ABSTRACT
The technological revolution in reproductive biology that started with artificial insemination procedures and embryo transfer led to the development of assisted reproduction techniques such as in vitro fertilization or even cloning of domestic animals by nuclear transfer from somatic cells. Currently, procedures of isolated immature ovarian follicles in vitro culture are becoming the prominent technology aimed to preserve or restore fertility especially of young oncological patients or those at risk of premature ovarian failure.Here, we describe a protocol that can be applied for in vitro growth of porcine, preantral ovarian follicles in three-dimensional (3D) culture conditions. After enzymatic isolation from the ovarian cortex, preantral follicles are suspended in a drop of medium and enclosed with fluorinated ethylene propylene (FEP) powder particles (microbioreactors). Such microbioreactors maintain the 3D structure of the follicles during the whole process of in vitro growth what is crucial to ensure proper folliculogenesis progression and their ability to survive.