Preparation and characterization of human decellularized ovarian scaffold based on supercritical carbon dioxide protocol.

Abstract

Background: Primary ovarian insufficiency affecting 1-3% of women under 40, causes premature menopause and estrogen deficiency. With increasing life expectancy, a large percentage of women also face estrogen-related symptoms. A bioengineered ovary is one of the strategies to replace or enhance ovarian tissue function. A key advancement in bioengineering is the development of ovarian decellularized extracellular matrix scaffolds that mimic natural ovarian niche. Recent studies indicate that supercritical carbon dioxide (scCo₂), with a density comparable to liquids and viscosity and diffusion coefficients properties similar to gases, holds substantial promise for application in engineered scaffolds. Therefore, we established a human decellularized ovarian scaffold based on a scCo₂ process, as an optimized protocol.

Methods: We evaluated two distinct pressure conditions (200 and 300 bar), while maintaining identical thermal (40 °C) and temporal (1.5 h) parameters, during the scCO₂ decellularization process. In addition, two modifications were implemented to identify the most optimal protocol for enhancing the decellularization process: the inclusion of 70% ethanol as a co-solvent and the application of 1% sodium dodecyl sulfate (SDS) as a pretreatment for 4 h while utilizing the scCO₂ system under the previously established conditions. Cell removal was confirmed by DNA quantification and H&E staining. Extracellular matrix structure evaluated by histological staining and scanning electron microscopy (SEM). Glycosaminoglycans (GAGs) content was quantified using a dimethyl methylene blue assay following extraction with HCL and MTT test was conducted to evaluate scaffold's cytocompatibility.

Results: Application of 1% SDS, while utilizing the scCO₂ system at 200 bar and 40 °C for 1.5 h, established an optimal protocol for preserving the essential characteristics of the ovarian ECM. This protocol is able to meet previously established decellularization criteria and histological staining and SEM showed that the ECM architecture was satisfactorily preserved. GAGs quantification indicated adequate preservation of GAGs content. Finally, MTT test presented the scaffolds had suitable cytocompatibility.

Conclusions: We propose an optimal protocol utilizing 1% SDS as a pretreatment, followed by the scCO₂ system. This protocol addresses common challenges associated with traditional decellularization methods and presents a promising avenue for advancing ovarian tissue engineering applications.