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. Maintien des cellules Caco-2 <ol><li> Utilisation plats réguliers de culture tissulaire <ol><li> Décongeler les cellules Caco-2 à partir d&#39;un flacon congelé en plaçant le flacon dans un bain d&#39;eau à 37 °, et tout de suite les ajouter à une boîte de culture tissulaire de 10 cm contenant 10 ml de milieu de croissance (sérum bovin fœtal préchauffé à 15% (FBS ) dans du milieu Eagle modifié de Dulbecco (DMEM)). <ol><li> Lorsque les cellules Caco-2 ont atteint 50-70% de confluence, les diviser 1: 6 par incubation des cellules avec 3 ml de 0,05% de trypsine / 0,53 mM d&#39;acide éthylènediaminetétraacétique (EDTA) à 37 ° C jusqu&#39;à ce qu&#39;ils soient détachés (15 min) . Pour éviter l&#39;agglutination des cellules, mélanger les cellules à plusieurs reprises par pipetage doux. Ajouter 0,5 ml des cellules trypsinisées à une boîte de culture tissulaire de 10 cm contenant 10 ml de milieu de culture préchauffé. </li></ol></li><li> Secouer doucement les plats à plusieurs reprises dans une direction vers l&#39;avant et vers l&#39;arrière suivie d&#39;une direction vers la gauche et vers la droite. Évitez les plats tourbillonnantcar il peut conduire à une dispersion de cellules inégales. </li><li> Placez les plats sur une surface plane dans un incubateur à 37 ° de fourni avec 5% de CO 2. Des surfaces inclinées peuvent également résulter en une dispersion de cellules inégales. </li><li> Modifiez les milieux de croissance d&#39;une journée après le début de l&#39;incubation. </li><li> Surveiller les cellules et enregistrer le jour où ils atteignent la confluence. Il faudra environ 1 semaine pour que les cellules atteignent la confluence de la journée, ils sont divisés. </li><li> Changer le milieu de croissance deux fois par semaine avant que les cellules atteignent la confluence. Une fois que les cellules ont atteint la confluence, modifier les milieux de croissance tous les autres jours. Après que les cellules sont 7-jours post-confluentes, changer le milieu de croissance quotidienne. Remarque: Les cellules sont prêtes pour les expériences quand ils sont 13 jours post-confluentes (figure 1). Puisque les cellules finiront par devenir moins efficace dans la production de lipoprotéines, utiliser des cellules qui sont entre 13 à 17 jours post-confluentes 8. Passez à l&#39;étape 3 sur la façon de mener l&#39;expérience.</li></ol></li><li> Utilisation du système de membrane perméable <ol><li> Ensemencer les cellules Caco-2 dans l&#39;insert de membrane perméable tel que décrit à l&#39;étape 1.1.1.1. ci-dessus. Évitez d&#39;utiliser des cellules à partir d&#39;un flacon congelé parce que leur croissance cellulaire est normalement plus lent (période de récupération). En bref, ajouter 0,5 ml des cellules trypsinisées à la chambre apicale (compartiment supérieur) contenant 10 ml de milieu de culture préchauffé. En outre, ajouter 10 ml de milieu de croissance préchauffé à la chambre basolatérale (compartiment inférieur). Remarque: Pour la meilleure pratique, ajouter les milieux de croissance à la chambre apicale d&#39;abord, puis à la chambre basolatérale. Lorsque vous retirez les anciens médias, retirez le support basolatérale devant les médias apical. Évitez piquer la membrane de polycarbonate comme il peut rompre la membrane. </li><li> En raison de la visibilité de la cellule pauvres à travers la membrane de polycarbonate, ensemencer les cellules Caco-2 dans un plat régulière de culture de tissu avec une densité similaire, et utiliser ce plat de juger de la confluence des cellules. </li><li> Suivez steps 1.1.2-1.1.6 ci-dessus. </li></ol></li></ol> 2. La surexpression Gene <ol><li> En utilisant l&#39;approche régulière de transfection <ol><li> Ensemencer les cellules Caco-2 sur une boîte de culture de tissu comme décrit dans 1.1.1.1.-1.1.4 ci-dessus. Remarque: Lorsque les cellules sont environ 40-50% de confluence, ils sont prêts à être transfectées. </li><li> Ajouter 67 ul du réactif de transfection de 472 ul du milieu de sérum réduit dans un tube de microcentrifugation. Eviter le contact du réactif de transfection non-dilué avec la paroi du tube. </li><li> Ajouter 23 pg de pLL3.7 protéine fluorescente verte améliorée (eGFP) 9 dans le réactif de transfection / réduit mélange des médias de sérum. </li><li> Incuber le mélange pendant 20 min à température ambiante. </li><li> Remplacer le milieu de croissance des cellules Caco-2 avec 8 ml de 10% de FBS dans du DMEM. </li><li> Ajouter le mélange / réactif de transfection d&#39;ADN médias / réduite de sérum goutte à goutte à l&#39;antenne. </li><li> Secouer doucement les plats à plusieurs reprises dans un avant et backward direction suivie par une direction vers la gauche et vers la droite. </li><li> Placer le plat dans le 37 ° C incubateur. </li><li> Remplacez le support dans le plat avec 10 ml de milieu de croissance par jour après le début de l&#39;incubation. </li><li> Surveiller l&#39;expression des gènes. <ol><li> Surveiller l&#39;expression des gènes sans 4 &#39;, 6-diamidino-2-phénylindole (DAPI). En utilisant un microscope à fluorescence, déterminer le pour cent de cellules qui sont en vert. Le pourcentage représente l&#39;efficacité de transfection. </li><li> Surveiller l&#39;expression génique avec DAPI. <ol><li> Laver les cellules avec une solution saline tamponnée phosphate (PBS) à trois reprises. </li><li> Fixer les cellules par addition de 10 ml de formaldehyde à 4% dans du PBS (10 minutes d&#39;incubation à la température ambiante). </li><li> Laver les cellules avec du PBS à trois reprises. </li><li> Incuber les cellules dans l&#39;obscurité pendant 15 min à température ambiante avec 8 ml de PBS contenant 1 500: 1, DAPI% de BSA, 0,01% de digitonine. </li><li> Laver les cellules avec du PBS à trois reprises. </li><li> Uchanter un microscope à fluorescence, déterminer le nombre de cellules qui sont vert / bleu par rapport à ceux qui sont bleu (panneau supérieur Figure 2). Le pourcentage calculé représente l&#39;efficacité de transfection. </li></ol></li></ol></li></ol></li><li> Utilisation du système de surexpression 10 lentivirus <ol><li> Seed humaines embryonnaires de rein (HEK), les cellules 293T dans une boîte de culture tissulaire de 15 cm (10% de FBS dans du DMEM que leur milieu de croissance). Remarque: Lorsque les cellules sont environ 60-70% de confluence, ils sont prêts à être transfectées. </li><li> Ajouter 162 ul de réactif de transfection à 1133 ul de médias de sérum réduit dans un tube de microcentrifugeuse 11. Eviter le contact du réactif de transfection non-dilué avec la paroi du tube. </li><li> Ajouter 24 pg de pLL3.7 eGFP (ou un autre produit d&#39;assemblage), 15,6 ug de l&#39;élément de réponse Rev (RRE), 6,0 ug de REV, et 8,4 pg du vésiculeuse glycoprotéine du virus de la stomatite (VSVG) dans le réactif de transfection / réduqué mélange des médias de sérum. </li><li> Incuber le mélange pendant 20 min à température ambiante. </li><li> Remplacez le support de croissance avec 16 ml de 10% de FBS dans du DMEM. </li><li> Ajouter le mélange / réactif de transfection d&#39;ADN médias / réduite de sérum goutte à goutte à l&#39;antenne. </li><li> Secouer doucement les plats à plusieurs reprises dans une direction vers l&#39;avant et vers l&#39;arrière suivie d&#39;une direction vers la gauche et vers la droite. Il est normal de voir quelques-unes des cellules HEK293T détachés à ce point. </li><li> Placer le plat dans l&#39;incubateur. </li><li> Après 24 heures d&#39;incubation, remplacez les médias dans le plat avec 25 ml de milieu de croissance. </li><li> Le lendemain, recueillir le milieu de croissance contenant le lentivirus (première collecte). </li><li> Répétez l&#39;étape 2.2.10 de recueillir plus de lentivirus (la deuxième collecte). </li><li> Mutualiser la première et la deuxième collection, et enlever les débris cellulaires par centrifugation (2500 g pendant 5 min). </li><li> Filtrer la collection avec un filtre sur flacon jetable (0,45 um pores), etconcentrer le virus par centrifugation la collection à 31 400 g pendant 2 heures à température ambiante (JA-20 rotor). Marquer le fond du tube de centrifugation où la pastille se trouve. Décanter le surnageant, et de laisser le tube inversé à température ambiante pendant environ 15 min. </li><li> Remettre en suspension le culot contenant le virus avec 100 ul de PBS 1X. Éviter les bulles. </li><li> Stocker le virus concentrée à -80 ° C. Éviter les cycles de gel-dégel. </li><li> Déterminer la quantité optimale de virus nécessaire pour transduire les cellules Caco-2 par titrage. Pour réduire les coûts, utiliser une plaque de 24 puits (0,3 ml de milieu de croissance / puits) à la place d&#39;une boîte de 10 cm de culture de tissus pour le titrage. <ol><li> Ajouter des quantités croissantes du virus concentré (0, 1, 2, 5, 10, 25, et 50 ul / puits) additionné de polybrène (concentration finale = 5 pg / ml) à 40-70% de la confluence des cellules Caco-2. Agiter la plaque de 24 puits doucement comme décrit dans 1.1.2. </li><li> Surveiller l&#39;expression de gène (étape 2.1.10.) (Figure 2 bas4 panneaux). </li></ol></li><li> Étant donné que l&#39;expression de transgène est généralement soutenue, maintenir les cellules transduites pour des expériences futures et surveiller en permanence leur expression des gènes (étape 1.2.10.). </li></ol></li></ol> 3. Stimuler la sécrétion des lipoprotéines <ol><li> Utilisation de la boîte de culture de tissu régulière <ol><li> Préparer un mélange lipidique par mélange 100X 50 mg d&#39;acide oléique, 40 mg de lécithine et 48 mg de taurocholate de sodium. Porter le volume à 900 pl avec du PBS (suffisant pour 8 plats). Vortexer le mélange lipidique vigoureusement. </li><li> Ajouter 900 ul du mélange lipidique 100X dans 89,1 ml de milieu de croissance. Mélanger et filtrer les médias contenant des lipides. Les concentrations finales (1 x) d&#39;acide oléique, la lécithine, et le taurocholate de sodium sont de 2,0 mM, 1,36 mM et 1,0 mM, respectivement. </li><li> Ajouter 10 ml d&#39;un milieu contenant le lipide aux cellules. </li><li> Incuber les cellules avec le support contenant des lipides pendant 4 heures dans un 37° C incubateur. </li><li> Laver les cellules avec du PBS à trois reprises. </li><li> Ajouter 10 ml de milieu de croissance pour les cellules afin de recueillir la sécrétion des lipoprotéines. </li><li> Incuber pendant 2 heures dans un incubateur à 37 ° C. </li><li> Recueillir les médias contenant des lipoprotéines. </li></ol></li><li> Utilisation du système de membrane perméable <ol><li> Suivre les étapes 3.1.1-3.1.2 pour préparer le mélange de lipides. </li><li> Ajouter 10 ml d&#39;un milieu contenant le lipide à la chambre apicale et 10 ml du milieu de culture à la chambre basolatérale. </li><li> Incuber pendant 4 heures dans un incubateur à 37 ° C. Recueillir les médias contenant des lipoprotéines dans la chambre basolatérale. </li></ol></li></ol> 4. Isolement des lipoprotéines (Figure 3) <ol><li> Retirer les débris cellulaires à partir du support contenant des lipoprotéines par centrifugation (2000 xg pendant 5 min). </li><li> Décanter les médias dans un tube de 50 ml. </li><li> Ajouter 5,95 g de chlorure de sodium pour les médias. </li><li> Si le SAmple sera analysée par le TEM, ajouter une protéase tablette de cocktail inhibiteur de maintenir l&#39;intégrité des lipoprotéines. </li><li> Porter le volume à 23 ml avec de l&#39;eau (1,2 g / ml de solution de NaCl densité). </li><li> Dissoudre les solutés complètement. </li><li> Décanter la totalité de la solution dans un tube d&#39;ultracentrifugation polycarbonate. </li><li> Superposer délicatement la solution de densité de 1,2 g / ml avec 0,5 ml d&#39;eau (1,0 g / ml densité). </li><li> Équilibrer le tube en poids et pas en volume. </li><li> Charger doucement les tubes dans le rotor de T865. </li><li> Faites tourner les échantillons dans l&#39;ultracentrifugation à 429 460 g pendant 24 heures à 4 ° C. </li><li> Immédiatement isoler le top 0,5 ml de solution par pipetage doux. Garder le tube aussi immobile que possible. </li></ol> 5. Analyse TEM <ol><li> Préparer 5 ml d&#39;acide phosphotungstique 2% (p / v) et ajuster le pH à 6,0. </li><li> Filtrer l&#39;acide phosphotungstique à 2% d&#39;un filtre à seringue (taille des pores: 0,2 um). </li><li> Déposer 20 ul de l&#39;échantillon de lipoprotéine sur un plat stérile. </li><li> Déposer 20 ul de l&#39;acide phosphotungstique à 2% filtrée à côté de l&#39;échantillon sur le même plat. </li><li> Laissez tomber doucement une grille d&#39;EM avec le côté mat reposant sur l&#39;échantillon. </li><li> Incuber à température ambiante pendant 1 min. </li><li> Tapoter doucement le côté de la grille EM sur un papier filtre pour retirer l&#39;échantillon de la grille. </li><li> Laissez tomber doucement la grille de EM avec le côté mat reposant sur l&#39;acide phosphotungstique à 2%. </li><li> Incuber à température ambiante pendant 1 min. </li><li> Tapoter doucement le côté de la grille EM sur un papier filtre pour éliminer l&#39;acide phosphotungstique de la grille. </li><li> Utilisation d&#39;un TEM, capturer les images des lipoprotéines (figure 4). </li></ol>

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

The author has nothing to disclose.

Dans ce document, les deux systèmes qui peuvent être utilisés pour maintenir les cellules Caco-2 sont décrits, à savoir, la boîte de culture de tissu normal et la membrane perméable. Les avantages de l&#39;utilisation du système de membrane perméable comprend la séparation de la apical et basolatéral des compartiments, et la capacité à incuber le mélange de lipides et de recueillir la sécrétion des lipoprotéines en même temps. Cependant, les inserts de membrane perméable sont chers, et leur membrane e...

Des études sur l&#39;absorption intestinale des graisses alimentaires, les médicaments et les vitamines et solubles dans les lipides peuvent être conduites in vivo en utilisant un modèle de la fistule lymphatique 1-4. Cependant, les techniques chirurgicales sont impliqués non seulement difficile, mais aussi coûteux. Bien que in vivo approches basées sur l&#39;analyse des fèces peuvent être utilisés, ils sont principalement utilisés pour déterminer l&#39;absorption pour cent par le tractus gastro-intestinal 2,5. Le modèle in vitro décrit dans le présent document est plus rentable, et les techniques impliquées sont sans doute moins difficile. Des études de modification génétique sont également plus économique et moins de temps lorsqu&#39;ils sont effectuées en utilisant ce modèle in vitro. Depuis matières solubles dans les lipides qui sont prises par les entérocytes sont emballés dans les lipoprotéines 6,7, l&#39;efficacité de ce modèle in vitro pour produire des lipoprotéines est crucial. Les deux intestina principalel lipoprotéines sont chylomicrons et les lipoprotéines de très faible densité (VLDL). Les chylomicrons, lipoprotéines définies comme ayant 80 nm ou plus de diamètre, sont fabriqués strictement par l&#39;intestin grêle quand lipides sont présents en abondance dans la lumière gastro-intestinale. Comme ils sont les plus grands lipoprotéines, chylomicrons sont vraisemblablement les transporteurs de lipides les plus efficaces. Ce modèle in vitro, qui est capable de produire des chylomicrons 8, peut être utilisé pour étudier l&#39;absorption de graisse alimentaire, la vitamine liposoluble absorption par l&#39;intestin, et la biodisponibilité orale du médicament lipophile. La présence de molécules solubles dans les lipides, des vitamines, des médicaments ou dans la fraction de lipoprotéine est un indicateur de leur absorption par l&#39;intestin grêle. Comme indiqué précédemment, ce modèle peut être utilisé pour améliorer la biodisponibilité des médicaments par voie orale lipophile 6. Ce document décrit comment cellules Caco-2 devraient être maintenus dans la membrane perméable ou plats réguliers de culture de tissus, la façon dont les mi lipidiquesxture pour stimuler la production de lipoprotéines doit être préparé, la façon dont le système d&#39;expression de lentivirus peut être utilisé pour réaliser une surexpression efficace, et la façon dont les lipoprotéines isolées doit être analysé.

<table border="0" cellpadding="0" cellspacing="0">
	<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&#160;</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><a href="https://www.addgene.org/11795/">https://www.addgene.org/11795/</a></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&#160;</td>
			<td>Fisher</td>
			<td>IC10214625</td>
			<td>Egg lecithin&#160;</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).

Figure 1 affiche post-confluentes cellules Caco-2 normales de 13 jours. L&#39;apparition de structures en forme de dôme et des gouttelettes lipidiques intracellulaires sont caractéristiques de Caco-2 différenciées des cellules. Lorsque les cellules Caco-2 ne sont pas dispersés aussi pendant les semis, ils vont agglutiner et proliférer dans certaines zones du plat; et il y aura quelques zones dans le plat sans cellules. Tourbillonnement et en plaçant le plat sur une surface inclinée doit être é...

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.

Utilisation de cellules Caco-2 à étudier Lipid Transport par l&#39;intestin

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

18.6K vues.  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.

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Recherche

Biologie

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated Stimuli modèle animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolement et purification de Drosophile Neurones périphériques en triant bille magnétique

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

En utilisant la réponse optocinétique pour étudier la fonction visuelle du poisson zèbre

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H+,K+-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb+ or Li+ transport by Na+,K+- or gastric H+,K+-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb+ (Li+) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb+ uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na+/2K+ transport stoichiometry of the Na+,K+-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H+,K+-ATPase. In principle, the assay is not limited to K+-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Measuring Cation Transport by Na,K- and H,K-ATPase in Xenopus Oocytes by Atomic Absorption Spectrophotometry: An Alternative to Radioisotope Assays

We describe a method to quantify the activity of K+-countertransporting P-type ATPases by heterologous expression of the enzymes in Xenopus oocytes and measuring Rb+ or Li+ uptake into individual cells by atomic absorption spectrophotometry. The method is a sensitive and safe alternative to radioisotope flux experiments facilitating complex kinetic studies.

Mesure de transport de cations Na, K et H, K-ATPase dans<em&gt; Xenopus</em&gt; Les ovocytes par spectrophotométrie d&#39;absorption atomique: une alternative à dosages radio-isotopes

Whereas cation transport by the electrogenic membrane transporter Na+,K+-ATPase can be measured by electrophysiology, the electroneutrally operating ...

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.

Méthodes pour l&#39;étude épithéliale Transport Fonction Protein et expression dans Natif Intestin et Caco-2 cellules cultivées en 3D

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

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

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

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

Cultures primaires mixtes de murine Petit Intestin destiné à l&#39;étude de la Gut hormone Sécrétion et imagerie de cellules vivantes de cellules entéroendocrines

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

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Membranes lipidiques bicouches attachées pour surveiller le transfert de chaleur entre les nanoparticules d’or et les membranes lipidiques

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) play an important role in the calcification process in aortic stenosis through the activation of their dedifferentiation program to osteoblast-like cells. Mouse VICs are a good in vitro tool for the elucidation of the signaling pathways driving the mineralization of the aortic valve cell. The method described herein, successfully used by these authors, explains how to obtain freshly isolated cells. A two-step collagenase procedure was performed with 1 mg/mL and 4.5 mg/mL. The first step is crucial to remove the endothelial cell layer and avoid any contamination. The second collagenase incubation is to facilitate the migration of VICs from the tissue to the plate. In addition, an immunofluorescence staining procedure for the phenotype characterization of the isolated mouse valve cells is discussed. Furthermore, the calcification assay was performed in vitro by using the calcium reagent measurement procedure and alizarin red staining. The use of mouse valve cell primary culture is essential for testing new pharmacological targets to inhibit cell mineralization in vitro.

Isolation of Mouse Interstitial Valve Cells to Study the Calcification of the Aortic Valve In Vitro

This article describes the isolation of mouse aortic valve cells by a two-step collagenase procedure. Isolated mouse valve cells are important for performing different assays, such as this in vitro calcification assay, and for investigating the molecular pathways leading to aortic valve mineralization.

Isolement des cellules valvulaires interstitielles de souris pour étudier la calcification de la valve aortique in vitro

The calcification of aortic valve cells is the hallmark of aortic stenosis and is associated with valve cusp fibrosis. Valve interstitial cells (VICs) ...

The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is limited by cost, throughput, and the caveats inherent to isotopic labeling of the food Fe. Thus, there exists a critical need for an approach that is high-throughput and cost-effective. The Caco-2 cell bioassay was developed to satisfy this need. The Caco-2 cell bioassay for Fe bioavailability utilizes simulated gastric and intestinal digestion coupled with culture of a human intestinal epithelial cell line known as Caco-2. In Caco-2 cells, Fe uptake stimulates the intracellular formation of ferritin, an Fe storage protein easily measured by enzyme-linked immunosorbent assay (ELISA). Ferritin forms in proportion to Fe uptake; thus, by measuring Caco-2 cell ferritin production, one can assess intestinal Fe uptake from simulated food digests into the enterocyte. 
Via this approach, the model replicates the key initial step that determines food Fe bioavailability. Since its inception in 1998, this model approach has been rigorously compared to factors known to influence human Fe bioavailability. Moreover, it has been applied in parallel studies, with three human efficacy studies evaluating Fe biofortified crops. In all cases, the bioassay correctly predicted the relative amounts of Fe bioavailability from the factors, crops, and overall diet. This paper provides detailed methods on Caco-2 cell culture coupled with the in vitro digestion process and cell ferritin ELISA necessary to conduct the Caco-2 cell bioassay for Fe bioavailability.

The Caco-2 cell bioassay for iron (Fe) bioavailability represents a cost-effective and versatile approach to assess Fe bioavailability from foods, food products, supplements, meals, and even diet regimens. Thoroughly validated to human studies, it represents the state of the art for studies of Fe bioavailability.

Le bioessai sur cellules Caco-2 pour la mesure de la biodisponibilité du fer alimentaire

Knowledge of Fe bioavailability is critical to the assessment of the nutritional quality of Fe in foods. In vivo measurement of Fe bioavailability is ...