An integrated microfluidic device for long-term culture of isolated single mammalian cells

We developed an integrated microfluidic chip for long-term culture of isolated single cells. This polydimethylsiloxane (PDMS) based device could accurately seed each single cell into different culture chambers, and isolate one chamber from each other with monolithically integrated pneumatic valves. We optimized the culture conditions, including the frequency of medium replacement and the introduction of conditioned medium, to keep the single cells alive for 4 days. We cultured a few hundred cells in a separated chamber on the same chip to continuously supply the conditioned medium into the culture chambers for single cells. This approach greatly facilitated the growth of single cells, and created a suitable microenvironment for observing cells’ autonomous process in situ without the interference of other adjacent cells. This single cell colony assay is expandable to higher throughput, fitting the needs in the studies of drug screening and stem cell differentiation.     

Stamp wound assay for studying coupled cell migration and cell debris clearance

A new method for studying wound healing under realistic conditions in vitro was developed. The method involves creating defined patterns of damaged cell debris with poly(dimethyl)siloxane (PDMS) stamping. This novel assay permitted the quantification of wound healing rates in the presence of cell debris. Experimental results with this assay suggest that cell migration in the presence of cell debris is a two step process requiring (1) non-muscle myosin II-dependent cell clearance followed by (2) cell migration into newly cleared wound areas. The novel stamp wound assay allows the study of coupled cell migration and debris clearance and is a more realistic wound healing assay in vitro.     

用于细胞三维培养的集成微柱阵列的微流控芯片设计与验证

设计并验证了一种用于细胞三维培养的集成微柱阵列的微流控芯片.芯片由一片聚二甲基硅氧烷(PDMS)沟道片和一片玻璃盖片组成, 在PDMS沟道片上集成了一个由两排微柱阵列围成的细胞培养室和两条用于输送培养基的侧沟道.微柱间距直接影响了芯片的使用性能, 是整个芯片设计的关键.基于数值模拟和实验验证, 本研究对微柱间距进行了优化设计.优化后的微流控芯片可以很好地实现细胞与细胞外基质模拟材料混合液的稳定注入、培养基中营养物质向培养室内的快速扩散和细胞代谢物的及时排出.在芯片上进行了神经干细胞的三维培养, 证明了芯片上构建的细胞体外微环境的稳定性.     

Characterization of the interaction between fibroblasts and tumor cells on a microfluidic co‐culture device

Fibroblasts and tumor cells have been involved in the process of cancer development, progression and therapy. Here, we present a simple microfluidic device which enables to study the interaction between fibroblasts and tumor cells by indirect contact co‐culture. The device is composed of multiple cell culture chambers which are connected by a parallel of cell migration regions, and it enables to realize different types of cells to communicate each other on the single device. In this work, human embryonic lung fibroblasts cells were observed to exhibit obvious migration towards tumor cells instead of normal epithelial cells on the co‐culture device. Moreover, transdifferentiation of human embryonic lung fibroblast cells was recognized by the specific expression of α‐smooth musle actin, indicating the effect of tumor cells on the behavior of fibroblasts. Furthermore, multiple types of cell co‐culture can be demonstrated on the single device which enables to mimic the complicated microenviroment in vivo. The device is simple and easy to operate, which enables to realize real‐time observation of cell migration after external stimulus. This microfluidic device allows for the characterization of various cellular events on a single device sequentially, faciliating the better understanding of interaction between heterotypic cells in a more complex microenvironment.     

An active bubble trap and debubbler for microfluidic systems

We present a novel, fully integrated microfluidic bubble trap and debubbler. The 2-layer structure, based on a PDMS valve design, utilizes a featured membrane to stop bubble progression through the device. A pneumatic chamber directly above the trap is evacuated, and the bubble is pulled out through the gas-permeable PDMS membrane. Normal device operation, including continuous flow at atmospheric pressure, is maintained during the entire trapping and debubbling process. We present a range of trap sizes, from 2 to 10 mm diameter, and can trap and remove bubbles up to 25 muL in under 3 h.     

Evaluating cell migration in vitro by the method based on cell patterning within microfluidic channels

Cell migration is an early-stage and critical step for cancer metastasis. The most common approach to monitor this process is wound-healing assay. However, this traditional method has some unavoidable limitations. We observed that simply scratching the monolayer of cultured cells might cause local cell damage around the injury line. The cells along the scratched border seemed to be irritated and exhibited abnormal distribution of cytoskeleton reassembly with protruding "cell islands" and "pseudopodia" during wound healing, which might potentially affect the assessment of cell migration behavior. Herein, we applied a microfluidic device that mechanically constrained cells seeded in a designed pattern inside microchannels, and monitored cell movement in a way of mimicking the natural microenvironment of cancerous tissues. We illustrated the capacity of this simple method to probe cellular migration behaviors and to screen some biological active agents that reflected in their influence on cellular motility.     

High-throughput droplet analysis and multiplex DNA detection in the microfluidic platform equipped with a robust sample-introduction technique

In this work, a simple, flexible and low-cost sample-introduction technique was developed and integrated with droplet platform. The sample-introduction strategy was realized based on connecting the components of positive pressure input device, sample container and microfluidic chip through the tygon tubing with homemade polydimethylsiloxane (PDMS) adaptor, so the sample was delivered into the microchip from the sample container under the driving of positive pressure. This sample-introduction technique is so robust and compatible that could be integrated with T-junction, flow-focus or valve-assisted droplet microchips. By choosing the PDMS adaptor with proper dimension, the microchip could be flexibly equipped with various types of familiar sample containers, makes the sampling more straightforward without trivial sample transfer or loading. And the convenient sample changing was easily achieved by positioning the adaptor from one sample container to another. Benefiting from the proposed technique, the time-dependent concentration gradient was generated and applied for quantum dot (QD)-based fluorescence barcoding within droplet chip. High-throughput droplet screening was preliminarily demonstrated through the investigation of the quenching efficiency of ruthenium complex to the fluorescence of QD. More importantly, multiplex DNA assay was successfully carried out in the integrated system, which shows the practicability and potentials in high-throughput biosensing.     

A single-view field filter device for rare tumor cell filtration and enumeration

In this work, we demonstrate a single-view field filter (SVFF) device for the efficient filtration and enumeration of rare tumor cells in the blood. In our device, the track-etched membrane is integrated within a low-cost polymer-film microfluidic chip, and multiplex microfiltration chambers are designed. Our device permits the performing of multiple sample tests on a single membrane and the dynamical observation of the entire filtration process in a single field of view. To characterize the device performance, our device is first tested using tumor cells, and three different cell behaviors are observed during the filtration process. Finally, we successfully apply our device for the separation of rare tumor cells from the lysed blood samples at various flow rates. The recovery rates of 93.3, 87.6, and 84.1% can be respectively achieved at the throughputs of 50, 100, and 150 μL/min. Our single-view field filter (SVFF) device offers the advantages of label-free filtration, efficient enumeration, easy integration, and low cost, and holds the potential to be used as an efficient tool for the filtration and enumeration of rare cells.     

Hydrogel microfluidic co‐culture device for photothermal therapy and cancer migration

We developed the photo‐crosslinkable hydrogel microfluidic co‐culture device to study photothermal therapy and cancer cell migration. To culture MCF7 human breast carcinoma cells and metastatic U87MG human glioblastoma in the microfluidic device, we used 10 w/v% gelatin methacrylate (GelMA) hydrogels as a semi‐permeable physical barrier. We demonstrated the effect of gold nanorod on photothermal therapy of cancer cells in the microfluidic co‐culture device. Interestingly, we observed that metastatic U87MG human glioblastoma largely migrated toward vascular endothelial growth factor (VEGF)‐treated GelMA hydrogel‐embedding microchannels. The main advantage of this hydrogel microfluidic co‐culture device is to simultaneously analyze the physiological migration behaviors of two cancer cells with different physiochemical motilities and study gold nanorod‐mediated photothermal therapy effect. Therefore, this hydrogel microfluidic co‐culture device could be a potentially powerful tool for photothermal therapy and cancer cell migration applications.     

Cell-based high content screening using an integrated microfluidic device

High content screening (HCS) has quickly established itself as a core technique in the early stage of drug discovery for secondary compound screening. It allows several independent cellular parameters to be measured in a single cell or populations of cells in a single assay. In this work, we describe high content screening for the multiparametric measurement of cellular responses in human liver carcinoma (HepG2) cells using an integrated microfluidic device. This device consists of multiple drug gradient generators and parallel cell culture chambers, in which the processes of liquid dilution and diffusion, micro-scale cell culture, cell stimulation and cell labeling can be integrated into a single device. The simple assay provides multiparametric measurements of plasma membrane permeability, nuclear size, mitochondrial transmembrane potential and intracellular redox states in anti-cancer drug-induced apoptosis of HepG2 cells. The established platform is able to rapidly extract the maximum of information from tumor cells in response to several drugs varying in concentration, with minimal sample and less time, which is very useful for basic biomedical research and cancer treatment.     

Imitation of drug metabolism in human liver and cytotoxicity assay using a microfluidic device coupled to mass spectrometric detection

In this work, we developed a microfluidic device for the imitation of drug metabolism in human liver and its cytotoxicity on cells. The integrated microfluidic device consists of three sections: (1) bioreactors containing poly(ethylene) glycol (PEG) hydrogel encapsulated human liver microsomes (HLMs); (2) cell culture chambers for cytotoxicity assay; and (3) integrated micro solid-phase extraction (SPE) columns to desalt and concentrate the products of enzymatic reaction. To verify the feasibility of the integrated microchip, we studied uridine 5'-diphosphate-glucuronosyltransferase (UGT) metabolism of acetaminophen (AP) and the cytotoxicity of products on HepG2 cells. The products of the reaction in one region of the device were injected into the cell culture chamber for cytotoxicity assay, while those in another region were directly detected online with an electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS) after micro-SPE pre-treatment. Semiquantitative analysis achieved in the experiments could be related to the drug-induced HepG2 cell cytotoxicity. Total analysis time for one product was about 30 min and only less than 4 μg HLM protein was required for one reaction region. The results demonstrated that the established platform could be used to imitate drug metabolism occurring in the human liver, thereby replacing animal experiments in the near future. In addition, the integrated microchip will be a useful tool for drug metabolism studies and cytotoxicity assays, which are pivotal in drug development.     

On-chip anticancer drug test of regular tumor spheroids formed in microwells by a distributive microchannel network

This paper proposes a new cytotoxicity assay in a microfluidic device with microwells and a distributive microfluidic channel network for the formation of cancer cell spheroids. The assay can generate rapid and uniform cell clusters in microwells and test in situ cytotoxicity of anticancer drugs including sequential drug treatments, long term culture of spheroids and cell viability assays. Inlet ports are connected to the microwells by a hydraulic resistance network. This uniform distribution of cell suspensions results in regular spheroid dimensions. Injected cancer cells were trapped in microwells, and aggregated into tumor spheroids within 3 days. A cytotoxicity test of the spheroids in microwells was subsequently processed in the same device without the extraction of cells. The in situ cytotoxicity assay of tumor spheroids in microwells was comparable with the MTT assay on hanging drop spheroids using a conventional 96-well plate. It was observed that the inhibition rate of the spheroids was less than that in the 2D culture dish and the effect on tumor spheroids was different depending on the anticancer drug. This device could provide a convenient in situ assay tool to assess the cytotoxicity of anticancer drugs on tumor spheroids, offering more information than the conventional 2D culture plate.     

Two-dimensional impedance imaging of cell migration and epithelial stratification

We present a miniaturized impedance imaging system, developed for 2D imaging of cell and tissue culture. The system is based on 16 microelectrodes (5 microm x 4 mm). An equivalent circuit for four-point (tetrapolar) impedance spectra was developed and validated. The system uses an Agilent 4294A impedance analyser combined with a front-end amplifier for the impedance measurements. Human epithelial stem cells (YF 29) were grown on the device surface. Cell migration speeds of 300 nm min(-1) following a "scratch" wound closure assay could be established. Using a commercial software developed for geophysical prospecting, we could generate impedance tomography images at 10 kHz revealing cell migration, increase of epithelial thickness and changes in tissue resistivity over a time course of several days.     

Three-dimensional interconnected microporous poly(dimethylsiloxane) microfluidic devices

This technical note presents a fabrication method and applications of three-dimensional (3D) interconnected microporous poly(dimethylsiloxane) (PDMS) microfluidic devices. Based on soft lithography, the microporous PDMS microfluidic devices were fabricated by molding a mixture of PDMS pre-polymer and sugar particles in a microstructured mold. After curing and demolding, the sugar particles were dissolved and washed away from the microstructured PDMS replica revealing 3D interconnected microporous structures. Other than introducing microporous structures into the PDMS replica, different sizes of sugar particles can be used to alter the surface wettability of the microporous PDMS replica. Oxygen plasma assisted bonding was used to enclose the microstructured microporous PDMS replica using a non-porous PDMS with inlet and outlet holes. A gas absorption reaction using carbon dioxide (CO(2)) gas acidified water was used to demonstrate the advantages and potential applications of the microporous PDMS microfluidic devices. We demonstrated that the acidification rate in the microporous PDMS microfluidic device was approximately 10 times faster than the non-porous PDMS microfluidic device under similar experimental conditions. The microporous PDMS microfluidic devices can also be used in cell culture applications where gas perfusion can improve cell survival and functions.     

Analysis of chemoresistance in lung cancer with a simple microfluidic device

Microchip-based systems have been developed rapidly due to their desirable advantages over conventional platforms. Higher level system integration and complex microdevices are emerging to satisfy the demand for high-throughput and large-scale applications. However, most of the devices need to be fabricated with complicated microvalves and micropumps, which, to some extent, limit the use of the novel technique. In this study, a simple microdevice was developed to perform chemotherapy resistance analysis in lung cancer cell line SPCA1. This device includes a PDMS chip for which a simple external small clip served as a microvalve to control the fluid flow so that the parallel control experiment could be carried out simultaneously, and a syringe pump, which supplied the cells with fresh medium mimicking the microenvironment in vivo. Cell culture, detection of drug resistance related protein P-glycoprotein (P-gp) and glutathione S-transferase-π (GST-π) and cell viability after VP-16 treatment on experimental (pretreated with corresponding inhibitors) and control groups were achieved. The results demonstrated that the cells could grow and spread well for at least 3 days. The expression of P-gp and GST-π was obviously downregulated by corresponding inhibitors. The percentage of apoptotic cells for P-gp inhibition group increased 2.9-fold compared with that of control group (23.7 ± 2.6 versus 8.1 ± 3.0%, p<0.05), while for GST-π inhibition, there was no obvious distinction between the experimental and control group. The simple microdevice is capable of integrating parallel operations involving cell culture and functional analysis, offering an easy and flexible platform for a stable long-term cell culture and comparison research.     

Activated T lymphocytes migrate toward the cathode of DC electric fields in microfluidic devices

Immune cell migration is a fundamental process that enables immunosurveillance and immune responses. Understanding the mechanism of immune cell migration is not only of importance to the biology of cells, but also has high relevance to cell trafficking mediated physiological processes and diseases such as embryogenesis, wound healing, autoimmune diseases and cancers. In addition to the well-known chemical concentration gradient based guiding mechanism (i.e. chemotaxis), recent studies have shown that lymphocytes can respond to applied physiologically relevant direct current (DC) electric fields by migrating toward the cathode of the fields (i.e. electrotaxis) in both in vitro and in vivo settings. In the present study, we employed two microfluidic devices allowing controlled application of electric fields inside the microfluidic channel for quantitative studies of lymphocyte electrotaxis in vitro at the single cell level. The first device is fabricated by soft-lithography and the second device is made in glass with integrated on-chip electrodes. Using both devices, we for the first time showed that anti-CD3/CD28 antibodies activated human blood T cells migrate to the cathode of the applied DC electric field. This finding is consistent with previous electrotaxis studies on other lymphocyte subsets suggesting electrotaxis is a novel guiding mechanism for immune cell migration. Furthermore, the characteristics of electrotaxis and chemotaxis of activated T cells in PDMS microfluidic devices are compared.     

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.     

Poly(dimethylsiloxane) microchip for precolumn reaction and micellar electrokinetic chromatography of biogenic amines

We have demonstrated that precolumn derivatization and capillary electrophoresis separation on a poly(dimethylsiloxane) (PDMS) microchip can be realized as efficient as those on glass microchips. In an optimized condition of micellar electrokinetic chromatography (MEKC), using 25 mM sodium borate buffer (pH 10.0) with 25 mM sodium dodecyl sulfate (SDS) and 5% v/v methanol, the electroosmotic flow in an oxidized PDMS microchip is stabilized within 3% for days. By employing a fluorometric derivatization with o-phthaldialdehyde (OPA) in an optimally designed reaction chamber, four most important biogenic amines occurring in foods, histamine, tyramine, putrescine, and tryptamine, are quantitatively determined in less than 1 min at the levels applicable to real samples. The migration behaviors of anionic OPA-derivatized biogenic amines under the MEKC conditions are analyzed, and it has been found that under our separation conditions, the electrophoretic mobility of the SDS micelles is significantly greater than those of the anions in the aqueous phase. The channel manifold in a PDMS substrate is fabricated using replica molding against a thick photoresist, SU-8, pattern generated by photolithography. The plate with the microchannel pattern is strongly, irreversibly bonded to another PDMS plate by using a new bonding technique, which employs surface oxidation by corona discharge generated from a cheap, handy source, Tesla coil.     

Small volume low mechanical stress cytometry using computer-controlled Braille display microfluidics

This paper describes a micro flow cytometer system designed for efficient and non-damaging analysis of samples with small numbers of precious cells. The system utilizes actuation of Braille-display pins for micro-scale fluid manipulation and a fluorescence microscope with a CCD camera for optical detection. The microfluidic chip is fully disposable and is composed of a polydimethylsiloxane (PDMS) slab with microchannel features sealed against a thin deformable PDMS membrane. The channels are designed with diffusers to alleviate pulsatile flow behaviors inherent in pin actuator-based peristaltic pumping schemes to maximize hydrodynamic focusing of samples with minimal disturbances in the laminar streams within the channel. A funnel connected to the microfluidic channel is designed for efficient loading of samples with small number of cells and is also positioned on the chip to prevent physical damages of the samples by the squeezing actions of Braille pins during actuation. The sample loading scheme was characterized by both computational fluidic dynamics (CFD) simulation and experimental observation. A fluorescein solution was first used for flow field investigation, followed by use of fluorescence beads with known relative intensities for optical detection performance calibration. Murine myoblast cells (C2C12) were exploited to investigate cell viability for the sample loading scheme of the device. Furthermore, human promyelocytic leukemia (HL60) cells stained by hypotonic DNA staining buffer were also tested in the system for cell cycle analysis. The ability to efficiently analyze cellular samples where the number of cells is small was demonstrated by analyzing cells from a single embryoid body derived from mouse embryonic stem cells. Consequently, the designed microfluidic device reported in this paper is promising for easy-to-use, small sample size flow cytometric analysis, and has potential to be further integrated with other Braille display-based microfluidic devices to facilitate a multi-functional lab-on-a-chip for mammalian cell manipulations.     

A Self‐Digitization Dielectrophoretic (SD‐DEP) Chip for High‐Efficiency Single‐Cell Capture,On‐Demand Compartmentalization,and Downstream Nucleic Acid Analysis

The design and fabrication of a self‐digitization dielectrophoretic (SD‐DEP) chip with simple components for single‐cell manipulation and downstream nucleic acid analysis is presented. The device employed the traditional DEP and insulator DEP to create the local electric field that is tailored to approximately the size of single cells, enabling highly efficient single‐cell capture. The multistep procedures of cell manipulation, compartmentalization, lysis, and analysis were performed in the integrated microdevice, consuming minimal reagents, minimizing contamination, decreasing lysate dilution, and increasing assay sensitivity. The platform developed here could be a promising and powerful tool in single‐cell research for precise medicine.