The overall goal of this protocol is to grow intestinal Caco-2 cells in three dimensions and to demonstrate the use of the Ussing chamber in studies on serotonin transporter regulation. The methods shown here can answer key questions in epithelial transport field, such as how intestinal serotonin transporter, SERT is regulated. The main advantage of the 3D Caco-2 is that they reflect cell-to-cell and cell-to-extracellular matrix interactions more accurately than monolayers.
And Ussing chamber technique enables precise measurements of transport function in the intestinal epithelium. The implication of 3D, the Caco cultures extends toward discovery of therapeutics because these models enable screening for agents against diseases associated with altered transport function. Though this method provides insight into serotonin transporter regulation, it can also be applied to studies of other electrolyte transporters such as sodium and chloride.
Visual demonstration of these techniques is critical, as stripping the seromuscular layer and mounting the intestinal mucosa in Ussing chambers are difficult to learn. Demonstrating the 3D Caco-2 cell procedures will be Ishita Chatterjee, instructor, and Anoop Kumar, a postdoc fellow from our group. Demonstrating the stripping of the seromuscular layer from mouse ileum will be Sangeeta Tyagi, a senior research specialist from my lab.
Also demonstrating the mounting of stripped mucosa and inserting them in Ussing chambers will be Shubha Priyamvada and Arivarasu Natarajan, instructors from my lab. To begin, thaw the growth factor reduced gelatinous protein solution overnight on ice in a four degree Celsius refrigerator. Once thawed, prepare one milliliter, or 500 microliter aliquots.
On the day of culture, precool the chambered slides on ice. Then, add 30 microliters of the gelatinous protein solution to each well of the eight-well chambered glass slide and spread it evenly. While plating the gelatinous mixture in the chamber slide or plates, care should be taken to avoid bubble formation as the cells may detach.
Place the cultured dish inside a 37 degree Celsius cell culture incubator for 15 to 30 minutes to allow the solution to solidify. Next, using trypsin, detach confluent Caco-2 cells from a culture flask. Then, count the cells in a hemocytometer and centrifuge them at 500 times g at four degrees Celsius for five minutes.
Re-suspend the resulting cell pellet in 3D Caco-2 medium to obtain the suspension of the desired density. Using the prepared the cell suspension, seed 4, 000 cells per well onto the glass chamber slides and allow them to grow for 12 to 14 days in a 5%carbon dioxide humidified incubator at 37 degrees Celsius. To stain the cells, first aspirate the medium and fix the cells with 400 microliters of 2%PFA.
Continue fixing the cells for 20 minutes at room temperature. After washing the cells twice in PBS, permeabilize them with 0.5%Triton solution in PBS for no longer than 15 minutes. Rinse the cells twice in PBS glycine buffer and then wash the permeabilized cells in IF buffer for ten minutes at room temperature.
Block the cells using 5%normal goat serum in IF buffer. Then, incubate the cells with 200 microliters of primary antibody diluted in IF buffer supplemented with 1%goat serum for one to two hours at room temperature. After incubation, wash cells thrice with IF buffer.
Incubate washed cells with 200 microliters of secondary antibody diluted in IF buffer supplemented with 1%goat serum for one hour at room temperature. Wash cells with IF buffer thrice for ten minutes. To detach the chamber for the glass slide, first place the slide in the slide base holder, then slide the white lifter through the holder until it contacts the edge of the wells.
Gently pull the chamber to remove it from the slide. Use a covered black box to protect the slides from light. Mount the slides with slow anti-fade medium and cover them with cover slips.
After allowing the slides to dry for ten minutes at room temperature, seal them with nail polish prior to imaging. Isolate mouse ileum following the procedure described in the text protocol. Then, using scissors, open the intestine longitudinally.
Incubate the resulting tissue section in ice-cold gassing KBR buffer containing one micromolar indomethacin for ten minutes. Pin an approximately one centimeter long intestinal section, mucosal side down, to a plate containing 0.5 centimeter thick 7%agarose or cured silicone elastomer. Using a dissection stereo microscope with bottom illumination, strip the seromuscular layers.
Then, cut the seromuscular layer with a feather scalpel blade. Use fine forceps to lift the edge of the layer along the longitudinal axis of the intestine. Finally, mount the mucosa carefully on the pins of the slider.
Hold the stripped mucosal tissue by the edges to avoid tearing. While mounting the stripped ilial mucosa on pins of the slider, precautions should be taken to prevent tearing the tissue at the pinheads and to minimize damaging the tissue's edges. Prior to tissue treatment, prepare Krebs solution gassed with 95%oxygen, 5%carbon dioxide.
Then, pour the solution into a Ussing chamber. Add 10 micromolars glucose as the energy substrate to the serosal bath and 10 micromolar mannitol for maintaining osmotic balance to the mucosal bath. Subsequently, insert the slider with the mounted mucosa into the chamber to expose both the apical and the basolateral sides of the tissue to the Krebs solution.
Equilibrate the ileal tissue in the bath for ten minutes. Then, pretreat the apical side with 10 micromolar fluoxetine for 30 minutes for the measurement of the mucosal to serosal flux. Ultimately, treat the basolateral side of the tissue with 10 nanograms per milliliter TGF beta 1 for one hour, and then incubate the apical side with 20 nanomolar tritiated 5-HT for 30 minutes.
To calculate mucosal to serosal flux rates, collect 0.75 milliliter aliquots from the serosal reservoir, replacing them with identical volumes of the bath medium to prevent hydrostatic pressure differences across the mucosa. To quantify the tritiated 5-HT accumulated in the tissue, remove the mucosa from the slider. Wash it once with ice-cold KRB buffer and place it in a glass culture tube.
Incubate the mucosa in 0.5 milliliters of 10%KOH overnight at 37 degrees Celsius to lyse the tissue. Then, measure the radioactivity in 150 microliter aliquots of the lysates in triplicate using a liquid scintillation counter. Using the Bradford method, measure the protein concentration in three to five microliter aliquots of the lysates.
Shown here are Caco-2 cysts grown in 3D culture stained for actin and Caco-2 3D cysts stained simultaneously for actin, nuclei and SERT protein. SERT is visible in the luminal membrane and in subapical compartments. To further confirm the advantage of Caco-2 3D spheres over 2D Caco-2 monolayers, western blot analysis of SERT protein expression was performed.
The results showed the enhanced expression of SERT protein in Caco-2 3D spheres compared to 2D Caco-2 cells. Finally, a Ussing chamber system was used to show the TGF beta increases mucosal to serosal flux, reflecting increased 5-HT uptake from the luminal membrane and enhanced 5-HT accumulation observed in the ileal mucosa. Such findings are supported by the observed sensitivity of 5-HT uptake to fluoxetine treatment that corresponds to SERT expression on the luminal membrane.
Once mastered, this technique of stripping and mounting the ileal mucosa can be completed in 15 minutes. While adopting this procedure, it's important to remember that upon removal from the animal, the extravio intestinal preparation has limited viability and it may last for up to three hours. 3D Caco-2 cysts could be utilized for various studies such as membrane trafficking events, gene expression, or protein-protein interaction of epithelial transporters.
After its development, the 3D Caco-2 technique paves the way for researchers in the field of intestinal transport to explore fluid movement and to study the pathophysiology of darial diseases. Do not forget that working with radioactivity can be extremely hazardous, and precautions, such as the use of PPE and proper decontamination procedures should always be taken. After watching this video, you will be able to grow Caco-2 cells in 3D cultures and to utilize these cultures to investigate epithelial transporter expression.
You will also be able to utilize the Ussing chamber to study serotonin transport function in the native mouse intestine.
