The ovarian tissue will undergo sequential culture steps to (hopefully) produce fertilisable mature oocytes: The tissue is then cultured to activate the primordial follicles and allow them to develop. The enzymes used, liberase DH and DNase, are produced by good manufacturing practice (GMP) to fully comply with GMP guidelines to ensure future application to patients. The enzymatic digestion process is inactivated every 30 minutes and the suspension is filtered to allow fully isolated follicles to be removed and reduce unnecessary enzyme exposure which may lead to damage of their basement membrane and their death. When recovering the isolated follicles, malignant cells may be inadvertently retrieved, which poses the risk of re-introducing malignant cells into the patient. To minimise the risk of contamination, the isolated follicles undergo a washing step which involves rinsing the follicles with fresh dissecting media, three times, to separate them from surrounding isolated cells.
Culturing the growing follicles within a 3D microenvironment The isolated follicles are then encapsulated within a 3D matrix and cultured for up to 4 weeks. The material used has to meet biosafety and clinically compatible standards, such as adequate protection and support of the follicles and adaptability to human body temperature, if artificial ovaries are to be transplanted into a patient. Potential materials are divided into
synthetic polymers and
natural polymers. Synthetic polymers tend to be more predictable than natural polymers in terms of their rate of degradation and their mechanical properties can be tailored to the specific clinical requirements. Although they contain no essential molecules for cell adhesion, bioactive factors can be incorporated to stimulate this. The only synthetic polymer utilised so far has been
poly(ethylene glycol), which developed immature mouse follicles into antral follicles and corpora lutea. Natural polymers have bioactive molecules which play a role in
cell adhesion, migration, proliferation and differentiation. However, they lack mechanical strength and the adaptability that synthetic polymers have. Unlike synthetic polymers, there has been a success with a wider range of natural polymers:
collagen,
plasma clots,
fibrin,
alginate and decellularized ovarian tissue. The microenvironment of the structure should mimic that of the natural ovary, so the artificial ovary should support the follicles structurally, but also cellularly. Ovarian
stromal cells are integrated into the microenvironment as they play an important role in early development of the follicles. They release various factors which positively regulate the transition of primordial follicles to primary follicles, but also release other cells which will differentiate into
theca cells; those that play a supportive role for growing follicles and produce
sex steroids such as
androstenedione and
testosterone. This can be achieved by isolating them from a second fresh ovarian biopsy once the patient has completed their cancer treatment, thus avoiding potential contamination.
Endothelial cells should also be co-transported as they are key to promoting
angiogenesis of the artificial ovary.
Oocyte culture The immature oocytes are retrieved from the artificial ovary and cultured in vitro for a further 24–48 hours, allowing them to mature oocytes which are ready for
IVF or
vitrification (cryopreservation). == Mouse models ==