Microfluidic wet spinning of chitosan-alginate microfibers and encapsulation of HepG2 cells in fibers

The successful encapsulation of human hepatocellular carcinoma (HepG2) cells would greatly assist a broad range of applications in tissue engineering. Due to the harsh conditions during standard chitosan fiber fabrication processes, encapsulation of HepG2 cells in chitosan fibers has been challenging. Here, we describe the successful wet-spinning of chitosan-alginate fibers using a coaxial flow microfluidic chip. We determined the optimal mixing conditions for generating chitosan-alginate fibers, including a 1:5 ratio of 2% (w∕w) water-soluble chitosan (WSC) solution to 2% (w∕w) alginate solution. Ratio including higher than 2% (w∕w) WSC solution increased aggregation throughout the mixture. By suspending cells in the WSC-alginate solution, we successfully fabricated HepG2 cell-laden fibers. The encapsulated HepG2 cells in the chitosan-alginate fibers were more viable than cells encapsulated in pure alginate fibers, suggesting that cross-linked chitosan provides a better environment for HepG2 cells than alginate alone. In addition, we found that the adhesion of HepG2 cells on the chitosan-alginate fiber is much better than that on the alginate fibers.     

Microfluidic fabrication of polymeric core-shell microspheres for controlled release applications

We report a facile and robust microfluidic method to fabricate polymeric core-shell microspheres as delivery vehicles for biomedical applications. The characteristics of core-shell microspheres can be precisely and easily tuned by manipulating the microfluidic double emulsion templates. The addition of a shell can significantly improve the versatility as well as functionality of these microspheres as delivery vehicles. We demonstrate that the nature of the shell material plays an important role in the properties of the core-shell delivery vehicles. The release kinetics is significantly influenced by the material of the shell and other characteristics such as the thickness. For example, by adding a poly(lactic-co-glycolic acid) (PLGA) shell to an alginate core, the encapsulation efficiency is enhanced and undesired leakage of hydrophilic actives is prevented. By contrast, adding an alginate shell to PLGA core can lead to a reduction of the initial release rate, thus extending the release period of hydrophobic actives. Microfluidic fabrication enables the generation of precisely controlled core-shell microspheres with a narrow size distribution, which enables the investigation of the relationship between the release kinetics of these microspheres and their characteristics. The approach of using core-shell particles as delivery vehicles creates new opportunities to customize the release kinetics of active ingredients.     

Microfluidic formulation of pectin microbeads for encapsulation and controlled release of nanoparticles

We report a method for formulation of pectin microbeads using microfluidics. The technique uses biocompatible ingredients and allows for controlled external gelation with hydrogen and calcium ions delivered from an organic phase of rapeseed oil. This method allows for encapsulation of nanoparticles into the microparticles of gel and for control of the rate of their release.     

Microfluidic droplet encapsulation of highly motile single zoospores for phenotypic screening of an antioomycete chemical

Highly motile Phytophthora sojae (P. sojae) zoospores of an oomycete plant pathogen and antioomycete candidate chemicals were encapsulated into microdroplets. Random fast self-motion of P. sojae zoospores was overcome by choosing an appropriate flow rate for a zoospore suspension. To influence stochastic loading of zoospores into a microfluidic channel, a zoospore suspension was directly preloaded into a microtubing with a largely reduced inner diameter. A relatively high single zoospore encapsulation rate of 60.5% was achieved on a most trivial T-junction droplet generator platform, without involving any specially designed channel geometry. We speculated that spatial reduction in the diameter direction of microtubing added a degree of zoospore ordering in the longitudinal direction of microtubing and thus influenced positively to change the inherent limitation of stochastic encapsulation of zoospores. Comparative phenotypic study of a plant oomycete pathogen at a single zoospore level had not been achieved earlier. Phenotypic changes of zoospores responding to various chemical concentration conditions were measured in multiple droplets in parallel, providing a reliable data set and thus an improved statistic at a low chemical consumption. Since each droplet compartment contained a single zoospore, we were able to track the germinating history of individual zoospores without being interfered by other germinating zoospores, achieving a high spatial resolution. By adapting some existing droplet immobilization and concentration gradient generation techniques, the droplet approach could potentially lead to a medium-to-high throughput, reliable screening assay for chemicals against many other highly motile zoospores of pathogens.     

Microfluidic cell fragmentation for mechanical phenotyping of cancer cells

Circulating tumor cells (CTCs) shed from the primary tumor undergo significant fragmentation in the microvasculature, and very few escape to instigate metastases. Inspired by this in vivo behavior of CTCs, we report a microfluidic method to phenotype cancer cells based on their ability to arrest and fragment at a micropillar-based bifurcation. We find that in addition to cancer cell size, mechanical properties determine fragmentability. We observe that highly metastatic prostate cancer cells are more resistant to fragmentation than weakly metastatic cells, providing the first indication that metastatic CTCs can escape rupture and potentially initiate secondary tumors. Our method may thus be useful in identifying phenotypes that succumb to or escape mechanical trauma in microcirculation.     

A microfluidic manifold with a single pump system to generate highly mono-disperse alginate beads for cell encapsulation

Cell encapsulation technology is a promising strategy applicable to tissue engineering and cell therapy. Many advanced microencapsulation chips that function via multiple syringe pumps have been developed to generate mono-disperse hydrogel beads encapsulating cells. However, their operation is difficult and only trained microfluidic engineers can use them with dexterity. Hence, we propose a microfluidic manifold system, driven by a single syringe pump, which can enable the setup of automated flow sequences and generate highly mono-disperse alginate beads by minimizing disturbances to the pump pressure. The encapsulation of P19 mouse embryonic carcinoma cells and embryonic body formation are demonstrated to prove the efficiency of the proposed system.     

Microfluidic electromanipulation with capacitive detection for the mechanical analysis of cells

The mechanical behavior of cells offers insight into many aspects of their properties. We propose an approach to the mechanical analysis of cells that uses a combination of electromanipulation for stimulus and capacitance for sensing. To demonstrate this approach, polystyrene spheres and yeast cells flowing in a 25 μm×100 μm microfluidic channel were detected by a perpendicular pair of gold thin film electrodes in the channel, spaced 25 μm apart. The presence of cells was detected by capacitance changes between the gold electrodes. The capacitance sensor was a resonant coaxial radio frequency cavity (2.3 GHz) coupled to the electrodes. The presence of yeast cells (Saccharomyces cerevisiae) and polystyrene spheres resulted in capacitance changes of approximately 10 and 100 attoFarad (aF), respectively, with an achieved capacitance resolution of less than 2 aF in a 30 Hz bandwidth. The resolution is better than previously reported in the literature, and the capacitance changes are in agreement with values estimated by finite element simulations. Yeast cells were trapped using dielectrophoretic forces by applying a 3 V signal at 1 MHz between the electrodes. After trapping, the cells were displaced using amplitude and frequency modulated voltages to produce modulated dielectrophoretic forces. Repetitive displacement and relaxation of these cells was observed using both capacitance and video microscopy.     

Microfluidic impedance cytometry of tumour cells in blood

The dielectric properties of tumour cells are known to differ from normal blood cells, and this difference can be exploited for label-free separation of cells. Conventional measurement techniques are slow and cannot identify rare circulating tumour cells (CTCs) in a realistic timeframe. We use high throughput single cell microfluidic impedance cytometry to measure the dielectric properties of the MCF7 tumour cell line (representative of CTCs), both as pure populations and mixed with whole blood. The data show that the MCF7 cells have a large membrane capacitance and size, enabling clear discrimination from all other leukocytes. Impedance analysis is used to follow changes in cell viability when cells are kept in suspension, a process which can be understood from modelling time-dependent changes in the dielectric properties (predominantly membrane conductivity) of the cells. Impedance cytometry is used to enumerate low numbers of MCF7 cells spiked into whole blood. Chemical lysis is commonly used to remove the abundant erythrocytes, and it is shown that this process does not alter the MCF7 cell count or change their dielectric properties. Combining impedance cytometry with magnetic bead based antibody enrichment enables MCF7 cells to be detected down to 100 MCF7 cells in 1 ml whole blood, a log 3.5 enrichment and a mean recovery of 92%. Microfluidic impedance cytometry could be easily integrated within complex cell separation systems for identification and enumeration of specific cell types, providing a fast in-line single cell characterisation method.     

Microfluidic based high throughput synthesis of lipid-polymer hybrid nanoparticles with tunable diameters

Core-shell hybrid nanoparticles (NPs) for drug delivery have attracted numerous attentions due to their enhanced therapeutic efficacy and good biocompatibility. In this work, we fabricate a two-stage microfluidic chip to implement a high-throughput, one-step, and size-tunable synthesis of mono-disperse lipid-poly (lactic-co-glycolic acid) NPs. The size of hybrid NPs is tunable by varying the flow rates inside the two-stage microfluidic chip. To elucidate the mechanism of size-controllable generation of hybrid NPs, we observe the flow field in the microchannel with confocal microscope and perform the simulation by a numerical model. Both the experimental and numerical results indicate an enhanced mixing effect at high flow rate, thus resulting in the assembly of small and mono-disperse hybrid NPs. In vitro experiments show that the large hybrid NPs are more likely to be aggregated in serum and exhibit a lower cellular uptake efficacy than the small ones. This microfluidic chip shows great promise as a robust platform for optimization of nano drug delivery system.     

Microfluidic separation of live and dead yeast cells using reservoir-based dielectrophoresis

Separating live and dead cells is critical to the diagnosis of early stage diseases and to the efficacy test of drug screening, etc. This work demonstrates a novel microfluidic approach to dielectrophoretic separation of yeast cells by viability. It exploits the cell dielectrophoresis that is induced by the inherent electric field gradient at the reservoir-microchannel junction to selectively trap dead yeast cells and continuously separate them from live ones right inside the reservoir. This approach is therefore termed reservoir-based dielectrophoresis (rDEP). It has unique advantages as compared to existing dielectrophoretic approaches such as the occupation of zero channel space and the elimination of any mechanical or electrical parts inside microchannels. Such an rDEP cell sorter can be readily integrated with other components into lab-on-a-chip devices for applications to biomedical diagnostics and therapeutics.     

Microfluidic production of monodisperse emulsions for cosmetics

Droplet-based microfluidic technology has enabled the production of emulsions with high monodispersity in sizes ranging from a few to hundreds of micrometers. Taking advantage of this technology, attempts to generate monodisperse emulsion drops with high drug loading capacity, ordered interfacial structure, and multi-functionality have been made in the cosmetics industry. In this article, we introduce the practicality of the droplet-based microfluidic approach to the cosmetic industry in terms of innovation in productivity and marketability. Furthermore, we summarize some recent advances in the production of emulsion drops with enhanced mechanical interfacial stability. Finally, we discuss the future prospects of microfluidic technology in accordance with consumers'' needs and industrial attributes.     

Microfluidic separation of viruses from blood cells based on intrinsic transport processes

Clinical analysis of acute viral infection in blood requires the separation of viral particles from blood cells, since the cytoplasmic enzyme inhibits the subsequent viral detection. To facilitate this procedure in settings without access to a centrifuge, we present a microfluidic device to continuously purify bionanoparticles from cells based on their different intrinsic movements on the microscale. In this device, a biological sample is layered on top of a physiological buffer, and both fluids are transported horizontally at the same flow rate in a straight channel under laminar flow. While the micron sized particles such as cells sediment to the bottom layer with a predictable terminal velocity, the nanoparticles move vertically by diffusion. As their vertical travel distances have a different dependence on time, the micro- and nanoparticles can preferentially reside in the bottom and top layers respectively after certain residence time, yielding purified viruses. We first performed numerical analysis to predicate the particle separation and then tested the theory using suspensions of synthetic particles and biological samples. The experimental results using dilute synthetic particles closely matched the numerical analysis of a two layer flow system containing different sized particles. Similar purification was achieved using diluted blood spiked with human immunodeficiency virus. However, viral purification in whole blood is compromised due to extensive bioparticle collisions. With the parallelization and automation potential offered by microfluidics, this device has the potential to function as an upstream sample preparation module to continuously provide cell depleted bio-nanoparticles for downstream analysis.     

Microfluidic size separation of cells and particles using a swinging bucket centrifuge

Biomolecular separation is crucial for downstream analysis. Separation technique mainly relies on centrifugal sedimentation. However, minuscule sample volume separation and extraction is difficult with conventional centrifuge. Furthermore, conventional centrifuge requires density gradient centrifugation which is laborious and time-consuming. To overcome this challenge, we present a novel size-selective bioparticles separation microfluidic chip on a swinging bucket minifuge. Size separation is achieved using passive pressure driven centrifugal fluid flows coupled with centrifugal force acting on the particles within the microfluidic chip. By adopting centrifugal microfluidics on a swinging bucket rotor, we achieved over 95% efficiency in separating mixed 20 μm and 2 μm colloidal dispersions from its liquid medium. Furthermore, by manipulating the hydrodynamic resistance, we performed size separation of mixed microbeads, achieving size efficiency of up to 90%. To further validate our device utility, we loaded spiked whole blood with MCF-7 cells into our microfluidic device and subjected it to centrifugal force for a mere duration of 10 s, thereby achieving a separation efficiency of over 75%. Overall, our centrifugal microfluidic device enables extremely rapid and label-free enrichment of different sized cells and particles with high efficiency.     

Microfluidic parallel circuit for measurement of hydraulic resistance

We present a microfluidic parallel circuit that directly compares the test channel of an unknown hydraulic resistance with the reference channel with a known resistance, thereby measuring the unknown resistance without any measurement setup, such as standard pressure gauges. Many of microfluidic applications require the precise transport of fluid along a channel network with complex patterns. Therefore, it is important to accurately characterize and measure the hydraulic resistance of each channel segment, and determines whether the device principle works well. However, there is no fluidic device that includes features, such as the ability to diagnose microfluidic problems by measuring the hydraulic resistance of a microfluidic component in microscales. To address the above need, we demonstrate a simple strategy to measure an unknown hydraulic resistance, by characterizing the hydraulic resistance of microchannels with different widths and defining an equivalent linear channel of a microchannel with repeated patterns of a sudden contraction and expansion.     

Microfluidic assessment of swimming media for motility-based sperm selection

Selection medium is important in sperm isolation for assisted reproductive technologies. Contrary to the naturally occurring human cervical mucus which has a high viscosity, most current practices for motility based sperm selection use a low viscosity medium. In this study, we used a microfluidic device to assess the effects of high viscosity media made with hyaluronic acid (HA) and methyl cellulose (MC) on bovine and human sperm motility and viability (sperm transferred directly from cryoprotectant). The microfluidic penetration test, viability, and motility were compared for sperm swimming in both HA and MC media with about 20cp viscosity (measured at 20 °C). Our resulted indicate that MC medium resulted in a significantly higher number of viable bovine sperm penetrating the medium as compared to HA. Furthermore, MC resulted in the selection of a sperm subpopulation with a 274% increase in sperm viability in comparison to the raw semen, while HA increased viability by only 133%. In addition to viability, bovine sperm motility parameters were significantly higher in the MC medium as compared with HA. Experiments with human sperm swimming in MC indicate that sperm swim slower and straighter at higher viscosities. In conclusion, the results indicate that in a micro-confined environment representative of the in vivo environment, MC is a preferred high viscosity medium to ensure the highest concentration of motile and viable sperm.     

Droplet based microfluidic fabrication of designer microparticles for encapsulation applications

Developing carriers of active ingredients with pre-determined release kinetics is a main challenge in the field of controlled release. In this work, we fabricate designer microparticles as carriers of active ingredients using droplet microfluidics. We show that monodisperse droplet templates do not necessarily produce monodisperse particles. Magnetic stirring, which is often used to enhance the droplet solidification rate, can promote breakup of the resultant microparticles into fragments; with an increase in the stirring time, microparticles become smaller in average size and more irregular in shape. Thus, the droplet solidification conditions affect the size, size distribution and morphology of the fabricated particles, and these attributes of the microparticles strongly influence their release kinetics. The smaller the average size of the microparticles is, the higher the initial release rate is. The release kinetics of drug carriers is strongly related to their characteristics. The understanding of this relationship enables the fabrication of tailor-designed carriers with a specified release rate, and even programmed release to meet the needs of applications that require a complex release profile of the active ingredients.     

Microfluidic chip for fast bioassays��evaluation of binding parameters

A seven channel polystyrene (PS) microchip has been constructed using a micromilling machine and a high-temperature assembling. Protein A (PA) has been immobilized by a passive sorption on the microchannel walls. Two bioaffinity assays with human immunoglobulin G (hIgG) as a ligand have been carried out. (i) PA as the receptor and fluorescently labeled hIgG (FITC-hIgG) as the ligand, (ii) PA as the receptor with hIgG as the quantified ligand and fluorescently labeled goat anti-human IgG (FITC-gIgG) as the secondary ligand. One incubation step of the assays took only 5 min instead of hours typical for enzyme-linked immunosorbent assay applications. Calibration curves of the dependence of a fluorescence signal on the hIgG concentration in a sample have been obtained in one step due to a parallel arrangement of microchannels. A mathematical model of the PA-FITC-hIgG complex formation in the chip has been developed. The values of the kinetic constant of the PA-FITC-hIgG binding (k_on=5.5 m³ mol⁻¹ s⁻¹) and the equilibrium dissociation constant of the formed complex (K_d≤3×10⁻⁶ mol m⁻³) have been obtained by fitting to experimental data. The proposed microchip enables fast evaluation of kinetic and equilibrium constants of ligand-receptor bioaffinity pairs and the ligand quantification. As the use of microfluidic chips for immunoassays is often limited by price, we used procedures and chemicals that allow for an inexpensive construction and operation of the microdevice, e.g., temperature assembling as a fabrication technique, detection via an ordinary digital camera, nonspecific polystyrene as a substrate, passive sorption of biomolecules as an immobilization technique, etc.     

Microfluidic device capable of medium recirculation for non-adherent cell culture

We present a microfluidic device designed for maintenance and culture of non-adherent mammalian cells, which enables both recirculation and refreshing of medium, as well as easy harvesting of cells from the device. We demonstrate fabrication of a novel microfluidic device utilizing Braille perfusion for peristaltic fluid flow to enable switching between recirculation and refresh flow modes. Utilizing fluid flow simulations and the human promyelocytic leukemia cell line, HL-60, non-adherent cells, we demonstrate the utility of this RECIR-REFRESH device. With computer simulations, we profiled fluid flow and concentration gradients of autocrine factors and found that the geometry of the cell culture well plays a key role in cell entrapping and retaining autocrine and soluble factors. We subjected HL-60 cells, in the device, to a treatment regimen of 1.25% dimethylsulfoxide, every other day, to provoke differentiation and measured subsequent expression of CD11b on day 2 and day 4 and tumor necrosis factor-alpha (TNF-α) on day 4. Our findings display perfusion sensitive CD11b expression, but not TNF-α build-up, by day 4 of culture, with a 1:1 ratio of recirculation to refresh flow yielding the greatest increase in CD11b levels. RECIR-REFRESH facilitates programmable levels of cell differentiation in a HL-60 non-adherent cell population and can be expanded to other types of non-adherent cells such as hematopoietic stem cells.     

磁性琼脂糖微球活化条件的研究

对环氧氯丙烷活化琼脂糖磁性微球的活化条件进行了研究。以微球表面的环氧基密度为指标,考察了活化时间、Na OH浓度、环氧氯丙烷体积分数、Na BH4浓度和活化温度等因素对琼脂糖磁性微球活性的影响。实验结果表明,环氧氯丙烷活化琼脂糖磁性微球的最优条件为活化时间为4 h,Na OH浓度为0.9 mol/L,环氧氯丙烷体积分数为40%,Na BH4的浓度为0.5g/L,活化温度为40℃。