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.

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
<ol>
	<li>Warm 50 mL of vaginal epithelial medium (VEM, see Table of Materials) to 37 &#176;C.</li>
	<li>...

Microfluidic Vagina Chip for Studying the Vaginal Microbiome

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

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...

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&#160;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&#39;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 (&#60;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.&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.22 &#181;m Steriflip</td>
			<td>Millipore&#160;</td>
			<td>SCGP00525</td>
			<td>To degas media</td>
		</tr>
		<tr>
			<td>2 channel chip</td>
			<td>Emulate</td>
			<td>BRK-S1-WER-24</td>
			<td>Part of the two-channel Chip kit</td>
		</tr>
		<tr>
			<td>200 &#956;L barrier tips (or filter tips)</td>
			<td>Thomas Scientific, SHARP</td>
			<td>1159M40</td>
			<td>Tips used for chip seeding</td>
		</tr>
		<tr>
			<td>Activation Reagent 1 (or ER-1 powder)&#160;</td>
			<td>Emulate</td>
			<td>Chip S1 Basic Research kit-24PK</td>
			<td>Part of the two-channel Chip kit; Storage temperature -20 &#176;C&#160;&#160;</td>
		</tr>
		<tr>
			<td>Activation Reagent 2 (or ER-2 solution)&#160;</td>
			<td>Emulate</td>
			<td>Chip S1 Basic Research kit-24PK</td>
			<td>Part of the two-channel Chip kit; Storage temperature 4 &#176;C</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma Aldrich&#160;</td>
			<td>A2786</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>Brucella blood agar plates</td>
			<td>VWR International Inc.&#160;</td>
			<td>89405-032</td>
			<td>with Hemin and Vitamin K; For the enumeration of Gardnerella vaginalis</td>
		</tr>
		<tr>
			<td>Ca2+ and Mg2+ free DPBS (DPBS (-/-)</td>
			<td>ScienCell</td>
			<td>303</td>
			<td>For washing cells</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich&#160;</td>
			<td>C5670</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>Calcium chloride (anhyd.)&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>499609</td>
			<td>Component of HBSS (LB/+G)</td>
		</tr>
		<tr>
			<td>Collagen I&#160;</td>
			<td>Corning</td>
			<td>354236</td>
			<td>For the coating solution for HVEC</td>
		</tr>
		<tr>
			<td>Collagen IV&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>C7521</td>
			<td>For the coating solution for HVEC</td>
		</tr>
		<tr>
			<td>Collagenase IV</td>
			<td>Gibco</td>
			<td>17104019</td>
			<td>For the dissociation of cells from the Vagina Chips</td>
		</tr>
		<tr>
			<td>Complete fibroblast medium&#160;</td>
			<td>ScienCell</td>
			<td>2301</td>
			<td>Media for the culture of HUF</td>
		</tr>
		<tr>
			<td>Complete vaginal epithelium medium</td>
			<td>Lifeline</td>
			<td>LL-0068</td>
			<td>Media for the culture of HVEC</td>
		</tr>
		<tr>
			<td>D-Glucose (dextrose)&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>158968</td>
			<td>Component of HBSS (LB/+G)</td>
		</tr>
		<tr>
			<td>DMEM (Low Glucose)&#160;</td>
			<td>Thermofisher</td>
			<td>12320-032</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>Dynamic Flow Module (or Zo&#235;)</td>
			<td>Emulate</td>
			<td>Zo&#235;-CM1</td>
			<td>Regulates the flow rate of the chips</td>
		</tr>
		<tr>
			<td>Ham&#39;s F12</td>
			<td>Thermofisher</td>
			<td>11765-054</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>Heat inactivated FBS&#160;</td>
			<td>Thermofisher&#160;</td>
			<td>10438018</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>Human uterine fibroblasts</td>
			<td>ScienCell</td>
			<td>7040</td>
			<td>HUF</td>
		</tr>
		<tr>
			<td>Human vaginal epithelial cells</td>
			<td>Lifeline</td>
			<td>FC-0083</td>
			<td>HVEC</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma Aldrich&#160;</td>
			<td>H0396</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>ITES</td>
			<td>Lonza</td>
			<td>17-839Z</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Thermofisher</td>
			<td>25030081</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>Sigma Aldrich&#160;</td>
			<td>M2393</td>
			<td>Component of HBSS (LB/+G)</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma Aldrich&#160;</td>
			<td>M1880</td>
			<td>Component of HBSS (LB/+G)</td>
		</tr>
		<tr>
			<td>MRS agar plates</td>
			<td>VWR International Inc.&#160;</td>
			<td>89407-214</td>
			<td>For enumeration of Lactobacillus</td>
		</tr>
		<tr>
			<td>O-phosphorylethanolamine</td>
			<td>Sigma Aldrich&#160;</td>
			<td>P0503</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>Pen/Strep</td>
			<td>Thermofisher&#160;</td>
			<td>15070063</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>pH strips</td>
			<td>Fischer-Scientific</td>
			<td>13-640-520</td>
			<td>For measurement of pH&#160;</td>
		</tr>
		<tr>
			<td>Pods (1/chip)&#160;</td>
			<td>Emulate</td>
			<td>BRK-S1-WER-24</td>
			<td>Part of the two-channel Chip kit</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>ScienCell</td>
			<td>403</td>
			<td>For the coating solution for HUFs</td>
		</tr>
		<tr>
			<td>Potassium chloride&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>P3911</td>
			<td>Component of HBSS (LB/+G)</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma Aldrich&#160;</td>
			<td>P0662</td>
			<td>Component of HBSS (LB/+G)</td>
		</tr>
		<tr>
			<td>Sterile 80% glycerol&#160;</td>
			<td>MP Biomedicals&#160;</td>
			<td>113055034</td>
			<td>For freezing bacterial samples</td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma Aldrich&#160;</td>
			<td>&#160;T6397</td>
			<td>Component of the Differentiation media</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution (0.4%)&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>T8154</td>
			<td>For counting live/dead cells</td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Thermofisher&#160;</td>
			<td>12605010</td>
			<td>For the dissociation of cells from the Vagina Chips</td>
		</tr>
		<tr>
			<td>Trypsin Neutralizing Solution (TNS)&#160;</td>
			<td>ScienCell</td>
			<td>113</td>
			<td>For neutralization of Trypsin</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA Solutiom (0.25%)</td>
			<td>ScienCell</td>
			<td>103</td>
			<td>For cell dissociation&#160;</td>
		</tr>
		<tr>
			<td>&#946;-estradiol&#160;</td>
			<td>Sigma Aldrich&#160;</td>
			<td>E2257</td>
			<td>Hormone for differentiation media</td>
		</tr>
	</tbody>
</table>

modeling healthy and dysbiotic vaginal microenvironments in a human vagina-on-a-chip

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.

Author Spotlight: Revolutionizing Research on Vaginal Microbiome Interactions Using a Vaginal Chip

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

We successfully developed a human vagina chip that supports spontaneous differentiation of squamous stratified vaginal epithelium, forms a strong barrier, responds to hormones, and generates a 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.

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 (<strong class="xfig...

Watch this Scientific Journal Video about Author Spotlight: Revolutionizing Research on Vaginal Microbiome Interactions Using a Vaginal Chip at JoVE.com

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.

Women's health, and particularly diseases of the female reproductive tract (FRT), have not received the attention they deserve, even though an unhealthy ...

modeling-healthy-dysbiotic-vaginal-microenvironments-human-vagina-on

Research

JoVE Journal

Bioengineering

Seeding Organ Chips with Human Uterine Fibroblasts (HUFs) for Studying Living Vaginal Microbiome

Connecting Human Uterine Fibroblast-Seeded Organ Chips to Pods and Differentiation of Vaginal Epithelial Cells for Studying Living Vaginal Microbiome

Bacterial Inoculation of Organ Chips with Differentiated Human Vaginal Epithelial Cells for Studying Living Vaginal Microbiome Under Microaerophilic Conditions

1.6K Views.  Harvard University.  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.

Microfluidic Vagina Chip for Studying the Vaginal Microbiome

Summary