Biology subjects

Kamei, K.-i.

Publications and source records attributed to Kamei, K.-i..

At least 19 recordsLinked to original sources

bioRxiv2025

High-Purity Production of Endothelial Cells from Human Pluripotent Stem Cells

Endothelial cells (ECs) are essential for vascular network formation and tissue homeostasis, yet the fields of tissue engineering and vascularized organoid generation still relies heavily on human umbilical vein ECs (HUVECs), which are venous, allogeneic, and difficult to mature fully. Human pluripotent stem cells (hPSCs) offer an autologous, developmentally flexible alternative, but most differentiation protocols require fluorescence-activated cell sorting, limiting scalability. Here we present a streamlined method that produces highly pure ECs directly from human embryonic stem cells (hESCs) without cell sorting. Extending Wnt activation with CHIR99021 to three days maximizes mesoderm induction, and brief Notch blockade with DAPT during specification suppresses smooth-muscle commitment. The result is over 90 % CD31+ CD144+ ECs that display classic cobblestone morphology, robust DiI-acetylated LDL uptake, and capillary-like sprouting comparable to HUVECs. Bulk RNA barcoding and sequencing segregates the hESC-derived ECs from both HUVECs and undifferentiated hESCs and uncovers an arterial-skewed transcriptome: NOTCH1, DLL4, and CXCR4 are up-regulated, whereas venous markers (EPHB4, NRP2) are reduced. Enrichment of Notch-responsive pathways further supports an arterial-like identity. Although several adult functional genes (e.g., vWF, NOS3) are expressed at lower levels than in HUVECs, the protocol delivers a scalable source of developmentally relevant ECs ideal for vascularizing organoids derived from the same hPSCs and for future applications in drug screening, disease modeling, and cell-based vascular therapies.

bioengineering↗

bioRxiv2024

Dual-targeted nanoparticulate drug delivery systems for enhancing triple-negative breast cancer treatment

The efficacy of DNA-damaging agents, such as the topoisomerase I inhibitor SN38, is often compromised by the robust DNA repair mechanisms in tumor cells, notably homologous recombination (HR) repair. Addressing this challenge, we introduce a novel nano-strategy utilizing binary tumor-killing mechanisms to enhance the therapeutic impact of DNA damage and mitochondrial dysfunction in cancer treatment. Our approach employs a synergistic drug pair comprising SN38 and the BET inhibitor JQ-1. We synthesized two prodrugs by conjugating linoleic acid (LA) to SN38 and JQ-1 via a cinnamaldehyde thioacetal (CT) bond, facilitating co-delivery. These prodrugs co-assemble into a nanostructure, referred to as SJNP, in an optimal synergistic ratio. SJNP was validated for its efficacy at both the cellular and tissue levels, where it primarily disrupts the transcription factor protein BRD4. This disruption leads to downregulation of BRCA1 and RAD51, impairing the HR process and exacerbating DNA damage. Additionally, SJNP releases cinnamaldehyde (CA) upon CT linkage cleavage, elevating intracellular ROS levels in a self-amplifying manner and inducing ROS-mediated mitochondrial dysfunction. Our results indicate that SJNP effectively targets murine triple-negative breast cancer (TNBC) with minimal adverse toxicity, showcasing its potential as a formidable opponent in the fight against cancer.

cancer biology↗

bioRxiv2024

Self-amplifying ROS-sensitive SN38 Dimeric Prodrug nanoparticles for Combined Chemotherapy and Ferroptosis in Cancer Treatment

Chemotherapy drugs are often limited by their own clinical shortcomings and serious adverse consequences. To solve these problems, we developed a self-amplifying reactive oxygen species (ROS)-sensitive dimeric prodrug nanoparticles, namely SN38-CA@FC NPs for tumor treatment. A ROS-sensitive 7-ethyl-10-hydroxycamptothecin (SN38) prodrug (SN38-CA) was synthesized by a thioacetal linker between SN38 and the ROS generator cinnamaldehyde (CA). The subsequent release of SN38 inflicts DNA damage, exerting chemotherapeutic effects, while the liberated CA intensifies ferroptosis through Fenton reaction-mediated disruption of the redox balance. This dual-action strategy not only leverages chemotherapy but also induces ferroptosis, establishing a synergistic therapeutic paradigm. The system is uniquely characterized by a positive feedback loop where ROS instigates the release of SN38/CA, which in turn promotes further ROS production. In experimental evaluations, this combination therapy exhibited potent antitumor activity against both A549 and LLC cancer cell lines, as well as in xenograft LLC-bearing C57BL/6 mouse models. Collectively, our findings introduce a transformative Nano-Drug Delivery System (NDDS) that holds significant promise for advancing cancer chemotherapy and ferroptosis-based therapies.

cancer biology↗

bioRxiv2023

SANTA FE OXA: Self-assembled oxaliplatin nanomicelle for enhanced cascade cancer chemotherapy via self-sensitized ferroptosis

The clinical utility of chemotherapy is often compromised by its limited efficacy and significant side effects. Addressing these concerns, we develop a self-assembled nanomicelle, namely SANTA FE OXA, which is composed of hyaluronic acid (HA) conjugated with ferrocene methanol (FC-OH), oxaliplatin prodrug (OXA({square})) and glycol-coupled linoleic acid (EG-LA). Targeted delivery is achieved as HA binds to the CD44 receptors that are overexpressed on tumor cells, facilitating drug uptake. Once internalized, hyaluronidase (HAase) catalyzes the digestions of the SANTA FE OXA, releasing FC and reducing OXA({square}) into an active form. Active OXA induces DNA damage, while simultaneously promoting intracellular hydrogen peroxide levels via cascade reactions. In parallel, FC disrupts the redox balance within tumor cells, inducing ferroptosis. The synergistic combination of cascade chemotherapy and self-sensitized ferroptosis therapy has demonstrated remarkable anti-cancer efficacy in both in vitro and in vivo models. Moreover, this SANTA FE OXA significantly mitigates the systemic toxicity commonly associated with platinum-based chemotherapeutics. Our findings suggest a compelling advancement in nanomedicine for enhanced cascade cancer therapy.

cancer biology↗

bioRxiv2023

Hybrid Adipocyte-Derived Exosome Nano Platform for Potent Chemo-Phototherapy in Targeted Hepatocellular Carcinoma

The high prevalence and severity of hepatocellular carcinoma (HCC) present a significant menace to human health. Despite the significant advancements in nanotechnology-driven antineoplastic agents, there remains a conspicuous gap in the development of targeted chemotherapeutic agents specifically designed for HCC. Consequently, there is an urgent need to explore potent drug delivery systems for effective HCC treatment. Here we have exploited the interplay between HCC and adipocyte to engineer a hybrid adipocyte-derived exosome platform, serving as a versatile vehicle to specifically target HCC and exsert potent antitumor effect. A lipid-like prodrug of docetaxel (DSTG) with a reactive oxygen species (ROS)-cleavable linker, and a lipid-conjugated photosensitizer (PPLA), spontaneously co-assemble into nanoparticles, functioning as the lipid cores of the hybrid exosomes (HEMPs and NEMPs). These nanoparticles are further encapsuled within adipocyte-derived exosome membranes, enhancing their affinity towards HCC cancer cells. As such, cancer cell uptakes of hybrid exosomes are increased up to 5.73-fold compared to lipid core nanoparticles. Our in vitro and in vivo experiments have demonstrated that HEMPs not only enhance the bioactivity of the prodrug and extend its circulation in the bloodstream but also effectively inhibit tumor growth by selectively targeting hepatocellular carcinoma tumor cells. Self-facilitated synergistic drug release subsequently promoting antitumor efficacy, inducing significant inhibition of tumor growth with minimal side effects. Our findings herald a promising direction for the development of targeted HCC therapeutics. Graphical abstractAn adipocyte-derived exosome nanoplatform has been developed for the combined therapy of both chemotherapy and photodynamic therapy. The hybrid adipocyte-derived exosome nanoplatform integrates the advantage of targeted co-delivery and combination therapy, which can stimulate the release of drugs under the tumor microenvironment, effectively enhanced targeted co-delivery in hepatocellular carcinoma cells, prolonged blood circulation time and improve the therapeutic effect of hepatocellular carcinoma tumors model. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/567027v2_ufig1.gif" ALT="Figure 1"> View larger version (83K): org.highwire.dtl.DTLVardef@195f031org.highwire.dtl.DTLVardef@cc2b02org.highwire.dtl.DTLVardef@1bbdd9org.highwire.dtl.DTLVardef@14d147d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Modeling dry eye with an air-liquid interface in corneal epithelium-on-a-chip

Dry eye syndrome (DES) is a complex ocular condition characterized by an unstable tear film and inadequate tear production, leading to tissue damage. Despite its common occurrence, there is currently no comprehensive in vitro model that accurately reproduce the cellular characteristics of DES. Here we modified a corneal epithelium-on-a-chip (CEpOC) model to recapitulate DES by subjecting HCE-T human corneal epithelial cells to an air-liquid (AL) interface stimulus. We then assessed the effects of AL stimulation both in the presence and absence of diclofenac (DCF). Transcriptomic analysis revealed distinct gene expression changes in response to AL and AL_DCF, affecting pathways related to development, epithelial structure, inflammation, and extracellular matrix remodeling. Both treatments upregulated PIEZO2, linked to corneal damage signaling, while downregulating OCLN, involved in cell-cell junctions. They increased the expression of inflammatory genes (e.g., IL6) and reduced mucin production genes (e.g., MUC16), reflecting dry eye characteristics. TGFB1, crucial for corneal wound healing, was slightly downregulated in AL_DCF, potentially affecting wound healing processes rather than reducing inflammation by DCF. Metabolomic analysis showed increased secretion of metabolites associated with cell damage and inflammation (e.g., methyl-2-oxovaleric acid, 3-methyl-2-oxobutanoic acid, lauroyl-carnitine) in response to AL and even more with AL_DCF, indicating a shift in cellular metabolism. This study showcases the utilization of AL stimulus within the CEpOC as a comprehensive approach to faithfully reproduce the cellular characteristics of DES.

bioengineering↗

bioRxiv2023

Brain organoid-on-a-chip to create multiple domains in forebrain organoids

Brain organoids are three-dimensionally reconstructed brain tissue derived from pluripotent stem cells in vitro. 3D tissue cultures have opened new avenues for exploring development and disease modeling. However, some physiological conditions, including signaling gradients in 3D cultures, have not yet been easily achieved. Here, we introduce Brain Organoid-on-a-Chip platforms that generate signaling gradients that in turn enable the induction of topographic forebrain organoids. This creates a more continuous spectrum of brain regions and provides a more complete mimic of the human brain for evaluating neurodevelopment and disease in unprecedented detail.

bioengineering↗

bioRxiv2023

Cyclic mechanical stretching stimuli promotes angiocrine signals during in vitro liver bud formation from human pluripotent stem cells

Liver organoids derived from human pluripotent stem cells (hPSCs) allow elucidation of liver development and have great potential for drug discovery. However, current methods for generating liver organoids using biochemical substances do not realize the vascular network of the liver lobule, due to the lack of knowledge of the role of in vivo mechanical environments during liver development. Here, we investigate the role of cyclic mechanical stretch (cMS) to angiocrine signals of hepatoblasts (HBs) and endothelial progenitor cells (EPCs) using an organ-on-a-chip platform to emulate in vivo-like mechanical environments and hPSCs to recapitulate hepatic differentiation. RNA sequencing revealed that the expression of angiocrine signal genes, such as HGF and matrix metallopeptidase 9 (MMP9), was increased by cMS in co-cultured HBs and EPCs. The secretion of HGF and MMP9 increased by 3.23-folds and 3.72-folds with cMS in the co-cultured HBs and EPCs but was not increased by cMS in the mono-cultured HBs and EPCs. Immunofluorescence micrographs with anti-KRT19, HGF, and MMP9 antibodies also revealed that cMS increased HGF and MMP9 expression when HBs and EPCs were co-cultured. cMS increased HGF and MMP9 expression and secretion when HBs and EPCs were co-cultured. Our findings provide new insights into the mechanical factors involved in the vascular network of human liver bud formation and liver organoid generation.

bioengineering↗

bioRxiv2022

Human blastoid from primed human embryonic stem cells

Artificial human blastoids are used to investigate early embryo development, pregnancy failures, and birth deficiencies, previously impossible without human embryos. Recent methods generating blastoids used human naive pluripotent stem cells, which are prone to genomic instability during in vitro culturing. Here, we introduce a simple, robust, and scalable method for generating human blastoids from more stable, primed human pluripotent stem cells (hPSC). Using a non-cell-adhesive hydrogel, hPSC aggregates formed an asymmetric blastoid structure with a cellular distribution similar to that of a human blastocyst, with in vitro implantation capability. Single-cell RNA-seq followed by cellular trajectory analysis revealed that hydrogel promoted differentiation to tri-lineage cells associated with a blastocyst. This model will allow studies on the underlying mechanisms of human pre- and postimplantation processes, consider elaborating on the potential implications of the model on assistive reproductive technology. One Sentence SummaryNo more than 125 characters and spaces.

bioengineering↗

bioRxiv2022

Gut-liver-axis microphysiological system for studying cellular fluidic shear stress and inter-tissue interaction

Gut-liver-axis (GLA) is a fundamental interaction between the gut and liver for maintaining human health. To clarify the physiological and pathological roles of GLA in the human body, a GLA microphysiological system (GLA-MPS) holds great potential. However, in current GLA-MPS, the importance of a physiologically relevant flow for gut and liver cells cultivation is not fully addressed. In addition, the integration of individual organ perfusion, circulation flow, and organ tissue functions in a single device has not been achieved. Here, we introduce a GLA-MPS by integrating two cell culture chambers with individually applied perfusion flows and a circulation channel with an on-chip pneumatic micropump under cell culture chambers via a porous membrane for interconnecting them. We analyzed the fluid shear stress (FSS) with computational fluid dynamics simulations and confirmed that the physiologically relevant FSS (i.e., 8 x 10-3 and 1.2 x 10-7 dyne cm-2) could be applied for the gut (Caco-2) and liver (HepG2) cells, respectively. Under physiologically relevant flow, the Caco-2 and HepG2 cells in the GLA-MPS maintained a cell survival rate of 95% and 92%, respectively; further, they enhanced the expression of functional proteins such as zonula occludens 1 (ZO-1) and albumin (ALB), respectively. Thus, the presented GLA-MPS can be adapted as an advanced in vitro model in a wide range of applications for disease modeling associated with inter-tissue interactions, such as fatty liver diseases.

bioengineering↗

bioRxiv2022

An efficient simplified method for the generation of corneal epithelial cells from human pluripotent stem cells

Corneal epithelial cells derived from human pluripotent stem cells (hPSCs) are an important cell source for preclinical models to test ophthalmic drugs. However, current differentiation protocols lack instructions regarding optimal culturing conditions, which hinders the quality of cells and limits scale-up. Here, we introduce a simplified small molecule-based corneal induction method (SSM-CI) to generate corneal epithelial cells from hPSCs. SSM-CI provides the advantage of minimizing cell culturing time using two defined culturing media containing TGF-{beta}, and Wnt/{beta}-catenin pathway inhibitors, and bFGF growth factor over 25 days. Compared to the conventional human corneal epithelial cell line (HCE-T) and human primary corneal epithelial cells (hPCEpCs), corneal epithelial cells generated by SSM-CI are well-differentiated and express relevant maturation markers, including PAX6 and CK12. RNA-seq analysis indicated the faithful differentiation of hPSCs into corneal epithelia, with significant upregulation of corneal progenitor and adult corneal epithelial phenotypes. Furthermore, despite the initial inhibition of TGF-{beta} and Wnt/{beta}-catenin, upregulation of these pathway-related transcripts was observed in the later stages, indicating their necessity in the generation of mature corneal epithelial cells. Moreover, we observed a shift in gene signatures associated with the metabolic characteristics of mature corneal epithelial cells, involving a decrease in glycolysis- and an increase in fatty acid oxidation. This was also attributed to the overexpression of metabolic enzymes and transporter-related transcripts responsible for fatty acid metabolism. Thus, SSM-CI provides a comprehensive method for the generation of functional corneal epithelial cells for use in preclinical models.

bioengineering↗

bioRxiv2021

Generation of iPSCs from endangered Grevy's zebra and comparative transcriptomic analysis of mammalian PSCs

Induced pluripotent stem cells (iPSCs) can provide a biological resource for functional and conservation research in various species. This expectation has led to generation of iPSCs from various species, including those identified as endangered species. However, the understanding of species variation in mammalian iPSCs is largely unknown. Here, to gain insight into the species variation in iPSCs, we the first generated iPSCs from the endangered species Grevys zebra (Equus grevyi; gz-iPSCs) for the first time in the world. We isolated primary fibroblasts cell from an individual that had died of natural causes at a zoo and reprogrammed the fibroblasts into iPSCs. We confirmed their pluripotency and differentiation potential and performed RNA sequencing analysis. The gz-iPSC transcriptome showed that the generated gz-iPSCs robustly expressed genes associated with pluripotency and reprogramming processes, including epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions. Comparative transcriptomics with other species revealed patterns of gene expression among mammalian PSCs and detected evolutionary conservation of pluripotency-associated genes and the plausible importance of the translation process. This study provides new insights into the evolution of mammalian PSCs, and the species conservation and variation of PSCs will advance our understanding of the early development of mammals.

cell biology↗

bioRxiv2021

Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration

Skeletal muscle regeneration requires extracellular matrix (ECM) remodeling including an acute and transient breakdown of collagen that produces gelatin. However, the physiological function of such a remodeling process on muscle tissue repair is unclear. Here we elaborate on a bi-phasic effect of gelatin in skeletal muscle regeneration, mediated by hormetic effects of reactive oxygen species (ROS). Low-dose gelatin stimulates ROS production from NADPH oxidase 2 (NOX2) and simultaneously upregulates antioxidant system for cellular defense, reminiscent of the adaptive compensatory process during mild stress. This response triggers the release of myokine IL-6 which stimulates myogenesis and facilitates muscle regeneration. By contrast, high-dose gelatin stimulates ROS overproduction from NOX2 and mitochondrial chain complex, and ROS accumulation by suppressing antioxidant system, triggering release of TNF, which inhibits myogenesis and regeneration. Our findings reveal gelatin-ROS-IL-6/TNF signaling cascades underlying a hormetic response of myogenic cells to gelatin.

physiology↗

bioRxiv2021

Evaluation of solvents used for fabrication of microphysiological systems

Microphysiological systems (MPSs) have shown great promise for the advancement of drug discovery and toxicological tests, and as an alternative to animal models. However, although several chips and systems have been reported, some important issues are yet to be addressed, such as the use of polydimethylsiloxane (PDMS). Cyclo olefin polymers (COPs) have advantages over other thermoplastic materials, but most COP-based MPSs use solvent bonding during fabrication, which can affect any cells they are used to culture. This study uses a photobonding process with vacuum ultraviolet (UVU) to produce MPSs without the need for solvents such as cyclohexane, dichloromethane, and toluene. This is then used for comparison to investigate the effects of solvents on cell cultures. Quantitative immunofluorescent assays show that the coating efficiencies of extracellular matrix proteins, such as Matrigel and collagen I, are reduced on solvent-treated COP surfaces, compared with those prepared using VUV photobonding. Furthermore, SH-SY5Y neuroblastoma cells are used to evaluate cytotoxicity. This shows that solvent-MPSs induce apoptosis, but VUV-MPSs do not. These results provide insights into solvent bonding for MPS fabrication so that undesirable reactions can be avoided. Moreover, this work may be used to standardize MPS protocols and establish good manufacturing practices.

bioengineering↗

bioRxiv2021

Spatiotemporal Determination of Metabolite Activities in Multiple Corneal Epithelium Barriers on a Chip

The corneal epithelial barrier maintains the metabolic activities of the ocular surface by regulating membrane transporters and metabolic enzymes responsible for the homeostasis of the eye as well as the pharmacokinetic behavior of drugs. Despite its importance, no established biomimetic in vitro methods are available to perform the spatiotemporal investigation of corneal metabolism and determine the transportation of endogenous and exogenous molecules. This study introduces multiple corneal epithelium barriers on a chip, namely, Cornea-Chip, which enables the spatiotemporal collection as well as analysis of micro-scaled extracellular metabolites from both the apical and basolateral sides of the barriers. Longitudinal samples collected during 48 h period were analyzed using untargeted liquid chromatography-mass spectrometry metabolomics method, and 104 metabolites were annotated. The shifts in extracellular metabolites and quantitative analysis of the mRNA associated with membrane transporters could allow the investigation of the correlation between the expression of and the secretion and transportation of metabolites across the polarized corneal epithelial barrier. The Cornea-Chip might provide a non-invasive, simple, and effectively informative method to determine pharmacokinetics and pharmacodynamics as well as to discover novel biomarkers for drug toxicological and safety tests as an alternative to animal experiments.

bioengineering↗

bioRxiv2020

In vitro nonalcoholic fatty liver disease model with cyclo-olefin-polymer-based microphysiological systems

Nonalcoholic fatty liver disease (NAFLD) is one of the common chronic liver conditions, whose treatment involves curing patients without liver transplantation. Understanding the mechanism of NAFLD initiation and progression would enable development of new diagnostic tools and drugs; however, until now, the underlying mechanisms of this condition remain largely unknown owing to the lack of experimental settings that can simplify the complicated NAFLD process in vitro. Microphysiological systems (MPSs) have long been used to recapture human pathophysiological conditions in vitro for applications in drug discovery. However, polydimethylsiloxane (PDMS) has been used in most of these MPSs as the structural material; it absorbs hydrophobic molecules, such as free fatty acids (FFAs), which are the key components that initiate NAFLD. Therefore, the current PDMS-based MPSs cannot be directly applied to in vitro NALFD modeling. In this work, we present an in vitro NAFLD model with an MPS made of cyclo-olefin polymer (COP), namely COP-MPS, to prevent absorption of FFAs. We demonstrated induction of the NAFLD-like phenotype in HepaRG hepatocyte-like cells cultured in the COP-MPS by introducing FFAs. The FFAs induced lipid accumulation in the HepaRG cells, resulting in inactivation of the apoptotic cells. We believe that the proposed COP-MPS can contribute toward investigations of NAFLD mechanisms and identification of new drugs to prevent the progression of liver disease and avoid liver transplantation.

bioengineering↗

bioRxiv2020

Untargeted LC-MS metabolomics for the analysis of micro-scaled extracellular metabolites from hepatocytes

Metabolome analysis in micro physiological models is a challenge due to the low volume of cell culture medium (CCM). Here, we report a LC-MS-based untargeted metabolomics protocol for the detection of hepatocyte extracellular metabolites from micro-scale samples of CCM. Using a single LC-MS method we have detected 57 metabolites of which 27 showed >2-fold shifts after 72-hours incubation. We demonstrate that micro-scale CCM samples can be used for modelling micro-physiological temporal dynamics in metabolite intensities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=62 SRC="FIGDIR/small/312520v3_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1b307e3org.highwire.dtl.DTLVardef@cada64org.highwire.dtl.DTLVardef@1c7fb70org.highwire.dtl.DTLVardef@4cb37f_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

bioengineering↗

bioRxiv2020

Cyclo olefin polymer-based solvent-free mass-productive microphysiological systems

A microphysiological system (MPS) holds a great promise for drug screening and toxicological testing as an alternative to animal models. However, this platform has several issues in terms of the materials used (e.g., polydimethylsiloxane), such as the absorbance of tested drug candidates and fluorescent dyes by the material, as well as the effect on cultured cellular status, thus misleading the results obtained from cell assays and fabrication processes. Hence, to eliminate the issues mentioned above, we developed a cyclo olefin polymer (COP)-based MPS via photobonding process using vacuum ultraviolet (VUV), named COP-VUV-MPS. COP-VUV-MPS showed better chemical resistance and avoided molecule absorption. COP-VUV-MPS could maintain the stemness of environmentally sensitive human-induced pluripotent stem cells without causing undesired cellular phenotypes and gene expression. These results suggested that COP-VUV-MPS might be broadly used for the advancement of MPS and applications in drug development and in vitro toxicological testing.Competing Interest StatementM.Y. is an employee of Ushio Inc. The part of this project was financially supported by Ushio Inc. Kyoto University (K.K.) and Ushio INC. (M.Y.) filed a Japanese patent application based on the research presented herein. The remaining authors declare no competing interests.View Full Text

bioengineering↗