of the chips and slide each tray into a DFM.
13. Set the following parameters on the DFM as: Top and Bottom - Liquid; Apical (Top Channel) Flow - 15 µL/h; Basal (Bottom Channel) Flow - 30 µL/h; Stretch = 0%; Frequency = 0 Hz.
14. Run the Regulate cycle on the DFM and allow flow overnight.
15. After 24 h, change the flow settings to 0 µL/h for the apical channel and keep the basal channel at 30 µL/h flow rate for another 24 h.
16. Prepare 500 mL of differentiation medium (DM) by adding 4 mM L-glutamine, 20 mM Hydrocortisone, 1x ITES, 20 nM Triiodothyronine, 100 µM O-Phosphoryl Ethanolamine, 180 µM Adenine, 3.2 mM Calcium chloride, 2% Heat-inactivated FBS, 1% Pen-strep, and 120 mL of Ham's F-12 media to Low glucose DMEM (see Table of Materials), and filter-sterilize.
17. Warm 50 mL of DM to 37 °C.
18. Add 20 µL of 10 µM Estradiol (see Table of Materials) to the 50 mL of DM, mix well, and degas under a sterile vacuum for 5 min.
19. Warm 50 mL of VEM to 37 °C in a water bath and degas under a sterile vacuum for 5 min.
20. Remove the trays from the DFM, place them in a BSC, and aspirate media from the pods, avoiding the ports in the reservoirs (Figure 1A). Then, add 2 mL of VEM to the apical channel inlet reservoir and 3 mL of DM to the basal channel inlet reservoir.
21. Return the trays to the DFM and set the apical channel flow to 15 µL/h and the basal channel flow to 30 µL/h.
22. Allow the DFM to flow for 4-7 h. Then, stop the apical channel flow by setting it to 0 µL/h. Let the basal channel flow continue at 30 µL/h.
23. Change the media following steps 6.16-6.19 every 48 h.
24. Flow media intermittently in the apical channel for 4-7 h each day for 5 additional days following steps 6.20-6.21.
25. Prepare Hanks' Balanced Salt Solution with low buffering capacity with Glucose (HBSS (LB/+G)) media by adding 1.26 mM Calcium chloride, 0.49 mM Magnesium chloride hexahydrate, 0.406 mM Magnesium sulfate, 5.33 mM Potassium chloride, 137.93 mM Sodium chloride, 0.441 mM Potassium phosphate monobasic, and 5.55 mM D- Glucose to dd H₂O (see Table of Materials); pH 4.8.
26. Prepare 500 mL of Pen-Strep-free DM by adding 4 mM L-glutamine, 20 mM Hydrocortisone, 1x ITES, 20 nM Triiodothyronine, 100 µM O-Phosphoryl Ethanolamine, 180 µM Adenine, 3.2 mM Calcium chloride, 2% Heat-inactivated FBS, and 120 mL of Ham's F-12 media to Low glucose DMEM, and filter-sterilize.
27. On day 6, replace the apical channel medium with (HBSS (LB/+G)) and the basal channel medium with Pen-Strep-free DM following steps 6.16-6.19.
28. Set the flow on the DFM to 15 µL/h for the apical channel and 30 µL/h for the basal channel for 24 h before proceeding with bacterial inoculation.

7. Bacterial inoculation of differentiated chips

NOTE: Perform the following steps in a Lab and BSC that comply with regulations to handle microbes.

1. Calculate the CFU/mL of each bacterial strain to be included in the inoculum. Mix the required amount of each bacterial strain to total up to ~5 x 10⁶ CFU/mL.
2. Centrifuge the mix at 7,000 x g for 7 min at 4 °C and carefully remove the supernatant. Resuspend the pellet in (HBSS (LB/+G)). This will be the bacterial inoculum.
3. Detach chips from pods. Plug the outlet of the basal channel with a pipette tip and add 200 µL of Pen-Strep-free DM to the basal channel inlet without pushing down on the pipette plunger. Release the pipette tip from the pipette and allow the medium to flow freely through the channel to the outlet by gravitational flow.
4. Add 37 µL of the bacterial inoculum to the apical channel inlet, while aspirating from the outlet. When about 2 µL is left in the pipette tip, pull the tip out and plug the apical channel inlet and outlet with pipette tips.
5. For the control (uninoculated) chips, repeat step 7.4 with 37 µL of (HBSS (LB/+G)).
6. Aspirate any media on the surface of the chip. Place the chips in a 150 mm Petri dish and incubate at 37 °C with 5% CO₂ for 24 h.
7. Place the pods (without chips) on the trays and place them in the incubator at 37 °C with 5% CO₂ for 24 h.
8. Warm 50 mL of (HBSS (LB/+G)) and 50 mL of Pen-Strep-free DM to 37 °C.
9. Add 20 µL of 10 µM Estradiol to the 50 mL of Pen-Strep-free DM, mix well, and degas under a sterile vacuum for 5 min. Degas the (HBSS (LB/+G)) under a vacuum for 5 min.
10. Carefully aspirate the media in the pods while avoiding the reservoir inlet and outlet ports.
11. Add 3 mL of degassed (HBSS (LB/+G)) to the apical inlet pod reservoir and 500 µL of degassed (HBSS (LB/+G)) to the apical outlet pod reservoir.
12. Add 3 mL of degassed Pen-Strep-free DM to the basal inlet pod reservoir and 500 µL of degassed antibiotic-free DM to the basal outlet pod reservoir.
13. Slide trays with the pods (without the chips) into the DFM and run the Prime cycle twice. Check for droplets coming out of each port on the underside of the pod.
    NOTE: If a droplet does not form after 4 "Prime" cycles, make direct contact with the port inside the outlet reservoir of the pod (Figure 1B) and pipette 200 µL of the respective medium to allow the medium to flow between the reservoir and the channel.
14. Remove pipette tips from chips and place a droplet of respective media over all the channel inlets and outlets.
15. Slide chips into pods and place pods into trays.
16. Aspirate media in the apical and basal outlet pod reservoirs and any media on the surface of the chips. Then, slide the trays into the DFM.
17. Set the following parameters on the DFM: Top and Bottom - Liquid; Apical (Top) Flow - 40 µL/h; Basal (Bottom) Flow - 40 µL/h; Stretch - 0%; Frequency - 0 Hz. Run the Regulate cycle.
18. Stop the flow after 4 h and collect the effluent, i.e., the media in the apical and basal outlet reservoirs.
19. Measure and record the effluent volumes.
20. Place the chips back into the DFM and set the apical flow rate to 0 µL/h and the basal flow to 30 µL/h. Start the flow to run overnight.
21. Aliquot the collected effluent for various planned assays and store them at appropriate temperatures.
    NOTE: For CFU measurement, add glycerol to a final concentration of 16% and immediately store at -80 °C.
22. Aspirate the medium in only the basal outlet reservoirs and set apical and basal flow rates to 40 µL/h in the DFM. Start the flow for 4 h and repeat steps 7.18-7.21. This will be the effluent collection for 48 h.
23. Repeat step 7.22 for 72 h or until the experiment ends.
24. At the endpoint of the experiment, collect the effluents following steps 7.18-7.19.
25. Prepare digestion solution by adding 1 mg/mL Collagenase IV in TrypLE express. Warm 10 mL of digestion solution to 37 °C.
26. Plug the outlet port of the basal channel with a pipette tip. Add 100 µL of digestion solution to the basal channel. Mix well, and using a pipette, collect all the solution from the channel in a tube labeled as 'Tube B'.
27. Plug the outlet port of the apical channel with a pipette tip. Add 100 µL of digestion solution to the apical channel. Mix well, and using a pipette, collect all the solution from the channel in a tube labeled as 'Tube A'.
28. Add another 100 µL of the digestion solution to both the apical and basal channel inlets while plugging outlets with pipette tips. Incubate the chips and Tubes A and B at 37 °C for 1-1.5 h.
29. Mix digestion contents well inside the channels using the plugged tips already placed in the inlets and outlets. Collect the contents of the apical and the basal channels to Tubes A and B, respectively. These are the chip apical and basal digests.
30. Count the cells in Tube A using a hemocytometer. Also, remove an aliquot from the chip digests for CFU measurement following step 7.21.

8. Analysis of chip effluents and digests

1. For the enumeration of bacteria from the effluents and digests, serial dilute the effluents or digests in sterile DPBS (-/-) and plate on a suitable media plate, incubate at 37 °C for 24-48 h, and count the colonies on the plate.
2. For the measurement of pH, take 10 μL of the 72 h effluent immediately after collection and use a pH strip to measure the pH of the effluent.
3. For the analysis of the cytokines, use the effluents to detect specific cytokines using Luminex-based assay, ELISA, or any other applicable technique^29,30.

Results

The human vagina is lined by a stratified epithelium that overlies a fibroblast-rich collagenous stroma. To model this, a tissue interface was created by culturing primary human vaginal epithelium and fibroblasts on opposite sides of a common porous membrane within a two-channel microfluidic Organ Chip device. Formation of the vaginal epithelium is monitored using bright field microscopic imaging, which reveals the formation of a continuous sheet of cells that progressively forms multiple cell layers (Figure 2A). Previous reports confirmed that this morphology correlates with the development of a fully stratified epithelium when viewed in cross-section²⁹. However, if the epithelial layer appears patchy and discontinuous (Figure 2B), the Vagina Chip may not be fit for use in experiments.

Figure 3 shows a schematic representation of the generation of the Vagina Chip. To validate the functionality of the Vagina Chip, the chips were inoculated with L. crispatus and G. vaginalis to model healthy and dysbiotic vaginal environments, respectively. G. vaginalis is the bacterium primarily involved in bacterial vaginosis. To check if healthy and dysbiotic bacteria engraft on the Vagina Chips, the bacterial load was quantified in the Vagina Chips inoculated with the different bacterial populations by plating channel effluents and digested cell layers on selective bacterial growth media (De Man-Rogosa-Sharpe (MRS) agar for L. crispatus and Brucella blood agar (BBA) for G. vaginalis)(see Table of Materials) and comparing them to similar cultures using the original inoculum. Colonies of L. crispatus and G. vaginalis were detected within 48 h of plating (Figure 2C), confirming that both healthy and dysbiotic bacteria engrafted in the Vagina Chips. However, if bacterial colonies are observed on the plates containing the inoculums but not observed on plates containing the effluent or digest after the required incubation, then it can be concluded that the bacteria did not engraft.

A healthy vaginal environment is acidic, and dysbiosis results in an increase in vaginal pH³¹. Therefore the pH of the effluent of the apical epithelial channel of the Vagina Chip was also analyzed. The pH of 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 (Figure 2D). If the pH of an uninfected Vagina Chip is observed to be high, it indicates that there is a problem, and these chips should not be used for experiments.

The inflammatory state of vaginal tissue is also sensitive to the composition of the vaginal microbiome, with a dysbiotic microbiome stimulating inflammation. Upon analysis of the pro-inflammatory cytokines in the apical channel of the Vagina Chip 3 days after inoculation with either L. crispatus or G. vaginalis, the pro-inflammatory response was similarly found to be higher with G. vaginalis compared to uninfected chips and chips inoculated with L. crispatus (Figure 2E). Taken together, these results show that the Vagina Chip closely mimics the human vaginal microenvironment in both healthy and dysbiotic states.

Figure 1: A two-channel chip and its pod. (A) Image of a two-channel PDMS chip depicting its channels and ports. (B) Image of a pod depicting the reservoirs and ports for apical and basal channels. (C) Schematic diagram showing the cross-section view of a Vagina Chip infected with microbes. Please click here to view a larger version of this figure.

Figure 2: Vagina Chip mimics healthy and dysbiotic human vaginal microenvironments. (A) Vaginal epithelial cells in the apical channel of a robust Vagina Chip. Scale bar represents 1 mm of the chip in the top image and 500 µm in the bottom image. (B) Vaginal epithelial cells in the apical channel of an inadequate Vagina Chip. Scale bar represents 1 mm of the chip in the top image and 500 µm in the bottom image. (C) Engraftment of L. crispatus (LC) and G. vaginalis (GV) in the Vagina Chip. (D) pH of Vagina Chips after 72 h incubation with L. crispatus (LC) and G. vaginalis (GV) as compared to uninfected (control) Vagina Chips. (E) Pro-inflammatory response of Vagina Chips to L. crispatus (LC) and G. vaginalis (GV) after 72 h incubation, as compared to uninfected (control) Vagina Chips. (C-E) Graphs depict mean ± SD for 4-6 chips; *p < 0.05, **p < 0.01, ***p < 0.001 as compared to control Vagina Chips; Each (●) represents data from 1 chip. Please click here to view a larger version of this figure.

Figure 3: Schematic representation of the protocol for the generation of the Vagina Chip. Schematic representation of the steps involved in seeding cells and generation of the Vagina Chip. HVECs - Human vaginal epithelial cells; HUFs - Human uterine fibroblasts; VEM - Vaginal epithelial medium; FM - Fibroblast media; DM - Differentiation medium; PS - Penicillin Streptomycin. Please click here to view a larger version of this figure.

Discussion

Past in vitro models of the human vagina do not faithfully replicate vaginal tissue structures, fluid flow, and host-pathogen interactions^19,22. Animal models are also limited by inter-species variation in microbiome and differences in the estrous or menstrual cycle^19,22. This manuscript describes a protocol to create an Organ Chip model of the human vagina that can effectively mimic human responses to healthy and dysbiotic microbial communities.

This protocol involves seeding vaginal epithelial and fibroblast cells on opposite sides of a shared porous membrane that separates parallel microchannels in a two-channel Organ Chip device that is commercially available (see Table of Materials). The porous membrane allows for the migration of growth factors and other forms of intercellular communication. However, the collagen coating and presence of the cell monolayers prevent the mixing of media between channels. Upon the formation of a vaginal epithelial cell monolayer in the apical channel, differentiation factors are introduced into the medium flowing in the basal channel, which passes through the interstitial space and thereby promotes differentiation of the vaginal epithelial cells to form a stratified epithelium. The density of the vaginal epithelial cells at the time of seeding is a crucial determinant of the health of the Vagina Chip at the end of the differentiation phase. Thus, the density of vaginal epithelial cells should be assessed before initiating differentiation, which should not be initiated until a monolayer is established. Exposure to the differentiation factors can be continued until the desired density of the vaginal epithelial cells is obtained and before bacterial inoculation. Further, it should be noted that the growth rate may vary for primary vaginal epithelial cells from different donors (or commercial sources), which could affect the quality of the Vagina Chip generated. In all microfluidic Organ Chip studies, it is of utmost importance to remove any bubbles that might form in the channels throughout the chip culture as they interfere with the medium flow and will eventually result in reduced nutrient availability and a loss of cell viability.

This protocol also describes how to use the Vagina Chip to establish bacterial communities on-chip that mimic either the healthy vaginal state or bacterial vaginosis. The Vagina Chip also can be used to study other vaginal diseases or disorders; however, care should be taken to understand the characteristics of each disease and the best means for correlating results with clinical findings when carrying out these studies. In summary, the human Vagina Chip opens new avenues to study a plethora of diseases and conditions related to the FRT, and it can be a valuable tool for investigating potential therapeutics.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 µm Steriflip	Millipore	SCGP00525	To degas media
2 channel chip	Emulate	BRK-S1-WER-24	Part of the two-channel Chip kit
200 μL barrier tips (or filter tips)	Thomas Scientific, SHARP	1159M40	Tips used for chip seeding
Activation Reagent 1 (or ER-1 powder)	Emulate	Chip S1 Basic Research kit-24PK	Part of the two-channel Chip kit; Storage temperature -20 °C
Activation Reagent 2 (or ER-2 solution)	Emulate	Chip S1 Basic Research kit-24PK	Part of the two-channel Chip kit; Storage temperature 4 °C
Adenine	Sigma Aldrich	A2786	Component of the Differentiation media
Brucella blood agar plates	VWR International Inc.	89405-032	with Hemin and Vitamin K; For the enumeration of Gardnerella vaginalis
Ca2+ and Mg2+ free DPBS (DPBS (-/-)	ScienCell	303	For washing cells
Calcium Chloride	Sigma Aldrich	C5670	Component of the Differentiation media
Calcium chloride (anhyd.)	Sigma Aldrich	499609	Component of HBSS (LB/+G)
Collagen I	Corning	354236	For the coating solution for HVEC
Collagen IV	Sigma Aldrich	C7521	For the coating solution for HVEC
Collagenase IV	Gibco	17104019	For the dissociation of cells from the Vagina Chips
Complete fibroblast medium	ScienCell	2301	Media for the culture of HUF
Complete vaginal epithelium medium	Lifeline	LL-0068	Media for the culture of HVEC
D-Glucose (dextrose)	Sigma Aldrich	158968	Component of HBSS (LB/+G)
DMEM (Low Glucose)	Thermofisher	12320-032	Component of the Differentiation media
Dynamic Flow Module (or Zoë)	Emulate	Zoë-CM1	Regulates the flow rate of the chips
Ham's F12	Thermofisher	11765-054	Component of the Differentiation media
Heat inactivated FBS	Thermofisher	10438018	Component of the Differentiation media
Human uterine fibroblasts	ScienCell	7040	HUF
Human vaginal epithelial cells	Lifeline	FC-0083	HVEC
Hydrocortisone	Sigma Aldrich	H0396	Component of the Differentiation media
ITES	Lonza	17-839Z	Component of the Differentiation media
L-glutamine	Thermofisher	25030081	Component of the Differentiation media
Magnesium chloride hexahydrate	Sigma Aldrich	M2393	Component of HBSS (LB/+G)
Magnesium sulfate heptahydrate	Sigma Aldrich	M1880	Component of HBSS (LB/+G)
MRS agar plates	VWR International Inc.	89407-214	For enumeration of Lactobacillus
O-phosphorylethanolamine	Sigma Aldrich	P0503	Component of the Differentiation media
Pen/Strep	Thermofisher	15070063	Component of the Differentiation media
pH strips	Fischer-Scientific	13-640-520	For measurement of pH
Pods (1/chip)	Emulate	BRK-S1-WER-24	Part of the two-channel Chip kit
Poly-L-lysine	ScienCell	403	For the coating solution for HUFs
Potassium chloride	Sigma Aldrich	P3911	Component of HBSS (LB/+G)
Potassium phosphate monobasic	Sigma Aldrich	P0662	Component of HBSS (LB/+G)
Sterile 80% glycerol	MP Biomedicals	113055034	For freezing bacterial samples
Triiodothyronine	Sigma Aldrich	T6397	Component of the Differentiation media
Trypan Blue Solution (0.4%)	Sigma Aldrich	T8154	For counting live/dead cells
TrypLE Express	Thermofisher	12605010	For the dissociation of cells from the Vagina Chips
Trypsin Neutralizing Solution (TNS)	ScienCell	113	For neutralization of Trypsin
Trypsin/EDTA Solutiom (0.25%)	ScienCell	103	For cell dissociation
β-estradiol	Sigma Aldrich	E2257	Hormone for differentiation media