Este estudo foi apoiado por subsídios SR1 CA 135.561, R01 CA151494, R01 CA174432 e P3013330 do National Institutes of Health. Gostaríamos de agradecer a Michele Houston, Elena Dhima e Dr. Anna Velcich por seus valiosos comentários no desenvolvimento do protocolo de isolamento cripta. Agradecemos também a Formação Diabetes e Centro do Albert Einstein College of Medicine apoiado pelo NIH P60DK20541 Research, e Dr. Michael Brownlee e Dr. Xue Liang-Du, que dirigem e operar as instalações do cavalo marinho, respectivamente.

Este estudo foi realizado de acordo com as recomendações do Guia para o Cuidado e Uso de Animais de Laboratório do National Institutes of Health. O protocolo foi aprovado pelo Comitê de Ética de Experimentação Animal do Albert Einstein College of Medicine. 1. Crypt Isolamento e Cultura <ol><li> O isolamento das criptas do intestino delgado: <ol><li> Isolar criptas intestinais de qualquer modelo de ratos de interesse. Euthanize os ratos com CO 2, seguida por deslocação cervical. </li><li> Abra o abdômen longitudinalmente e encher o intestino delgado (SI) com soro fisiológico frio gelo fosfato tamponada, PBS (-Ca 2+; Mg 2+) com 2x antibiótico-antimicótico (anti-anti). Rapidamente isolar intestino delgado. </li><li> Completamente dissecar livre de gordura mesentérica usando um bisturi. Tenha cuidado para não perfurar o tecido - em todos os momentos manter o tecido úmido com PBS gelado. Abra-se inteStine longitudinalmente e lavar cuidadosamente com PBS gelado. </li><li> Corte intestino delgado em duas seções e achatar usando uma ponta de algodão molhado, raspe fora as vilosidades usando uma lâmina pré-resfriada. Lave vigorosamente em PBS gelado várias vezes. </li><li> Incubar 3 min, em 20 ml de 1x PBS por 3 mM de ácido etilenodiaminotetracético (EDTA) /0.5 mM de ditiotreitol (DTT) para a dissociação não-enzimática do tecido. </li><li> Corte o tecido em pedaços pequenos (2-4 mm), usando uma lâmina de barbear em uma lâmina pré-resfriada. Transferem-se as peças em um tubo de 50 ml contendo 20 ml de PBS gelado. </li><li> Pipeta cima e para baixo suavemente 10x utilizando uma pipeta de 10 ml descartável estéril. Vamos fragmentos de tecido sedimentar por gravidade. Retirar o sobrenadante com uma pipeta. Repita mais três vezes; certifique-se sobrenadante é clara. </li><li> Adicionar 20 ml de PBS por EDTA 2 mM. Redemoinho e incubar a 4 ° C durante 30 min com agitação suave. </li><li> Deixe sedimento de tecido e descartar o sobrenadante. Adicionar 15 ml de PBS gelado e pipetacima e para baixo de 5x com uma pipeta de 10 ml. Deixe os fragmentos de tecido de sedimentos e recolher o sobrenadante como Fração 1 (F1). Repetir recolha F2-F5, mantidas separadamente cada fracção. </li><li> Adicionar 15 ml de PBS gelado e desta vez agitar vigorosamente à mão por 15 segundos. Colete F6 e repita para F7, F8. Se pedaços de tecido começar a flutuar, toque para ajudá-los a resolver. </li><li> Inspeccionar uma aliquota de cada fracção sob o microscópio. Reunir as fracções contendo criptas. </li><li> Passar as fracções reunidas através de um filtro de células de 70 mm de nylon, recolhendo criptas em um tubo de 50 ml. </li><li> Centrifugar a 100 xg durante 5 min a 4 ° C, descartar o sobrenadante e ressuspender o pellet em 10 ml de PBS gelado com criptas anti-anti 2x e transferência para um tubo de 15 ml. Repetir a lavagem mais uma vez. </li><li> Lave as criptas uma vez com 10 ml gelada ADF, Advanced DMEM / F-12 (Dulbecco &quot;s Modified Eagle Medium / Ham&quot; s F-12) - 2x anti-anti. </li><li> Lavam-se as criptas uma vez com 10 ml UmaDF - 1x anti-anti. </li><li> Conte as criptas utilizando um hemocitômetro. Ajustar o volume da solução cripta e transferir para um tubo de 1,5 ml, de modo que a concentração final quando cripta ressuspensas será 100-500 criptas por cada 50 uL de mistura de proteína gelatinosa (Matrigel). Centrifugar a 100 xg durante 5 min a 4 ° C e colocar a mistura em criptas proteína gelatinosa. </li><li> Placa 50 ul de suspensão cripta - mistura gelatinosa proteína por poço numa placa de 24 poços (área de crescimento: 2 cm2), colocando cuidadosamente gota a suspensão no centro de cada poço. Manter a placa numa CO 2, 37 ° C incubadora durante ~ 30 min, até que a suspensão se solidifica. </li><li> Adicionar 500 ul meios de gelo frio de cultura completo (Avançada DMEM / F12 com 1x anti - anti, 10 mM ácido 4- (2-hidroxietil) -1-piperazinoetanossulfónico (HEPES), GlutaMAX 1x, 1x suplemento B27, 1x suplemento N2, 1 mM N-acetil-L-cisteína (NAC), / ml de factor de crescimento epidérmico 50 ng (EGF), 100 ng / mlNoggin, 500 ng / ml de R-espondina). <ol><li> Reconstituir factores de crescimento em 0,1% de Albumina de Soro Bovino (BSA) em PBS. </li></ol></li></ol></li><li> Crypt organóide Cultura e Passage: <ol><li> Passagem Organóides criptas 14-21 dias após sementeira (ou conforme necessário) como se segue: <ol><li> Alterar mídia toda sexta-feira e segunda-feira. Às quartas-feiras, substitua metade da media com meio fresco. </li></ol></li><li> Remover o meio de cultura. Lava-se a amostra duas vezes com 500 ul de PBS a intervalos de 5 min. </li><li> Remover PBS e quebrar delicadamente até a mistura gelatinosa de proteína usando um P1000 estéril micro ponteira. </li><li> Adicionar 1 ml de meio de cultura completo para um único poço e ressuspender as Organóides em 1 ml de meio. </li><li> Gentilmente interromper as Organóides usando um P1000 por pipetagem cima e para baixo 20-30x (verifique sob o microscópio para a dissociação organóide adequada com um bom rendimento cripta até uma técnica consistente é desenvolvido). </li><li> Transferem-se as criptas para um tubo de microcentrífuga de 1,5 ml;centrifugar a 100 xg a 4 ° C, 5 min. </li><li> Lava-se a pelete duas vezes com 1 ml de meio de cultura completo. </li><li> Dividir a uma razão de 1: 3 ou 1: 6, conforme necessário, ressuspensão criptas em 50 ul por poço de Matrigel. Proceda como descrito acima (seção 1.1.16-18). </li></ol></li><li> Crypt congelamento organóide: <ol><li> Remover o meio de cultura. Lavar a amostra em 500 mL de PBS. </li><li> Dispersa-se a mistura gelatinosa de proteína utilizando uma ponta de pipeta P1000. </li><li> Adicionar 1 ml de mídia para um único poço e voltar a suspender as Organóides em 1 ml de mídia. </li><li> Cuidadosamente, quebre os Organóides usando um P1000 por pipetagem cima e para baixo 20-30x. </li><li> Transfira os Organóides a um tubo de 1,5 ml. Centrifuga-se a 100 xg a 4 ° C, 5 min. </li><li> Lava-se a pelete duas vezes com 1 ml de meio de cultura completo. </li><li> Criptas Ressuspender em 500 mL de mídia congelamento. </li><li> Criptas transferência para um tubo criogénico, colocar o tubo em um recipiente e loja de congelação de -80 ° C congelador overnight. </li><li> Transferir para criptas N2 líquido do tanque. <ol><li> Recuperar Organóides cripta congeladas pelo rápido degelo em um banho de água a 37 ° C. Lava-se com 500 ul meios de cultura completo uma vez, centrifugar a 100 xg, 5 min e ressuspender em mistura gelatinosa de proteína, e da cultura como descrito acima. Para uma melhor recuperação, adicione inibidor ROCHA (Y-27632) para a mídia congelamento. </li></ol></li></ol></li></ol> 2. Crypt organóide Metabolismo Ensaio <ol><li> 24 poços Preparação Plate: <ol><li> Isolar e contar criptas de acordo com o protocolo (ver secção 1.1 para isolamento cripta e 1.2 para a passagem cripta). </li><li> Criptas Ressuspender em mistura de proteína gelatinosa (100-200 criptas por 20 l). </li><li> Placa de suspensão mistura de proteínas cripta-gelatinoso numa placa de 24 poços de ensaio (certificar-se de que há pelo menos três repetições para cada amostra) e permitir que a suspensão para solidificar a 37 ° C num CO 2incubadora; em seguida, adicione 500 mL de meios de cultura completo. </li><li> Criptas Cultura (conforme descrito na seção 1.1) e observar ao microscópio até criptas crescer em Organóides totalmente desenvolvidos. </li></ol></li><li> Extracelular Flux Ensaio: <ol><li> Hidrato de cartucho durante a noite num não-CO 2 (0% de CO2), 37 ° C incubadora. </li><li> Remover o meio de cultura e lavar Organóides duas vezes com 500 ul de DMEM (sem: glucose, L-glutamina, piruvato de sódio e bicarbonato de sódio; e com: vermelho de fenol). Espere 5 min. </li><li> Adicionar 675 ul por poço meio de ensaio (DMEM com 2 mM de L-glutamina e 5 mM de D-glicose) a cada poço. </li><li> Verificar cripta Organóides morfologia microscópica para garantir que Organóides e a mistura gelatinosa proteína estão intactos depois as lavagens. Incubar 1 hora em um não-CO 2, 37 ° C incubadora. </li><li> Preparar 10 uM compostos injectáveis ​​(oligomicina, carbonil-cianeto de p-trifluoro-metoxi-fenil-hidrazona (FCCP), rotenone) no meio de ensaio. </li><li> Set-up do cartucho através do carregamento de 75 mL de 10 mM compostos injetáveis ​​nas portas da sequencialmente cartucho: Port A - oligomicina; Porto B - FCCP; e Port C - rotenona (As concentrações finais no ensaio serão de 1? M). </li><li> Incubar cartucho 30 min - 1 hr a 37 ° C numa incubadora com CO 2 não. </li><li> Ao mesmo tempo, ligue o XF Analyzer e criar o modelo de protocolo de ensaio. </li><li> Coloque cartucho e placa utilidade em XF Analyzer e executar &quot;calibrar&quot; cartucho. </li><li> Verifique Organóides cripta microscopicamente para se certificar de que eles estão ligados à mistura de proteína gelatinosa. </li><li> Célula de carga em placa de cultura de protocolo do ensaio instrumento e correr. </li></ol></li></ol>

<ol>
	<li>Jemal, 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> CA Cancer J Clin. 58, 71-96 (2008).</li><li>Slattery, M. L., Boucher, K. M., Caan, B. J., Potter, J. D., Ma, K. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eating+patterns+and+risk+of+colon+cancer.">Eating patterns and risk of colon cancer.</a> Am J Epidemiol. 148, 4-16 (1998).</li><li>Warburg, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer+cells.">On the origin of cancer cells.</a> Science. 123, 309-314 (1956).</li><li>Coles, N. W., Johnstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+metabolism+in+Ehrlich+ascites-carcinoma+cells.">Glutamine metabolism in Ehrlich ascites-carcinoma cells.</a> Biochem J. 83, 284-291 (1962).</li><li>Medina, M. A., Castro I, N. u. n. e. z. d. e. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutaminolysis+and+glycolysis+interactions+in+proliferant+cells.">Glutaminolysis and glycolysis interactions in proliferant cells.</a> Int J Biochem. 22, 681-683 (1990).</li><li>Anso, 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=Metabolic+changes+in+cancer+cells+upon+suppression+of+MYC.">Metabolic changes in cancer cells upon suppression of MYC.</a> Cancer Metab. 1, 7 (2013).</li><li>Malmgren, 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=Coordinate+changes+in+histone+modifications,+mRNA+levels,+and+metabolite+profiles+in+clonal+INS-1+832/13+&#946;-cells+accompany+functional+adaptations+to+lipotoxicity.">Coordinate changes in histone modifications, mRNA levels, and metabolite profiles in clonal INS-1 832/13 &#946;-cells accompany functional adaptations to lipotoxicity.</a> J Biol Chem. 288 (17), 11973-11987 (2013).</li><li>Whitehead, R. H., 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=Establishment+of+conditionally+immortalized+epithelial+cell+lines+from+both+colon+and+small+intestine+of+adult+H-2Kb-tsA58+transgenic+mice.">Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice.</a> Proc Natl Acad Sci U S A. 90 (2), 587-591 (1993).</li><li>Roig, A. 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=Immortalized+epithelial+cells+derived+from+human+colon+biopsies+express+stem+cell+markers+and+differentiate+in+vitro.">Immortalized epithelial cells derived from human colon biopsies express stem cell markers and differentiate in vitro.</a> Gastroenterology. 138 (3), 1012-1021 (2010).</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, 262-265 (2009).</li><li>Sato, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+self-organizing+mini-guts+from+a+single+intestinal+stem+cell:+mechanism+and+applications.">Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications.</a> Science. 340, 1190-1194 (2013).</li><li>Yilmaz, O. H., 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=mTORC1+in+the+Paneth+cell+niche+couples+intestinal+stem-cell+function+to+calorie+intake.">mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.</a> Nature. 486, 490-495 (2012).</li></ol>

Não há divulgações.

Testamos a taxa de consumo de oxigénio (OCR) e a taxa de acidificação extracelular (ECAR) de criptas isolados a partir de 8 meses de idade e ratinhos cultivadas em Organóides ex vivo. Após a medição da taxa basal, metabolismo cripta foi avaliada adicionando oligomicina, cianeto de carbonilo -p-Trifluormetoxifenilidrazona (FCCP) e rotenona, sequencialmente. Basal OCR e ECAR basal foram gravadas 0-29 min (Figura 2A e 2B). No dia 29 m...

O câncer colorretal (CCR) é a terceira principal causa de mortes relacionadas ao câncer nos Estados Unidos. Câncer de cólon esporádico - ou seja, que resulta mais tarde na vida (&gt; 50 anos de idade) e sem fatores predisponentes genéticos claras - contas para ~ 80% de todos os casos, com incidência fortemente influenciada por padrões alimentares a longo prazo 1,2. Estes tumores exibem um desvio metabólico no sentido para a glicólise oxidativo, conhecido como o efeito de Warburg, que pode, em parte, tornar as concentrações mais elevadas de blocos de construção celulares e energia disponíveis (através glutaminolysis) para permitir a conduzir e, talvez, as altas taxas de proliferação de células tumorais 3-5 . Estudos de câncer de cólon, bem como outros cânceres gastrointestinais, incluindo cânceres do intestino delgado fornecer informação importante sobre a causa da formação de tumores. Investigando as diferenças metabólicas entre os estados normais, pró-tumorigênicos e tumorigênicos de sistemas de órgãos gastrointestinais podem ajudar determination de risco relativo para o desenvolvimento do tumor, bem como a detecção precoce de neoplasia. Além disso, a compreensão do metabolismo bioenergético envolvendo respiração mitocondrial e da glicólise irá fornecer informações fundamentais sobre a forma como a fisiologia celular, o envelhecimento e as doenças estado perturba a homeostase intestinal. A utilização da tecnologia de ensaio para análise bioenergética fluxo extracelular pode avaliar as taxas de respiração mitocondrial e, simultaneamente, a glicólise em células que crescem em cultura em tempo real, 6,7. Até recentemente, os estudos in vitro de intestino delgado foram limitadas a linhas celulares derivadas de tumores benignos ou malignos ou 8,9 e não representam a fisiologia do epitélio intestinal normal e a influência do microambiente em que residem. Em 2009, Sato et al. 10 introduziu um sistema de cultura ex vivo para crescer tridimensional do mouse (3D) Organóides intestinais epiteliais, ou epithElial &quot;mini-coragem&quot;, adequado para investigações experimentais, diagnósticos e terapêuticos 10,11. Além disso, criptas isolados de ratos sob restrição calórica manter suas propriedades de crescimento alterados como Organóides em tais culturas 12. Em comparação com linhas celulares transformadas, culturas organï¿½de criptas pode ser usado para gerar os dados fisiologicamente relevantes que apresentem um modelo muito melhor para compreender o estado in vivo. Nós adaptamos a tecnologia de análise bioenergética para ensaio de metabolismo energético de Organóides cripta intestinal. Rato cripta intestinal Organóides foram cultivadas ex vivo para desenvolver os estudos do metabolismo energético organóide cripta apresentados. A taxa de consumo de oxigénio (OCR) e a taxa de acidificação extracelular (ECAR) de Organóides criptas foram medidos na ausência e na presença de dois diferentes inibidores metabólicos (oligomicina, rotenona) e um transportador de iões (carbonil-cianeto de p-Trifluormetoxifenilidrazona). A cripta orgaresposta metabólica noid a estes compostos químicos foram refletidos com sucesso através da mudança ECAR e valores de OCR. Estudos bioenergéticos celulares irá elucidar as interações recíprocas entre o estado metabólico e risco de doença e fenótipo em câncer, obesidade, diabetes, distúrbios metabólicos e doenças mitocondriais e ajudar métodos de rastreio antecipadamente com implicações diretas para a medicina translacional. Aqui, nós descrevemos um protocolo detalhado para isolar pequenas criptas intestinais e Organóides cripta cultura. Além disso, introduzimos um novo método de usar culturas organóide cripta para ensaios metabólicos.

<table border="0" cellpadding="0" cellspacing="0" width="1633">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">BD Matrigel Basement Membrane Matrix, GFR, Phenol Red-free, LDEV-free</td>
			<td style="width:229px;">BD Biosciences</td>
			<td style="width:135px;">356231</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">PBS (phosphate buffered saline), no magnesium, no calcium, pH 7.2</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">20012-027</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Advanced DMEM/F-12 (1X)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12634-028</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Dulbecco&#8242;s Modified Eagle&#8242;s Medium without glucose, L-glutamine, Phenol Red, sodium pyruvate and sodium bicarbonate</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">D5030</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Phenol Red sodium salt&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">P4758</td>
			<td style="width:456px;">Final Concentration 15 mg / L in DMEM (D5030) - step 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Antibiotic-Antimycotic, 100X, 100ml</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15240-062&#160;</td>
			<td style="width:456px;">Final Concentration 1 X or 2 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Penicilin-Streptomycin, liquid</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15140-122</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; GlutaMAX&#8482; supplement</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">35050061</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 1 M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15630-080</td>
			<td style="width:456px;">Final Concentration 10 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">N-Acetyl-L-Cysteine, 25g</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A9165-25G</td>
			<td style="width:456px;">Final Concentration 1 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">100X N-2 supplement, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">17502-048</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">50X B-27&#174; supplement minus Vitamin A, liquid</td>
			<td style="width:229px;">Invitrogen</td>
			<td style="width:135px;">12587-010</td>
			<td style="width:456px;">Final Concentration 1 X</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Mouse R-Spondin 1, CF, 50ug</td>
			<td style="width:229px;">R&#38;D Systems</td>
			<td style="width:135px;">3474-RS-050&#160;</td>
			<td style="width:456px;">Final Concentration 500 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine EGF, 100ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">315-09&#160;</td>
			<td style="width:456px;">Final Concentration 50 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recombinant Murine Noggin, 20ug&#160;</td>
			<td style="width:229px;">Peprotech</td>
			<td style="width:135px;">250-38</td>
			<td style="width:456px;">Final Concentration 100 ng / mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; L-glutamine, 200 mM</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">25030-081</td>
			<td style="width:456px;">Final Concentration 2 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Gibco&#174; Glucose powder</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">15023-021</td>
			<td style="width:456px;">Final Concentration 5 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Ambion&#174; 0.5 M EDTA (Ethylenediaminetetraacetic acid), pH 8.0</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">AM9260G</td>
			<td style="width:456px;">Final Concentration 3 mM for step 1.1.5; 2 mM for step 1.1.8&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">DTT (Dithiothreitol), 1M</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">P2325</td>
			<td style="width:456px;">Final Concentration 3 mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Albumin from bovine serum (BSA)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">A2058</td>
			<td style="width:456px;">0.1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">16000-044</td>
			<td style="width:456px;">1 % in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Recovery&#8482; Cell Culture Freezing Medium</td>
			<td style="width:229px;">Life Technologies</td>
			<td style="width:135px;">12648-010</td>
			<td style="width:456px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">ROCK inhibitor (Y-27632)&#160;</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">Y0503</td>
			<td style="width:456px;">Final Concentration 10 &#181;M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Oligomycin</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">O4876</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">C2920</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Rotenone</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">R8875</td>
			<td style="width:456px;">Final Concentration 1 &#181;M&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">Sodium hydroxide</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:135px;">221465</td>
			<td style="width:456px;">Final Concentration 0.1 N in PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:815px;">XF24 Extracellular Flux Analyzer (XF Analyzer)</td>
			<td style="width:229px;">Seahorse Bioscience</td>
			<td style="width:135px;"></td>
			<td style="width:456px;"></td>
		</tr>
	</tbody>
</table>

real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures

A mucosa do intestino delgado apresenta uma arquitetura repetitivo organizado em duas estruturas fundamentais: vilosidades, projetando para o lúmen intestinal e composto por enterócitos maduros, células caliciformes e células enteroendócrinas; e criptas, residentes proximal para a submucosa e muscularis, abrigando estaminais adultas e nas células progenitoras e células de Paneth madura, bem como do estroma e as células imunes do microambiente cripta. Até aos últimos anos, estudos in vitro do intestino delgado foi limitado a linhas celulares derivadas de tumores benignos ou malignos, e não representam a fisiologia do epitélio intestinal normal e a influência do microambiente em que residem. Aqui, demonstramos um método adaptado de Sato et al. (2009) para a cultura de Organóides das criptas intestinais do rato primárias derivadas de ratinhos C57BL / 6. Além disso, apresentamos a utilização de culturas organóide cripta para analisar o perfil metabólico cripta em tempo real por medidamento do consumo basal de oxigênio, a taxa glicolítica, a produção de ATP e da capacidade respiratória. Organóides manter as propriedades definidas por sua origem e reter os aspectos de sua adaptação metabólica refletido pelo consumo de oxigênio e taxas de acidificação extracelular. Estudos metabólicos em tempo real no presente cripta organï¿½de sistema de cultura são uma ferramenta poderosa para estudar o metabolismo da energia organï¿½de cripta, e como ela pode ser modulado por factores nutricionais e farmacológicas.

Organóides da cripta foram estabelecidos a partir de oito meses de idade camundongos C57BL / 6 alimentados com dieta purificada roedor Instituto Americano de Nutrição 76A (AIN76A). Organóides criptas intestinais podem ser cultivadas em cultura durante longos períodos de tempo a partir de um único cripta (Figura 1A, única seta vermelha). Organóides crescer para fora da cripta estruturas semelhantes em 18-20 dias em cultura (Figura 1B, setas vermelhas). Criptas foram passadas a ca...

Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Pequenas Organóides cripta intestinal cultivadas ex vivo, proporcionam um sistema de cultura de tecidos que recapitula o crescimento das criptas dependentes de células-tronco e seu nicho. Foi estabelecido um método para ensaiar o perfil metabólico em tempo real em Organóides das criptas do rato primária. Encontramos Organóides manter as propriedades fisiológicas definidas por sua origem.

Tempo real Análise do Perfil metabólico em<em&gt; Ex Vivo</em&gt; Culturas mouse Intestinal Crypt organóide

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

real-time-analysis-metabolic-profile-ex-vivo-mouse-intestinal-crypt

Research

JoVE Journal

Biology

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

16.1K visualizações.  Albert Einstein College of Medicine.  Pequenas Organóides cripta intestinal cultivadas ex vivo, proporcionam um sistema de cultura de tecidos que recapitula o crescimento das criptas dependentes de células-tronco e seu nicho. Foi estabelecido um método para ensaiar o perfil metabólico em tempo real em Organóides das criptas do rato primária. Encontramos Organóides manter as propriedades fisiológicas definidas por sua origem. 

Vídeo: Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Análise de Perfil eucarióticas Polyribosome

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Biologia

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

In vivo de imagens e tratamentos terapêuticos em um modelo de camundongo ortotópico de câncer de ovário

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Análise do Perfil Metabólico de embriões Zebrafish

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Metabólica Labeling e Membrana Fracionamento para Comparative Analysis proteômica de<em&gt; Arabidopsis thaliana</em&gt; Culturas suspensão de células

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Um Protocolo de vídeo de infecção retroviral na Primária Intestinal organóide Cultura

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Medindo a rigidez<em&gt; Ex Vivo</em&gt; Mouse aortas Usando Microscopia de Força Atômica

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Culturas primárias mistas de Murino Intestino Delgado destinadas ao estudo de Gut hormonal Secreção e imagens de células vivas de Células Enteroendócrinas

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Gravação em tempo real na Vivo de Arabidopsis sinais de cálcio durante a alimentação de insetos usando um biossensor fluorescente

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Um Ensaio Ex Vivo Choroid de Angiogênese Microvascular Ocular

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

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.

Monocameira epitelial derivada organoide: um modelo in vitro clinicamente relevante para a função da barreira intestinal

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