A Versatile Protein and Cell Patterning Method Suitable for Long-Term Neural Cultures.

Herein, we present an easy-to-use protein and cell patterning method relying solely on pipetting, rinsing steps and illumination with a desktop lamp, which does not require any expensive laboratory equipment, custom-built hardware or delicate chemistry. This method is based on the adhesion promoter poly(allylamine)-grafted perfluorophenyl azide, which allows UV-induced cross-linking with proteins and the antifouling molecule poly(vinylpyrrolidone). Versatility is demonstrated by creating patterns with two different proteins and a polysaccharide directly on plastic well plates and on glass slides, and by subsequently seeding primary neurons and C2C12 myoblasts on the patterns to form islands and mini-networks. Patterning characterization is done via immunohistochemistry, Congo red staining, ellipsometry, and infrared spectroscopy. Using a pragmatic setup, patterning contrasts down to 5 µm and statistically significant long-term stability superior to the gold standard poly(l-lysine)-grafted poly(ethylene glycol) could be obtained. This simple method can be used in any laboratory or even in classrooms and its outstanding stability is especially interesting for long-term cell experiments, e.g., for bottom-up neuroscience, where well-defined microislands and microcircuits of primary neurons are studied over weeks.

Modular microstructure design to build neuronal networks of defined functional connectivity.

Theoretical and in vivo neuroscience research suggests that functional information transfer within neuronal networks is influenced by circuit architecture. Due to the dynamic complexities of the brain, it remains a challenge to test the correlation between structure and function of a defined network. Engineering controlled neuronal networks in vitro offers a way to test structural motifs; however, no method has achieved small, multi-node networks with stable, unidirectional connections. Here, we screened ten different microchannel architectures within polydimethylsiloxane (PDMS) devices to test their potential for axonal guidance. The most successful design had a 92% probability of achieving strictly unidirectional connections between nodes. Networks built from this design were cultured on multielectrode arrays and recorded on days in vitro 9, 12, 15 and 18 to investigate spontaneous and evoked bursting activity. Transfer entropy between subsequent nodes showed up to 100 times more directional flow of information compared to the control. Additionally, directed networks produced a greater amount of information flow, reinforcing the importance of directional connections in the brain being critical for reliable communication. By controlling the parameters of network formation, we minimized response variability and achieved functional, directional networks. The technique provides us with a tool to probe the spatio-temporal effects of different network motifs.

Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks.

Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm2 down to isolated cells at 1,000 cells/cm2. Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development.

Local Chemical Stimulation of Neurons with the Fluidic Force Microscope (FluidFM).

Physiological communication between neurons is dependent on the exchange of neurotransmitters at the synapses. Although this chemical signal transmission targets specific receptors and allows for subtle adaptation of the action potential, in vitro neuroscience typically relies on electrical currents and potentials to stimulate neurons. The electric stimulus is unspecific and the confinement of the stimuli within the media is technically difficult to control and introduces large artifacts in electric recordings of the activity. Here, we present a local chemical stimulation platform that resembles in vivo physiological conditions and can be used to target specific receptors of synapses. Neurotransmitters were dispensed using the force-controlled fluidic force microscope (FluidFM) nanopipette, which provides exact positioning and precise liquid delivery. We show that controlled release of the excitatory neurotransmitter glutamate induces spiking activity in primary rat hippocampal neurons, as measured by concurrent electrical and optical recordings using a microelectrode array and a calcium-sensitive dye, respectively. Furthermore, we characterized the glutamate dose response of neurons by applying stimulation pulses of glutamate with concentrations from 0 to 0.5 mm. This new stimulation approach, which combines FluidFM for gentle and precise positioning with a microelectrode array read-out, makes it possible to modulate the activity of individual neurons chemically and simultaneously record their induced activity across the entire neuronal network. The presented platform not only offers a more physiological alternative compared with electrical stimulation, but also provides the possibility to study the effects of the local application of neuromodulators and other drugs.

Mechanical force induces mitochondrial fission.

Eukaryotic cells are densely packed with macromolecular complexes and intertwining organelles, continually transported and reshaped. Intriguingly, organelles avoid clashing and entangling with each other in such limited space. Mitochondria form extensive networks constantly remodeled by fission and fusion. Here, we show that mitochondrial fission is triggered by mechanical forces. Mechano-stimulation of mitochondria - via encounter with motile intracellular pathogens, via external pressure applied by an atomic force microscope, or via cell migration across uneven microsurfaces - results in the recruitment of the mitochondrial fission machinery, and subsequent division. We propose that MFF, owing to affinity for narrow mitochondria, acts as a membrane-bound force sensor to recruit the fission machinery to mechanically strained sites. Thus, mitochondria adapt to the environment by sensing and responding to biomechanical cues. Our findings that mechanical triggers can be coupled to biochemical responses in membrane dynamics may explain how organelles orderly cohabit in the crowded cytoplasm.

Easy to Apply Polyoxazoline-Based Coating for Precise and Long-Term Control of Neural Patterns.

Arranging cultured cells in patterns via surface modification is a tool used by biologists to answer questions in a specific and controlled manner. In the past decade, bottom-up neuroscience emerged as a new application, which aims to get a better understanding of the brain via reverse engineering and analyzing elementary circuitry in vitro. Building well-defined neural networks is the ultimate goal. Antifouling coatings are often used to control neurite outgrowth. Because erroneous connectivity alters the entire topology and functionality of minicircuits, the requirements are demanding. Current state-of-the-art coating solutions such as widely used poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) fail to prevent primary neurons from making undesired connections in long-term cultures. In this study, a new copolymer with greatly enhanced antifouling properties is developed, characterized, and evaluated for its reliability, stability, and versatility. To this end, the following components are grafted to a poly(acrylamide) (PAcrAm) backbone: hexaneamine, to support spontaneous electrostatic adsorption in buffered aqueous solutions, and propyldimethylethoxysilane, to increase the durability via covalent bonding to hydroxylated culture surfaces and antifouling polymer poly(2-methyl-2-oxazoline) (PMOXA). In an assay for neural connectivity control, the new copolymer's ability to effectively prevent unwanted neurite outgrowth is compared to the gold standard, PLL-g-PEG. Additionally, its versatility is evaluated on polystyrene, glass, and poly(dimethylsiloxane) using primary hippocampal and cortical rat neurons as well as C2C12 myoblasts, and human fibroblasts. PAcrAm-g-(PMOXA, NH2, Si) consistently outperforms PLL-g-PEG with all tested culture surfaces and cell types, and it is the first surface coating which reliably prevents arranged nodes of primary neurons from forming undesired connections over the long term. Whereas the presented work focuses on the proof of concept for the new antifouling coating to successfully and sustainably prevent unwanted connectivity, it is an important milestone for in vitro neuroscience, enabling follow-up studies to engineer neurologically relevant networks. Furthermore, because PAcrAm-g-(PMOXA, NH2, Si) can be quickly applied and used with various surfaces and cell types, it is an attractive extension to the toolbox for in vitro biology and biomedical engineering.

Controlled single-cell deposition and patterning by highly flexible hollow cantilevers.

Single-cell patterning represents a key approach to decouple and better understand the role and mechanisms of individual cells of a given population. In particular, the bottom-up approach of engineering neuronal circuits with a controlled topology holds immense promises to perceive the relationships between connectivity and function. In order to accommodate these efforts, highly flexible SU-8 cantilevers with integrated microchannels have been fabricated for both additive and subtractive patterning. By directly squeezing out single cells onto adhesive surfaces, controlled deposition with a spatial accuracy of 5 µm could be achieved, while subtractive patterning has been realized by selective removal of targeted single cells. Complex cell patterns were created on substrates pre-patterned with cell-adhesive and repulsive areas, preserving the original pattern geometry for long-term studies. For example, a circular loop with a diameter of 530 µm has been realized using primary hippocampal neurons, which were fully connected to their respective neighbors along the loop. Using the same cantilevers, the versatility of the technique has also been demonstrated via in situ modification of already mature neuronal cultures by both detaching individual cells of the population and adding fresh ones, incorporating them into the culture.