Microfluidic depletion of endothelial cells, smooth muscle cells, and fibroblasts from heterogeneous suspensions

Interactions between ligands and cell surface receptors can be exploited to design adhesion-based microfluidic cell separation systems. When ligands are immobilized on the microfluidic channel surfaces, the resulting cell capture devices offer the typical advantages of small sample volumes and low cost associated with microfluidic systems, with the added benefit of not requiring complex fabrication schemes or extensive operational infrastructure. Cell-ligand interactions can range from highly specific to highly non-specific. This paper describes the design of an adhesion-based microfluidic separation system that takes advantage of both types of interactions. A 3-stage system of microfluidic devices coated with the tetrapeptides arg-glu-asp-val (REDV), val-ala-pro-gly (VAPG), and arg-gly-asp-ser (RGDS) is utilized to deplete a heterogeneous suspension containing endothelial cells, smooth muscle cells, and fibroblasts. The ligand-coated channels together with a large surface area allow effective depletion of all three cell types in a stagewise manner.     

In vitro model on glass surfaces for complex interactions between different types of cells

This report establishes an in vitro model on glass surfaces for patterning multiple types of cells to simulate cell-cell interactions in vivo. The model employs a microfluidic system and poly(ethylene glycol)-terminated oxysilane (PEG-oxysilane) to modify glass surfaces in order to resist cell adhesion. The system allows the selective confinement of different types of cells to realize complete confinement, partial confinement, and no confinement of three types of cells on glass surfaces. The model was applied to study intercellular interactions among human umbilical vein endothelial cells (HUVEC), PLA 801 C and PLA801 D cells.     

Patterning cells and shear flow conditions: convenient observation of endothelial cell remoulding, enhanced production of angiogenesis factors and drug response

We present a method that allows patterning cells and shear flow conditions for endothelial cell based assays. This method is novel in combining (1) cell culture on the surface of a substrate both topographically and chemically patterned; (2) multi-shear flow assays after covering the cell substrate with a microfluidic cover plate containing microchannels of different channel widths, and (3) conventional immunostaining assays after removal of the cover plate. This method has the advantage of performing cell cultures and immunoassays in standard cell biology environments with open access, facilitating the formation of confluent cell layers and the observation of cell responses to shear-flow and drug stimulations. To obtain multi-shear stress conditions, a single channel with stepwise increasing channel widths was patterned on the surfaces of both the substrate and the microfluidic cover plate. As results, we observed excellent viability of endothelial cells in the whole range of applied shear stresses (0-25 dyn cm(-2)) and shear stress dependent cytoskeleton remoulding, activation of von Willebrand factor (vWF), and re-organisation of angiogenesis factors such as tetra peptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) of endothelial cells. To validate this approach for drug analysis, we also studied drug effects under shear stress conditions. Our results indicate that the drug effect of combretastatin A-4, an anti-tumour vascular targeting drug, could be significantly enhanced under shear flow conditions.     

Effect of surface nanotopography on immunoaffinity cell capture in microfluidic devices

Immunoaffinity microfluidic devices have recently become a popular choice to isolate specific cells for many applications. To increase cell capture efficiency, several groups have employed capture beds with nanotopography. However, no systematic study has been performed to quantitatively correlate surface nanopatterns with immunoaffinity cell immobilization. In this work, we controlled substrate topography by depositing close-packed arrays of silica nanobeads with uniform diameters ranging from 100 to 1150 nm onto flat glass. These surfaces were functionalized with a specific antibody and assembled as the base in microfluidic channels, which were then used to capture CD4+ T cells under continuous flow. It is observed that capture efficiency generally increases with nanoparticle size under low flow rate. At higher flow rates, cell capture efficiency becomes increasingly complex; it initially increases with the bead size then gradually decreases. Surprisingly, capture yield plummets atop depositions of some particle diameters. These dips likely stem from dynamic interactions between nanostructures on the substrate and cell membrane as indicated by roughness-insensitive cell capture after glutaraldehyde fixing. This systematic study of surface nanotopography and cell capture efficiency will help optimize the physical properties of microfluidic capture beds for cell isolation from biological fluids.     

Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity

Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.     

Monitoring the status of T-cell activation in a microfluidic system

Leukocyte adhesion to the endothelium through surface molecules such as E-selectin and intercellular adhesion molecule-1 (ICAM-1) is a critical cellular event reflecting the physiological status of both cell types. Here we present a microfluidic system that can not only easily monitor the interaction between leukocytes and endothelial cells under physiological conditions, but also screen drug candidates for potential modulation of this interaction. Shear stress, which is an important factor for the binding of activated T cells to tumor necrosis factor-alpha (TNF-α)-treated human umbilical vein endothelial cells (HUVECs), was easily controlled by adjusting the flow rate in the microfluidic system. Whole blood of patients with systemic lupus erythematosus (SLE) who have auto-reactive T cells were infused into the activated HUVECs which subsequently showed a higher level of binding compared to a control blood sample from a person without SLE. When these autoreactive T cells were treated with immunosuppressors tacrolimus and cyclosporin A, the binding of the T cells to HUVECs was dramatically decreased. Therefore, this microfluidic system is capable of differentiating the physiological status of T cells or endothelial cells representing different disease conditions, as well as being useful for the identification of novel reagents that modulate the functions of leukocytes or endothelial cells.     

Label-free, microfluidic separation and enrichment of human breast cancer cells by adhesion difference

A label-free microfluidic method for separation and enrichment of human breast cancer cells is presented using cell adhesion as a physical marker. To maximize the adhesion difference between normal epithelial and cancer cells, flat or nanostructured polymer surfaces (400 nm pillars, 400 nm perpendicular, or 400 nm parallel lines) were constructed on the bottom of polydimethylsiloxane (PDMS) microfluidic channels in a parallel fashion using a UV-assisted capillary moulding technique. The adhesion of human breast epithelial cells (MCF10A) and cancer cells (MCF7) on each channel was independently measured based on detachment assays where the adherent cells were counted with increasing flow rate after a pre-culture for a period of time (e.g., one, two, and four hours). It was found that MCF10A cells showed higher adhesion than MCF7 cells regardless of culture time and surface nanotopography at all flow rates, resulting in label-free separation and enrichment of cancer cells. For the cell types used in our study, an optimum separation was found for 2 hours pre-culture on the 400 nm perpendicular line pattern followed by flow-induced detachment at a flow rate of 200 microl min(-1). The fraction of MCF7 cells was increased from 0.36 +/- 0.04 to 0.83 +/- 0.04 under these optimized conditions.     

A palmtop-sized microfluidic cell culture system driven by a miniaturized infusion pump

A palmtop-sized microfluidic cell culture system is presented. The system consists of a microfluidic device and a miniaturized infusion pump that possesses a reservoir of culture medium, an electrical control circuit, and an internal battery. The footprint of the system was downsized to 87 × 57 mm, which is, to the best of our knowledge, the smallest integrated cell culture system. Immortalized human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC) were cultured in the system. HMEC-1 in the system proliferated at the same speed as cells in a microchannel perfused by a syringe pump and cells in a culture flask. HUVEC in the system oriented along the direction of the fluid flow. Claudin-5, a tight junction protein, was localized along the peripheries of the HUVEC. We expect that the present system is applicable to various cell types as a stand-alone and easy-to-use system for microfluidic bioanalysis.     

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.     

High-density microfluidic arrays for cell cytotoxicity analysis

In this paper, we report on the development of a multilayer elastomeric microfluidic array platform for the high-throughput cell cytotoxicity screening of mammalian cell lines. Microfluidic channels in the platform for cell seeding are orthogonal to channels for toxin exposure, and within each channel intersection is a circular chamber with cell-trapping sieves. Integrated, pneumatically-actuated elastomeric valves within the device isolate the microchannel array within the device into parallel rows and columns for cell seeding and toxin exposure. As a demonstration of the multiplexing capability of the platform, a microfluidic array containing 576 chambers was used to screen three cell types (BALB/3T3, HeLa, and bovine endothelial cells) against a panel of five toxins (digitonin, saponin, CoCl(2), NiCl(2), acrolein). Evaluation of on-chip cell morphology and viability was carried out using fluorescence microscopy, with outcomes comparable to microtiter plate cytotoxicity assays. Using this scalable platform, cell seeding and toxin exposure can be carried out within a single microfluidic device in a multiplexed format, enabling high-density parallel cytotoxicity screening while minimizing reagent consumption.     

Polystyrene-coated micropallets for culture and separation of primary muscle cells

Despite identification of a large number of adult stem cell types, current primary cell isolation and identification techniques yield heterogeneous samples, making detailed biological studies challenging. To identify subsets of isolated cells, technologies capable of simultaneous cell culture and cloning are necessary. Micropallet arrays, a new cloning platform for adherent cell types, hold great potential. However, the microstructures composing these arrays are fabricated from an epoxy photoresist 1002F, a growth surface unsuitable for many cell types. Optimization of the microstructures’ surface properties was conducted for the culture of satellite cells, primary muscle cells for which improved cell isolation techniques are desired. A variety of surface materials were screened for satellite cell adhesion and proliferation and compared to their optimal substrate, gelatin-coated Petri dishes. A 1-μm thick, polystyrene copolymer was applied to the microstructures by contact printing. A negatively charged copolymer of 5% acrylic acid in 95% styrene was found to be equivalent to the control Petri dishes for cell adhesion and proliferation. Cells cultured on control dishes and optimal copolymer-coated surfaces maintained an undifferentiated state and showed similar mRNA expression for two genes indicative of cell differentiation during a standard differentiation protocol. Experiments using additional contact-printed layers of extracellular matrix proteins collagen and gelatin showed no further improvements. This micropallet coating strategy is readily adaptable to optimize the array surface for other types of primary cells.     

A microfluidic "baby machine" for cell synchronization

Common techniques used to synchronize eukaryotic cells in the cell cycle often impose metabolic stress on the cells or physically select for size rather than age. To address these deficiencies, a minimally perturbing method known as the "baby machine" was developed previously. In the technique, suspension cells are attached to a membrane, and as the cells divide, the newborn cells are eluted to produce a synchronous population of cells in the G1 phase of the cell cycle. However, the existing "baby machine" is only suitable for cells which can be chemically attached to a surface. Here, we present a microfluidic "baby machine" in which cells are held onto a surface by pressure differences rather than chemical attachment. As a result, our method can in principle be used to synchronize a variety of cell types, including cells which may have weak or unknown surface attachment chemistries. We validate our microfluidic "baby machine" by using it to produce a synchronous population of newborn L1210 mouse lymphocytic leukemia cells in G1 phase.     

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.     

Continuous sorting of magnetic cells via on-chip free-flow magnetophoresis

The ability to separate living cells is an essential aspect of cell research. Magnetic cell separation methods are among some of the most efficient methods for bulk cell separation. With the development of microfluidic platforms within the biotechnology sector, the design of miniaturised magnetic cell sorters is desirable. Here, we report the continuous sorting of cells loaded with magnetic nanoparticles in a microfluidic magnetic separation device. Cells were passed through a microfluidic chamber and were deflected from the direction of flow by means of a magnetic field. Two types of cells were studied, mouse macrophages and human ovarian cancer cells (HeLa cells). The deflection was dependent on the magnetic moment and size of the cells as well as on the applied flow rate. The experimentally observed deflection matched well with calculations. Furthermore, the separation of magnetic and non-magnetic cells was demonstrated using the same microfluidic device.     

Human plasma protein adsorption onto dextranized surfaces: a two-dimensional electrophoresis and mass spectrometry study

Protein adsorption is fundamental to thrombosis and to the design of biocompatible materials. We report a two-dimensional electrophoresis and mass spectrometry study to characterize multiple human plasma proteins adsorbed onto four different types of model surfaces: silicon oxide, dextranized silicon, polyurethane and dextranized polyurethane. Dextran was grafted onto the surfaces of silicon and polyurethane to mimic the blood-contacting endothelial cell glycocalyx surface. Surface topography and hydrophobicity/hydrophilicity were determined and analyzed using atomic force microscopy and water contact angle measurements, respectively. Using two-dimensional electrophoresis, we show that, relative to the unmodified surfaces, dextranization significantly inhibits the adsorption of several human plasma proteins including IGHG1 protein, fibrinogen, haptoglobin, Apo A-IV, Apo A-I, immunoglobulin, serum retinal-binding protein and truncated serum albumin. We further demonstrate the selectivity of plasma protein adsorbed onto the different functionalized surfaces and the potential to control and manipulate proteins adsorption on the surfaces of medical devices, implants and microfluidic devices. This result shows that adsorption experiments using a single protein or a binary mixture of proteins are consistent with competitive protein adsorption studies. In summary, these studies indicate that coating blood-contacting biomedical applications with dextran is an effective route to reduce thrombo-inflammatory responses and to surface-direct biological activities.     

An Enzyme-Loaded Metal–Organic Framework-Assisted Microfluidic Platform Enables Single-Cell Metabolite Analysis

Colonization of cancer cells at secondary sites, a decisive step in tumor metastasis, is strongly dependent on the formation of metastatic microenvironments regulated by intrinsic single-cell metabolism traits. Herein, we report a single-cell microfluidic platform for high-throughput dynamic monitoring of tumor cell metabolites to evaluate tumor malignancy. This microfluidic device empowers efficient isolation of single cells (>99 %) in a squashed state similar to tumor extravasation, and employs enzyme-packaged metal–organic frameworks to catalyze tumor cell metabolites for visualization. The microfluidic evaluation was confirmed by in vivo assays, suggesting that the platform allowed predicting the tumorigenicity of captured tumor cells and screening metabolic inhibitors as anti-metastatic drugs. Furthermore, the platform efficiently detected various aggressive cancer cells in unprocessed whole blood samples with high sensitivity, showing potential for clinical application.     

Microfluidic biofunctionalisation protocols to form multi‐valent interactions for cell rolling and phenotype modification investigations

In this study, we propose a fast, simple method to biofunctionalise microfluidic systems for cellomic investigations based on micro‐fluidic protocols. Many available processes either require expensive and time‐consuming protocols or are incompatible with the fabrication of microfluidic systems. Our method differs from the existing since it is applicable to an assembled system, uses few microlitres of reagents and it is based on the use of microbeads. The microbeads have specific surface moieties to link the biomolecules and couple cell receptors. Furthermore, the microbeads serve as arm spacer and offer the benefit of the multi‐valent interaction. Microfluidics was adapted together with topology and biochemistry surface modifications to offer the microenvironment for cellomic studies. Based on this principle, we exploit the streptavidin–biotin interaction to couple antibodies to the biofunctionalised microfluidic environment within 5 h using 200 μL of reagents and biomolecules. We selected the antibodies able to form complexes with the MHC class I (MHC‐I) molecules present on the cell membrane and involved in the immune surveillance. To test the microfluidic system, tumour cell lines (RMA) were rolled across the coupled antibodies to recognise and strip MHC‐I molecules. As result, we show that cell rolling performed inside a microfluidic chamber functionalised with beads and the opportune antibody facilitate the removal of MHC class I molecules. We showed that the level of median fluorescent intensity of the MHC‐I molecules is 300 for cells treated in a not biofunctionalised surface. It decreased to 275 for cells treated in a flat biofunctionalised surface and to 250 for cells treated on a surface where biofunctionalised microbeads were immobilised. The cells with reduced expression of MHC‐I molecules showed, after cytotoxicity tests, susceptibility 3.5 times higher than normal cells.     

Microfluidic lithography of SAMs on gold to create dynamic surfaces for directed cell migration and contiguous cell cocultures

A straightforward, flexible, and inexpensive method to create patterned self-assembled monolayers (SAMs) on gold using microfluidics-microfluidic lithography-has been developed. Using a microfluidic cassette, alkanethiols were rapidly patterned on gold surfaces to generate monolayers and mixed monolayers. The patterning methodology is flexible and, by controlling the solvent conditions and thiol concentration, permeation of alkanethiols into the surrounding PDMS microfluidic cassette can be advantageously used to create different patterned feature sizes and to generate well-defined SAM surface gradients with a single microfluidic chip. To demonstrate the utility of microfluidic lithography, multiple cell experiments were conducted. By patterning cell adhesive regions in an inert background, a combination of selective masking of the surface and centrifugation achieved spatial and temporal control of patterned cells, enabling the design of both dynamic surfaces for directed cell migration and contiguous cocultures. Cellular division and motility resulted in directed, dynamic migration, while the centrifugation-aided seeding of a second cell line produced contiguous cocultures with multiple sites for heterogeneous cell-cell interactions.     

Parallel single-cell analysis microfluidic platform

We report a PDMS microfluidic platform for parallel single-cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non-invasive analysis schemes are performed. First, we report single-cell trapping in a fast (2-5 min) and reproducible manner with a single-cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF-7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF-7 cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin-labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF-7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)-based extraction of the cell content. It is validated here using calcein-loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content.     

Design, fabrication and implementation of a novel multi-parameter control microfluidic platform for three-dimensional cell culture and real-time imaging

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigation. While the importance of the extracellular microenvironment is clear, the ability to investigate the effects of physiologically relevant biophysical and biochemical factors is restricted in traditional cell culture platforms. Moreover, the versatility for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small population is lacking. Here we introduce a microfluidic platform for 3D cell culture in biologically derived or synthetic hydrogels with the capability to monitor cellular dynamics in response to changes in their microenvironment. Direct scaffold microinjection, was employed to incorporate 3D matrices into microfluidic devices. Our system geometry permits a unique window for studying directional migration, e.g. sprouting angiogenesis, since sprouts grow predominantly in the microscopic viewing plane. In this study, we demonstrate the ability to generate gradients (non-reactive solute), surface shear, interstitial flow, and image cells in situ. Three different capillary morphogenesis assays are demonstrated. Human adult dermal microvascular endothelial cells (HMVEC-ad) were maintained in culture for up to 7 days during which they formed open lumen-like structures which was confirmed with confocal microscopy and by perfusion with fluorescent microspheres. In the sprouting assay, time-lapse movies revealed cellular mechanisms and dynamics (filopodial projection/retraction, directional migration, cell division and lumen formation) during tip-cell invasion of underlying 3D matrix and subsequent lumen formation.