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. Manutenzione delle cellule Caco-2 <ol><li> Utilizzando regolari piastre di coltura tissutale <ol><li> Scongelare le cellule Caco-2 da una fiala congelata ponendo la provetta in un bagno d&#39;acqua a 37 °, e subito aggiungerli a un piatto di coltura di tessuto al 10 cm contenente 10 ml di terreni di crescita (siero bovino fetale pre-riscaldato 15% (FBS ) in Dulbecco modificato (DMEM)). <ol><li> Quando le cellule Caco-2 hanno raggiunto 50-70% di confluenza, dividerle 1: 6 incubando le cellule con 3 ml di 0,05% tripsina / 0.53 mM di acido etilendiamminotetraacetico (EDTA) a 37 ° C fino a quando sono staccati (15 min) . Per evitare aggregazione cellulare, mescolare le cellule più volte delicatamente pipettando. Aggiungere 0,5 ml di cellule trypsinized ad un piatto di coltura di tessuto al 10 cm contenente 10 ml di terreni di crescita pre-riscaldato. </li></ol></li><li> Agitare delicatamente i piatti più volte in avanti e indietro, seguito da una direzione verso sinistra e verso destra. Evitare vorticoso i piattiin quanto può provocare una dispersione delle cellule disuguale. </li><li> Porre le capsule su una superficie piana in un incubatore a 37 ° in dotazione con il 5% di CO 2. Superfici inclinate possono anche provocare una dispersione cellulare disuguale. </li><li> Modificare i media di crescita di un giorno dopo l&#39;avvio l&#39;incubazione. </li><li> Monitorare le cellule e registrare la giornata raggiungono confluenza. Ci vorrà circa 1 settimana per le cellule di raggiungere confluenza dal giorno in cui sono divisi. </li><li> Modificare i mezzi di crescita due volte la settimana prima che le cellule raggiungono confluenza. Una volta che le cellule hanno raggiunto confluenza, modificare i mezzi di crescita ogni altro giorno. Dopo che le cellule sono 7 giorni post-confluenti, modificare i mezzi di crescita giornaliera. Nota: Le cellule sono pronte per esperimenti quando sono 13 giorni post-confluenti (Figura 1). Dal momento che le cellule diventeranno meno efficace nel produrre lipoproteine, usare le cellule che si trovano tra i 13 ei 17 giorni dopo confluenti 8. Andare al punto 3 su come condurre l&#39;esperimento.</li></ol></li><li> Utilizzando il sistema di membrana permeabile <ol><li> Seme le cellule Caco-2 nel foglietto membrana permeabile come descritto al punto 1.1.1.1. sopra. Evitare l&#39;uso di cellule da un flaconcino congelato perché la loro crescita cellulare è normalmente più lento (periodo di recupero). Brevemente, aggiungere 0,5 ml delle cellule trypsinized alla camera apicale (scomparto superiore) contenente 10 ml di terreni di crescita pre-riscaldato. Inoltre, aggiungere 10 ml di terreni di crescita pre-riscaldato alla camera basolaterale (scomparto inferiore). Nota: per le migliori prassi, aggiungere i media di crescita alla camera apicale e poi alla camera basolaterale. Durante la rimozione dei vecchi media, rimuovere il supporto basolaterale prima che i media apicali. Evitare colpendo la membrana di policarbonato in quanto potrebbe rompere la membrana. </li><li> Grazie alla visibilità delle cellule poveri attraverso la membrana in policarbonato, seminare le cellule Caco-2 in un piatto di coltura tissutale regolare con densità simile, e utilizzare questo piatto per giudicare la confluenza delle cellule. </li><li> Segui steps 1.1.2-1.1.6 sopra. </li></ol></li></ol> 2. Gene Overexpression <ol><li> Utilizzando l&#39;approccio trasfezione regolare <ol><li> Seme le cellule Caco-2 su un piatto di coltura di tessuti, come descritto nella 1.1.1.1.-1.1.4 sopra. Nota: Quando le cellule sono circa il 40-50% confluenti, sono pronti per essere transfettate. </li><li> Aggiungere 67 ml di reagente trasfezione a 472 l di media siero ridotti in una provetta. Evitare il contatto del reagente di trasfezione diluito con la parete del tubo. </li><li> Aggiungere 23 mg di pLL3.7 rafforzata proteina fluorescente verde (eGFP) 9 in / ridotto miscela Mezzi di siero di reagenti di trasfezione. </li><li> Incubare la miscela per 20 minuti a RT. </li><li> Sostituire mezzi di crescita delle cellule Caco-2 &#39;con 8 ml di 10% FBS in DMEM. </li><li> Aggiungere il composto DNA / reagente trasfezione / ridotto supporto siero goccia a goccia al piatto. </li><li> Agitare delicatamente i piatti più volte in un avanti e badirezione ckward seguita da una direzione verso sinistra e verso destra. </li><li> Porre la capsula nel C incubatore 37 °. </li><li> Sostituire il supporto nel piatto con 10 ml di terreni di crescita giorno dopo l&#39;avvio l&#39;incubazione. </li><li> Monitorare l&#39;espressione genica. <ol><li> Monitorare l&#39;espressione genica senza 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) colorazione. Utilizzando un microscopio a fluorescenza, determinare la percentuale di cellule che sono verde. La percentuale rappresenta l&#39;efficienza di trasfezione. </li><li> Monitorare l&#39;espressione genica con colorazione DAPI. <ol><li> Lavare le cellule con tampone fosfato salino (PBS) tre volte. </li><li> Fissare le cellule aggiungendo 10 ml di formaldeide al 4% in PBS (10 min di incubazione a RT). </li><li> Lavare le cellule con PBS per tre volte. </li><li> Incubare le cellule al buio per 15 minuti a RT con 8 ml di PBS contenente 1: 500 DAPI, 1% BSA, 0,01% digitonina. </li><li> Lavare le cellule con PBS per tre volte. </li><li> Ucantare un microscopio a fluorescenza, determinare il numero di cellule che sono verde / blu rispetto a quelli che sono blu (Figura 2 pannello superiore). La percentuale calcolata rappresenta l&#39;efficienza di trasfezione. </li></ol></li></ol></li></ol></li><li> Utilizzando il sistema sovraespressione lentivirus 10 <ol><li> Seme umano rene embrionale (HEK) le cellule 293T in un 15 centimetri piatto di coltura tissutale (10% FBS in DMEM come loro mezzi di crescita). Nota: Quando le cellule sono circa il 60-70% confluenti, sono pronti per essere transfettate. </li><li> Aggiungere 162 ml di reagente trasfezione di 1.133 l di media siero ridotti in una provetta 11. Evitare il contatto del reagente di trasfezione diluito con la parete del tubo. </li><li> Aggiungere 24 mg di pLL3.7 eGFP (o un altro costrutto), 15,6 mg di elemento risposta Rev (RRE), 6.0 mg di REV, e 8,4 mg di vescicolare glicoproteina virus della stomatite (VSVG) nel reagente trasfezione / reduced miscela Mezzi di siero. </li><li> Incubare la miscela per 20 minuti a RT. </li><li> Sostituire il supporto di crescita con 16 ml di 10% FBS in DMEM. </li><li> Aggiungere il composto DNA / reagente trasfezione / ridotto supporto siero goccia a goccia al piatto. </li><li> Agitare delicatamente i piatti più volte in avanti e indietro, seguito da una direzione verso sinistra e verso destra. E &#39;normale vedere alcune delle cellule HEK293T distaccati a questo punto. </li><li> Porre la capsula in incubatrice. </li><li> Dopo 24 ore di incubazione, sostituire il supporto nel piatto con 25 ml di terreni di crescita. </li><li> Il giorno seguente, raccogliere i mezzi di crescita contenente il lentivirus (prima raccolta). </li><li> Ripetere il punto 2.2.10 per raccogliere più lentivirus (la seconda raccolta). </li><li> PISCINA La prima e la seconda raccolta, e rimuovere detriti cellulari mediante centrifugazione (2500 xg per 5 min). </li><li> Filtrare la collezione con un filtro a perdere per bottiglia (0,45 micron pori), econcentrare il virus mediante centrifugazione dalla collezione 31.400 xg per 2 ore a temperatura ambiente (JA-20 rotore). Contrassegnare il fondo della provetta in cui si trova il pellet. Decantare il surnatante, e lasciare la provetta capovolta a temperatura ambiente per circa 15 min. </li><li> Risospendere il pellet contenente il virus con 100 ml di PBS 1X. Evitare bolle. </li><li> Conservare il virus concentrato a -80 ° C. Evitare cicli di congelamento-scongelamento. </li><li> Determinare la quantità ottimale di virus necessari per trasdurre cellule Caco-2 mediante titolazione. Per risparmiare sui costi, utilizzare una piastra da 24 pozzetti (0,3 ml di crescita media / pozzo) invece di un piatto di coltura di tessuti 10 centimetri per la titolazione. <ol><li> Aggiungere quantità crescenti del virus concentrati (0, 1, 2, 5, 10, 25, e 50 ml / pozzetto) supplementato con polibrene (concentrazione finale = 5 mg / ml) al 40-70% confluenti cellule Caco-2. Agitare la piastra da 24 pozzetti dolcemente, come descritto al punto 1.1.2. </li><li> Monitorare la loro espressione genica (Fase 2.1.10.) (Figura 2 bottom4 pannelli). </li></ol></li><li> Dal momento che l&#39;espressione del transgene è generalmente sostenuta, di mantenere le cellule trasdotte per futuri esperimenti e monitorare continuamente la loro espressione genica (Step 2.1.10.). </li></ol></li></ol> 3. stimolare la secrezione Lipoprotein <ol><li> Utilizzando la normale piatto di coltura tissutale <ol><li> Preparare una miscela di lipidi 100X mescolando 50 mg di acido oleico, 40 mg di lecitina, e 48 mg di sodio taurocolato. Portare il volume a 900 ml con PBS (sufficiente per 8 piatti). Vortex la miscela lipidica vigorosamente. </li><li> Aggiungere 900 ml di miscela lipidica 100X in 89,1 ml di terreni di crescita. Mescolare e filtrare i media lipidi contenenti. Le concentrazioni finali (1X) di acido oleico, lecitina e sodio taurocolato sono 2,0 mM, 1,36 mM e 1,0 mM, rispettivamente. </li><li> Aggiungere 10 ml di media-lipidico contenente alle cellule. </li><li> Incubare le cellule con i media lipidi contenenti per 4 ore a 37° C incubatore. </li><li> Lavare le cellule con PBS per tre volte. </li><li> Aggiungere 10 ml di mezzi di crescita per le cellule a raccogliere la secrezione delle lipoproteine. </li><li> Incubare per 2 ore in un incubatore a 37 °. </li><li> Raccogliere la media-lipoproteine ​​contenenti. </li></ol></li><li> Utilizzando il sistema di membrana permeabile <ol><li> Seguire i passaggi 3.1.1-3.1.2 per preparare la miscela lipidica. </li><li> Aggiungere 10 ml di media-lipidico contenente alla camera apicale e 10 ml di mezzi di crescita alla camera basolaterale. </li><li> Incubare per 4 ore in un incubatore a 37 °. Raccogliere la media-lipoproteine ​​contenenti nella camera basolaterale. </li></ol></li></ol> 4. Lipoprotein Isolation (Figura 3) <ol><li> Rimuovere i detriti cellulari dal supporto lipoproteine ​​contenenti mediante centrifugazione (2000 xg per 5 min). </li><li> Decantare i mezzi di comunicazione in un tubo da 50 ml. </li><li> Aggiungere 5,95 g di cloruro di sodio ai media. </li><li> Se la sample sarà analizzata dal TEM, aggiungere 1 inibitore della proteasi cocktail della tavoletta per garantire l&#39;integrità delle lipoproteine. </li><li> Portare il volume a 23 ml con acqua (1,2 g / ml NaCl soluzione densità). </li><li> Sciogliere completamente soluti. </li><li> Decantare tutta la soluzione in un tubo di policarbonato ultracentrifuga. </li><li> Sovrapporre delicatamente la soluzione densità di 1,2 g / ml con 0,5 ml di acqua (1,0 g / ml densità). </li><li> Bilanciare il tubo in peso e non in volume. </li><li> Caricare delicatamente i tubi nel rotore T865. </li><li> Spin i campioni nel ultracentrifuga a 429.460 xg per 24 ore a 4 ° C. </li><li> Immediatamente isolare la parte superiore di 0,5 ml di soluzione delicatamente pipettando. Mantenere il tubo più fermi possibile. </li></ol> Analisi 5. TEM <ol><li> Preparare 5 ml di 2% di acido fosfotungstico (w / v) e regolare il pH a 6,0. </li><li> Filtrare l&#39;acido fosfotungstico 2% con un filtro a siringa (dimensione dei pori: 0,2 um). </li><li> Goccia 20 ml di campione lipoproteina su un piatto sterile. </li><li> Goccia 20 ml di 2% di acido fosfotungstico filtrato accanto al campione sullo stesso piatto. </li><li> Cadere delicatamente una griglia EM con il lato opaco appoggiata sul campione. </li><li> Incubare a temperatura ambiente per 1 min. </li><li> Battere delicatamente il lato della griglia EM su una carta da filtro per rimuovere il campione dalla griglia. </li><li> Rilasciare delicatamente la griglia EM con il lato opaco appoggiato sul acido fosfotungstico 2%. </li><li> Incubare a temperatura ambiente per 1 min. </li><li> Battere delicatamente il lato della griglia EM su una carta da filtro per rimuovere l&#39;acido fosfotungstico dalla rete. </li><li> Utilizzando un TEM, catturare le immagini delle lipoproteine ​​(Figura 4). </li></ol>

<ol>
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The author has nothing to disclose.

In questo documento, i due sistemi che possono essere utilizzati per mantenere cellule Caco-2 sono descritte, cioè, il piatto di coltura tissutale regolare e la membrana permeabile. I vantaggi di utilizzare il sistema di membrana permeabile includono la separazione del apicale e basolaterale compartimenti, e la capacità di incubare la miscela lipidica e raccogliere la secrezione lipoproteina simultaneamente. Tuttavia, gli inserti membrana permeabile sono costosi, e la loro membrana di policarbonato non consentono una ...

Studi di assorbimento intestinale dei grassi alimentari e farmaci e vitamine liposolubili possono essere condotti in vivo, utilizzando un modello di fistola linfatica 1-4. Tuttavia, le tecniche chirurgiche coinvolte non sono solo una sfida, ma anche costoso. Sebbene in vivo approcci basati sull&#39;analisi fecale possono essere utilizzati, sono utilizzati principalmente per determinare la percentuale di assorbimento dal tratto gastrointestinale 2,5. Il modello in vitro descritto in questo documento è più conveniente, e le tecniche coinvolte sono forse meno impegnativo. Studi modificazione genetica sono anche più economici e quando sono condotti utilizzando questo modello in vitro in termini di tempo di meno. Poiché i materiali liposolubili che sono presi da enterociti sono confezionati in lipoproteine ​​6,7, l&#39;efficacia di questo modello in vitro per produrre lipoproteine ​​è cruciale. Le due intestina principalel lipoproteine ​​sono chilomicroni e molto lipoproteine ​​a bassa densità (VLDL). Chilomicroni, definiti come lipoproteine ​​a 80 nm o più di diametro, sono prodotti rigorosamente dal piccolo intestino quando sono abbondantemente presenti nel lume gastrointestinale lipidi. Dal momento che sono i più grandi lipoproteine, chilomicroni sono plausibilmente i trasportatori lipidici più efficienti. Questo modello in vitro, che è capace di produrre chilomicroni 8, può essere usato per studiare l&#39;assorbimento grassi, liposolubile vitamina assorbimento da parte dell&#39;intestino e biodisponibilità orale lipofilo. La presenza di molecole liposolubili, vitamine o farmaci nella frazione lipoproteina è un indicatore della loro assorbimento da parte dell&#39;intestino tenue. Come discusso in precedenza, questo modello può essere utilizzato per migliorare la biodisponibilità orale farmaco lipofila 6. Questo documento descrive come cellule Caco-2 dovrebbero essere mantenute in membrana permeabile o regolari piastre di coltura dei tessuti, come le mi lipidixture per stimolare la produzione di lipoproteine ​​dovrebbe essere preparato, come il sistema di espressione lentivirus può essere impiegato per realizzare sovraespressione efficace, e come dovrebbe essere analizzato lipoproteine ​​isolate.

<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 visualizza normali 13 giorni post-confluenti cellule Caco-2. La comparsa di strutture a forma di cupola e gocce lipidiche intracellulari sono caratteristici di differenziate cellule Caco-2. Quando le cellule Caco-2 non sono dispersi ugualmente durante la semina, si ciuffo e invadere in alcune zone del piatto; e ci saranno alcune aree nel piatto, senza tutte le cellule. Vorticoso e mettendo il piatto su una superficie inclinata deve essere evitata. E &#39;anche importante notare che il post-conf...

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.

Utilizzando cellule Caco-2 allo Studio Lipid Trasporto dall&#39;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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.7K Views.  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.

Video: 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.

Efflusso test Colesterolo

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 ...

Ricerca

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.

Modelli e metodi per valutare il trasporto di Drug Delivery Systems attraverso le barriere cellulari

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

Bioingegneria

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.

Esplorare la regolazione del catabolismo delle goccioline lipidiche attraverso la lipofagia

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

Ricerca sul cancro

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.

Un metodo di co-coltura di indagare il Crosstalk tra raggi x irradiati PBMC e cellule 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 ...

Immunologia e infezioni

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.

Saggio di scambio lipidico in cellule viventi

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

Biochimica

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.

Un saggio basato sulla fluorescenza per la caratterizzazione e la quantificazione della formazione di gocciolini di lipidi negli organiidi intestinali umani

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 Proteoliposome-Based Efflux Assay per determinare le proprietà singola molecola di Cl<sup&gt; -</sup&gt; Canali e trasportatori

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.

Saggio di transcitosi delle cellule endoteliali come modello in vitro per valutare la permeabilità della barriera emato-retinica interna

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.

Metodi per studiare epiteliale Trasporti funzione delle proteine ​​e di espressione in nativo Intestino e Caco-2 cellule coltivate in 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.

Utilizzando cellule Caco-2 allo Studio Lipid Trasporto dall'intestino

Riepilogo

Esplora Altri Video

Capitoli in questo video

Efflusso test Colesterolo

Modelli e metodi per valutare il trasporto di Drug Delivery Systems attraverso le barriere cellulari

Esplorare la regolazione del catabolismo delle goccioline lipidiche attraverso la lipofagia

Un metodo di co-coltura di indagare il Crosstalk tra raggi x irradiati PBMC e cellule Caco-2

Saggio di scambio lipidico in cellule viventi

Un saggio basato sulla fluorescenza per la caratterizzazione e la quantificazione della formazione di gocciolini di lipidi negli organiidi intestinali umani

A Proteoliposome-Based Efflux Assay per determinare le proprietà singola molecola di Cl

Saggio di transcitosi delle cellule endoteliali come modello in vitro per valutare la permeabilità della barriera emato-retinica interna

Metodi per studiare epiteliale Trasporti funzione delle proteine e di espressione in nativo Intestino e Caco-2 cellule coltivate in 3D

In Vitro e In Vivo approcci per determinare la permeabilità intestinale delle cellule epiteliali

Utilizzando cellule Caco-2 allo Studio Lipid Trasporto dall'intestino

Riepilogo

Esplora Altri Video

Capitoli in questo video

Efflusso test Colesterolo

Modelli e metodi per valutare il trasporto di Drug Delivery Systems attraverso le barriere cellulari

Esplorare la regolazione del catabolismo delle goccioline lipidiche attraverso la lipofagia

Un metodo di co-coltura di indagare il Crosstalk tra raggi x irradiati PBMC e cellule Caco-2

Saggio di scambio lipidico in cellule viventi

Un saggio basato sulla fluorescenza per la caratterizzazione e la quantificazione della formazione di gocciolini di lipidi negli organiidi intestinali umani

A Proteoliposome-Based Efflux Assay per determinare le proprietà singola molecola di Cl

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