Research Program
Cell Surface Interactions

Objectives
The principal objective of this project is to design, evaluate and validate compartmented cultures suitable for automated imaging of axonal organelle transport under conditions that model neural diseases.  This design will allow assessment of transport in regions of the axon subjected to oxidative stress or oxygen/glucose deficiency and also to assess the effects of this damage on adjacent regions of the axon proximal and distal to the injury zone.  A second goal is to explore and evaluate novel microfluidic strategies to deliver soluble biomolecules to cells growing in culture at a user-determined position and a user-defined time.  

Methods
Surface patterning of poly-L-lysine has been employed to control rat hippocampal neuron attachment and axonal growth, using photopatterning and liftoff techniques combined with sacrificial fluidic housings to pattern axonal “tracks” as small as 2 microns in width emanating from somal attachment zones with characteristic widths on the order of 50 microns.

Poly-dimethyl siloxane compartmented chambers have been fabricated using replicate molding, affixed to glass substrates, and used to control the distribution of solutes.  Quantitative measurements of solute distribution indicate that solute transport between chambers is reduced 10,000-fold by the presence of the compartments.  These allow localized soluble factors (e.g., neurotrophic factors, ATP, oxygen or its absence) to be correlated with imaging of organelle transport.

Nanoporous, nanometer-thick Si membranes have been integrated into a plastic device via thermal encapsulation, and we have demonstrated that the fluids on one side of these membranes can be addressed fluidically within seconds.  This will allow membranes to be grown on nanoporous substrates in concert with real-time adjustment of the solute chemistry permeating the substrate.

Summary
Our initial objective has been to optimize geometry and substrate preparation in order to confine neuronal cell bodies to small “landing pads” on a substrate and to induce the formation of long axons along lines leading to additional microfluidic compartments.  Our present strategy involves plating neurons into narrow wells formed with sacrificial microfluidic housings, which can be removed after the cells attach.  Axons grow out along parallel, micropatterned lines of polylysine, which facilitates neurite elongation.  Using this approach, axons extend up to 4 mm after 1 week in culture, long enough to allow imaging in 3 or 4 different fluid compartments.  Presently, we are investigating variations in substrate preparation and cell culture conditions to maximize the consistency of these results.  Once we have accomplished this, we plan to integrate compartments that will allow axons to grow out from a central well along microetched lines that lead beneath a PDMS barrier and into additional microfluidically controlled compartments.

Accomplishments

• A protocol for photopatterning poly-L-lysine on glass wafers has been developed and shown to control the geometry of rat hippocampal neuron axons.
• Photolithographic patterning of PLL has been shown to isolate single axons that grow as long as 4mm after 7 days in culture.
• Compartments have been fabricated and shown to isolate solutes.
• Silicon nanomembranes have been integrated into microdevices and microfluidic addressing of backside fluids has been demonstrated.