the-fabrication-operation-continuous-flow-micro-electroporation

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Rutgers, the State University of New Jersey

Proper brain function relies on the precise arrangement and flow of information between diverse neural subtypes. Developing improved human cell-based models which faithfully mimic biologically relevant connectivity patterns may improve drug screening efforts given the limited success of animal models to predict safety and efficacy of therapeutics in human clinical trials. To address this need, we have developed experimental models of defined neural circuitries through the compartmentalization of neuronal cell subtypes in a 96 well plate-based platform where each microwell is divided into two compartments connected by microchannels allowing high-throughput screening (HTS) of small molecules. We demonstrate that we can generate subtype-specific excitatory and inhibitory induced neuronal cells (iNs) from human stem cell lines and that these neurons form robust functional circuits with defined connectivity. Through the use of the genetically encoded calcium indicator GCaMP6f, we monitor calcium ion transients generated during neuronal firing between and within compartments. We further demonstrate functionality of the circuit by perturbing network activity through the addition of glutamate receptor blockers using automated liquid handling. Lastly, we show that we can stimulate network activity in defined neuronal subtypes through the expression of the designer receptor exclusively activated by designer drugs (DREADD) hM3Dq and application of the ligand clozapine-N-oxide (CNO). Our results demonstrate the formation of functional neural circuits in a high-throughput platform that is compatible with compound screening, representing an important step towards developing new screening platforms for studying and ultimately treating psychiatric brain disorders that arise from disordered neural circuit function.

Development of a high-throughput arrayed neural circuitry platform using human induced neurons for drug screening applications.

Electroporation is an electro-physical, non-viral approach to perform DNA, RNA, and protein transfections of cells. Upon application of an electric field, the cell membrane is compromised, allowing the delivery of exogenous materials into cells. Cell viability and electro-transfection efficiency (eTE) are dependent on various experimental factors, including pulse waveform, vector concentration, cell type/density, and electroporation buffer properties. In this work, the effects of buffer composition on cell viability and eTE were systematically explored for plasmid DNA encoding green fluorescent protein following electroporation of 3T3 fibroblasts. A HEPES-based buffer was used in conjunction with various salts and sugars to modulate conductivity and osmolality, respectively. Pulse applications were chosen to maintain constant applied electrical energy (J) or total charge flux (C/m). The energy of the pulse application primarily dictated cell viability, with Mg-based buffers expanding the reversible electroporation range. The enhancement of viability with Mg-based buffers led to the hypothesis that this enhancement is due to ATPase activation via re-establishing ionic homeostasis. We show preliminary evidence for this mechanism by demonstrating that the enhanced viability is eliminated by introducing lidocaine, an ATPase inhibitor. However, Mg also hinders eTE compared to K-based buffers. Collectively, the results demonstrate that the rational selection of pulsing conditions and buffer compositions are critical for the design of electroporation protocols to maximize viability and eTE.

Jeffrey D. Zahn

The Department of Biomedical Engineering

Rutgers, the State University of New Jersey

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Jeffrey D. Zahn

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Department of Biomedical Engineering

Rutgers, the State University of New Jersey

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

The Department of Biomedical Engineering