Anja Kunze

Micropatterning neural cell cultures in 3D with a multi-layered scaffold

Cortical neurons, in their native state, are organized in six different cell layers; and the thickness of the cell layer ranges from 0.12 mm to 0.4 mm. The structure of cell layers plays an important role in neurodegenerative diseases or corticogenesis. We developed a 3D microfluidic device for creating physiologically realistic, micrometer scaled neural cell layers. Using this device, we demonstrated that (1) agarose-alginate mixture can be gelled thermally, thus an excellent candidate for forming multi-layered scaffolds for micropatterning embedded cells; (2) primary cortical neurons were cultured successfully for up to three weeks in the micropatterned multi-layered scaffold; (3) B27 concentration gradient enhanced neurite outgrowth. In addition, this device is compatible with optical microscopy, the dynamic process of neural growth can be imaged, and density and number of neurites can be quantified. This device can potentially be used for drug development, as well as research in basic neural biology.

Advances in High-Throughput Single-Cell Microtechnologies

Micro-scale biological tools that have allowed probing of individual cells--from the genetic, to proteomic, to phenotypic level--have revealed important contributions of single cells to direct normal and diseased body processes. In analyzing single cells, sample heterogeneity between and within specific cell types drives the need for high-throughput and quantitative measurement of cellular parameters. In recent years, high-throughput single-cell analysis platforms have revealed rare genetic subpopulations in growing tumors, begun to uncover the mechanisms of antibiotic resistance in bacteria, and described the cell-to-cell variations in stem cell differentiation and immune cell response to activation by pathogens. This review surveys these recent technologies, presenting their strengths and contributions to the field, and identifies needs still unmet toward the development of high-throughput single-cell analysis tools to benefit life science research and clinical diagnostics.

Astrocyte–neuron co-culture on microchips based on the model of SOD mutation to mimic ALS

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease. ALS is believed to be a non-cell autonomous condition, as other cell types, including astrocytes, have been implicated in disease pathogenesis. Hence, to facilitate the development of therapeutics against ALS, it is crucial to better understand the interactions between astrocytes and neural cells. Furthermore, cell culture assays are needed that mimic the complexity of cell to cell communication at the same time as they provide control over the different microenvironmental parameters. Here, we aim to validate a previously developed microfluidic system for an astrocyte-neuron cell culture platform, in which astrocytes have been genetically modified to overexpress either a human wild-type (WT) or a mutated form of the super oxide dismutase enzyme 1 (SOD1). Cortical neural cells were co-cultured with infected astrocytes and studied for up to two weeks. Using our microfluidic device that prevents direct cell to cell contact, we could evaluate neural cell response in the vicinity of astrocytes. We showed that neuronal cell density was reduced by about 45% when neurons were co-cultured with SOD-mutant astrocytes. Moreover, we demonstrated that SOD-WT overexpressing astrocytes reduced oxidative stress on cortical neurons that were in close metabolic contact. In contrast, cortical neurons in metabolic contact with SOD-mutant astrocytes lost their synapsin protein expression after severe glutamate treatment, an indication of the toxicity potentiating effect of the SOD-mutant enzyme.

Induction of Calcium Influx in Cortical Neural Networks by Nanomagnetic Forces

Nanomagnetic force stimulation with ferromagnetic nanoparticles was found to trigger calcium influx in cortical neural networks without observable cytotoxicity. Stimulated neural networks showed an average of 20% increment in calcium fluorescence signals and a heightened frequency in calcium spiking. These effects were also confined spatially to areas with engineered high magnetic field gradients. Furthermore, blockage of N-type calcium channels inhibited the stimulatory effects of the nanomagnetic forces, suggesting the role of mechano-sensitive ion channels in mediating calcium influx.

Engineering cortical neuron polarity with nanomagnets on a chip.

Intra- and extracellular signaling play critical roles in cell polarity, ultimately leading to the development of functional cell-cell connections, tissues, and organs. In the brain, pathologically oriented neurons are often the cause for disordered circuits, severely impacting motor function, perception, and memory. Aside from control through gene expression and signaling pathways, it is known that nervous system development can be manipulated by mechanical stimuli (e.g., outgrowth of axons through externally applied forces). The inverse is true as well: intracellular molecular signals can be converted into forces to yield axonal outgrowth. The complete role played by mechanical signals in mediating single-cell polarity, however, remains currently unclear. Here we employ highly parallelized nanomagnets on a chip to exert local mechanical stimuli on cortical neurons, independently of the amount of superparamagnetic nanoparticles taken up by the cells. The chip-based approach was utilized to quantify the effect of nanoparticle-mediated forces on the intracellular cytoskeleton as visualized by the distribution of the microtubule-associated protein tau. While single cortical neurons prefer to assemble tau proteins following poly-L-lysine surface cues, an optimal force range of 4.5-70 pN by the nanomagnets initiated a tau distribution opposed to the pattern cue. In larger cell clusters (groups comprising six or more cells), nanoparticle-mediated forces induced tau repositioning in an observed range of 190-270 pN, and initiation of magnetic field-directed cell displacement was observed at forces above 300 pN. Our findings lay the groundwork for high-resolution mechanical encoding of neural networks in vitro, mechanically driven cell polarization in brain tissues, and neurotherapeutic approaches using functionalized superparamagnetic nanoparticles to potentially restore disordered neural circuits.

Synergistic NGF/B27 Gradients Position Synapses Heterogeneously in 3D Micropatterned Neural Cultures

Native functional brain circuits show different numbers of synapses (synaptic densities) in the cerebral cortex. Until now, different synaptic densities could not be studied in vitro using current cell culture methods for primary neurons. Herein, we present a novel microfluidic based cell culture method that combines 3D micropatterning of hydrogel layers with linear chemical gradient formation. Micropatterned hydrogels were used to encapsulate dissociated cortical neurons in laminar cell layers and neurotrophic factors NGF and B27 were added to influence the formation of synapses. Neurotrophic gradients allowed for the positioning of distinguishable synaptic densities throughout a 3D micropatterned neural culture. NGF and B27 gradients were maintained in the microfluidic device for over two weeks without perfusion pumps by utilizing a refilling procedure. Spatial distribution of synapses was examined with a pre-synaptic marker to determine synaptic densities. From our experiments, we observed that (1) cortical neurons responded only to synergistic NGF/B27 gradients, (2) synaptic density increased proportionally to synergistic NGF/B27 gradients; (3) homogeneous distribution of B27 disturbed cortical neurons in sensing NGF gradients and (4) the cell layer position significantly impacted spatial distribution of synapses.

Flexible and Stretchable Micromagnet Arrays for Tunable Biointerfacing

A process to surface pattern polydimethylsiloxane (PDMS) with ferromagnetic structures of varying sizes (micrometer to millimeter) and thicknesses (>70 μm) is developed. Their flexibility and magnetic reach are utilized to confer dynamic, additive properties to a variety of substrates, such as coverslips and Eppendorf tubes. It is found that these substrates can generate additional modes of magnetic droplet manipulation, and can tunably steer magnetic-cell organization.

The Age of Cortical Neural Networks Affects Their Interactions with Magnetic Nanoparticles.

Despite increasing use of nanotechnology in neuroscience, the characterization of interactions between magnetic nanoparticles (MNPs) and primary cortical neural networks remains underdeveloped. In particular, how the age of primary neural networks affects MNP uptake and endocytosis is critical when considering MNP-based therapies for age-related diseases. Here, primary cortical neural networks are cultured up to 4 weeks and with CCL11/eotaxin, an age-inducing chemokine, to create aged neural networks. As the neural networks are aged, their association with membrane-bound starch-coated ferromagnetic nanoparticles (fMNPs) increases while their endocytic mechanisms are impaired, resulting in reduced internalization of chitosan-coated fMNPs. The age of the neurons also negates the neuroprotective effects of chitosan coatings on fMNPs, attributing to decreased intracellular trafficking and increased colocalization of MNPs with lysosomes. These findings demonstrate the importance of age and developmental stage of primary neural cells when developing in vitro models for fMNP therapeutics targeting age-related diseases.

Research highlights: microtechnologies for engineering the cellular environment

In this issue we highlight recent microtechnology-enabled approaches to control the physical and biomolecular environment around cells: (1) developing micropatterned surfaces to quantify cell affinity choices between two adhesive patterns, (2) controlling topographical cues to align cells and improve reprogramming to a pluripotent state, and (3) controlling gradients of biomolecules to maintain pluripotency in embryonic stem cells. Quantitative readouts of cell-surface affinity in environments with several cues should open up avenues in tissue engineering where self-assembly of complex multi-cellular structures is possible by precisely engineering relative adhesive cues in three dimensional constructs. Methods of simple and local epigenetic modification of chromatin structure with microtopography and biomolecular gradients should also be of use in regenerative medicine, as well as in high-throughput quantitative analysis of external signals that impact and can be used to control cells. Overall, approaches to engineer the cellular environment will continue to be an area of further growth in the microfluidic and lab on a chip community, as the scale of the technologies seamlessly matches that of biological systems. However, because of regulations and other complexities with tissue engineered therapies, these micro-engineering approaches will likely first impact organ-on-a-chip technologies that are poised to improve drug discovery pipelines.

A virtual valve for smooth contamination-free flow switching

We present a channel geometry that allows for clean switching between different inlets of a microchip without any contamination of the inlets or the downstream flow. We drive this virtual valve with a pneumatic pressure setup that minimizes disturbance of the downstream flow during the switching procedure by simultaneous variation of the pressures applied to the different inlets. We assess the efficiency of the setup by spectroscopic measurement of downstream dye concentrations, and demonstrate its practical utility by sequentially constructing multiple layers of alginate hydrogel. The method is potentially useful for a whole series of further applications, such as changing perfusion liquids for cell culture and cell analysis, metering, chemical-reaction initiation and multi-sample chromatography, to name a few.