Microfluidic construction of minimalistic neuronal co-cultures.

In this paper we present compartmentalized neuron arraying (CNA) microfluidic circuits for the preparation of neuronal networks using minimal cellular inputs (10-100-fold less than existing systems). The approach combines the benefits of microfluidics for precision single cell handling with biomaterial patterning for the long term maintenance of neuronal arrangements. A differential flow principle was used for cell metering and loading along linear arrays. An innovative water masking technique was developed for the inclusion of aligned biomaterial patterns within the microfluidic environment. For patterning primary neurons the technique involved the use of meniscus-pinning micropillars to align a water mask for plasma stencilling a poly-amine coating. The approach was extended for patterning the human SH-SY5Y neuroblastoma cell line using a poly(ethylene glycol) (PEG) back-fill and for dopaminergic LUHMES neuronal precursors by the further addition of a fibronectin coating. The patterning efficiency Epatt was >75% during lengthy in chip culture, with ∼85% of the outgrowth channels occupied by neurites. Neurons were also cultured in next generation circuits which enable neurite guidance into all outgrowth channels for the formation of extensive inter-compartment networks. Fluidic isolation protocols were developed for the rapid and sustained treatment of the different cellular and sub-cellular compartments. In summary, this research demonstrates widely applicable microfluidic methods for the construction of compartmentalized brain models with single cell precision. These minimalistic ex vivo tissue constructs pave the way for high throughput experimentation to gain deeper insights into pathological processes such as Alzheimer and Parkinson Diseases, as well as neuronal development and function in health.

[1]  D. Janasek,et al.  A microfluidic array with cellular valving for single cell co-culture. , 2011, Lab on a chip.

[2]  Jonathan West,et al.  Plasma stencilling methods for cell patterning , 2009, Analytical and bioanalytical chemistry.

[3]  G. Whitesides,et al.  Soft lithography in biology and biochemistry. , 2001, Annual review of biomedical engineering.

[4]  Nitish Thakor,et al.  Axon Myelination and Electrical Stimulation in a Microfluidic, Compartmentalized Cell Culture Platform , 2012, NeuroMolecular Medicine.

[5]  Digant P. Dave,et al.  Neuro-optical microfluidic platform to study injury and regeneration of single axons. , 2009, Lab on a chip.

[6]  Tanja Waldmann,et al.  Rapid, complete and large‐scale generation of post‐mitotic neurons from the human LUHMES cell line , 2011, Journal of neurochemistry.

[7]  R. Campenot,et al.  Development of sympathetic neurons in compartmentalized cultures. II. Local control of neurite survival by nerve growth factor. , 1982, Developmental biology.

[8]  G. Whitesides,et al.  Patterning proteins and cells using soft lithography. , 1999, Biomaterials.

[9]  N. Lemon,et al.  Dopamine D1/D5 Receptors Gate the Acquisition of Novel Information through Hippocampal Long-Term Potentiation and Long-Term Depression , 2006, The Journal of Neuroscience.

[10]  Jean-Louis Viovy,et al.  Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers. , 2011, Lab on a chip.

[11]  Charless C. Fowlkes,et al.  Quantitative analysis of axonal transport by using compartmentalized and surface micropatterned culture of neurons. , 2012, ACS chemical neuroscience.

[12]  C. Cotman,et al.  β-Amyloid impairs axonal BDNF retrograde trafficking , 2011, Neurobiology of Aging.

[13]  D. Beebe,et al.  PDMS bonding by means of a portable, low-cost corona system. , 2006, Lab on a chip.

[14]  Hiroyuki Fujita,et al.  Constraining the connectivity of neuronal networks cultured on microelectrode arrays with microfluidic techniques: a step towards neuron-based functional chips. , 2006, Biosensors & bioelectronics.

[15]  Shoji Takeuchi,et al.  A trap-and-release integrated microfluidic system for dynamic microarray applications , 2007, Proceedings of the National Academy of Sciences.

[16]  Sung June Kim,et al.  Low-density neuronal networks cultured using patterned poly-l-lysine on microelectrode arrays , 2007, Journal of Neuroscience Methods.

[17]  Luke P. Lee,et al.  Dynamic single cell culture array. , 2006, Lab on a chip.

[18]  Jan G Hengstler,et al.  High fidelity neuronal networks formed by plasma masking with a bilayer membrane: analysis of neurodegenerative and neuroprotective processes. , 2011, Lab on a chip.

[19]  M. Théry,et al.  Micropatterning as a tool to decipher cell morphogenesis and functions , 2010, Journal of Cell Science.

[20]  C. Cotman,et al.  A microfluidic culture platform for CNS axonal injury, regeneration and transport , 2005, Nature Methods.

[21]  Jan G Hengstler,et al.  The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening. , 2010, Lab on a chip.

[22]  James J. Hickman,et al.  Developmental Neurobiology Implications from Fabrication and Analysis of Hippocampal Neuronal Networks on Patterned Silane-Modified Surfaces , 1998 .

[23]  Nitish Thakor,et al.  Circular compartmentalized microfluidic platform: Study of axon-glia interactions. , 2010, Lab on a chip.

[24]  Noo Li Jeon,et al.  A Microfluidic Chamber for Analysis of Neuron-to-Cell Spread and Axonal Transport of an Alpha-Herpesvirus , 2008, PloS one.

[25]  M. Meyer,et al.  Aligned microcontact printing of micrometer-scale poly-L-Lysine structures for controlled growth of cultured neurons on planar microelectrode arrays , 2000, IEEE Transactions on Biomedical Engineering.

[26]  Rui Liu,et al.  Spatiotemporally controlled and multifactor involved assay of neuronal compartment regeneration after chemical injury in an integrated microfluidics. , 2012, Analytical chemistry.

[27]  M. Textor,et al.  Surface engineering approaches to micropattern surfaces for cell-based assays. , 2006, Biomaterials.

[28]  Noo Li Jeon,et al.  Patterned cell culture inside microfluidic devices. , 2005, Lab on a chip.

[29]  J. Sturm,et al.  Continuous Particle Separation Through Deterministic Lateral Displacement , 2004, Science.

[30]  Albert Folch,et al.  Integration of topographical and biochemical cues by axons during growth on microfabricated 3-D substrates. , 2005, Experimental cell research.

[31]  Luke P. Lee,et al.  Single-cell enzyme concentrations, kinetics, and inhibition analysis using high-density hydrodynamic cell isolation arrays. , 2006, Analytical chemistry.

[32]  D Kleinfeld,et al.  Controlled outgrowth of dissociated neurons on patterned substrates , 1988, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[33]  Erin M. Schuman,et al.  Microfluidic Local Perfusion Chambers for the Visualization and Manipulation of Synapses , 2010, Neuron.

[34]  N. Thakor,et al.  Varicella-Zoster Virus (VZV) Infection of Neurons Derived from Human Embryonic Stem Cells: Direct Demonstration of Axonal Infection, Transport of VZV, and Productive Neuronal Infection , 2011, Journal of Virology.

[35]  Anja Kunze,et al.  Co-pathological connected primary neurons in a microfluidic device for Alzheimer studies. , 2011, Biotechnology and bioengineering.

[36]  Jean-Louis Viovy,et al.  Wallerian-Like Degeneration of Central Neurons After Synchronized and Geometrically Registered Mass Axotomy in a Three-Compartmental Microfluidic Chip , 2010, Neurotoxicity Research.

[37]  Andreas Offenhäusser,et al.  Synaptic plasticity in micropatterned neuronal networks. , 2005, Biomaterials.

[38]  Mark A. Scott,et al.  Identification of small molecules that Enhance Synaptogenesis using Synapse Microarrays , 2011, Nature communications.

[39]  Wael Mismar,et al.  Examination of axonal injury and regeneration in micropatterned neuronal culture using pulsed laser microbeam dissection. , 2010, Lab on a chip.

[40]  Bruce C. Wheeler,et al.  Microelectrode Array Recordings of Patterned Hippocampal Neurons for Four Weeks , 2000 .

[41]  Gengfeng Zheng,et al.  Detection, Stimulation, and Inhibition of Neuronal Signals with High-Density Nanowire Transistor Arrays , 2006, Science.

[42]  Jianrong Li,et al.  Multi-compartment neuron-glia co-culture platform for localized CNS axon-glia interaction study. , 2012, Lab on a chip.

[43]  M. Toner,et al.  Microengineering of cellular interactions. , 2000, Annual review of biomedical engineering.

[44]  A. Lander,et al.  Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules , 1985, The Journal of cell biology.

[45]  Shy Shoham,et al.  Rapid neurotransmitter uncaging in spatially defined patterns , 2005, Nature Methods.

[46]  Luke P. Lee,et al.  Mammalian electrophysiology on a microfluidic platform. , 2005, Proceedings of the National Academy of Sciences of the United States of America.