Biomedical subjects

Albert Folch

Publications and source records attributed to Albert Folch.

13 recordsLinked to original sources

PubMed2006

A high-performance elastomeric patch clamp chip.

Ion channels play key roles in cell physiology and underlie a broad spectrum of disorders. To this day, the gold standard for studying ion channels is the patch clamp technique. Patch clamping involves careful positioning of a fine-tipped glass micropipette onto the surface of the cell to form a high-resistance (>1 Gohms) seal ("gigaseal"), a procedure that is laborious, vibration-sensitive, and not easily amenable to automation. In addition, the solution inside the pipette cannot be easily exchanged. Recently reported patch clamp chips offer the potential of increased throughput, but to date the overall per-cell performance of most designs has been very low when compared to pipettes, and/or the fabrication process is prohibitively expensive. Here we demonstrate a replica-molded elastomeric patch clamp chip incorporating nanofabricated constrictions, which delivers high-stability gigaseals, with success rates comparable to those of pipettes, using rat basophilic leukemia (RBL) cells. The high stability enables exchanges of both the extracellular and intracellular solution during whole-cell recordings. In a sample of 103 experiments, 66 cells (64%) were successfully immobilized at the patch aperture; 38 cells (58% of immobilized cells, 37% of all cells) were successfully gigasealed; and 25 cells (65% of gigasealed cells, 34% of immobilized cells, 24% of all cells) were successfully perforated for whole-cell access. In the last group of 27 experiments, 79% of the cells could be immobilized, of which 68% could be gigasealed and 46% perforated for whole-cell access, indicating that dexterity is important.

Animals↗

PubMed2006

Measurement of cell migration in response to an evolving radial chemokine gradient triggered by a microvalve.

We describe a novel chemotaxis assay based on the microvalve-actuated release of a chemoattractant from a cell-free microchamber into a cell-containing microchamber. The microvalve chemotaxis device (microVCD) was placed on the stage of a conventional inverted microscope to obtain time-lapse micrographs of neutrophils migrating in a radially-symmetric evolving gradient of the chemotactic factor CXCL8/Interleukin-8. A fluorescent tracer was added to the CXCL8 solution to visualize the evolution of the gradient profile, so that at each time point the cell positions could be assigned CXCL8 concentration values. Tracking of individual neutrophils for 90 minutes showed that (a) the neutrophil migratory response is, on average, radially directed towards the CXCL8 source; (b) significant non-radial displacements occur frequently; and (c) there is considerable heterogeneity in the migration speeds and directions amongst the neutrophil population. A custom-made imaging analysis tool was used to extract measurements of migratory behavior such as speed, velocity along the gradient's radial axis, and the cosine of the turning angle as a function of CXCL8 concentration. The microVCD can be easily adapted to study the migratory behavior of cultured cells other than neutrophils.

Chemotaxis, Leukocyte↗

PubMed2006

A nanofabricated planar aperture as a mimic of the nerve-muscle contact during synaptogenesis.

The release of synaptogenic factors by the nerve terminal plays a central role in the aggregation of neurotransmitter receptors at the postsynaptic membrane, a precisely timed and localized process that is essential for the correct formation and functioning of the synapse. This process has been difficult to re-capitulate in cell culture because present cell stimulation methods do not have sufficient spatiotemporal control of the delivery of soluble signals. We cultured myotubes atop nanofabricated planar apertures (2-8 microm diameter) to focally stimulate the muscle cell membrane with neural agrin, a synaptogenic factor released by motor neurons during development. Focal agrin delivery through the apertures after myotube fusion results in local aggregation of acetylcholine receptors (AChRs) in the vicinity of the apertures, a process reminiscent of AChR clustering at innervation sites. Since the apertures are spatially organized in microarrays, multiple experiments can be run in parallel on one device. The technique has wide applicability in cell-cell communication studies and cell-based bioassays.

Animals↗

PubMed2005

Local induction of acetylcholine receptor clustering in myotube cultures using microfluidic application of agrin.

During neuromuscular synaptogenesis, the exchange of spatially localized signals between nerve and muscle initiates the coordinated focal accumulation of the acetylcholine (ACh) release machinery and the ACh receptors (AChRs). One of the key first steps is the release of the proteoglycan agrin focalized at the axon tip, which induces the clustering of AChRs on the postsynaptic membrane at the neuromuscular junction. The lack of a suitable method for focal application of agrin in myotube cultures has limited the majority of in vitro studies to the application of agrin baths. We used a microfluidic device and surface microengineering to focally stimulate muscle cells with agrin at a small portion of their membrane and at a time and position chosen by the user. The device is used to verify the hypothesis that focal application of agrin to the muscle cell membrane induces local aggregation of AChRs in differentiated C2C12 myotubes.

Agrin↗

PubMed2005

Integration of topographical and biochemical cues by axons during growth on microfabricated 3-D substrates.

During embryonic neural development, axon tips ("growth cones") are guided through a dynamic three-dimensional (3-D) landscape by soluble chemotropic factors and by immobilized, growth-permissive or growth-inhibiting contact cues present in the extracellular matrix and on the surface of surrounding cells. It has been difficult to probe the search algorithms of growth cones in response to multiple contact cues during 3-D navigation using traditional two-dimensional (2-D) substrates. Here, we present an in vitro study in which the axons of murine embryonic cortical neurons are challenged with competing growth options, using 3-D substrates that feature variations in permissiveness and microtopography. As 3-D substrates, we used poly-D-lysine (PDL) coatings on microfabricated steps of polydimethylsiloxane (PDMS) and complementary features of Matrigel. We found that axons display a preference for PDL over Matrigel and for the straightest path within a distance consistent with the exploratory range of the growth cone. When these two preferences are in conflict, axons choose to grow straight into Matrigel; when the straight path is not permissive, the axon turns in the direction that minimizes the turning angle. These results suggest that growth cones make 3-D navigation decisions by integrating permissiveness and topographical cues.

Animals↗

PubMed2005

Automated identification of axonal growth cones in time-lapse image sequences.

The isolation and purification of axon guidance molecules has enabled in vitro studies of the effects of axon guidance molecule gradients on numerous neuronal cell types. In a typical experiment, cultured neurons are exposed to a chemotactic gradient and their growth is recorded by manual identification of the axon tip position from two or more micrographs. Detailed and statistically valid quantification of axon growth requires evaluation of a large number of neurons at closely spaced time points (e.g. using a time-lapse microscopy setup). However, manual tracing becomes increasingly impractical for recording axon growth as the number of time points and/or neurons increases. We present a software tool that automatically identifies and records the axon tip position in each phase-contrast image of a time-lapse series with minimal user involvement. The software outputs several quantitative measures of axon growth, and allows users to develop custom measurements. For, example analysis of growth velocity for a dissociated E13 mouse cortical neuron revealed frequent extension and retraction events with an average growth velocity of 0.05 +/- 0.14 microm/min. Comparison of software-identified axon tip positions with manually identified axon tip positions shows that the software's performance is indistinguishable from that of skilled human users.

Animals↗

PubMed2005

Large-scale single-cell trapping and imaging using microwell arrays.

High-throughput single-cell measurements of cellular responses are of great importance for a variety of applications including drug testing, toxicology, and basic cell biology. We present an optimization study for trapping single cells with high efficiency in large arrays of microwells. The method is compatible with standard fluorescence microscopy equipment and is not dependent on, but is compatible with, cell adherence. We have characterized microwell occupancy by cells for a range of microwell dimensions and seeding parameters and optimized it for fibroblasts (as a model of adherent cell) and rat basophilic leukemia cells (as a model of nonadherent cell). We have been able to obtain phase-contrast and fluorescence micrographs with more than 18,000 single cells per image using a 4x objective.

Animals↗

PubMed2005

Parallel mixing of photolithographically defined nanoliter volumes using elastomeric microvalve arrays.

Portable microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed a monolithic polydimethylsiloxane (PDMS) microdevice that allows for storing and mixing subnanoliter volumes of aqueous solutions at various mixing ratios. Filling and mixing is controlled via two integrated PDMS microvalve arrays. The volumes of the microchambers are entirely defined by photolithography, hence volumes from picoliter to nanoliter can be fabricated with high precision. Because the microvalves do not require an energy input to stay closed, fluid can be stored in a highly portable fashion for several days. We have confirmed the mixing precision and predictability using fluorescence microscopy. We also demonstrate the application of the device for calibrating fluorescent calcium indicators. Due to the biocompatibility of PDMS, the device will have broad applications in miniaturized diagnostic assays as well as basic biological studies.

Calcium↗

PubMed2004

Differentiation-on-a-chip: a microfluidic platform for long-term cell culture studies.

Here we demonstrate a microfluidic perfusion system suitable for a long-term (>2 week) culture of muscle cells spanning the whole process of differentiation from myoblasts to myotubes. Cell-adhesive surface microdomains alternating with a robust cell-repellent coating mimic in vivo spatial cues for muscle cell assembly and allow for confining the fusion of myoblasts into aligned, isolated multinucleated myotubes. The microfluidic system provides accurate control of the perfusion rates and biochemical composition of the environment surrounding the cells. Comparing muscle cell-specific differentiation markers and the timing of fusion, we observed no differences in differentiation between microfluidic and traditional cultures. All differentiation assays were fully microfluidic, i.e. they were performed by sequentially changing the fluids in the micro-channels. By delivering fluorescent markers using heterogeneous laminar flows, it was possible to confine a membrane receptor labeling assay to a region smaller than a myotube. Our method can serve as an improved in vitro model for studying muscle cell differentiation and for characterizing extracellular molecules and mechanisms involved in neuromuscular differentiation.

Animals↗

PubMed2004

"Microcanals" for micropipette access to single cells in microfluidic environments.

We demonstrate the fabrication and operation of "microcanals"(i.e. open-air microfluidic channels without a roof), which enable micropipette manipulation and probing of cells within a microfluidic environment. The microcanal devices are fabricated in PDMS on glass substrates using a PDMS membrane transferring technique. Here we show patch-clamp electrophysiological recording and intracellular dye injection performed on cells seeded in microcanals.

Animals↗

PubMed2004

Combinatorial mixing of microfluidic streams.

We have devised a microfluidic mixer design that produces all the mixture combinations of a given number of dilutions of the input compounds. As proof of the concept, we present a device that generates four titrations of two dye solutions, blue and yellow, and combinatorially mixes the blue titrations with the yellow titrations to deliver the sixteen mixture combinations in separate outlet microchannels. Our device features four different flow levels made by stacking nine laser-cut Mylar laminates. The fluidic network has a symmetric design that guarantees that the flow rates are the same at all the outlets, with deviations attributable to imperfections in the fabrication, assembly, or perfusion processes. Design rules for scaling up the number of compounds and/or dilutions are presented. The mixing scheme has broad applicability in high-throughput combinatorial testing applications such as drug screening, cell-based biochemical assays, lab-on-a-chip devices, and biosensors.

Equipment Design↗

PubMed2003

Gray-scale photolithography using microfluidic photomasks.

The ability to produce three-dimensional (3D) microstructures is of increasing importance in the miniaturization of mechanical or fluidic devices, optical elements, self-assembling components, and tissue-engineering scaffolds, among others. Traditional photolithography, the most widely used process for microdevice fabrication, is ill-suited for 3D fabrication, because it is based on the illumination of a photosensitive layer through a "photomask" (a transparent plate that contains opaque, unalterable solid-state features), which inevitably results in features of uniform height. We have devised photomasks in which the light-absorbing features are made of fluids. Unlike in conventional photomasks, the opacity of the photomask features can be tailored to an arbitrary number of gray-scale levels, and their spatial pattern can be reconfigured in the time scale of seconds. Here we demonstrate the inexpensive fabrication of photoresist patterns that contain features of multiple and/or smoothly varying heights. For a given microfluidic photomask, the developed photoresist pattern can be predicted as a function of the dye concentrations and photomask dimensions. For selected applications, microfluidic photomasks offer a low-cost alternative to present gray-scale photolithography approaches.

Journal Article↗

PubMed2003

Biology on a chip: microfabrication for studying the behavior of cultured cells.

The ability to culture cells in vitro has revolutionized hypothesis testing in basic cell and molecular biology research and has become a standard methodology in drug screening and toxicology assays. However, the traditional cell culture methodology--consisting essentially of the immersion of a large population of cells in a homogeneous fluid medium--has become increasingly limiting, both from a fundamental point of view (cells in vivo are surrounded by complex spatiotemporal microenvironments) and from a practical perspective (scaling up the number of fluid handling steps and cell manipulations for high-throughput studies in vitro is prohibitively expensive). Microfabrication technologies have enabled researchers to design, with micrometer control, the biochemical composition and topology of the substrate, the medium composition, as well as the type of neighboring cells surrounding the microenvironment of the cell. In addition, microtechnology is conceptually well suited for the development of fast, low-cost in vitro systems that allow for high-throughput culturing and analysis of cells under large numbers of conditions. Here we review a variety of applications of microfabrication in cell culture studies, with an emphasis on the biology of various cell types.

Animals↗