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Whole-ovary decellularization generates an effective 3D bioscaffold for ovarian bioengineering.
Journal of Assisted Reproduction and Genetics ( IF 3.1 ) Pub Date : 2020-05-02 , DOI: 10.1007/s10815-020-01784-9
Georgia Pennarossa 1 , Matteo Ghiringhelli 1 , Fulvio Gandolfi 2 , Tiziana A L Brevini 1
Affiliation  

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.

中文翻译:

全卵巢脱细胞化为卵巢生物工程生成有效的 3D 生物支架。

目的 开发一种新的全卵巢脱细胞方案,用于生产适用于体外/离体研究和生物工程卵巢重建的 3D 生物支架。方法 对猪卵巢进行脱细胞处理(DECELL;n = 20),其中包括冻融循环,然后依次在 0.5% SDS 中孵育 3 小时、1% Triton X-100 9 小时和 2% 脱氧胆酸盐12小时。未经处理的卵巢用作对照(CTR;n = 6)。对两组进行分析以评估细胞和 DNA 去除以及 ECM 保存。评估 DECELL 生物支架的细胞毒性和细胞归巢能力。结果 DECELL卵巢保持形状和均质性,无任何变形,颜色由红色变为白色。组织学染色和 DNA 定量证实,与天然组织 (CTR) 相比,DNA 含量降低了 98.11%。组织化学评估表明,去细胞过程后完整的 ECM 微结构得以保存。胶原蛋白、弹性蛋白和 GAG 含量的定量分析也证实了这一点。DECELL生物支架在共培养中没有表现出细胞毒性作用,并且当用同源成纤维细胞重新接种时,促进快速细胞粘附和迁移,重新填充的细胞数量增加并聚集成簇状结构,这与其维持细胞粘附的能力一致、增殖和分化。结论所描述的协议允许生成 3D 生物支架,该支架可能构成卵巢细胞和卵泡离体培养的合适模型,以及重建生物工程卵巢的有前途的工具。
更新日期:2020-05-02
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