Towards more realistic cultivated meat by rethinking bioengineering approaches.

Cultivated meat (CM) refers to edible lab-grown meat that incorporates cultivated animal cells. It has the potential to address some issues associated with real meat (RM) production, including the ethical and environmental impact of animal farming, and health concerns. Recently, various biomanufacturing methods have been developed to attempt to recreate realistic meat in the laboratory. We therefore overview recent achievements and challenges in the production of CM. We also discuss the issues that need to be addressed and suggest additional recommendations and potential criteria to help to bridge the gap between CM and RM from an engineering standpoint.

Fabrication of oxygen-releasing dextran microgels by droplet-based microfluidic method.

In the tissue engineering field, the supply of oxygen to three-dimensional (3D) tissues is an important aspect to avoid necrosis due to hypoxia. Although oxygen-releasing bulk materials containing calcium peroxide (CaO2, CP) have attracted much attention, micrometer-sized oxygen-releasing soft materials would be advantageous because of their highly controllable structures, which can be applied for cell scaffolds, injectable materials, and bioink components in 3D bioprinting. In this study, oxygen-releasing microgels were fabricated via a droplet-based microfluidic system. Homogeneous, monodisperse and stable oxygen-releasing microgels were obtained by photo-crosslinking of droplets composed of biocompatible dextran modified with methacrylate groups and CP nanoparticles as an oxygen source. We also used our microfluidic system for the in situ amorphous calcium carbonate (CaCO3, ACC) formation on the surface of CP nanoparticles to achieve the controlled release of oxygen from the microgel. Oxygen release from an ACC-CP microgel in a neutral cell culture medium was suppressed because incorporation of CP in the ACC suppressed the reaction with water. Strikingly, stimuli to dissolve ACC such as a weak acidic conditions triggered the oxygen release from microgels loaded with ACC-CP, as the dissolution of CaCO3 allows CP to react. Taken together, applications of this new class of biomaterials for tissue engineering are greatly anticipated. In addition, the developed microfluidic system can be used for a variety of oxygen-releasing microgels by changing the substrates of the hydrogel network.

Cationic polymer effect on brown adipogenic induction of dedifferentiated fat cells.

Obesity and its associated comorbidities place a substantial burden on public health. Given the considerable potential of brown adipose tissue in addressing metabolic disorders that contribute to dysregulation of the body's energy balance, this area is an intriguing avenue for research. This study aimed to assess the impact of various polymers, including collagen type I, fibronectin, laminin, gelatin, gellan gum, and poly-l-lysine (PLL), on the in vitro brown adipogenic differentiation of dedifferentiated fat cells within a fibrin gel matrix. The findings, obtained through RT-qPCR, immunofluorescent imaging, ELISA assay, and mitochondria assessment, revealed that PLL exhibited a significant browning-inducing effect. Compared to fibrin-only brown-like drops after two weeks of incubation in brown adipogenic medium, PLL showed 6 (±3) times higher UCP1 gene expression, 5 (±2) times higher UCP1 concentration by ELISA assay, and 2 (±1) times higher mitochondrial content. This effect can be attributed to PLL's electrostatic properties, which potentially facilitate the cellular uptake of crucial brown adipogenic inducers such as the thyroid hormone, triiodothyronine (T3), and insulin from the induction medium.

Promoting biological similarity by collagen microfibers in 3D colorectal cancer-stromal tissue: Replicating mechanical properties and cancer stem cell markers.

The extracellular matrix (ECM) of cancer tissues is rich in dense collagen, contributing to the stiffening of these tissues. Increased stiffness has been reported to promote cancer cell proliferation, invasion, metastasis, and prevent drug delivery. Replicating the structure and mechanical properties of cancer tissue in vitro is essential for developing cancer treatment drugs that target these properties. In this study, we recreated specific characteristics of cancer tissue, such as collagen density and high elastic modulus, using a colorectal cancer cell line as a model. Using our original material, collagen microfibers (CMFs), and a constructed three-dimensional (3D) cancer-stromal tissue model, we successfully reproduced an ECM highly similar to in vivo conditions. Furthermore, our research demonstrated that cancer stem cell markers expressed in the 3D cancer-stromal tissue model more closely mimic in vivo conditions than traditional two-dimensional cell cultures. We also found that CMFs might affect an impact on how cancer cells express these markers. Our 3D CMF-based model holds promise for enhancing our understanding of colorectal cancer and advancing therapeutic approaches. STATEMENT OF SIGNIFICANCE: Reproducing the collagen content and stiffness of cancer tissue is crucial in comprehending the properties of cancer and advancing anticancer drug development. Nonetheless, the use of collagen as a scaffold material has posed challenges due to its poor solubility, hindering the replication of a cancer microenvironment. In this study, we have successfully recreated cancer tissue-specific characteristics such as collagen density, stiffness, and the expression of cancer stem cell markers in three-dimensional (3D) colorectal cancer stromal tissue, utilizing a proprietary material known as collagen microfiber (CMF). CMF proves to be an ideal scaffold material for replicating cancer stromal tissue, and these 3D tissues constructed with CMFs hold promise in contributing to our understanding of cancer and the development of therapeutic drugs.

Polyelectrolyte nanofilms on cell surface can induce brown adipogenic differentiation of DFATs.

Obesity and its related health issues significantly burden public health systems. Brown adipose tissue holds promise for addressing metabolic disorders and balancing the body's energy, making it a key research focus. Stimulating brown adipogenesis from stem cells could advance regenerative medicine and healthcare. In our previous research, we discovered that poly-l-lysine (PLL) significantly stimulates brown adipogenesis in three-dimensional differentiation of dedifferentiated fat cells (DFATs) within fibrin gels. In this study, we evaluated polyelectrolyte (PE) nanofilms made of PLL and dextran sulfate, applied directly to DFAT surfaces to improve brown adipogenic differentiation through an innovative approach. This approach involved coating the DFAT surfaces with PE nanofilms, forming a multilayer structure that not only provided a supportive matrix but also facilitated the adsorption of essential molecules like T3 and insulin for brown adipogenesis. DFATs coated with three PE layers and encapsulated in fibrin gel showed a significant increase in the adipogenic marker UCP1 gene expression and content. This PLL-based PE nanofilm coating on DFAT surfaces can be a novel and crucial technology for promoting brown adipogenesis in regenerative medicine and healthcare.

Fabrication of Fully Positively Charged Layer-by-Layer Polyelectrolyte Nanofilms with pH-Dependent Swelling Properties.

Nanofilms fabricated by layer-by-layer (LbL) assembly from polyelectrolytes (PEs) are important materials for various applications. However, PE films cannot retain the charges along the polymer chains during fabrication, resulting in a low charge density. In this study, the preparation of LbL nanofilms with preserved positive charges via a controllable and efficient approach was achieved. To fabricate fully positively charged (FPC) LbL nanofilms, a polycation, poly-l-lysine, was partially grafted with azide and alkyne groups. Through copper-catalyzed azide-alkyne cycloaddition and the LbL procedure, nanofilms were fabricated with all of the individual layers covalently bonded, improving the pH stability of the nanofilms. Because the resulting nanofilms had a high charge density with positive charges both inside and on the surface, they showed unique pH-dependent swelling properties and adsorption of negatively charged molecules compared with those of traditional polyelectrolyte LbL nanofilms. This kind of FPC nanofilm has great potential for use in sensors, diagnostics, and filter nanomaterials in the biomedical and environmental fields.

In situ fabrication of fully negatively-charged layer-by-layer nanofilms on a living cell surface.

Fully negatively-charged (FNC) layer-by-layer nanofilms were successfully assembled on a living cell surface for the first time using only poly(acrylic acid) (PAA) by introducing strain-promoted click chemistry to crosslink PAA layers. The resulting nanofilms retained their negative charges and showed higher adsorption of positively-charged molecules without affecting the cell viability.

Cellular memory function from 3D to 2D: Three-dimensional high density collagen microfiber cultures induce their resistance to reactive oxygen species.

Cell properties generally change when the culture condition is changed. However, mesenchymal stem cells cultured on a hard material surface maintain their differentiation characteristics even after being cultured on a soft material surface. This phenomenon suggests the possibility of a cell culture material to memorize stem cell function even in changing cell culture conditions. However, there are no reports about cell memory function in three-dimensional (3D) culture. In this study, colon cancer cells were cultured with collagen microfibers (CMF) in 3D to evaluate their resistance to reactive oxygen species (ROS) in comparison with a monolayer (2D) culture condition and to understand the effect of 3D-culture on cell memory function. The ratio of ROS-negative cancer cells in 3D culture increased with increasing amounts of CMF and the highest amount of CMF was revealed to be 35-fold higher than that of the 2D condition. The ROS-negative cells ratio was maintained for 7 days after re-seeding in a 2D culture condition, suggesting a 3D-memory function of ROS resistance. The findings of this study will open up new opportunities for 3D culture to induce cell memory function.

Construction of Tough Hydrogel Cross-Linked via Ionic Interaction by Protection Effect of Hydrophobic Domains.

Most hydrogels have poor mechanical properties, severely limiting their potential applications, and numerous approaches have been introduced to fabricate more robust and durable examples. However, these systems consist of nonbiodegradable polymers which limit their application in tissue engineering. Herein, we focus on the fabrication and investigate the influence of hydrophobic segments on ionic cross-linking properties for the construction of a tough, biodegradable hydrogel. A biodegradable, poly(γ-glutamic acid) polymer conjugated with a hydrophobic amino acid, l-phenylalanine ethyl ester (Phe), together with an ionic cross-linking group, alendronic acid (Aln) resulting in γ-PGA-Aln-Phe, was initially synthesized. Rheological assessments through time sweep oscillation testing revealed that the presence of hydrophobic domains accelerated gelation. Comparing gels with and without hydrophobic domains, the compressive strength of γ-PGA-Aln-Phe was found to be six times higher and exhibited longer stability properties in ethylenediaminetetraacetic acid solution, lasting for up to a month. Significantly, the contribution of the hydrophobic domains to the mechanical strength and stability of ionic cross-linking properties of the gel was found to be the dominant factor for the fabrication of a tough hydrogel. As a result, this study provides a new strategy for mechanical enhancement and preserves ionic cross-linked sites by the addition of hydrophobic domains. The development of tough, biodegradable hydrogels reported herein will open up new possibilities for applications in the field of biomaterials.

Fluorophore-Probed Curdlan Polysaccharide Chemosensor: "Turn-On" Oligosaccharide Sensing in Aqueous Media.

The ability to sense saccharides in aqueous media has attracted much attention in multidisciplinary sciences because the detection of ultrahigh concentrations of sugar chains associated with serious diseases could lead to further health promotion. However, there are notable challenges. In this study, a rhodamine-modified Curdlan (Rhod-Cur) chemosensor was synthesized that exhibited distinctive fluorescence "turn-on" responses. Rhod-Cur exhibited simultaneous sensitive and selective sensing of clinically useful acarbose with a good limit of detection (5 µM) from among those of the saccharides examined. The (chir)optical properties of Rhod-Cur were elucidated using UV/vis, fluorescence, excitation, and circular dichroism spectroscopies; lifetime measurements and morphological studies using atomic force and confocal laser scanning microscopy and dynamic light scattering techniques revealed that the fluorescence "turn-on" behavior originates from globule-to-coaggregation conversion upon insertion of the oligosaccharides in the dynamic Cur backbone.

In vitro throughput screening of anticancer drugs using patient-derived cell lines cultured on vascularized three-dimensional stromal tissues.

The development of high-throughput anticancer drug screening methods using patient-derived cancer cell (PDC) lines that maintain their original characteristics in an in vitro three-dimensional (3D) culture system poses a significant challenge to achieving personalized cancer medicine. Because stromal tissue plays a critical role in the composition and maintenance of the cancer microenvironment, in vitro 3D-culture using reconstructed stromal tissues has attracted considerable attention. Here, a simple and unique in vitro 3D-culture method using heparin and collagen together with fibroblasts and endothelial cells to fabricate vascularized 3D-stromal tissues for in vitro culture of PDCs is reported. Whereas co-treatment with bevacizumab, a monoclonal antibody against vascular endothelial growth factor, and 5-fluorouracil significantly reduced the survival rate of 3D-cultured PDCs to 30%, separate addition of each drug did not induce comparable strong cytotoxicity, suggesting the possibility of evaluating the combined effect of anticancer drugs and angiogenesis inhibitors. Surprisingly, drug evaluation using eight PDC lines with the 3D-culture method resulted in a drug efficacy concordance rate of 75% with clinical outcomes. The model is expected to be applicable to in vitro throughput drug screening for the development of personalized cancer medicine. STATEMENT OF SIGNIFICANCE: To replicate the cancer microenvironment, we constructed a cancer-stromal tissue model in which cancer cells are placed above and inside stromal tissue with vascular network structures derived from vascular endothelial cells in fibroblast tissue using CAViTs method. Using this method, we were able to reproduce the invasion and metastasis processes of cancer cells observed in vivo. Using patient-derived cancer cells, we assessed the possibility of evaluating the combined effect with an angiogenesis inhibitor. Further, primary cancer cells also grew on the stromal tissues with the normal medium. These data suggest that the model may be useful for new in vitro drug screening and personalized cancer medicine.

Biofabrication of engineered blood vessels for biomedical applications.

To successfully engineer large-sized tissues, establishing vascular structures is essential for providing oxygen, nutrients, growth factors and cells to prevent necrosis at the core of the tissue. The diameter scale of the biofabricated vasculatures should range from 100 to 1,000 µm to support the mm-size tissue while being controllably aligned and spaced within the diffusion limit of oxygen. In this review, insights regarding biofabrication considerations and techniques for engineered blood vessels will be presented. Initially, polymers of natural and synthetic origins can be selected, modified, and combined with each other to support maturation of vascular tissue while also being biocompatible. After they are shaped into scaffold structures by different fabrication techniques, surface properties such as physical topography, stiffness, and surface chemistry play a major role in the endothelialization process after transplantation. Furthermore, biological cues such as growth factors (GFs) and endothelial cells (ECs) can be incorporated into the fabricated structures. As variously reported, fabrication techniques, especially 3D printing by extrusion and 3D printing by photopolymerization, allow the construction of vessels at a high resolution with diameters in the desired range. Strategies to fabricate of stable tubular structures with defined channels will also be discussed. This paper provides an overview of the many advances in blood vessel engineering and combinations of different fabrication techniques up to the present time.

This review covers several aspects and advancements of engineered blood vessel biofabrication, which are essential for establishment of large-sized tissues in different areas of biomedical applications.

Improving stability of human three dimensional skin equivalents using plasma surface treatment.

In developing three-dimensional (3D) human skin equivalents (HSEs), preventing dermis and epidermis layer distortion due to the contraction of hydrogels by fibroblasts is a challenging issue. Previously, a fabrication method of HSEs was tested using a modified solid scaffold or a hydrogel matrix in combination with the natural polymer coated onto the tissue culture surface, but the obtained HSEs exhibited skin layer contraction and loss of the skin integrity and barrier functions. In this study, we investigated the method of HSE fabrication that enhances the stability of the skin model by using surface plasma treatment. The results showed that plasma treatment of the tissue culture surface prevented dermal layer shrinkage of HSEs, in contrast to the HSE fabrication using fibronectin coating. The HSEs from plasma-treated surface showed significantly higher transepithelial electrical resistance compared to the fibronectin-coated model. They also expressed markers of epidermal differentiation (keratin 10, keratin 14 and loricrin), epidermal tight junctions (claudin 1 and zonula occludens-1), and extracellular matrix proteins (collagen IV), and exhibited morphological characteristics of the primary human skins. Taken together, the use of plasma surface treatment significantly improves the stability of 3D HSEs with well-defined dermis and epidermis layers and enhanced skin integrity and the barrier functions.

Technology for the formation of engineered microvascular network models and their biomedical applications.

Tissue engineering and regenerative medicine have made great progress in recent decades, as the fields of bioengineering, materials science, and stem cell biology have converged, allowing tissue engineers to replicate the structure and function of various levels of the vascular tree. Nonetheless, the lack of a fully functional vascular system to efficiently supply oxygen and nutrients has hindered the clinical application of bioengineered tissues for transplantation. To investigate vascular biology, drug transport, disease progression, and vascularization of engineered tissues for regenerative medicine, we have analyzed different approaches for designing microvascular networks to create models. This review discusses recent advances in the field of microvascular tissue engineering, explores potential future challenges, and offers methodological recommendations.

Controlled Release of Oxygen from Calcium Peroxide in a Weak Acidic Condition by Stabilized Amorphous Calcium Carbonate Nanocoating for Biomedical Applications.

Calcium peroxide (CaO2) has recently attracted much attention as an oxygen-releasing biomaterial for tissue engineering. CaO2 has also been used in cancer therapies, such as photodynamic therapy. However, the uncontrollability of oxygen release after immersion in water is a challenge. Furthermore, the nanoscale surface chemistry of CaO2 on the oxygen release properties under cell culture conditions has not been taken into account in these applications. Herein, we report the stabilized amorphous calcium carbonate (ACC) nanocoating on CaO2 in a cell culture medium, which suppressed the reaction between CaO2 and water. Stabilized ACC was produced by the reaction between calcium hydroxide (Ca(OH)2) derived from CaO2 and sodium hydrogen carbonate (NaHCO3) including sodium dihydrogen phosphate (NaH2PO4) in a cell culture medium. In contrast, surface modification of CaO2 by calcium carbonate crystals was difficult due to the crystallization process via dissolution-reprecipitation. Strikingly, ACC-CaO2 showed pH-dependent oxygen release in a cell culture medium probably because of the dissolution of ACC under weak acidic condition. Since the environments in ischemic tissue and cancer are weakly acidic, our findings should be important for understanding and designing properties related to biomaterials and drugs using CaO2.

Analysis of Homo- and Heterotriple Helix Formation of Collagen Model Peptides and Evaluation of Their Stability in a Biological Environment.

Self-assembled materials have attracted attention and have been extensively studied because the reversibility of noncovalent interactions allows them to possess various properties, such as stimulus responsiveness and self-healing. Collagen model peptides have an amino acid sequence characteristic of the triple helix region of collagen and exhibit repeatable triple helix formation. Many studies of their applications have used homotrimers, and although some studies on heterotrimers have been reported, few have clarified the details. If the characteristics of heterotrimers can be revealed, they are expected to be applied as new self-assembled materials. In this study, we analyzed the detailed self-assembling properties of hetero- and homohelices formed by (proline-proline-glycine)10 (PPG)10 and (proline-hydroxyproline-glycine)10 (POG)10 to evaluate the potential of the helices for biomedical application. Fluorescein isothiocyanate-labeled (PPG)10 (F(PPG)10) and (POG)10 (F(POG)10) were synthesized to analyze the heterotriple helix formation using concentration quenching based on triple helix formation. When (PPG)10 was added to F(POG)10, the fluorescence intensity did not reach a plateau, while the fluorescence intensity reached about 100% in the other pairs such as (POG)10-F(POG)10, (PPG)10-F(PPG)10, and (POG)10-F(PPG)10. The critical triple helix formation concentration was 7 µM for the heterotrimer prepared under 1:2 mixing conditions of (PPG)10 and (POG)10, 320 µM for [(PPG)10]3, and 4 µM for [(POG)10]3, indicating that the triple helix formation concentration of the heterotrimer is almost half that of [(POG)10]3 but 45 times higher than [(PPG)10]3. Furthermore, the heterotrimer formed at 37 °C was stable after 5 days, which was the same as [(POG)10]3. These results suggest that heterotrimers have different association properties from homotrimers and are expected to be applied in nanotechnology and biomaterials as new self-assembled materials.

Recent Developments in Layer-by-Layer Assembly for Drug Delivery and Tissue Engineering Applications.

Surfaces with biological functionalities are of great interest for biomaterials, tissue engineering, biophysics, and for controlling biological processes. The layer-by-layer (LbL) assembly is a highly versatile methodology introduced 30 years ago, which consists of assembling complementary polyelectrolytes or biomolecules in a stepwise manner to form thin self-assembled films. In view of its simplicity, compatibility with biological molecules, and adaptability to any kind of supporting material carrier, this technology has undergone major developments over the past decades. Specific applications have emerged in different biomedical fields owing to the possibility to load or immobilize biomolecules with preserved bioactivity, to use an extremely broad range of biomolecules and supporting carriers, and to modify the film's mechanical properties via crosslinking. In this review, the focus is on the recent developments regarding LbL films formed as 2D or 3D objects for applications in drug delivery and tissue engineering. Possible applications in the fields of vaccinology, 3D biomimetic tissue models, as well as bone and cardiovascular tissue engineering are highlighted. In addition, the most recent technological developments in the field of film construction, such as high-content liquid handling or machine learning, which are expected to open new perspectives in the future developments of LbL, are presented.

ECM proteins and cationic polymers coating promote dedifferentiation of patient-derived mature adipocytes to stem cells.

Reprogramming of mature adipocytes is an attractive research area due to the plasticity of these cells. Mature adipocytes can be reprogrammed in vitro, transforming them into dedifferentiated fat cells (DFATs), which are considered a new type of stem cell, and thereby have a high potential for use in tissue engineering and regenerative medicine. However, there are still no reports or findings on in vitro controlling the dedifferentiation. Although ceiling culture performed in related studies is a relatively simple method, its yield is low and does not allow manipulation of mature adipocytes to increase or decrease the dedifferentiation. In this study, to understand the role of physicochemical surface effects on the dedifferentiation of patient-derived mature adipocytes, the surfaces of cell culture flasks were coated with extracellular matrix, basement membrane proteins, and cationic/anionic polymers. Extracellular matrix such as fibronectin and collagen type I, and basement membrane proteins such as collagen type IV and laminin strongly promoted dedifferentiation of mature adipocytes, with laminin showing the highest effect with a DFAT ratio of 2.98 (±0.84). Interestingly, cationic polymers also showed a high dedifferentiation effect, but anionic polymers did not, and poly(diallyl dimethylammonium chloride) showed the highest DFAT ratio of 2.27 (±2.8) among the cationic polymers. Protein assay results revealed that serum proteins were strongly adsorbed on the surfaces of the cationic polymer coating, including inducing high mature adipocyte adhesion. This study demonstrates for the first time the possibility of regulating the transformation of mature adipocytes to DFAT stem cells by controlling the physicochemical properties of the surface of conventional cell culture flasks.

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

Collagen is the most abundant protein in the human body and one of the main components of stromal tissues in tumors which have a high elastic modulus of over 50 kPa. Although collagen has been widely used as a cell culture scaffold for cancer cells, there have been limitations when attempting to fabricate a tough collagen gel with cells like a cancer stroma. Here, rapid gelation of a collagen solution within a few minutes by transition metal complexation is demonstrated. Type I collagen solution at neutral pH shows rapid gelation with a transparency of 81% and a high modulus of 1,781 kPa by mixing with K2 PtCl4 solution within 3 min. Other transition metal ions also show the same rapid gelation, but not basic metal ions. Interestingly, although type I to IV collagen molecules show rapid gelation, other extracellular matrices  do not exhibit this phenomenon. Live imaging of colon cancer organoids in 3D culture indicates a collective migration property with modulating high elastic modulus, suggesting activation for metastasis progress. This technology will be useful as a new class of 3D culture for cells and organoids due to its facility for deep-live observation and mechanical stiffness adjustment.

Control of blood capillary networks and holes in blood-brain barrier models by regulating elastic modulus of scaffolds.

The blood-brain barrier (BBB) is a type of capillary network characterized by a highly selective barrier, which restricts the transport of substances between the blood and nervous system. Numerous in vitro models of the BBB have been developed for drug testing, but a BBB model with controllable capillary structures remains a major challenge. In this study, we report for the first time a unique method of controlling the blood capillary networks and characteristic holes formation in a BBB model by varying the elastic modulus of a three-dimensional scaffold. The characteristic hole structures are formed by the migration of endothelial cells from the model surface to the interior, which have functions of connecting the model interior to the external environment. The hole depth increased, as the elastic modulus of the fibrin gel scaffold increased, and the internal capillary network length increased with decreasing elastic modulus. Besides, internal astrocytes and pericytes were also found to be important for inducing hole formation from the model surface. Furthermore, RNA sequencing indicated up-regulated genes related to matrix metalloproteinases and angiogenesis, suggesting a relationship between enzymatic degradation of the scaffolds and hole formation. The findings of this study introduce a new method of fabricating complex BBB models for drug assessment.