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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 chorioamnionitis2,3,4,5. These diseases, in turn, increase a woman's chances of acquiring sexually transmitted diseases and pelvic inflammatory disease6,7,8,9. They also pose a higher risk for pre-term birth and miscarriages in pregnant women10,11,12 and have also been implicated in infertility13,14,15,16.

Although efforts have been made to model vaginal dysbiosis using vaginal epithelial cells cultured in static, two-dimensional (2D) culture systems17,18, 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 unclear19,20,21. To counteract these issues, organoids and Transwell insert models of human vaginal tissue also have been used to study host-pathogen interactions in the FRT19,22,23,24. 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 microbiome25,26,27,28. 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% CO2. 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 technique29,30.

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 (

Discussion

Past in vitro models of the human vagina do not faithfully replicate vaginal tissue structures, fluid flow, and host-pathogen interactions19,22. Animal models are also limited by inter-species variation in microbiome and differences in the estrous or menstrual cycle19,22. This manuscript describes a protocol to create an Organ Chip model of the human vagina that can effectively mimic human respon...

Disclosures

Donald Ingber is a founder, board member, scientific advisory board chair, and equity holder in Emulate. The other authors declare that they have no competing interests.

Acknowledgements

This research was sponsored by funding from the Bill and Melinda Gates Foundation (INV-035977) and the Wyss Institute for Biologically Inspired Engineering at Harvard University. We also thank Gwenn E. Merry, Wyss Institute, for editing this manuscript. The diagram in Figure 1 has been created with BioRender.

Materials

NameCompanyCatalog NumberComments
0.22 µm SteriflipMillipore SCGP00525To degas media
2 channel chipEmulateBRK-S1-WER-24Part of the two-channel Chip kit
200 μL barrier tips (or filter tips)Thomas Scientific, SHARP1159M40Tips used for chip seeding
Activation Reagent 1 (or ER-1 powder) EmulateChip S1 Basic Research kit-24PKPart of the two-channel Chip kit; Storage temperature -20 °C  
Activation Reagent 2 (or ER-2 solution) EmulateChip S1 Basic Research kit-24PKPart of the two-channel Chip kit; Storage temperature 4 °C
AdenineSigma Aldrich A2786Component of the Differentiation media
Brucella blood agar platesVWR International Inc. 89405-032with Hemin and Vitamin K; For the enumeration of Gardnerella vaginalis
Ca2+ and Mg2+ free DPBS (DPBS (-/-)ScienCell303For washing cells
Calcium ChlorideSigma Aldrich C5670Component of the Differentiation media
Calcium chloride (anhyd.) Sigma Aldrich 499609Component of HBSS (LB/+G)
Collagen I Corning354236For the coating solution for HVEC
Collagen IV Sigma Aldrich C7521For the coating solution for HVEC
Collagenase IVGibco17104019For the dissociation of cells from the Vagina Chips
Complete fibroblast medium ScienCell2301Media for the culture of HUF
Complete vaginal epithelium mediumLifelineLL-0068Media for the culture of HVEC
D-Glucose (dextrose) Sigma Aldrich 158968Component of HBSS (LB/+G)
DMEM (Low Glucose) Thermofisher12320-032Component of the Differentiation media
Dynamic Flow Module (or Zoë)EmulateZoë-CM1Regulates the flow rate of the chips
Ham's F12Thermofisher11765-054Component of the Differentiation media
Heat inactivated FBS Thermofisher 10438018Component of the Differentiation media
Human uterine fibroblastsScienCell7040HUF
Human vaginal epithelial cellsLifelineFC-0083HVEC
HydrocortisoneSigma Aldrich H0396Component of the Differentiation media
ITESLonza17-839ZComponent of the Differentiation media
L-glutamineThermofisher25030081Component of the Differentiation media
Magnesium chloride hexahydrateSigma Aldrich M2393Component of HBSS (LB/+G)
Magnesium sulfate heptahydrateSigma Aldrich M1880Component of HBSS (LB/+G)
MRS agar platesVWR International Inc. 89407-214For enumeration of Lactobacillus
O-phosphorylethanolamineSigma Aldrich P0503Component of the Differentiation media
Pen/StrepThermofisher 15070063Component of the Differentiation media
pH stripsFischer-Scientific13-640-520For measurement of pH 
Pods (1/chip) EmulateBRK-S1-WER-24Part of the two-channel Chip kit
Poly-L-lysineScienCell403For the coating solution for HUFs
Potassium chloride Sigma Aldrich P3911Component of HBSS (LB/+G)
Potassium phosphate monobasicSigma Aldrich P0662Component of HBSS (LB/+G)
Sterile 80% glycerol MP Biomedicals 113055034For freezing bacterial samples
TriiodothyronineSigma Aldrich  T6397Component of the Differentiation media
Trypan Blue Solution (0.4%) Sigma Aldrich T8154For counting live/dead cells
TrypLE ExpressThermofisher 12605010For the dissociation of cells from the Vagina Chips
Trypsin Neutralizing Solution (TNS) ScienCell113For neutralization of Trypsin
Trypsin/EDTA Solutiom (0.25%)ScienCell103For cell dissociation 
β-estradiol Sigma Aldrich E2257Hormone for differentiation media

References

  1. Smith, S. B., Ravel, J. The vaginal microbiota, host defence and reproductive physiology. J Physiol. 595 (2), 451-463 (2017).
  2. Van De Wijgert, J., Jespers, V. The global health impact of vaginal dysbiosis. Res Microbiol. 168 (9-10), 859-864 (2017).
  3. Ralph, S. G., Rutherford, A. J., Wilson, J. D. Influence of bacterial vaginosis on conception and miscarriage in the first trimester: Cohort study. BMJ. 319 (7204), 220-223 (1999).
  4. Goldenberg, R. L., Hauth, J. C., Andrews, W. W. Intrauterine infection and preterm delivery. N Engl J Med. 342 (20), 1500-1507 (2000).
  5. Han, Y., Liu, Z., Chen, T. Role of vaginal microbiota dysbiosis in gynecological diseases and the potential interventions. Front Microbiol. 12, 643422 (2021).
  6. Leitich, H., Kiss, H. Asymptomatic bacterial vaginosis and intermediate flora as risk factors for adverse pregnancy outcome. Best Pract Res Clin Obstet Gynaecol. 21 (3), 375-390 (2007).
  7. Torcia, M. G. Interplay among vaginal microbiome, immune response and sexually transmitted viral infections. Int J Mol Sci. 20 (2), 266 (2019).
  8. Van Oostrum, N., De Sutter, P., Meys, J., Verstraelen, H. Risks associated with bacterial vaginosis in infertility patients: A systematic review and meta-analysis. Hum Reprod. 28 (7), 1809-1815 (2013).
  9. Lewis, F. M. T., Bernstein, K. T., Aral, S. O. Vaginal microbiome and its relationship to behavior, sexual health, and sexually transmitted diseases. Obstet Gynecol. 129 (4), 643-654 (2017).
  10. Hong, X., et al. The association between vaginal microbiota and female infertility: A systematic review and meta-analysis. Arch Gynecol Obstet. 302 (3), 569-578 (2020).
  11. Peelen, M. J., et al. The influence of the vaginal microbiota on preterm birth: A systematic review and recommendations for a minimum dataset for future research. Placenta. 79, 30-39 (2019).
  12. Smith, P. P., et al. Outcomes in prevention and management of miscarriage trials: A systematic review. BJOG. 126 (2), 176-189 (2019).
  13. Harp, D. F., Chowdhury, I. Trichomoniasis: Evaluation to execution. Eur J Obstet Gynecol Reprod Biol. 157 (1), 3-9 (2011).
  14. Pastorek, J. G., Cotch, M. F., Martin, D. H., Eschenbach, D. A. Clinical and microbiological correlates of vaginal trichomoniasis during pregnancy. The vaginal infections and prematurity study group. Clin Infect Dis. 23 (5), 1075-1080 (1996).
  15. Petrin, D., Delgaty, K., Bhatt, R., Garber, G. Clinical and microbiological aspects of trichomonas vaginalis. Clin Microbiol Rev. 11 (2), 300-317 (1998).
  16. Edwards, T., Burke, P., Smalley, H., Hobbs, G. Trichomonas vaginalis: Clinical relevance, pathogenicity and diagnosis. Crit Rev Microbiol. 42 (3), 406-417 (2016).
  17. Eade, C. R., et al. Identification and characterization of bacterial vaginosis-associated pathogens using a comprehensive cervical-vaginal epithelial coculture assay. PLoS One. 7 (11), e50106 (2012).
  18. Fichorova, R. N., Yamamoto, H. S., Delaney, M. L., Onderdonk, A. B., Doncel, G. F. Novel vaginal microflora colonization model providing new insight into microbicide mechanism of action. mBio. 2 (6), e00168 (2011).
  19. Herbst-Kralovetz, M. M., Pyles, R. B., Ratner, A. J., Sycuro, L. K., Mitchell, C. New systems for studying intercellular interactions in bacterial vaginosis. J Infect Dis. 214, S6-S13 (2016).
  20. Johnson, A. P., et al. A study of the susceptibility of three species of primate to vaginal colonization with gardnerella vaginalis. Br J Exp Pathol. 65 (3), 389-396 (1984).
  21. Yildirim, S., et al. Primate vaginal microbiomes exhibit species specificity without universal lactobacillus dominance. ISME J. 8 (12), 2431-2444 (2014).
  22. Edwards, V. L., et al. Three-dimensional models of the cervicovaginal epithelia to study host-microbiome interactions and sexually transmitted infections. Pathog Dis. 80 (1), 026 (2022).
  23. Zhu, Y., et al. Ex vivo 2D and 3D HSV-2 infection model using human normal vaginal epithelial cells. Oncotarget. 8 (9), 15267-15282 (2017).
  24. Barrila, J., et al. Modeling host-pathogen interactions in the context of the microenvironment: Three-dimensional cell culture comes of age. Infect Immun. 86 (11), e00282 (2018).
  25. Bein, A., et al. Microfluidic organ-on-a-chip models of human intestine. Cell Mol Gastroenterol Hepatol. 5 (4), 659-668 (2018).
  26. Jalili-Firoozinezhad, S., et al. A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip. Nat Biomed Eng. 3 (7), 520-531 (2019).
  27. Valiei, A., Aminian-Dehkordi, J., Mofrad, M. R. K. Gut-on-a-chip models for dissecting the gut microbiology and physiology. APL Bioeng. 7 (1), 011502 (2023).
  28. Izadifar, Z., et al. Mucus production, host-microbiome interactions, hormone sensitivity, and innate immune responses modeled in human endo- and ecto-cervix chips. bioRxiv. , (2023).
  29. Mahajan, G., et al. Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip. Microbiome. 10 (1), 201 (2022).
  30. Masson, L., et al. Inflammatory cytokine biomarkers to identify women with asymptomatic sexually transmitted infections and bacterial vaginosis who are at high risk of HIV infection. Sex Transm Infect. 92 (3), 186-193 (2016).
  31. Amsel, R., et al. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med. 74 (1), 14-22 (1983).

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