Paper-based patterned 3D neural cultures as a tool to study network activity on multielectrode arrays

Dermutz, Harald; Thompson-Steckel, Greta; Forró, Csaba; Lange, Victoria de; Dorwling-Carter, Livie; Vörös, János; Demkó, László

doi:10.1039/c7ra00971b

Cited by 12 publications

(27 citation statements)

References 93 publications

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“…Whatman filter paper was previously shown to promote extensive neuronal growth into the cellulose fiber matrix, creating a dense 3D network especially within the Whatman Grade 2 membranes, which are characterized to have a porosity of 8 μm (Dermutz et al, 2017 ). We adapted the culture technique for astrocytic populations by seeding primary embryonic cortical cells onto laser-cut paper membrane rings and culturing them in astrocyte-promoting serum-based media.…”

Section: Resultsmentioning

confidence: 99%

“…Culturing on the side creates a non-uniform diffusion gradient of trophic factors across the experimental culture. Cellulose paper has been established as a cell culture platform that is permeable and does not act as a complete physical barrier (Derda et al, 2009 ; Dermutz et al, 2017 ). The 3D fiber matrix also more closely resembles the physiological conditions in tissues.…”

Section: Discussionmentioning

confidence: 99%

“…Efforts toward meaningful cell patterning have helped to increase the physiological relevance of the experimental culture compared to organized, 3D populations in vivo (Goubko and Cao, 2009 ; Roy et al, 2013 ; Matsusaki et al, 2014 ; Albers et al, 2015 ; Tomba and Villard, 2015 ; Aebersold et al, 2016 ; Alagapan et al, 2016 ; Honegger et al, 2016 ). Techniques exist both in 2D, with methods such as microcontact printing, and in 3D, with the development of novel 3D culture substrates (Birgersdotter et al, 2005 ; Huh et al, 2011 ; Edmondson et al, 2014 ; Knight and Przyborski, 2015 ; Ravi et al, 2015 ; Dermutz et al, 2017 ). Functionalizing culture substrates with extracellular matrix proteins and other key factors is necessary not only for basic cell adhesion and viability but also for creating versatile, defined environments.…”

Section: Introductionmentioning

confidence: 99%

“…Cellulose filter paper has been demonstrated to be a mechanically stable material to use as a 3D culture substrate (Derda et al, 2009 , 2011 ; Dermutz et al, 2017 ). The material is commercially available, inexpensive, biocompatible, bioinert, and ion and nutrient permeable (Akram et al, 2015 ).…”

Section: Introductionmentioning

confidence: 99%

“…The material is commercially available, inexpensive, biocompatible, bioinert, and ion and nutrient permeable (Akram et al, 2015 ). Various cell types, including primary neurons, readily adhere and integrate into the 3D porous fiber matrix, which can be functionalized with standard adhesion-promoting proteins (Derda et al, 2009 , 2011 ; Dermutz et al, 2017 ). In addition, major advantages of using paper as a culture substrate are that the material is easy to handle and is compatible with standard sample preparation methods.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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

et al. 2017

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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.

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Three-dimensional highly porous hydrogel scaffold for neural circuit dissection and modulation

Yan¹,

Wang²,

Wu³

et al. 2023

Acta Biomaterialia

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Paper-based patterned 3D neural cultures as a tool to study network activity on multielectrode arrays

Abstract: High-throughput platform targeting activity patterns of 3D neural cultures with arbitrary topology, by combining network-wide intracellular and local extracellular signals.

Cited by 12 publications

References 93 publications

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

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

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

Three-dimensional highly porous hydrogel scaffold for neural circuit dissection and modulation

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