Ovarian Cell Encapsulation in an Enzymatically Crosslinked Silk-Based Hydrogel with Tunable Mechanical Properties

An artificial ovary is a promising approach for preserving fertility in prepubertal girls and women who cannot undergo current cryopreservation strategies. However, this approach is in its infancy, due to the possible challenges of creating a suitable 3D matrix for encapsulating ovarian follicles and stromal cells. To maintain the ovarian stromal cell viability and proliferation, as a first step towards developing an artificial ovary, in this study, a double network hydrogel with a high water swelling capacity (swelling index 15–19) was developed, based on phenol conjugated chitosan (Cs-Ph) and silk fibroin (SF) through an enzymatic crosslinking method using horseradish peroxidase. The addition of SF (1%) to Cs (1%) decreased the storage modulus (G’) from 3500 Pa (Cs1) to 1600 Pa (Cs-SF1), and the hydrogels with a rapid gelation kinetic produced a spatially homogeneous distribution of ovarian cells that demonstrated 167% proliferation after 7 days. This new Cs-SF hydrogel benefits from the toughness and flexibility of SF, and phenolic chemistry could provide the potential microstructure for encapsulating human ovarian stromal cells.

How to Support the In Vitro Culture of Human Ovarian Cells and Follicles? A Preliminary Step for Assembling an Artificial Ovary

BackgroundThe first step in assembling an artificial ovary is supporting the in vitro growth of ovarian cells/follicles. Therefore, proliferation of granulosa or cumulus cells (CCs) is important and due to limited proliferation, improving the mediums for in vitro propagation of these cells is helpful. ObjectiveThis study aimed to characterized the appropriate media and supplements for in vitro culture of ovarian cells (OCs) and CCs. Material & MethodsCortical, medullar and hilar cells of ovary were cultured and their conditioned medium (CMs) collected. The expression of GDF9, as a key factor for ovarian follicular growth, was evaluated. CCs were collected from healthy women, who referred due to male factor infertility. To choose the optimum basal medium, a mixture of OCs was cultured with basal mediums, supplemented with two concentrations of fetal bovine serum (FBS) and human serum albumin (HSA). The cocktails were as follows: [Serum free mediums], [mediums+10%FBS], [mediums+20%FBS], [mediums+1%Alb)], [mediums+2%Alb], [mediums+10%FBS+1%Alb], [mediums+10%FBS+2%Alb], [mediums+20%FBS+1%Alb] & [mediums+20%FBS+2%Alb]. The same process was repeated for CCs. Also, the effect of various concentrations of L-Glutamine, bovine serum albumin (BSA), HSA, insulin transferrin selenium (ITS), Follitropin alfa® and Pregnyl® was evaluated on the growth of CCs. As well, CCs were treated with various concentrations of follicular fluids (FFs) and CMs. CMs were collected from ovarian, testicular, adipose & amniotic derived and ovarian carcinoma cells. FFs were collected from women with poor responders, endometriosis, advanced age, polycystic ovarian syndrome, male factor and unknown infertility. Then, CCs morphology, proliferation capacity and culture duration were evaluated. ResultsAll the ovarian cells expressed GDF9. αMEM+20%FBS and DMEMF12+20%FBS were the most suitable cocktails for OCs and CCs, respectively. 20%FBS was superior to 10% for both OCs and CCs. HSA could not support the growth of OCs and CCs, alone. The cocktail of mediums with 20%FBS superior to the others. The CMs of cortical and (hillar+medullar) cells and FFs from male factor and unknown infertility patients caused higher CCs proliferation. 17 mM/l L-Glutamine, 24 mg/ml BSA, 20 mg/ml HSA, 10 ng/ml ITS, 300 mIU/ml Follitropinα and 3.5 IU/ml Pregnyl led to higher proliferation of CCs. ConclusionCMs, serums and FFs can support the CCs growth alongside with basal mediums, supplemented with hormones, ITS and L-Glutamine, which are cheaper and more accessible.

Bioengineering of Human Artificial Ovary From Cryopreserved Tissue: Optimization of the Follicles Enzymatic Isolation.

Background: This study aims to evaluate the effectiveness of Tumor Dissociation Enzyme (TDE) for the isolation of cryopreserved human ovarian cortex tissues, proposing an optimized applicable for artificial ovary.Methods: This is a prospective experimental study. We present a comparative analysis among the outputs of follicles isolated from cryopreserved ovarian biopsies of ten young women undergoing laparoscopy. Follicles were isolated by Tumor Dissociation Enzyme (TDE) or Liberase Dispase High (DH) enzymatic digestion. Follicles were assessed by evaluating the number, viability, morphology, oxidative stress. Moreover, the follicle growth and viability were analyzed after eight days of in vitro culture (IVC) between each protocol.Results: The recovery rate of follicles in Group 1 was significantly higher by 78 follicles compare to Group 2 (p < 0.05), while the difference in weight and volume of biopsies in both groups was no significant (p > 0.05). Group 1 allowed to isolate more primordial follicles (p < 0.05) and smaller diameter of follicles (p < 0.01) than Group 2. Group 1 allowed to isolate 7% more of bright red follicles than Group 2 (p < 0.01). We also found that Group 1 had isolated a significantly higher percent of viable follicles (p < 0.05), more morphologically normal follicles (p < 0.05), and lower oxidative stress levels compared with Group 2 (p < 0.01). The velocity of follicles growth from Day 0 to Day 8 in Group 1 was significantly higher (p < 0.05) than in Group 2. The viability of follicles on Day 8 of in vitro culture in Group 1 was significantly higher than Group 2 (p < 0.05).Conclusions: TDE treatment can be an alternative for Liberase DH, allows the isolation of highly viable follicles from the cryopreserved human ovarian cortex, with an intact morphology and low oxidative stress, and with high proliferation potential after culture in vitro.

FERTILITY PRESERVATION: Construction and use of artificial ovaries

Increasing numbers of patients are now surviving previously fatal malignant diseases, so for women of childbearing age, fertility concerns are paramount once they are cured. However, the treatments themselves, namely chemo- and radiotherapy, can cause considerable damage to endocrine and reproductive functions, often leaving these women unable to conceive. When such gonadotoxic therapy cannot be postponed due to the severity of the disease or for prepubertal girls, the only way to preserve fertility is cryobanking their ovarian tissue for future use. Unfortunately, with some types of cancer, there is a risk of reimplanting malignant cells together with the frozen-thawed tissue, so it is not recommended. A safer approach involves grafting isolated preantral follicles back to their native environment inside a specially created transplantable artificial ovary for their protection. This bioengineered ovary must mimic the natural organ and therefore requires an appropriate scaffold to encapsulate not only isolated follicles, but also autologous ovarian cells, which are needed for follicles to survive and develop. Here we review the indications for use of this artificial ovary and advances in the field that are bringing us ever closer to clinical implementation.