Ovarian Decellularized Bioscaffolds Provide an Optimal Microenvironment for Cell Growth and Differentiation In Vitro

Abstract

Ovarian failure is the most common cause of infertility. Although numerous strategies have been proposed, a definitive solution for recovering ovarian functions and restoring fertility is currently unavailable. One innovative alternative may be represented by the development of an “artificial ovary” that could be transplanted in patients for re-establishing reproductive activities. Here, we describe a novel approach for successful repopulation of decellularized ovarian bioscaffolds in vitro. Porcine whole ovaries were subjected to a decellularization protocol that removed the cell compartment, while maintaining the macrostructure and microstructure of the original tissue. The obtained bioscaffolds were then repopulated with porcine ovarian cells or with epigenetically erased porcine and human dermal fibroblasts. The results obtained demonstrated that the decellularized extracellular matrix (ECM)-based scaffold may constitute a suitable niche for ex vivo culture of ovarian cells. Furthermore, it was able to properly drive epigenetically erased cell differentiation, fate, and viability. Overall, the method described represents a powerful tool for the in vitro creation of a bioengineered ovary that may constitute a promising solution for hormone and fertility restoration. In addition, it allows for the creation of a suitable 3D platform with useful applications both in toxicological and transplantation studies.

Impact of Aging on the Ovarian Extracellular Matrix and Derived 3D Scaffolds

“…When a whole-organ decellularization protocol, previously developed in our laboratory, [ 23 , 24 , 25 ] was applied to aged ovaries, we were able to derive 3D ECM-based biological scaffolds that represent a viable alternative to the use of synthetic matrix such as hydrogels [ 49 ]. The decellularized scaffolds preserve all the changes that are described above and that are distinctive of the senescent organ.…”

“…Young and aged whole-ovaries were decellularized according to the protocol previously developed in our laboratory [ 23 , 24 , 25 ]. Briefly, the entire organs were frozen at –80 °C for at least 24 h. They were then thawed at 37 °C in a water bath for 30 min, followed by an incubation with 0.5% sodium dodecyl sulfate (SDS; Bio-Rad, Milan, Italy) in deionized water (dd-H 2 O) for 3 h. Ovaries were then treated overnight with 1% Triton X-100 (Sigma, Milan, Italy) in dd-H 2 O, extensively washed in dd-H 2 O for 9 h, and, subsequently, immersed in 2% deoxycholate in dd-H 2 O (Sigma, Milan, Italy) for 12 h. Lastly, decellularized whole-ovaries were washed in dd-H 2 O for 6 h, with changes every 2 h. All steps were carried out at room temperature using an orbital shaker at 200 rpm.…”

Synergistic Effect of miR-200 and Young Extracellular Matrix-based Bio-scaffolds to Reduce Signs of Aging in Senescent Fibroblasts

“…18 ovaries from 6-month ( n = 9, Young) and 5 years old ( n = 9, Aged) swine were collected at the local abattoir and subjected to whole-organ decellularization protocol [ 2 , 30 – 32 ]. Briefly, organs were frozen at − 80 °C for at least 24 h, thawed in a 37 °C water bath for 30 min, treated with 0.5% sodium dodecyl sulfate (SDS; Bio-Rad) for 3 h, and immersed overnight in 1% Triton X-100 (Sigma-Aldrich).…”

Construction of Artificial Ovaries with Decellularized Porcine Scaffold and Its Elicited Immune Response after Xenotransplantation in Mice

“…Accordingly, we decellularized ovaries using combined multiple decellularization strategies (mechanical, chemical and biological reagents). Other research groups have also used combined methods, among which SDS, Triton X-100, and SDC were the most frequently used [ 15 , 19 ]. Other reagents such as sodium lauryl ester sulfate, NaOH, and propanol-2 were also used to decellularize ovaries [ 12 , 20 , 21 ].…”