In vitro induction of in vivo-relevant stellate astrocytes in 3D brain-derived, decellularized extracellular matrices.

In vitro fabrication of 3D cell culture systems that could provide in vivo tissue-like, structural, and biochemical environments to neural cells is essential not only for fundamental studies on brain function and behavior, but also for tissue engineering and regenerative medicine applicable to neural injury and neurodegenerative diseases. In particular, for astrocytes-which actively respond to the surroundings and exhibit varied morphologies based on stimuli (e.g., stiffness and chemicals) in vitro, as well as physiological or pathological conditions in vivo-it is crucial to establish an appropriate milieu in in vitro culture platforms. Herein, we report the induction of in vivo-relevant, stellate-shaped astrocytes derived from cortices of Rattus norvegicus by constructing the 3D cell culture systems of brain-derived, decellularized extracellular matrices (bdECMs). The bdECM hydrogels were mechanically stable and soft, and the bdECM-based 3D scaffolds supplied biochemically active environments that astrocytes could interact with, leading to the development of in vivo-like stellate structures. In addition to the distinct morphology with actively elongated endfeet, the astrocytes, cultured in 3D bdECM scaffolds, would have neurosupportive characteristics, indicated by the accelerated neurite outgrowth in the astrocyte-conditioned media. Furthermore, next-generation sequencing showed that the gene expression profiles of astrocytes cultured in bdECMs were significantly different from those cultured on 2D surfaces. The stellate-shaped astrocytes in the bdECMs were analyzed to have reached a more mature state, for instance, with decreased expression of genes for scaffold ECMs, actin filaments, and cell division. The results suggest that the bdECM-based 3D culture system offers an advanced platform for culturing primary cortical astrocytes and their mixtures with other neural cells, providing a brain-like, structural and biochemical milieu that promotes the maturity and in vivo-like characteristics of astrocytes in both form and gene expression. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrices (dECMs) have emerged as strong candidates for the construction of three-dimensional (3D) cell cultures in vitro, owing to the potential to provide native biochemical and physical environments. In this study, we fabricated hydrogels of brain-derived dECMs (bdECMs) and cultured primary astrocytes within the bdECM hydrogels in a 3D context. The cultured astrocytes exhibited a stellate morphology distinct from conventional 2D cultures, featuring tridimensionally elongated endfeet. qRT-PCR and NGS-based transcriptomic analyses revealed gene expression patterns indicative of a more mature state, compared with the 2D culture. Moreover, astrocytes cultured in bdECMs showed neurosupportive characteristics, as demonstrated by the accelerated neurite outgrowth in astrocyte-conditioned media. We believe that the bdECM hydrogel-based culture system can serve as an in vitro model system for astrocytes and their coculture with other neural cells, holding significant potential for neural engineering and therapeutic applications.

Cell-in-Catalytic-Shell Nanoarchitectonics: Catalytic Empowerment of Individual Living Cells by Single-Cell Nanoencapsulation.

Cell-in-shell biohybrid structures, synthesized by encapsulating individual living cells with exogenous materials, have emerged as exciting functional entities for engineered living materials, with emergent properties outside the scope of biochemical modifications. Artificial exoskeletons have, to date, provided physicochemical shelters to the cells inside in the first stage of technological development, and further advances in the field demand catalytically empowered, cellular hybrid systems that augment the biological functions of cells and even introduce completely new functions to the cells. This work describes a facile and generalizable strategy for empowering living cells with extrinsic catalytic capability through nanoencapsulation of living cells with a supramolecular metal-organic complex of Fe3+ and benzene-1,3,5-tricarboxylic acid (BTC). A series of enzymes are embedded in situ, without loss of catalytic activity, in the Fe3+ -BTC shells, not to mention the superior characteristics of cytocompatible and rapid shell-forming processes. The nanoshell enhances the catalytic efficiency of multienzymatic cascade reactions by confining reaction intermediates to its internal voids and the nanoencapsulated cells acquire exogenous biochemical functions, including enzymatic cleavage of lethal octyl-ß-d-glucopyranoside into d-glucose, with autonomous cytoprotection. The system will provide a versatile, nanoarchitectonic tool for interfacing biological cells with functional materials, especially for catalytic bioempowerment of living cells.

Inexpensive water soluble methyl methacrylate-functionalized hydroxyphthalimide: variations of the mycophenolic acid core for selective live cell imaging of free cysteine.

Evident from numerous studies, cysteine plays a crucial role in cellular function. Reactions with analyte also enables for molecular recognition to adhere to molecular therapeutic potential; integration between synthetic probes therefore allows for a potentially deep therapy-related interogation of biological systems (theranostics). The development of molecular cysteine probes with extremely accurate detection is still a key challenge for the field. The development of water-soluble organic molecular fluorescent probes able to efficiently distinguish common biothiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) by chemical recognition means i.e. by (binding, cleavage) in biological systems is a greatly sought research challenge due to the similarity of the small sulfhydryl-containing species. Herein, we have developed a water-soluble and highly cell viable fluorescent organic molecule (log P = 0.82) for the selective detection of cysteine. The probe (Myco-Cys) shows a "turn-on" response with the cleavage ester linkage of the methacrylate as cysteine is encountered in solution. The probe shows strong fluorescence enhancement (16.5-fold) when treated with Cys (1 equiv., 10 µM) compared to closely related species such as amino acids, including HCy/GSH, and the limit of detection was determined as 45.0 nM. DFT calculations helped confirm the photomechanism of Myco-Cys. Furthermore, the sensing ability of the probe was demonstrated by living cell assays through the use of confocal fluorescence microscopy. Myco-Cys could selectively detect cysteine among biothiols. Myco-Cys was able to monitor the cysteine level, apart from the oxidative stress present in the form of H2O2 in A549 cells.

Ascorbic acid-mediated reductive disassembly of Fe3+-tannic acid shells in degradable single-cell nanoencapsulation.

Rapid degradation of Fe3+-tannic acid films is achieved under mild conditions via ascorbic acid-mediated Fe3+ reduction, which overcomes the problems in the disassembly of a metal-organic complex including slow reaction rates and reaction incompatibility with living cells. The strategy of reductive disassembly is applied to degradable single-cell nanoencapsulation, providing an advanced tool for tightly controlling and manipulating the cell-material interface.

Fabrication and Characterization of Neurocompatible Ulvan-Based Layer-by-Layer Films.

Construction of extracellular matrix-mimetic nanofilms has considerable potential in biomedical and nanomedicinal fields. In this work, we fabricated neurocompatible layer-by-layer (LbL) films based on ulvan (ULV), a highly sulfated polysaccharide having compositional similarity to glycosaminoglycans that play important functional roles in the brain. ULV was durably assembled as a film with chitosan, another marine-derived polysaccharide, and the film enabled the stable adhesion of primary hippocampal neurons with high viability, comparable to the conventional poly-d-lysine surface. Notably, the ULV-based LbL films accelerated neurite outgrowth and selectively suppressed the adhesion of astrocytes, highlighting its potential as an advanced platform for neural implants and devices.

Neuro-taxis: Neuronal movement in gradients of chemical and physical environments.

Environmental chemical and physical cues dynamically interact with migrating neurons and sprouting axons, and in particular, the gradients of environmental cues are regarded as one of the factors intimately involved in the neuronal movement. Since a growth cone was first described by Cajal, more than one century ago, chemical gradients have been suggested as one of the mechanisms by which the neurons determine proper paths and destinations. However, the gradients of physical cues, such as stiffness and topography, which also interact constantly with the neurons and their axons as a component of the extracellular environments, have rarely been noted regarding the guidance of neurons, despite their gradually increasingly reported influences in the case of nonneuronal-cell migration. In this review, we discuss chemical (i.e., chemo- and hapto-) and physical (i.e., duro-) taxis phenomena on the movement of neurons including axonal elongation. In addition, we suggest topotaxis, the most recently proposed physical-taxis phenomenon, as another potential mechanism in the neuronal movement, based on the reports of neuronal recognition of and responses to nanotopography.

Single-Cell Nanoencapsulation: From Passive to Active Shells.

Single-cell nanoencapsulation is an emerging field in cell-surface engineering, emphasizing the protection of living cells against external harmful stresses in vitro and in vivo. Inspired by the cryptobiotic state found in nature, cell-in-shell structures are formed, which are called artificial spores and which show suppression or retardation in cell growth and division and enhanced cell survival under harsh conditions. The property requirements of the shells suggested for realization of artificial spores, such as durability, permselectivity, degradability, and functionalizability, are demonstrated with various cytocompatible materials and processes. The first-generation shells in single-cell nanoencapsulation are passive in the operation mode, and do not biochemically regulate the cellular metabolism or activities. Recent advances indicate that the field has shifted further toward the formation of active shells. Such shells are intimately involved in the regulation and manipulation of biological processes. Not only endowing the cells with new properties that they do not possess in their native forms, active shells also regulate cellular metabolism and/or rewire biological pathways. Recent developments in shell formation for microbial and mammalian cells are discussed and an outlook on the field is given.

Thickness-Tunable Eggshell Membrane Hydrolysate Nanocoating with Enhanced Cytocompatibility and Neurite Outgrowth.

The eggshell membrane is one of the easily obtainable natural biomaterials, but has been neglected in the biomaterial community, compared with marine biomaterials and discarded as a food waste. In this work, we utilized the ESM hydrolysate (ESMH), which was obtained by the enzymochemical method, as a bioactive functional material for interfacial bioengineering, exemplified by thickness-tunable, layer-by-layer (LbL) nanocoating with the Fe(III)-tannic acid (TA) complex. [Fe(III)-TA/ESMH] LbL films, ending with the ESMH layer, showed great cytocompatiblility with HeLa cells and even primary hippocampal neuron cells. More importantly, the films were found to be neurochemically active, inducing the acceleration of neurite outgrowth for the long-term neuron culture. We believe that the ability for building cytocompatible ESMH films in a thickness-tunable manner would be applicable to a broad range of different nanomaterials in shape and size and would be utilized with multimodal functionalities for biomedical applications, such as bioencapsulation, theranostics, and regenerative medicine.

Iron Gall Ink Revisited: In Situ Oxidation of Fe(II)-Tannin Complex for Fluidic-Interface Engineering.

The ancient wisdom found in iron gall ink guides this work to a simple but advanced solution to the molecular engineering of fluidic interfaces. The Fe(II)-tannin coordination complex, a precursor of the iron gall ink, transforms into interface-active Fe(III)-tannin species, by oxygen molecules, which form a self-assembled layer at the fluidic interface spontaneously but still controllably. Kinetic studies show that the oxidation rate is directed by the counteranion of Fe(II) precursor salts, and FeCl2 is found to be more effective than FeSO4 -an ingredient of iron gall ink-in the interfacial-film fabrication. The optimized protocol leads to the formation of micrometer-thick, free-standing films at the air-water interface by continuously generating Fe(III)-tannic acid complexes in situ. The durable films formed are transferable, self-healable, pliable, and postfunctionalizable, and are hardened further by transfer to the basic buffer. This O2 -instructed film formation can be applied to other fluidic interfaces that have high O2 level, demonstrated by emulsion stabilization and concurrent capsule formation at the oil-water interface with no aid of surfactants. The system, inspired by the iron gall ink, provides new vistas on interface engineering and related materials science.

Enzymatic film formation of nature-derived phenolic amines.

An enzyme-instructed method is developed for material-independent, cytocompatible coating of phenolic amines, inspired by melanogenesis found in nature. Tyrosinase-based film formation proceeds smoothly in an aqueous solution at neutral pH, and can use various phenolic amines including catecholamines, such as tyrosine, tyramine, dopamine, norepinephrine, and DOPA, as a coating precursor. Compared with polydopamine coating, the method is fast and efficient, and forms uniform films. Its high cytocompatibility is advantageously applied to cell-surface engineering, where chemically labile Jurkat cells are coated individually without any noticeable decrease in viability. Considering the huge potential of polyphenolic-based coatings, the strategy developed herein will provide an advanced tool for manipulating biological entities, including living cells, in biomedical and medicinal applications.

Cytoprotective Encapsulation of Individual Jurkat T Cells within Durable TiO2 Shells for T-Cell Therapy.

Lymphocytes, such as T cells and natural killer (NK) cells, have therapeutic promise in adoptive cell transfer (ACT) therapy, where the cells are activated and expanded in vitro and then infused into a patient. However, the in vitro preservation of labile lymphocytes during transfer, manipulation, and storage has been one of the bottlenecks in the development and commercialization of therapeutic lymphocytes. Herein, we suggest a cell-in-shell (or artificial spore) strategy to enhance the cell viability in the practical settings, while maintaining biological activities for therapeutic efficacy. A durable titanium oxide (TiO2 ) shell is formed on individual Jurkat T cells, and the CD3 and other antigens on cell surfaces remain accessible to the antibodies. Interleukin-2 (IL-2) secretion is also not hampered by the shell formation. This work suggests a chemical toolbox for effectively preserving lymphocytes in vitro and developing the lymphocyte-based cancer immunotherapy.

Modulation of Heterotypic and Homotypic Cell-Cell Interactions via Zwitterionic Lipid Masks.

Since the pioneering work by Whitesides, innumerable platforms that aim to spatio-selectively seed cells and control the degree of cell-cell interactions in vitro have been developed. These methods, however, have generally been technically and methodologically complex, or demanded stringent materials and conditions. In this work, we introduce zwitterionic lipids as patternable, cell-repellant masks for selectively seeding cells. The lipid masks are easily removed with a routine washing step under physiological conditions (37 °C, pH 7.4), and are used to create patterned cocultures, as well as to conduct cell migration studies. We demonstrate, via patterned cocultures of NIH 3T3 fibroblasts and HeLa cells, that HeLa cells proliferate far more aggressively than NIH 3T3 cells, regardless of initial population sizes. We also show that fibronectin-coated substrates induce cell movement akin to collective migration in NIH 3T3 fibroblasts, while the cells cultured on unmodified substrates migrate independently. Our lipid mask platform offers a rapid and highly biocompatible means of selectively seeding cells, and acts as a versatile tool for the study of cell-cell interactions.

Artificial Spores: Cytocompatible Coating of Living Cells with Plant-Derived Pyrogallol.

Cell nanoencapsulation, generating cell-in-shell structures ("artificial spores"), provides a chemical toolbox for controlling the cellular behaviors and functional characteristics of individual cells. Among the shell materials studied so far, naturally occurring polyphenolic compounds, including polydopamine and tannic acid, have intensively been employed in cell-surface engineering, because their material-independent coating property eliminates an extra priming step for inducing subsequent shell formation. Albeit successful in generating cell-in-shell structures, the coating of polyphenolic compounds generally requires alkaline conditions and/or high salt conditions, which are not compatible with certain cell types. In this work, we demonstrate that the nanocoating of individual cells with a plant-derived phenolic compound, pyrogallol (1,2,3-trihydroxybenzene), occurs at mildly alkaline pH of 7.8 in an isotonic buffer. Three different cell types (anucleate, microbial, and mammalian cells) are coated with pyrogallol without noticeable decrease in cell viability. The protocol developed in this work could be applied to other polyphenolic compounds, and, considering the many polyphenols identified as a coating material, provides an advanced chemical tool in cell-surface engineering.