# Nanoscale Topographies for Corneal Endothelial Regeneration
## 1 Introduction
Despite the current efforts to optimize the primary expansion of hCECs, these cells tend to undergo phenotypical alterations already after two passages [11; 12; 13], suggesting that a longer culture period in a non-physiological environment leads to seemingly irreversible changes in cell behavior, therefore limiting the number of cells that can be derived from a donor cornea. Moreover, most successful cultures are from donors aged less than 40 years. Current standards for evaluating hCECs in vitro culture consist of assessing cell morphology and a panel of markers, such as Na+/K+-ATPase (an ion transporter assessed as functionality marker, Figure 2B), zona occludens-1 (ZO-1) (a tight junction protein, Figure 2C) and CD166 (a transmembrane protein ligand of CD6), none of which can be considered highly specific when analyzed independently. As an alternative to primary culture of hCECs, scientists have explored the differentiation of pluripotent stem cells to hCECs. Nevertheless, such differentiation protocols are still at an early developmental stage and need further improvements to prove that the genotype, phenotype and functionality of the differentiated cells resembles that of native hCECs [14; 15; 16]. Overall, the challenges associated with the primary culture of hCECs remain a barrier to their therapeutic application. Thus, there is a need for more advanced approaches that perhaps combine biophysical and biochemical factors in order to better control hCECs' phenotype over a prolonged period of time. Novel bioengineering tools have the potential to generate more defined and more physiological cell culture conditions also for hCECs. To support the expansion and differentiation of cells in vitro, advanced, bioengineered cell culture substrates have been increasingly employed as a means to mimic biophysical cues that are originally present in native ECM environments [17; 18; 19]. These can be obtained by altering substrate features, such as stiffness, wettability, surface energy and topography through suitable fabrication and surface modification processes. Micrometer scale and nanometer scale topographies' (microtopographies and nanotopographies') structures on cell culture surfaces have been shown to modulate cell behavior, for example by increasing cell adhesion, proliferation, migration, survival, differentiation and phenotype maintenance in many applications [17; 19; 20; 21; 22]. The addition of instructive topographies on cell culture scaffolds could, therefore, provide significant advantages compared to conventional flat (smooth) 2D substrates, as the first can offer biophysical cues that more closely replicate those present in the native ECM. Surface topographies have already been shown to modulate mesenchymal-to-endothelial/epithelial transition [23; 24; 25; 26] and vice versa [27; 28; 29; 30] in several others applications. However, research focusing on the interaction between hCECs and surface topography of the culture substrate remains relatively less explored compared to other (corneal) cell types, despite the fact that growing evidence (as reported in the next sections as well as from preliminary work by our group) is showing that subcellular topographies can also have a significant impact on hCECs' expansion and differentiation. In addition, although systematic data on how a defined microenvironment can be used to better guide corneal endothelial cells are essential to design/improve future studies, these are not yet available. In this review, we briefly describe the range of nanofabrication techniques that can potentially be used to create well-defined cellular or subcellular topographic features for the culture of primary hCECs. We also summarize current knowledge and understanding of the effect of substrate topography on hCEC responses based on the current literature.