精巣上体から分離した一次上皮細胞の全細胞パッチクランプ記録

The overall goal of this procedure is to isolate epithelial cells from the rat epididymis and to measure the electrical properties on these primarily dissociated cells using whole-cell patch-clamp method. This method can help answer key questions in the reproductive biology field, such as the function of epididymis, which is important for sperm maturation and inheritance of paternal traits. This technique allows the user to cauterize the electrical properties of isolated epididymal epithelial cells so that unknown physiological function of these cells can then be illustrated.

Demonstrating the procedure will be Bao Li Zhang, a grad student from my laboratory. To begin the procedure, disinfect the dissection tools by immersion in 70%ethanol. Then, let them air-dry.

Next, prepare the full IMDM. Afterward, create 50 ml aliquots in aseptic conditions, seal with Parafilm, and store at four degrees celsius. Following that, prepare a collagenous enzyme digestion solution by dissolving collagenase type I and collagenase type II in RPMI, resulting in one milligram per milliliter of each collagenase in the solution.

Filter the solution through a 0.22 micrometer membrane. Then, mark it as collagenase solution, and keep the solution at room temperature until use. Next, adjust the volume of the enzyme solution based on the weight of the enzyme.

The minimal volume required for both cauda epididymides from a single rat is two milliliters. To dissect the rat cauda epididymides, sacrifice the animal by either using sodium pentobarbital via IP injection or using an isoflurane chamber until the animal does not respond to tail pinching stimulation, followed by cervical dislocation. Afterward, disinfect the lower abdomen by wiping it with 70%ethanol.

Gently push the two testes down to the lower abdomen and then open the lower abdomen near the scrotum. Subsequently, remove the epididymal fat and dissect out all the reproductive organs. Afterward, transfer them to the dish with RPMI.

In a clean, airflow controlled working station, dissect out the cauda epididymides from the connective and fatty tissues and the epididymal capsule. Place one epididymis with 0.2 milliliters of collagenase solution in a 1.5 milliliter tube. To dissociate single cells from the cauda epididymides, cut the epididymides in the collagenase solution using fine scissors until the tissue becomes a paste-like fluid.

Then, rinse the scissors gently with the rest of the enzyme solution in the tube. Following that, place the tube on a metal thermo-mixer for 30 minutes at 37 degrees celsius with a shaking speed of 1, 000 rpm. Then, centrifuge the enzyme tissue mixture at room temperature for three minutes.

After that, decant the sticky supernatant that contains mostly the sperm. Next, resuspend the pellet in one milliliter of full IMDM to quench all the enzymatic activity. Subsequently, transfer the cell suspension to a 50 milliliter tube containing 49 milliliters of RPMI.

Centrifuge the cell mixture and decant the supernatant. Resuspend the pellet in one milliliter of full IMDM with gentle trituration for at least five minutes to dissociate single cells from the enzymatic treated epididymal tissue mixture. Separate the epithelial cells from other cells under aseptic conditions, and culture the cell suspension on a 10 centimeter Petri dish containing full IMDM for at least eight hours or overnight in an incubator at 32 degrees celsius.

The next morning, prepare the sterile cover slips by immersion in 100%alcohol. Air-dry and then dip them in a small amount of culture medium. Then, place the cover slips in six-centimeter culture dishes or in single wells of a 24-well plate.

Now, harvest the dissociated epithelial cells by gently collecting the cell suspension from the Petri dish which consists mostly of epithelial cells. Centrifuge the cell suspension at 30 times G in room temperature for five minutes, and then decant the supernatant. Following that, resuspend the cell pellet in two milliliters of full IMDM.

Seed 0.2 milliliters of the harvested cell suspension onto the center of each sterile cover slip. Let the cell suspension settle in the liquid droplet for at least 10 minutes to allow the cells to loosely adhere to the glass cover slips. Then, carefully add one milliliter of full IMDM at the edge of a six-centimeter dish or 0.3 milliliters of full IMDM to each well of a 24-well plate, without disturbing the cells.

Keep the isolated single cells on cover slips in the incubator at 32 degrees celsius until the patch-clamp experiments. On the day of the patch-clamp experiment, thaw one aliquot of intracellular solution on ice and keep it chilled during the patch-clamp experiment to prevent degradation. Next, transfer the culturing epithelial cells on the glass cover slip to the recording chamber filled with standard PSS at room temperature.

Carefully change the bathing PSS at least two times using a pipette before any patch-clamp experiments. To establish the whole-cell configuration, immerse the pipette into the bath solution at the highest speed of the micromanipulator. Find the pipette tip on the screen connected to the digital camera.

Then, slow down the micromanipulator speed to the medium-high mode. Quickly check the micropipette resistance using the data acquisition interface command by applying a voltage step generated from the computer controlled amplifier. Change to a new micropipette if the resistance is significantly out of this range.

And set the liquid junction potential between the pipette and bath solution to zero using the pipette offset command in the commander interface of the software. Monitor the resistance with the membrane test. If the resistance is larger than 500 megaohms, but smaller than one gigaohm, apply a negative potential to help form the gigaseal.

In the meantime, compensate the transient capacitive current of the micropipette. At this moment, apply a 10 millivolt hyperpolarizing step from a holding potential of zero millivolts for the seal resistance determination. Immediately after achieving a successful whole-cell configuration, apply a 10 millivolts hyperpolarizing step from a holding potential of negative 60 millivolts.

Switch the voltage mode to the zero current mode and perform a 10 second gap-free recording to measure the resting membrane potential of the cell. Afterward, quickly switch back to, and remain in, the voltage mode. Apply the voltage protocols according to the planned experiments and measure the current responses.

Here are the examples of the epithelial cells isolated from the rat cauda epididymides before and after reharvest of overnight culture on the dish prior to the patch-clamp experiments. This figure shows the typical whole-cell currents recorded from single epididymal epithelial cells using a pulse-eliciting protocol. The dotted line indicated the zero current levels.

Here is the current voltage relationship of the current responses measured at the indicated time points. These are the representative tracings of the resting membrane potential of the principal cells dialyzed with a pipette solution, either with ATP or without ATP. And this bar graph shows no significant difference between the initial resting membrane potential of plus ATP and minus ATP.

The numbers in brackets within the bars indicate the number of tested cells from at least three animals. And here are the correlation analyses of the seal resistance and the input resistance of all the cells, or the principal cells only versus the resting potentials measured at the zero current clamp shortly after the whole-cell establishment and the current magnitudes measured at the hyperpolarizing step to negative 100 millivolts from holding potential. Once mastered, this technique can be done in nine hours if it is performed properly.

While attempting this procedure, it's important to remember to stop the enzymatic digestion immediately during cell isolation steps. Otherwise, overdigestion may occur which may lead to difficult formation of gigaohm seal and failure of patch-clamp experiments. To maintain the health of the culture, it's important to spin down the enzymatically dissociated cell mixture in the low speed of 30 G for two to three minutes, and then decant the sticky supernatant which contains mostly sperm cells.

Following these procedures, other methods such as live cell imaging and a one-cell culture techniques can be performed in order to answer additional questions, such as the resting membrane potential in naive epithelial cells might regulate the extracellular behavior under controlled conditions. This technique paves the way for researchers in the field of reproductive biology to explore the electrophysiological properties of epithelial cells of epididymis for the regulation of sperm maturation and paternal acquired traits inheritance. After watching this video, you should have good understanding on how to isolate epithelial cells from rat epididymis and how to measure the electrical properties on these cells using whole-cell patch-clamp method.