RESUMEN
Stem-cell-based therapeutics have shown immense potential in treating various diseases that are currently incurable. In particular, partial recovery of Parkinson's disease, which occurs due to massive loss or abnormal functionality of dopaminergic (DAnergic) neurons, through the engraftment of stem-cell-derived neurons ex vivo is reported. However, precise assessment of the functionality and maturity of DAnergic neurons is still challenging for their enhanced clinical efficacy. Here, a novel conductive cell cultivation platform, a graphene oxide (GO)-incorporated metallic polymer nanopillar array (GOMPON), that can electrochemically detect dopamine (DA) exocytosis from living DAnergic neurons, is reported. In the cell-free configuration, the linear range is 0.5-100 µm, with a limit of detection of 33.4 nm. Owing to its excellent biocompatibility, a model DAnergic neuron (SH-SY5Y cell) can be cultivated and differentiated on the platform while their DA release can be quantitatively measured in a real-time and nondestructive manner. Finally, it is showed that the functionality of the DAnergic neurons derived from stem cells can be precisely assessed via electrochemical detection of their DA exocytosis. The developed GOMPON is highly promising for a wide range of applications, including real-time monitoring of stem cell differentiation into neuronal lineages, evaluating differentiation protocols, and finding practical stem cell therapies.
Asunto(s)
Grafito , Neuroblastoma , Humanos , Polímeros , Dopamina , Pirroles , Oro , Neuronas , Técnicas ElectroquímicasRESUMEN
Deoxyribonuclease-I (DNase-I), a representative endonuclease, is an important biomarker for the diagnosis of infectious diseases and cancer progression. However, enzymatic activity decreases rapidly ex vivo, which highlights the need for precise on-site detection of DNase-I. Here, a localized surface plasmon resonance (LSPR) biosensor that enables the simple and rapid detection of DNase-I is reported. Moreover, a novel technique named electrochemical deposition and mild thermal annealing (EDMIT) is applied to overcome signal variations. By taking advantage of the low adhesion of gold clusters on indium tin oxide substrates, both the uniformity and sphericity of gold nanoparticles are increased under mild thermal annealing conditions via coalescence and Ostwald ripening. This ultimately results in an approximately 15-fold decrease in LSPR signal variations. The linear range of the fabricated sensor is 20-1000 ng mL-1 with a limit of detection (LOD) of 127.25 pg mL-1 , as demonstrated by spectral absorbance analyses. The fabricated LSPR sensor stably measured DNase-I concentrations from samples collected from both an inflammatory bowel disease (IBD) mouse model, as well as human patients with severe COVID-19 symptoms. Therefore, the proposed LSPR sensor fabricated via the EDMIT method can be used for early diagnosis of other infectious diseases.
Asunto(s)
Técnicas Biosensibles , COVID-19 , Nanopartículas del Metal , Animales , Ratones , Humanos , Resonancia por Plasmón de Superficie/métodos , Oro/química , Nanopartículas del Metal/química , Técnicas Biosensibles/métodos , DesoxirribonucleasasRESUMEN
Graphene, a two-dimensional plane-structured carbon allotrope, has outstanding properties. Owing to their unique features, graphene-based materials including graphene derivatives have recently emerged as an ideal material and been used in various fields. Especially, in terms of specific advantages of graphene including great electrical conductivity, high potential to conjugate with biomolecules, and applicability to three-dimensional structures, neurogenesis-based stem cell therapies using graphene-based materials have been reported to be a candidate of treatment for neurodegenerative disease (e.g., Parkinson's disease, Alzheimer's disease, and Huntington's disease). To date, extensive studies on neurogenesis-based stem cell therapies including enhanced neural differentiation and monitoring stem cells behavior have been conducted using graphene-based materials. Herein, we have summarized recent various studies of neurogenesis using graphene-based materials in depth and focused on effect of graphene on functional improvement of neural stem cells and monitoring of differentiation into neural linages.
Asunto(s)
Grafito , Células-Madre Neurales , Enfermedades Neurodegenerativas , Diferenciación Celular , Grafito/química , Humanos , Enfermedades Neurodegenerativas/terapia , NeurogénesisRESUMEN
Biocompatible platforms, wherein cells attach and grow, are important for controlling cytoskeletal dynamics and steering stem cell functions, including differentiation. Among various components, membrane integrins play a key role in focal adhesion of cells (18-20 nm in size) and are, thus, highly sensitive to the nanotopographical features of underlying substrates. Hence, it is necessary to develop a platform/technique that can provide high flexibility in controlling nanostructure sizes. We report a platform modified with homogeneous nanohole patterns, effective in guiding neurogenesis of mouse neural stem cells (mNSCs). Sizes of nanoholes were easily generated and varied using laser interference lithography (LIL), by changing the incident angles of light interference on substrates. Among three different nanohole patterns fabricated on conductive transparent electrodes, 500 nm-sized nanoholes showed the best performance for cell adhesion and spreading, based on F-actin and lamellipodia/filopodia expression. Enhanced biocompatibility and cell adhesion of these nanohole patterns ultimately resulted in the enhanced neurogenesis of mNSCs, based on the mRNAs expression level of the mNSCs marker and several neuronal markers. Therefore, platforms modified with homogeneous nanohole patterns fabricated by LIL are promising for the precise tuning of nanostructures in tissue culture platforms and useful for controlling various differentiation lineages of stem cells.
Asunto(s)
Células-Madre Neurales/citología , Neurogénesis , Técnicas de Cultivo de Tejidos/instrumentación , Actinas/metabolismo , Animales , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Ratones , Nanoestructuras , Células-Madre Neurales/metabolismo , Tamaño de la Partícula , Seudópodos/metabolismoRESUMEN
Non-invasive, non-destructive, and label-free sensing techniques are required to monitor real-time stem cell differentiation. However, conventional analysis methods, such as immunocytochemistry, polymerase chain reaction, and Western blot, involve invasive processes and are complicated and time-consuming. Unlike traditional cellular sensing methods, electrochemical and optical sensing techniques allow non-invasive qualitative identification of cellular phenotypes and quantitative analysis of stem cell differentiation. In addition, various nano- and micromaterials with cell-friendly properties can greatly improve the performance of existing sensors. This review focuses on nano- and micromaterials that have been reported to improve sensing capabilities, including sensitivity and selectivity, of biosensors towards target analytes associated with specific stem cell differentiation. The information presented aims to motivate further research into nano-and micromaterials with advantageous properties for developing or improving existing nano-biosensors to achieve the practical evaluation of stem cell differentiation and efficient stem cell-based therapies.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/métodos , Diferenciación CelularRESUMEN
Stem cell therapy has shown great potential in treating various incurable diseases using conventional chemotherapy. Parkinson's disease (PD)-a neurodegenerative disease-has been reported to be caused by quantitative loss or abnormal functionality of dopaminergic neurons (DAnergic neurons). To date, stem cell therapies have shown some potential in treating PD throughex vivoengraftment of stem-cell-derived neurons. However, accurately identifying the differentiation and non-invasively evaluating the functionality and maturity of DAnergic neurons are formidable challenges in stem cell therapies. These strategies are important in enhancing the efficacy of stem cell therapies. In this study, we report a novel cell cultivation platform, that is, a nanocrater-like electrochemical nanoelectrode array (NCENA) for monitoring dopamine (DA) release from neurons to detect exocytotic DA release from DAnergic neurons. In particular, the developed NCENA has a nanostructure in which three-dimensional porous gold nanopillars are uniformly arranged on conductive electrodes. The developed NCENA exhibited great DA sensing capabilities with a linear range of 0.39-150µM and a limit of detection of 1.16µM. Furthermore, the nanotopographical cues provided by the NCENA are suitable for cell cultivation with enhanced cellular adhesion. Finally, we successfully analysed the functionality and maturity of differentiated neurons on the NCENA through its excellent sensing ability for exocytotic DA.
RESUMEN
Direct detection of cellular redox signals has shown immense potential as a novel living cell analysis tool. However, the origin of such signals remains unknown, which hinders the widespread use of electrochemical methods for cellular research. In this study, the authors found that intracellular metabolic pathways that generate adenosine triphosphate (ATP) are the main contributors to extracellularly detectable electrochemical signals. This is achieved through the detection of living cells (4,706 cells/chip, linearity: 0.985) at a linear range of 7,466-48,866. Based on this discovery, the authors demonstrated that the cellular signals detected by differential pulse voltammetry (DPV) can be rapidly amplified with a developed medium containing metabolic activator cocktails (MACs). The DPV approach combined with MAC treatment shows a remarkable performance to detect the effects of the anticancer drug CPI-613 on cervical cancer both at a low drug concentration (2 µm) and an extremely short treatment time (1 hour). Furthermore, the senescence of mesenchymal stem cells could also be sensitively quantified using the DPV+MAC method even at a low passage number (P6). Collectively, their findings unveiled the origin of redox signals in living cells, which has important implications for the characterization of various cellular functions and behaviors using electrochemical approaches.
Asunto(s)
Técnicas Biosensibles , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Transducción de SeñalRESUMEN
The advancement of science and technology has led to the recent development of highly sensitive pathogen biosensing techniques. The effective treatment of pathogen infections requires sensing technologies to not only be sensitive but also render results in real-time. This review thus summarises the recent advances in optical surface plasmon resonance (SPR) sensor technology, which possesses the aforementioned advantages. Specifically, this technology allows for the detection of specific pathogens by applying nano-sized materials. This review focuses on various nanomaterials that are used to ensure the performance and high selectivity of SPR sensors. This review will undoubtedly accelerate the development of optical biosensing technology, thus allowing for real-time diagnosis and the timely delivery of appropriate treatments as well as preventing the spread of highly contagious pathogens.
Asunto(s)
Técnicas Biosensibles , Nanoestructuras , Técnicas Biosensibles/métodos , Resonancia por Plasmón de Superficie/métodosRESUMEN
Stable and continuous supply of essential biomolecules is critical to mimic in vivo microenvironments wherein spontaneous generation of various cell types occurs. Here, we report a new platform that enables highly efficient neuronal cell generation of neural stem cells using single metal-organic framework (MOF) nanoparticle-embedded nanopit arrays (SMENA). By optimizing the physical parameters of homogeneous periodic nanopatterns, each nanopit can confine single nMOFs (UiO-67) that are specifically designed for long-term storage and release of retinoic acid (RA). The SMENA platform successfully inhibited physical interaction with cells, which contributed to remarkable stability of the nMOF (RAâUiO-67) structure without inducing nanoparticle-mediated toxicity issues. Owing to the continuous and long-term supply of RA, the neural stem cells showed enhanced mRNA expressions of various neurogenesis-related activities. The developed SMENA platform can be applied to other stem cell sources and differentiation lineages and is therefore useful for various stem cell-based regenerative therapies.
RESUMEN
Direct messenger ribonucleic acid (mRNA) delivery to target cells or tissues has revolutionized the field of biotechnology. However, the applicability of regenerative medicine is limited by the technical difficulties of various mRNA-loaded nanocarriers. Herein, we report a new conductive hybrid film that could guide osteogenic differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) via electrically controlled mRNA delivery. To find optimal electrical conductivity and mRNA-loading capacity, the polypyrrole-graphene oxide (PPy-GO) hybrid film was electropolymerized on indium tin oxide substrates. We found that the fluorescein sodium salt, a molecule partially mimicking the physical and chemical properties of mRNAs, can be effectively absorbed and released by electrical stimulation (ES). The hADMSCs cultivated on the PPy-GO hybrid film loaded with pre-osteogenic mRNAs showed the highest osteogenic differentiation under electrical stimulation. This platform can load various types of RNAs thus highly promising as a new nucleic acid delivery tool for the development of stem cell-based therapeutics. Electronic Supplementary Material: Supplementary material (electrochemical and FT-IR analysis on the film, additional SEM, AFM and C-AFM images of the film, optical and fluorescence images of cells, and the primers used for RT-qPCR analysis) is available in the online version of this article at 10.1007/s12274-022-4613-y.
RESUMEN
ABSTRACT: We retrospectively compared the central corneal thickness (CCT) obtained by ultrasound pachymetry (USP; SP-3000, Tomey Corp., Nagoya, Japan), non-contact tonopachy (TP) (NT-530P, Nidek Co., Ltd., Gamagori, Japan), Pentacam HR (OCULUS Inc., Wetzlar, Germany), and RTVue optical coherence tomography (OCT) (Optovue Inc., Fremont, CA, USA) in 78 eyes of 78 healthy subjects with myopia. Agreement between the measurement methods was evaluated using 95% confidence intervals for the limits of agreement (LoA). The mean CCT values were 546.9 ± 34.7, 548.1â±â33.5, 559.2â±â34.0, and 547.2â±â34.8âµm for USP, non-contact TP, Pentacam, and RTVue, respectively. The thickest and the thinnest mean CCT values corresponded to those obtained by Pentacam HR and USP, respectively. Plots of the differences against the means showed the best agreement between USP and RTVue (LoA, 10.14-10.70âµm), while the largest discrepancy was observed between RTVue and Pentacam systems (LoA, -25.47-1.44âµm). Our data showed that CCT measurements using these 4 instruments were well correlated. However, the results from Pentacam differed significantly from those of the other instruments.
Asunto(s)
Córnea/diagnóstico por imagen , Paquimetría Corneal/estadística & datos numéricos , Miopía/diagnóstico por imagen , Tomografía de Coherencia Óptica/estadística & datos numéricos , Tonometría Ocular/estadística & datos numéricos , Adolescente , Adulto , Córnea/patología , Paquimetría Corneal/métodos , Femenino , Voluntarios Sanos , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Reproducibilidad de los Resultados , Estudios Retrospectivos , Tomografía de Coherencia Óptica/métodos , Tonometría Ocular/métodos , Ultrasonografía , Adulto JovenRESUMEN
Graphene derivatives are highly promising materials for use in stem-cell-based regenerative therapies, particularly for bone regeneration. Herein, we report a graphene oxide (GO)-based hybrid platform (GOHP) that is highly effective for guiding the osteogenesis of human adipose-derived mesenchymal stem cells (hAMSCs). A GO-coated indium tin oxide (ITO) substrate was electrochemically modified with Au nanostructures (GNSs), following which a cysteine-modified quadruple-branched arginine-glycine-aspartic acid was self-assembled on the ITO-GO-GNS hybrid via Au-S bonds. The synthesized GOHP, with the highest density of GNSs (deposition time of 120â¯s), exhibited the highest osteogenic differentiation efficiency based on the osteogenic marker expression level, osteocalcin expression, and osteoblastic mineralisation. Remarkably, although GO is known to be less efficient than the high-quality pure graphene synthesised via chemical vapour deposition (CVD), the fabricated GOHP exhibited an efficiency similar to that of CVD-grown graphene in guiding the osteogenesis of hAMSCs. The total RNA sequencing results revealed that CVD graphene and GOHP induced the osteogenesis of hAMSCs by upregulating the transcription factors related to direct osteogenesis, Wnt activation, and extracellular matrix deposition. Considering that GO is easy to produce, cost-effective, and biocompatible, the developed GOHP is highly promising for treating various diseases/disorders, including osteoporosis, rickets, and osteogenesis imperfecta.
Asunto(s)
Grafito , Células Madre Mesenquimatosas , Nanoestructuras , Diferenciación Celular , Células Cultivadas , Oro , Humanos , Osteogénesis , PéptidosRESUMEN
Dopamine is a key neurotransmitter that plays essential roles in the central nervous system, including motor control, motivation, arousal, and reward. Thus, abnormal levels of dopamine directly cause several neurological diseases, including depressive disorders, addiction, and Parkinson's disease (PD). To develop a new technology to treat such diseases and disorders, especially PD, which is currently incurable, dopamine release from living cells intended for transplantation or drug screening must be precisely monitored and assessed. Owing to the advantages of miniaturisation and rapid detection, numerous electrical techniques have been reported, mostly in combination with various nanomaterials possessing specific nanoscale geometries. This review highlights recent advances in electrical biosensors for dopamine detection, with a particular focus on the use of various nanomaterials (e.g., carbon-based materials, hybrid gold nanostructures, metal oxides, and conductive polymers) on electrode surfaces to improve both sensor performance and biocompatibility. We conclude that this review will accelerate the development of electrical biosensors intended for the precise detection of metabolite release from living cells, which will ultimately lead to advances in therapeutic materials and techniques to cure various neurodegenerative disorders.