A Microfluidic High-Capacity Screening Platform for Neurological Disorders.

Compartmentalized cell cultures (CCCs) provide the possibility to study mechanisms of neurodegenerative diseases, such as spreading of misfolded proteins in Alzheimer's or Parkinson's disease or functional changes in, e.g., chronic pain, in vitro. However, many CCC devices do not provide the necessary capacity for identifying novel mechanisms, targets, or drugs in a drug discovery context. Here, we present a high-capacity cell culture microtiter microfluidic plate compliant with American National Standard Institute of the Society for Laboratory Automation and Screening (ANSI/SLAS) standards that allows to parallelize up to 96 CCCs/experimental units, where each experimental unit comprises three microchannel-connected compartments. The plate design allows the specific treatment of cells in individual compartments through the application of a fluidic barrier. Moreover, the compatibility of the plate with neuronal cultures was confirmed with rodent primary as well as human-induced pluripotent stem cell-derived neurons of the central or peripheral nervous system for up to 14 days in culture. Using immunocytochemistry, we demonstrated that the plate design restricts neuronal soma to individual compartments, while axons, but not dendrites, can grow through the connecting microchannels to neighboring compartments. In addition, we show that neurons are spontaneously active and, as deemed by the appearance of synchronous depolarizations in neighboring compartments, are synaptically coupled. In summary, the design of the microfluidic plate allows for both morphological and functional studies of neurological in vitro cultures with increased capacity to support identification of novel mechanisms, targets, or drugs.

Microfluidic technologies in drug discovery.

Microfluidic systems are increasingly used as tools in various stages of the drug discovery process. Microscale systems offer several obvious advantages, such as low sample consumption and significantly reduced analysis or experiment time. These technologies raise the possibility of massive parallelization and concomitant reduction in cost per acquired data point. In addition, fluids in confined spaces display unique behaviors that can be used to acquire information not accessible using macroscopic systems. This article will focus on the implementation of microfluidic systems and technologies in the process of drug discovery.

High-throughput transfection of differentiated primary neurons from rat forebrain.

Primary neurons in culture are considered to be a highly relevant model in the study of neuronal development and activity. They can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. To address this problem, a capillary electroporation system called Cellaxess Elektra was developed for efficient and reproducible transfection of primary cortical and hippocampal neurons without significant impact on cell morphology and viability. The cells are transfected in any stage of differentiation and development, directly in cell culture plates. Genetic material is delivered in situ to as many as 384 samples at a time, which enables both high-throughput and high-quality screening for hard-to-transfect primary cells, meaning that gene function can be studied on a genome-wide scale in cells previously inaccessible to genetic manipulation.

A biohybrid dynamic random access memory.

We report that GABA(A) receptors in a patch-clamped biological cell form a short-term memory circuit when integrated with a scanning-probe microfluidic device. Laminar patterns of receptor activators (agonists) provided by the microfluidic device define and periodically update the data input which is read and stored by the receptors as state distributions (based on intrinsic multistate kinetics). The memory is discharged over time and lasts for seconds to minutes depending on the input function. The function of the memory can be represented by an equivalent electronic circuit with striking similarity in function to a dynamic random access memory (DRAM) used in electronic computers. Multiplexed biohybrid memories may form the basis of large-scale integrated biocomputational/sensor devices with the curious ability to use chemical signals including odorants, neurotransmitters, chemical and biological warfare agents, and many more as input signals.

Microfluidic gradient-generating device for pharmacological profiling.

We describe an on-chip microfluidic gradient-generating device that generates concentration gradients spanning nearly 5 orders of magnitude starting from a single concentration. The exiting stream of drugs held at different concentrations remains laminar in a recording chamber and can be presented as 24 discrete solutions to a cell-based sensor. The high-performance characteristics of the device are demonstrated by pharmacological screening of voltage-gated K+ channels (hERG) and ligand-gated GABA(A) receptors using scanning-probe patch-clamp measurements. Multiple data point dose-response curves and IC50 and EC50 values were rapidly obtained, typically in less than 30 min, through its combined functionality of gradient generation and open-volume laminar flow. The device facilitates rapid pharmacological profiling of ion channel and GPCR effectors and enables the acquisition of large numbers of data points with minute sample consumption and handling.

A microfluidics approach to the problem of creating separate solution environments accessible from macroscopic volumes.

We report on a microfluidic device that generates separate solution environments in macroscopic volumes. Spatially distinct patterns are created by emitting fluids from 16 different sources (closely spaced microchannels) into a solution-filled macroscopic chamber. The fluid in neighboring microchannels couples viscously in the macroscopic container, generating one single interdigitated stream. Scanning nanoelectrode amperometry was used for characterizing the concentration landscape and the diffusion zones between solutions running in parallel at different coordinates in the stream. These experiments were complemented by finite element simulations of the Navier-Stokes and mass transport equations to describe the velocity distributions and the diffusion behavior. For in channel flow velocities of 50 mm.s(-1), patterns could persist on the order of millimeters to centimeters in the open volume. The most narrow diffusion zones with widths less than 10 microm (5-95% concentration change) were found some tens of micrometers out in the macroscopic container. We demonstrate that a 14-microm-diameter nearly spherical object (biological cell) attached to a micropipet can be moved from one solution environment to another by a lateral displacement of only 8 microm. The device is suitable for applications where the solution environment around a microscopic or nanoscopic sensor needs to be changed multiple times, i.e., in order to build layered structures, for obtaining binding isotherms, and kinetic information, for example, on ion channels, enzymes, and receptors as well as in applications where different loci on an object need to be exposed to different environments or where complex solution environments need to be created for studies of interfacial chemistry between two streaming layers.

A cell-based bar code reader for high-throughput screening of ion channel-ligand interactions.

This paper presents a microfluidics-patch clamp platform for performing high-throughput screening and rapid characterization of weak-affinity ion channel-ligand interactions. This platform integrates a microfluidic chip consisting of multiple channels entering an open volume with standard patch clamp equipment. The microfluidic chip is placed on a motorized scanning stage and the method relies on the ability to scan rapidly, on the order of milliseconds, a patch-clamped cell across discrete zones of different solutions created in the open volume. Under ideal conditions, this method has the capacity to obtain kinetically resolved patch clamp measurements and dose-response curves of up to 10(3) ligand solutions in a single day.