# Nanoscale Topographies for Corneal Endothelial Regeneration
## 2 Nanofabrication Techniques to Design Instructive Cell Substrates  
A systematic study on the effect of subcellular topographies was so far difficult to realize also due to the complexity and high costs to produce large-scale nanopatterned areas for extensive cell studies. However, to gain a better understanding of the tools available to conduct such assays with hCECs, a methodic description of the corresponding nanofabrication techniques is essential. Some of the current advanced nanofabrication techniques that have demonstrated to generate instructive cell substrates [31] are shown in Figure 3. Recapitulating key architectural features of the Descemet's membrane could be one of the most effective approaches for controlling hCECs expansion and phenotype in vitro. Nevertheless, fabricating multi-scale and hierarchical structures with a precise control over porosity, fiber diameter and the overlaying hexagonal honeycomb feature, as in in vivo corneas, is challenging to achieve by a single technique and would likely require a combination of multiple nanofabrication techniques, some of which have not been widely employed in this field. Various nanofabrication techniques that are already well established in the semiconductor and micro-electronics industry have been adopted and are constantly growing into the tissue engineering and regenerative medicine field [32]. Many of these techniques rely  
Figure 3: Examples of advanced fabrication tools that can be used for producing bio-instructive substrates for the culture of hCECs. (**A**) Photolithography for transferring a pattern into a photosensitive material. A photoresist is selectively exposed to UV light through a partially light-blocking photomask placed between the UV source and the resist; (**B**) 3D direct laser writing technique based on two-photon polymerization (2 PP) lithography. This technique can generate 3D structures from photosensitive materials with resolutions <100 nm; (**C**) nanoimprint lithography. Surface features are transferred onto a polymer material through a mold (stamp) by means of heat or UV light; (**D**) electrospinning for producing continuous nanofibers. A high-voltage electric field is applied between a needle (connected to a polymeric solution reservoir) and a collector on which polymeric nanofibers are attracted. on conventional light-/photon- or particle radiation-based lithography mostly in polymeric \"(photo)resists\" [33; 34], or the same/similar methods followed by a transfer of the lithographic pattern through etching or electroplating/galvanoforming. These techniques were not originally developed for biological applications, and the materials commonly used are not dedicated/designed biomaterials or in some cases even cytotoxic, therefore, an increasing body of work is being (and needs to be further) carried out to identify suitable biomaterials and fabrication procedures for biomedical applications. Additionally, nano-lithographic \"master\" structures are typically too time consuming and costly concerning their (clean room) fabrication to be directly used as disposable culture substrates. Therefore, nanostructures for cell studies are usually created by copying/replicating the pattern(s) of the master into another material [33], directly or via an intermediate template. This replication can be achieved via several techniques, such as soft-lithography [35; 36; 37], nanoimprint lithography (NIL) (including hot-embossing/thermal NIL and UV/photo-NIL) [38] and (micro-)injection molding [39; 40; 41]. These processes enable not only the replication of structures onto more biocompatible or active materials, but also large-scale production at low cost. Table 1 shows representative studies that combine advanced nanofabrication methods and pattern replication techniques to obtain features <1 um, a size regime that is of specific interest for cell studies.