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

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

Studies of intestinal absorption of dietary fat, and lipid-soluble drugs and vitamins can be conducted in vivo by using a lymph fistula model ^1–4. However, the surgical techniques involved are not only challenging, but also costly. Although in vivo approaches based on fecal analysis may be utilized, they are used mainly to determine the percent uptake by the gastrointestinal tract ^2,5. The in vitro model described in this paper is more cost effective, and the techniques involved are arguably less challenging. Genetic modification studies are also more economical and less time-consuming when t....

Protocol

1. Maintenance of the Caco-2 Cells

1. Using regular tissue culture dishes
   1. Thaw the Caco-2 cells from a frozen vial by placing the vial in a 37 °C water bath, and immediately add them to a 10 cm tissue culture dish containing 10 ml of pre-warmed growth media (15% fetal bovine serum (FBS) in Dulbecco’s Modified Eagle Medium (DMEM)).
      1. When the Caco-2 cells have reached 50-70% confluence, split them 1:6 by incubating the cells with 3 ml of 0.05% Trypsin / 0.53 mM ethylenediaminetetraacetic acid (EDTA) at 37 °C until they are detached (15 min). To avoid cell clumping, mix the cells several times by gentle pipettin....

Results

Figure 1 displays normal 13-days post-confluent Caco-2 cells. The appearance of dome-shaped structures and intracellular lipid droplets are characteristic of differentiated Caco-2 cells. When the Caco-2 cells are not dispersed equally during seeding, they will clump and overgrow in certain areas of the dish; and there will be a few areas in the dish without any cells. Swirling and placing the dish on a slanted surface should be avoided. It is also important to note that post-confluent Caco-2 cells are mo.......

Discussion

In this paper, the two systems that can be used to maintain Caco-2 cells are described, namely, the regular tissue culture dish and the permeable membrane. The benefits of using the permeable membrane system include the separation of the apical and the basolateral compartments, and the ability to incubate the lipid mixture and collect the lipoprotein secretion simultaneously. However, the permeable membrane inserts are expensive, and their polycarbonate membrane does not allow for good cell visibility. One of the advanta.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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