Fundación Instituto Valenciano de Infertilidad (FIVI), Instituto Universitario IVI/INCLIVA, Valencia, Spain.
Instituto de Ciencia y Tecnología Animal, Universitat Politècnica de València a, C/Camino de Vera s/n, 46022 Valencia, Spain.
Acta Biomater. 2019 Apr 15;89:126-138. doi: 10.1016/j.actbio.2019.03.004. Epub 2019 Mar 5.
In the last decades, the decellularization (DC) of organs has become an established technique in the field of regenerative medicine to yield complex and vascularized bioscaffolds. Furthermore, it has been demonstrated in vitro that these decellularized scaffolds retain their native tissue-specificity. This is also the case when this tissue-specific extracellular matrix (ECM) is solubilized and used as hydrogels or coatings to create a biomimetic environment. In this study we investigated if this specificity not only remains when applied to distinct tissues but even more, that these differences can be distinguished within the same tissue at different stages of proliferation. To address this question, a sensitive in vitro animal model was used: rabbit embryos at the third day of development were cultured on coatings made from acellular endometrium that was non-proliferating (non-synchronous, NS) and proliferating (synchronous with the embryo, S) and their development was compared. For this, we obtained whole NS and S rabbit uteri and subjected them to an adapted decellularization protocol. The acellular endometrium was carefully separated by microdissection and converted into a pre-gel solution to be used as hydrogels and coatings for in vitro assays. First, the characteristics of these NS and S hydrogels were investigated by proteomic analysis, electron microscopy and gelling kinetics. When used as substrata for day 3 embryos culture, it became apparent that only the acellular ECM from synchronous endometrial coating achieved similar results to the gold standard culture protocols and conditions, possibly because of the slow release of growth factors present in the synchronous/proliferating endometrium. STATEMENT OF SIGNIFICANCE: It has been shown by in vitro culture of stem cells, progenitor cells and primary culture cells that decellularized tissues retain their specific functions and biochemical and structural compositions. The present work demonstrates that using a mild SDS and perfusion based decellularization (DC) protocol not only effectively decellularize whole rabbit uteri, adding to the growing field of reproductive tissue engineering, but more importantly that the differences in the proliferating endometrium are translated after DC. This implies that DC not only retains the interspecificity of tissues but also the intraspecificity of a developing hormonally stimulated tissue. For the first time, we demonstrate that the coating from decellularized synchronous endometrium acts as a biological support for in vitro embryo development, achieving comparable results with the current gold standard that only uses serum-containing media.
在过去的几十年中，器官的去细胞化（DC）已成为再生医学领域中的一种成熟技术，可产生复杂且血管化的生物支架。此外，体外研究表明，这些去细胞化的支架保留了其天然的组织特异性。当将这种组织特异性细胞外基质（ECM）溶解并用作水凝胶或涂层以创建仿生环境时，也是如此。在这项研究中，我们研究了当应用于不同组织时，这种特异性是否不仅保持不变，而且更重要的是，即使在同一组织的不同增殖阶段，这些差异也可以区分。为了解决这个问题，我们使用了一种敏感的体外动物模型：发育第三天的兔胚胎在无增殖（非同步，NS）和增殖（与胚胎同步，S）的去细胞化子宫内膜涂层上培养，并比较它们的发育情况。为此，我们获得了整个非增殖和增殖的兔子宫，并对其进行了适应性去细胞化处理。通过显微解剖小心地分离无细胞子宫内膜，并将其转化为预凝胶溶液，用作体外测定的水凝胶和涂层。首先，通过蛋白质组学分析、电子显微镜和胶凝动力学研究了这些 NS 和 S 水凝胶的特性。当用作第 3 天胚胎培养的基底时，明显的是，只有来自同步子宫内膜涂层的无细胞 ECM 才能达到与黄金标准培养方案和条件相似的结果，这可能是因为同步/增殖子宫内膜中存在的生长因子的缓慢释放。意义声明：已经通过体外培养干细胞、祖细胞和原代细胞表明，去细胞化组织保留了其特定的功能以及生化和结构组成。本工作表明，使用温和的 SDS 和基于灌注的去细胞化（DC）方案不仅可以有效地去细胞化整个兔子宫，这为生殖组织工程领域增添了新的研究方向，而且更重要的是，在 DC 后可以转化增殖子宫内膜的差异。这意味着 DC 不仅保留了组织的种间特异性，而且保留了发育中受激素刺激的组织的种内特异性。我们首次证明，去细胞化同步子宫内膜的涂层可作为体外胚胎发育的生物支持，其效果可与仅使用含血清培养基的当前黄金标准相媲美。
