Primary human mesenchymal stem cell culture under xeno-free conditions using surface-modified cellulose nanofiber scaffolds.

Stem cell culture often requires various animal-derived components such as serum and collagen. This limits its practical use. Therefore, xeno-free (xenogeneic component-free) culture systems are receiving increased attention. Herein, we propose xeno-free, plant-derived cellulose nanofibers (CNFs) with different surface chemistry: 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO)-oxidized CNFs (TOCNFs) with carboxy groups and surface-sulfated CNFs (S-CNFs) for the proliferation of human mesenchymal stem cells (hMSCs) under various serum conditions. We cultured bone marrow-derived hMSCs on CNF scaffolds with various fiber lengths and functional group contents. Original CNFs were bioinert materials that did not contribute to cell adhesion. In contrast, the surface-modified CNFs facilitated the proliferation of immortalized hMSCs under normal and low-serum conditions. The TOCNFs (COONa: 1.47 mmol g-1; length: 0.53 µm), the S-CNFs (OSO3Na: 0.64 mmol g-1; 0.61 µm), and a combination of the two (1:1 by weight) enabled immortalized hMSCs to maintain their multipotency, even under serum-free conditions. Primary cultured hMSCs proliferated well on the TOCNF/S-CNF scaffolds in a completely serum-free medium, comparable to animal-derived type I collagen, although few hMSCs adhered to the standard polystyrene substrate. Our strategy of using surface-modified CNFs will inform the development of xeno-free culture systems to avoid the use of animal-derived materials for both cell culture media and scaffolds.

Injectable cell-laden hydrogels fabricated with cellulose and chitosan nanofibers for bioprinted liver tissues.

Bio-based hydrogels as three-dimensional (3D) constructs have attracted attention in advanced tissue engineering. Compared with conventional two-dimensional (2D) cell culture, cells grown in 3D scaffolds are expected to demonstrate the inherent behavior of living organisms of cellular spheroids. Herein, we constructed cell-laden nanofiber-based hydrogels in combination with 2,2,6,6-tetramethylpiperidine 1-oxyl-oxidized cellulose nanofiber (TOCNF) and chitosan nanofiber (CsNF) for bioadaptive liver tissue engineering. The carboxylates of TOCNF and amines of CsNF were directly crosslinked via EDC/NHS chemistry. The rheological properties of the solutions for the nanofibers and hydrogels revealed sufficient physical properties for the injection, printing, and plotting process, as well as significant encapsulation of living cells. As-designed hydrogels exhibited excellent viscoelastic properties with typical shear-thinning behavior, and had a storage modulus of 1234 Pa ± 68 Pa, suitable for cell culture. Non-cytotoxicity was confirmed using a live/dead assay with mouse-derived fibroblast NIH/3T3 cells. Human hepatocellular carcinoma HepG2 cells could be cultured on a gel surface (2D environment) and encapsulated in the gel structure (3D environment), which enabled 10 d growth with high gene expression level of albumin of HepG2 spheroids in the 3D gels. The biodegradable cell-laden hydrogels are expected to mimic the cellular microenvironment and provide potential for bioadaptive 3D cell cultures in biomedical applications.

Polysaccharide Nanofiber-Stabilized Pickering Emulsion Microparticles Induce Pyroptotic Cell Death in Hepatocytes and Kupffer Cells.

The intracellular uptake and interaction behavior of emulsion microparticles in liver cells critical to host defense and inflammation is significant to understanding their potential cytotoxicity and biomedical applications. In this study, the cell death responses of fibroblastic, hepatocyte, and Kupffer cells (KCs) induced by four types of emulsion particles that are stabilized by polysaccharide nanofibers (cellulose or chitin), an inorganic nanoparticle (ß-tricalcium phosphate), or surfactants are compared. Pickering emulsion (PE) microparticles stabilized by polysaccharide nanofibers or inorganic nanoparticles have a droplet size of 1-3 µm, while the surfactant-stabilized emulsion has a diameter of ≈190 nm. Polysaccharide nanofiber-stabilized PEs (PPEs) markedly induce lactate dehydrogenase release in all cell types. Additionally, characteristic pyroptotic cell death, which is accompanied by cell swelling, membrane blebbing, and caspase-1 activation, occurs in hepatocytes and KCs. These PE microparticles are co-cultured with lipopolysaccharide-primed KCs associated with cytokine interleukin-1ß release, and the PPEs demonstrate biological activity as a mediator of the inflammation response. Well-designed PPE microparticles induce pyroptosis of liver cells, which may provide new insight into regulating inflammation-related diseases for designing potent anticancer drugs and vaccine adjuvants.

One-step synthesis of cellooligomer-conjugated gold nanoparticles in a water-in-oil emulsion system and their application in biological sensing.

Monodisperse gold nanoparticles (GNPs) were synthesized in a water-in-oil emulsion system (reverse micelles) composed of 80% N-methylmorpholine N-oxide (NMMO)/20% H2O and dodecane, stabilized with an anionic surfactant: bis(2-ethylhexyl)sulfosuccinate sodium salt. Cellooligomers with a degree of polymerization of 6 or 15 (ßGlc6 or ßGlc15, respectively), which were labeled at each reducing end group with thiosemicarbazide (TSC) and dissolved in the aqueous NMMO phase, were successfully conjugated to the surfaces of GNPs in situ during spontaneous NMMO-mediated gold reduction. As-synthesized ßGlc6-GNPs and ßGlc15-GNPs had average diameters of 11.3 ± 2.1 and 10.5 ± 0.7 nm, respectively, while their surface sugar densities were 0.21 and 0.51 chains nm-2, respectively. Concanavalin A (ConA), a lectin that recognizes non-reducing end groups of glucose residues, aggregated with ßGlc15-GNPs with higher sensitivity than it did with ßGlc6-GNPs, possibly as a result of the sugar density on the GNP surfaces. The aggregates were rapidly re-suspended by adding methyl-ß-d-glucopyranoside as a binding inhibitor. Other lectins and proteins showed no interaction with ßGlc-GNPs. Therefore, clustering of glucose non-reducing ends on the GNP surfaces via strong intermolecular association of cellooligomers, possibly led to high affinity for ConA. This facile synthesis route to structural carbohydrate-decorated GNPs has potential applications in carbohydrate-nanometal conjugate nano-biosensor development.

Insight into metabolic diversity of the brown-rot basidiomycete Postia placenta responsible for sesquiterpene biosynthesis: semi-comprehensive screening of cytochrome P450 monooxygenase involved in protoilludene metabolism.

A wide variety of sesquiterpenoids have been isolated from basidiomycetes, and their bioactive properties have attracted significant attention in an effort to understand biosynthetic machineries. As both sesquiterpene synthases and cytochrome P450 monooxygenases play key roles in the diversification of sesquiterpenoids, it is important to widely and mutually understand their biochemical properties. In this study, we performed genome-wide annotation and functional characterization of sesquiterpene synthases from the brown-rot basidiomycete Postia placenta. Using RT-PCR, we isolated 16 sesquiterpene synthases genes as full-length cDNAs. Heterologous expression revealed that the sesquiterpene synthases could produce a series of sesquiterpene scaffolds with distinct metabolic profiles. Based on metabolic studies, we identified 25 sesquiterpene scaffolds including Δ6-protoilludene produced by the sesquiterpene synthases. In particular, a protoilludene synthase from the brown-rot basidiomycete was characterized for the first time. Furthermore, we conducted a semi-comprehensive functional screening of cytochrome P450 monooxygenases from P. placenta to elucidate biosynthetic machineries involved in metabolisms of Δ6-protoilludene. Coexpression of protoilludene synthase and 184 isoforms of cytochrome P450 monooxygenases enabled the identification of CYP5344B1, CYP5348E1 and CYP5348J3, which catalysed the hydroxylation reaction of Δ6-protoilludene to produce Δ6-protoilludene-8-ol and Δ6-protoilludene-5-ol. Furthermore, structural isomers of Δ7-protoilludene-6-ol were obtained from incubation of Δ6-protoilludene-8-ol in acidic culture medium.

Ethylene-gibberellin signaling underlies adaptation of rice to periodic flooding.

Most plants do poorly when flooded. Certain rice varieties, known as deepwater rice, survive periodic flooding and consequent oxygen deficiency by activating internode growth of stems to keep above the water. Here, we identify the gibberellin biosynthesis gene, SD1 (SEMIDWARF1), whose loss-of-function allele catapulted the rice Green Revolution, as being responsible for submergence-induced internode elongation. When submerged, plants carrying the deepwater rice-specific SD1 haplotype amplify a signaling relay in which the SD1 gene is transcriptionally activated by an ethylene-responsive transcription factor, OsEIL1a. The SD1 protein directs increased synthesis of gibberellins, largely GA4, which promote internode elongation. Evolutionary analysis shows that the deepwater rice-specific haplotype was derived from standing variation in wild rice and selected for deepwater rice cultivation in Bangladesh.

Ultraselective Gas Separation by Nanoporous Metal-Organic Frameworks Embedded in Gas-Barrier Nanocellulose Films.

Metal-organic frameworks (MOFs) are synthesized at carboxy groups on crystalline TEMPO-oxidized cellulose nanofibers (TOCNs). MOF-TOCN films coated on a paper filter have a hierarchical structure from the nano- to macroscale, and demonstrate a high CO2 /CH4 selectivity, over 120 for CO2 at a high gas flux, by the combination of the nanoporous MOFs and the gas-barrier TOCNs, which have strong affinity with each other.

Hybrid immobilization of galactosyl lactose and cellobiose on a gold substrate to modulate biological responses.

Bioactive O-ß-d-galactopyranosyl-(1→4)-O-ß-d-galactopyranosyl-(1→4)-d-glucopyranose (4'-galactosyl lactose) was site-selectively modified at a reducing end with thiosemicarbazide (TSC). As-synthesized 4'-galactosyl lactose-TSC was immobilized on a gold substrate with cellobiose-TSC as a spacer through spontaneous self-assembly chemisorption via SAu bonding. Quartz crystal microbalance analysis suggested the successful formation of self-assembled monolayers (SAMs) of 4'-galactosyl lactose-TSC and/or cellobiose-TSC. Galactose-binding lectin exhibited the highest affinity for hybrid SAMs with an equimolar ratio of the two oligosaccharide-TSCs, while glucose-binding lectin showed decreasing adsorption with a decrease in cellobiose-TSC ratios. Human hepatocellular carcinoma cells, which recognize galactose residues, efficiently adhered to the hybrid SAMs. Higher enzymatic deethoxylation of ethoxyresorufin via cytochrome P450 appeared on hybrid SAMs. These results suggested that clustering of the bioactive sugars was involved in the cellular responses, possibly via biological carbohydrate-protein interactions. This approach to designing carbohydrate-based scaffolds should provide a basis for the functional development of glyco-decorated biointerfaces for cell culture applications.

A novel double-coating carrier produced by solid-in-oil and solid-in-water nanodispersion technology for delivery of genes and proteins into cells.

A novel intracellular delivery method both for genes and proteins is one of the most coveted systems in the drug delivery field. In the present study, we developed a double-coating carrier loaded with gene and protein produced by solid-in-oil and solid-in-water nanodispersion technology. The double-coating carriers did not require electrostatic interactions during the preparation so were able to encapsulate plasmid DNA, ovalbumin (pI 4.5), horseradish peroxidase (pI 7.2), and cytochrome-c (pI 10.5) in a consistent manner. The carriers had practical encapsulation efficiencies and release profiles for genes and proteins. Furthermore, effective gene expression and cellular uptakes of both anionic and cationic proteins were achieved by modification of carriers with functional molecules. These findings indicate that the double-coating carrier has high potential for cellular delivery of various drugs and is a novel, superior method for both gene and protein delivery into cells.