Selective Cell Capture Research Articles

Barcoding cells using cell-surface programmable DNA-binding domains

We develop here a novel approach to barcode large numbers of cells through cell-surface expression of programmable zinc-finger DNA-binding domains (sZFs). We show sZFs enable double-stranded DNA to sequence-specifically label living cells, and also develop a sequential tagging approach to in situ image >3 cell types using just 3 fluorophores. Finally we demonstrate their broad versatility through ability to serve as surrogate reporters and facilitate selective cell capture and targeting.

Biosensing using straight long-range surface plasmon waveguides

Straight long-range surface plasmon waveguides are demonstrated as biosensors for the detection of cells, proteins and changes in the bulk refractive index of solutions. The sensors consist of 5 μm wide 22 nm thick Au stripes embedded in polymer (CYTOP™) with microfluidic channels etched into the top cladding. Bulk sensing is demonstrated by sequentially injecting six solutions of different refractive indices in 2 × 10(-3) RIU increments; such index steps were detected with a signal-to-noise ratio of ~1000. Selective capture of cells is demonstrated using Au waveguides functionalized with antibodies against blood group A, and red blood cells of group A and O in buffer as positive and negative analyte. Bovine serum albumin in buffer was used to demonstrate protein sensing. A monolayer of bovine serum albumin physisorbed on a carboxyl-terminated self-assembled monolayer on Au was detected with a signal-to-noise ratio of ~300. Overall, the biosensor demonstrated a good capability for detecting bulk changes in solution and for sensing analyte over a very wide range of mass (from cells to proteins). The biosensors are compact, inexpensive to fabricate, and may find use over a wide range of cost-sensitive sensing and detection applications.

Selective cell capture and analysis using shallow antibody-coated microchannels

Demand for analysis of rare cells such as circulating tumor cells in blood at the single molecule level has recently grown. For this purpose, several cell separation methods based on antibody-coated micropillars have been developed (e.g., Nagrath et al., Nature 450, 1235-1239 (2007)). However, it is difficult to ensure capture of targeted cells by these methods because capture depends on the probability of cell-micropillar collisions. We developed a new structure that actively exploits cellular flexibility for more efficient capture of a small number of cells in a target area. The depth of the sandwiching channel was slightly smaller than the diameter of the cells to ensure contact with the channel wall. For cell selection, we used anti-epithelial cell adhesion molecule antibodies, which specifically bind epithelial cells. First, we demonstrated cell capture with human promyelocytic leukemia (HL-60) cells, which are relatively homogeneous in size; in situ single molecule analysis was verified by our rolling circle amplification (RCA) method. Then, we used breast cancer cells (SK-BR-3) in blood, and demonstrated selective capture and cancer marker (HER2) detection by RCA. Cell capture by antibody-coated microchannels was greater than with negative control cells (RPMI-1788 lymphocytes) and non-coated microchannels. This system can be used to analyze small numbers of target cells in large quantities of mixed samples.

Antibody-Functionalized Fluid-Permeable Surfaces for Rolling Cell Capture at High Flow Rates

Adhesion-based cell capture on surfaces in microfluidic devices forms the basis of numerous biomedical diagnostics and in vitro assays. However, the performance of these platforms is partly limited by interfacial phenomena that occur at low Reynolds numbers. In contrast, cell homing to porous vasculature is highly effective in vivo during inflammation, stem cell trafficking, and cancer metastasis. Here, we show that a porous, fluid-permeable surface functionalized with cell-specific antibodies promotes efficient and selective cell capture in vitro. This architecture is advantageous due to enhanced transport as streamlines are diverted toward the surface. Moreover, specific cell-surface interactions are promoted due to reduced shear, allowing gentle cell rolling and arrest. Together, these synergistic effects enable highly effective cell capture at flow rates more than an order of magnitude larger than those provided by existing devices with solid surfaces.

Controlled viable release of selectively captured label-free cells in microchannels.

Selective capture of cells from bodily fluids in microchannels has broadly transformed medicine enabling circulating tumor cell isolation, rapid CD4(+) cell counting for HIV monitoring, and diagnosis of infectious diseases. Although cell capture methods have been demonstrated in microfluidic systems, the release of captured cells remains a significant challenge. Viable retrieval of captured label-free cells in microchannels will enable a new era in biological sciences by allowing cultivation and post-processing. The significant challenge in release comes from the fact that the cells adhere strongly to the microchannel surface, especially when immuno-based immobilization methods are used. Even though fluid shear and enzymes have been used to detach captured cells in microchannels, these methods are known to harm cells and affect cellular characteristics. This paper describes a new technology to release the selectively captured label-free cells in microchannels without the use of fluid shear or enzymes. We have successfully released the captured CD4(+) cells (3.6% of the mononuclear blood cells) from blood in microfluidic channels with high specificity (89% ± 8%), viability (94% ± 4%), and release efficiency (59% ± 4%). We have further validated our system by specifically capturing and controllably releasing the CD34(+) stem cells from whole blood, which were quantified to be 19 cells per million blood cells in the blood samples used in this study. Our results also indicated that both CD4(+) and CD34(+) cells released from the microchannels were healthy and amenable for in vitro culture. Manual flow based microfluidic method utilizes inexpensive, easy to fabricate microchannels allowing selective label-free cell capture and release in less than 10 minutes, which can also be used at the point-of-care. The presented technology can be used to isolate and purify a broad spectrum of cells from mixed populations offering widespread applications in applied biological sciences, such as tissue engineering, regenerative medicine, rare cell and stem cell isolation, proteomic/genomic research, and clonal/population analyses.

Label-free impedance detection of low levels of circulating endothelial progenitor cells for point-of-care diagnosis

This paper presents a novel microfluidic system for rapid label-free detection of endothelial progenitor cells (EPCs) from small volumes of white blood cells samples, to obtain a bedside cardiovascular diagnostic solution. The system was built on a single 1 cm 2 microelectrode array silicon chip, integrated with negative dielectrophoresis for cell trapping, surface immunochemistry for selective cell capture, and fluidics for cell washing and impedance detection. The level of circulating EPC level in blood is a biomarker of clinical interest, linked to the assessment of risk factors in cardiovascular diseases which are a major global concern. Rare EPCs are usually detected through in vitro culture or flow cytometry, which are too time-consuming to bring timely reports in acute diseases. Although microfluidics approaches have enabled reduced processing time and enhanced portability, their sensitivity and processing volumes are still inadequate for rare cell detection at a bedside setting. Using small highly sensitive microelectrodes, our novel integrated system achieved the detection of 720 EPCs in a small 12 μl sample of 72,000 peripheral blood mononuclear cells (PBMC), i.e. equivalent to a concentration of EPCs of 0.1% of 100 μl blood. This demonstrated that clinically significant level of EPCs (<0.5% of PBMC) could be detected for the first time on a detection system at bedside set-up, showing great potential in applications for point-of-care diagnosis.

Aptamer-Based Microfluidic Device for Enrichment, Sorting, and Detection of Multiple Cancer Cells

The ability to diagnose cancer based on the detection of rare cancer cells in blood or other bodily fluids is a significant challenge. To address this challenge, we have developed a microfluidic device that can simultaneously sort, enrich, and then detect multiple types of cancer cells from a complex sample. The device, which is made from poly(dimethylsiloxane) (PDMS), implements cell-affinity chromatography based on the selective cell-capture of immobilized DNA-aptamers and yields a 135-fold enrichment of rare cells in a single run. This enrichment is achieved because the height of the channel is on the order of a cell diameter. The sorted cells grow at the comparable rate as cultured cells and are 96% pure based on flow cytometry determination. Thus, by using our aptamer based device, cell capture is achieved simply and inexpensively, with no sample pretreatment before cell analysis. Enrichment and detection of multiple rare cancer cells can be used to detect cancers at the early stages, diagnose metastatic relapse, stratify patients for therapeutic purposes, monitor response to drugs and therapies, track tumor progression, and gain a deeper understanding of the biology of circulating tumor cells (CTCs).