Neuron-cell culture is widely used for studies in basic research, drug discovery, and toxicity testing. Traditional random cultures allow limited access to subcellular compartments (axons, dendrites, synapses) due to extensive and haphazard growth of neurons. Our long-term goal is to provide robust, user-friendly, and cost effective culture platforms that can optically, fluidically, and biochemically access neurons and their subcellular compartments. Data acquired through sales of prototype platforms developed and patented by our team show a large and increasing demand. Customer feedback also indicates that technological improvements would make our platforms more accessible and user-friendly. Such improvements include ensuring greater uniformity of the device, reducing end-user assembly procedures, and enhancing viability for the long-term culturing period that is often needed for neurons. Thus, our first aim focuses on addressing these issues by (1) incorporating cell loading ports into the platform to reduce cell loading errors, (2) improving feature uniformit through the development of high-resolution, durable metal molds, (3) developing methods to reduce evaporative losses that impair neuron viability, (4) increasing the wettability and biocompatibility of the device material through surface modification, and (5) covalently linking extracellular matrix proteins onto glass to minimize end-user assembly. The successful completion of this aim will result in a consistent, cost-effective, and ready-to-use neuron-cell culture platform. Our next aim focuses on the development of novel cell-based tools to study synapses, the cornerstone of neuroscience. There is considerable demand for methods to isolate synapses and there are limitations in existing techniques.
In Aim 2 we will develop user-friendly synapse isolation tools, expanding on core technological advancements from Aim 1.
Aim 2 will involve (1) development of stable, artificial synaptic bead targets allowing novel investigations of the presynaptic compartment and (2) development of a three-compartment synapse isolation platform that exploits device geometry and synaptic beads to encourage synapse formation within an isolated synaptic compartment. The successful completion of this aim will result in a user-friendly, accessible, and innovative cell-based tool to optically and biochemically probe synapses. The significance of the proposed work is to improve research scientists'ability to visualize, manipulate, and measure cultured neurons leading to greater understanding of the underlying causes of neurological diseases. This research is innovative because we seek to shift current research paradigms through the development of novel cell-based tools to isolate synapses that maintain intact cell morphology in the absence of somata or glia.
The proposed research is relevant to public health because technology developed in this proposal will facilitate investigations of neurological illnesses and injury. The project is relevant to NIH's mission because it will provide a user-friendly and novel tool for cellular neuroscience, advancing the study of neuronal and synaptic function.
|Pinto, Maria J; Alves, Pedro L; Martins, Luís et al. (2016) The proteasome controls presynaptic differentiation through modulation of an on-site pool of polyubiquitinated conjugates. J Cell Biol 212:789-801|
|Kamande, J W; Wang, Y; Taylor, A M (2015) Cloning SU8 silicon masters using epoxy resins to increase feature replicability and production for cell culture devices. Biomicrofluidics 9:036502|
|Niedringhaus, Mark; Dumitru, Raluca; Mabb, Angela M et al. (2015) Transferable neuronal mini-cultures to accelerate screening in primary and induced pluripotent stem cell-derived neurons. Sci Rep 5:8353|
|Gordon, Kent R; Wang, Yuli; Allbritton, Nancy L et al. (2015) Magnetic Alignment of Microelements Containing Cultured Neuronal Networks for High-Throughput Screening. J Biomol Screen 20:1091-100|
|Menon, Shalini; Boyer, Nicholas Patrick; Winkle, Cortney Chelise et al. (2015) The E3 Ubiquitin Ligase TRIM9 Is a Filopodia Off Switch Required for Netrin-Dependent Axon Guidance. Dev Cell 35:698-712|
|Taylor, A M; Menon, S; Gupton, S L (2015) Passive microfluidic chamber for long-term imaging of axon guidance in response to soluble gradients. Lab Chip 15:2781-9|