Ultra-thin, transparent, porous substrates as 3D culture scaffolds for engineering ASC spheroids for high-magnification imaging
Ultra-thin, transparent, porous substrates as 3D culture scaffolds for engineering ASC spheroids for high-magnification imaging
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DOI：
10.1039/d0tb00723d
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发表时间：
2020-08-21
影响因子：
7
通讯作者：
Fujie, Toshinori
中科院分区：
文献类型：
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作者：
Suematsu, Yoshitaka;Tsai, Ya An;Fujie, Toshinori
三维培养有望比单层培养更有代表性地复制生物组织，这对于药物筛选等体外研究具有重要意义。近来，基于防止细胞粘附，已经开发了用于球体工程的各种细胞培养基质。然而，尽管这些基板提供了扩展的可用性，但它们在具有高放大率透镜的球状体的光学显微镜成像方面仍然受到限制。这是因为几乎所有通过纳米压印产生的基底都由于其厚度和表面结构导致的低光学透明度而阻碍了通过它们的光。在这项研究中，我们实现了从脂肪组织来源的干细胞（ASCs）的球状体的制备上的独立的多孔聚合物纳米片（“多孔纳米片”）组成的聚（d，l-乳酸）（PDLLA）的厚度为120 nm，平均孔径为4 μ m。ASC在多孔纳米片上迁移，导致通过单层细胞自发组织固定的球状体。与用于3D细胞培养的常规纳米压印基底（NanoCulture Dish）相比，多孔纳米片还在共聚焦和全息显微镜观察中提供了超过两倍的光学透明度。在40倍放大倍数下可以清楚地观察到有序球体的内部结构。此外，工程球体显示生物活性指示成纤维细胞生长因子（FGF-2）和血管内皮生长因子（VEGF）的mRNA表达。因此，多孔纳米片提供了一种独特的细胞培养基质，不仅用于从干细胞工程化3D细胞组织，而且用于使用光学显微镜成像详细结构。
Three-dimensional (3D) culture is expected to reproduce biological tissues more representatively than monolayer culture, which is important forin vitroresearch such as drug screening. Recently, various cell culture substrates for spheroid engineering have been developed based on the prevention of cell adhesion. However, despite the expanded usability these substrates provide, they remain limited in terms of optical microscopy imaging of spheroids with high magnification lenses. This is because almost all substrates generated by nanoimprinting hamper the light passing through them owing to their low optical transparency caused by the thickness and surface structure. In this study, we achieved the preparation of spheroids from adipose-tissue derived stem cells (ASCs) on free-standing porous polymeric ultrathin films ("porous nanosheets") consisting of poly(d,l-lactic acid) (PDLLA) with thickness of 120 nm and average pore diameter of 4 mu m. ASCs migrated on the porous nanosheet, leading to the spontaneous organization of spheroids anchoredviaa cell monolayer. The porous nanosheet also provided more than twice the optical transparency in confocal and holographic microscopy observation compared to conventional nanoimprinted substrates for 3D cell culture (NanoCulture Dish). The internal structure of the organized spheroids could be clearly observed with 40x magnification. In addition, the engineered spheroids showed bioactivities indicated by mRNA expression of fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF). Thus, porous nanosheets offer a unique cell culture substrate, not only for engineering 3D cellular organization from stem cells, but also for imaging detailed structure using light microscopy.