Nacre inspires researchers by combining stiffness with toughness by its unique microstructure of aligned aragonite platelets. This brick‐and‐mortar structure of reinforcing platelets separated with thin organic matrix has been replicated in numerous mimics that can be divided into two categories: microcomposites with aligned metal oxide microplatelets in polymer matrix, and nanocomposites with self‐assembled nanoplatelets—usually clay or graphene oxide—and polymer. While microcomposites have shown exceptional fracture toughness, current fabrication methods have limited nacre‐mimetic nanocomposites to thin films where fracture properties remained unexplored. Yet, fracture resistance is the defining property of nacre, therefore centrally important in any mimic. Furthermore, to make use of these properties in applications, bulk materials are required. Here, up to centimeter‐thick nacre‐mimetic clay/polymer nanocomposites are produced by the lamination of self‐assembled films. The aligned clay nanoplatelets are separated by poly(vinyl alcohol) matrix, with 106–107 nanoplatelets on top of each other in the bulk plates. Fracture testing shows crack deflection and a fracture toughness of 3.4 MPa m1/2, not far from nacre. Flexural tests show high stiffness (25 GPa) and strength (220 MPa) that, despite the hydrophilic constituents, are not substantially affected by exposure to humidity.

Human pluripotent stem cell-derived retinal pigment epithelium (RPE) transplantation is currently under evaluation as treatment for macular degeneration. For therapeutic applications, cryostorage during cell production is typically needed with potential consequences to cell functionality. We have previously shown that the culture substrate affects human embryonic stem cell-derived RPE (hESC-RPE) properties in fresh cultures. Here, we aimed to further identify the role of RPE basement membrane proteins type IV collagen (Col-IV), laminin (LN), and nidogen-1 in the maturation and functionality of hESC-RPE after cryopreservation. In addition to cell attachment and morphology, transepithelial electrical resistance, expression of key RPE proteins, phagocytosis capacity and Ca2+ signalling were analysed. After cryostorage, attachment of hESC-RPE on culture surfaces coated with Col-IV alone was poor. Combining Col-IV and LN with or without nidogen-1 significantly improved cell attachment and barrier properties of the epithelium. Furthermore, functional homogeneity of the hESC-RPE monolayer was enhanced in the presence of nidogen-1. Our results suggest that the choice of coating proteins for the cell culture may have implications to the functional properties of these cells after cryostorage cell banking.

Calcium is one of the most important second messengers in cells and thus involved in a variety of physiological processes. In retinal pigment epithelium (RPE), Ca 2+ and its ATP-dependent signaling pathways play important roles in the retina maintenance functions. Changes in intracellular Ca 2+ concentration can be measured from living cells by Ca 2+ imaging. Combining these measurements with quantitative analysis of Ca 2+ response properties enables studies of signaling pathways affecting RPE functions. However, robust tools for response analysis from large cell populations are lacking. We developed MATLAB-based analysis tools for single cell level Ca 2+ response data recorded from large fields of intact RPE monolayers. The analysis revealed significant heterogeneity in ATP-induced Ca 2+ responses inside cell populations regarding magnitude and response kinetics. Further analysis including response grouping and parameter correlations allowed us to characterize the populations at the level of single cells.

Background Transplantation of human pluripotent stem cell-derived retinal pigment epithelium (RPE) is an urgently needed treatment for the cure of degenerative diseases of the retina. The transplanted cells must tolerate cellular stress caused by various sources such as retinal inflammation and regain their functions rapidly after the transplantation. We have previously shown the maturation level of the cultured human embryonic stem cell-derived RPE (hESC-RPE) cells to influence for example their calcium (Ca2+) signaling properties. Yet, no comparison of the ability of hESC-RPE at different maturity levels to tolerate cellular stress has been reported. Methods Here, we analyzed the ability of the hESC-RPE populations with early (3 weeks) and late (12 weeks) maturation status to tolerate cellular stress caused by chemical cell stressors protease inhibitor (MG132) or hydrogen peroxide (H2O2). After the treatments, the functionality of the RPE cells was studied by transepithelial resistance, immunostainings of key RPE proteins, phagocytosis, mitochondrial membrane potential, Ca2+ signaling, and cytokine secretion. Results The hESC-RPE population with late maturation status consistently showed improved tolerance to cellular stress in comparison to the population with early maturity. After the treatments, the early maturation status of hESC-RPE monolayer showed impaired barrier properties. The hESC-RPE with early maturity status also exhibited reduced phagocytic and Ca2+ signaling properties, especially after MG132 treatment. Conclusions Our results suggest that due to better tolerance to cellular stress, the late maturation status of hESC-RPE population is superior compared to monolayers with early maturation status in the transplantation therapy settings.