This cell culture model allow us to study characteristics of estradiol-producing bovine granulosa cells in vitro such as thyroid factors or conditions. The main advantage of this protocol is the cryopreservation technique that make cell culture work independently of direct tissue supply. The procedure will be demonstrated by Veronica Schreiter, a technician in my laboratory.
Before starting cell isolation procedure, wash bovine ovaries in antibiotic supplemented 1X PBS in a beaker to remove blood from the surface. Discard the PBS and repeat the wash three to four times until the ovaries are clean from any remaining blood. Use a lab wipe soaked in 70%ethanol to wipe one ovary.
Use a three milliliter syringe with an 18 gauge needle to puncture small to medium sized follicles. And then aspirate the follicular fluid which contains the granulosa cells. Transfer the follicular fluid to a 50 milliliter centrifuge tube.
After puncturing several follicles, use one 1X PBS to rinse the syringe and needle for intermediate cleaning to prevent the needle from blocking with cells. Repeat with puncturing and aspirating the follicles to obtain as much cells as possible with the follicular fluid. To set aside appropriate number of freshly isolated GCs as reference samples for RNA, DNA, and protein analyses, place several aliquots of the reference cells in fresh 1.5 milliliter tubes.
Centrifuge for one minute at 12, 000 times G at room temperature. Discard the supernatant and shock free the pellets in liquid nitrogen and store at minus 80 degrees Celsius. To begin cryopreservation, gently pellet the GCs by centrifuging them at 500 times G at room temperature for three minutes.
Carefully remove the supernatant, and gently re-suspend the cell pellet in 100%fetal calf serum, or FCS, to remove all visible clumps. Then add one volume of the previously prepared freezing medium, mix gently, and transfer the mixture to a cryogenic vial. Place the vial in a specialized freezing container that prevents rapid freezing, so that the cooling rate does not exceed minus one degree Celsius per minute.
Then freeze the container at minus 80 degrees Celsius for a minimum of four hours. Quickly thaw the cells in the cryogenic vial in a 37 degree water bath for three to four minutes and transfer the cell suspension into 37 degree, pre-warmed Alpha MEM without hormone supplement to achieve 10 milliliter final volume. Centrifuge the cells at 500 times G at room temperature for three minutes.
Discard the supernatant and add the pre-warmed supplemented Alpha MEM to the cell pellet and re-suspend carefully. Seed the GCs in a 24 well plate in a final volume of 500 microliters per well, making sure to include replicates of at least three wells per condition. Finally, incubate the cells at 37 degrees Celsius and with 5%CO2 for eight days, and then proceed with cell analysis.
After culturing GCs at a normal density of one times 10 to the fifth cells per well, cells displayed a fibroblast like phenotype typical for GCs. Cells cultured at high plating density of one times 10 to the sixth didn't change their morphology, but formed large cell clusters more frequently. After culturing the cells at a density of one times 10 to the fifth, gene expression of several evaluated marker transcripts revealed no difference between cultured cells with or without initial cryopreservation.
When GCs were cultured at high cell density, estradiol concentration decreased significantly, whereas progesterone concentration increased when compared to cells grown at normal density. Bovine GCs cultured under serum free conditions for eight days revealed a significant difference in regulation of several selected marker genes in high versus normal density cultures as measured with QPCR. While using this cell culture model of estradiol-producing bovine granulosa cells it's important to remember factors that influenced the cell physiology such as, cell density.
Following this procedure, other treatment protocols can be performed in order to answer questions such as, which molecules or signaling pathways are involved in bovine granulosa cell differentiation. The cryopreservation technique paved the way for more structured and organized cell culture work and might be easily transferred to other species.
