이 연구는 인간 세포 사용에 대한 기관 지침에 따라 수행되었습니다. 세포는 상업적으로 얻어졌다( 재료 표 참조). 모든 단계는 생물안전 작업대(BSC)에서 무균 상태로 수행해야 합니다. 이 프로토콜에는 필터(또는 배리어) 피펫 팁만 사용하십시오.
1. 인간 질 상피 세포 배양
<ol><li>50mL의 질 상피 배지(VEM, 재료 표 참조)를 37°C로 데웁니다.<...

질 마이크로바이옴(Vaginal Microbiome)을 연구하기 위한 Microfluidic Vagina Chip

<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>

과거의 인간 질의 체외 모델은 질 조직 구조, 유체 흐름 및 숙주-병원체 상호 작용을 충실하게 복제하지 못했다19,22. 동물 모델은 또한 미생물군집의 종간 변이와 발정기 또는 월경 주기의 차이에 의해 제한된다19,22. 이 원고는 건강하고 불균형한 미생물 군집에 대한 인간의 반응을 효과적으로 모방할 ?...

산성 미세환경을 유지하는 데 도움이 되는 Lactobacillus spp.가 지배하는 질 마이크로바이옴은 여성의 생식 건강을 유지하는 데 중요한 역할을 합니다1. 그러나 때때로 마이크로바이옴을 구성하는 미생물 군집의 구성에 변화가 있을 수 있으며, 이로 인해 질 박테리아의 다양성이 증가할 수 있습니다. 이러한 불균형적 변화는 종종 락토바실러스가 지배하는 상태에서 보다 다양한 혐기성 박테리아 종(예: Gardnerella vaginalis)이 지배하는 상태로 전환되는 결과를 낳으며, 세균성 질염, 위축성 질염, 요로 감염, 외음부 칸디다증, 요도염 및 융모막염과 같은 생식계의 다양한 질병과 관련이 있습니다 2,3,4,5 . 이러한 질병은 여성이 성병과 골반 염증성 질환에 걸릴 가능성을 높입니다 6,7,8,9. 또한 임산부의 조산 및 유산 위험이 더 높으며10,11,12 불임과도 관련이 있다 13,14,15,16.
정적인 2차원(2D) 배양 시스템에서 배양된 질 상피 세포를 사용하여 질 미생물 불균형을 모델링하려는 노력이 이루어졌지만17,18, 질 미세환경의 생리학적 및 복잡성을 효과적으로 모방하지는 못한다19. 동물 모델은 또한 질 미생물 불균형을 연구하는 데 사용되었습니다. 그러나 이들의 월경 단계와 숙주-마이크로바이옴 상호작용은 인간과 크게 다르기 때문에 이러한 연구 결과의 생리학적 관련성은 불분명합니다 19,20,21. 이러한 문제에 대응하기 위해 인간 질 조직의 오가노이드 및 Transwell 삽입 모델도 FRT 19,22,23,24에서 숙주-병원체 상호 작용을 연구하는 데 사용되었습니다. 그러나 이들은 정적 배양이기 때문에 짧은 기간(<16-24시간) 동안만 살아있는 미생물과 인간 세포의 공동 배양을 지원할 수 있으며, 점액 생성 및 유체 흐름과 같은 인간 질 미세환경의 다른 잠재적으로 중요한 다른 많은 물리적 특징이 부족합니다22.
장기 칩(Organ Chip)은 동적 유체 흐름 하에서 배양된 살아있는 세포가 늘어선 하나 이상의 평행한 중공 미세채널을 포함하는 3차원(3D) 미세유체 배양 시스템입니다. 2채널 칩은 두 개의 평행 채널을 분리하는 다공성 멤브레인의 반대쪽에서 서로 다른 세포 유형(예: 상피 및 기질 섬유아세포 또는 상피와 혈관 내피)을 배양하여 장기 수준의 조직-조직 인터페이스를 재현할 수 있습니다(그림 1). 두 조직 모두 유체 흐름에 독립적으로 노출될 수 있으며, 복잡한 마이크로바이옴 25,26,27,28과 공동 배양을 가능하게 하는 미세 호기성 조건을 경험할 수도 있습니다. 이 접근법은 최근 호르몬에 민감한 원발성 질 상피와 기저 기질 섬유아세포로 둘러싸인 인간 질 칩을 개발하는 데 활용되었으며, 이는 상피 내강에서 낮은 생리적 산소 농도를 유지하고 시험관 내에서 최소 3일 동안 건강한 디스바이오틱 마이크로바이옴과 공동 배양을 가능하게 합니다 29. 질 칩은 최적(건강한) L. crispatus 컨소시엄에 의한 집락화를 연구하고 최적이 아닌(건강하지 않은) G. vaginalis 함유 컨소시엄으로 인한 염증 및 부상을 감지하는 데 사용할 수 있음을 입증했습니다. 여기에서는 인간 질 칩을 만들고 건강하고 불균형적인 박테리아 커뮤니티를 온칩에 구축하는 데 사용되는 방법을 자세히 설명합니다.

<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

여성의 건강, 특히 여성 생식관 질환(FRT)은 건강하지 않은 생식기관이 생명을 위협하는 질병, 불임 또는 임신 중 부정적인 결과를 초래할 수 있음에도 불구하고 마땅히 받아야 할 관심을 받지 못했습니다. 이 분야의 한 가지 장벽은 FRT의 생리학 및 병태생리학을 충실하게 모방하는 전임상 실험 모델이 부족하다는 것입니다. 현재의 체외 및 동물 모델은 호르몬 변화, 미세 호기성 조건 및 질 마이크로바이옴과의 상호 작용을 완전히 요약하지 못합니다. 조직-조직 계면, 혈관 관류, 간질액 흐름 및 인간 장기의 주요 하위 단위의 물리적 미세환경을 모방할 수 있는 Organ-on-a-Chip(Organ Chip) 미세유체 배양 기술의 출현은 잠재적으로 이 문제에 대한 해결책이 될 수 있습니다. 최근에는 인간 질 기질과 연결되어 역동적인 유체 흐름을 경험하는 원발성 인간 질 상피와 인간 질 미생물 컨소시엄의 공동 배양을 지원하는 human Vagina Chip이 개발되었습니다. 이 칩은 건강하고 불균형한 미생물 군집에 대한 인간 질의 생리적 반응을 복제합니다. 인간 질 칩을 만들기위한 자세한 프로토콜이이 기사에 설명되어 있습니다.

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.

저자 스포트라이트: 질 칩을 사용한 질 마이크로바이옴 상호 작용에 대한 연구의 혁명

인간 Vagina-on-a-Chip에서 건강하고 dysbiotic 질 미세환경 모델링

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.

인간의 질은 섬유아세포가 풍부한 콜라겐 기질 위에 있는 성층상피(stratified epithelium)로 둘러싸여 있습니다. 이를 모델링하기 위해 2채널 미세유체 장기 칩 장치 내에서 공통 다공성 막의 반대쪽에 있는 원발성 인간 질 상피와 섬유아세포를 배양하여 조직 인터페이스를 만들었습니다. 질 상피의 형성은 명시야 현미경 이미징을 사용하여 모니터링되며, 이를 통해 점진적으로 여러 세포층을 형성하?...

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

이 기사에서는 미세공기성 조건에서 살아있는 질 마이크로바이옴과 인간 숙주의 상호 작용을 연구할 수 있는 미세유체 질 온 칩(Vagina Chip) 배양 장치를 만들기 위한 프로토콜에 대해 설명합니다. 이 칩은 질 질환을 조사하고 잠재적인 치료 대책을 개발 및 테스트하는 도구로 사용할 수 있습니다.

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

우리는 편평 성층 질 상피의 자발적 분화를 지원하고, 강력한 장벽을 형성하고, 호르몬에 반응하고, 산소 구배를 지원하는 마이크로바이옴을 생성하는 인간 질 칩을 성공적으로 개발했습니다. 2D 및 오가노이드 배양은 현재 질 미생물 불균형을 연구하는 데 사용되지만 정적 특성으로 인해 질 미세환경의 생리학적 복잡성을 모방할 수 없습니다. 동물 모델 역시 인간의 월경 주기에 존재하는 동일한 호르몬 변화를 모델링할 수 없으며, 동물 종과 인간 간의 질 마이크로바이옴은 다릅니다.우리는 장기 칩 기술을 사용하여 인간 질 마이크로바이옴 상호 작용의 전임상 모델을 개발할 수 있는지 여부를 조사하기 시작했으며, 이는 잠재적인 마이크로바이옴 기반 치료법의 발견 및 평가에 잠재적으로 사용될 수 있습니다. 질 칩은 질 상피와 기질을 가로지르는 동적 유체 흐름을 허용하여 질 미생물 컨소시엄과의 공동 배양을 연장하고 생리학적으로 관련된 미세환경을 유지합니다. 인간 버자이너 온 어 칩(Human Vagina-on-a-Chip)은 여성 생식관의 다양한 질병을 연구하고 새로운 바이오 치료제를 검증하는 데 사용할 수 있는 흥미로운 도구입니다.

인간 Vagina-on-a-Chip에서 건강하고 dysbiotic 질 미세환경 모델링

Abstract