User:CarmillaEarp/sandbox

This is my sandbox - this is where I draft content first

Culture methods using mouse ovarian tissue
There are several culture systems which can be employed to investigate ovarian and follicular growth and development:


 * Whole ovarian culture - The culture of intact ovaries supports the formation and development of primordial follicles. Ovaries are dissected from neonatal mouse pups (postnatal day 0-5) and placed into ovarian culture medium (Bovine Serum Albumen (BSA) dissolved in α-Minimal Essential Media (αMEM)) which is changed every 2 days. The cultures are maintained in a 37℃, 5% CO2 incubator for up to 6 days and then the ovaries are frozen or fixed to facilitate further study.
 * Follicle culture
 * Individual - This method of culturing supports the growth of individual follicles from late pre-antral to pre-ovulatory stage. This system allows follicle growth and hormone production to be studied. The ovaries of young mice (19-23 days) are removed and halved, and follicles are identified under a microscope. Late pre-antral follicles are identified as having a diameter of 180-200µm and containing 2-3 layers of granulosa cells. Follicles are manually dissected and then examined for suitability to culture. Follicles are chosen for culture only if they are healthy (diameter of 190 ± 10µm; translucent; without dark atretic areas; intact basal lamina.) Wells containing follicle culture medium (α-Minimal Essential Media, recombinant human follicle stimulating hormone, ascorbic acid and adult female mouse serum) is overlaid with sterilised silicon oil, which prevents medium evaporation. A follicle is placed at the bottom of each well and maintained in a 37℃, 5% CO2 incubator, being moved into a well containing fresh medium for up to 6 days. If growth measurements are being taken visually the distortion due to the oil layer must be accounted for. Follicles are frozen or fixed so further analysis can be performed.
 * Paired - By culturing 2 follicles in close proximity, follicle-follicle interactions can be examined. The follicles may grow together to form a two-follicle unit. The follicles are dissected from the ovaries as above, then placed in contact with each other in pairs, in a well with follicle culture medium and sterilised silicon oil. Follicles from different genetic sources can be co-cultured so that tissue origins can be differentiated within the co-culture. The medium is replaced every 2 days and after 6 days the culture is fixed or frozen for further processing.
 * Follicle-Ovary Co-culture - This method allows follicle-ovary interactions to be studied.  The ovaries and follicles are dissected as above and then one follicle is placed in contact with one pole of a neonatal ovary on a plate. The follicle-ovary plate is cultured in follicle culture medium at 37°C, 5% CO2 for up to 5 days. At this point the co-culture is frozen or fixed before further processing. To facilitate differentiation between tissue origins the ovary and the follicle should be from different genetic sources.