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. Wartung der Caco-2-Zellen <ol><li> Verwenden von regulären Gewebekulturschalen <ol><li> Tauen die Caco-2-Zellen, die aus einem gefrorenen Fläschchen durch Platzieren des Gefäßes in einem 37 ° C Wasserbad, und fügen Sie sie sofort auf eine 10 cm Gewebekulturschale mit 10 ml vorgewärmten Wachstumsmedien (15% fötalem Rinderserum (FBS ) in Dulbeccos modifiziertem Eagle Medium (DMEM)). <ol><li> Wenn die Caco-2-Zellen 50-70% Konfluenz erreicht, spalteten sich 1: 6, indem die Zellen mit 3 ml 0,05% Trypsin / 0,53 mM Ethylendiamintetraessigsäure (EDTA) bei 37 ° C, bis sie abgenommen werden (15 min) . Zu Zelle Verklumpung zu vermeiden, mischen Sie die Zellen mehrfach durch vorsichtiges Pipettieren. 0,5 ml der Trypsin-Zellen auf eine 10 cm-Gewebekulturschale mit 10 ml vorgewärmtem Nährmedium. </li></ol></li><li> Schütteln Sie das Geschirr mehrmals in einer Vorwärts- und Rückwärtsrichtung, gefolgt von einer linken und rechten Richtung. Vermeiden Sie wirbeln die Gerichtewie es in einer ungleichen Zelldispersion führen. </li><li> Die Schalen werden auf eine flache Oberfläche in einem 37 ° C Inkubator mit 5% CO 2 zugeführt wird. Geneigten Flächen kann auch in einer ungleichen Zelldispersion führen. </li><li> Ändern Sie den Wachstumsmedien einen Tag nach Beginn der Inkubation. </li><li> Überwachen Sie die Zellen, und notieren Sie den Tag ihrer Zusammenfluss zu erreichen. Es wird ca. 1 Woche dauern, bis die Zellen bis zur Konfluenz ab dem Tag sie aufgeteilt werden zu erreichen. </li><li> Ändern Sie den Wachstumsmedien zweimal pro Woche, bevor die Zellen Konfluenz erreichen. Sobald die Zellen Konfluenz erreicht, ändern Sie die Wachstumsmedien jeden zweiten Tag. Nachdem die Zellen 7 Tage post-konfluente, ändern Sie die Wachstumsmedien täglich. Anmerkung: Die Zellen sind bereit für Experimente, wenn sie 13 Tage nach der Konfluenz (Abbildung 1). Da die Zellen werden schließlich weniger wirksam in der Herstellung von Lipoproteinen, verwenden Zellen, die zwischen 13 bis 17 Tage post-konfluente 8 sind. Zum, wie man das Experiment durchführen Schritt 3.</li></ol></li><li> Verwendung der permeablen Membransystem <ol><li> Samen der Caco-2-Zellen in der durchlässigen Membran Einsatz wie in Schritt 1.1.1.1 beschrieben. über. Vermeiden Sie Zellen aus einem zugefrorenen Fläschchen, weil ihre Zellwachstum ist in der Regel langsamer (Erholungszeit). Kurz gesagt, werden 0,5 ml der Zellen mit Trypsin behandelt, um die apikale Kammer (Oberkammer), die mit 10 ml vorgewärmtem Nährmedium enthält. Außerdem werden 10 ml des vorgewärmten Nährmedium zur basolateralen Kammer (unteres Fach). Hinweis: Für Best Practice, fügen Sie die Wachstumsmedien zur apikalen Kammer und dann zur basolateralen Kammer. Beim Entfernen der alten Medien, entfernen Sie den basolateralen Medien vor den apikalen Medien. Vermeiden Stossen die Polycarbonatmembran, da es die Membran reißen. </li><li> Aufgrund der schlechten Sicht durch die Zelle Polycarbonatmembran, Saatgut die Caco-2-Zellen in einem normalen Gewebekulturschale mit ähnlicher Dichte, und verwenden Sie dieses Gericht, das Zellkonfluenz beurteilen. </li><li> Folgen Sie sTEPs 1.1.2-1.1.6 oben. </li></ol></li></ol> 2. Genüberexpression <ol><li> Mit Hilfe der regulären Transfektionsansatz <ol><li> Samen der Caco-2-Zellen auf einer Gewebekulturschale wie oben in 1.1.1.1.-1.1.4 beschrieben. Hinweis: Wenn die Zellen etwa 40-50% konfluent, bereit, transfiziert werden, sind sie. </li><li> Hinzuzufügen 67 ul des Transfektionsreagenz auf 472 &amp; mgr; l der reduzierten Serum-Medium in einem Reaktionsgefäß. Kontakt mit unverdünntem Transfektionsreagenz mit der Rohrwand zu vermeiden. </li><li> Hinzuzufügen 23 ug pLL3.7 enhanced green fluorescent protein (eGFP) 9 in das Transfektionsreagenz / reduziert Serum Mediengemisch. </li><li> Inkubieren des Gemisches für 20 min bei RT. </li><li> Ersetzen Sie die Caco-2-Zellen &quot;Wachstumsmedien mit 8 ml 10% FBS in DMEM. </li><li> Fügen Sie die DNA / Transfektionsreagenz / reduziert Serum Medien Mischung tropfenweise in die Schale. </li><li> Schütteln Sie das Geschirr mehrmals in einer Vorwärts- und backward Richtung, gefolgt von einer linken und rechten Richtung. </li><li> Die Schale in der 37 ° C-Inkubator. </li><li> Ersetzen Sie die Medien in der Schale mit 10 ml Wachstumsmedium einen Tag nach Beginn der Inkubation. </li><li> Überwachen Sie die Genexpression. <ol><li> Überwachen Sie die Genexpression ohne 4 &#39;, 6-Diamidino-2-phenylindol (DAPI) Färbung. Verwendung eines Fluoreszenzmikroskops, bestimmen den Prozentsatz der Zellen, die grün sind. Der Anteil stellt die Transfektionseffizienz. </li><li> Überwachen Sie die Genexpression mit DAPI-Färbung. <ol><li> Mit phosphatgepufferter Salzlösung (PBS) dreimal Waschen der Zellen. </li><li> Fixieren die Zellen durch Zugabe von 10 ml von 4% Formaldehyd in PBS (10 min Inkubation bei RT). </li><li> Dreimal mit PBS waschen der Zellen. </li><li> Die Zellen im Dunkeln inkubieren für 15 min bei RT mit 8 ml PBS, das 1: 500 DAPI, 1% BSA, 0,01% Digitonin. </li><li> Dreimal mit PBS waschen der Zellen. </li><li> Usingen ein Fluoreszenzmikroskop, bestimmen die Anzahl der Zellen, die grün / blau im Vergleich zu denen, die blaue (Abbildung 2 oberes Feld) sind. Der errechnete Prozent stellt die Transfektionseffizienz. </li></ol></li></ol></li></ol></li><li> Verwenden des Lentivirus Expression System 10 <ol><li> Seed menschliche embryonale Nieren (HEK) 293T-Zellen in einer 15 cm Gewebekulturschale (10% FBS in DMEM als Wachstumsmedien). Hinweis: Wenn die Zellen etwa 60-70% konfluent, bereit, transfiziert werden, sind sie. </li><li> Fügen Sie 162 ul der Transfektionsreagenz auf 1.133 &amp; mgr; l der reduzierten Serum-Medien in ein Mikrozentrifugenröhrchen 11. Kontakt mit unverdünntem Transfektionsreagenz mit der Rohrwand zu vermeiden. </li><li> In 24 ug pLL3.7 eGFP (oder ein anderes Konstrukt), 15,6 ug Rev-Response-Element (RRE), 6,0 &amp; mgr; g REV, und 8,4 ug des vesikulären Stomatitis-Virus-Glykoprotein (VSVG) in das Transfektionsreagenz / reduced Serum Medienmischung. </li><li> Inkubieren des Gemisches für 20 min bei RT. </li><li> Ersetzen Sie das Wachstumsmedium mit 16 ml 10% FBS in DMEM. </li><li> Fügen Sie die DNA / Transfektionsreagenz / reduziert Serum Medien Mischung tropfenweise in die Schale. </li><li> Schütteln Sie das Geschirr mehrmals in einer Vorwärts- und Rückwärtsrichtung, gefolgt von einer linken und rechten Richtung. Es ist normal, einige der HEK293T-Zellen freistehend an dieser Stelle zu sehen. </li><li> Die Schale in den Inkubator. </li><li> Nach 24 Stunden Inkubation, ersetzen Sie die Medien in der Schale mit 25 ml Wachstumsmedium. </li><li> Am folgenden Tag, sammeln das Wachstumsmedium, das die Lentivirus (erste Kollektion). </li><li> Wiederholen Sie Schritt 2.2.10 mehr Lentivirus (die zweite Sammlung) zu sammeln. </li><li> Bündeln die erste und die zweite Sammlung, und entfernen Zelltrümmer durch Zentrifugation (2500 × g für 5 min). </li><li> Filtern Sie die Sammlung mit einem Einweg-Flaschenaufsatz-Filter (0,45 um Poren) undKonzentrat, das Virus durch Zentrifugation der Sammlung 31.400 × g für 2 Stunden bei RT (JA-20-Rotor). Markieren den Boden des Zentrifugenrohrs, wo das Pellet entfernt. Den Überstand umfüllen, und lassen Sie das Rohr bei RT invertiert für etwa 15 min. </li><li> Resuspendieren virushaltige Pellet mit 100 ul 1X PBS. Vermeiden Sie Luftblasen. </li><li> Bewahren Sie die konzentrierte Virus bei -80 ° C. Vermeiden Einfrieren und Auftauen. </li><li> Bestimmung der optimalen Menge des Virus benötigt, um die Caco-2-Zellen durch Titration zu transduzieren. Um Kosten zu sparen, verwenden Sie eine 24-Well-Platte (0,3 ml Wachstumsmedium / well) anstelle eines 10 cm Gewebekulturschale für die Titration. <ol><li> Hinzuzufügen zunehmenden Mengen des konzentrierten Virus (0, 1, 2, 5, 10, 25, und 50 ul / Vertiefung), ergänzt mit Polybren (Endkonzentration = 5 &amp; mgr; g / ml) auf 40-70% konfluent Caco-2-Zellen. Schütteln Sie die Platte mit 24 Vertiefungen vorsichtig wie in 1.1.2 beschrieben. </li><li> Überwachung ihrer Genexpression (Schritt 2.1.10.) (Abbildung 2 unten4 Platten). </li></ol></li><li> Da die Expression des Transgens in der Regel aufrechterhalten, pflegen die transduzierten Zellen für zukünftige Experimente und ihre Genexpression kontinuierlich zu überwachen (Schritt 2.1.10.). </li></ol></li></ol> 3. Förderung der Lipoproteinsekretion <ol><li> Mit Hilfe der regulären Gewebekulturschale <ol><li> Vorbereitung einer 100X Lipidgemisch durch Mischen von 50 mg Ölsäure 40 mg Lecithin und 48 mg Natriumtaurocholat. Bringen Sie die Lautstärke auf 900 ul mit PBS (ausreichend für 8 Geschirr). Mischen Sie den Lipidmischung kräftig. </li><li> Add 900 ul 100X Lipidgemisch in 89,1 ml Wachstumsmedium. Zu mischen, und Filtern der lipidhaltigen Medien. Die Endkonzentrationen (1X) Ölsäure, Lecithin und Natriumtaurocholat sind 2,0 mM, 1,36 mM und 1,0 mM. </li><li> 10 ml des lipidhaltigen Medien zu den Zellen. </li><li> Die Zellen mit dem lipidhaltigen Medien Inkubieren 4 Stunden in eine 37° C Inkubator. </li><li> Dreimal mit PBS waschen der Zellen. </li><li> 10 ml Wachstumsmedium zu den Zellen, um die Lipoproteinsekretion sammeln. </li><li> Inkubieren für 2 Stunden in einem 37 ° C Inkubator. </li><li> Sammeln Sie die Lipoprotein-haltigen Medien. </li></ol></li><li> Verwendung der permeablen Membransystem <ol><li> Führen Sie die Schritte, um den Lipid 3.1.1-3.1.2 Mischung herzustellen. </li><li> 10 ml des lipidhaltigen Medien zur apikalen Kammer und 10 ml des Wachstumsmediums zur basolateralen Kammer. </li><li> Inkubieren für 4 Stunden in einem 37 ° C Inkubator. Sammeln Sie die Lipoprotein enthaltenden Medien in der basolateralen Kammer. </li></ol></li></ol> 4. Lipoprotein Isolation (Abbildung 3) <ol><li> Entfernen der Zelltrümmer aus der Lipoprotein enthaltenden Medien durch Zentrifugation (2.000 × g für 5 min). </li><li> Dekantieren Sie das Medium in einen 50-ml-Tube. </li><li> Hinzuzufügen 5,95 g Natriumchlorid zu den Medien. </li><li> Wenn der sample wird durch die TEM untersucht werden, fügen 1 Proteaseinhibitor-Cocktail-Tablette, die Integrität der Lipoproteine ​​aufrechtzuerhalten. </li><li> Bringen Sie die Lautstärke auf 23 ml mit Wasser (1,2 g / ml NaCl-Lösung Dichte). </li><li> Lösen sich die gelösten Stoffe vollständig. </li><li> Dekantiert die gesamte Lösung in ein Ultrazentrifugenröhrchen aus Polycarbonat. </li><li> Überlagern Sie vorsichtig die 1,2 g / ml Dichte Lösung mit 0,5 ml Wasser (1,0 g / ml Dichte). </li><li> Gleichgewicht der Röhre beträgt und nicht auf das Volumen. </li><li> Die Rohre in dem T865 Rotor sanft zu laden. </li><li> Dreh die Proben in der Ultrazentrifuge bei 429.460 xg für 24 h bei 4 ° C. </li><li> Sofort isolieren die Spitze 0,5 ml Lösung durch vorsichtiges Pipettieren. Halten Sie das Rohr so ​​ruhig wie möglich. </li></ol> 5. TEM-Analyse <ol><li> Vorbereitung 5 ml 2% Phosphorwolframsäure (w / v) und der pH-Wert auf 6,0. </li><li> Filtern Sie die 2% Phosphorwolframsäure mit einem Spritzenfilter (Porengröße: 0,2 um). </li><li> Drop 20 ul der Probe auf einem Lipoprotein sterile Schüssel. </li><li> 20 ul Tropfen der filtrierten 2% Phosphorwolframsäure neben der Probe auf der gleichen Schale. </li><li> Fallen sanft ein EM Gitter mit der stumpfen Seite auf der Probe aufliegt. </li><li> Inkubieren bei Raumtemperatur für 1 min. </li><li> Klopfen Sie leicht die Seite des EM Gitter auf einem Filterpapier, um die Probe aus dem Netz zu entfernen. </li><li> Drop Sie vorsichtig die EM Gitter mit der stumpfen Seite auf der 2% Phosphorwolframsäure ruhen. </li><li> Inkubieren bei Raumtemperatur für 1 min. </li><li> Klopfen Sie leicht die Seite des EM Gitter auf einem Filterpapier, um die Phosphorwolframsäure aus dem Netz zu entfernen. </li><li> Verwendung eines TEM, erfassen die Bilder der Lipoproteine ​​(Abbildung 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.

In diesem Papier, um die beiden Systeme, die verwendet werden können, zu erhalten Caco-2-Zellen beschrieben werden, nämlich die normale Gewebekulturschale und die durchlässige Membran. Die Vorteile der Verwendung der permeablen Membransystem umfassen die Trennung der apikalen und basolateralen Kompartimenten, und die Fähigkeit, das Lipid-Mischung zu inkubieren, und gleichzeitig sammelt die Lipoproteinsekretion. Jedoch sind die durchlässigen Membraneinsätze teuer, und ihre Polycarbonat-Membran nicht für eine gute ...

4 - Studien der intestinalen Aufnahme von Nahrungsfett und lipidlösliche Arzneimittel und Vitamine können in vivo unter Verwendung eines Lymphfistel Modell 1 durchgeführt werden. Jedoch sind die Operationstechniken involviert nicht nur anspruchsvoll, sondern auch kostspielig. Obwohl in vivo-Ansätzen auf fäkale Analysen können verwendet werden, basieren, sind sie hauptsächlich verwendet, um die prozentuale Aufnahme durch den Magen-Darm-2,5 bestimmen. Die in diesem Dokument beschriebenen in vitro-Modell ist kostengünstiger, und die Techniken beteiligt sind wohl weniger anspruchsvoll. Genetische Veränderung Studien sind auch wirtschaftlicher und weniger zeitaufwendig, wenn sie mit diesen in-vitro-Modell durchgeführt. Da lipidlöslichen Materialien, die von Enterozyten genommen werden, in Lipoproteinen 6,7 verpackt ist, wird die Wirksamkeit dieser in vitro-Modell zu erzeugen Lipoproteine ​​entscheidend. Die zwei Haupt intestinal Lipoproteine ​​Chylomikronen und sehr geringer Dichte (VLDL). Chylomikronen, definiert als Lipoproteine ​​mit 80 nm oder mehr im Durchmesser, werden ausschließlich durch den Dünndarm produziert, wenn Lipide in Fülle im Magen-Lumen vorhanden sind. Da sie die größte Lipoproteine ​​Chylomikronen sind denkbar die effizientesten Lipidtransportern. Dieses in vitro-Modell, das in der Lage ist Chylomikronen 8 kann verwendet werden, um Nahrungsfettabsorption, lipidlösliches Vitamin Absorption durch den Darm und die orale Bioverfügbarkeit lipophiles Arzneimittel zu untersuchen. Die Anwesenheit von lipidlösliche Moleküle, Vitamine oder Drogen in der Lipoproteinfraktion ist ein Indikator für deren Absorption durch den Dünndarm. Wie zuvor erläutert, kann dieses Modell verwendet werden, um die orale Bioverfügbarkeit lipophilen Arzneimittel 6 zu verbessern. Dieses Papier beschreibt, wie Caco-2-Zellen sollten in durchlässige Membran oder regelmäßige Gewebekulturschalen, wie die Lipid-mi beibehalten werdenBefestigung zur Stimulierung Lipoproteinsynthese sollten darauf vorbereitet sein, wie die Lentivirus-Expressionssystem verwendet werden, um effektiv eine Überexpression zu erreichen, und wie die isolierte Lipoproteine ​​sollten analysiert werden.

<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).

Abbildung 1 zeigt die normale 13-Tage nach der konfluenten Caco-2-Zellen. Das Aussehen des kuppelförmigen Strukturen und intrazellulären Lipidtröpfchen Charakteristik unterschieden Caco-2-Zellen. Wenn die Caco-2-Zellen werden nicht in gleicher Weise während der Aussaat dispergiert werden sie verklumpen und wuchern in bestimmten Bereichen der Schale; und es gibt ein paar Bereiche in der Schale ohne Zellen sein. Wirbelnden und Platzieren der Schale auf einer schrägen Fläche zu vermeiden. Es ist auch...

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.

Verwenden von Caco-2-Zellen, die Lipidtransport Studie der Darm

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 Aufrufe.  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.

Serie: 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.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

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

Forschung

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-Tiermodell Stimuli

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.

Isolierung und Reinigung von Drosophila Peripheren Neuronen durch Magnetic Bead Sorting

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.

Mit der optokinetischen Reaktion auf visuelle Funktion des Zebrafisch-Studie

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.

Mess-Cation Transport von Na, K-und H, K-ATPase in<em&gt; Xenopus</em&gt; Oozyten mittels Atomabsorptionsspektrometrie: Eine Alternative zu Radioisotopen Assays

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.

Methoden Epithelial Transportprotein Funktion und Expression in Mutterdarm und Caco-2-Zellen gezüchtet in 3D zu studieren

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.

Gemischte Primärkulturen von Murine Duenndarm bestimmt zur Untersuchung von Gut Hormonausschüttung und Live Cell Imaging von enteroendokrinen Zellen

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.

Angebundene Doppelschicht-Lipidmembranen zur Überwachung der Wärmeübertragung zwischen Goldnanopartikeln und Lipidmembranen

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.

Isolierung von interstitiellen Klappenzellen der Maus zur Untersuchung der Verkalkung der Aortenklappe 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.

Der Caco-2-Zell-Bioassay zur Messung der Eisenbioverfügbarkeit von Lebensmitteln

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