The overall goal of this procedure is to isolate highly purified primary Sertoli cells and peritubular cells, or PTCs, from rat testes. This method can help answer key questions in the male reproductive immunology field such as how do Sertoli cells influence testicular macrophages, or how is autophagy regulated in Sertoli cells? Harvesting Sertoli cells and peritubular cells is tedious and requires expensive reagents.
The isolated primary cells retain significantly more qualitative in vivo properties during cell culture, as compared to cell lines. Implications of this technique extend to our diagnosis of male infertility, as Sertoli cells play crucial role in the development of the spermatogenic cells. Generally, individuals new to this method will struggle, because it can be difficult to assess whether the seminiferous tubules have been properly digested.
Visual demonstration of this method is critical, as the entire procedure involves many steps that require proper execution. Small mistakes can add up to failure. Begin by disinfecting the abdomens of ten exsanguinated male Wistar rats with 70%ethanol.
Then, for each animal in turn, use forceps to lift the skin from the abdominal muscles, and cut out an oval-shaped skin lobe extending from the pubic symphysis to the sternum. Lift the skin upwards onto the chest. Then grasp the abdominal muscles, and make a medial incision from the pubic symphysis to the sternum.
Now squeeze the abdomen with the thumbs from the pelvis upwards to push the testes out of the lower pelvis. Using forceps, pull on the epididymal fat pad to extend the testes further. Then cut the spermatic cord, leaving the tunica albuginea intact.
And collect the testes in 20 milliliters of PBS in a 50 milliliter conical tube. After collecting all of the testes, disinfect the organs with an equal volume of 1%iodine in ethanol. And decant the supernatant immediately.
Quickly wash the testes twice with 25 milliliters of PBS each, then transfer the organs to a Petri dish containing 15 milliliters of fresh PBS. Next, holding each testis firmly with forceps at one end, make a small incision into the tunica albuginea at the opposite end. Squeeze out the tubules using closed scissor blades as a scraping tool.
The tubules should still exhibit a compact testicle-shaped bundle, with only a few tubules extending into the saline solution. When all of the testes have been decapsulated, transfer the denuded organs into a 100 milliliter screw cap bottle containing 10 milliliters of trypsin-DNase 1 solution, and digest the decapsulated testes in a shaking water bath at 32 degrees Celsius. After four minutes, evaluate the tubules with the naked eye for the dispersion of the tubules.
As soon as the tubules begin to deseminate into the solution, immediately stop the digestion with five milliliters of Trypsin Inhibitor Solution A, pipetted up and down three to four times. Transfer the tissue slurry to a 50 milliliter conical tube. And let the tubules settle by unit gravity.
After five minutes, use the same 10 milliliter pipette to carefully remove the supernatant, then add 10 milliliters of Trypsin Inhibitor B solution, pipetting the tubules up and down two to three times to completely stop the digestion. To remove the contaminating interstitial cells, use a 25 milliliter pipette to resuspend the tubules in 25 milliliters of PBS. Then let the tubules settle by gravity for 12 minutes.
And repeat the PBS wash nine more times as just demonstrated. To isolate the peritubular cells, transfer the PBS washed tubules into a 100 milliliter screw cap bottle containing a collagenase hyaluronidase DNAse 1 solution, and digest them for another ten minutes in a shaken water bath at 32 degrees Celsius. At the end of the incubation, transfer a drop of the suspension onto a glass slide, and quickly check the tubules under an inverted light microscope at a 100x magnification.
The tubules will appear shortened in length with rough edges, indicative of the release of the peritubular cells. Using the same 25 milliliter pipette, transfer the digested tubules into a 50 milliliter conical tube, and rinse the 100 milliliter bottle with 10 milliliters of PBS. Add the PBS wash to the suspension.
After allowing the isolated tubule fragments to settle for 10 minutes, use the 25 milliliter pipette to carefully transfer the supernatant, containing the PTCs, into a new conical tube. To prevent the carry over of any tubules, use a five milliliter pipette to aspirate the last few milliliters of solution. Now mix 20 milliliters of RPMI 1640 medium supplemented with 10%heat inactivated FPS, with the supernatant.
And spin down the cells. Carefully decant the supernatant, and resuspend the peritubular cell pellet in 20 milliliters of fresh medium. Then seed four milliliters of PTCs in each of five T75 flasks containing 16 milliliters of medium at 37 degrees Celsius and 5%carbon dioxide.
To isolate the Sertoli cells, use a new 25 milliliter pipette to resuspend the settled tubules thoroughly in 25 milliliters of PBS. After allowing the tubules to settle for another 12 minutes, use the same pipette to wash the tubules three more times in the same way. Then transfer the tubules into a new 100 milliliter screw cap bottle.
And add the hyaluronidase DNase 1 solution. Further digest the tubules in a shaking water bath at 32 degrees Celsius for five minutes. Take an aliquot of the suspension by light microscope inspection at a 100x magnification.
Short tubules, tubular aggregates, and released cells should be visible. Allow the tubular aggregates to settle for 10 minutes, then use a new 25 milliliter pipette to remove the supernatant and wash the aggregates four times in 25 milliliters of PBS, with ten minutes of sedimentation between each wash. The contaminating PTC population will have been reduced.
After the last wash, add 20 milliliters of RPMI 1640 medium to the cells, and pass the suspension through an 18G needle mounted on a 20 milliliter syringe 10 times. Next, quickly confirm that all of the aggregates have been dissociated, and filter the cell suspension through a 70 micron cell strainer to obtain a pure single-cell suspension. Spin down the filtrate.
Using the 25 milliliter pipette to carefully aspirate the supernatant. Then resuspend the isolated Sertoli cells in 40 milliliters of serum-free RPMI 1640 medium. Then count the number of viable cells by Trypan blue exclusion, and adjust the cell concentration to three times ten to the six cells per milliliter.
Finally, feed one milliliter of cells per well in a six-well plate. The purity of the isolated Sertoli cells is greater than 95%as demonstrated by Vimentin immunolabeling. On day six of culture, most of the Sertoli cells have acquired lipid droplets, with a single large vacuole observed in the majority of cells.
The neutrolipic content of the droplets can be easily visualized by Oil Red O staining. The isolated Sertoli cell culture is the least contaminated with germ and peritubular cells around day seven, so experiments should be performed as close to this day as possible. The PTCs can be cultured as well.
Reaching a purity of 95%by day five after isolation, as confirmed by smooth muscle actin immunolabeling. The procedure can be completed in one day. Using ten rats provides enough Sertoli cells for six or seven six-well plates, and enough peritubular cells for ten conifer T75 flasks.
When attempting this step for the first time, remember it is important to monitor carefully the three enzymatic digestion, with either naked eye or an inverted light microscope. Initially, it is also important to assess the purity of the isolated Sertoli cells and PTCs using immunolabeling with Vimentin and smooth muscle actin antibodies, respectively. In presence of serum, the peritubular cells proliferate relatively fastly, whereas Sertoli cells stop dividing.
Keep in mind that Sertoli cell and peritubular cells culture are contaminated with germ cell that have to be removed by washing or passaging, respectively. This technique has paved the way for endocrinologists and immunologists to explore the interactions between testicular cells and hormones, to establish testicular cell lines, and to investigate the testicular immune privilege. Well, after watching this video, you should have a good understanding of how to isolate rat Sertoli cells and peritubular cells, and how to avoid technique pitfalls like over-digestion of tubules by Trypsin DNase 1.
