이 작업은 국립 보건원 보조금 R01DK120986 (KPM에)의 지원을 받았습니다.

확립된 쥐 대장에 대한 장 염증의 상피 효과

<ol>
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N., Mizoguchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colitis+in+transgenic+and+knockout+animals+as+models+of+human+inflammatory+bowel+disease.">Colitis in transgenic and knockout animals as models of human inflammatory bowel disease.</a> Immunological Reviews. 169, 195-207 (1999).</li><li>Okayasu, I., 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+novel+method+in+the+induction+of+reliable+experimental+acute+and+chronic+ulcerative+colitis+in+mice.">A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice.</a> Gastroenterology. 98 (3), 694-702 (1990).</li><li>Dotti, I., Salas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+use+of+human+stem+cell-derived+intestinal+organoids+to+study+inflammatory+bowel+diseases.">Potential use of human stem cell-derived intestinal organoids to study inflammatory bowel diseases.</a> Inflammatory Bowel Diseases. 24 (12), 2501-2509 (2018).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Santos, A. J. M., Lo, Y. H., Mah, A. T., Kuo, C. 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+intestinal+stem+cell+niche:+Homeostasis+and+adaptations.">The intestinal stem cell niche: Homeostasis and adaptations.</a> Trends in Cell Biology. 28 (12), 1062-1078 (2018).</li><li>Yin, 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=Niche-independent+high-purity+cultures+of+Lgr5++intestinal+stem+cells+and+their+progeny.">Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny.</a> Nature Methods. 11 (1), 106-112 (2014).</li><li>Wilson, S. 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=Optimized+culture+conditions+for+improved+growth+and+functional+differentiation+of+mouse+and+human+colon+organoids.">Optimized culture conditions for improved growth and functional differentiation of mouse and human colon organoids.</a> Frontiers in Immunology. 11, 547102 (2021).</li><li>Kim, J. J., Shajib, M. S., Manocha, M. M., Khan, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+intestinal+inflammation+in+DSS-induced+model+of+IBD.">Investigating intestinal inflammation in DSS-induced model of IBD.</a> Journal of Visualized Experiments. (60), e3678 (2012).</li><li>Cunningham, K. E., 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=Peroxisome+proliferator-activated+receptor-&#947;+coactivator+1-&#945;+(PGC1&#945;)+protects+against+experimental+murine+colitis.">Peroxisome proliferator-activated receptor-&#947; coactivator 1-&#945; (PGC1&#945;) protects against experimental murine colitis.</a> Journal of Biological Chemistry. 291 (19), 10184-10200 (2016).</li><li>Miyoshi, H., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+and+genetic+modification+of+gastrointestinal+stem+cells+in+spheroid+culture.">In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.</a> Nature Protocols. 8 (12), 2471-2481 (2013).</li><li>Sato, T., 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=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Julian, M. W., 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=Intestinal+epithelium+is+more+susceptible+to+cytopathic+injury+and+altered+permeability+than+the+lung+epithelium+in+the+context+of+acute+sepsis.">Intestinal epithelium is more susceptible to cytopathic injury and altered permeability than the lung epithelium in the context of acute sepsis.</a> International Journal of Experimental Pathology. 92 (5), 366-376 (2011).</li><li>Haberman, 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=Ulcerative+colitis+mucosal+transcriptomes+reveal+mitochondriopathy+and+personalized+mechanisms+underlying+disease+severity+and+treatment+response.">Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response.</a> Nature Communications. 10, 38 (2019).</li><li>Yang, 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=Mitochondrial+transcription+factor+A+plays+opposite+roles+in+the+initiation+and+progression+of+colitis-associated+cancer.">Mitochondrial transcription factor A plays opposite roles in the initiation and progression of colitis-associated cancer.</a> Cancer Communications. 41 (8), 695-714 (2021).</li><li>Maidji, E., Somsouk, M., Rivera, J. M., Hunt, P. W., Stoddart, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+CMV+in+the+gut+of+HIV-infected+individuals+and+epithelial+barrier+dysfunction.">Replication of CMV in the gut of HIV-infected individuals and epithelial barrier dysfunction.</a> PLoS Pathogen. 13, 1006202 (2017).</li><li>Noel, 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=A+primary+human+macrophage-enteroid+co-culture+model+to+investigate+mucosal+gut+physiology+and+host-pathogen+interactions.">A primary human macrophage-enteroid co-culture model to investigate mucosal gut physiology and host-pathogen interactions.</a> Scientific Reports. 7, 452-470 (2017).</li><li>Grondin, J., Kwon, Y., Far, P., Haq, S., Khan, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucins+in+Intestinal+mucosal+defense+and+inflammation:+Learning+from+clinical+and+experimental+studies.">Mucins in Intestinal mucosal defense and inflammation: Learning from clinical and experimental studies.</a> Frontiers in Immunology. 11, 2054 (2020).</li><li>Metcalfe, C., Kljavin, N. M., Ybarra, R., de Sauvage, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lgr5++stem+cells+are+indispensable+for+radiation-induced+intestinal+regeneration.">Lgr5+ stem cells are indispensable for radiation-induced intestinal regeneration.</a> Cell Stem Cell. 14 (2), 149-159 (2014).</li><li>Yui, 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=YAP/TAZ-Dependent+reprogramming+of+colonic+epithelium+links+ECM+remodeling+to+tissue+regeneration.">YAP/TAZ-Dependent reprogramming of colonic epithelium links ECM remodeling to tissue regeneration.</a> Cell Stem Cell. 22 (1), 35-49 (2018).</li><li>Komiya, Y., Habas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signal+transduction+pathways.">Wnt signal transduction pathways.</a> Organogenesis. 4 (2), 68-75 (2008).</li></ol>

기고 저자는 공개 할 것이 없습니다.

오가노이드 개발은 과학계가 접시에 담긴 동물이나 인간의 세포 구조와 기능을 부분적으로 요약할 수 있는 능력으로 체외에서 장기 시스템을 연구하는 방식에 혁명을 일으켰습니다. 또한, 질병이 있는 인간에게서 파생된 오가노이드 시스템은 치료 의사 결정을 안내할 수 있는 개인화된 의학을 위한 유망한 도구를 제공합니다. 여기에서는 잘 작동하는 크립트 격리 프로토콜에 대해 설명하?...

염증성 장 질환(IBD)은 염증 및 임상적 정지 에피소드를 특징으로 하는 만성, 완화 및 재발성 질환입니다. IBD의 병인은 다인성이지만, 이 질환의 주요 특징은 상피 구획 내에서 활성화된 전염증성 신호 캐스케이드 외에도 장벽 기능 결함과 장 상피의 투과성 증가를 포함한다 1,2. 염증의 세포 배양 및 쥐 모델을 포함하여 IBD 동안 상피 반응을 요약하기 위해 여러 시험관 내 및 생체 내 모델이 사용되었습니다3. 그러나, 이들 모든 시스템은 IBD4 동안 상피 변화를 요약하는 능력을 제한하는 중요한 단점을 가지고 있다. IBD 연구에 사용되는 대부분의 세포주는 형질전환되고, 단층을 형성할 수 있는 능력을 가지며,3개를 분화시킬 수 있지만 숙주에서 형질전환되지 않은 장 상피 세포와 본질적으로 다르게 증식합니다. 염증의 여러 다른 쥐 모델이 IBD를 연구하는 데 사용되며, 그 중 일부는 녹아웃 모델, 감염성 모델, 화학적 염증 모델 및 T 세포 전달 모델 5,6,7,8을 포함합니다. 각각은 유전적 소인, 장벽 기능 장애, 면역 조절 완화 및 미생물군집과 같은 IBD의 특정 병인학적 측면을 연구할 수 있지만 질병의 다인성 특성을 연구하는 능력에는 한계가 있습니다.
엔테로이드 및 콜로노이드를 포함한 장 오가노이드는 지난 10년 동안 장 줄기 세포의 역학뿐만 아니라 장 항상성 및 질병에서 장 상피의 역할, 장벽 무결성 및 기능을 연구하기 위한 유용한 시험관 내 모델로 확립되었습니다. 이러한 실체는 IBD9의 발병기전에 대한 우리의 이해에 크게 기여했으며 개인화된 의학을 위한 새로운 기회를 열었습니다. 대장에서 줄기 세포 유래 자가 조직화 배양물인 콜로노이드(Colonoids)는 쥐와 인간 조직 모두에서 개발되어 장 선와(intestinal crypt) 내에 위치한 줄기세포가 번식하고 무기한 유지될 수 있도록 하는 과정이다10. 생체 내 줄기 세포 틈새는 성장을 지원하기 위해 세포외 인자, 특히 표준 Wnt 신호 전달 및 뼈 형태 형성 단백질 신호 전달 경로에 의존합니다11. 이러한 인자의 추가는 콜로노이드의 건강과 수명을 촉진할 뿐만 아니라 자가 재생 세포와 말단 분화 세포 모두로 구성된 생체 내 상피 세포 구조를 반영하지 않는 줄기 세포와 유사한 상태로 배양을 유도합니다12,13. 장 상피의 기능은 줄기 세포 구획과 분화된 세포 사이의 지속적인 누화에 의존하지만, 결장 배양 시스템에서 둘 다를 갖는 능력은 상당히 제한적입니다. 이러한 한계에도 불구하고 오가노이드 배양 시스템은 생체 외 상피의 고유 특성을 연구하기 위한 황금 표준으로 남아 있습니다. 그럼에도 불구하고 당면한 과학적 질문에 답하기 위해 대안적 문화 전략을 고려해야 할 수도 있습니다.
덱스트란 황산나트륨(DSS)을 7일 동안 지속적으로 투여한 쥐는 상피 염증과 장벽 기능 장애를 모두 발생시키는 것으로 나타났다14. 또한, 인간 IBD에서 명백한 것으로 밝혀진 장 상피 내의 미토콘드리아 생물 발생 실패 및 대사 재프로그래밍도 대장염의 DSS 모델에서도 포착되었습니다15. 그러나 우리의 예비 데이터는 미토콘드리아 생물 발생 실패의 특성이 DSS 처리 동물의 선와에서 파생된 콜로노이드에서 유지되지 않는다는 것을 보여줍니다(보충 그림 1). 따라서 쥐 장 염증 동안 염증이 상피 변화를 유발하는 방법을 조사할 때 대체 배양 방법을 사용해야 합니다. 여기에서 우리는 1) 쥐 콜로노이드의 확립을 위해 전체 결장 조직에서 선와를 분리하는 방법, 2) 생체 내 세포 집단을 반영하기 위해 이 세포 집단을 최종적으로 분화하는 방법, 3) 이 시험관 내 모델에서 염증을 유도하는 방법. 염증이 있는 상피 내에서 약물 상호작용을 연구하기 위해 우리는 염증 매개체로 기저적으로 치료하고 약물 요법으로 정점으로 치료할 수 있는 쥐 결장에서 2-디멘소날(2D) 단층을 설정하는 프로토콜을 개발했습니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4-&#956;M transparent transwell, 24-well</td>
			<td>Greiner Bio-one</td>
			<td>662-641</td>
			<td></td>
		</tr>
		<tr>
			<td>15-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#956;M cell strainer</td>
			<td>VWR</td>
			<td>76327-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-010</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>B-27 supplement&#160;</td>
			<td>Invitrogen</td>
			<td>12587-010</td>
			<td>Stock Concentration (50x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Chopsticks Electrode Set for EVO</td>
			<td>World Precision Instruments</td>
			<td>STX2</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel GFR Membrane Mix</td>
			<td>Corning</td>
			<td>354-230</td>
			<td>Stock Concentration (100%); Final Concentration (100%)</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-5G</td>
			<td>Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Thermo Fisher</td>
			<td>11960-069</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline without Calcium and Magnesium</td>
			<td>Gibco&#160;</td>
			<td>14190-144</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (ETDA)</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td>Stock Concentration (0.5 M); Final Concentration (30 mM)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Bio-Techne</td>
			<td>S11150H</td>
			<td>Stock Concentration (100%); Final Concentration (1%)</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>InvivoGen</td>
			<td>ant-ga-1</td>
			<td>Final Concentration (400 &#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>Gentamicin Reagent</td>
			<td>Gibco/Fisher</td>
			<td>15750-060</td>
			<td>Stock Concentration (50 mg/mL); Final Concentration (250 &#956;g/mL)</td>
		</tr>
		<tr>
			<td>GlutaMAX-1</td>
			<td>Fisher Scientific</td>
			<td>35050-061</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>Stock Concentration (1 M); Final Concentration (10 mM)</td>
		</tr>
		<tr>
			<td>hIFN&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF</td>
			<td>Stock Concentration (1000 ng/&#181;L); Final Concentration (10 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hIL-1&#946;</td>
			<td>R&#38;D Systems</td>
			<td>201-LB</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (20 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hTNF&#945;</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (40 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide&#160;</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media)</td>
		</tr>
		<tr>
			<td>Hygromycin B Gold</td>
			<td>InvivoGen</td>
			<td>ant-hg-1</td>
			<td>Final Concentration (400 &#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>L-WRN Cell Line</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>mEGF</td>
			<td>Novus</td>
			<td>NBP2-35176</td>
			<td>Stock Concentration (0.5 &#181;g/&#181;L); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA)</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma&#160;</td>
			<td>A9165-5G</td>
			<td>Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>Stock Concentration (0.1 ng/&#181;L); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Microplates, 24 well tissue culture treated, sterile</td>
			<td>Greiner Bio-one</td>
			<td>5666-2160</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Spondin</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>Stock Concentration (0.25 &#181;g/&#181;L); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA)</td>
		</tr>
		<tr>
			<td>Tryp LE Express</td>
			<td>Thermo Fisher</td>
			<td>12604-013</td>
			<td>Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA)</td>
		</tr>
		<tr>
			<td>UltraPure Water&#160;</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Y-27632 dihyddrochloride&#160;</td>
			<td>Abcam</td>
			<td>ab120129</td>
			<td>Stock Concentration (10 mM); Final Concentration (10 &#181;M); Solvent (UltraPure Water)</td>
		</tr>
	</tbody>
</table>

studying the epithelial effects of intestinal inflammation in vitro on established murine colonoids

장 상피는 인체 건강에 필수적인 역할을 하며 숙주와 외부 환경 사이에 장벽을 제공합니다. 이 매우 역동적인 세포층은 미생물과 면역 집단 사이의 첫 번째 방어선을 제공하고 장 면역 반응을 조절하는 데 도움이 됩니다. 상피 장벽의 파괴는 염증성 장 질환(IBD)의 특징이며 치료 표적화에 관심이 있습니다. 3차원 결장 배양 시스템은 IBD 발병기전에서 장 줄기 세포 역학 및 상피 세포 생리학을 연구하는 데 매우 유용한 시험관 내 모델입니다. 이상적으로는 동물의 염증이 있는 상피 조직에서 콜로노이드를 확립하는 것이 질병에 대한 유전적 및 분자적 영향을 평가하는 데 가장 유익할 것입니다. 그러나, 우리는 생체 내 상피 변화가 급성 염증이 있는 마우스에서 확립된 콜로노이드에서 반드시 유지되는 것은 아니라는 것을 보여주었습니다. 이러한 한계를 해결하기 위해 우리는 IBD 동안 일반적으로 상승하는 염증 매개체의 칵테일로 콜로노이드를 치료하는 프로토콜을 개발했습니다. 이 시스템은 다양한 배양 조건에 유비쿼터스로 적용될 수 있지만, 이 프로토콜은 차별화된 콜로노이드와 확립된 콜로노이드에서 파생된 2차원 단층 모두에 대한 처리를 강조합니다. 전통적인 배양 환경에서 콜로노이드는 장내 줄기 세포가 풍부하여 줄기 세포 틈새를 연구하기에 이상적인 환경을 제공합니다. 그러나 이 시스템은 장벽 기능과 같은 장 생리학의 특징을 분석할 수 없습니다. 또한, 전통적인 콜로노이드는 전염증성 자극에 대한 말단적으로 분화된 상피 세포의 세포 반응을 연구할 기회를 제공하지 않습니다. 여기에 제시된 방법은 이러한 제한 사항을 해결하기 위한 대체 실험 프레임워크를 제공합니다. 2차원 단층 배양 시스템은 또한 생체 외에서 치료 약물 스크리닝을 위한 기회를 제공합니다. 이 편광된 세포층은 세포의 기저부에 있는 염증 매개체로 치료할 수 있으며 IBD 치료에서의 유용성을 결정하기 위해 정점적으로 추정 치료제와 병행할 수 있습니다.

Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing disease characterized by episodes of inflammation and clinical quiescence. The etiology of IBD is multifactorial, but key characteristic features of the disease include defective barrier function and increased permeability of the intestinal epithelium, in addition to proinflammatory signaling cascades activated within the epithelial compartment1,2. Several in vitro and in vivo models have been used to recapitulate the epithelial response during IBD, including cell culture and murine models of inflammation3. However, all these systems have important shortcomings that limit their ability to recapitulate the epithelial changes during IBD4. Most cell lines used to study IBD are transformed, have the ability to form a monolayer, and can differentiate3 but intrinsically propagate differently than non-transformed intestinal epithelial cells in the host. Several different murine models of inflammation are used to study IBD, some of which include knockout models, infectious models, chemical inflammatory models, and T-cell transfer models5,6,7,8. While each can study certain etiological aspects of IBD, such as genetic predispositions, barrier dysfunction, immune deregulation, and the microbiome, they are limited in their ability to study the multifactorial nature of the disease.
Intestinal organoids, including enteroids and colonoids, have been established over the last decade as a useful in vitro model for studying not only the dynamics of intestinal stem cells but also their role the barrier integrity and function of the intestinal epithelium play in intestinal homeostasis and disease. These entities have significantly contributed to our understanding of the pathogenesis of IBD9 and have opened new opportunities for personalized medicine. Colonoids, or stem cell-derived, self-organizing tissue cultures from the colon, have been developed from both murine and human tissue in a process that allows stem cells located within intestinal crypts to propagate and be maintained indefinitely10. The stem cell niche in vivo relies on extracellular factors to support its growth, notably the canonical Wnt signaling and bone-morphogenic protein signaling pathways11. The addition of these factors promotes the health and longevity of colonoids but also drives the culture toward a stem cell-like state that is not reflective of the in vivo epithelial cellular architecture, which consists of both self-renewing and terminally differentiated cells12,13. While the functionality of the intestinal epithelium is dependent upon the continual crosstalk between the stem cell compartment and differentiated cells, the ability to have both in a colonoid culture system is fairly limited. Despite these limitations, the organoid culture system remains the gold standard to study the intrinsic properties of the epithelium ex vivo. Nonetheless, alternative culture strategies may need to be considered to answer the scientific question at hand.
It has been shown that mice on a continuous 7 day regimen of dextran sodium sulfate (DSS) develop both epithelial inflammation and barrier dysfunction14. Furthermore, mitochondrial biogenesis failure and metabolic reprogramming within the intestinal epithelium, which have been shown to be evident in human IBD, have also been captured in this DSS model of colitis15. However, our preliminary data demonstrate that the characteristics of mitochondrial biogenesis failure are not retained in colonoids derived from the crypts of DSS-treated animals (Supplementary Figure 1). Thus, alternative culture methods must be used when examining how inflammation drives epithelial changes during murine intestinal inflammation. Here, we outline a protocol we have developed that describes 1) how to isolate crypts from whole colonic tissue for the establishment of murine colonoids, 2) how to terminally differentiate this cell population to reflect the cell population as it stands in vivo, and 3) how to induce inflammation in this in vitro model. To study drug interactions within the inflamed epithelium, we have developed a protocol to establish 2-dimensonal (2D) monolayers from murine colonoids that can be basally treated with inflammatory mediators and apically treated with drug therapies.

저자 스포트라이트: 확립된 쥐 결장체에 대한 장 염증의 체외 상피 효과 연구  

확립된 쥐 콜로노이드에 대한 시험관 내 장 염증의 상피 효과 연구

Through our research, we aim to explore how murine colonoids could be modified from a conventional colonoid culture system, to study certain aspects of intestinal physiology and their alteration in the presence of an inflamed disease state. Studying barrier function and intestinal physiology in disease states like inflammatory bowel disease is limited when using a traditional murine colonoid culture system that reflects only stem cell physiology. Our protocol is advantageous in that it provides the reader with multiple techniques to study how inflammatory mediators affect the intestinal epithelium, by either terminally differentiating them, or creating a 2D monolayer system.In the future, our laboratory will concentrate on comprehending how the human colonoid culture system can be altered to investigate different aspects of intestinal physiology and diseases, particularly inflammatory bowel disease.

3D 장 결장 배양 시스템은 장 점막 항상성에 대한 상피의 본질적인 기여를 연구하는 데 매우 중요한 도구입니다. 설명된 프로토콜은 8주령에 C57BL/6J(WT) 마우스에서 크립트를 분리하고 여러 다운스트림 애플리케이션을 위해 조작할 수 있는 장기 콜로노이드 배양 시스템을 구축하는 방법에 대한 자세한 지침을 제공합니다. 기저막 매트릭스에서 크립트를 분리하고 도금할 때, 크립드는 명시야 현미경...

Watch this Scientific Journal Video about Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids at JoVE.com

우리는 3차원 결장류의 발달을 위한 쥐 결장 선와(miline colonic crypt)의 분리를 자세히 설명하는 프로토콜을 설명합니다. 확립된 콜로노이드는 염증성 챌린지를 받거나 상피 단층을 형성하도록 지시되기 전에 숙주 상피의 세포 구성을 반영하도록 최종적으로 분화될 수 있습니다.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

우리의 연구를 통해 우리는 쥐 콜로노이드가 기존의 콜로노이드 배양 시스템에서 어떻게 변형될 수 있는지 탐구하고, 장 생리학의 특정 측면과 염증이 있는 질병 상태가 있는 경우 그 변화를 연구하는 것을 목표로 합니다. 염증성 장 질환과 같은 질병 상태에서 장벽 기능 및 장 생리학을 연구하는 것은 줄기 세포 생리학만을 반영하는 전통적인 쥐 결장 배양 시스템을 사용할 때 제한적입니다. 우리의 프로토콜은 염증 매개체가 장 상피에 어떻게 영향을 미치는지 연구할 수 있는 여러 기술을 독자에게 제공한다는 점에서 유리합니다.앞으로 우리 연구실은 장 생리학 및 질병, 특히 염증성 장 질환의 다양한 측면을 조사하기 위해 인간 대장 배양 시스템을 어떻게 변경할 수 있는지 이해하는 데 집중할 것입니다.

studying-epithelial-effects-intestinal-inflammation-vitro-on

Research

JoVE Journal

Biology

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

738 조회수.  UPMC Children’s Hospital of Pittsburgh. 우리의 연구를 통해 우리는 쥐 콜로노이드가 기존의 콜로노이드 배양 시스템에서 어떻게 변형될 수 있는지 탐구하고, 장 생리학의 특정 측면과 염증이 있는 질병 상태가 있는 경우 그 변화를 연구하는 것을 목표로 합니다. 염증성 장 질환과 같은 질병 상태에서 장벽 기능 및 장 생리학을 연구하는 것은 줄기 세포 생리학만을 반영하는 전통적인 쥐 결장 배양 시스템을 사용할 ...

비디오: 저자 스포트라이트: 확립된 쥐 결장체에 대한 장 염증의 체외 상피 효과 연구  

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

연구

생물학

Intestinal Epithelial Monolayers Derived from Established Murine Colonoids

Begin by preparing all the reagents prior to the experiment and thaw the basement membrane matrix on ice. Then prepare the murine monolayer medium. Dilute the thawed basement membrane one to 20 with the cold murine monolayer medium, and keep it on ice.Place a 0.4 micrometer transparent cell culture insert, using sterile forceps, into a single well of a 24 well tissue culture plate. Grab a corner prong of the insert and transfer it into the well. Repeat until the appropriate number of cell culture inserts are available for culture.To cover the apical surface of each cell culture insert with diluted basement membrane matrix, place the tip of the P 200 pipette in the center of the insert and expel 150 microliters matrix. Then, transfer the plate to a sterile 37 degrees incubator with 5%carbon dioxide, for at least one hour. At the end of the incubation, rotate the 24 well plate at a 45 degree angle.Place a single finger on the corner of the prong to stabilize the insert. Gently place the P 200 pipette tip along the bottom side of the insert and remove the basement matrix. Remove any excess residue by washing each cell culture insert with 150 microliters of sterile DPBS without calcium or magnesium ions and allow them to dry in a biological safety cabinet for a minimum of 10 minutes.Once the inserts are dried, add 400 microliters of the murine monolayer medium to the bottom, and 75 microliters on top of the cell culture insert. Repeat until all the cell culture inserts are immersed. Leave the plate in the biological safety cabinet or the incubator until needed.Then remove the 24 well plate of mature intestinal colonoids from the incubator and place it under the biological safety cabinet. Aspirate the medium using sterile tips and wash each well with one milliliter of sterile DPBS at room temperature. Using sterile tips, remove the DPBS without calcium or magnesium ions.Digest each plug of the basement membrane matrix by adding 500 microliters of enzymatic dissociation reagent in each well to be passaged. To break up the plug, rotate the 24 well plate at a 45 degree angle. Place the tip of the pipette at the edge of the plug and gently dislodge it by pipetting each plug approximately six to 10 times.Place the 24 well plate back in a sterile incubator at 37 degrees with 5%carbon dioxide, for three to four minutes. After incubation, pipette the trypsin up and down five to seven times for each well under a biological safety cabinet. Transfer the dissociated colonoids from each well into a sterile 15 milliliter conical tube and bring up to a volume of 10 milliliters with an ice cold mouse wash medium.Next, centrifuge the partially digested colonoids at 300 G for five minutes at four degrees Celsius in a swinging bucket centrifuge. Under the biological safety cabinet, remove the supernatant from the pellet using a 10 milliliter pipette. Also, remove any remaining medium using a P 200 pipette.Then, re-suspend the colonoid pellet by adding 75 microliters of murine monolayer medium. Dispense 75 microliters of the colonoid suspension to the center of each cell culture insert. Gently pipette the suspension once or twice before adding it to the well to ensure uniform platting.Place the 24 well plate with cell culture inserts on a rotating platform for 10 minutes to allow for uniform dispersion across the cell culture insert, before placing the plate in a sterile 37 degree Celsius in 5%carbon dioxide incubator. At the end of the incubation, dislodged the unattached colonoids fragments by placing the tip alongside the inside of the cell culture and pipetting the medium three to five times with a P 200 pipette. Avoid disrupting the attached intestinal cells.Remove the medium and colonoid mixture immediately. Repeat until all the cell culture inserts have been cleaned. Now add 150 microliters of pre-warmed murine monolayer medium to the apical side of each cell culture insert.Add 400 microliters of warmed murine monolayer medium to each well of a new 24 well plate. Transfer the cell culture insert to the new plate by grabbing its prong using sterile forceps. Change the medium every other day until the desired confluency is attained.The enzymatically disrupted colonoids plated on the cell culture inserts initially appeared in cellular clusters ranging from 15 to 150 cells. On day three, the cells flattened out and slowly covered the cell culture insert, generally reaching confluency. Over the next couple of days, monolayers continued to grow and formed a continuous barrier that can be quantitatively measured.