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Continuous analysis of two dyes loaded into single mammalian cells using laser-based lysis combined with electrophoretic separation was developed and characterized on microfluidic chips. The devices employed hydrodynamic flow to transport cells to a junction where they were mechanically lysed by a laser-generated cavitation bubble. An electric field then attracted the analyte into a separation channel while the membranous remnants passed through the intersection towards a waste reservoir. Phosphatidylcholine (PC)-supported bilayer membrane coatings (SBMs) provided a weakly negatively charged surface and prevented cell fouling from interfering with device performance. Cell lysis using a picosecond-pulsed laser on-chip did not interfere with concurrent electrophoretic separations. The effect of device parameters on performance was evaluated. A ratio of 2  :  1 was found to be optimal for the focusing-channel  :  flow-channel width and 3  :  1 for the flow-channel :  separation-channel width. Migration times decreased with increased electric field strengths up to 333 V cm−1, at which point the field strength was sufficient to move unlysed cells and cellular debris into the electrophoretic channel. The migration time and full width half-maximum (FWHM) of the peaks were independent of cell velocity for velocities between 0.03 and 0.3 mm s−1. Separation performance was independent of the exact lysis location when lysis was performed near the outlet of the focusing channel. The migration time for cell-derived fluorescein and fluorescein carboxylate was reproducible with <10% RSD. Automated cell detection and lysis were required to reduce peak FWHM variability to 30% RSD. A maximum throughput of 30 cells min−1was achieved. Device stability was demonstrated by analyzing 600 single cells over a 2 h time span. [ABSTRACT FROM AUTHOR]

We present an efficient, yet inexpensive, approach for isolating viable single cells or colonies from a mixed population. This cell microarray platform possesses innovations in both the array manufacture and the manner of target cell release. Arrays of microwells with bases composed of detachable concave elements, termed microrafts, were fabricated by a dip-coating process using a polydimethylsiloxane mold as the template and the array substrate. This manufacturing approach enabled the use of materials other than photoresists to create the array elements. Thus microrafts possessing low autofluorescence could be fabricated for fluorescence-based identification of cells. Cells plated on the microarray settled and attached at the center of the wells due to the microrafts' concavity. Individual microrafts were readily dislodged by the action of a needle inserted through the compliant polymer substrate. The hard polymer material (polystyrene or epoxy resin) of which the microrafts were composed protected the cells from damage by the needle. For cell analysis and isolation, cells of interest were identified using a standard inverted microscope and microrafts carrying target cells were dislodged with the needle. The released cells/microrafts could be efficiently collected, cultured and clonally expanded. During the separation and collection procedures, the cells remained adherent and provided a measure of protection during manipulation, thus providing an extremely high single-cell cloning rate (>95%). Generation of a transfected cell line based on expression of a fluorescent protein demonstrated an important application for performing on-chip cell separations. [ABSTRACT FROM AUTHOR]

There is a need in biological research for tools designed to manipulate the environment surrounding microscopic regions of tissue. In the current work, a device for the oriented capture of an important and under-studied tissue, the colon crypt, has been designed and tested. The objective of this work is to create a BioMEMs device for biological assays of living colonic crypts. The end goal will be to subject the polarized tissue to user-controlled fluidic microenvironments in a manner that recapitulates the in vivostate. Crypt surrogates, polymeric structures of similar dimensions and shape to isolated colon crypts, were used in the initial design and testing of the device. Successful capture of crypt surrogates was accomplished on a simple device composed of an array of micron-scale capture sites that enabled individual structures to be captured with high efficiency (92 ± 3%) in an ordered and properly oriented fashion. The device was then evaluated using colon crypts isolated from a murine animal model. The capture efficiency attained using the fixed biologic sample was 37 ± 5% due to the increased variability of the colon crypts compared with the surrogate structures, yet 94 ± 3% of the captured crypts were properly oriented. A simple approach to plug the remaining capture sites in the array was performed using inert glass beads. Blockage of unfilled capture sites is an important feature to establish a chemical gradient across the arrayed crypts. A chemical concentration gradient (Cluminal/Cbasal> 10) was demonstrated across the arrayed crypts for over 8 h. Finally unfixed colon crypts were demonstrated to be effectively captured by the micromesh array and to remain viable on the capture sites at 5 h after mouse sacrifice. The present study demonstrates the feasibility and potential for rationally microengineered technologies to address the specific needs of the biologic researcher. [ABSTRACT FROM AUTHOR]

Electrically addressable cell traps were integrated with capillary electrophoresis for the analysis of the contents of single adherent cells. Electrodes composed of indium tin oxide were patterned on a glass surface followed by formation of topographical cell traps using 1002F photoresist. Single cells trapped in the holes could be lysed in less than 66 ms by applying a brief electric field (10 ms) across the electrode beneath the cell and the ground electrode placed in the aqueous media above the cell traps. The gas formed during cell lysis remained localized within the cavity formed by the 1002F photoresist. The retention of the gas in the cell trap enabled the cell traps to be coupled to an overlying capillary without blockage of the capillary. Single cells cultured in the traps were loaded with fluorescein and Oregon Green and then electrically lysed. By simultaneous application of an electric field to the capillary, the cell's contents were loaded into the capillary and electrophoretically separated. Orgeon Green and fluorescein from a single cell were fully resolved in less than two minutes. The use of a single patterned electrode beneath the 1002F cell trap yielded a simple easily fabricated design that was robust when immersed in aqueous solutions. Moreover, the design can easily be scaled up to create arrays of adherent cells for serial analyses using a single capillary or for parallel analysis by mating to an array of capillaries. Enhancing the rate of analysis of single adherent cells would enable a greater understanding of cellular physiology. [ABSTRACT FROM AUTHOR]

Arrays of releasable micropallets with surrounding walls of poly(ethylene glycol) (PEG) were fabricated for the patterning and sorting of adherent cells. PEG walls were fabricated between the SU-8 pallets using a simple, mask-free strategy. By utilizing the difference in UV-transmittance of glass and SU-8, PEG monomer was selectively photopolymerized in the space surrounding the pallets. Since the PEG walls are composed of a cross-linked structure, the stability of the walls is independent of the pallet array geometry and the properties of the overlying solution. Even though surrounded with PEG walls, the individual pallets were detached from the array by the mechanical force generated by a focused laser pulse, with a release threshold of 6 μJ. Since the PEG hydrogels are repellent to protein adsorption and cell attachment, the walls localized cell growth to the pallet top surface. Cells grown in the microwells formed by the PEG walls were released by detaching the underlying pallet. The released cells/pallets were collected, cultured and clonally expanded. The micropallet arrays with PEG walls provide a platform for performing single cell analysis and sorting on chip. [ABSTRACT FROM AUTHOR]

A goal of modern biology is to understand the molecular mechanisms underlying cellular function. The ability to manipulate and analyze single cells is crucial for this task. The advent of microengineering is providing biologists with unprecedented opportunities for cell handling and investigation on a cell-by-cell basis. For this reason, lab-on-a-chip (LOC) technologies are emerging as the next revolution in tools for biological discovery. In the current discussion, we seek to summarize the state of the art for conventional technologies in use by biologists for the analysis of single, mammalian cells, and then compare LOC devices engineered for these same single-cell studies. While a review of the technical progress is included, a major goal is to present the view point of the practicing biologist and the advances that might increase adoption by these individuals. The LOC field is expanding rapidly, and we have focused on areas of broad interest to the biology community where the technology is sufficiently far advanced to contemplate near-term application in biological experimentation. Focus areas to be covered include flow cytometry, electrophoretic analysis of cell contents, fluorescent-indicator-based analyses, cells as small volume reactors, control of the cellular microenvironment, and single-cell PCR. [ABSTRACT FROM AUTHOR]