STL-files for 3D-printed grid holders described in Fäßler F, Zens B, et al.; 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy

Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.

冷冻电子显微镜技术（cryo-electron microscopy，cryo-EM）对细胞样品的观察，使得我们能够在原生状态下深入理解生物过程与结构。然而，在为后续的cryo-EM分析进行高效且可重复的粘附细胞制备方面，仍存在一项重大挑战。这主要归因于众多细胞样品对电子显微镜实验所需的种子培养和培养条件的敏感性，细胞材料的有限数量，以及电子显微镜网格及其基底的脆弱性。在本研究中，我们提出了一种低成本且可重复使用的3D打印网格支架，旨在提升直接在网格上培养具有挑战性的细胞样品时的样品制备质量。所描述的网格支架能够提升细胞培养的可重复性和效率，并降低细胞培养所需的资源。我们展示了网格支架可以整合到多种cryo-EM工作流程中，包括微图案化方法以控制网格上的细胞播种，以及用于生成冷冻聚焦离子束研磨和冷冻电子断层扫描实验的样品。其可适应性设计使得能够根据多种应用需求定制专业化的网格支架。