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The elasticity, topography, and chemical composition of cell culture substrates influence cell behavior. However, the cellular responses to in vivo extracellular matrix (ECM), a hydrogel of proteins (mainly collagen) and polysaccharides, remain unknown as there is no substrate that preserves the key features of native ECM. This study introduces novel collagen hydrogels that can combine elasticity, topography, and composition and reproduce the correlation between collagen concentration (C) and elastic modulus (E) in native ECM. A simple reagent-free method based on radiation-cross-linking altered ECM-derived collagen I and hydrolyzed collagen (gelatin or collagen peptide) solutions into hydrogels with tunable elastic moduli covering a broad range of soft tissues (E = 1–236 kPa) originating from the final collagen density in the hydrogels (C = 0.3%–14%) and precise microtopographies (⩾1 μm). The amino acid composition ratio was almost unchanged by this method, and the obtained collagen hydrogels maintained enzyme-mediated degradability. These collagen hydrogels enabled investigation of the responses of cell lines (fibroblasts, epithelial cells, and myoblasts) and primary cells (rat cardiomyocytes) to soft topographic cues such as those in vivo under the positive correlation between C and E. These cells adhered directly to the collagen hydrogels and chose to stay atop or spontaneously migrate into them depending on E, that is, the density of the collagen network, C. We revealed that the cell morphology and actin cytoskeleton organization conformed to the topographic cues, even when they are as soft as in vivo ECM. The stiffer microgrooves on collagen hydrogels aligned cells more effectively, except HeLa cells that underwent drastic changes in cell morphology. These collagen hydrogels may not only reduce in vivo and in vitro cell behavioral disparity but also facilitate artificial ECM design to control cell function and fate for applications in tissue engineering and regenerative medicine.

ZEP520A (1:1 copolymer of α-chloromethacrylate and α-methylstyrene, ZEON) is a main-chain scission-type positive-tone resist used for electron beam (EB) lithography and is known for its high sensitivity and high resolution. In this study, ZEP520A was irradiated using a 100 kV EB, and the decomposition mechanisms were analyzed by gel permeation chromatography, Fourier transform IR spectroscopy, NMR spectroscopy, and pulse radiolysis. Chlorines were confirmed to easily dissociate as Cl- ions (dissociative electron attachment, DEA) and ZEP520A underwent β-scission. Multiple channels could be considered for the main-chain scission, including DEA and the charge transfer complex between phenyl radical cations and Cl- ions.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

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