Abstract—

Currently, biological scaffolds composed of extracellular matrix (ECM) are being actively examined for the needs of regenerative medicine. ECM substrates are prepared by decellularization and used to deliver cells to damaged tissue. Native scaffolds of ECM have an advantage over bioengineered ones because ECM retains natural biologic cues that provide efficient reparative cell functions. Mesenchymal stromal cells (MSCs) have a multipotent potential of differentiation and secrete a wide range of bioactive molecules. In this regard, MSCs are valuable intermediaries for tissue repair. The ECM as a critical component of the MSCs niche modulates their functional activity, including migration, proliferation, and differentiation, and supports their potential for self-renewal. In vitro investigations would be useful in elucidation of how biological scaffolds can affect the reparative functions of MSCs. There are several different protocols for decellularization. Since ECM of various cell types differs qualitatively and quantitatively, these protocols should be optimized for each specific case. In the present study we compared the effectiveness of approach to prepare decellularized ECM (dcECM) of adipose-derived MSC (adMSC): Triton X-100/NH₄OH solution in phosphate buffered solution or H₂O, and the possibility of using dcECM after spheroids were formed. ECM-derived substrates were analyzed with immunocytochemistry and scanning electron microscopy. During long-term culture, MSСs produced a well-developed EСM, which maintained a structure close to the native one after treatment with phosphate buffered solution of Triton X‑100/NH₄OH. It was impossible to receive a uniform dcECM layer, when water solution of Triton X-100/NH₄OH was used. On the scanning electron microscopy images single fiber of ECM were revealed in this case. Fragments of ECM and cells after spheroids formation with RGD peptides were detected. Therefore, this method was not effective for obtaining dcECM of adMSCs.

中文翻译：

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制备人脂肪组织间充质基质细胞脱细胞细胞外基质的最佳方案的选择

摘要-

目前，正在积极研究由细胞外基质（ECM）组成的生物支架对再生医学的需求。ECM底物通过脱细胞作用制备，并用于将细胞递送至受损组织。ECM的天然支架比生物工程的支架更具优势，因为ECM保留了提供有效修复细胞功能的天然生物学线索。间充质基质细胞（MSCs）具有多能分化潜能，并分泌多种生物活性分子。在这方面，MSC是用于组织修复的有价值的中介。ECM作为MSC小生境的关键组成部分，可调节其功能活动，包括迁移，增殖和分化，并支持其自我更新的潜力。体外研究将有助于阐明生物支架如何影响MSC的修复功能。有几种不同的脱细胞方案。由于各种细胞类型的ECM在质量和数量上都不同，因此应对每种具体情况优化这些方案。在本研究中，我们比较了制备脂肪来源的MSC（adMSC）的脱细胞ECM（dcECM）的方法的有效性：Triton X-100 / NH在磷酸盐缓冲溶液或H ₂ O中加入₄ OH溶液，并在形成球状体后使用dcECM。用免疫细胞化学和扫描电子显微镜分析ECM来源的底物。在长期培养过程中，MSСs产生了发达的EСM，在用Triton X‑100 / NH ₄ OH的磷酸盐缓冲溶液处理后，MSСs的结构与天然的接近。当使用Triton X-100 / NH ₄ OH的水溶液时，不可能获得均匀的dcECM层。在这种情况下，在扫描电子显微镜图像上显示了ECM的单纤维。用RGD肽形成球体后，检测到ECM和细胞的片段。因此，该方法对于获得adMSC的dcECM无效。