Protein Patterning for directed in vitro biological neural networks

The formation of a nervous system begins when young neurons migrate to specific positions within the body. Once the cells find their appropriate position in the nervous system, the neurons mature and neurites extend away from the cell body to probe the surrounding environment. Initiated in part by extracellular cues, intracellular signaling mechanisms cause a particular neurite to develop into the axon. The axon will serve as the process which transmits electrical and chemical communications to other neurons, and the rest of the neurites will become dendrites to accept incoming communications. The growth cone on the end of the axon will guide the axon towards its intended target to create a synapse with a neighboring cell. The field of neuroscience is based largely on understanding how these fundamental phenomena occur both within the context of a single cell and within the nervous system as a whole. As we learn more about the chemical and physical cues that affect neuronal growth and behavior, greater opportunities arise to use these cues to engineer neural systems. These neural systems can, in turn, be used to further study neural development and investigate ways to repair damaged nervous tissue. Nervous tissue used to be considered unrepairable after damage, but recent evidence suggests that neuroregeneration could be possible [1, 2].

There are currently numerous methods used to control the microenvironment of a cell, including chemical [3-5], physical [6], electrical [7], and optical [8]. Each of these methods offers the ability to affect and/or detect the activity of a cell, but none are yet capable of truly controlling the growth of a complex neural network.

Under the mentorship of Luke Lee and Mu-ming Poo at UC Berkeley, I have invented a new way of patterning proteins and small molecules in extremely complex patterns onto planar substrates using microfluidics. This new method (manuscript under preparation) offers the ability to fill circuitous microfluidic channels with extremely high aspect ratios as well as dead-end channels. The patterning resolution has been demonstrated to be less than 2 µm. The key advantage of this technique is the ability to pattern multi-component protein patterns at sub-cellular resolution with a single step. Currently, there is no other practical method to create such a pattern. Other techniques either rely on multiple stamps with complicated alignment steps, or simply are unable to create patterns with this resolution. We have used this technique to create large arrays (up to 2,850 repeated elements on a single coverslip) of three bioactive molecules embedded in a polypeptide matrix. Rat hippocampal neurons plated onto these substrates aligned into ordered arrays, initiated axons at a specified location, and sprouted neurites that followed predetermined paths (see Figure). This is a first step towards guiding the formation of simple neuronal circuits in vitro, and this technologically integrated approach towards neuroscience will hopefully yield new insight into nerve cell physiology.

This figure is an overview of using novel microfluidic patterning to create multi-component patterns of neural guidance cues on a substrate. (A) Schematic of the pattern deposited onto a glass substrate, which includes an Axonal Growth Factor (AGF), a Cell Adhesion Protein (CAP), and a Dendritic Growth Factor (DGF). (B) A sample of fluorescent images of primary rat hippocampus cells cultured on patterned substrates. The neurons are stained red and the patterns are stained green. Scale bars are about 30 µm. The axon is shown to prefer the patterned regions which contain axonal growth factors.

References:

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