The protocol demonstrated here enables the labeling and isolation of specific cell lineages that are present in ovarian follicles beginning at early antral stages, thereby enabling the high resolution analysis and culture. This highly precise approach utilizes combinations of fluorophore conjugated antibodies to target cell surface markers that are robustly and specifically expressed among discrete granulosa, stroma, and theca lineages. Begin by placing the ovary in a sterile petri dish, and pour a chilled medium with it to hydrate the tissue, ensuring that the ovary is half-submerged in the medium.
Remove any sutures or other material, and dissect the residual surrounding tissue away from the ovary. To isolate the antral follicles, identify individual follicles and choose healthy follicles. Exclude any blood-filled, dark, or non-symmetric follicles.
Isolate the chosen antral follicles from the ovary using scalpels, keeping the antrum intact and excising the cortical tissue encompassing the follicle. Place each excised intact antral follicle in one well of the six well plate. Using a ruler placed underneath the dish, determine the follicle diameter for descriptive purposes.
Using a scalpel, bisect the intact antral follicle to assess the antral cavity and add six milliliters of enzymatic cell detachment medium before incubating the plate in a humidified incubator for 10 minutes. Following incubation, add six milliliters of available medium containing 20%fetal bovine serum, or FBS, and flush with the medium by repeated vigorous pipetting over the bisected follicles with a P-1000 micropipette. Next, collect the medium and pass it through a 100-millimeter filter.
After centrifuging the filtered supernatant at 300 G and four degrees Celsius for three minutes before, aspirate the supernatant. Place the remaining tissue in a mixture of 100 units per milliliter collagenase and one unit per milliliter dispase diluted in a balanced saline solution before 30 minutes of incubation. mechanically disrupt the tissue by triturating and vigorous pipetting twice after 10 and 20 minutes.
Once done, recover the tissue and flush via pipetting, through a P-1000 micropipette tip, and strain through a 100-milliliter filter. Centrifuge the strained tissue at 300 G and four degrees Celsius for three minutes before putting the cell pellet enriched with the theca cells and stroma cells on ice for labeling and FACS. To identify granulosa cells and theca cells, resuspend the cell pellets in a blocking solution containing directly conjugated antibodies and incubate at four degrees Celsius for 10 minutes.
After washing and centrifuging the cells, resuspend the cells in FACS buffer containing 4-6 diamidino-2-phenylindole, or DAPI, and run the cell suspension through a FACS instrument to capture a small population of the cells, using the fluorescent intensity of fluorophore conjugated antibodies for gating. Bisect the remainder of the ovary. Excise the medulla using curved fine scissors and scalpels, avoiding damage to the ovarian cortex.
Continue processing and thinning of the ovarian cortex by gentle scraping until minimal medulla remains. Divide the cortical tissue into two to three millimeter wide strips along the ovary length. For ovarian tissue freezing, place one cortical strip into each chilled cryogenic vial containing the freezing solution.
Shake the cryogenic vials containing the cortical strips on a rotating plate for 20 minutes at four degrees Celsius. Nucleate ice crystal formation by touching each cryo vial with a cotton tip briefly immersed in liquid nitrogen. The ovarian cells were collected and the cell fractions were sorted from the antral follicles FACS to more than 95%purity.
CD99 specifically labeled granulosa cells in PVRL1, or nectin, was increasingly localized to the oophorus granulosa cell compartment as the antral follicles increased in size. It was seen that the antibodies against endoglin or CD55 specifically demarcated all stroma and theca cells and that alanine aminopeptidase was specifically expressed by androgenic theca cells. The cells were labeled with an antibody directed against CD99 to identify granulosa cells and antibodies against CD45 were used to exclude hematopoietic cells.
Antibodies against PVRL1 separated the granulosa cell population into oophorus and mural compartments. Following enzymatic digestion with collagenase dispase, the cell preparations labeled with antibodies against CD55, CD34, and alanine aminopeptidase enabled the capture of all the stroma or theca cells or androgenic theca cells with the exclusion of endothelial cells from the theca cells'populations. Take particular care to avoid disrupting the antrum of follicles during initial follicle isolation by cutting within a safe margin around follicle borders.
Downstream of this procedure, the cells can be utilized for molecular or diagnostic analysis or alternatively cultured in vitro for toxicology studies, experimental analysis, or recapitulation of the follicle microenvironment. To date, these methods were used to model endocrine disorders involving the ovary and to identify novel subpopulations of follicle associated cells.
