Valve-Based Microfluidic Compression Platform: Single Axon Injury and Regrowth

Transcription

1 Supplementary Material (ESI) for Lab on a Chip This Journal is The Royal Society of Chemistry 2011 Valve-Based Microfluidic Compression Platform: Single Axon Injury and Regrowth Suneil Hosmane a, Adam Fournier b, Rika Wright b, Labchan Rajbhandari c, Rezina Siddique a, In Hong Yang a, K.T. Ramesh b, Arun Venkatesan c*, and Nitish Thakor a* ESI Fig. 1 A cross-sectional view of device construction. The AIM device was assembled by (A1) first pouring a thick layer of PDMS over the control template and baking until fully cured. (A2) The cured PDMS on the control wafer was removed and access ports were punched. (B) At the same time, the neuronal template was spin-coated with a thin-film of uncured PDMS followed by a complete bake. (C) Both the control devices and the coated neuronal template were oxygen plasma treated, aligned, bonded, and baked overnight to facilitate fusion between adjacent layers. (D) Composite devices were then removed and fluidic access ports to the neuronal layer were punched. (E) Devices were cleaned, bonded to glass-bottom Petri dishes, and seeded with neurons prior to injury experiments. Figure not to scale. 1

2 ESI Fig. 2 (Left) The assembled AIM device consists of a cell body, injury, and axonal compartment (not to scale). (Right) A subsection of the finite element model depicts variable mesh sizes for the injury pad, thin-film membrane, and glass substrate. Scale bar 30 μm. ESI Fig. 3 PDMS stress-stretch curve fitting literature and experimentally obtained data. 2

3 ESI Fig. 4 Representative images demonstrated the response of a single axon immediately before, during (~1 s), and after (~1 m) a 67 kpa compressive injury. Injury pad deformation was confirmed as the pad initially began out-of-focus, became in-focus during injury, and went out-of-focus after pressure was released. Axons (green) were false colored to enhance contrast and allow clear visualization. Scale bar 25 μm. ESI Fig. 5 (A) A 3-D schematic of the AIM platform under pressure application and microchannel deflection. (B) The same AIM device shown in Figure 2 was imaged at the microchannel interface to determine the extent of microchannel deflection. Under loads greater than that required to bring the injury pad in contact with the glass substrate (> 68.5 kpa), a > 3 μm gap could be seen. This was sufficient to allow unperturbed axon outgrowth (diameter < 2 μm) before, during, and after injury. 3

4 ESI Fig. 6 Percentage of injury pad deflection as a function of input pressure for a membrane thickness of 55 μm. ESI Fig. 7 Regrowing axon growth rates were 40% faster than control axon counterparts. *p-value < 0.05, unpaired 1-way Welch s t-test. Error bars on graphs correspond to standard errors. ESI Movie 1 Time-lapse of an axon prior to, during, and after crush by the injury pad. ESI Movie 2 Time-lapse of a healthy axon growing before and after a mild injury. ESI Movie 3 Time-lapse of a degenerating axon before and after a moderate injury. ESI Movie 4 Time-lapse of a regrowing axon before and after a severe injury that resulted in transection. 4

5 ESI Movie 5 Time-lapse of a regrowing axon before and after a severe injury. Axons were labeled with a tau-tdt (tandem tomato; red) fusion protein. ESI Table 1. Device Geometries Feature Figure Length (L) Width (W) Height (H) Resist microchannels 1A Top 500 μm 10 μm μm 3005 pad clearance 1A Mid mm mm 20 μm 3025 pad height 1A Bot. 1.8 mm 30 μm 30 μm 3025 control pad 1B 2 mm 1 mm 100 μm 3050 ESI Table 2. Mooney-Rivlin coefficients for PDMS material model Coefficients (Pa) Estimated Lower Limit Estimated Upper Limit C e e e+05 C e e e+05 ESI Table3. Mesh Density Sensitivity Element Length (μm)* Total Contact Force (mn) *Aspect ratio of elements in the compression pad and glass substrate remain constant as mesh density is increased 5

6 ESI Table 4. Coefficients for Eqn. 3 relating contact pressure, input pressure, and membrane thickness Coefficients Estimated Lower Limit Estimated Upper Limit p p p