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

The overall goal of this procedure is to study the transport of lipid-soluble molecules by the intestine. This is accomplished by first placing differentiated caco two cells in the permeable membrane.Insert. Next, the Caco two cells are incubated with a lipid mixture and lipoprotein secretion is stimulated.

Then the lipoproteins are isolated by using sodium chloride density gradient ultracentrifugation. Finally, the isolated lipoproteins are negatively stained with phospho toic acid. Ultimately, particle size analysis is used to show the relative abundance of chylomicrons to very low density lipoproteins.

Though this method can provide insight into the transport of dietary lipids by the intestine, it can also be applied to other systems such as determining the bioavailability of oral lipic drugs. After implementing gene overexpression and monitoring according to the text protocol, maintain the KCO two cells that sustain the gene overexpression. Using cells that have reached 50 to 70%confluence, split the cells one to six by adding three milliliters of 0.05%trypsin 0.53, millimolar A DTA, and incubating a 37 degree Celsius until the cells have detached gently pipette the cells several times to avoid clumping.

Then after transferring 0.5 milliliters of the cells to the apical or upper chamber of a permeable membrane, insert containing 10 milliliters of prewarm growth Medium. Add 10 milliliters of warm growth medium to the lower paso lateral chamber. Gently shake the dishes several times in a forward and backward motion.

Avoid swirling the dishes as this may result in unequal cell dispersion. Place the dishes on a flat surface in a 37 degree Celsius and 5%carbon dioxide incubator. One day later, change the growth medium and continue to change the medium according to the text protocol.

Prepare a 100 x lipid mixture by adding 900 microliters of PBS to 50 milligrams of oleic acid vortex. The mixture vigorously transfer the entire oleic acid mixture to a tube containing 40 milligrams of Han and 48 milligrams of sodium tarot. Coate dissolve the lipid mixture by vigorous vortexing.

Add the 900 microliters of lipid mixture to 89.1 milliliters of growth medium and mix filtered the solution for the final concentrations in the medium shown here. Using 13 day post confluent caco, two cells grown on permeable membrane inserts. Add 10 milliliters of the lipid containing medium to the apical chamber, and 10 milliliters of the growth medium to the basal lateral chamber.

Incubate for four hours at 37 degrees Celsius before collecting the lipoprotein containing medium in the basolateral chamber. To isolate the lipoproteins, remove the cell debris from the lipoprotein containing medium by centrifuging at 2000 times G for five minutes. Decant the medium into a 50 milliliter tube, then add 5.95 grams of sodium chloride, and if analyzing by transmission electron microscopy or TEM, add one protease inhibitor tablet.

Use water to bring the volume to 23 milliliters and completely dissolve the solutes. Then decant the solution into a polycarbonate ultracentrifuge tube. Use 0.5 milliliters of water to gently overlay the 1.2 gram per milliliter density solution.

Then balance the tube by weight. Load the tubes into a T 8 65 rotor and carry out ultracentrifugation at 429, 460 times G and four degree Celsius for 24 hours. Then while keeping the tube as still as possible, immediately isolate the top 0.5 milliliters of solution.

To carry out TEM analysis, prepare five milliliters of 2%phospho toic acid and adjust to pH 6.0. Using a syringe filter with a pore size of 0.2 micrometers, filter the solution. Drop 20 microliters of the lipoprotein sample on a sterile dish.

Then drop 20 microliters of the filtered phospho toic acid next to the sample on the same dish with the dull side towards the sample. Gently drop an EM grid onto the lipoprotein drop incubate at room temperature for one minute. Then gently tap the side of the grid on a filter paper to remove excess sample from the grid.

Then gently drop the grid with the same side resting on the phospho tongue acid drop and incubate for one minute before gently tapping the grid on filter paper to remove the excess solution. Finally, perform TEM to capture images of lipoproteins. Shown here are normal 13 days post confluent kco two cells displaying dome-shaped structures and intracellular lipid droplets that are characteristic of differentiated caco two cells.

At this point, new medium should be added gently because cells are more susceptible to detachment. Using the sodium chloride density gradient ultracentrifugation method, the lipoproteins secreted by the caco two cells were isolated and analyzed by TEM. Some of the chylomicrons, which are lipoproteins larger than 80 nanometers in diameter are depicted here.

Smaller v LDLs are also present. A high percentage of chylomicron particles relative to the total number of lipoprotein particles indicates efficient lipid transport After its development. This technique paved the way for researchers in the field of pharmaceutical sciences to explore more extensively the bioavailability of oral lipophilic drugs.