Biology subjects

Matsusaki, M.

Publications and source records attributed to Matsusaki, M..

17 recordsLinked to original sources

bioRxiv2026

Collagen Rope Trick: Cell-Laden Fibre Assembly at 1 Liquid Interfaces

Summary ParagraphTissues and organs in living organisms represent centimeter-scale hierarchical architectures comprising nano-to microscale, uniaxially aligned extracellular matrix (ECM) fibres with high mechanical strength, integrated with cellular components, as exemplified in tendon, skin, cartilage, bone, and blood vessels1. Here, we present a liquid-liquid interfacial spinning method to produce highly uniaxially aligned, centimeter-scale collagen fibres. The dried fibres exhibit exceptional mechanical properties, with fracture strength of 280 MPa, Youngs modulus of 6 GPa, and toughness of 17 MJ m-3, comparable to spider silk and tendon collagen, and exceeding supramolecular and double-network hydrogels1. Incorporating living cells into the collagen solution yielded centimeter-scale, cell-laden aligned fibres, with densely adherent, uniaxially aligned cells and over 80% viability. Myoblast-laden fibres recapitulate biological features of fibrotic muscle tissues, as observed in type II diabetes2. Interfacial collagen assembly further enables fabrication of dimension-controlled constructs, like 2D sheets, 0D capsules, and 1D tubes, thus providing modular building blocks for centimeter-scale 3D tissues and organ-like structures. This approach offers a versatile platform to engineer mechanically robust, cell-laden tissues with controlled hierarchical architecture.

bioengineering↗

bioRxiv2025

Differentiation of THP-1 Cells in 3D Cancer-Stromal Tissue by Collagen Microfiber for Replicating of Tumor Microenvironment

Abstract textIn three-dimensional (3D) cancer tissue models, the low solubility of type I collagen (Col I) is a limitation in reproducing cancer tissue with high collagen density. we previously reported a "sedimentation culture method" using collagen microfibers (CMF) homogenized Col I powder. This 3D cancer tissue model is expected to reproduce tumor microenvironment comparable to that in tumors bearing mice owing to its high collagen density. However, this 3D cancer tissue model including immune cells such as macrophages has not been reported. In this study, we fabricated a 3D culture system including normal human dermal fibroblasts, human umbilical vein endothelial cells, and cancer cells human monocytes cell line; THP-1 cells by sedimentation culture using CMF. 3D cancer tissue models co-cultured with human colorectal cancer cells for 5 days increased the expression levels of CD14 and CD206, markers of macrophages and M2 macrophages, in THP-1 cells, suggesting differentiation induction toward macrophages and polarization toward M2-like macrophages. We could also demonstrate the applicability of drug efficacy evaluation systems by evaluating the cytotoxicity of poly(vinyl alcohol)-ursodeoxycholic acid15 that induce cell death in response to weak acidic tumor microenvironment in 3D cancer tissue models with macrophages. This 3D cancer tissue models with macrophages are expected to be applied as tools for evaluating the efficacy of cancer therapeutic molecules and for understanding in detail the behavior of immune cells within the tumor microenvironment.

bioengineering↗

bioRxiv2025

Meat the Fat: From High Lipid Content to Nutritional Enhancement to Reconstruct Wagyu Fat with Tunable Rheology and Organoleptic Fidelity for Cultivated Meat

Cultivated meat holds promise as a sustainable and ethical alternative to conventional meat, but reproducing the rich flavor, texture, and lipid composition of premium beef, such as Wagyu, remains a major hurdle. In this study, we redefine bovine adipogenesis by systematically optimizing edible biomaterials, fatty acid delivery systems, and culture conditions to engineer high-fat cultivated tissues with authentic sensory properties. Among various candidates, 1% alginate emerged as the optimal scaffold, providing mechanical softness compatible with adipogenic differentiation and mimicking the rheological properties of native fat. Supplementation with oleic acid complexed to edible albumins, especially whey protein (WP), significantly enhanced lipid accumulation and monounsaturated fatty acid content, particularly oleic acid, which reached up to 80.6% of total fatty acids. Engineered fat tissues displayed soft, spreadable textures and organoleptic characteristics consistent with high-quality beef fat. Fatty acid profiling revealed a bovine-like omega-6 signature, dominated by linoleic, arachidonic, and dihomo-{gamma}-linolenic acids, alongside detectable omega-3 subtypes including alpha-linolenic acid and trace levels of EPA and DHA. This composition not only validates the physiological relevance of the engineered fat, but also highlights its potential to deliver bioactive lipids rarely found in terrestrial animal products. Additionally, the WP-oleic acid system supported robust adipogenesis even under lower serum conditions. These findings establish a food-grade, tunable platform for producing physiologically and nutritionally enhanced fat tissues, advancing the realism, functionality, and health potential of next-generation cultivated meat.

bioengineering↗

bioRxiv2025

Escape from Cell Uptake: Drug-Free Cancer Therapeutics Regulated Hydrophobicity and Negative Charge

Self-aggregation and inducing cell membrane disruption in response to tumor microenvironment-stimuli is expected to be a promising approach for cancer treatment, but is limited by its insufficient stimuli-responsive cytotoxicity due to a lack of in-depth understanding of molecular characteristics, resulting in low selectivity of cell death induction. In this study, we focused on engineering polymer aggregation in detail to further improve tumor microenvironment-responsive cytotoxicity. PVA-U with grafting degrees (G.D.) of 3% (PVA-U3), 15% (PVA-U15), and 25% (PVA-U25) were synthesized and their aggregation properties cytotoxicity was evaluated. The difference in half maximal inhibitory concentration (IC50) values between pH 7.4 and pH 6.5 for PVA-U15 was 4.3-fold, which was greater than that of PVA-U25 at 2.8-fold, suggesting that tumor microenvironment-responsive cytotoxicity could be regulated by controlling G.D. of UDCA. Interestingly, PVA-U15 formed aggregates in the pericellular environment and adsorbed on the cell, effectively inducing cell death whereas PVA-U3 and PVA-U25 showed internalization in the cell. These results indicated that the balance of the surface charge and hydrophobicity could contribute to the adsorption on the cell membrane. These findings are expected to contribute to the development of membrane disruption strategies to control the aggregation properties and cell membrane interaction. Table of ContentsCancer therapeutics strategy targeting tumor microenvironment and cell adsorption of PVA-U with varying granting degree (G.D.). PVA-U is expected to aggregate and disrupt cell membrane in response to tumor microenvironment. PVA-U could regulate cell membrane adsorption by its negative charge and hydrophobicity due to controlling G.D. of UDCA, resulting in optimized tumor microenvironment selectivity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/671160v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@e8d4acorg.highwire.dtl.DTLVardef@1e27a1borg.highwire.dtl.DTLVardef@1beb5d8org.highwire.dtl.DTLVardef@1948d51_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Cultivated Beef Meat Has the Potential to Maintain Original Characteristics of Beef Meat with Customizable Features

Cultivated meat (CM) is currently attracting much attention because of its promise to become a solution to issues of environmental sustainability, animal welfare, and decarbonization. While the fabrication process and materials are still the main focus, it is not yet clear what the characteristics of the constructed CMs are and how they take on the original characteristics of beef meat. In this study, the biological, physicochemical and sensory characteristics of three types of beef (Wagyu, Crossbreed and Holstein) meat were systematically analyzed and compared to muscle and fat fibers constructed by three-dimensional printing using satellite and adipose-derived stem cells isolated from these beef meats. The different characteristics of each beef meat were largely taken on by the CM fibers composed of bSC and bADSC of each meat. In addition, some differing properties from those of the respective beef meat were observed in CM fibers such as one of the omega-3 fatty acids, docosahexaenoic acid (DHA; Wagyu fat fibers showed the highest amount). Furthermore, we also found the important possibility to increase the composition of oleic acid to over 80% in monounsaturated fatty acid (Wagyu has around 50% oleic acid). This study revealed the importance of using cells isolated from each beef meat to provide CM that closely resemble the original texture and taste of each meat, and further the possibility of more carefully arranging their properties.

bioengineering↗

bioRxiv2025

OptoChaperone - A biohybrid tool for regulating protein condensates in cells and in vitro

Protein condensates formed via liquid-liquid phase separation (LLPS) are increasingly recognized as key players in diverse cellular processes, including those associated with disease. Despite extensive efforts to characterize their formation and function, tools that enable precise, reversible, and spatiotemporal control of LLPS remain limited. Here, we report OptoChaperone, a light-activatable molecular system designed to manipulate protein condensates both in vitro and in living cells. This biohybrid system leverages photoresponsive switching to control chaperone activity: blue light triggers the suppressive function, leading to the dissolution of protein condensates, whereas UV light deactivates the system, allowing condensate formation. We demonstrate the efficacy of OptoChaperone in regulating several disease-related protein condensates, such as fused in sarcoma, TAR DNA-binding protein 43, and heat shock factor 1. Importantly, the system exhibits reversible and robust control over droplet dynamics without requiring chemical additives or genetic modifications of the client proteins. Given the reversibility and efficiency of OptoChaperone in the manipulation of protein condensates, this tool offers a powerful platform for dissecting the roles of protein condensation in cellular physiology and pathology. This strategy also holds potential for broader applications in synthetic biology, biomolecular engineering, and therapeutic modulation of aberrant phase separation.

biochemistry↗

bioRxiv2025

A Disease-Associated Mutation Impedes PPIA Through Allosteric Dynamics Modulation

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron degeneration. Peptidylprolyl cis-trans isomerase A (PPIA) is a molecular chaperone involved in protein folding, and its dysfunction has been linked to ALS pathogenesis as proline is recognized as a key residue for maintaining proper folding of ALS-related proteins. A recent study identified a K76E mutation in PPIA in sporadic ALS patients, but its effects on protein function and structure remain unclear. In this study, we used biochemical and biophysical techniques to investigate the structural and functional consequences of the K76E mutation. Our results show that K76E significantly reduces enzyme activity without affecting structure, monodispersity, or substrate recognition. Significant effects of K76E mutation were identified by relaxation dispersion NMR experiments, showing that K76E disrupts key protein dynamics and alters an allosteric network essential for isomerase activity. Corroborated by theoretical kinetic analysis, this dynamics data, revealing the exchange process for K76E approximately one-tenth that of the wild type, explain the reduced cis-trans isomerase activity of the K76E mutant. These findings suggest that the pathogenic effect of K76E arises primarily from impaired protein dynamics rather than direct structural disruption. Our study provides new insights into the molecular mechanisms underlying ALS-associated mutations and their impact on protein function. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/653011v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@82f81corg.highwire.dtl.DTLVardef@6f0a04org.highwire.dtl.DTLVardef@1fabe12org.highwire.dtl.DTLVardef@d49d11_HPS_FORMAT_FIGEXP M_FIG For Table of Contents C_FIG

biochemistry↗

bioRxiv2025

Emergence of a Cell-Guided Multivalent Ligand of Enzymes on Cancer Cells Triggered by Click Reaction between Hetero Nano-Assemblies

Stimuli-responsive nanomaterials with multivalent ligands have attracted significant attention in the context of cancer chemotherapy and imaging. However, challenges remain such as non-selective stimuli response and/or existence of triggers in healthy regions in addition to the intrinsic heterogeneity of cancer that causes insufficient target recognition. In-spired by the expression of precise and diverse functions of biological machineries triggered by specific protein-protein complexation and conformational change, we report an artificial system where a bioorthogonal click reaction between hetero-nano-assemblies triggers their complexation and conformational changes, resulting in emergence of multivalent ligands for cancer-associated enzymes: carbonic anhydrase IX (CAIX). We also demonstrated that the multivalent ligands selectively inhibited the proliferation of cancer cells overexpressing CAIX under hypoxic conditions. Additionally, the click reaction between nano-assemblies in the presence of target cells provided a higher efficacy of the emerged multivalent ligands than that pre-formed in the absence of cells. Our study provides a basis for the development of multivalent ligands displaying adaptive binding interfaces for target cancer cells with high selectivity and affinity to thus potentially overcome tumor heterogeneity.

biochemistry↗

bioRxiv2024

Ca2+-driven PDIA6 phase separation to ensure proinsulin quality control

The endoplasmic reticulum (ER) plays key roles in protein quality control1,2 and dynamic Ca2+ storage3,4 in eukaryotic cells. However, the protein homeostasis (proteostasis) system that regulates these ER functions is still incompletely characterised. Previous study revealed the importance of oligomerization in the function PDIA1, an ER-resident disulfide isomerase and molecular chaperone, regulates oligomeric states in accordance with client folding5. This result suggests that at least some of the 20 members of other PDI family may undergo regulated self-assembly in order to optimally function. Here, we show that Ca2+ triggers the phase separation of PDIA6 into liquid-like condensates. In contrast to the condensation mechanism observed for proteins containing low-complexity domains, our results indicate that transient but specific electrostatic interactions occur between the first and the third folded thioredoxin-like domains of PDIA6. We further show that the Ca2+-driven condensation of PDIA6 recruits PDIA3 and proinsulin, thus increasing their local concentrations. This process results in the 30-fold enhancement of proinsulin folding and in the inhibition of proinsulin aggregation. Our findings shed light on a condensation-driven Ca2+-mediated proteostasis cascade in the ER by revealing how the efficiency of the protein folding process can be enhanced within quality control granules.

biochemistry↗

bioRxiv2024

Conserved loop of a phase modifier endows protein condensates with fluidity

Dipeptide repeats (DPRs) that are gene products from abnormal hexanucleotide repeat expansion in C9orf72 trigger amyotrophic lateral sclerosis (ALS) through unknown mechanism. This study highlights, importin Karyopherin{beta}2 (Kap{beta}2), which is responsible for nuclear transport and phase modification of RNA-binding proteins (RBPs), as a major DPR target. We demonstrate DPR accumulation in the nucleus via Kap{beta}2-mediated transport, which results in dose-dependent toxicity observed in nematode and yeast models. In vitro interaction studies exploiting chemical probe arrays and biophysical measurements reveal multivalent DPR binding to Kap{beta}2, including at the conserved acidic loop. Refractive index and fluorescence imaging coupled with biochemical assays unveiled that binding of excess DPRs to the acidic loop turns a phase modifier Kap{beta}2 into phase disrupter, resulting more condensed and viscous RBP condensates. Our findings provides molecular insight into C9orf72-ALS related to age and repeat expansion.

biochemistry↗

bioRxiv2024

Client recognition differences between PDI and ERp46 to guide oxidative folding

Endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) family members function not only as disulfide bond-catalysts but also as chaperones for the oxidative folding of client proteins. However, due to the scarcity of structural data, the client recognition mechanism remains poorly understood. We report the distinct recognition mechanisms for PDI/ERp46 proteins to recruit a reduced and denatured bovine pancreatic trypsin inhibitor (BPTI). NMR data demonstrated that PDI recognizes a broad region consisting of [~]30 amino acid residues in unfolded BPTI in order to function as a chaperone, while ERp46 interacts nonspecifically and weakly with unfolded BPTI to promiscuously and rapidly introduce disulfide bonds. These two distinct client recognition modes are likely important for the functional modulation of the disulfide-catalyst and chaperone against client proteins. Client recognition differences between PDI family proteins likely contribute to determining whether disulfide bonds are introduced into the client protein or aggregates are reduced to an unfolded state during the early folding step. Thus, the mammalian ER ensures concerted protein quality control through the differences in the client recognition modes of PDI family proteins, resulting in the production of large quantities of multiple-disulfide bonded proteins.

biochemistry↗

bioRxiv2023

Ultra-Rapid and Specific Gelation of Collagen Molecules for Transparent and Tough Gels by Transition Metal Complexation

Collagen is one of the main components of tumor stromal tissues with a high elastic modulus, but there have been limitations when attempting to fabricate a tough collagen gel with cells like a cancer stroma. Here, we demonstrate the rapid and specific formation of collagen gels with high transparency and high elastic modulus by transition metal complexation within minutes. Transition metal ions such as K2PtCl4 exhibited rapid gelation due to the formation of a cross-linked network of the collagen triple-helix by Pt- O and/or Pt-N bonds. Interestingly, type I to IV collagens showed rapid gelation, while other extracellular matrices and DNA did not exhibit this phenomenon, suggesting the importance of intermolecular interaction in a rigid triple-helix structure. Live imaging of colon cancer organoids in three-dimensional culture indicated a collective migration property with modulating high elastic modulus, suggesting activation for metastasis progress. This technology that facilitates deep-live observation and mechanical stiffness adjustment will be useful as a new class of scaffolds. TeaserTransparent collagen gels with tunable mechanical properties allow deep-live observation of cells cultured in a tough environment like our bodies.

bioengineering↗

bioRxiv2022

Fabrication of Molecular Blocks with High Responsiveness to the Cancer Microenvironment by Ursodeoxycholic Acid

In cancer therapy, drug delivery system (DDS) has been widely studied to achieve selective drug accumulation at the tumor site. However, DDS still has a major drawback in that it requires multi-step processes for intracellular delivery, resulting in low efficiency of drug delivery. To overcome this problem, we recently reported a molecular block (MB) that disrupts cancer cell membranes in the cancer microenvironment using deoxycholic acid (DCA). However, the MB showed considerable cytotoxicity even at neutral pH possibly due to the structural hydrophobic property of DCA. Herein, we focused on selecting the most suitable bile acid for an MB that possessed high responsiveness to the cancer microenvironment without cytotoxicity at neutral pH. Cell viabilities of the free bile acids such as DCA, chenodeoxycholic acid (CDCA), cholic acid (CA) and ursodeoxycholic acid (UDCA) were evaluated at neutral pH (pH = 7.4) and a cancer acidic environment (pH = 6.3 [~] 6.5). The half-maximal inhibition concentration (IC50) value of UDCA at pH = 7.4 showed an approximately 7.5-fold higher IC50 value than that at pH = 6.3, whereas the other bile acids yielded less than a 4-fold IC50 value difference between the same pHs. Biocompatible poly(vinyl alcohol) (PVA) was functionalized with UDCA (PVA-UDCA) for synthesis of higher responsiveness to the cancer microenvironment without cytotoxicity at neutral pH. Importantly, 56% pancreatic cancer cell death was observed at pH = 6.5, whereas only 10% was detected at neutral pH by the PVA-UDCA treatment. However, PVA-DCA indicated almost the same cancer cell death property independent of pH condition. These results suggest PVA-UDCA shows great potential for a new class of MB.

bioengineering↗

bioRxiv2022

Fabrication of a Polymeric Inhibitor of Proximal Metabolic Enzymes in Hypoxia for Synergistic Inhibition of Cancer Cell Proliferation, Survival and Migration

Since conventional molecular targeted drugs often result in side effect, the development of novel molecular targeted drugs with both high efficacy and selectivity are desired. Simultaneous inhibition of metabolically and spatiotemporally related proteins/enzymes is a promising strategy for improving therapeutic interventions in cancer treatment. Herein, we report a poly--L-glutamate-based polymer inhibitor that simultaneously targets proximal transmembrane enzymes under hypoxia, namely carbonic anhydrase IX (CAIX) and zinc-dependent metalloproteinases. A polymer incorporating two types of inhibitors more effectively inhibited the proliferation and migration of human breast cancer cells than a combination of two polymers functionalized exclusively with either inhibitor. Synergistic inhibition of cancer cells would occur owing to the hetero-multivalent interactions of the polymer with proximate enzymes on the cancer cell membrane. Our results highlight the potential of polymer-based cancer therapeutics. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/512469v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1ae7cdborg.highwire.dtl.DTLVardef@8734aeorg.highwire.dtl.DTLVardef@93a258org.highwire.dtl.DTLVardef@8770f0_HPS_FORMAT_FIGEXP M_FIG For Table of Contents only C_FIG

bioengineering↗

bioRxiv2021

Oxidative phase transition of heat shock factor-1

Heat shock factor 1 (Hsf1) was found as a central upregulator of molecular chaperones in stress adaptation, but it has recently been rediscovered as a major component of persistent nuclear stress bodies (nSBs). When the persistently stressed cells undergo apoptosis, the phase transition of nSBs from fluid to gel-like states is proposed to be an important event in switching the cell fate from survival to death. Nonetheless, how the phase separation and transition of nSBs are driven remain unanswered. In this study, we discovered that Hsf1 formed liquid-liquid phase separation droplets in vitro, causing the assembly of Hsf1 to drive nSBs formation. Under oxidative conditions, disulfide-bonded and oligomerized Hsf1 formed gel-like and more condensed droplets, confirmed through fluorescence recovery, refractive index imaging, and light scattering. Then, on the basis of our results, we proposed that Hsf1 undergoes oxidative phase transition by sensing redox conditions potentially to drive the cell fate decision by nSBs.

biochemistry↗

bioRxiv2020

Injectable prevascularized mature adipose tissues (iPAT) to achieve long-term survival in soft tissues regeneration

Soft tissue regeneration remains a challenge in reconstructive surgery. Current autologous fat implantations lead to high fat absorption ratios, while artificial implants can be associated with lymphoma occurrence. To overcome these limitations, our aim was to reproduce adipose tissue vasculature structure before implantation. Here, we developed injectable prevascularized adipose tissues (iPAT), using physiological collagen microfibers (CMF) mixed with human mature adipocytes, adipose-derived stem cells (ADSC) and human umbilical vein endothelial cells (HUVEC). Following murine subcutaneous implantation, higher cell survival (84{+/-}6% viability) and volume maintenance were shown after 3 months for the iPAT (up to twice heavier than the non-prevascularized balls). This higher survival can be explained by the greater amount of blood vessels (up to 1.6 folds increase), with balanced host anastomosis (51{+/-}1% of human/mouse lumens), also involving infiltration by the lymphatic and neural vasculature networks. These iPAT tissues allowed non-invasive soft tissue reconstruction for long-term outcomes, and the ability to cryopreserve them with maintained viability and functionality also enables a later reinjection usually required before reaching the final patient desired graft volume.

bioengineering↗

bioRxiv2020

A unique adhesive motif of protein disulfide isomerase P5 supports its function via dimerization

P5, also known as PDIA6, is a PDI-family member that plays an important role in the ER quality control. Herein, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. This motif is apparently similar to, but radically different from conventional leucine-zipper motifs, in that the former includes a periodic repeat of leucine or valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel -helices, unlike the latter of which the leucine residues appear every two helical turns on [~]30-residue parallel -helices at dimer interfaces. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as an aberrant protein that induces the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1 via reduction of intermolecular disulfide bonds and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and physiological function of P5.

biochemistry↗