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. Maintenance of the Caco-2 Cells
<ol>
	<li>Using regular tissue culture dishes
	<ol>
		<li>Thaw the Caco-2 cells from a frozen vial by placing the vial in a 37 &#176;C water bath, and immediately add them to a 10 cm tissue culture dish containing 10 ml&#160;of pre-warmed growth media (15% fetal bovine serum (FBS) in Dulbecco&#8217;s Modified Eagle Medium (DMEM)).
		<ol>
			<li>When the Caco-2 cells have reached 50-70% confluence, split them 1:6 by incubating the cells with 3 ml&#160;of 0.05% Trypsin / 0.53 mM ethylenediaminetetraacetic acid (EDTA) at 37 &#176;C until they are detached (15 min). To avoid cell clumping, mix the cells several times by gentle pipetting. Add 0.5 ml&#160;of the trypsinized cells to a 10 cm tissue culture dish containing 10 ml&#160;of pre-warmed growth media.</li>
		</ol>
		</li>
		<li>Gently shake the dishes several times in a forward and backward direction followed by a leftward and rightward direction. Avoid swirling the dishes as it may result in an unequal cell dispersion.</li>
		<li>Place the dishes on a flat surface in a 37 &#176;C incubator supplied with 5% CO2. Slanted surfaces may also result in an unequal cell dispersion.</li>
		<li>Change the growth media a day after starting the incubation.</li>
		<li>Monitor the cells and record the day they reach confluence. It will take about 1 week for the cells to reach confluence from the day they are split.</li>
		<li>Change the growth media twice a week before the cells reach confluence. Once the cells have reached confluence, change the growth media every other day. After the cells are 7-days post-confluent, change the growth media daily. 
		Note: The cells are ready for experiments when they are 13-days post-confluent (Figure 1). Since the cells will eventually become less effective in producing lipoproteins, use cells that are between 13 to 17-days post-confluent 8. Go to step 3 on how to conduct the experiment.</li>
	</ol>
	</li>
	<li>Using the permeable membrane system
	<ol>
		<li>Seed the Caco-2 cells in the permeable membrane insert as described in step 1.1.1.1. above. Avoid using cells from a frozen vial because their cell growth is normally slower (recovery period). Briefly, add 0.5 ml&#160;of the trypsinized cells to the apical chamber (upper compartment) that contains 10 ml&#160;of pre-warmed growth media. Also, add 10 ml&#160;of pre-warmed growth media to the basolateral chamber (lower compartment). 
		Note: For best practice, add the growth media to the apical chamber first and then to the basolateral chamber. When removing the old media, remove the basolateral media before the apical media. Avoid poking the polycarbonate membrane as it may rupture the membrane.</li>
		<li>Due to poor cell visibility through the polycarbonate membrane, seed the Caco-2 cells in a regular tissue culture dish with similar density, and use this dish to judge the cell confluence.</li>
		<li>Follow steps 1.1.2-1.1.6 above.</li>
	</ol>
	</li>
</ol>
2. Gene Overexpression
<ol>
	<li>Using the regular transfection approach
	<ol>
		<li>Seed the Caco-2 cells on a tissue culture dish as described in 1.1.1.1.-1.1.4 above. 
		Note: When the cells are about 40-50% confluent, they are ready to be transfected.</li>
		<li>Add 67 &#956;l&#160;of the transfection reagent to 472 &#956;l&#160;of the reduced serum media in a microcentrifuge tube. Avoid contact of the undiluted transfection reagent with the tube wall.</li>
		<li>Add 23 &#956;g of pLL3.7 enhanced green fluorescent protein (eGFP) 9 into the transfection reagent/reduced serum media mixture.</li>
		<li>Incubate the mixture for 20 min&#160;at RT.</li>
		<li>Replace the Caco-2 cells&#8217; growth media with 8 ml&#160;of 10% FBS in DMEM.</li>
		<li>Add the DNA/transfection reagent/reduced serum media mixture drop-wise to the dish.</li>
		<li>Gently shake the dishes several times in a forward and backward direction followed by a leftward and rightward direction.</li>
		<li>Place the dish in the 37 &#176;C incubator.</li>
		<li>Replace the media in the dish with 10 ml of growth media a day after starting the incubation.</li>
		<li>Monitor the gene expression.
		<ol>
			<li>Monitor the gene expression without 4&#8217;,6-diamidino-2-phenylindole (DAPI) staining. Using a fluorescent microscope, determine the percent of cells that are green. The percentage represents the transfection efficiency.</li>
			<li>Monitor the gene expression with DAPI staining.&#160;
			<ol>
				<li>Wash the cells with phosphate buffered saline (PBS) three times.</li>
				<li>Fix the cells by adding 10 ml&#160;of 4% formaldehyde in PBS (10 min incubation at RT).</li>
				<li>Wash the cells with PBS three times.</li>
				<li>Incubate the cells in the dark for 15 min&#160;at RT with 8 ml&#160;of PBS containing 1:500 DAPI, 1% BSA, 0.01% digitonin.</li>
				<li>Wash the cells with PBS three times.</li>
				<li>Using a fluorescent microscope, determine the number of cells that are green/blue relative to those that are blue (Figure 2 top panel). The calculated percentage represents the transfection efficiency.</li>
			</ol>
			</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Using the lentivirus overexpression system 10
	<ol>
		<li>Seed human embryonic kidney (HEK)293T cells in a 15 cm tissue culture dish (10% FBS in DMEM as their growth media). 
		Note: When the cells are about 60-70% confluent, they are ready to be transfected.</li>
		<li>Add 162 &#956;l&#160;of the transfection reagent to 1,133 &#956;l&#160;of the reduced serum media in a microcentrifuge tube 11. Avoid contact of the undiluted transfection reagent with the tube wall.</li>
		<li>Add 24 &#956;g of pLL3.7 eGFP (or another construct), 15.6 &#956;g of Rev response element (RRE), 6.0 &#956;g of REV, and 8.4 &#956;g of vesicular stomatitis virus glycoprotein (VSVG) into the transfection reagent/reduced serum media mixture.</li>
		<li>Incubate the mixture for 20 min&#160;at RT.</li>
		<li>Replace the growth media with 16 ml&#160;of 10% FBS in DMEM.</li>
		<li>Add the DNA/transfection reagent/reduced serum media mixture drop-wise to the dish.</li>
		<li>Gently shake the dishes several times in a forward and backward direction followed by a leftward and rightward direction. It is normal to see some of the HEK293T cells detached at this point.</li>
		<li>Place the dish in the incubator.</li>
		<li>After 24 hr&#160;of incubation, replace the media in the dish with 25 ml&#160;of growth media.</li>
		<li>On the following day, collect the growth media containing the lentivirus (first collection).</li>
		<li>Repeat step 2.2.10 to collect more lentivirus (the second collection).</li>
		<li>Pool the first and the second collection, and remove cell debris by centrifugation (2,500 x g for 5 min).</li>
		<li>Filter the collection with a disposable bottle-top filter (0.45 &#956;m pore), and concentrate the virus by centrifuging the collection at 31,400 x g for 2 hr&#160;at RT (JA-20 rotor). Mark the bottom of the centrifuge tube where the pellet is located. Decant the supernatant, and leave the tube inverted at RT for about 15 min.</li>
		<li>Resuspend the virus-containing pellet with 100 &#956;l&#160;of 1X PBS. Avoid bubbles.</li>
		<li>Store the concentrated virus at -80 &#176;C. Avoid freeze-thaw cycles.</li>
		<li>Determine the optimal amount of virus needed to transduce the Caco-2 cells by titration. To save cost, use a 24-well plate (0.3 ml&#160;of growth media/well) instead of a 10 cm tissue culture dish for titration.
		<ol>
			<li>Add increasing amounts of the concentrated virus (0, 1, 2, 5, 10, 25, and 50 &#956;l/well) supplemented with polybrene (final concentration = 5 &#956;g/ml) to the 40-70% confluent Caco-2 cells. Shake the 24-well plate gently as described in 1.1.2.</li>
			<li>Monitor their gene expression (Step 2.1.10.) (Figure 2 bottom 4 panels).</li>
		</ol>
		</li>
		<li>Since the expression of transgene is generally sustained, maintain the transduced cells for future experiments and continuously monitor their gene expression (Step 2.1.10.).</li>
	</ol>
	</li>
</ol>
3. Stimulating the Lipoprotein Secretion
<ol>
	<li>Using the regular tissue culture dish
	<ol>
		<li>Prepare a 100X lipid mixture by mixing 50 mg of oleic acid, 40 mg of lecithin, and 48 mg of sodium taurocholate. Bring the volume to 900 &#956;l&#160;with PBS (enough for 8 dishes). Vortex the lipid mixture vigorously.</li>
		<li>Add 900 &#956;l&#160;of the 100X lipid mixture into 89.1 ml&#160;of growth media. Mix, and filter the lipid-containing media. The final concentrations (1X) of oleic acid, lecithin, and sodium taurocholate are 2.0 mM, 1.36 mM, and 1.0 mM, respectively.</li>
		<li>Add 10 ml&#160;of the lipid-containing media to the cells.</li>
		<li>Incubate the cells with the lipid-containing media for 4 hr&#160;in a 37 &#176;C incubator.</li>
		<li>Wash the cells with PBS three times.</li>
		<li>Add 10 ml&#160;of the growth media to the cells to collect the lipoprotein secretion.</li>
		<li>Incubate for 2 hr&#160;in a 37 &#176;C incubator.</li>
		<li>Collect the lipoprotein-containing media.</li>
	</ol>
	</li>
	<li>Using the permeable membrane system
	<ol>
		<li>Follow steps 3.1.1-3.1.2 to prepare the lipid mixture.</li>
		<li>Add 10 ml&#160;of the lipid-containing media to the apical chamber and 10 ml&#160;of the growth media to the basolateral chamber.</li>
		<li>Incubate for 4 hr&#160;in a 37 &#176;C incubator. Collect the lipoprotein-containing media in the basolateral chamber.</li>
	</ol>
	</li>
</ol>
4. Lipoprotein Isolation (Figure 3)
<ol>
	<li>Remove the cell debris from the lipoprotein-containing media by centrifugation (2,000 x g for 5 min).</li>
	<li>Decant the media into a 50 ml&#160;tube.</li>
	<li>Add 5.95 g of sodium chloride to the media.</li>
	<li>If the sample will be analyzed by the TEM, add 1 protease inhibitor cocktail tablet to maintain the integrity of the lipoproteins.</li>
	<li>Bring the volume to 23 ml&#160;with water (1.2 g/ml&#160;NaCl density solution).</li>
	<li>Dissolve the solutes completely.</li>
	<li>Decant all of the solution into a polycarbonate ultracentrifuge tube.</li>
	<li>Gently overlay the 1.2 g/ml&#160;density solution with 0.5 ml&#160;water (1.0 g/ml&#160;density).</li>
	<li>Balance the tube by weight and not by volume.</li>
	<li>Gently load the tubes in the T865 rotor.</li>
	<li>Spin the samples in the ultracentrifuge at 429,460 x g for 24 hr&#160;at 4 &#176;C.</li>
	<li>Immediately isolate the top 0.5 ml&#160;solution by gentle pipetting. Keep the tube as still as possible.</li>
</ol>
5. TEM Analysis
<ol>
	<li>Prepare 5 ml&#160;of 2% phosphotungstic acid (w/v) and adjust the pH to 6.0.</li>
	<li>Filter the 2% phosphotungstic acid with a syringe filter (pore size: 0.2 &#181;m).</li>
	<li>Drop 20 &#956;l&#160;of the lipoprotein sample on a sterile dish.</li>
	<li>Drop 20 &#956;l&#160;of the filtered 2% phosphotungstic acid next to the sample on the same dish.</li>
	<li>Gently drop an EM grid with the dull side resting on the sample.</li>
	<li>Incubate at RT for 1 min.</li>
	<li>Gently tap the side of the EM grid on a filter paper to remove the sample from the grid.</li>
	<li>Gently drop the EM grid with the dull side resting on the 2% phosphotungstic acid.</li>
	<li>Incubate at RT for 1 min.</li>
	<li>Gently tap the side of the EM grid on a filter paper to remove the phosphotungstic acid from the grid.</li>
	<li>Using a TEM, capture the images of the lipoproteins (Figure 4).</li>
</ol>

<ol><li>Drover, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD36+deficiency+impairs+intestinal+lipid+secretion+and+clearance+of+chylomicrons+from+the+blood%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">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/pubmed?term=((CD36+is+important+for+chylomicron+formation+and+secretion+and+may+mediate+cholesterol+uptake+in+the+proximal+intestine%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">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/pubmed?term=((Intestinal+alkaline+phosphatase+release+is+not+associated+with+chylomicron+formation%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">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/pubmed?term=((Why+does+the+gut+choose+apolipoprotein+B48+but+not+B100+for+chylomicron+formation%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">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/pubmed?term=((A+novel%2C+noninvasive+method+for+the+measurement+of+intestinal+fat+absorption%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">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/pubmed?term=((Intestinal+transport+as+a+potential+determinant+of+drug+bioavailability%5BTitle%5D)+AND+%22Curr+Clin+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Enterocyte+fatty+acid+uptake+and+intestinal+fatty+acid-binding+protein%5BTitle%5D)+AND+%22Biochem+Soc+Trans%22%5BJournal%5D)">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/pubmed?term=((Chylomicrons+produced+by+Caco-2+cells+contained+ApoB-48+with+diameter+of+80-200+nm%5BTitle%5D)+AND+%22Physiol+Rep%22%5BJournal%5D)">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/pubmed?term=((A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells%2C+stem+cells+and+transgenic+mice+by+RNA+interference%5BTitle%5D)+AND+%22Nat+Genet%22%5BJournal%5D)">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/pubmed?term=((Production+of+lentiviral+vectors+for+transducing+cells+from+the+central+nervous+system%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Insulin+biosynthetic+interaction+network+component%2C+TMEM24%2C+facilitates+insulin+reserve+pool+release%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">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/pubmed?term=((Effects+of+saturated%2C+mono-%2C+and+polyunsaturated+fatty+acids+on+the+secretion+of+apo+B+containing+lipoproteins+by+Caco-2+cells%5BTitle%5D)+AND+%22Atherosclerosis%22%5BJournal%5D)">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., Ménard, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caco-2+cells+and+human+fetal+colon%3A+a+comparative+analysis+of+their+lipid+transport%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">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/pubmed?term=((Assembly+and+secretion+of+chylomicrons+by+differentiated+Caco-2+cells.+Nascent+triglycerides+and+preformed+phospholipids+are+preferentially+used+for+lipoprotein+assembly%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">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 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...

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&#8211;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 they are conducted using this in vitro model.
Since lipid-soluble materials that are taken up by enterocytes are packaged into lipoproteins 6,7, the effectiveness of this in vitro model to produce lipoproteins is crucial. The two main intestinal lipoproteins are chylomicrons and very low-density lipoproteins (VLDL). Chylomicrons, defined as lipoproteins with 80 nm or more in diameter, are produced strictly by the small intestine when lipids are abundantly present in the gastrointestinal lumen. Since they are the largest lipoproteins, chylomicrons are conceivably the most efficient lipid transporters. This in vitro model, which is capable of producing chylomicrons 8, can be used to study dietary fat absorption, lipid-soluble vitamin absorption by the gut, and oral lipophilic drug bioavailability. The presence of lipid-soluble molecules, vitamins, or drugs in the lipoprotein fraction is an indicator of their absorption by the small intestine. As previously discussed, this model can be used to improve oral lipophilic drug bioavailability 6.
This paper describes how Caco-2 cells should be maintained in permeable membrane or regular tissue culture dishes, how the lipid mixture for stimulating lipoprotein production should be prepared, how the lentivirus expression system can be employed to achieve effective overexpression, and how the isolated lipoproteins should be analyzed.

<table><tbody><tr><td>DMEM</td><td>VWR</td><td>16750-112</td><td>Pre-warm the growth media in individual tissue culture dish before adding cells</td></tr><tr><td>FBS</td><td>Fisher</td><td>3600511</td><td>We do not heat inactivate our serum</td></tr><tr><td>Trypsin</td><td>VWR</td><td>45000-660</td><td>Cells can be washed with PBS prior to trypsin treatment&amp;nbsp;</td></tr><tr><td>Permeable membrane system</td><td>Fisher</td><td>7200173</td><td>10-cm dish, 3-mm pore size, polycarbonate membrane</td></tr><tr><td>10 cm dish</td><td>Fisher</td><td>08-772-E</td><td>Tissue culture dish</td></tr><tr><td>15 cm dish</td><td>Fisher</td><td>08-772-24</td><td>Tissue culture dish</td></tr><tr><td>24 well plate</td><td>Fisher</td><td>12565163</td><td>Tissue culture plate</td></tr><tr><td>Lipid-based transfection reagent</td><td>Fisher</td><td>PRE2691</td><td>Can be substituted with other transfection reagent</td></tr><tr><td>Reduced serum media</td><td>Invitrogen</td><td>11058021</td><td>For transfection</td></tr><tr><td>pLL3.7 eGFP</td><td>Addgene</td><td>11795</td><td>&lt;a href=&quot;https://www.addgene.org/11795/&quot;&gt;https://www.addgene.org/11795/&lt;/a&gt;</td></tr><tr><td>Bottle-top filter</td><td>Fisher</td><td>9761120</td><td>0.45 mm pore</td></tr><tr><td>Polybrene</td><td>Fisher</td><td>NC9840454</td><td>10 mg/ml</td></tr><tr><td>Oleic acid</td><td>Sigma</td><td>01383-5G</td><td>Prevent freeze-thaw cycle</td></tr><tr><td>Lecithin&amp;nbsp;</td><td>Fisher</td><td>IC10214625</td><td>Egg lecithin&amp;nbsp;</td></tr><tr><td>Sodium taurocholate</td><td>Fisher</td><td>NC9620276</td><td>Product discontinued; alternative catalog number: 50-121-7956</td></tr><tr><td>Protease inhibitor cocktail tablet (EDTA-free)</td><td>Fisher</td><td>5892791001</td><td>Used mainly for samples that need TEM analysis</td></tr><tr><td>Polycarbonate ultracentrifuge tube</td><td>Fisher</td><td>NC9696153</td><td>Reusing it multiple times will collapse the tube</td></tr><tr><td>Lid for ultracentrifuge tube</td><td>Fisher</td><td>NC9796914</td><td>A tool is required to remove the tube/lid from rotor</td></tr><tr><td>Syringe</td><td>Fisher</td><td>50-949-261</td><td>Disposable</td></tr><tr><td>Syringe filter</td><td>Fisher</td><td>09-719C</td><td>Pore size = 0.2 mm; nylon</td></tr><tr><td>Phosphotungstic acid</td><td>Fisher</td><td>AC208310250</td><td>For preparing 2% phosphotungstic acid, pH 6.0</td></tr><tr><td>Tweezer</td><td>Fisher</td><td>50-238-62</td><td>Extra fine and strong tips</td></tr><tr><td>Formvar/carbon grid</td><td>Fisher</td><td>50-260-34</td><td>Formvar/carbon film square grid 400 Copper</td></tr></tbody></table>

using caco-2 cells to study lipid transport by the intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

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...

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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.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

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18.8K Views.  California Northstate University. Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

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

Cholesterol Efflux Assay

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of cellular membranes, apoptosis and necrosis 1. Cells can source cholesterol from intracellular synthesis and from plasma lipoproteins, both sources are sufficient to fully satisfy cells' requirements for cholesterol. The processes of cholesterol synthesis and uptake are tightly regulated and deficiencies of cholesterol are rare 2. Excessive cholesterol is more common problem 3. With the exception of hepatocytes and to some degree adrenocortical cells, cells are unable to degrade cholesterol. Cells have two options to reduce their cholesterol content: to convert cholesterol into cholesteryl esters, an option with limited capacity as overloading cells with cholesteryl esters is also toxic, and cholesterol efflux, an option with potentially unlimited capacity. Cholesterol efflux is a specific process that is regulated by a number of intracellular transporters, such as ATP binding cassette transporter proteins A1 (ABCA1) and G1 (ABCG1) and scavenger receptor type B1. The natural acceptor of cholesterol in plasma is high density lipoprotein (HDL) and apolipoprotein A-I. 
The cholesterol efflux assay is designed to quantitate the rate of cholesterol efflux from cultured cells. It measures the capacity of cells to maintain cholesterol efflux and/or the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of the following steps. Step 1: labelling cellular cholesterol by adding labelled cholesterol to serum-containing medium and incubating with cells for 24-48 h. This step may be combined with loading of cells with cholesterol. Step 2: incubation of cells in serum-free medium to equilibrate labelled cholesterol among all intracellular cholesterol pools. This stage may be combined with activation of cellular cholesterol transporters. Step 3: incubation of cells with extracellular acceptor and quantitation of movement of labelled cholesterol from cells to the acceptor. If cholesterol precursors were used to label newly synthesized cholesterol, a fourth step, purification of cholesterol, may be required.
The assay delivers the following information: (i) how a particular treatment (a mutation, a knock-down, an overexpression or a treatment) affects the capacity of cell to efflux cholesterol and (ii) how the capacity of plasma acceptors to accept cholesterol is affected by a disease or a treatment. This method is often used in context of cardiovascular research, metabolic and neurodegenerative disorders, infectious and reproductive diseases.

The cholesterol assay is designed to quantitate the rate of cholesterol efflux from cultured cells and the capacity of plasma acceptors to accept cholesterol released from cells. The assay consists of labelling cells with cholesterol, equilibration of cholesterol among intracellular pools and release of cholesterol to an extracellular acceptor.

Cholesterol content of cells must be maintained within the very tight limits, too much or too little cholesterol in a cell results in disruption of ...

Medicine

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

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

Bioengineering

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

Macroautophagy, commonly referred to as autophagy, is a highly conserved cellular process responsible for the degradation of cellular components. This process is particularly prominent under conditions such as fasting, cellular stress, organelle damage, cellular damage, or aging of cellular components. During autophagy, a segment of the cytoplasm is enclosed within double-membrane vesicles known as autophagosomes, which then fuse with lysosomes. Following this fusion, the contents of autophagosomes undergo non-selective bulk degradation facilitated by lysosomes. However, autophagy also exhibits selective functionality, targeting specific organelles, including mitochondria, peroxisomes, lysosomes, nuclei, and lipid droplets (LDs). Lipid droplets are enclosed by a phospholipid monolayer that isolates neutral lipids from the cytoplasm, protecting cells from the harmful effects of excess sterols and free fatty acids (FFAs). Autophagy is implicated in various conditions, including neurodegenerative diseases, metabolic disorders, and cancer. Specifically, lipophagy -- the autophagy-dependent degradation of lipid droplets -- plays a crucial role in regulating intracellular FFA levels across different metabolic states. This regulation supports essential processes such as membrane synthesis, signaling molecule formation, and energy balance. Consequently, impaired lipophagy increases cellular vulnerability to death stimuli and contributes to the development of diseases such as cancer. Despite its significance, the precise mechanisms governing lipid droplet metabolism regulated by lipophagy in cancer cells remain poorly understood. This article aims to describe confocal imaging acquisition and quantitative imaging analysis protocols that enable the investigation of lipophagy associated with metabolic changes in cancer cells. The results obtained through these protocols may shed light on the intricate interplay between autophagy, lipid metabolism, and cancer progression. By elucidating these mechanisms, novel therapeutic targets may emerge for combating cancer and other metabolic-related diseases.

Lipophagy is a selective form of autophagy that involves the degradation of lipid droplets. Dysfunctions in this process are associated with cancer development. However, the precise mechanisms are not yet fully understood. This protocol describes quantitative imaging approaches to better understand the interplay between autophagy, lipid metabolism, and cancer progression.

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

Cancer Research

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between colorectal tumor cells and the immune system. Colorectal carcinoma is among the most common type of cancer, typically treated by surgery, chemotherapy, and radiotherapy. Advantages of radiotherapy in targeting the tumor are well known. However, even limited exposures of healthy tissues are of great concern, particularly regarding the effects on the intestinal barrier and the immune system. The adopted setup allows to study the interplay between two cell populations in a condition more similar to the physiological one, when compared to normal cell cultures. For this purpose, we resort to different techniques and we used an in vitro co-culture model, based on Caco-2 cells differentiated as a monolayer and PBMC, sharing the same culture medium. This protocol has been developed to focus on both macroscopic effects, i.e. cell viability and Trans-Epithelial Electrical Resistance (TEER), and, through western blot, molecular alterations, i.e. the activation of inflammatory pathway in immune cells and the tight junction protein expression in Caco-2 cells. Initial evaluation of radiation effects on Caco-2 cell viability was assessed via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Trypan blue assays, while TEER was measured at fixed time intervals through an ohmmeter specifically designed for co-culture systems. In this way, the effects due to radiation, the presence of Peripheral Blood Mononuclear Cells (PBMC), and eventually their synergistic effect, can be demonstrated. Through these complementary techniques, we observed a high radio-resistance of Caco-2 within the range of 2 - 10 Gy of X-rays and an increased Caco-2 monolayer permeability when PBMCs were added. In particular, PBMC presence was found to be associated with the variation in the tight junction scaffold proteins expression.

We present a protocol to investigate the crosstalk between X-ray-irradiated Caco-2 and peripheral blood mononuclear cells (PBMC). The protocol starts with Caco-2 irradiation and set-up of the co-culture with PBMC; subsequently, trans-epithelial electrical resistance is measured regularly over 48 h and western blot performed in both Caco-2 and PBMC.

The protocol adopted in this work aims at unraveling how X-rays perturb the functioning of the intestinal barrier, focusing on the interplay between ...

Immunology and Infection

Lipid Exchange Assay in Living Cells

Lipid rafts are dynamic, ordered domains in the plasma membrane often formed during membrane protein clustering and signaling. The lipid identity of the outer leaflet drives the membrane&#39;s propensity to form lipid rafts. The transient nature of lipid rafts makes it difficult to study in living cells. Therefore, methods that add or remove raft-forming lipids at&#160;the outer leaflet of living cells facilitate studying the characteristics of rafts, such as their effects on membrane proteins. Lipid exchange experiments developed in our lab utilize lipid-loaded cyclodextrins to remove and add exogenous phospholipids to change the lipid constitution of the plasma membrane. Substituting the membrane with a raft or non-raft-forming lipid can aid in studying the effects on transmembrane protein activity. Here, we describe a method for lipid exchange on the outer leaflet of the plasma membrane using lipid-loaded cyclodextrin. We demonstrate the preparation of the exchange media and the subsequent treatment of attached mammalian cells. We also showcase how to measure the efficiency of exchange using HP-TLC. This protocol yields a nearly complete replacement of the outer leaflet with exogenous lipids without altering cellular viability, permitting further experimentation on modified intact plasma membranes.

We describe a method for using cyclodextrin to mediate exchange between lipids of the plasma membrane with exogenous lipids. This technique can be paired with experiments studying transmembrane proteins, which behave differently in lipid raft-like environments than they do in non-raft-like environments.

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

Biochemistry

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) molecules, before they are packaged into chylomicrons for transport to the lymph or into cytosolic lipid droplets (LDs) for intracellular storage. A crucial step for the formation of LDs is the catalytic activity of diacylglycerol acyltransferases (DGAT) in the final step of TG synthesis. LDs are important to buffer toxic lipid species and regulate cellular metabolism in different cell types. Since the human intestinal epithelium is regularly confronted with high concentrations of lipids, LD formation is of great importance to regulate homeostasis. Here we describe a simple assay for the characterization and quantification of LD formation (LDF) upon stimulation with the most common unsaturated fatty acid, oleic acid, in human intestinal organoids. The LDF assay is based on the LD-specific fluorescent dye LD540, which allows for quantification of LDs by confocal microscopy, fluorescent plate reader, or flow cytometry. The LDF assay can be used to characterize LD formation in human intestinal epithelial cells, or to study human (genetic) disorders that affect LD metabolism, such as DGAT1 deficiency. Furthermore, this assay can also be used in a high-throughput pipeline to test novel therapeutic compounds, which restore defects in LD formation in intestinal or other types of organoids.

This protocol describes an assay for the characterization of lipid droplet (LD) formation in human intestinal organoids upon stimulation with fatty acids. We discuss how this assay is used for quantification of LD formation, and how it can be used for high throughput screening for drugs that affect LD formation.

Dietary lipids are taken up as free fatty acids (FAs) by the intestinal epithelium. These FAs are intracellularly converted into triglyceride (TG) ...

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

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.

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

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

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

Summary

Explore More Videos

Cholesterol Efflux Assay

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

Exploring the Regulation of Lipid Droplet Catabolism through Lipophagy

A Co-culture Method to Investigate the Crosstalk Between X-ray Irradiated Caco-2 Cells and PBMC

Lipid Exchange Assay in Living Cells

A Fluorescence-based Assay for Characterization and Quantification of Lipid Droplet Formation in Human Intestinal Organoids

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl^- Channels and Transporters

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability