Bead-bound Cells Research Articles

Automated electrotransformation of Escherichia coli on a digital microfluidic platform using bioactivated magnetic beads.

This paper reports on the use of a digital microfluidic platform to perform multiplex automated genetic engineering (MAGE) cycles on droplets containing Escherichia coli cells. Bioactivated magnetic beads were employed for cell binding, washing, and media exchange in the preparation of electrocompetent cells in the electrowetting-on-dieletric (EWoD) platform. On-cartridge electroporation was used to deliver oligonucleotides into the cells. In addition to the optimization of a magnetic bead-based benchtop protocol for generating and transforming electrocompetent E. coli cells, we report on the implementation of this protocol in a fully automated digital microfluidic platform. Bead-based media exchange and electroporation pulse conditions were optimized on benchtop for transformation frequency to provide initial parameters for microfluidic device trials. Benchtop experiments comparing electrotransformation of free and bead-bound cells are presented. Our results suggest that dielectric shielding intrinsic to bead-bound cells significantly reduces electroporation field exposure efficiency. However, high transformation frequency can be maintained in the presence of magnetic beads through the application of more intense electroporation pulses. As a proof of concept, MAGE cycles were successfully performed on a commercial EWoD cartridge using variations of the optimal magnetic bead-based preparation procedure and pulse conditions determined by the benchtop results. Transformation frequencies up to 22% were achieved on benchtop; this frequency was matched within 1% (21%) by MAGE cycles on the microfluidic device. However, typical frequencies on the device remain lower, averaging 9% with a standard deviation of 9%. The presented results demonstrate the potential of digital microfluidics to perform complex and automated genetic engineering protocols.

Design, synthesis, and application of OB2C combinatorial peptide and peptidomimetic libraries.

The "one-bead two-compound" (OB2C) combinatorial library is constructed on topologically segregated trifunctional bilayer beads such that each bead has a fixed cell-capturing ligand and a random library compound co-displayed on its surface and a chemical coding tag (bar code) inside the bead. An OB2C library containing thousands to millions of compounds can be synthesized and screened concurrently within a short period of time. When live cells are incubated with such OB2C libraries, every bead will be coated with a monolayer of cells. The cell membranes of the captured cells facing the bead surface are exposed to the library compounds tethered to each bead. A specific biochemical or cellular response can be detected with an appropriate reporter system. The OB2C method enables investigators to rapidly discover synthetic molecules that not only interact with cell-surface receptors but can also stimulate or inhibit downstream cell signaling. To demonstrate this powerful method, one OB2C peptide library and two OB2C peptidomimetic libraries were synthesized and screened against Molt-4 lymphoma cells to discover "death ligands." Apoptosis of the bead-bound cells was detected with immunocytochemistry using horseradish peroxidase (HRP)-conjugated anti-cleaved caspase-3 antibody and 3,3'-diaminobenzidine as a substrate. Two novel synthetic "death ligands" against Molt-4 cells were discovered using this OB2C library approach.

High-Throughput Immunomagnetic Cell Detection Using a Microaperture Chip System

We report a microchip system based on a combination of immunomagnetic separation, microfluidics, and size-based filtration for high-throughput detection of rare cells. In this system, target cells bind to magnetic beads in vitro and flow parallel to a microchip with flow rates of milliliters/minute. A magnetic field draws the bead-bound cells toward the microchip, which contains apertures that allow passage of unbound beads while trapping the target cells. The cells captured on the chip can be investigated clearly under a microscope and released from the chip for further analysis. We first characterize the system by detecting cancer cell lines (MCF-7 and A549) in culture media. We then demonstrate detection of 100 MCF-7 cells spiked in 7.5 mL of human blood to simulate detection of circulating tumor cells present in cancer patient blood samples. On average, 85% of the spiked cells were detected. We expect this system to be highly useful in a wide variety of clinical as well as other applications that seek rare cells.

From the rat to the beta cell: a fast and effective technique of separation of Langerhans islets and direct purification of pancreatic beta cells

Isolated Langerhans islets represent a useful model for the study of the endocrine pancreas. The possibility to purify pancreatic beta cells from a mixed Langerhans islet cell population may lead towards a dedicated focus on beta cell research. We describe an effective and rapid immunomagnetic technique for the direct purification of beta cells from isolated Langerhans islets of rat. After the sacrifice of the rat, the Langerhans islets were separated by ductal injection of the pancreas with collagenase, altered to a mixed Langerhans islet cell population and incubated with conditioned immunomagnetic beads targeted to the beta cell surface. The beads were previously coated with a specific antibody against the surface of the beta cell, namely K14D10. The suspension of mixed Langerhans islet cells and immunomagnetic K14D10-conditioned beads was pelleted by a magnetic particle concentrator to isolate the bead-bound cells, which were finally suspended in a culture medium. The purified cells were immunoreactive for insulin and no glucagon-positive cells were detected at immunocytochemistry. Real Time PCR confirmed the purification of the pancreatic beta cells. This immunomagnetic technique allows a rapid, effective and consistent purification of beta cells from isolated Langerhans islets in a direct manner by conditioning the immunomagnetic beads only. This technique is easy, fast and reproducible. It promises to be a reliable method for providing purified beta cells for in vitro research.

Isolation and Expansion of Antigen-Specific Mouse T cells using Dynabeads® (85.9)

Abstract Background: CD134 (OX-40) and CD137 (4-1BB), both members of the TNFR-family, functions as costimulatory molecules promoting proliferation and survival of activated T cells. CD134 and CD137 also identifies recently activated CD8+ and CD4+ T cell and represent a promising approach for the isolation and expansion of viable T cells of a defined specificity. Results: Dynabeads® Mouse CD134 isolates activated antigen-specific T cells by use of an agonistic anti-CD134 antibody conjugated to Dynabeads®. The bead-bound cells are separated on the magnet promoting a prolonged activating signal to the T cells during culture. Dynabeads® Mouse T-Activator CD3/CD28/CD137 enables an easy expansion of the isolated T cells to be used for downstream analysis. Discussion: Dynabeads® Mouse CD134 allows for specific isolation of T cell with defined antigen specificity directly from a crude sample (e.g. splenocytes or tissue from immunized mice) in an easy-to-use in vitro procedure. Its use in combination with Dynabeads® Mouse T-Activator CD3/CD28/CD137 enables easy and reliable isolation and expansion of T cells of a defined specificity. With Dynabeads® Technology, pure, viable T cells can be conveniently isolated for downstream analysis.

Caspase-8 and c-FLIPL Associate in Lipid Rafts with NF-κB Adaptors during T Cell Activation

Humans and mice lacking functional caspase-8 in T cells manifest a profound immunodeficiency syndrome due to defective T cell antigen receptor (TCR)-induced NF-kappaB signaling and proliferation. It is unknown how caspase-8 is activated following T cell stimulation, and what is the caspase-8 substrate(s) that is necessary to initiate T cell cycling. We observe that following TCR ligation, a small portion of total cellular caspase-8 and c-FLIP(L) rapidly migrate to lipid rafts where they associate in an active caspase complex. Activation of caspase-8 in lipid rafts is followed by rapid cleavage of c-FLIP(L) at a known caspase-8 cleavage site. The active caspase.c-FLIP complex forms in the absence of Fas (CD95/APO1) and associates with the NF-kappaB signaling molecules RIP1, TRAF2, and TRAF6, as well as upstream NF-kappaB regulators PKC theta, CARMA1, Bcl-10, and MALT1, which connect to the TCR. The lack of caspase-8 results in the absence of MALT1 and Bcl-10 in the active caspase complex. Consistent with this observation, inhibition of caspase activity attenuates NF-kappaB activation. The current findings define a link among TCR, caspases, and the NF-kappaB pathway that occurs in a sequestered lipid raft environment in T cells.

IC/Microfluidic Hybrid System for Magnetic Manipulation of Biological Cells

This paper introduces an integrated circuit (IC)/microfluidic hybrid system for magnetic manipulation of biological cells. The hybrid system consists of an IC and a microfluidic system fabricated on top. Biological cells attached to magnetic beads are suspended inside the microfluidic system that maintains biocompatibility. The IC contains a microcoil array circuit that produces spatially-patterned microscopic magnetic fields. Programmable, rapid reconfiguration of the field pattern made possible by the IC allows an efficient simultaneous manipulation of multiple individual bead-bound cells with precise position control. Two prototypes, SiGe/microfluidic and CMOS/microfluidic hybrid systems, validate the proposed approach.

A procedure for selecting and culturing subpopulations of neurons from rat dorsal root ganglia using magnetic beads

Current protocols for preparing primary sensory neuron cultures are inadequate when studying individual subpopulations of dorsal root ganglion (DRG) neurons. The DRG is made up of a heterogeneous population of cells, making it difficult to study treatment effects on any given population in mass cultures. Thus, we describe a procedure using magnetic beads from Dynal™ to select and plate viable populations of neurons based on expression of specific cell surface markers. We show that, by the use of the lectin IB4, we can select a highly enriched viable subpopulation of GDNF-responsive DRG neurons, leaving a viable population of non-selected IB4−ve, Trk+ve neurons. Key factors for successful cultures are (i) quick and careful dissection of DRGs from 4- to 5-week-old Sprague–Dawley rats, (ii) adequate removal of debris and non-neuronal contamination and (iii) gentle handling of bead-bound cells during selection.

Cholesterol-independent Interactions with CD47 Enhance αvβ3 Avidity

Expression in OV10 cells of either wild-type CD47 or its extracellular IgV domain linked to a glycosylphosphatidylinositol anchor-(IgV-GPI) enhanced ligand-induced alpha(v)beta(3) activation as detected by the binding of LIBS1 and LIBS6 mAbs. The amplitude of LIBS binding was greater with both CD47 and IgV-GPI expression, indicating an increase in the population of "activable" integrin molecules. Expression of either CD47 species also increased alpha(v)beta(3)-mediated adhesion to vitronectin, and to surfaces coated with the anti-beta(3) antibody AP3, because of enhanced clustering of alpha(v)beta(3) as confirmed by chemical cross-linking. Cholesterol depletion with methyl-beta-cyclodextrin did not prevent the increase in anti-LIBS binding, but reduced cell adhesion to vitronectin and AP3. However, cells expressing CD47 were partially insulated against this disruption, and IgV-GPI was even more effective. Both CD47 and IgV-GPI were found in cholesterol-rich rafts prepared in the absence of detergent, but only CD47 could recruit alpha(v)beta(3) and its associated signaling molecules to these domains. Thus CD47-alpha(v)beta(3) complexes in cholesterol-rich raft domains appear to engage in G(i)-dependent signaling whereas CD47-alpha(v)beta(3) interactions that lead to integrin clustering are also detergent resistant, but are insensitive to cholesterol depletion and do not require the transmembrane region of CD47.

Rapid purification of glial cells using immunomagnetic separation

By purifying glial cells from brain tissue containing a heterogeneous cell population, a number of interactions that define glial cell diversification and function within the central nervous system have been determined. The current methods for purifying glial cells, however, can be time consuming and costly. In the following study we have adapted the technique of immunomagnetic separation to separately enrich 0-2A progenitor cells and astrocytes from the rat central nervous system (CNS). In this procedure, tissue from the CNS was enzymatically dissociated and incubated in a primary antibody specific to a surface antigen found on the target cell type (e.g. A2B5 or RAN-2). The target cells were then immunologically coupled to magnetic beads, which were precoated with a secondary antibody specific to the primary, and then separated out from the heterogeneous cell population using a magnetic field. We found that the immunomagnetic separation procedure, which was completed within 2 h, produced a near pure population of glial cells (>99%). This was confirmed by the absence of unbound cells in the bead-bound fraction. The identification and viability of bead-bound cells were established by culturing these cells and subsequently examining their morphology and antigenic expression. This study shows that glial cell types can be separated out of brain tissue to near purity using immunomagnetic separation. This simple procedure is reliable, inexpensive, and achieves levels of purity and viability comparable with currently available techniques of immunopanning and fluorescence-activated cell sorting, within a fraction of the time.