A miniaturized multipurpose platform for rapid, label-free, and simultaneous separation, patterning, and in vitro culture of primary and rare cells.

Given that current cell isolation techniques are expensive, time consuming, yield low isolation purities, and/or alter target cell properties, a versatile, cost effective, and easy-to-operate microchip with the capability to simultaneously separate, capture, pattern, and culture rare and primary cells in vitro is developed. The platform is based on target cell adhesion onto the micro-fabricated interfaces produced by microcontact printing of cell-specific antibodies. Results show over 95% separation efficiency in less than 10 min for the separation of oligodendrocyte progenitor cells (OPCs) and cardiomyocytes from rat brain and heart mixtures, respectively. Target cell attachment and single cell spreading can be precisely controlled on the basis of the designed patterns. Both cell types can maintain their biofunctionality. Indeed, isolated OPCs can proliferate and differentiate into mature oligodendrocytes, while isolated cardiomyocytes retain their contractile properties on the separation platform. Successful separation of two dissimilar cell types present in varying concentrations in their respective cell mixtures and the demonstration of their integrity after separation open new avenues for time and cost-effective sorting of various cell types using the developed miniaturized platform.

High throughput multilayer microfluidic particle separation platform using embedded thermoplastic-based micropumping.

We present an integrated thermoplastic elastomer (TPE) based multilayer microfluidic device with an embedded peristaltic micropump and through-holes membrane for high throughput particle sorting and separation. Fluidic and pneumatic layers of the device were fabricated using hot-embossing lithography and commercially available polycarbonate membranes were succcessfully sandwiched between two thermoplastic elastomer fluidic layers integrated to a peristaltic micropumping layer. The integrated peristaltic micropump induces turbulence at the top-microfluidic layer ring which successfully avoids particle aggregation and membrane blocking even at nanorange size. We present herein the general design of the device structure and pumping characteristics for three devices with membrane pore sizes of 10 µm, 5 µm and 800 nm. By using this design we have successfully demonstrated a separation efficiency as high as 99% of polystyrene microbeads with different sizes and most importantly the separation of 390 nm particles from 2 µm beads was achieved. Using this device, we were also able to separate red blood cells with size of about 6-8 µm from osteoblasts typically larger than 10 µm to demonstrate the potential applicability of this platform for biological samples. The produced microfluidic chip operating at flow rates up to 100 µl min(-1) allows us to achieve efficient high-throughput sorting and separation of target particles/cells.

Separation of rare oligodendrocyte progenitor cells from brain using a high-throughput multilayer thermoplastic-based microfluidic device.

Despite the advances made in the field of regenerative medicine, the progress in cutting-edge technologies for separating target therapeutic cells are still at early stage of development. These cells are often rare, such as stem cells or progenitor cells that their overall properties should be maintained during the separation process for their subsequent application in regenerative medicine. This work, presents separation of oligodendrocyte progenitor cells (OPCs) from rat brain primary cultures using an integrated thermoplastic elastomeric (TPE)- based multilayer microfluidic device fabricated using hot-embossing technology. OPCs are frequently used in recovery, repair and regeneration of central nervous system after injuries. Indeed, their ability to differentiate in vitro into myelinating oligodendrocytes, are extremely important for myelin repair. OPCs form 5-10% of the glial cells population. The traditional macroscale techniques for OPCs separation require pre-processing of cells and/or multiple time consuming steps with low efficiency leading very often to alteration of their properties. The proposed methodology implies to separate OPCs based on their smaller size compared to other cells from the brain tissue mixture. Using aforementioned microfluidic chip embedded with a 5 µm membrane pore size and micropumping system, a separation efficiency more than 99% was achieved. This microchip was able to operate at flow rates up to 100 µl/min, capable of separating OPCs from a confluent 75 cm(2) cell culture flask in less than 10 min, which provides us with a high-throughput and highly efficient separation expected from any cell sorting techniques.

Generating multiplex gradients of biomolecules for controlling cellular adhesion in parallel microfluidic channels.

Here we present a microfluidic platform to generate multiplex gradients of biomolecules within parallel microfluidic channels, in which a range of multiplex concentration gradients with different profile shapes are simultaneously produced. Nonlinear polynomial gradients were also generated using this device. The gradient generation principle is based on implementing parrallel channels with each providing a different hydrodynamic resistance. The generated biomolecule gradients were then covalently functionalized onto the microchannel surfaces. Surface gradients along the channel width were a result of covalent attachments of biomolecules to the surface, which remained functional under high shear stresses (50 dyn/cm(2)). An IgG antibody conjugated to three different fluorescence dyes (FITC, Cy5 and Cy3) was used to demonstrate the resulting multiplex concentration gradients of biomolecules. The device enabled generation of gradients with up to three different biomolecules in each channel with varying concentration profiles. We were also able to produce 2-dimensional gradients in which biomolecules were distributed along the length and width of the channel. To demonstrate the applicability of the developed design, three different multiplex concentration gradients of REDV and KRSR peptides were patterned along the width of three parallel channels and adhesion of primary human umbilical vein endothelial cell (HUVEC) in each channel was subsequently investigated using a single chip.

Patterning multiplex protein microarrays in a single microfluidic channel.

The development of versatile biofunctional surfaces is a fundamental prerequisite in designing Lab on a Chip (LOC) devices for applications in biosensing interfaces and microbioreactors. The current paper presents a rapid combinatorial approach to create multiplex protein patterns in a single microfluidic channel. This approach consists of coupling microcontact printing with microfluidic patterning, where microcontact printing is employed for silanization using (3-Aminopropyl) triethoxysilane (APTES), followed by microfluidic patterning of multiple antibodies. As a result, the biomolecules of choice could be covalently attached to the microchannel surface, thus creating a durable and highly resistant functional interface. Moreover, the experimental procedure was designed to create a microfluidic platform that maintains functionality at high flow rates. The functionalized surfaces were characterized using X-ray photoelectron spectroscopy (XPS) and monitored with fluorescence microscopy at each step of functionalization. To illustrate the possibility of patterning multiple biomolecules along the cross section of a single microfluidic channel, microarrays of five different primary antibodies were patterned onto a single channel and their functionality was evaluated accordingly through a multiplex immunoassay using secondary antibodies specific to each patterned primary antibody. The resulting patterns remained stable at shear stresses of up to 50 dyn/cm(2). The overall findings suggest that the developed multiplex functional interface on a single channel can successfully lead to highly resistant multiplex functional surfaces for high throughput biological assays.

Adhesion based detection, sorting and enrichment of cells in microfluidic Lab-on-Chip devices.

The detection, isolation and sorting of cells are important tools in both clinical diagnostics and fundamental research. Advances in microfluidic cell sorting devices have enabled scientists to attain improved separation with comparative ease and considerable time savings. Despite the great potential of Lab-on-Chip cell sorting devices for targeting cells with desired specificity and selectivity, this field of research remains unexploited. The challenge resides in the detection techniques which has to be specific, fast, cost-effective, and implementable within the fabrication limitations of microchips. Adhesion-based microfluidic devices seem to be a reliable solution compared to the sophisticated detection techniques used in other microfluidic cell sorting systems. It provides the specificity in detection, label-free separation without requirement for a preprocessing step, and the possibility of targeting rare cell types. This review elaborates on recent advances in adhesion-based microfluidic devices for sorting, detection and enrichment of different cell lines, with a particular focus on selective adhesion of desired cells on surfaces modified with ligands specific to target cells. The effect of shear stress on cell adhesion in flow conditions is also discussed. Recently published applications of specific adhesive ligands and surface functionalization methods have been presented to further elucidate the advances in cell adhesive microfluidic devices.