El uso de células Caco-2 para el Estudio de lípidos Transporte por el Intestino
Summary
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.
Abstract
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).
Introduction
Los estudios de absorción intestinal de grasas en la dieta, y las drogas y vitaminas solubles en lípidos pueden llevar a cabo in vivo mediante el uso de un modelo de fístula linfática de 1 – 4. Sin embargo, las técnicas quirúrgicas que participan no sólo son un reto, pero también costoso. Aunque in vivo enfoques basados en el análisis fecal pueden ser utilizados, que se utilizan principalmente para determinar el porcentaje de absorción por el tracto gastrointestinal 2,5. El modelo in vitro descrito en este artículo es más rentable, y las técnicas utilizadas son sin duda menos difícil. Estudios de modificación genética también son más económicos y menos tiempo cuando se llevaron a cabo utilizando este modelo in vitro.
Dado que los materiales solubles en lípidos que son absorbidos por los enterocitos son empaquetados en lipoproteínas de 6,7, la eficacia de este modelo in vitro para producir las lipoproteínas es crucial. Los dos intestina principall lipoproteínas son quilomicrones y lipoproteínas de muy baja densidad (VLDL). Los quilomicrones, definidos como lipoproteínas con 80 nm de diámetro o más, se producen estrictamente por el intestino delgado cuando los lípidos son abundantemente presentes en el lumen gastrointestinal. Puesto que son las lipoproteínas más grandes, los quilomicrones son posiblemente los transportadores de lípidos más eficientes. Este modelo in vitro, que es capaz de producir los quilomicrones 8, se puede utilizar para estudiar la absorción de grasa dietética, soluble en lípidos vitamina absorción por el intestino, y la biodisponibilidad oral de fármaco lipofílico. La presencia de moléculas solubles en lípidos, vitaminas, medicamentos o en la fracción de lipoproteína es un indicador de su absorción por el intestino delgado. Como se discutió anteriormente, este modelo se puede utilizar para mejorar la biodisponibilidad oral de fármaco lipofílico 6.
Este artículo describe cómo se deben mantener células Caco-2 en la membrana permeable o platos regulares de cultivo de tejidos, cómo las millas de lípidosxture para estimular la producción de lipoproteínas debe prepararse, cómo el sistema de expresión lentivirus puede ser empleado para lograr la sobreexpresión efectiva, y cómo se debe analizar las lipoproteínas aisladas.
Protocol
Representative Results
Discussion
En este papel, los dos sistemas que se pueden utilizar para mantener las células Caco-2 se describen, a saber, el plato de cultivo de tejido normal y la membrana permeable. Los beneficios de usar el sistema de membrana permeable incluyen la separación de la apical y basolateral de los compartimentos, y la capacidad de incubar la mezcla de lípidos y recoger la secreción de lipoproteínas de forma simultánea. Sin embargo, los insertos de membrana permeable son caros, y su membrana de policarbonato no permite una buen…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| DMEM | VWR | 16750-112 | Pre-warm the growth media in individual tissue culture dish before adding cells |
| FBS | Fisher | 3600511 | We do not heat inactivate our serum |
| Trypsin | VWR | 45000-660 | Cells can be washed with PBS prior to trypsin treatment |
| Permeable membrane system | Fisher | 7200173 | 10-cm dish, 3-mm pore size, polycarbonate membrane |
| 10 cm dish | Fisher | 08-772-E | Tissue culture dish |
| 15 cm dish | Fisher | 08-772-24 | Tissue culture dish |
| 24 well plate | Fisher | 12565163 | Tissue culture plate |
| Lipid-based transfection reagent | Fisher | PRE2691 | Can be substituted with other transfection reagent |
| Reduced serum media | Invitrogen | 11058021 | For transfection |
| pLL3.7 eGFP | Addgene | 11795 | https://www.addgene.org/11795/ |
| Bottle-top filter | Fisher | 9761120 | 0.45 mm pore |
| Polybrene | Fisher | NC9840454 | 10 mg/mL |
| Oleic acid | Sigma | 01383-5G | Prevent freeze-thaw cycle |
| Lecithin | Fisher | IC10214625 | Egg lecithin |
| Sodium taurocholate | Fisher | NC9620276 | Product discontinued; alternative catalog number: 50-121-7956 |
| Protease inhibitor cocktail tablet (EDTA-free) | Fisher | 5892791001 | Used mainly for samples that need TEM analysis |
| Polycarbonate ultracentrifuge tube | Fisher | NC9696153 | Reusing it multiple times will collapse the tube |
| Lid for ultracentrifuge tube | Fisher | NC9796914 | A tool is required to remove the tube/lid from rotor |
| Syringe | Fisher | 50-949-261 | Disposable |
| Syringe filter | Fisher | 09-719C | Pore size = 0.2 mm; nylon |
| Phosphotungstic acid | Fisher | AC208310250 | For preparing 2% phosphotungstic acid, pH 6.0 |
| Tweezer | Fisher | 50-238-62 | Extra fine and strong tips |
| Formvar/carbon grid | Fisher | 50-260-34 | Formvar/carbon film square grid 400 Copper |
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