Cryopreservation of Zebrafish Spermatogonia by Whole Testes Needle Immersed Ultra-Rapid Cooling

The main purpose of this study was to adapt the needle immersed vitrification (NIV) procedure to cryopreserve whole zebrafish testes. Additionally, the repeatability of the method in five different zebrafish strains was tested.

Cryopreservation offers the possibility of maintaining live cells and tissues at low temperatures for an infinite period of time, at least in human terms. Vitrification is a form of cryopreservation which involves ultra-rapid cooling into a glassy, amorphous state without the formation of crystalline ice and without the damages associated. In this video, we present the protocol for the vitrification of whole zebrafish testes, which allows their safe storage until their later use as a source of germ cells for transplantation into suitable recipients.

The current vitrification protocol has one significant advantage over the slow-rate freezing protocols in regard to germ stem cell manipulations. And that is that after vitrification and warming, the spermatozoa die and they get adjusted so they are not present in the cell suspension. Therefore, there is no need for isolation of germ cells after warming.

And that readily facilitates the downstream manipulations. Euthanize the fish by placing it into a dish containing 200 milligrams per liter of MS-222 at pH seven. Before continuing with the dissection, make sure that the gills have stopped moving, and at least 10 minutes have passed since that moment to ensure death by hypoxia.

Dry the fish by patting it on a paper towel and place it on its dorsal side on a dissecting mat. Firstly, cut off the pelvic and pectoral fins for easier dissection. Horizontally snip the skin on the belly of the fish between the pectoral fins, and cut the skin and underlying muscle along the belly until the anal fin.

Open the body cavity of the fish and pin the left and right body wall to the dissection mat with needles. Testes are located above the gastrointestinal organs on both sides of the swimming bladder. To prevent contamination, do not take out the gastrointestinal organs, but gently push them to one side and remove the testes from the opposing side.

Testes are connected to the body wall, so remove them carefully and cut the connecting peritoneum by micro scissors. Additionally, in order to prevent contamination, firstly sterilize the testes by putting them into 70%ethanol for two seconds, and then place them in L-15 on 96-well plate on ice. Mark needles with appropriate labels, depending on the sample type, and prepare the equilibration and vitrification solutions.

Transfer the testes to a larger-well plate or dish and pin three testes or more, depending on the size of the needle, on a sterile acupuncture needle. Firstly, position the testes on top of curved tweezers. Place the point of the needle in the middle of the testis and carefully pull the testis upward with the tweezers.

After puncturing the testis, gently pull it upwards towards the top of the needle. Make sure the testes are separated from each other and they are not falling off or sliding down. Place the needles with pinned testes in a Tubulator Eppendorf tube filled with L-15 until vitrification at 25 degrees.

The storage time should not exceed one hour. Transfer the needle with the testes into the equilibration solution and incubate for five minutes at 25 degrees. Transfer the needle into the vitrification solution and incubate for 30 seconds at 25 degrees.

Remove the needle from the vitrification solution. Quickly and gently absorb the remaining solution from the tissue by a sterile paper towel. Use caution to avoid the testes sticking to the paper towel or falling off.

Quickly place the needle into liquid nitrogen in a styrofoam box. Keep the needle in liquid nitrogen in a closed styrofoam box for five to 10 minutes. Pre-cool an opened 4.5 milliliter cryotube and its cap in the same box.

After five to 10 minutes, transfer each needle into a separate cryotube under the surface of liquid nitrogen and close the cryotube. Place the cryotube onto a metal cane and place it in the cryobank canister as fast as possible. Store the samples in a canister storage drawer until further use.

Warm each needle separately. All warming solutions should be at 25 degrees. Detach the cryotube from the metal cane and plunge it into liquid nitrogen stored in a styrofoam box.

Open the cryotube in the liquid nitrogen vapor using forceps, and release the needle into the liquid nitrogen. Transfer the needle very fast into the first warming solution and incubate for one minute. Make sure to transfer the needle quickly to prevent it warming in the air during transfer.

Transfer the needle into the second warming solution, and incubate for three minutes. Transfer the needle into the third warming solution and incubate for five minutes. Release the testes from the needle by sliding them downwards with curved tweezers.

Place each warmed testis individually into a separate well filled with 250 microliters of L-15, supplemented with 10%FPS. Make sure that the wells are labeled according to the sample. Keep the well plate on ice until all samples are warmed, and until further work.

Prepare 500 microliters of the dissociation solution for each sample in separate two-milliliter tubes. Label the tubes according to the code of the sample. Incubate the prepared dissociation solution for five minutes at room temperature.

Add the warmed tissue into the digestion solution, and cut it into small pieces by at least 30 movements of small scissors. Incubate the tissue on a shaking plate for 90 minutes at 24 degrees. Stop the digestion process by adding 400 microliters of L-15 and 100 microliters of 10%FPS.

Briefly shake the solution and incubate for one minute at room temperature. Filter the obtained solution through 50-micron filters into a 1.5 milliliter tube. Label the tubes accordingly.

Centrifuge the filtered solution at 200 G for 10 minutes at 25 degrees. Carefully remove the supernatant and re-suspend the pellet in 20 microliters of L-15, supplemented with 10%FPS. Manually shake the tube until the pellet is completely dissolved.

Keep the cell suspension on ice or at four degrees until further use. Mix five microliters of cell suspension and five microliters of 0.4%trypan blue solution in an appropriately-labeled 0.2 milliliter PCR tube. If different volumes are used, make sure that the dilution ratio of one to one remains.

Incubate this suspension for one to three minutes at 25 degrees. Check the viability of each sample in a hemocytometer under a microscope. Count the number of live cells unstained by trypan blue in 15 fields.

Calculate the total number of cells in 20 microliters of the cell suspension. The suspension is now ready for any downstream application. On average, the number of early-stage germ cells isolated from a single fresh zebrafish testis vary between 40, 000 and 200, 000 cells, depending on the size of the fish.

In all zebrafish lines used in this study, the current vitrification protocol yielded average viability rates of 50%and higher. This indicates favorable viability and repeatability of the presented protocol. When digesting fresh zebrafish testes in all five strains, besides the early-stage germ cells, numerous spermatozoa were found as well.

After digestion of cryo-preserved testes, there were far less spermatozoa, indicating that they do not survive this vitrification protocol, and that they are most likely eliminated during the digestion process. The needle-immersed vitrification protocol presented in this study offers several advantages in the cryopreservation of testicular pieces. Firstly, several pieces can be pinned to one needle, and therefore they can simultaneously be exposed to cryoprotectants and to the liquid nitrogen.

Furthermore, this method enables that very low amounts of cryoprotectants are attached to the tissue pieces. And therefore, cooling and warming rates can be higher. One significant advantage is that spermatozoa do not survive this protocol, and therefore in the end, the cell suspensions are enriched spermatogonia, which will facilitate the downstream applications.

But one thing that has to be taken into account is the possibility of cross-contamination through nitrogen. Therefore when using this method, liquid nitrogen should not be reused.