Radiotherapy or chemotherapy for cancer treatment may deplete the pool of follicles and lead to premature ovarian failure in women and children. For fertility preservation, Ovarian Tissue Cryopreservation (OTC) is proposed to preserve their fertility. Still, transplantation of ovarian tissue is an experimental method, with a risk to reintroduce malignant cells. To overcome this, we aim to optimize an in vitro culture system for the production of mature, functional oocytes starting from primordial follicles. In addition, we will try to isolate oogonial stem cells (OSCs), as their existence in the human ovary remains debatable and requires elucidation. In parallel to this trajectory, we will optimize the in vitro maturation process starting from pre-antral and small antral follicles isolated from the human ovarian medulla tissue. We will create 3D models in order to understand cell-cell communications and mimic in vivo – like culture conditions. Long-term in vitro cultures can lead to genetic and epigenetic aberrations of the growing populations. Since our ultimate goal is to produce mature oocytes, their functionality and (epi-)genetic and transcriptomic status will be tested to ensure safety for possible future clinical use.