RESUMEN
Stem cells are crucial in tissue engineering, and their microenvironment greatly influences their behavior. Among the various dental stem cell types, stem cells from the apical papilla (SCAPs) have shown great potential for regenerating the pulp-dentin complex. Microenvironmental cues that affect SCAPs include physical and biochemical factors. To research optimal pulp-dentin complex regeneration, researchers have developed several models of controlled biomimetic microenvironments, ranging from in vivo animal models to in vitro models, including two-dimensional cultures and three-dimensional devices. Among these models, the most powerful tool is a microfluidic microdevice, a tooth-on-a-chip with high spatial resolution of microstructures and precise microenvironment control. In this review, we start with the SCAP microenvironment in the regeneration of pulp-dentin complexes and discuss research models and studies related to the biological process.
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Papila Dental , Dispositivos Laboratorio en un Chip , Células Madre , Humanos , Células Madre/citología , Papila Dental/citología , Animales , Microambiente Celular , Pulpa Dental/citología , Ingeniería de Tejidos/instrumentación , Nicho de Células Madre , Dentina/citologíaRESUMEN
Oxygen levels vary in the environment. Oxygen availability has a major effect on almost all organisms, and oxygen is far more than a substrate for energy production. However, less is known about related biological processes under hypoxic conditions and about the adaptations to changing oxygen concentrations. The yeast Saccharomyces cerevisiae can adapt its metabolism for growth under different oxygen concentrations and can grow even under anaerobic conditions. Therefore, we developed a microfluidic device that can generate serial, accurately controlled oxygen concentrations for single-cell studies of multiple yeast strains. This device can construct a broad range of oxygen concentrations, [O2] through on-chip gas-mixing channels from two gases fed to the inlets. Gas diffusion through thin polydimethylsiloxane (PDMS) can lead to the equilibration of [O2] in the medium in the cell culture layer under gas cover regions within 2 min. Here, we established six different and stable [O2] varying between ~0.1 and 20.9% in the corresponding layers of the device designed for multiple parallel single-cell culture of four different yeast strains. Using this device, the dynamic responses of different yeast transcription factors and metabolism-related proteins were studied when the [O2] decreased from 20.9% to serial hypoxic concentrations. We showed that different hypoxic conditions induced varying degrees of transcription factor responses and changes in respiratory metabolism levels. This device can also be used in studies of the aging and physiology of yeast under different oxygen conditions and can provide new insights into the relationship between oxygen and organisms. Integration, innovation and insight: Most living cells are sensitive to the oxygen concentration because they depend on oxygen for survival and proper cellular functions. Here, a composite microfluidic device was designed for yeast single-cell studies at a series of accurately controlled oxygen concentrations. Using this device, we studied the dynamic responses of various transcription factors and proteins to changes in the oxygen concentration. This study is the first to examine protein dynamics and temporal behaviors under different hypoxic conditions at the single yeast cell level, which may provide insights into the processes involved in yeast and even mammalian cells. This device also provides a base model that can be extended to oxygen-related biology and can acquire more information about the complex networks of organisms.
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Oxígeno , Saccharomyces cerevisiae , Análisis de la Célula Individual , Oxígeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Dimetilpolisiloxanos/química , Dispositivos Laboratorio en un Chip , Proteínas de Saccharomyces cerevisiae/metabolismo , Diseño de Equipo , Técnicas Analíticas Microfluídicas/instrumentación , MicrofluídicaRESUMEN
Cells can respond and adapt to complex forms of environmental change. Budding yeast is widely used as a model system for these stress response studies. In these studies, the precise control of the environment with high temporal resolution is most important. However, there is a lack of single-cell research platforms that enable precise control of the temperature and form of cell growth. This has hindered our understanding of cellular coping strategies in the face of diverse forms of temperature change. Here, we developed a novel temperature-controlled microfluidic platform that integrates a microheater (using liquid metal) and a thermocouple (liquid metal vs. conductive PDMS) on a chip. Three forms of temperature changes (step, gradient, and periodical oscillations) were realized by automated equipment. The platform has the advantages of low cost and a simple fabrication process. Moreover, we investigated the nuclear entry and exit behaviors of the transcription factor Msn2 in yeast in response to heat stress (37 °C) with different heating modes. The feasibility of this temperature-controlled platform for studying the protein dynamic behavior of yeast cells was demonstrated.
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Respuesta al Choque Térmico , Técnicas Analíticas Microfluídicas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Análisis de la Célula Individual , Temperatura , Análisis de la Célula Individual/instrumentación , Proteínas de Saccharomyces cerevisiae/metabolismo , Técnicas Analíticas Microfluídicas/instrumentación , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción del Choque Térmico/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/citología , Dispositivos Laboratorio en un Chip , Dimetilpolisiloxanos/química , Diseño de Equipo , Factores de TranscripciónRESUMEN
AIM: Three-dimensional (3D) cell culture systems perform better in resembling tissue or organism structures compared with traditional 2D models. Organs-on-chips (OoCs) are becoming more efficient 3D models. This study aimed to create a novel simplified dentin-on-a-chip using microfluidic chip technology and tissue engineering for screening dental materials. METHODOLOGY: A microfluidic device with three channels was designed for creating 3D dental tissue constructs using stem cells from the apical papilla (SCAP) and gelatin methacrylate (GelMA). The study investigated the effect of varying cell densities and GelMA concentrations on the layer features formed within the microfluidic chip. Cell viability and distribution were evaluated through live/dead staining and nuclei/F-actin staining. The osteo/odontogenic potential was assessed through ALP staining and Alizarin red staining. The impact of GelMA concentrations (5 %, 10 %) on the osteo/odontogenic differentiation trajectory of SCAP was also studied. RESULTS: The 3D tissue constructs maintained high viability and favorable spreading within the microfluidic chip for 3-7 days. A cell seeding density of 2 × 104 cells/µL was found to be the most optimal choice, ensuring favorable cell proliferation and even distribution. GelMA concentrations of 5 % and 10 % proved to be most effective for promoting cell growth and uniform distribution. Within the 5 % GelMA group, SCAP demonstrated higher osteo/odontogenic differentiation than that in the 10 % GelMA group. CONCLUSION: In 3D culture, GelMA concentration was found to regulate the osteo/odontogenic differentiation of SCAP. The study recommends a seeding density of 2 × 104 cells/µL of SCAP within 5 % GelMA for constructing simplified dentin-on-a-chip. CLINICAL SIGNIFICANCE: This study built up the 3D culture protocol, and induced odontogenic differentiation of SCAP, thus forming the simplified dentin-on-a-chip and paving the way to be used as a well-defined biological model for regenerative endodontics. It may serve as a potential testing platform for cell differentiation.
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Diferenciación Celular , Proliferación Celular , Supervivencia Celular , Papila Dental , Dentina , Gelatina , Dispositivos Laboratorio en un Chip , Ingeniería de Tejidos , Ingeniería de Tejidos/métodos , Humanos , Papila Dental/citología , Células Madre/citología , Odontogénesis , Osteogénesis/fisiología , Metacrilatos , Técnicas de Cultivo de Célula , Microfluídica/métodos , Microfluídica/instrumentación , Técnicas de Cultivo Tridimensional de Células/métodos , Técnicas de Cultivo Tridimensional de Células/instrumentación , Células CultivadasRESUMEN
Differential equation models are widely used to describe genetic regulations, predict multicomponent regulatory circuits, and provide quantitative insights. However, it is still challenging to quantitatively link the dynamic behaviors with measured parameters in synthetic circuits. Here, we propose a dynamic delay model (DDM) which includes two simple parts: the dynamic determining part and the doses-related steady-state-determining part. The dynamic determining part is usually supposed as the delay time but without a clear formula. For the first time, we give the detail formula of the dynamic determining function and provide a method for measuring all parameters of synthetic elements (include 8 activators and 5 repressors) by microfluidic system. Three synthetic circuits were built to show that the DDM can notably improve the prediction accuracy and can be used in various synthetic biology applications.
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Proper microtubule dynamics are critical for neuronal morphogenesis and functions, and their dysregulation results in neurological disorders and regeneration failure. Superior cervical ganglion-10 (SCG10, also known as stathmin-2 or STMN2) is a well-known regulator of microtubule dynamics in neurons, but its functions in the peripheral nervous system remain largely unknown. Here, we show that Scg10 knockout mice exhibit severely progressive motor and sensory dysfunctions with significant sciatic nerve myelination deficits and neuromuscular degeneration. Additionally, increased microtubule stability, shown by a significant increase in tubulin acetylation and decrease in tubulin tyrosination, and decreased axonal transport were observed in Scg10 knockout dorsal root ganglion (DRG) neurons. Furthermore, SCG10 depletion impaired axon regeneration in both injured mouse sciatic nerve and cultured DRG neurons following replating, and the impaired axon regeneration was found to be induced by a lack of SCG10-mediated microtubule dynamics in the neurons. Thus, our results highlight the importance of SCG10 in peripheral axon maintenance and regeneration.
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Axones , Tubulina (Proteína) , Animales , Ratones , Axones/fisiología , Ganglios Espinales , Regeneración Nerviosa/genética , Neuronas , Estatmina/genéticaRESUMEN
A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the minimalist known angiosperm to overcome this challenge and provide a model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the plant-on-chip culture system and demonstrate the application of advanced technologies such as single-nucleus RNA-sequencing, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can decipher the core regulatory mechanisms of plant morphogenesis.
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As an absolute quantification method at the single-molecule level, digital PCR (dPCR) offers the highest accuracy. In this work, we developed a 3D scalable chamber-array chip that multiplied the number of partitions by stacking chamber-array layers and realized digital loop-mediated isothermal amplification to quantify DNA molecules. It greatly increases the number of partitions to improve the performance of dPCR without increasing the chip size, the operation workflow complicity, and operation time. For the three-chamber-array-layer chip which contains 200,000 reactors of a 0.125 nL volume, it has been proved that the reagent filling and partition were finished within 3 min, and the whole detection could be finished within 1 h. The method demonstrated that it could be scalable to a six-chamber-array layer, which contains 400,000 reactors without increasing the size of the chip and the complication of filling/partition workflow but only takes an additional hour for scanning. Due to its potential for high throughput, low cost, and simple operation, our device may significantly expand the clinical application range of dPCR.
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ADN , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN/genética , Nanotecnología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
Super-resolution microscopy is rapidly developed in recent years, allowing biologists to extract more quantitative information on subcellular processes in live cells that is usually not accessible with conventional techniques. However, super-resolution imaging is not fully exploited because of the lack of an appropriate and multifunctional experimental platform. As an important tool in life sciences, microfluidics is capable of cell manipulation and the regulation of the cellular environment because of its superior flexibility and biocompatibility. The combination of microfluidics and super-resolution microscopy revolutionizes the study of complex cellular properties and dynamics, providing valuable insights into cellular structure and biological functions at the single-molecule level. In this perspective, an overview of the main advantages of microfluidic technology that are essential to the performance of super-resolution microscopy are offered. The main benefits of performing super-resolution imaging with microfluidic devices are highlighted and perspectives on the diverse applications that are facilitated by combining these two powerful techniques are provided.
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Microfluídica , Microscopía , Microfluídica/métodos , NanotecnologíaRESUMEN
Cells constantly respond to environmental changes by modulating gene expression programs. These responses may demand substantial costs and, thus, affect cell growth. Understanding the regulation of these processes represents a key question in biology and biotechnology. Here, we studied the responses to osmotic stress in glucose-limited environments. By analyzing seventeen osmotic stress-induced genes and stress-activated protein kinase Hog1, we found that cells exhibited stronger osmotic gene expression response and larger integral of Hog1 nuclear localization during adaptation to osmotic stress under glucose-limited conditions than under glucose-rich conditions. We proposed and verified that in glucose-limited environment, glycolysis intermediates (representing "reserve flux") were limited, which required cells to express more glycerol-production enzymes for stress adaptation. Consequently, the regulatory mechanism of osmoresponse was derived in the presence and absence of such reserve flux. Further experiments suggested that this reserve flux-dependent stress-defense strategy may be a general principle under nutrient-limited environments.
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Escherichia coli is a common chassis for synthetic gene circuit studies. In addition to the dose-response of synthetic gene circuits, the analysis of dynamic responses is also an important part of the future design of more complicated synthetic systems. Recently, microfluidic-based methods have been widely used for the analysis of gene expression dynamics. Here, we established a two-layered microfluidic platform for the systematic characterization of synthetic gene circuits (eight strains in eight different culture environments could be observed simultaneously with a 5 min time resolution). With this platform, both dose responses and dynamic responses with a high temporal resolution could be easily derived for further analysis. A controlled environment ensures the stability of the bacterial growth rate, excluding changes in gene expression dynamics caused by changes of the growth dilution rate. The precise environmental switch and automatic micrograph shooting ensured that there was nearly no time lag between the inducer addition and the data recording. We studied four four-node incoherent-feedforward-loop (IFFL) networks with different operators using this device. The experimental results showed that as the effect of inhibition increased, two of the IFFL networks generated pulselike dynamic gene expressions in the range of the inducer concentrations, which was different from the dynamics of the two other circuits with only a simple pattern of rising to the platform. Through fitting the dose-response curves and the dynamic response curves, corresponding parameters were derived and introduced to a simple model that could qualitatively explain the generation of pulse dynamics.
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Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Sintéticos , Redes Reguladoras de Genes/genética , Proteínas de Escherichia coli/genética , Dispositivos Laboratorio en un ChipRESUMEN
Living erythrocyte (red blood cell, RBC) membranes present rich ultrastructural and dynamic details, which require synchronous super-resolution imaging and single-molecule tracking to be revealed. Yet, it poses a serious challenge to achieve these dual functions in a single probe, due to the rigid and conflicting photophysical demands of the different techniques. Herein, we rationally developed a far-red boron dipyrromethene membrane probe with blinking capability and persistent single-molecule emission, and constructed a microfluidic platform for noninvasive trapping and long-term imaging of RBCs. By combining them, multi-dimensional super-resolution reconstructions and single-molecule tracking were achieved at the molecular scale on living human RBC membranes in a high-throughput manner. Our integrated system defines a quantitative method for analyzing RBC membranes under physiological and pathological conditions, improving precision and revealing new perspectives for future disease diagnostics.
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Colorantes Fluorescentes , Microfluídica , Humanos , Colorantes Fluorescentes/química , Microfluídica/métodos , Membrana Eritrocítica , Imagen Individual de Molécula/métodos , EritrocitosRESUMEN
To reveal the underlying mechanism of the biological function of multicellular systems, it is important to obtain comprehensive spatial gene expression profiles. Among the emerging single-cell spatial-omics techniques, immunofluorescence (IF)-based iterative multiplexed imaging is a promising approach. However, the conventional method is usually costly, time-consuming, labor-intensive, and has low throughput. Moreover, it has yet to be demonstrated in intact multicellular organisms. Here, we developed an integrated microfluidic system to overcome these challenges for quantitatively measuring multiple protein profiles sequentially in situ in the same Drosophila embryo. We designed an array of hydrodynamic trapping sites to automatically capture over ten Drosophila embryos with orientation selectivity at more than 90% trapping rates. We also optimized the geometry of confinement and the on-chip IF protocol to achieve the same high signal-to-noise ratio as the off-chip traditional IF experiments. Moreover, we developed an efficient de-staining protocol by combining on-chip antibody stripping and fluorophore bleaching. Using the same secondary antibody to sequentially stain different genes, we confirmed that the de-stained genes have no detectable interference with the subsequently stained genes, and the gene expression profiles are preserved after multiple cycles of staining and de-staining processes. This preliminary test shows that our newly developed integrated microfluidic system can be a powerful tool for multiplexed imaging of Drosophila embryos. Our work opens a new avenue to design microfluidic chips for multicellular organisms and single-cell spatial-omics techniques.
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Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas , Animales , Drosophila/genética , Microfluídica , Hidrodinámica , Expresión GénicaRESUMEN
Aging is a major risk factor for chronic diseases and disability in humans. Nowadays, no effective anti-aging treatment is available clinically. In this study, oridonin was selected based on the drug screening strategy similar to Connectivity MAP (CMAP) but upon transcriptomes of 102 traditional Chinese medicines treated cell lines. Oridonin is a diterpenoid isolated from Rabdosia rubescens. As reported, Oridonin exhibits a variety of pharmacological activities, including antitumor, antibacterial and anti-inflammatory activities. Here, we found that oridonin inhibited cellular senescence in human diploid fibroblasts (2BS and WI-38), manifested by decreased senescence-associated ß-galactosidase (SA-ß-gal) staining. Compared with the elderly control group, the positive cell rate in the oridonin intervention group was reduced to 48.5%. Notably, oridonin prolonged the lifespan of yeast by 48.9%, and extended the average life span of naturally aged mice by 21.6%. Our mice behavior experiments exhibited that oridonin significantly improved the health status of naturally aged mice. In addition, oridonin also delayed doxorubicin-induced cellular senescence and mouse senescence. Compared with the model group, the percentage of SA-ß-gal positive cells in the oridonin treatment group was reduced to 59.8%. It extended the average lifespan of mice by 53.8% and improved healthspan. Mechanistically, we showed that oridonin delayed aging through the AKT signaling pathway and reversed the genetic changes caused by doxorubicin-induced cell senescence. Therefore, oridonin is a potential candidate for the development of anti-aging drugs.
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The mechanical properties of cells are of great significance to their normal physiological activities. The current methods used for the measurement of a cell's mechanical properties have the problems of complicated operation, low throughput, and limited measuring range. Based on micropipette technology, we designed a double-layer micro-valve-controlled microfluidic chip with a series of micropipette arrays. The chip has adjustment pressure ranges of 0.03-1 and 0.3-10 kPa and has a pressure stabilization design, which can achieve a robust measurement of a single cell's mechanical properties under a wide pressure range and is simple to operate. Using this chip, we measured the mechanical properties of the cells treated with different concentrations of paraformaldehyde (PFA) and observed that the viscoelasticity of the cells gradually increased as the PFA concentration increased. Then, this method was also used to characterize the changes in the mechanical properties of the differentiation pathways of stem cells from the apical papilla to osteogenesis.
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Circulating tumor cells (CTCs) survive in the bloodstream and then seed and invade to foster tumor metastasis. The arrest of cancer cells is favored by permissive flow forces and geometrical constraints. Through the use of high-throughput microfluidic devices designed to mimic capillary-sized vessels, we applied pressure differences to cancer cells (MCF-7 cell line) and recorded the cell traverse-vessel behaviors. Our results showed that cancer cells transform from a Newtonian droplet state to an adhesion/migration state when cancer cells traverse artificial vessels. To explain these phenomena, a modified Newtonian droplet model was also proposed. These phenomena and the modified model may reveal how CTCs in the blood seed and invade vessels under suitable conditions.
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Células Neoplásicas Circulantes , Línea Celular Tumoral , Humanos , Dispositivos Laboratorio en un Chip , Células MCF-7 , Células Neoplásicas Circulantes/patologíaRESUMEN
Synthetic Biology aims to create predictable biological circuits and fully operational biological systems. Although there are methods to create more stable oscillators, such as repressilators, independently controlling the oscillation of reporter genes in terms of their amplitude and period is only on theoretical level. Here, we introduce a new oscillator circuit that can be independently controlled by two inducers in Escherichia coli. Some control components, including σECF11 and NahR, were added to the circuit. By systematically tuning the concentration of the inducers, salicylate and IPTG, the amplitude and period can be modulated independently. Furthermore, we constructed a quantitative model to forecast the regulation results. Under the guidance of the model, the expected oscillation can be regulated by choosing the proper concentration combinations of inducers. In summary, our work achieved independent control of the oscillator circuit, which allows the oscillator to be modularized and used in more complex circuit designs.
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Relojes Biológicos/genética , Redes Reguladoras de Genes/genética , Modelos Genéticos , Biología Sintética/métodos , Escherichia coli/genética , Genes Reporteros/genética , Análisis de la Célula IndividualRESUMEN
The causes and consequences of aging have always been a concern. In recent studies, changes in the stress response capacity of cells during aging were quantitatively analyzed. It was found that aging was accompanied by a decline in response capacity. When the response capacity decreased to a critical value, which we assumed was the internal noise level, the cell soon died. To survive, the response capacity should be, at minimum, sufficiently strong to resist intracellular noise. Here, we discuss the role of stress response capacity in aging and conjecture that lifespan might be extended by enhancing stress response capacity.
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Envejecimiento/genética , Replicación del ADN/genética , Longevidad/genética , Proteínas de Saccharomyces cerevisiae/genética , Envejecimiento/patología , Regulación Fúngica de la Expresión Génica/genética , Humanos , Saccharomyces cerevisiae/genética , Estrés Fisiológico/genéticaRESUMEN
Isolation and analysis of circulating tumor cells (CTCs) from peripheral blood provides a potential way to detect and characterize cancer. Existing technologies to separate or capture CTCs from whole blood still have issues with sample throughput, separation efficiency or stable efficiency at different flow rates. Here, we proposed a new concept to capture rare CTCs from blood by integrating a triangular prism array-based capture apparatus with streamline-based focus-separation speed reduction design. The focus-separation design could focus and maintain CTCs, while removing a considerable proportion of liquid (98.9%) containing other blood cells to the outlet, therefore, a high CTC capture efficiency could be achieved in the trap arrays with a high initial flow rate. It is worth mentioning that the new design works well over a wide range of flow rates, so it does not require the stability of the flow rate. The results showed that this novel integrated chip can achieve a sample throughput from 5 to 40 mL h-1 with a stable and high CTC capture efficiency (up to 94.8%) and high purity (up to 4 log white blood cells/WBC depletion). The clinical experiment showed that CTCs including CTC clusters were detected in 11/11 (100.0%) patients (mean = 31 CTCs mL-1, median = 25 CTCs mL-1). In summary, our chip enriches and captures CTCs based on physical properties, and it is simple, cheap, fast, and efficient and has low requirements on flow rate, which is very suitable for large-scale application of CTC testing in clinics.