Whole-ovary decellularization generates an effective 3D bioscaffold for ovarian bioengineering

Abstract

Purpose: To develop a new protocol for whole-ovary decellularization for the production of a 3D bioscaffold suitable for in vitro/ex vivo studies and for the reconstruction of a bioengineered ovary.

Methods: Porcine ovaries were subjected to the decellularization process (DECELL; n = 20) that involved a freeze-thaw cycle, followed by sequential incubations in 0.5% SDS for 3 h, 1% Triton X-100 for 9 h, and 2% deoxycholate for 12 h. Untreated ovaries were used as a control (CTR; n = 6). Both groups were analyzed to evaluate cell and DNA removal as well as ECM preservation. DECELL bioscaffolds were assessed for cytotoxicity and cell homing ability.

Results: DECELL ovaries maintained shape and homogeneity without any deformation, while their color turned from red to white. Histological staining and DNA quantification confirmed a decrease of 98.11% in DNA content, compared with the native tissue (CTR). Histochemical assessments demonstrated the preservation of intact ECM microarchitecture after the decellularization process. This was also confirmed by quantitative analysis of collagen, elastin, and GAG contents. DECELL bioscaffold showed no cytotoxic effects in co-culture and, when re-seeded with homologous fibroblasts, encouraged a rapid cell adhesion and migration, with repopulating cells increasing in number and aggregating in cluster-like structures, consistent with its ability to sustain cell adherence, proliferation, and differentiation.

Conclusion: The protocol described allows for the generation of a 3D bioscaffold that may constitute a suitable model for ex vivo culture of ovarian cells and follicles, as well as a promising tool for the reconstruction of a bioengineered ovary.

Keywords: 3D bioscaffold; Decellularization; Extracellular matrix; Porcine; Whole ovary.

Fig. 1

Macro/microscopic evaluations and DNA quantification in untreated (CTR) and decellularized (DECELL) ovaries. a, b CTR and DECELL ovaries display comparable shapes and homogeneity, while their color turns from red (CTR; a) to white (DECELL; b). c Chronological macroscopic images illustrating the decellularization process. d, e Hematoxylin-eosin staining shows the presence of both basophilic (cell nuclei) and eosinophilic (cell cytoplasm and ECM) staining in untreated tissue (CTR; d), while cell nuclei and the related basophilic staining are absent in DECELL ovaries (e). f, g DAPI staining displays the presence of nuclei in CTR ovaries (f) and their disappearance after the decellularization process (DECELL; g). h Cell density demonstrates a significantly lower number of nuclei in DECELL tissues compared with that in the untreated ones (CTR). Data are expressed as the mean ± standard error of the mean (SEM), *p < 0.05. i DNA quantification analysis showed a significant decrease in the DNA content of DECELL ovaries compared with that of the native tissue (CTR). Data are expressed as the mean ± standard error of the mean (SEM). *p < 0.05

Fig. 2

Scanning electron microphotographs of decellularized (DECELL) ovaries. a Decellularized hemiovary section. b, c Efficient cell removal, preservation of 3D microarchitecture, and ECM integrity are revealed after the decellularization process. Porous structures once populated by different cell types and complex fiber network are visible. d–h The ovarian surface epithelium and well-organized collagen fibers within pore walls are distinguishable

Fig. 3

ECM microarchitecture and composition in untreated (CTR) and decellularized (DECELL) ovaries. a, b Masson’s trichrome staining shows the persistence of collagen fibers (blue) and their comparable distribution between CTR (a) and DECELL (b) tissues. c, d Mallory’s trichrome staining demonstrates the maintenance of intact collagen (blue) and elastic fibers (pink) after the decellularization process (DECELL; d). e, f Gomori’s aldehyde-fuchsin staining confirms that DECELL tissues (f) retain elastic fibers scattered throughout the ovary, similarly to CTR ovaries (e). g, h Alcian blue staining reveals GAG retention in DECELL tissues (h). i, j Alcian blue/PAS staining indicates comparable distribution of neutral (magenta) and acid (blue) GAGs between DECELL (j) and CTR tissue (i). k Collagen content analysis demonstrates no significant differences between CTR and DECELL groups. Data are expressed as the mean ± standard error of the mean (SEM) (p > 0.05). l Elastin quantification shows comparable amount of the protein before (CTR) and after the decellularization process (DECELL). Data are expressed as the mean ± standard error of the mean (SEM) (p > 0.05). m Total GAG analysis contents display no significant reductions in DECELL ovaries compared with CTR ones. Data are expressed as the mean ± standard error of the mean (SEM) (p > 0.05)

Fig. 4

Cytotoxicity and re-seeding of decellularized ovarian tissue. a MTT assay demonstrates no significant differences in OD values between cells co-cultured with DECELL and those of control (CTR) at the different time points analyzed. Data are expressed as the mean ± standard error of the mean (SEM) (p > 0.05). b Images illustrating the scaffold before re-seeding. c Re-seeded porcine fibroblasts rapidly migrate into the bioscaffolds within 24 h (day 1). An increasing number of cells and the formation of cluster-like structures are visible at day 3 and steadily maintained at day 7. d H&E staining demonstrates the presence of cells into the bioscaffolds after 24 h of co-culture (day 1), with an increment during the following days (day 3 and day 7). e DAPI staining confirms the positivity for nuclei from 24 h onward. f Cell density shows bioscaffold re-population after 24 h (day 1), with a higher cell number at day 3 and day 7. Data are expressed as the mean ± standard error of the mean (SEM). Different superscripts denote significant differences (p < 0.05). g DNA quantification analysis demonstrates the presence of cells at day 1, which increases in number at day 3 and is steadily maintained at day 7. Data are expressed as the mean ± standard error of the mean (SEM). Different superscripts denote significant differences (p < 0.05)