Membrane-integrated microfluidic device for high-resolution live cell imaging

The design and fabrication of a membrane-integrated microfluidic cell culture device (five layers,≤500 μm total thickness) developed for high resolution microscopy is reported here. The multi-layer device was constructed to enable membrane separated cell culture for tissue mimetic in vitro model applications and pharmacodynamic evaluation studies. The microdevice was developed via a unique combination of low profile fluidic interconnect design, substrate transfer methodology, and wet silane bonding. To demonstrate the unique high resolution imaging capability of this device, we used oil immersion microscopy to image stained nuclei and mitochondria in primary hepatocytes adhered to the incorporated membrane     

A microfluidic device for uniform-sized cell spheroids formation,culture, harvesting and flow cytometry analysis

Culture of cells as three-dimensional (3D) aggregates, named spheroids, possesses great potential to improve in vitro cell models for basic biomedical research. However, such cell spheroid models are often complicated, cumbersome, and expensive compared to conventional Petri-dish cell cultures. In this work, we developed a simple microfluidic device for cell spheroid formation, culture, and harvesting. Using this device, cells could form uniformly sized spheroids due to strong cell–cell interactions and the spatial confinement of microfluidic culture chambers. We demonstrated cell spheroid formation and culture in the designed devices using embryonic stem cells, carcinoma cells, and fibroblasts. We further scaled up the device capable of simultaneously forming and culturing 5000 spheroids in a single chip. Finally, we demonstrated harvesting of the cultured spheroids from the device with a simple setup. The harvested spheroids possess great integrity, and the cells can be exploited for further flow cytometry assays due to the ample cell numbers.     

Flip channel: A microfluidic device for uniform-sized embryoid body formation and differentiation

This paper reports a two-layered polydimethylsiloxane microfluidic device—Flip channel, capable of forming uniform-sized embryoid bodies (EBs) and performing stem cell differentiation within the same device after flipping the microfluidic channel. The size of EBs can be well controlled by designing the device geometries, and EBs with multiple sizes can be formed within a single device to study EB size-dependent stem cell differentiation. During operation of the device, cells are positioned in the designed positions. As a result, observation and monitoring specific population of cells can be achieved for further analysis. In addition, after flipping the microfluidic channel, stem cell differentiation from the EBs can be performed on an unconfined flat surface that is desired for various differentiation processes. In the experiments, murine embryonic stem cells (ES-D3) are cultured and formed EBs inside the developed device. The size of EBs is well controlled inside the device, and the neural differentiation is performed on the formed EBs after flipping the channel. The EB size-dependent stem cell differentiation is studied using the device to demonstrate its functions. The device provides a useful tool to study stem cell differentiation without complicated device fabrication and tedious cell handling under better-controlled microenvironments.     

A microfluidic device enabling high-efficiency single cell trapping

Single cell trapping increasingly serves as a key manipulation technique in single cell analysis for many cutting-edge cell studies. Due to their inherent advantages, microfluidic devices have been widely used to enable single cell immobilization. To further improve the single cell trapping efficiency, this paper reports on a passive hydrodynamic microfluidic device based on the “least flow resistance path” principle with geometry optimized in line with corresponding cell types. Different from serpentine structure, the core trapping structure of the micro-device consists of a series of concatenated T and inverse T junction pairs which function as bypassing channels and trapping constrictions. This new device enhances the single cell trapping efficiency from three aspects: (1) there is no need to deploy very long or complicated channels to adjust flow resistance, thus saving space for each trapping unit; (2) the trapping works in a “deterministic” manner, thus saving a great deal of cell samples; and (3) the compact configuration allows shorter flowing path of cells in multiple channels, thus increasing the speed and throughput of cell trapping. The mathematical model of the design was proposed and optimization of associated key geometric parameters was conducted based on computational fluid dynamics (CFD) simulation. As a proof demonstration, two types of PDMS microfluidic devices were fabricated to trap HeLa and HEK-293T cells with relatively significant differences in cell sizes. Experimental results showed 100% cell trapping and 90% single cell trapping over 4 × 100 trap sites for these two cell types, respectively. The space saving is estimated to be 2-fold and the cell trapping speed enhancement to be 3-fold compared to previously reported devices. This device can be used for trapping various types of cells and expanded to trap cells in the order of tens of thousands on 1-cm² scale area, as a promising tool to pattern large-scale single cells on specific substrates and facilitate on-chip cellular assay at the single cell level.     

Transport and shear in a microfluidic membrane bilayer device for cell culture

Microfluidic devices have been established as useful platforms for cell culture for a broad range of applications, but challenges associated with controlling gradients of oxygen and other soluble factors and hemodynamic shear forces in small, confined channels have emerged. For instance, simple microfluidic constructs comprising a single cell culture compartment in a dynamic flow condition must handle tradeoffs between sustaining oxygen delivery and limiting hemodynamic shear forces imparted to the cells. These tradeoffs present significant difficulties in the culture of mesenchymal stem cells (MSCs), where shear is known to regulate signaling, proliferation, and expression. Several approaches designed to shield cells in microfluidic devices from excessive shear while maintaining sufficient oxygen concentrations and transport have been reported. Here we present the relationship between oxygen transport and shear in a "membrane bilayer" microfluidic device, in which soluble factors are delivered to a cell population by means of flow through a proximate channel separated from the culture channel by a membrane. We present an analytical model that describes the characteristics of this device and its ability to independently modulate oxygen delivery and hemodynamic shear imparted to the cultured cells. This bilayer configuration provides a more uniform oxygen concentration profile that is possible in a single-channel system, and it enables independent tuning of oxygen transport and shear parameters to meet requirements for MSCs and other cells known to be sensitive to hemodynamic shear stresses.     

Magnetophoretic-based microfluidic device for DNA isolation

This paper presents a continuous flow microfluidic device for the separation of DNA from blood using magnetophoresis for biological applications and analysis. This microfluidic bio-separation device has several benefits, including decreased sample handling, smaller sample and reagent volumes, faster isolation time, and decreased cost to perform DNA isolation. One of the key features of this device is the use of short-range magnetic field gradients, generated by a micro-patterned nickel array on the bottom surface of the separation channel. In addition, the device utilizes an array of oppositely oriented, external permanent magnets to produce strong long-range field gradients at the interfaces between magnets, further increasing the effectiveness of the device. A comprehensive simulation is performed using COMSOL Multiphysics to study the effect of various parameters on the magnetic flux within the separation channel. Additionally, a microfluidic device is designed, fabricated, and tested to isolate DNA from blood. The results show that the device has the capability of separating DNA from a blood sample with a purity of 1.8 or higher, a yield of up to 33 μg of polymerase chain reaction ready DNA per milliliter of blood, and a volumetric throughput of up to 50 ml/h.     

Embryonic stem cell bioprinting for uniform and controlled size embryoid body formation

Embryonic stem cells (ESCs) are pluripotent with multilineage potential to differentiate into virtually all cell types in the organism and thus hold a great promise for cell therapy and regenerative medicine. In vitro differentiation of ESCs starts with a phase known as embryoid body (EB) formation. EB mimics the early stages of embryogenesis and plays an essential role in ESC differentiation in vitro. EB uniformity and size are critical parameters that directly influence the phenotype expression of ESCs. Various methods have been developed to form EBs, which involve natural aggregation of cells. However, challenges persist to form EBs with controlled size, shape, and uniformity in a reproducible manner. The current hanging-drop methods are labor intensive and time consuming. In this study, we report an approach to form controllable, uniform-sized EBs by integrating bioprinting technologies with the existing hanging-drop method. The approach presented here is simple, robust, and rapid. We present significantly enhanced EB size uniformity compared to the conventional manual hanging-drop method.     

A polystyrene-based microfluidic device with three-dimensional interconnected microporous walls for perfusion cell culture

In this article, we present a simple, rapid prototyped polystyrene-based microfluidic device with three-dimensional (3D) interconnected microporous walls for long term perfusion cell culture. Patterned 3D interconnected microporous structures were created by a chemical treatment together with a protective mask and the native hydrophobic nature of the microporous structures were selectively made hydrophilic using oxygen plasma treatment together with a protective mask. Using this polystyrene-based cell culture microfluidic device, we successfully demonstrated the support of four days perfusion cell culture of hepatocytes (C3A cells).     

Bile canaliculi formation by aligning rat primary hepatocytes in a microfluidic device

In this study, we propose a microfluidic cell culture device mimicking the microscopic structure in liver tissue called hepatic cords. The cell culture area of the device was designed to align hepatocytes in two lines in a similar way to hepatic cords. Thanks to the structural design together with a cell seeding procedure, rat primary hepatocytes were successfully aligned in two lines and cultured under perfusion condition. It is shown that aligned hepatocytes gradually self-organize and form bile canaliculi along the hepatic cord-like structure. The present technique to culture hepatocytes with functional bile canaliculi could be used as an alternative to animal testing in the field of drug discovery and toxicological studies, and also be beneficial to tissue engineering applications.     

A modular cell culture device for generating arrays of gradients using stacked microfluidic flows

Microfluidics has become increasingly important for the study of biochemical cues because it enables exquisite spatiotemporal control of the microenvironment. Well-characterized, stable, and reproducible generation of biochemical gradients is critical for understanding the complex behaviors involved in many biological phenomena. Although many microfluidic devices have been developed which achieve these criteria, the ongoing challenge for these platforms is to provide a suitably benign and physiologically relevant environment for cell culture in a user-friendly format. To achieve this paradigm, microfluidic designs must consider the full scope of cell culture from substrate preparation, cell seeding, and long-term maintenance to properly observe gradient sensing behavior. In addition, designs must address the challenges associated with altered culture conditions and shear forces in flow-based devices. With this consideration, we have designed and characterized a microfluidic device based on the principle of stacked flows to achieve highly stable gradients of diffusible molecules over large areas with extremely low shear forces. The device utilizes a benign vacuum sealing strategy for reversible application to pre-established cell cultures. We apply this device to an existing culture of breast cancer cells to demonstrate the negligible effect of its shear flow on migratory behavior. Lastly, we extend the stacked-flow design to demonstrate its scalable architecture with a prototype device for generating an array of combinatorial gradients.     

Phaseguide-assisted blood separation microfluidic device for point-of-care applications

We propose a blood separation microfluidic device suitable for point-of-care (POC) applications. By utilizing the high gas permeability of polydimethylsiloxane (PDMS) and phaseguide structures, a simple blood separation device is presented. The device consists of two main parts. A separation chamber with the phaseguide structures, where a sample inlet, a tape-sealed outlet, and a dead-end ring channel are connected, and pneumatic chambers, in which manually operating syringes are plugged. The separation chamber and pneumatic chambers are isolated by a thin PDMS wall. By manually pulling out the plunger of the syringe, a negative pressure is instantaneously generated inside the pneumatic chamber. Due to the gas diffusion from the separation chamber to the neighboring pneumatic chamber through the thin permeable PDMS wall, low pressure can be generated, and then the whole blood at the sample inlets starts to be drawn into the separation chamber and separated through the phaseguide structures. Reversely, after removing the tape at the outlet and manually pushing in the plunger of the syringe, a positive pressure will be created which will cause the air to diffuse back into the ring channel, and therefore allow the separated plasma to be recovered at the outlet on demand. In this paper, we focused on the study of the plasma separation and associated design parameters, such as the PDMS wall thickness, the air permeable overlap area between the separation and pneumatic chambers, and the geometry of the phaseguides. The device required only 2 μl of whole blood but yielding approximately 0.38 μl of separated plasma within 12 min. Without any of the requirements of sophisticated equipment or dilution techniques, we can not only separate the plasma from the whole blood for on-chip analysis but also can push out only the separated plasma to the outlet for off-chip analysis.     

A cell electrofusion microfluidic device integrated with 3D thin-film microelectrode arrays

A microfluidic device integrated with 3D thin film microelectrode arrays wrapped around serpentine-shaped microchannel walls has been designed, fabricated and tested for cell electrofusion. Each microelectrode array has 1015 discrete microelectrodes patterned on each side wall, and the adjacent microelectrodes are separated by coplanar dielectric channel wall. The device was tested to electrofuse K562 cells under a relatively low voltage. Under an AC electric field applied between the pair of the microelectrode arrays, cells are paired at the edge of each discrete microelectrode due to the induced positive dielectrophoresis. Subsequently, electric pulse signals are sequentially applied between the microelectrode arrays to induce electroporation and electrofusion. Compared to the design with thin film microelectrode arrays deposited at the bottom of the side walls, the 3D thin film microelectrode array could induce electroporation and electrofusion under a lower voltage. The staggered electrode arrays on opposing side walls induce inhomogeneous electric field distribution, which could avoid multi-cell fusion. The alignment and pairing efficiencies of K562 cells in this device were 99% and 70.7%, respectively. The electric pulse of low voltage (～9 V) could induce electrofusion of these cells, and the fusion efficiency was about 43.1% of total cells loaded into the device, which is much higher than that of the convectional and most existing microfluidics-based electrofusion devices.     

Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.     

An integrated microfluidic device for rapid serodiagnosis of amebiasis

A microfluidic device was successfully fabricated for the rapid serodiagnosis of amebiasis. A micro bead-based immunoassay was fabricated within integrated microfluidic chip to detect the antibody to Entamoeba histolytica in serum samples. In this assay, a recombinant fragment of C terminus of intermediate subunit of galactose and N-acetyl-_D-galactosamine-inhibitable lectin of Entamoeba histolytica (C-Igl, aa 603-1088) has been utilized instead of the crude antigen. This device was validated with serum samples from patients with amebiasis and showed great sensitivity. The serodiagnosis can be completed within 20 min with 2 μl sample consumption. The device can be applied for the rapid and cheap diagnosis of other infectious disease, especially for the developing countries with very limited medical facilities.Entamoeba histolytica is the causative agent of amebiasis and is globally considered a leading parasitic cause of human mortality.¹ It has been estimated that 50 × 10⁶ people develop invasive disease such as amebic dysentery and amebic liver abscess, resulting in 100 000 deaths per annum.^{2, 3} High sensitive diagnosis method for early stage amebiasis is quite critical to prevent and cure this disease. To date, various serological tests have been used for the immune diagnosis of amebiasis, such as the indirect fluorescent antibody test (IFA) and enzyme-linked immunosorbent assay (ELISA).We have recently identified a 150-kDa surface antigen of E. histolytica as an intermediate subunit (Igl) of galactose and N-acetyl-_D-galactosamine-inhibitable lectin.^{4, 5} In particular, it has been shown that the C-terminus of Igl (C-Igl, aa 603-1088) was an especially useful antigen for the serodiagnosis of amebiasis. ELISA using C-Igl is more specific than the traditional ELISA using crude antigen.⁶ However, the ELISA process usually takes several hours, which is still labor-intensive and requires experienced operators to perform. More economic and convenient filed diagnosis methods are still in need, especially for the developing countries with limited medical facilities.Among all the bioanalytical techniques, microfluidics has been attracting more and more attention because of its low reagent/power consumption, the rapid analysis speed as well as easy automation.^{7, 8, 9, 10, 11} Especially with the development of the fabrication technique, microfluidics chip can include valves, mixers, pumps, heating devices, and even micro sensors, so many traditional bioanalytical methods can be performed in the microfluidics. Qualitative and quantitative immune analysis on the microfluidic chip was successfully proved by plenty of research with improved sensitivity, shorten reaction time, and less sample consumption.^{8, 10, 11, 12, 13, 14, 15, 16, 17} Moreover, with the intervention of other physical, chemical, biology, and electronic technology, microfluidic technique has been successfully utilized in protein crystallization, protein and gene analysis, cell capture and culturing and analysis as well as in the rapid and quantitative detection of microbes.^{13, 14, 15, 16, 17, 18, 19, 20}Herein, we report a new integrated microfluidic device, which is capable of rapid serodiagnosis of amebiasis with little sample consumption. The microfluidic device was fabricated from polydimethysiloxane (PDMS) following standard soft lithography.^{21, 22} The device was composed of two layers (shown in Figure ​Figure1)1) including upper fluidic layer (in green and blue) and bottom control layer (in red).Open in a separate windowFigure 1Structure illustration of microfluidic chip.To create the fluidic layer and the control layer, two different molds with different patterns have fabricated by photolithographic processes. The mold to create the fluidic channels was made by positive photoresist (AZ-50 XT), while the control pneumatic mold was made by negative photoresist (SU8 2025). For the chip fabrication, the fluidic layer is made from PDMS (RTV 615 A: B in ratio 5:1), and the pattern was transferred from the respective mold. The control layer is made from PDMS (RTV 615 A:B in ratio 20:1). The two layers were assembled and bonded together accurately, and there is elastic PDMS membrane about 30 μm thick between the fluidic layer channels and control layer.^{21, 22} The elastic membrane at the intersection can deform to block the fluid inside the fluidic channels, functioning as valves under the pressures introduced though control channels. There are two types of channels in fluidic layer, the rectangular profiled (in green, 200 μm wide, 35 μm thick) channel and round profiled channels (in blue, 200 μm wide, 25 μm center height). Because of the position of the valves on the fluidic channels, two types of valves (Figure ​(Figure2a)2a) were built, working as a standard valve and a sieve valve. The standard valves (on blue fluidic channels) can totally block the fluid because of the round profile of fluidic channel; the sieve valve can only half close because of the rectangular profile. The sieve valve can be used to trap the microspheres (beads) filled inside the green fluidic channels, while letting the fluid pass through. By this sieve valve, a micro column (in green) is constructed, where the entire ELISA reaction happens. The micrograph of the fabricated micro device is shown in Figure ​Figure2b.2b. The channels were filled with food dyes in different colors to show the relative positions of the channels. The pressures though different control channels are individually controlled by solenoid valves, connected to a computer through relay board. By programming the status (on/off) of various valves at different time periods, all the microfluidic chip operation can be digitally controlled by the computer in manual, semi-automatic, or automatic manner.Open in a separate windowFigure 2(a) Structure illustration of micro column, standard valve and sieve valve; (b) photograph of the microfluidic chip.To validate this device, 12 patient serum samples were collected. Sera from 9 patients (Nos. 1–9) with an amebic liver abscess or amebic colitis were used as symptomatic cases. The diagnosis of these patients was based on their clinical symptoms, ultrasound examination (liver abscess) and endoscopic or microscopic examination (colitis). We also identified the clinical samples using PCR amplification of rRNA genes.²⁴ As negative control, sera obtained from 3 healthy individuals with no known history of amebiasis were mixed into pool sera. The serum was positive for E. histolytica with a titer of 1:64 (borderline positive), as determined by an indirect fluorescent-antibody (IFA) test.^{23, 24} In our previously study, the sensitivity and specificity of the recombinant C-Igl in the ELISA were 97% and 99%.^{6, 25} In the current study, the serodiagnosis of amebiasis was also examined by ELISA using C-Igl.²⁶ The cut-off for a positive result was defined as an ELISA value > 3 SD above the mean for healthy negative controls²⁷ (shown in Figure ​Figure3).3). The seropositivity to C-Igl was 100% in patients with amebiasis.Open in a separate windowFigure 3ELISA reactivity of sera from patients against C-Igl. ELISA plate was coated with 100 ng per well of C-Igl. Serum samples from patients and healthy controls were used at 1:400 dilutions. The dashed line indicates the cut-off value. Data are representative of results from three independent experiments.In the diagnosis process with microfluidic chip, the 4 micro immuno-columns filled with C-Igl-coated microspheres were the key components of the device. The C-Igl was prepared in E. coli as inclusion bodies. After expression, the recombinant protein was purified and analyzed by SDS-PAGE. The apparent molecular mass was 85 kDa.²⁶The immune-reaction mechanism is illustrated in Figure ​Figure4.4. The anti-His monocolonal antibody was immobilized onto the microspheres (beads, 9 μm diameter) coated with protein A. The C-Igl was then immobilized onto the beads through the binding between the His tag and C-Igl. For the diagnosis, the microspheres immobilized with C-Igl and blocked by 5% BSA were preloaded into the columns for the rapid analysis of the patient serum samples. Generally, serum samples which were diluted 100 times were first loaded into the reaction column and incubated at room temperature for 5 min. After being washed by PBS buffer, FITC-conjugated goat anti-human polyclonal antibody was added into the column for 4 min incubation. The fluorescence image can be collected by the fluorescence microscope after the micro column was washed with PBS buffer. From loading diluted serum samples into column to collecting fluorescence images, the total time to complete the immunoassay is less than 10 min. The final fluorescence results were analyzed by Image Pro Plus 6.0.Open in a separate windowFigure 4Schematic representation of the ELISA in the chip.Different reaction conditions have been investigated to find the optimized ones. For each patient, 2 μl sample is enough for the analysis. The designed microfluidic chip with 4 micro columns is capable for 4 parallel analyses at the same time. More micro columns can be integrated into the device if more parallel tests are needed.Different incubating time for the diagnosis has also been investigated and no significant difference has been found for various time periods. It is enough to incubate the chip for only 5 min. The total diagnosis time for one sample is less than 10 min. The detection result appeared as the fluorescence intensity of the reaction column. As shown in Figure ​Figure5,5, the negative sample showed relatively low fluorescence intensity, because little FITC-conjugated goat anti-human polyclonal antibody could attach to the surface of microspheres; on the contrast, the positive sample showed much brighter fluorescence. The fluorescence intensity can be transferred to digital data (Table

On-chip three-dimensional tumor spheroid formation and pump-less perfusion culture using gravity-driven cell aggregation and balanced droplet dispensing

This paper presents a spheroid chip in which three-dimensional (3D) tumor spheroids are not only formed by gravity-driven cell aggregation but also cultured at the perfusion rates controlled by balanced droplet dispensing without fluidic pumps. The previous spheroid chips require additional off-chip processes of spheroid formation and extraction as well as bulky components of fluidic pumps. However, the present spheroid chip, where autonomous medium droplet dispensers are integrated on a well array, achieves the on-chip 3D tumor spheroid formation and perfusion culture using simple structure without bulky fluidic pumps. In the experimental study, we demonstrated that the spheroid chip successfully forms 3D tumor spheroids in the wide diameter range of 220 μm–3.2 mm (uniformity > 90%) using H358, H23, and A549 non-small cell lung cancer cells. At the pump-less perfusion culture (Q = 0.1–0.3 μl/min) of spheroids, the number of H358 cells in the spheroid increased up to 50% from the static culture (Q = 0 μl/min) and the viability of the cultured cells also increased about 10%. Therefore, we experimentally verified that the perfusion environment created by the spheroid chip offers a favourable condition to the spheroids with high increase rate and viability. The present chip achieves on-chip 3D tumor spheroid formation and pump-less perfusion culture with simple structure, thereby exhibiting potential for use in integrated in-vivo-like cell culture systems.     

Microbridge structures for uniform interval control of flowing droplets in microfluidic networks

Precise temporal control of microfluidic droplets such as synchronization and combinatorial pairing of droplets is required to achieve a variety range of chemical and biochemical reactions inside microfluidic networks. Here, we present a facile and robust microfluidic platform enabling uniform interval control of flowing droplets for the precise temporal synchronization and pairing of picoliter droplets with a reagent. By incorporating microbridge structures interconnecting the droplet-carrying channel and the flow control channel, a fluidic pressure drop was derived between the two fluidic channels via the microbridge structures, reordering flowing droplets with a defined uniform interval. Through the adjustment of the control oil flow rate, the droplet intervals were flexibly and precisely adjustable. With this mechanism of droplet spacing, the gelation of the alginate droplets as well as control of the droplet interval was simultaneously achieved by additional control oil flow including calcified oleic acid. In addition, by parallel linking identical microfluidic modules with distinct sample inlet, controlled synchronization and pairing of two distinct droplets were demonstrated. This method is applicable to facilitate and develop many droplet-based microfluidic applications, including biological assay, combinatorial synthesis, and high-throughput screening.     

Probing the mechanical properties of brain cancer cells using a microfluidic cell squeezer device

Despite being invasive within surrounding brain tissues and the central nervous system, little is known about the mechanical properties of brain tumor cells in comparison with benign cells. Here, we present the first measurements of the peak pressure drop due to the passage of benign and cancerous brain cells through confined microchannels in a “microfluidic cell squeezer” device, as well as the elongation, speed, and entry time of the cells in confined channels. We find that cancerous and benign brain cells cannot be differentiated based on speeds or elongation. We have found that the entry time into a narrow constriction is a more sensitive indicator of the differences between malignant and healthy glial cells than pressure drops. Importantly, we also find that brain tumor cells take a longer time to squeeze through a constriction and migrate more slowly than benign cells in two dimensional wound healing assays. Based on these observations, we arrive at the surprising conclusion that the prevailing notion of extraneural cancer cells being more mechanically compliant than benign cells may not apply to brain cancer cells.     

A microfluidic device for simultaneous electrical and mechanical measurements on single cells

This paper presents a microfluidic device for simultaneous mechanical and electrical characterization of single cells. The device performs two types of cellular characterization (impedance spectroscopy and micropipette aspiration) on a single chip to enable cell electrical and mechanical characterization. To investigate the performance of the device design, electrical and mechanical properties of MC-3T3 osteoblast cells were measured. Based on electrical models, membrane capacitance of MC-3T3 cells was determined to be 3.39±1.23 and 2.99±0.82 pF at the aspiration pressure of 50 and 100 Pa, respectively. Cytoplasm resistance values were 110.1±37.7 kΩ (50 Pa) and 145.2±44.3 kΩ (100 Pa). Aspiration length of cells was found to be 0.813±0.351 μm at 50 Pa and 1.771±0.623 μm at 100 Pa. Quantified Young's modulus values were 377±189 Pa at 50 Pa and 344±156 Pa at 100 Pa. Experimental results demonstrate the device's capability for characterizing both electrical and mechanical properties of single cells.     

A microfluidic cell for studying the formation of regenerated silk by synchrotron radiation small- and wide-angle X-ray scattering

A tube-in-square-pipe microfluidic glass cell has been developed for studying the aggregation and fiber formation from regenerated silk solution by in-situ small-angle X-ray scattering using synchrotron radiation. Acidification-induced aggregation has been observed close to the mixing point of the fibroin and buffer solution. The fibrous, amorphous material is collected in a water bath. Micro-wide-angle X-ray scattering of the dried material confirms its β-sheet nature.     

A tapered channel microfluidic device for comprehensive cell adhesion analysis, using measurements of detachment kinetics and shear stress-dependent motion

We have developed a method for studying cellular adhesion by using a custom-designed microfluidic device with parallel non-connected tapered channels. The design enables investigation of cellular responses to a large range of shear stress (ratio of 25) with a single input flow-rate. For each shear stress, a large number of cells are analyzed (500–1500 cells), providing statistically relevant data within a single experiment. Besides adhesion strength measurements, the microsystem presented in this paper enables in-depth analysis of cell detachment kinetics by real-time videomicroscopy. It offers the possibility to analyze adhesion-associated processes, such as migration or cell shape change, within the same experiment. To show the versatility of our device, we examined quantitatively cell adhesion by analyzing kinetics, adhesive strength and migration behaviour or cell shape modifications of the unicellular model cell organism Dictyostelium discoideum at 21 °C and of the human breast cancer cell line MDA-MB-231 at 37 °C. For both cell types, we found that the threshold stresses, which are necessary to detach the cells, follow lognormal distributions, and that the detachment process follows first order kinetics. In addition, for particular conditions’ cells are found to exhibit similar adhesion threshold stresses, but very different detachment kinetics, revealing the importance of dynamics analysis to fully describe cell adhesion. With its rapid implementation and potential for parallel sample processing, such microsystem offers a highly controllable platform for exploring cell adhesion characteristics in a large set of environmental conditions and cell types, and could have wide applications across cell biology, tissue engineering, and cell screening.