This method can help answer key questions in the field of endocrinology regarding how pituitary cells function and interact in a controlled environment. The main advantages of this technique are that it promotes physiologically meaningful results for medaka and provides a reference guide for making primary cell cultures from other species. Begin by securing the head of the medaka to the wax plate with one fine needle inserted over the mouth and one on each side of the head behind the brain.
Place the fish under a dissection microscope and use fine forceps with an angled tip to gently scrape the scales from the top of the head. Carefully insert the forceps under the skin of the side of the head and slowly move the forceps towards the mouth to remove the skull roof. Cut the spinal cord completely off with the forceps and carefully flip the brain over toward the mouth to expose the pituitary.
Harvest the gland with clean forceps with a straight tip into a plastic tube containing 1.5 milliliters of modified DPBS on ice. When all of the pituitaries have been collected, spin down the tissues with a microcentrifuge for one to two seconds at room temperature and use a glass pipette to carefully remove the DPBS taking care not to disturb the pituitary pellet. Wash the tissue with one milliliter of trypsin solution for another one to two seconds in the microcentrifuge and use the glass pipette to aspirate most of the supernatant.
When all of the trypsin has been removed, add another milliliter of trypsin to the sample and incubate the tissue in a 26 degrees Celsius water bath for 30 minutes with occasional gentle flicking. At the end of the incubation, centrifuge the pituitaries for one to two seconds and use a glass pipette to remove most of the trypsin without disturbing the tissue. Then, wash the sample with one milliliter of trypsin inhibitor solution to inactivate the trypsin and centrifuge for one to two seconds.
After removing the supernatant, add another milliliter of trypsin inhibitor solution to the tube and incubate the sample in a 26 degree Celsius water bath for 20 minutes with flicking. While the tissue is incubating, add two milliliters of L-15 medium to a 35 millimeter poly-D-lysine coded cell culture dish with a central glass bottom and equilibrate the plate at 26 degrees Celsius and 1%carbon dioxide for at least 10 minutes. At the end of the incubation, centrifuge the pituitaries for one to two seconds and carefully aspirate all of the trypsin inhibitor solution from the tube.
In a laminar flow hood, add one milliliter of ice cold modified DPBS to the pituitary tissue pieces and use a fire-polished glass pipette to gently triturate the tissues six to seven times on ice taking care to avoid bubbles. After another one to two seconds centrifugation, gently transfer about one milliliter of the dissociated cell containing upper layer of DPBS into a new 15 milliliter tube on ice. Repeat the trituration and dissociated cell collection nine more times until all 10 milliliters of the DPBS has been used starting with the pipette tip with a medium sized opening and switching to a pipette tip with the smaller opening for the last three to four milliliter of DPBS.
The mechanical dissociation with a glass pipette is a critical step of this protocol and requires some training to achieve a good result. Pipette the cell solution gently to avoid damaging the fragile dissociated cells. After the 10th trituration, mark the side of each tube where the pellet will be and spin down the sample in a pre-cooled centrifuge.
At the end of the centrifugation, carefully remove the tube from the centrifuge and use a glass pipette to immediately but gently transfer the supernatant into a plastic 15 milliliter tube taking care to remove most of the supernatant without disturbing the small, frequently invisible pellet, then carefully resuspend the pellet in 50 to 100 microliters of L-15 culture medium. Carefully drip the dissociated cells into the middle of the previously prepared cell culture dish and allow the cells to sink to the bottom of the dish for approximately five minutes before moving the dish to the incubator to avoid having the cells spread outside of the sunken area of the glass bottom. After 30 minutes at 26 degrees Celsius and 1%carbon dioxide, check the cells by light microscopy to confirm that they have attached to the bottom of the dish and return the dissociated pituitary cell culture to the incubator for no more than seven days.
Harvesting around 10 adult medaka pituitaries usually results in a density of around 50, 000 to 100, 000 cells per dish after seeding. The number of dying cells is nearly constant over time with over 95%of the cells viable after one day and over 90%still alive after three days with a small decline in viability observed at one week of culture. Electrophysiological recordings of GFP expressing luteinizing hormone gonadotrope cells demonstrate that the dissociated pituitary cells are able to fire action potentials spontaneously after four days in culture.
Further, upon stimulation of the gonadotropin releasing hormone, an initial transient hyperpolarization of the cell membrane is followed by a dramatic increase in firing frequency that is concomitant with the depolarization and increased duration of the action potentials. While attempting this procedure, it's important to remember to work in a precise and efficient manner to maintain the pituitary cells in a healthy state. Following this procedure, other methods like qPCR or advanced imaging techniques can be performed to answer additional questions regarding how specific treatments may alter pituitary cell physiology.
