Name: real-time measurement of epithelial barrier permeability in human intestinal organoids
Uploaded: 2017-12-18T15:00:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

저자 박사 스테파니 Spohn과 바젤 Abuaita organoid microinjection에 많은 유용한 토론에 감사 하 고 싶습니다. 주니어 장 줄기 세포 컨소시엄 (U01DK103141), 공동 연구 프로젝트는 국립 연구소의 당뇨병과 소화와 신장 질병 (NIDDK) 알레르기의 국가 학회 및 감염 증 (NIAID)에 의해 자금 지원 됩니다. 주니어 및 VBY NIAID 소설, 장의 질병 (NAMSED) 컨소시엄 (U19AI116482)에 대 한 대안 모델 시스템에 의해 지원 됩니다. DRH 지원 메커니즘의 미생물 병 인 교육 부여 알레르기의 국가 학회 및 감염 증 (NIAID, T32AI007528)와 임상, 번역 상 과학 상 미시간 연구소 임상 및 건강에 대 한 연구 (UL1TR000433)입니다.
완전 한 데이터 파일 및 데이터 분석 코드가이 원고에 사용 된 https://github.com/hilldr/HIO_microinjection에서 사용할 수 있습니다.

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저자 아무 경쟁 금융 관심사 또는 다른 충돌 공개할 수 있다.

이 프로토콜 실시간으로 hPSC 파생 HIOs 및 조직 파생 장 organoids의 microinjection 및 상피 장벽 침투성의 측정을 위한 범용 메서드를 설정합니다. 우리는 또한 우리의 접근 방식을 분석 하 고 이러한 메서드를 사용 하 여 생성 하는 데이터의 해석 설명 했다. 준 장 organoids의 성장 채택 모델 시스템16,20,,2128 및 장의 장벽 침투성으로 순수 관련 기능에 오랜 관심 결과3,,45,6, 우리는이 분야에서 일하고 다른 사람 적용 하 고 이러한 방법에 따라 만들 수 있을 것입니다 예상.
이 기술의 응용 프로그램에 중요 한 몇 가지 단계가 있습니다. 고품질 hPSC-또는 파생된 HIO 조직 microinjection와 광범위 한 실험 전에 설치 되어야 하는 조직에 접근. HIO macrostructure 조직 정체성과 세포 형태학은 높은 재현성 HIOs24를 생성 하기 위해 설립 된 방법론을 활용 하면 이기종, 크기와 모양, 변형 될 수 있습니다. 구형 HIOs 단일 반투명 루멘 이루어져 고 직경에서 약 1 m m microinjection 및 실시간 luminal 형광의 측정에 이상적입니다. 경우에 따라 microinjection는 하이 오의 붕괴 또는 주입 된 물자의 분명 한 누설의 결과로 실패 합니다. 실패 한 HIOs 표준 micropipette를 사용 하 여 사용자의 판단에 따라 잘 문화에서 제거할 수 있습니다. Microinjection 및 이미징 HIOs 선택할 때 이미징 플랫폼에서 사용할 수 있는 대물 렌즈를 고려 하십시오. 일반적으로 2-4 X 렌즈는 완전 한 HIO 형광 신호 캡처에 이상적 저전력 렌즈는 사용할 수 없거나 사용 가능한 HIOs 10 X 목표 사용할 수 있지만 &#60; 직경에서 1 m m. 이미징 소프트웨어 시간이 지남에 정의 된 지점에서 형광 이미지의 자동된 캡처 허용 해야 합니다.
이 프로토콜의 여러 수정 실험 요구 사항에 맞게 하기 위해 수 있습니다. 예를 들어 장벽 기능 테스트 결과 사용43 화합물의 분자 크기에 따라 있을 수 있습니다 그리고 그것은 다양 한 분자량의 dextran 준비를 테스트 하려면 적절 한 수 있습니다. 또한, 명시 이미징 조직25의 전반적인 구조적 무결성의 지표로 서 형광 이미징 뿐만 아니라 수행할 수 있습니다. 그것은 페니실린을 추가 해야 할 수 있습니다 라이브 박테리아25,28,31,32,,3344의 microinjection를 수행할 때와 스 또는 gentamicin HIO 문화 미디어 이전에 또는 microinjection 후에. microcapillary의 외부 세균성 문화 중단 작성 하는 동안 오염 될 것 이다 하 고이 하이 오 미디어에 전송 될 수 있습니다. 또는, microinjection HIOs 미디어 하지 않고, 세포 외 기질 (예를 들어, Matrigel)에 정지에 microinjection 완료 되는 미디어를 추가에 수행할 수 있습니다. 이 기질을 하는 오의 외부 얼굴 오염을 제한할 수 있습니다. 미생물 성장 분석 실험을 계획할 때 그것을 감속 하거나 방지 microinjected 생물의 성장을 피하기 위해 1-2 h 후 미디어에서 항생제를 제거 필요할 수 있습니다.
마지막으로, 모든 연구원 생체 외에서 영상에 적합 한 현미경 장비에 접근이 있을 것을 인식, 그것은 촬영 한 이미지에 형광 데이터를 수집 하기 위해이 프로토콜에서 설명 하는 절차를 적용할 수 있습니다 지적 하는 것이 중요 자동된 이미지 캡처 또는 환경 컨트롤 없이 표준 epifluorescent 현미경 검사 법을 사용 하 여 고정된 timepoints에서. 이 방법의 예는 레슬리와 황 외 에 의해 보고서에서 찾을 수 있습니다. 25, 누가 hPSC 파생 장 organoids, 그리고 Karve 및 Pradan 외. C. 남과 어울리지 않는 독 소 활동 검사 44, 유사한 hPSC 파생 장 organoids 라이브 대장균을 microinjected에 상피 장벽 침투성을 검사 들. 이미징 장비의 수동 조작 큰 변화와 어려움 형광 신호를 정상화 될 수 있습니다. HIOs 주입 FITC dextran의 수동 이미징 수행 때 고정된 배율, 형광 흥분 강도, 형광 강도 측정을 왜곡 하지 않도록 하는 실험 내내 노출 시간을 유지 하기 위해 필수적 이다.

장 상피 형성 선택적 장벽 영양소, H2O, 이온, 및 폐기물의 방향 전송 루멘 사이 다른 입자의 일반적인 보급 중재 교환 최소화 하면서 중재 하 고 중간 엽 조직 또는 혈액 공급1,2. 장 상피 방 벽의 일반적인 침투성 오래 간주 되었습니다 건강 및 질병3,,45,6의 속도 반영 하는 주요 기능 매개 변수 작은 분자 paracellular 공간 통해 상피에 걸쳐 확산 상피 장벽 침투성의 측정에서 지휘 될 수 있다 동물 모델7 과 lactulose의 섭취를 통해 인간의 환자8 , 위장, 그리고 이후의 컬렉션에 없는 특정 전송이 그리고 주변 혈액에 있는 농도 lactulose의 측정입니다. 장벽 기능 붙일 레이블된 탄수화물 등의 대체 섭취 마커 사용 가능한9,10도 있습니다. 이 방법은 장 상피 세포 배양 Transwell 지원11, 실험 제어에 대 한만 비보 의 가난한 예언자로 강평 되었다 또한 단순화 된 접근에 대 한 적응 되었습니다. 차별화 된 상피 하위 및 조직 구조12의 부재로 인해 침투성. 챔버를 사용 하 여 또 다른 접근 방식을 나타내고 전체 장 점 막 전 비보13상피 장벽 기능 측정에 대 한 허용. 이 기술의 응용 프로그램은 자주 조직의 가용성 및 조건13,14에 의해 제한 됩니다. 따라서 새로운 방법 생리 관련성 재현성 및 처리량의 균형은 필요 합니다.
Organogenesis 생체 외에서 최근 개발 업과 복잡 한 조직15,16,17의 역학에 대 한 정교한 플랫폼으로 3 차원 조직 배양 모델 시스템의 도입을 이끈 ,18,19,20,21,,2223. 특히, 인간 만능 줄기 세포 (hPSC) 파생 된 인간의 창 자 organoids (HIOs)19,24 호스트 미생물 상호 작용의 연구에 대 한 재현 하 고 실험적으로 세공 모델 시스템으로 등장 하 고 상피 장벽은 역학25,26,,2728. 마찬가지로, 인간의 조직 파생 된 organoids (일컬어 enteroids) 간단한 생 검 절차에서 파생 될 수 있다 고 연구 인간 생리학 및 질병15,,2930세공 시스템으로 사용할 수 있습니다. 인간의 장 organoids의 microinjection 실험적인 화합물25 의 배달에 대 한 허용 또는 미생물25,31,,3233 는 꼭대기에 상피 라이브 organoid 루멘의 표면입니다. 레슬리와 황 외. 25 는 최근 HIOs fluorescein isothiocyanate (FITC) 세균 독 소에 노출 다음 dextran을 표시와 함께 microinjected에 장벽 침투성 측정에이 기술을 적응.
이 프로토콜은 hPSC 파생 HIOs 및 형광 현미경 검사 법을 사용 하 여 조직 파생 HIOs 상피 장벽 침투성의 측정을 위한 가이드 것입니다. 사소한 수정, 그것은 또한 실험적인 화합물과 HIOs의 microinjection에 대 한 일반적인 입문서로 사용할 수 있습니다. 사용자가 microinjection 후 추가 또는 대체 다운스트림 응용 프로그램이 프로토콜을 보충 수 있습니다.

<table><tbody><tr><td>EGTA 0.5 M sterile (pH 8.0)</td><td>Bioworld</td><td>405200081</td><td></td></tr><tr><td>Cell matrix solution (Matrigel)</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Deltavision RT live cell imaging system</td><td>GE Life Sciences</td><td>29065728</td><td>http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/brands/deltavision/</td></tr><tr><td>Camera</td><td>GE Life Sciences</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>softWoRx Imaging software</td><td>GE Life Sciences</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>Biosafety cabinet</td><td>Labconco</td><td>Cell Logic+</td><td>http://www.labconco.com/product/purifier-cell-logic-class-ii-type-a2-biosafety-cabinets-2/4262</td></tr><tr><td>1X PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Advanced DMEM-F12</td><td>Life Technologies</td><td>12634-010</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>B27 supplement (50X)</td><td>Life Technologies</td><td>17504044</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>L-glutamine (100X)</td><td>Life Technologies</td><td>25030-081</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>HEPES buffer</td><td>Life Technologies</td><td>15630080</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Manipulator</td><td>Narshge</td><td>UM-3C</td><td></td></tr><tr><td>Micromanipulator</td><td>Narshge</td><td>UM-1PF</td><td></td></tr><tr><td>Pipette Holder</td><td>Narshge</td><td>UP-1</td><td>Alternate to Xenoworks pipette holder</td></tr><tr><td>Magnetic stand</td><td>Narshge</td><td>GJ-1</td><td></td></tr><tr><td>Dissecting scope</td><td>Olympus</td><td>SX61</td><td>Recommended scope, although other models are likely compatible</td></tr><tr><td>Olympus IX71 Fluorescent microscope</td><td>Olympus</td><td>IX71</td><td>Included with Deltavision system</td></tr><tr><td>CoolSNAP HQ2</td><td>Photometrics</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>Recombinant &lt;em&gt;C. difficile&lt;/em&gt; Toxin A/TcdA Protein</td><td>R&amp;amp;D Systems</td><td>8619-GT-020</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D Systems</td><td>236-EG</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>R-spondin 1</td><td>R&amp;amp;D Systems</td><td>4645-RS</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Noggin</td><td>R&amp;amp;D Systems</td><td>6057-NG</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410</td><td></td></tr><tr><td>FITC-dextran (4 kDa)</td><td>Sigma-Aldrich</td><td>46944</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>P-30</td><td></td></tr><tr><td>Nunc Lab-Tek II Chamber Slides</td><td>ThermoFisher Scientific</td><td>154526PK</td><td></td></tr><tr><td>Glass filaments</td><td>WPI</td><td>TW100F-4</td><td></td></tr><tr><td>Micropipette holder</td><td>Xenoworks</td><td>BR-MH2</td><td>Preferred device</td></tr><tr><td>Analog Tubing kit</td><td>Xenoworks</td><td>BR-AT</td><td></td></tr><tr><td>1/16 in clear ferrule</td><td>Xenoworks</td><td>V001104</td><td></td></tr><tr><td>1-1.2 mm O-ring</td><td>Xenoworks</td><td>V300450</td><td></td></tr></tbody></table>

real-time measurement of epithelial barrier permeability in human intestinal organoids

장 조직 생 검을 통해 얻은 또는 감독된 차별화를 통해 만능 줄기 세포에서 생성의 3D 문화 발전 장 점 막의 정교한 생체 외에서 모델에서 이어지고 있다. 이러한 신흥 모델 시스템을 활용 하 여 도구와 2D 문화 시스템 및 동물을 위해 개발 된 기술의 적응이 필요 합니다. 여기, 우리가 실시간으로 인간의 장 organoids에 상피 장벽 침투성을 측정 하기 위한 기술을 설명 합니다. 이것은 붙일 표시 된 dextran 및 epifluorescent 필터를 장착 한 거꾸로 한 현미경에 이미징의 microinjection에 의해 수행 됩니다. 장 organoids에 장벽 침투성의 실시간 측정이이 기술은 고정된 timepoint 이미징 접근에도 적용할 수 있지만 인간의 창 자 상피 조직에서 고해상도 임시 데이터의 생성을 촉진 한다. 이 프로토콜은 쉽게 상피 장벽 침투성 pharmacologic 대리인, 세균성 제품 또는 독 소, 또는 라이브 미생물에 노출 다음의 측정에 대 한 적응력입니다. 약간의 수정이이 프로토콜도 장 organoids의 microinjection에 대 한 일반적인 입문서로 사용할 수 있습니다와 사용자 추가 또는 대체 다운스트림 응용 프로그램 microinjection 다음이 프로토콜을 보완 하도록 선택할 수 있습니다.

HIOs 인간의 pluripotent 줄기 세포에서 분화 되었다 고 설명한19,24셀 매트릭스 솔루션으로 이전에 양식. 4 주 후에 문화, 여 HIOs microinjection 수 있도록 충분히 확장 했다. HIOs 4 kDa FITC 활용 된 dextran PBS 또는 Clostridium 남과 어울리지 않는 독 소 TcdA를 포함 하는 PBS에와 microinjected 했다. C. 남과 어울리지 않 는 HIOs25독 소 중재 상피 독성을 전시 하는 기회 위장 병원 체 이다. 긍정적인 컨트롤로 EGTA HIOs PBS와 FITC dextran 주입의 하위 집합에서 하이 오 문화 미디어에 추가 되었습니다. EGTA 말라 골격42의 급속 한 탈 중 합을 일으키는 칼슘 chelator입니다. FITC 형광은 제어 환경 챔버 내에서 라이브 영상 현미경에 실시간으로에서 모니터링 하 고 이미지에서 10 분 간격으로 체포 되었다.
영상 데이터의 post hoc 분석 FITC 형광 (그림 5)의 보존에 상당한 차이 밝혔다. 그러나 HIOs PBS와 주입 거의 모든 t 에 형광 신호 유지 = 0, 또한 주입 했다의 TcdA와 EGTA 치료 HIOs 8 시간 후 microinjection (그림에 의해 형광 강도에 상당한 감소를 전시 5A). 영상 데이터는 각 실험 조건 (그림 5B) 시간이 지남에 형광 강도 상대적인 변화를 나타내는 고해상도 데이터 집합을 생성 하는 모든 시간 지점에서 모든 HIOs에 대 한 계량 했다. 상피 침투성에 차이 각 치료 그룹 (표 1) FITC의 평균 제거 시간 (t1/2 )를 계산 하 고 t t1/2 그룹 간의 차이 비교 하 여 평가 했다 학생의 t를 사용 하 여-테스트. 제어 처리 HIOs 유지 16 시간 이상 FITC 형광 신호의 대부분 (t1/2 = 17 ± 0.3 h). EGTA 치료 크게 제어 HIOs 상대적인 FITC dextran 제거 시간 감소 (1/2 t = 2.6 ± 0.2 h; P = 10-8x 1.3). 일치 결과 이전에 게시25, TcdA의 microinjection 크게 증가 제어 치료 기준으로 상피 장벽 침투성 (t1/2 = 7.6 ± 0.6 h; P = 5.4 x 10-4). 따라서, 외부 (EGTA) 및 microinjected (TcdA) 화합물 HIOs에서 상피 장벽 침투성에 유도 하는 중요 한 변경 할 수 있다. 이러한 결과 상피 장벽 기능에 다양 한 약리학 적인 대리인, 대사 산물, 세균성 제품, cytokines, 성장 요인, 및 다른 화합물의 효과 평가 될 수이 접근을 사용 하 여 것이 좋습니다.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>치료</td>
			<td>반감기 (h)</td>
			<td>SEM (h)</td>
			<td>낮은 95 %CI (h)</td>
			<td>위 95 %CI (h)</td>
			<td>n</td>
		</tr>
		<tr>
			<td>제어</td>
			<td>17.11</td>
			<td>0.3</td>
			<td>16.45</td>
			<td>17.78</td>
			<td>11</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>2.58</td>
			<td>0.19</td>
			<td>1.78</td>
			<td>3.38</td>
			<td>3</td>
		</tr>
		<tr>
			<td>TcdA</td>
			<td>7.56</td>
			<td>0.63</td>
			<td>5.93</td>
			<td>9.18</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
표 1: 제거 시간을 의미 (t1/2) FITC-dextran HIOs에서 EGTA 또는 TcdA 치료에 대 한. 단위는 시간 후 microinjection.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56960/56960fig1.jpg"> 
그림 1: microinjector와 micromanipulator HIO microinjection에 대 한 기본 레이아웃. 이 이미지는 장비 HIOs의 microinjection를 수행 하는 데 사용의 완전 한 보완을 보여줍니다. 주요 구성 요소는 표시 하 고 주문 정보 자료 테이블에서 찾을 수 있습니다. <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56960/56960fig2.jpg"> 
그림 2: Micropipette 끌어당기는. 구리 난방 코일 hc, 탑 클램프 c1, 하단 클램프 (c2), 끌어당기는 팔 (pa), 열 선택 토글 (ht) 화살표로 식별 됩니다. 열 1과 풀에 대 한 올바른 설정은 빨간색 텍스트로 표시 됩니다. 삽입 된 난방에 대 한 준비가 제대로 탑재 유리 필 라 멘 트를 보여 줍니다. <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56960/56960fig3.jpg"> 
그림 3: 조직의 파생 HIO FITC dextran 주사의 대표 이미지. (A) 이미지 하이 오와 microinjection에 삽입 직전 유리 microcapillary의 위치를 보여주는. (B)는 조직에서 파생 된 인간 창 자 organoids FITC dextran의 microinjection 후의 명시 이미지. 형광 신호는 특정 필터 집합의 사용 없이 명백한 참고. 이 채색 microinjection 정밀 에이즈. 3 배 확대 <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig3large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56960/56960fig4.jpg"> 
그림 4: 라이브 이미징 워크플로. (A) 스크린샷 현미경 매개 변수를 설정, 슬라이드에는 HIOs 찾을 이미지 수를 각 하이 오에 대 한 무대의 위치를 저장 하는 데 필요한 단계를 설명. 각 위치에서 일정 한 간격 FITC 채널 캡처에 대 한 (B) 사전 영상 설정을 DV 포맷 데이터의 대답 (C) 후 이미징 수출에 시간 포인트 당 HIO 당 한 TIFF 이미지를 TIFF 이미지 파일 지정.<a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56960/56960fig5.jpg"> 
그림 5: 대표적인 결과. (A) 줄기 세포 파생 인간의 창 자 organoids (HIO) 2 mg/mL FITC dextran와 microinjected (4 kDa) 20 시간에 대 한 군데. HIOs 했다 또한 PBS (제어) 또는 Clostridium 남과 어울리지 않는 독 소 TcdA microinjected (12.8 ng / µ L) 2mm EGTA 외부 문화 미디어에 추가 된 취급. 4 X 확대입니다. (B) 줄거리 HIOs에서 시간이 지남에 의미 정규화 FITC 강도의 PBS (제어), TcdA, 또는 EGTA 취급. 오차 막대를 나타내는 S.E.M.과 n = 11 HIOs (제어), 3 HIOs (EGTA), 6 HIOs (TcdA). <a href="//ecsource.jove.com/files/ftp_upload/56960/56960fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>

Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

이 프로토콜 상피 장벽 침투성 형광 현미경 검사 법을 사용 하 여 인간의 창 자 organoids에서 실시간 다음 약리학 적인 치료에서의 측정을 설명 하 고 라이브 셀 현미경 검사 법.

인간의 장 Organoids에 상피 장벽 침투성의 실시간 측정

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

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Research

JoVE Journal

Developmental Biology

14.1K 조회수.  University of Michigan. 이 프로토콜 상피 장벽 침투성 형광 현미경 검사 법을 사용 하 여 인간의 창 자 organoids에서 실시간 다음 약리학 적인 치료에서의 측정을 설명 하 고 라이브 셀 현미경 검사 법.

비디오: Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

예 생체 내 창자 주머 니나는 위장 질환 모델에서 점막 투과성을 평가하는

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

연구

의학

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

인간 오가노이드와 장기 온 칩 기술을 결합하여 장 영역별 기능 모델링

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

생체공학

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

장 내 투과성 및 상피 세포 흘리기 성능을 평가하기 위한 인트라바이탈 현미경 기반 접근법

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

면역학 및 감염병학

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

정점 장내구 모델에서 루시퍼 옐로우를 사용한 장 투과성 측정

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

살아있는 오르가노이드의 장 장벽 고장 조사

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

태아 조직 유래 장내 투과성 테스트

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

생체 외에서 그리고 Vivo에서 접근 장 상피 세포 침투성을 결정 하

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

생물학

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

오가노이드 유래 상피 단층: 장 장벽 기능을 위한 임상적으로 관련된 시험관내 모델

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

소 장 오가노이드로부터 2D Monolayer Cultures 개발

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

저자 스포트라이트: 병원체 상호작용 연구를 위한 소 오가노이드의 2D 장 단층 모델 개발 및 특성화

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Small and Large Intestinal Monolayer interface를 사용한 소 오가노이드 기술의 발전

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...