preparation-of-neuronal-co-cultures-with-single-cell-precision

Preparation of Neuronal Co-cultures with Single Cell Precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms ...

ISAS

University College London

We present a highly parallel microfluidic approach for contacting single cell pairs. The approach combines a differential fluidic resistance trapping method with a novel cellular valving principle for homotypic and heterotypic single cell co-culturing. Differential fluidic resistance was used for sequential single cell arraying, with the adhesion and flattening of viable cells within the microstructured environment acting to produce valves in the open state. Reversal of the flow was used for the sequential single cell arraying of the second cell type. Plasma stencilling, along the linear path of least resistance, was required to confine the cells within the trap regions. Prime flow conditions with minimal shear stress were identified for highly efficient cell arraying (∼99%) and long term cell culture. Larger trap dimensions enabled the highest levels of cell pairing (∼70%). The single cell co-cultures were in close proximity for the formation of connexon structures and the study of contact modes of communication. The research further highlights the possibility of using the natural behaviour of cells as the working principle behind responsive microfluidic elements.

A microfluidic array with cellular valving for single cell co-culture.

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research.

Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits.

A pinched-flow deflection technology was developed for rapid single cell switching between biochemical microenvironments. Millisecond switching was used to stimulate and preserve epidermal growth factor receptor (EGFR) autophosphorylation transitions. Intramolecular phosphorylation initiates signal transduction, is silenced by phosphatase activity until EGFR dimerization enables intermolecular phosphorylation to initiate downstream signalling.

Ultrafast cell switching for recording cell surface transitions: new insights into epidermal growth factor receptor signalling.

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.

Microfluidic construction of minimalistic neuronal co-cultures.

This paper describes a microfluidic quenched flow platform for the investigation of ligand-mediated cell surface processes with unprecedented temporal resolution. A roll-slip behavior caused by cell-wall-fluid coupling was documented and acts to minimize the compression and shear stresses experienced by the cell. This feature enables high-velocity (100-400 mm/s) operation without impacting the integrity of the cell membrane. In addition, rotation generates localized convection paths. This cell-driven micromixing effect causes the cell to become rapidly enveloped with ligands to saturate the surface receptors. High-speed imaging of the transport of a Janus particle and fictitious domain numerical simulations were used to predict millisecond-scale biochemical switching times. Dispersion in the incubation channel was characterized by microparticle image velocimetry and minimized by using a horizontal Hele-Shaw velocity profile in combination with vertical hydrodynamic focusing to achieve highly reproducible incubation times (CV = 3.6%). Microfluidic quenched flow was used to investigate the pY1131 autophosphorylation transition in the type I insulin-like growth factor receptor (IGF-1R). This predimerized receptor undergoes autophosphorylation within 100 ms of stimulation. Beyond this demonstration, the extreme temporal resolution can be used to gain new insights into the mechanisms underpinning a tremendous variety of important cell surface events.

Article	Year
A microfluidic array with cellular valving for single cell co-culture. Lab on a chip\| PubMed ID: 20978708	2011
Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits. Lab on a chip\| PubMed ID: 21079873	2011
Ultrafast cell switching for recording cell surface transitions: new insights into epidermal growth factor receptor signalling. Lab on a chip\| PubMed ID: 23385220	2013
Microfluidic construction of minimalistic neuronal co-cultures. Lab on a chip\| PubMed ID: 23403713	2013
Whole cell quenched flow analysis. Analytical chemistry\| PubMed ID: 24295019	2013

Ya-Yu Chiang

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