Name: using caco-2 cells to study lipid transport by the intestine
Uploaded: 2015-08-20T14:00:00.000+00:00
Duration: 7 min
Description: Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

This work was supported by the Seed Grant Award from California Northstate University College of Pharmacy (to AMN). The authors would like to thank California Northstate University College of Pharmacy for covering the publication cost of this article, and George Talbott for his help in editing this manuscript.

카코 2 셀 1. 유지 보수 <ol><li> 일반 조직 배양 접시를 사용하여 <ol><li> (FBS를 37 ℃의 수조에서 바이알을 배치하여 냉동 바이알로부터의 카코 -2 세포를 해동하고, 즉시 예열 된 성장 배지 (15 % 소 태아 혈청 10 ㎖를 함유 10cm 조직 배양 접시에 추가 ) 둘 베코의 수정 이글 중간 (DMEM))에서. <ol><li> 카코 -2 세포 50~70% 합류점에 도달 한 경우, 이들을 1 분할 : 그들은 분리 될 때까지 37 ℃에서 0.05 % 트립신 / 0.53 mM의 에틸렌 디아민 테트라 아세트산 (EDTA) 3 ㎖와 함께 세포를 배양하여 6 (15 분) . 세포 응집을 방지 부드러운 피펫 팅하여 세포를 여러 번 혼합합니다. 예열 성장 배지 10 ㎖를 함유 10cm 조직 배양 접시에 세포를 트립신 0.5ml를 추가. </li></ol></li><li> 부드럽게 좌우 방향으로 이어 전후 방향으로 요리를 여러 번 흔들어. 요리를 소용돌이 피으로는 불평등 한 세포 분산 될 수 있습니다. </li><li> 5 % CO 2와 함께 제공되는 37 ° C의 배양기에서 평평한 표면에 요리를 놓습니다. 경사면은 불평등 세포 분산 될 수 있습니다. </li><li> 배양을 시작한 후 성장 미디어에게 일을 변경합니다. </li><li> 세포를 모니터링하고 그들이 합류에 도달 하루를 기록한다. 셀들이 분할 일로부터 합류에 도달하는 것은 약 1 주일 정도 걸릴 것이다. </li><li> 세포가 합류에 도달하기 전에 일주일에 두 번 성장 미디어를 변경합니다. 세포가 합류에 도달하면, 매일 성장 미디어를 변경합니다. 세포가 7 일 후 합류 한 후 매일 성장 미디어를 변경합니다. 주 :이 13 일 후 합류 (그림 1) 때 세포 실험에 대한 준비가되어 있습니다. 세포는 결국 지질 단백질 생산에 덜 효과적이 될 것이기 때문에, 8 포스트 합류 13-17 - 일 사이에 세포를 사용합니다. 실험을 수행하는 방법에 대한 3 단계로 이동합니다.</li></ol></li><li> 투과 막 시스템을 사용하여 <ol><li> 단계 1.1.1.1에 기재된 투과성 막 인서트 카코 -2 세포를 시드. 위. 자신의 휴대 성장 (회복 기간) 일반적으로 느린 때문에 냉동 유리 병에서 세포를 사용하지 마십시오. 간단히, 미리 예열 성장 배지 10 ㎖를 포함하는 혀끝 챔버 (상단 구획)에 트립신 세포의 0.5 ml를 추가합니다. 또한, 기저 챔버 (낮은 구획)에 미리 예열 성장 배지 10 ㎖를 추가합니다. 참고 : 가장 좋은 방법은, 기저 챔버 먼저 다음 혀끝 챔버로 성장 매체를 추가 할 수 있습니다. 이전 용지를 제거 할 때, 혀끝의 미디어하기 전에 기저 용지를 제거합니다. 이 막 파열 될 수 있으므로 폴리 카보네이트 막 파고하지 마십시오. </li><li> 인하여 폴리 카보네이트 막을 통해 열악한 셀 가시성 유사한 밀도 정규 조직 배양 접시에 카코 -2 세포를 시드 및 셀 합류 판단이 접시를 사용한다. </li><li> 의에 따라TEPS 1.1.2-1.1.6 위. </li></ol></li></ol> 2. 유전자 과발현 <ol><li> 일반 형질 전환 방법을 사용하여 <ol><li> 상술 1.1.1.1.-1.1.4에 기술 된 바와 같이 조직 배양 접시에 카코 -2 세포를 시드. 주 : 세포가 40-50 %의 컨 플루 언트 될 때, 형질 감염이 될 준비가된다. </li><li> microcentrifuge 관에서 감소 된 혈청 미디어의 472 μL에 형질 전환 시약의 67 μl를 추가합니다. 튜브 벽 희석 형질 전환 시약의 접촉을 피하십시오. </li><li> pLL3.7 23 μg의이 형질 전환 시약 / 감소 혈청 미디어 혼합물에 녹색 형광 단백질 (eGFP는) 9 향상된 추가합니다. </li><li> RT에서 20 분 동안 혼합물을 인큐베이션. </li><li> DMEM에 10 % FBS 8 mL로 카코 2 세포의 성장 매체를 교체합니다. </li><li> 드롭 현명한 접시에 DNA / 형질 전환 시약 / 감소 혈청 미디어 혼합물을 추가합니다. </li><li> 조심스럽게 앞으로 바에서 요리를 여러 번 흔들어좌우 방향으로 이어 ckward 방향. </li><li> 37 ° C 배양기에서 접시를 놓습니다. </li><li> 배양을 시작한 후 성장 배지 10 ml의 하루 접시에 미디어를 교체합니다. </li><li> 유전자 발현을 모니터링합니다. <ol><li> 4 &#39;, 6-diamidino -2- 페닐 인돌 (DAPI) 얼룩없이 유전자 발현을 모니터한다. 형광 현미경을 사용하여, 녹색 세포의 비율을 결정한다. 비율은 형질 전환 효율을 나타낸다. </li><li> DAPI 염색과 유전자 발현을 모니터링합니다. <ol><li> 인산염 완충 식염수 (PBS)로 3 회 세포를 씻으십시오. </li><li> PBS (RT에서 10 분 동안 배양)에서 4 % 포름 알데히드를 10 ㎖를 첨가하여 세포를 고정. </li><li> PBS로 3 회 세포를 씻으십시오. </li><li> DAPI (500), 1 % BSA, 0.01 %의 디기 토닌 : 1을 함유하는 PBS의 8 mL를 실온에서 15 분 동안 어둠 속에서 세포를 배양한다. </li><li> PBS로 3 회 세포를 씻으십시오. </li><li> 유형광 현미경을 노래 블루 (그림 2 상단 패널) 된 것을 녹색 / 청색 상대적 세포의 수를 결정한다. 계산 된 비율은 형질 감염 효율을 나타낸다. </li></ol></li></ol></li></ol></li><li> 렌티 바이러스 과발현 시스템 (10)을 사용하여 <ol><li> 15cm 조직 배양 접시에 씨앗 인간 배아 신장 (HEK) 293T 세포 (DMEM에 10 % FBS 성장 매체로). 주 : 세포가 60-70 %의 컨 플루 언트 될 때, 형질 감염이 될 준비가된다. </li><li> microcentrifuge 관 (11)에 감소 된 혈청 매체의 1,133 μL에 형질 전환 시약 162 μl를 추가합니다. 튜브 벽 희석 형질 전환 시약의 접촉을 피하십시오. </li><li> 추가 24 pLL3.7 eGFP는 (또는 다른 구조물)의 μg의 15.6 레브 반응 요소 μg의 (RRE) REV 6.0 μg의 한 형질 감염 시약 / R로 포성 구내염 바이러스 당 단백질 8.4 μg의 (VSVG)educed 혈청 미디어 혼합물. </li><li> RT에서 20 분 동안 혼합물을 인큐베이션. </li><li> DMEM에 10 % FBS 16 mL로 성장 매체를 교체합니다. </li><li> 드롭 현명한 접시에 DNA / 형질 전환 시약 / 감소 혈청 미디어 혼합물을 추가합니다. </li><li> 부드럽게 좌우 방향으로 이어 전후 방향으로 요리를 여러 번 흔들어. 이 시점에서 분리 HEK293T 세포의 일부를 참조하는 정상이다. </li><li> 인큐베이터에서 접시를 놓습니다. </li><li> 배양 24 시간 후, 성장 배지 25 mL를 접시에 미디어를 교체합니다. </li><li> 다음 날, 렌티 바이러스 (제 컬렉션) 함유 증식 배지를 수집한다. </li><li> 단계를 반복 2.2.10 더 렌티 바이러스 (두 번째 컬렉션)를 수집합니다. </li><li> (5 분 동안 2,500 XG) 제 1 및 제 2 집합을 수영장, 원심 분리에 의해 세포 파편을 제거한다. </li><li> 일회용 병 톱 필터 (0.45 μm의 기공)과 컬렉션을 필터링하고,RT (JA-20 로터)에서 2 시간 동안 31,400 XG에서 컬렉션을 원심 분리하여 바이러스를 농축시킨다. 펠렛이있는 원심 분리 튜브의 하단을 표시한다. 상등액을 경사 분리하고, 약 15 분 동안 RT에서 반전 튜브를 떠난다. </li><li> 1X PBS 100 ㎕와 바이러스 - 함유 펠렛을 재현 탁. 거품을 피하십시오. </li><li> -80 ° C에서 집중 바이러스를 저장합니다. 동결 - 해동 사이클을 피하십시오. </li><li> 적정에 의해 카코 2 세포를 형질 도입에 필요한 바이러스의 최적의 양을 결정합니다. 비용을 절약하기 위해, 24 웰 플레이트 (물론 성장 미디어 / 0.3 ml)을 대신 적정 10cm 조직 배양 접시를 사용합니다. <ol><li> 농축 된 바이러스의 양을 증가시키는 추가의 (0, 1, 2, 5, 10, 25, 50 μL / 웰) 40~70% 합류 카코 -2 세포에 폴리 브렌 (최종 농도 = 5 μg의 / ㎖)으로 보충. 1.1.2에 설명 된 바와 같이 부드럽게 24- 웰 플레이트를 흔들어. </li><li> 자신의 유전자 발현 모니터 (단계 2.1.10를.) (그림 2 바닥4 패널). </li></ol></li><li> 형질 전환 유전자의 발현은 일반적으로 유지되어 있기 때문에, 미래의 실험 형질 세포를 유지하고 지속적으로 유전자 발현을 모니터한다 (단계를 2.1.10.). </li></ol></li></ol> 3. 지단백질 분비를 자극 <ol><li> 일반 조직 배양 접시를 사용하여 <ol><li> 올레산 50 ㎎, 레시틴 40 mg의 나트륨 타우로 콜산 48 mg을 혼합하여 100X 지질 혼합물을 제조한다. (8 요리에 대한 충분한) PBS로 900 μL에 볼륨을 가져와. 적극적으로 지질 혼합물을 소용돌이. </li><li> 성장 미디어의 89.1 ml의에 100X 지질 혼합물의 900 μl를 추가합니다. 혼합 및 지질 함유 미디어를 필터링 할 수 있습니다. 올레산, 레시틴, 및 나트륨 타우로 콜산의 최종 농도 (1X)는 2.0mm의 1.36 mm의 각각 1.0 mm의이다. </li><li> 세포에 지질 함유 미디어의 10 ML을 추가합니다. </li><li> (37)에서 4 시간 동안 지질 함유 매체를 사용하여 세포를 품어C 배양기를 °. </li><li> PBS로 3 회 세포를 씻으십시오. </li><li> 지질 단백질의 분비를 수집 세포 성장 배지 10 ㎖를 추가한다. </li><li> 37 ° C 배양기에서 2 시간 동안 품어. </li><li> 지질 단백질 함유 미디어를 수집합니다. </li></ol></li><li> 투과 막 시스템을 사용하여 <ol><li> 단계에 따라 3.1.1-3.1.2 지질 혼합물을 제조. </li><li> 혀끝 챔버 지질 함유 미디어 10 ㎖ 및 기저 챔버로 성장 매체의 10 ML을 추가합니다. </li><li> 37 ° C 배양기에서 4 시간 동안 품어. 기저 챔버에서 지질 단백질 함유 미디어를 수집합니다. </li></ol></li></ol> 4. 지단백 분리 (그림 3) <ol><li> 원심 분리 (5 분 2,000 XG)에 의해 지질 단백질 함유 매체에서 세포 파편을 제거합니다. </li><li> 50 ㎖ 튜브에 미디어를 가만히 따르다. </li><li> 미디어에 염화나트륨 5.95 g을 넣고. </li><li> SA의 경우mple이 TEM에 의해 분석되며, 지단백질의 무결성을 유지하기 위해 1, 프로테아제 억제제 칵테일 정제를 추가한다. </li><li> 물 23 ㎖의에 볼륨 (1.2 ㎍ / ㎖의 NaCl 농도 솔루션)를 준비한다. </li><li> 완전히 용질을 녹여. </li><li> 폴리 카보네이트 초 원심 분리기 튜브에 모든 용액을 경사 분리한다. </li><li> 부드럽게 0.5 ml의 물 (1.0 ㎍ / ㎖ 농도)와 1.2 ㎍ / ㎖ 밀도 솔루션을 오버레이. </li><li> 부피 중량이 아닌 튜브를 균형. </li><li> 조심스럽게 T865 회 전자에 튜브를로드합니다. </li><li> 4 ℃에서 24 시간 동안 429,460 XG에서 초 원심 분리기에서 샘플을 스핀. </li><li> 즉시 부드러운 피펫으로 최고 0.5 ml의 솔루션을 격리 할 것. 가능한 한 여전히 튜브를 유지합니다. </li></ol> 5. TEM 분석 <ol><li> (W / V) 및 6.0의 pH를 조절 2 % 포스 포 텅스텐 산을 5 ml를 준비한다. </li><li> (0.2 ㎛의 공극 크기) 주사기 필터와 2 % 인 텅스텐 산 필터. </li><li> 멸균 접시에 지질 단백질 샘플의 20 μl를 삭제합니다. </li><li> 다음 같은 접시에 샘플로 필터링 2 % 인 텅스텐 산의 20 μl를 삭제합니다. </li><li> 조심스럽게 무딘면이 샘플에 휴식과 EM 그리드를 놓습니다. </li><li> 1 분 동안 실온에서 품어. </li><li> 조심스럽게 그리드에서 샘플을 제거하기 위해 필터 종이에 EM 그리드의 측면을 누릅니다. </li><li> 조심스럽게 무딘면이 2 % 인 텅스텐 산에 휴식과 EM 그리드를 놓습니다. </li><li> 1 분 동안 실온에서 품어. </li><li> 조심스럽게 그리드에서 포스 산을 제거하기 위해 필터 종이에 EM 그리드의 측면을 누릅니다. </li><li> TEM을 사용하여, 지단백질 (도 4)의 이미지를 캡쳐. </li></ol>

<ol>
	<li>Drover, V. 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=CD36+deficiency+impairs+intestinal+lipid+secretion+and+clearance+of+chylomicrons+from+the+blood.">CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood.</a> J Clin Invest. 115 (5), 1290-1297 (2005).</li><li>Nauli, A. M., 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=CD36+is+important+for+chylomicron+formation+and+secretion+and+may+mediate+cholesterol+uptake+in+the+proximal+intestine.">CD36 is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine.</a> Gastroenterology. 131 (4), 1197-1207 (2006).</li><li>Nauli, A. M., Zheng, S., Yang, Q., Li, R., Jandacek, R., Tso, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+alkaline+phosphatase+release+is+not+associated+with+chylomicron+formation.">Intestinal alkaline phosphatase release is not associated with chylomicron formation.</a> Am J Physiol Gastrointest Liver Physiol. 284 (4), G583-G587 (2003).</li><li>Lo, C. -. M., 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=Why+does+the+gut+choose+apolipoprotein+B48+but+not+B100+for+chylomicron+formation.">Why does the gut choose apolipoprotein B48 but not B100 for chylomicron formation.</a> Am J Physiol Gastrointest Liver Physiol. 294 (1), G344-G352 (2008).</li><li>Jandacek, R. J., Heubi, J. E., Tso, P. <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,+noninvasive+method+for+the+measurement+of+intestinal+fat+absorption.">A novel, noninvasive method for the measurement of intestinal fat absorption.</a> Gastroenterology. 127 (1), 139-144 (2004).</li><li>Nauli, A. M., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+transport+as+a+potential+determinant+of+drug+bioavailability.">Intestinal transport as a potential determinant of drug bioavailability.</a> Curr Clin Pharmacol. 8 (3), 247-255 (2013).</li><li>Tso, P., Nauli, A., Lo, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enterocyte+fatty+acid+uptake+and+intestinal+fatty+acid-binding+protein.">Enterocyte fatty acid uptake and intestinal fatty acid-binding protein.</a> Biochem Soc Trans. 32 (Pt 1), 75-78 (2004).</li><li>Nauli, A. M., Sun, Y., Whittimore, J. D., Atyia, S., Krishnaswamy, G., Nauli, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chylomicrons+produced+by+Caco-2+cells+contained+ApoB-48+with+diameter+of+80-200+nm.">Chylomicrons produced by Caco-2 cells contained ApoB-48 with diameter of 80-200 nm.</a> Physiol Rep. 2 (6), (2014).</li><li>Rubinson, D. 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=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33 (3), 401-406 (2003).</li><li>Li, M., Husic, N., Lin, Y., Snider, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system.">Production of lentiviral vectors for transducing cells from the central nervous system.</a> J Vis Exp. (63), e4031 (2012).</li><li>Pottekat, 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=Insulin+biosynthetic+interaction+network+component,+TMEM24,+facilitates+insulin+reserve+pool+release.">Insulin biosynthetic interaction network component, TMEM24, facilitates insulin reserve pool release.</a> Cell Rep. 4 (5), 921-930 (2013).</li><li>Van Greevenbroek, M. M., van Meer, G., Erkelens, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+saturated,+mono-,+and+polyunsaturated+fatty+acids+on+the+secretion+of+apo+B+containing+lipoproteins+by+Caco-2+cells.">Effects of saturated, mono-, and polyunsaturated fatty acids on the secretion of apo B containing lipoproteins by Caco-2 cells.</a> Atherosclerosis. 21 (1), 139-150 (1996).</li><li>Levy, E., Yotov, W., Seidman, E. G., Garofalo, C., Delvin, E., M&#233;nard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caco-2+cells+and+human+fetal+colon:+a+comparative+analysis+of+their+lipid+transport.">Caco-2 cells and human fetal colon: a comparative analysis of their lipid transport.</a> Biochim Biophys Acta. 1439 (3), 353-362 (1999).</li><li>Luchoomun, J., Hussain, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+and+secretion+of+chylomicrons+by+differentiated+Caco-2+cells.+Nascent+triglycerides+and+preformed+phospholipids+are+preferentially+used+for+lipoprotein+assembly.">Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly.</a> J Biol Chem. 274 (28), 19565-19572 (1999).</li></ol>

The author has nothing to disclose.

본 논문에서는 사용될 수 개의 시스템 카코 -2 세포, 즉, 정규 조직 배양 접시 투과 막을 기재되어 유지한다. 투과성 막 시스템을 사용할 때의 이점은 정점의 분리 및 기저 외측 구획하고, 지질 혼합물을 배양하는 동시에 지단백질 분비를 수집하는 기능을 포함한다. 다만, 투과 막 인서트 비싸고, 그 폴리 카보네이트 막 좋은 셀 가시성을 허용하지 않는다. 이 시험 관내 모델의 장점 중 하나는...

4 -식이 지방과 지용성 약물과 비타민의 장내 흡수 연구는 림프 누공 모델 1을 사용하여 생체 내에서 수행 할 수있다. 그러나 관련된 수술 기법은 도전뿐만 아니라 비용이 많이 드는되지 않습니다. 생체 이용 될 수 분변 분석에 기반 방식이지만, 이들은 위장관 2,5- 의해 흡수 퍼센트를 결정 주로 사용된다. 이 문서에 설명 된 체외 모델은 비용 효과적이며, 관련 기술은 틀림없이 덜 도전이다. 유전자 변형 연구는 시간이 많이 걸리는 사람들이 체외 모델을 사용하여 수행 할 때 더 경제적이고 저렴합니다. 장 세포에 의해 흡수되는 지용성 물질 지단백질 -6,7-로 포장되어 있기 때문에, 리포 단백질을 생산하기 위해이 시험 관내 모델의 효과는 매우 중요하다. 두 가지 주요 intestinaL 지단백질은 카일로 마이크론과 매우 저밀도 지단백질 (VLDL)입니다. 지질은 위장 루멘에 풍부하게 존재하는 경우 직경이 80 nm 이상과 지질 단백질로 정의 카일로 마이크론은, 소장에 의해 엄격하게 생산된다. 그들이 가장 큰 지단백질이기 때문에, 카일로 마이크론은 생각할 가장 효율적인 지질 수송이다. 킬로 미크론 (8)을 제조 할 수있는 이러한 시험 관내 모델은,식이 지방 흡수에 의한 소화관 지용성 비타민의 흡수 및 지용성 약물 경구 생체 이용률을 연구하는데 사용될 수있다. 지단백질 분획에 지용성 분자, 비타민, 또는 약물의 존재는 소장 흡수하여 자신의 지표이다. 전술 한 바와 같이,이 모델은 경구 지용성 약물 생체 이용율 (6)을 향상 시키는데 사용될 수있다. 이 논문은 카코 2 세포 투과 막 또는 일반 조직 배양 접시, 어떻게 지질 마일에서 유지되어야한다 방법을 설명합니다지질 단백질 생산을 자극 xture는 렌티 바이러스 발현 시스템이 효율적인 과발현을 달성하기 위해 이용 될 수있는 방법을 준비해야하며, 절연 지단백질은 어떻게 분석되어야한다.

<table><tbody><tr><td>DMEM</td><td>VWR</td><td>16750-112</td><td>Pre-warm the growth media in individual tissue culture dish before adding cells</td></tr><tr><td>FBS</td><td>Fisher</td><td>3600511</td><td>We do not heat inactivate our serum</td></tr><tr><td>Trypsin</td><td>VWR</td><td>45000-660</td><td>Cells can be washed with PBS prior to trypsin treatment&amp;nbsp;</td></tr><tr><td>Permeable membrane system</td><td>Fisher</td><td>7200173</td><td>10-cm dish, 3-mm pore size, polycarbonate membrane</td></tr><tr><td>10 cm dish</td><td>Fisher</td><td>08-772-E</td><td>Tissue culture dish</td></tr><tr><td>15 cm dish</td><td>Fisher</td><td>08-772-24</td><td>Tissue culture dish</td></tr><tr><td>24 well plate</td><td>Fisher</td><td>12565163</td><td>Tissue culture plate</td></tr><tr><td>Lipid-based transfection reagent</td><td>Fisher</td><td>PRE2691</td><td>Can be substituted with other transfection reagent</td></tr><tr><td>Reduced serum media</td><td>Invitrogen</td><td>11058021</td><td>For transfection</td></tr><tr><td>pLL3.7 eGFP</td><td>Addgene</td><td>11795</td><td>&lt;a href=&quot;https://www.addgene.org/11795/&quot;&gt;https://www.addgene.org/11795/&lt;/a&gt;</td></tr><tr><td>Bottle-top filter</td><td>Fisher</td><td>9761120</td><td>0.45 mm pore</td></tr><tr><td>Polybrene</td><td>Fisher</td><td>NC9840454</td><td>10 mg/ml</td></tr><tr><td>Oleic acid</td><td>Sigma</td><td>01383-5G</td><td>Prevent freeze-thaw cycle</td></tr><tr><td>Lecithin&amp;nbsp;</td><td>Fisher</td><td>IC10214625</td><td>Egg lecithin&amp;nbsp;</td></tr><tr><td>Sodium taurocholate</td><td>Fisher</td><td>NC9620276</td><td>Product discontinued; alternative catalog number: 50-121-7956</td></tr><tr><td>Protease inhibitor cocktail tablet (EDTA-free)</td><td>Fisher</td><td>5892791001</td><td>Used mainly for samples that need TEM analysis</td></tr><tr><td>Polycarbonate ultracentrifuge tube</td><td>Fisher</td><td>NC9696153</td><td>Reusing it multiple times will collapse the tube</td></tr><tr><td>Lid for ultracentrifuge tube</td><td>Fisher</td><td>NC9796914</td><td>A tool is required to remove the tube/lid from rotor</td></tr><tr><td>Syringe</td><td>Fisher</td><td>50-949-261</td><td>Disposable</td></tr><tr><td>Syringe filter</td><td>Fisher</td><td>09-719C</td><td>Pore size = 0.2 mm; nylon</td></tr><tr><td>Phosphotungstic acid</td><td>Fisher</td><td>AC208310250</td><td>For preparing 2% phosphotungstic acid, pH 6.0</td></tr><tr><td>Tweezer</td><td>Fisher</td><td>50-238-62</td><td>Extra fine and strong tips</td></tr><tr><td>Formvar/carbon grid</td><td>Fisher</td><td>50-260-34</td><td>Formvar/carbon film square grid 400 Copper</td></tr></tbody></table>

using caco-2 cells to study lipid transport by the intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

1 표시 정상 13 일 이후 합류 카코 2 세포를 그림. 돔형 구조 및 세포 내 지질 액 적의 모습 분화 카코 -2 세포의 특성이다. 카코 2 세포를 파종시에 동일하게 분산되지 않을 때, 그들은 덩어리와 접시의 특정 지역에서 자라다 것이다; 어떤 세포없이 접시에 몇 가지 영역이 될 것입니다. 소용돌이 경사면에 접시를 배치하는 것은 피해야한다. 이 후 합류 카코 2 세포가 새로운 ?...

Watch this Scientific Journal Video about Using Caco-2 Cells to Study Lipid Transport by the Intestine at JoVE.com

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

소장에 의해 지질 전송을 연구하기 위해 카코 2 셀을 사용하여

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

using-caco-2-cells-to-study-lipid-transport-by-the-intestine

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.7K 조회수.  California Northstate University. Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

비디오: Using Caco-2 Cells to Study Lipid Transport by the Intestine

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

콜레스테롤 유출 분석

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

연구

의학

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

모델 및 세포 장벽에 걸쳐 약물 전달 시스템의 전송을 평가하는 방법

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

생체공학

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

Lipophagy를 통한 지질 방울 이화작용의 조절 탐구

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This ...

암 연구학

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

X-선 사이 누화를 조사 하는 공동 문화 방법을 반구 Caco-2 셀 및 PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

면역학 및 감염병학

Lipid Exchange Assay in Living Cells

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane&#39;s propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at&#160;the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

살아있는 세포의 지질 교환 분석

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the ...

생화학

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

인간 장 유기체에서 지질 방울 형성의 특성화 및 정량화를 위한 형광 기반 분석

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Proteoliposome 기반 유출 분석은 CL의 단일 분자의 속성을 확인하는<sup&gt; -</sup&gt; 채널 및 전송기

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

생물학

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

내피 세포 트랜스사이토시스 분석법(Endothelial Cell Transcytosis assay)은 내부 혈액-망막 장벽 투과성을 평가하기 위한 시험관 내 모델로서 분석합니다.

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

방법은 3D로 재배 원주민 소장과 카코 2 세포에서 상피 전송 단백질의 기능과 발현을 연구하는

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

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.

소장에 의해 지질 전송을 연구하기 위해 카코 2 셀을 사용하여

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