Henn Itai, Atkins Ayelet, Markus Amos, Shpun Gal, Barad Hannah-Noa, Farah Nairouz, Mandel Yossi
Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.
Bar-Ilan Institute for Nanotechnology and Advanced Materials (BINA), Bar-Ilan University, Ramat Gan, Israel.
Dev Neurobiol. 2020 Sep;80(9-10):305-315. doi: 10.1002/dneu.22707. Epub 2019 Jul 25.
Tissue and neural engineering for various regenerative therapies are rapidly growing fields. Of major interest is studying the complex interface between cells and various 3D structures by scanning electron microscopy with focused ion beam. Notwithstanding its unrivaled resolution, the optimal fixation, dehydration, and staining protocols of the samples while preserving the complex cell interface in its natural form, are highly challenging. The aim of this work was to compare and optimize staining and sample drying procedures in order to preserve the cells in their "life-like state" for studying the cell interface with either 3D well-like structures or gold-coated mushroom-shaped electrodes. The process involved chemical fixation using a combination of glutaraldehyde and formaldehyde, followed by gentle drying techniques in which we compared four methods: (critical point drying, hexamethyldisiloxane, repeats of osmium tetroxide-thiocarbohydrazide [OTOTO], and resin) in order to determine the method that best preserves the cell and cell interface morphology. Finally, to visualize the intracellular organelles and membrane, we compared the efficacy of four staining techniques: osmium tetroxide, osmium tetroxide and salts, osmium and uranyl acetate, and OTOTO. Experiments were performed on embryonic stem cell-derived photoreceptor precursors, neural cells, and a human retinal pigment epithelial cell line, which revealed that the optimal processing combination was resin drying and OTOTO staining, as manifested by preservation of cell morphology, the lowest percentage of cellular protrusion breakage as well as a high-quality image. The obtained results pave the way for better understanding the cell interface with various structures for enhancing various biomedical applications.
用于各种再生疗法的组织工程和神经工程是快速发展的领域。通过聚焦离子束扫描电子显微镜研究细胞与各种三维结构之间的复杂界面是主要研究兴趣所在。尽管其具有无与伦比的分辨率，但要在以自然形式保留复杂细胞界面的同时，对样品进行最佳的固定、脱水和染色方案极具挑战性。这项工作的目的是比较和优化染色及样品干燥程序，以便将细胞保持在“逼真状态”，用于研究细胞与三维孔状结构或镀金蘑菇形电极的界面。该过程包括使用戊二醛和甲醛组合进行化学固定，随后采用温和干燥技术，我们比较了四种方法：（临界点干燥、六甲基二硅氧烷、四氧化锇 - 硫代碳酰肼重复处理[OTOTO]和树脂），以确定最能保留细胞和细胞界面形态的方法。最后，为了可视化细胞内细胞器和细胞膜，我们比较了四种染色技术的效果：四氧化锇、四氧化锇和盐、锇和醋酸铀，以及OTOTO。对胚胎干细胞衍生的光感受器前体、神经细胞和人视网膜色素上皮细胞系进行了实验，结果表明最佳处理组合是树脂干燥和OTOTO染色，表现为细胞形态得以保留、细胞突起断裂百分比最低以及图像质量高。所得结果为更好地理解细胞与各种结构的界面以增强各种生物医学应用铺平了道路。
