Name: using caco-2 cells to study lipid transport by the intestine
Uploaded: 2015-08-20T14:00:00.000+00:00
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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.

1. A manutenção das células Caco-2 <ol><li> Usando regulares pratos de cultura de tecidos <ol><li> Descongelar as células Caco-2 a partir de um frasco congelado colocando o frasco num banho de água a 37 C °, e adicioná-los imediatamente para uma placa de 10 cm de cultura de tecidos contendo 10 ml de meio de crescimento (15% de soro bovino fetal pré-aquecido (FBS ) em Dulbecco Modified Eagle Médium (DMEM)). <ol><li> Quando as células Caco-2 atingiram 50-70% de confluência, dividi-los 1: 6 por incubação das células com 3 ml de 0,05% de tripsina / 0,53 mM de ácido etilenodiaminotetracético (EDTA) a 37 ° C até que eles são destacados (15 min) . Para evitar aglomeração de células, mistura as células várias vezes por pipetagem suave. Adicionar 0,5 ml das células tripsinizadas a uma placa de 10 cm de cultura de tecidos contendo 10 ml de meio de crescimento pré-aquecido. </li></ol></li><li> Agite ligeiramente os pratos várias vezes para a frente e para trás seguido por uma direção para a esquerda e para a direita. Evite rodar os pratosuma vez que pode resultar numa dispersão celular desigual. </li><li> Colocar as cápsulas sobre uma superfície plana em uma incubadora a 37 ° C fornecido com 5% de CO 2. Superfícies inclinadas também podem resultar numa dispersão celular desigual. </li><li> Troque a mídia de crescimento de um dia após o início da incubação. </li><li> Monitorar as células e gravar no dia em que chegar a confluência. Isso levará cerca de uma semana para as células para alcançar confluência do dia eles estão divididos. </li><li> Mudar o meio de crescimento, duas vezes por semana antes de as células atingirem confluência. Uma vez que as células atingiram a confluência, muda os meios de crescimento a cada dois dias. Depois que as células são 7-dias pós-confluentes, alterar a mídia de crescimento diário. Nota: As células estão prontas para experiências quando eles são de 13 dias pós-confluentes (Figura 1). Uma vez que as células acabará por se tornar menos eficaz na produção de lipoproteínas, utilização de células que se encontram entre 13 a 17 dias pós-confluentes 8. Vá para a etapa 3 sobre como conduzir o experimento.</li></ol></li><li> Usando o sistema de membrana permeável <ol><li> Semente as células Caco-2 no inserto membrana permeável tal como descrito no passo 1.1.1.1. acima. Evite o uso de células a partir de um frasco congelado porque o seu crescimento celular é normalmente mais lenta (período de recuperação). Resumidamente, adicionar 0,5 ml das células tripsinizadas para a câmara de apical (compartimento superior) que contém 10 ml de meio de crescimento pré-aquecido. Além disso, adicionar 10 ml de meio de crescimento pré-aquecido para a câmara basolateral (compartimento inferior). Nota: Para as melhores práticas, adicione os meios de crescimento para a câmara apical primeiro e depois para a câmara basolateral. Ao remover a velha mídia, remova a mídia basolateral perante a mídia apical. Evitar picar a membrana de policarbonato, uma vez que pode romper a membrana. </li><li> Devido à visibilidade pobre célula através da membrana de policarbonato, semear as células Caco-2 em um prato de cultura de tecidos regular com densidade semelhante, e usar este prato para julgar a confluência celular. </li><li> Siga sPTE 1.1.2-1.1.6 acima. </li></ol></li></ol> 2. Gene Overexpression <ol><li> Usando a abordagem de transfecção regulares <ol><li> Semente as células Caco-2 em um prato de cultura de tecidos como descrito no 1.1.1.1.-1.1.4 supra. Nota: Quando as células são cerca de 40-50% confluentes, eles estão prontos para ser transfectada. </li><li> Adicionar 67 ul de reagente de transfecção de 472 ul de meio de soro reduzido os num tubo de microcentrífuga. Evitar o contacto do reagente de transfecção não diluída com a parede do tubo. </li><li> Adicionar 23 g de pLL3.7 reforçada proteína verde fluorescente (EGFP) 9 para o reagente de transfecção / meio de soro reduzido mistura. </li><li> Incubar a mistura durante 20 min à temperatura ambiente. </li><li> Substituir o meio de crescimento das células Caco-2 &#39;com 8 ml de 10% de FBS em meio DMEM. </li><li> Adicionar a mistura de ADN / reagente de transfecção / meio de soro reduzido, gota a gota para o prato. </li><li> Agite ligeiramente os pratos várias vezes em uma frente e badirecção ckward seguido por um sentido para a esquerda e para a direita. </li><li> Coloque o prato no 37 ° C incubadora. </li><li> Substitua a mídia no prato com 10 ml de meio de crescimento por dia após o início da incubação. </li><li> Monitorar a expressão do gene. <ol><li> Monitorar a expressão de genes sem 4 &#39;, 6-diamidino-2-fenilindole (DAPI) coloração. Usando um microscópio de fluorescência, determinar a percentagem de células que são verdes. A percentagem representa a eficiência da transfecção. </li><li> Monitorar a expressão gênica com coloração DAPI. <ol><li> Lavam-se as células com solução salina tamponada com fosfato (PBS) três vezes. </li><li> Fixar as células por adição de 10 ml de formaldeído a 4% em PBS (10 minutos de incubação à TA). </li><li> Lavam-se as células com PBS três vezes. </li><li> Incubar as células no escuro durante 15 min à temperatura ambiente com 8 ml de PBS contendo 1: 500 DAPI, 1% de BSA, 0,01% de digitonina. </li><li> Lavam-se as células com PBS três vezes. </li><li> VCcante um microscópio de fluorescência, determinar o número de células que são verde / azul em relação àqueles que são azuis (Figura 2 painel superior). A percentagem calculada representa a eficiência da transfecção. </li></ol></li></ol></li></ol></li><li> Usando o sistema de sobre-expressão de lentivírus 10 <ol><li> Rim embrionário (HEK) Semear as células em um humanos 293T 15 centímetros prato de cultura de tecidos (10% de FBS em DMEM como seu meio de crescimento). Nota: Quando as células são cerca de 60-70% confluentes, eles estão prontos para ser transfectada. </li><li> Adicionar 162 ul de reagente de transfecção de 1133 ul de os meio de soro reduzido em um tubo de microcentrífuga de 11. Evitar o contacto do reagente de transfecção não diluída com a parede do tubo. </li><li> Adicionar 24 g de pLL3.7 eGFP (ou outra construção), 15,6? G de elemento de resposta a Rev (RRE), 6,0 ug de REV, e 8,4? G de glicoproteína do vírus da estomatite vesicular (VSVG) para o reagente de transfecção / reduced mistura meio de soro. </li><li> Incubar a mistura durante 20 min à temperatura ambiente. </li><li> Substituir o meio de crescimento com 16 ml de 10% de FBS em DMEM. </li><li> Adicionar a mistura de ADN / reagente de transfecção / meio de soro reduzido, gota a gota para o prato. </li><li> Agite ligeiramente os pratos várias vezes para a frente e para trás seguido por uma direção para a esquerda e para a direita. É normal ver algumas das células HEK293T destacadas neste momento. </li><li> Coloque o prato na incubadora. </li><li> Após 24 horas de incubação, substituir a mídia no prato com 25 ml de meio de crescimento. </li><li> No dia seguinte, recolher o meio de crescimento contendo o lentivírus (primeira coleção). </li><li> Repita o passo 2.2.10 para coletar mais lentivírus (a segunda cobrança). </li><li> Reúnem-se os primeiro e o segundo recolha, e remover os detritos celulares por centrifugação (2500 xg durante 5 min). </li><li> Filtrar a coleção com um filtro descartável garrafa-top (0,45 poro), econcentra-se o vírus por centrifugação a recolha a 31.400 xg durante 2 horas à temperatura ambiente (rotor JA-20). Marcar a parte inferior do tubo de centrifugação em que o pelete é localizado. Decantar o sobrenadante, e deixar o tubo invertido à TA durante cerca de 15 min. </li><li> Ressuspender o sedimento contendo vírus com 100 ul de PBS 1X. Evite bolhas. </li><li> Armazenar o vírus concentrada a -80 ° C. Evite ciclos de congelamento e descongelamento. </li><li> Determinar a quantidade óptima de vírus necessária para transduzir as células Caco-2 por meio de titulação. Para poupar custos, utilizar uma placa de 24 poços (0,3 ml de meio de crescimento / poço) em vez de um 10 centímetros prato de cultura de tecidos para a titulação. <ol><li> Adicionar quantidades crescentes do vírus concentrado (0, 1, 2, 5, 10, 25, e 50 uL / ​​poço) suplementado com polibreno (concentração final = 5 ug / ml) para a 40-70% confluentes células Caco-2. Agitar a placa de 24 poços como descrito em suavemente 1.1.2. </li><li> Monitore sua expressão gênica (Passo 2.1.10.) (Figura 2 fundo4 painéis). </li></ol></li><li> Desde a expressão de transgene é geralmente sustentado, manter as células transduzidas para experiências futuras e monitorar continuamente a sua expressão gênica (Passo 2.1.10.). </li></ol></li></ol> 3. Estimular o Lipoproteína Secreção <ol><li> Usando o prato de cultura de tecido normal <ol><li> Prepara-se uma mistura de lípidos de 100X através da mistura de 50 mg de ácido oleico, 40 mg de lecitina, e 48 mg de taurocolato de sódio. Levar o volume a 900 mL com PBS (o suficiente para pratos 8). Vortex vigorosamente a mistura de lípidos. </li><li> Adicionar 900 ul da mistura de lípidos 100X em 89,1 ml de meio de crescimento. Misturar e filtrar a mídia contendo lípidos. As concentrações finais (1x) de ácido oleico, lecitina, e taurocolato de sódio é 2,0 mM, 1,36 mM e 1,0 mM, respectivamente. </li><li> Adicionar 10 ml de meio contendo lípido para as células. </li><li> Incubam-se as células com meio contendo o lípido durante 4 horas num 37° C incubadora. </li><li> Lavam-se as células com PBS três vezes. </li><li> Adicionam-se 10 ml do meio de crescimento para as células para recolher a secreção de lipoproteínas. </li><li> Incubar durante 2 horas num banho a 37 ° C incubadora. </li><li> Recolhe a mídia contendo lipoproteína. </li></ol></li><li> Usando o sistema de membrana permeável <ol><li> Siga os passos 3.1.1-3.1.2 para preparar a mistura de lípidos. </li><li> Adicionar 10 ml de meio contendo lípido para a câmara de apical e 10 ml de meio de crescimento para a câmara basolateral. </li><li> Incuba-se durante 4 horas numa incubadora de 37 ° C. Recolhe a mídia contendo lipoproteína na câmara basolateral. </li></ol></li></ol> 4. Isolamento de lipoproteína (Figura 3) <ol><li> Remover os restos celulares do meio contendo lipoproteína por centrifugação (2000 xg durante 5 min). </li><li> Decantar a mídia em um tubo de 50 ml. </li><li> Adicionar 5,95 g de cloreto de sódio para a mídia. </li><li> Se a sample serão analisados ​​pelo MET, adicionar um inibidor da protease de cocktail comprimido para manter a integridade das lipoproteínas. </li><li> Levar o volume a 23 ml com água (1,2 g / ml de solução de NaCl a densidade). </li><li> Dissolve-se os solutos completamente. </li><li> Decantar a totalidade da solução para um tubo de ultracentrífuga policarbonato. </li><li> Suavemente sobrepor a solução densidade 1,2 g / ml com 0,5 ml de água (1,0 g / ml de densidade). </li><li> Equilibrar o tubo por peso e não por volume. </li><li> Carregue suavemente os tubos no rotor T865. </li><li> Rodar as amostras na ultracentrífuga a 429.460 xg durante 24 horas a 4 ° C. </li><li> Isolar imediatamente o topo 0,5 ml de solução por pipetagem suave. Mantenha o tubo o mais imóvel possível. </li></ol> 5. Análise TEM <ol><li> Prepare 5 ml de ácido fosfotúngstico a 2% (w / v) e ajustar o pH a 6,0. </li><li> Filtra-se o ácido fosfotúngstico a 2% com um filtro de seringa (tamanho de poro: 0,2 um). </li><li> Gota de 20 ul da amostra em um prato de lipoproteína estéril. </li><li> Gota de 20 ul de ácido fosfotúngstico a 2% filtrada ao lado da amostra no mesmo prato. </li><li> Suavemente soltar uma grade EM com o lado maçante descansando na amostra. </li><li> Incubar à temperatura ambiente durante 1 min. </li><li> Bater levemente o lado da grade de EM num papel de filtro para remover a amostra da rede. </li><li> Suavemente soltar a grade EM com o lado maçante descansando no ácido fosfotúngstico 2%. </li><li> Incubar à temperatura ambiente durante 1 min. </li><li> Bater levemente o lado da grade de EM num papel de filtro para remover o ácido fosfotúngstico a partir da rede. </li><li> Usando uma ETM, capturar as imagens das lipoproteínas (Figura 4). </li></ol>

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The author has nothing to disclose.

Neste artigo, os dois sistemas que podem ser utilizados para manter células Caco-2 são descritos, a saber, o prato de cultura de tecidos normal e a membrana permeável. Os benefícios de usar o sistema de membrana permeável incluem a separação do apical e basolateral os compartimentos, e a capacidade de incubar a mistura de lípidos e recolher a secreção de lipoproteínas simultaneamente. No entanto, as pastilhas de membranas permeáveis ​​são caros, e a sua membrana de policarbonato não permite uma boa vis...

Estudos de absorção intestinal de gordura da dieta, e drogas e vitaminas solúveis em lípidos pode ser realizada in vivo usando um modelo de fistula linfática 1-4. No entanto, as técnicas cirúrgicas envolvidas não são apenas um desafio, mas também caro. Embora abordagens in vivo com base na análise fecal podem ser utilizados, eles são utilizados principalmente para determinar a percentagem de absorção pelo tracto gastrointestinal 2,5. O modelo in vitro descrito neste trabalho é mais rentável, e as técnicas envolvidas são, indiscutivelmente, menos desafiador. Estudos de modificação genética são também mais económico e menos quando eles são conduzidos usando este modelo in vitro demorado. Uma vez que os materiais solúveis em lípidos que são absorvidos por enterócitos são empacotados em lipoproteínas de 6,7, a eficácia deste modelo in vitro para a produção de lipoproteínas é crucial. Os dois intestina principall lipoproteínas são quilomicrons e lipoproteínas de muito baixa densidade (VLDL). Os quilomicrons, lipoproteínas como definidas com 80 nm ou mais de diâmetro, são produzidos de forma rigorosa o intestino delgado, quando lípidos estão abundantemente presentes no lúmen gastrointestinal. Uma vez que eles são os maiores lipoproteínas, chylomicrons são concebivelmente os transportadores lipídicos mais eficientes. Este modelo in vitro, que é capaz de produzir quilomicrons 8, pode ser usado para estudar a absorção de gordura dietética, a vitamina solúvel em lípidos absorção pelo intestino, e biodisponibilidade oral da droga lipofílica. A presença de moléculas solúveis em lípidos, vitaminas ou drogas na fracção de lipoproteínas é um indicador da sua absorção pelo intestino delgado. Como discutido anteriormente, este modelo pode ser usado para melhorar a biodisponibilidade oral da droga lipofílica 6. Este artigo descreve como células Caco-2 devem ser mantidas na membrana permeável ou regular pratos de cultura de tecidos, como os lipídios mixture para estimular a produção de lipoproteína deve ser preparado, como o sistema de expressão de lentivírus pode ser empregue para atingir a sobre-expressão eficaz, e como as lipoproteínas isolados devem ser analisados.

<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).

Figura 1 apresenta normais de 13 dias pós-confluentes células Caco-2. O aparecimento de estruturas em forma de cúpula e gotículas lipídicas intracelulares são características diferenciadas de células Caco-2. Quando as células Caco-2 não são dispersos igualmente durante a sementeira, eles vão aglutinar-se e crescer demais em certas áreas do prato; e haverá algumas áreas no prato sem quaisquer células. Roda e colocando o prato numa superfície inclinada deve ser evitada. Também é importa...

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.

Usando Caco-2 em células para estudo LIPID Transportes pelo intestino

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

Research

JoVE Journal

Biology

18.7K visualizações.  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.

Vídeo: 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.

Ensaio de efluxo do colesterol

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 ...

Medicina

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.

Modelos e Métodos para avaliar Transporte de Drug Delivery Systems através das barreiras celulares

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

Bioengenharia

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.

Explorando a regulação do catabolismo de gotículas lipídicas por meio da lipofagia

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

Pesquisa do câncer

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.

Um método de co-cultura para investigar o Crosstalk entre raio-x irradiados PBMC e células Caco-2

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

Imunologia e Infecção

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.

Ensaio de Troca Lipídica em Células Vivas

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

Bioquímica

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.

Um ensaio à base de fluorescência para caracterização e quantificação da formação de gotas lipídicas em organóides intestinais humanos

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.

A proteolipossoma-Based Efluxo Ensaio para determinar as propriedades de uma única molécula de Cl<sup&gt; -</sup&gt; Canais e Transportadores

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 ...

Biologia

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.

Ensaio de Transcitose de Células Endoteliais como Modelo In Vitro para Avaliar a Permeabilidade da Barreira Interna Sangue-Retina

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.

Métodos de estudo epitelial Transporte função das proteínas e Expressão em Native Intestino e Caco-2 células cultivadas em 3D

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.

Usando Caco-2 em células para estudo LIPID Transportes pelo intestino

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