Functional Ovarian Tissue Construct with Engineered Multi-Layered Follicles That Secrete Sex Hormones

Background: Ovarian hormone-deprived conditions result in a host of physiological conditions ranging from osteoporosis to urogenital disease. Hormone replacement therapy (HRT) is often prescribed to alleviate ovarian hormone cessation; however, pharmacological delivery methods consistently lead to increased serum concentrations and clinical complications such as cancer and heart disease.The present study aimed to overcome these drawbacks by delivering sex hormones by means of a functional ovarian tissue construct containing engineered ovarian follicles. The cell-based platform would permit interaction with the innate hypothalamic-pituitary-ovarian axis, allowing the regulated delivery of sex hormones at more physiologically-relevant levels.

Methods: Granulosa and theca cells were isolated from ovaries of 21-day old rats and used to generate three different types of ovarian tissue constructs. In the first scheme, both cell types were mixed together at a ratio of 3:1 granulosa cells to theca cells and subsequently encapsulated in a collagen cell carrier (referred to as co-cultured non-spheroid constructs). In the second scheme, prior to encapsulation, the mixed cells were formed into 3D spheroids through forced microwell aggregation (referred to as non-layered spheroid constructs). In the third scheme, a compartmentalized spheroid design was used (referred to as multi-layered spheroid constructs). First, spheroids composed solely of granulosa cells were formed. Theca cells were than aggregated over these spheroids, resulting in compartmentalized spheroids. To assess compartmentalization, spheroids were stained for FSHR (granulosa cell marker) and LHR (theca cell marker). The viability of encapsulated cells was determined using a live/dead assay. To evaluate the endocrine function of tissue constructs, the levels of 17 β-estradiol (E2) and progesterone (P4) were measured in the culture media using ELISA kits.

Results: Immunofluorescent staining of the cellular spheroids revealed that microwell aggregation is capable of generating distinctly compartmentalized spheroids with structural architecture similar to that of native ovarian follicles. Live/dead staining of tissue constructs indicated that the viability of cells in 3D spheroids encapsulated in collagen are maintained over the course of 30 days in culture. Measurements of the amount of sex hormones in the culture media showed that non-layered spheroid constructs secrete significantly higher levels of E2 and P4 than co-cultured non-spheroid constructs (p < 0.05), and that multi-layered spheroid constructs secrete significantly higher levels of E2 and P4 than non-layered spheroid constructs (p < 0.05).

Conclusion: Our results demonstrate the feasibility of using a microwell-aggregation system to generate large numbers of homogenous follicle-like structures from primary endocrine cells, and the ability to leverage these engineered follicles to fabricate functional ovarian tissue constructs capable of sustained release of vital sex hormones. Furthermore, multi-layered spheroids that recapitulate the structural architecture of native ovarian follicles emphasize the importance spatial arrangement plays in maximizing the viability and function of ovarian endocrine cells. This study thus offers a simple but potent strategy for facilitating cell-based HRT and underlines the integral role tissue engineering strategies play in optimizing systems for cell therapeutics.