This method can help answer key questions in enteric neuroscience and gastrointestinal physiology, such as, what are the mechanisms of enterochromaffin cell excitability and response to luminal stimuli, and what are the mechanisms of gastrointestinal hormone release? The main advantage of this technique is it allows us to study in detail enterochromaffin cells using single-cell techniques, like electrophysiology. Demonstrating the procedure will be co-first authors Kaitlyn Knutson and Peter Strege, the outstanding technologists who developed these techniques.
To begin this procedure, place a six-milliliter syringe needle filled with ice-cold PBS into the lumen of the extracted intestinal segment. Use the microdissection forceps, clamp and seal the intestines around the syringe needle, and gently flush six milliliters of PBS through the lumen to rinse away any fecal matter. Next, invert the intestinal tissue by gently weaving the microdissection forceps through the lumen, pinching an intestinal wall, and retracting the tissue proximal to the tip of the forceps.
Repeat this process until the segment is inside-out with the lumen facing outward. Using microdissection scissors, cut the tissue segments into one-centimeter pieces. Then transfer the tissue segments into a 50-milliliter beaker filled with five milliliters of sterile PBS.
Subsequently, mince the tissue pieces to a liquified consistency. Transfer the minced tissue and 15 milliliters of ice-cold PBS to a clean 50-milliliter tube. Triturate two times with the transfer pipette.
Wait for the tissue to settle. Afterward, remove 15 milliliter of the PBS supernatant and replace with fresh PBS. Repeat this process three times, until the PBS is clear.
For the first digestion, from the water bath, retrieve one of the 50-milliliter tubes containing 10 milliliters of pre-warmed digestion media. Next, add 250 microliters of the PBS collagenase solution in the tube with the minced intestinal pieces. Finally, minced intestinal tissue pieces were added to the digestion media and PBS collagenase solution.
Then, place the sample in a water bath for five minutes at 37 degrees Celsius. Slowly titrate the tissue once with a 10-milliliter serological pipette and briefly let the tissue pieces settle. Use a transfer pipette to remove 10 milliliters of the supernatant.
For the second digestion, add 250 microliters of the PBS collagenase solution to the tissue and place it in a water bath at 37 degrees Celsius. Afterward, slowly titrate the solution once with a 10-milliliter serological pipette. Then remove 10 milliliters of the supernatant.
For the third digestion, add 250 microliters of the PBS collagenase solution in the tube. Place it in a 37-degree Celsius water bath for 10 minutes. Then shake the tube two to three times per minute at a higher intensity than digestions one and two.
Subsequently, slowly pipette the tissue up and down with a 10-milliliter serological pipette and allow the tissue pieces to settle for a short time. Transfer 10 milliliters of the supernatant into a new 15-milliliter tube. Add two milliliters of warmed EC cell complete culture media and invert once to mix.
Next, spin the sample at 100 g for five minutes at room temperature. After five minutes, remove the supernatant and re-suspend the remaining pellet in 2.5 milliliters of warmed EC cell complete culture media. For the fourth digestion, repeat the digestion procedures, but increase the incubation time by 15 minutes for colon, and remain at 10 minutes for jejunum.
To culture the cells, use a transfer pipette to combine the cell suspensions collected from digestions three and four into a new 15-milliliter tube. Remove a 10-microliter aliquot of cells and count with a hemocytometer. Then spin the remaining cell suspension at 100 g for five minutes at room temperature.
After five minutes, remove the supernatant and add EC cell complete culture media using a 5-milliliter serological pipette at a density of one million cells per milliliter. Re-suspend the tissue using a transfer pipette. Subsequently, remove the coated glass-bottom culture dishes from the incubator.
Using a P1000 pipette, replace the extra cellular matrix from each culture dish with 250 microliters of the final cell suspension. To achieve a whole cell gigaseal, lower the electrode into the bath solution. With the micromanipulator, maneuver the electrode tip in plane with and directly horizontal from the cell.
Then expel 0.2 milliliters of air from the syringe. Gently move the electrode horizontally along the X axis to touch the cell. Watch for a dimple to appear on the EC cell, and an increase in membrane resistance on the seal tests.
Apply 0.1 to 0.2 milliliters of suction on the syringe and hold the plunger steady until 100 mega-ohms is achieved. Then release the grip from the plunger. Wait for the cell to gigaseal.
Then gently disconnect the syringe. To record whole-cell voltage-gated sodium current, apply a quick tap of suction on a syringe. Repeat until the whole cell access is achieved before gently disconnecting the syringe.
Next, turn on the whole cell capacitance compensation and adjust the capacitance and series resistance. Turn on the series resistance compensation. With lag set at 60 microseconds, adjust the series resistance compensation and close the seal test.
Record an average of five runs of whole-cell voltage-gate sodium current. Quickly take note of two parameters of the voltage-gated sodium current:The voltage at window current, and the peak sodium current density. To record the elicited action potentials, switch the mode from voltage clamp to current clamp.
Load the episodic current clamp protocol. Adjust the holding current to zero picoamp here and turn on the external command. Record the elicited action potentials.
Note the least amount of current injected to fire off an action potential. To record spontaneous action potentials, turn off the external command. Load a gap-free current clamp protocol.
Change the holding current to the amount of current injected in the last sweep in the elicited protocol that did not fire an action potential. Subsequently, turn on the external command. Double check that the predicted holding current brings the membrane potential below the threshold to fire an action potential.
Adjust the holding current if necessary and record the spontaneous activity. Cultures that have been optimized for electrophysiology consisted of single cells and small clumps with bright CFP signal. When culture conditions have not been optimized, the epithelial cell culture consisted of large clumps, floating cellular debris, damaged membranes, and weak CFP signal in the EC cells.
EC whole cells were obtained at 30 plus or minus 7%of attempts from colon cultures, and 41 plus or minus 3%of attempts from jejunum culture. By whole cell electrophysiology, sodium currents were recorded from 81.3 plus or minus 4.0%of TPH-1 CPF positive cells from jejunum and 64.1 plus or minus 9.2%of them from colon. Of 59 EC cells from jejunum cultures, 19 EC cells in current clamp mode fired spontaneous AP, 29 EC cells fired AP only when elicited by a step protocol in current clamp mode, and 11 EC cells did not fire AP spontaneously or by step protocol.
While attempting this procedure, it's important to remember that the optimal digestion protocol for primary epithelial cultures may be variable between uses, so it's important to determine the optimal amount of agitation to obtain single cells. Following this procedure, other methods, like single cell PCR and fluorescence, can be performed to answer additional questions, like what enterochromaffin cells express and how stimuli are coupled to intercellular signaling. After this development, this technique paved the way for the researches in gastrointestinal physiology to explore how enterochromaffin cells work and how they regulate gastrointestinal motility and secretion in mice.
