Direct patterning of coplanar polyethylene glycol alkylsilane monolayers by deep-ultraviolet photolithography as a general method for high fidelity, long-term cell patterning and culture

Abstract

This manuscript details a general method for patterning coplanar alkylsilane monolayers using deep-ultraviolet photolithography that has broad application for high fidelity patterning of cells of varying phenotype in long-term cultures. A polyethylene glycol monolayer was formed on a silica substrate and then patterned using 193 nm light from an ArF excimer laser. The regions of photoablation were then rederivatized with (3-trimethoxysilyl propyl) diethyltriamine (DETA), yielding high contrast cytophilic islands that promoted cell adhesion and growth. Rat hippocampal neurons, motoneurons, and myoblasts were then cultured in a defined, serum-free medium on the patterned surfaces for periods in excess of 40 days. This approach has been shown to be useful as a general method for the long-term culture of multiple cell types in highly defined spatial patterns and can be used for supporting complex cocultures for creating in vitro models for biological systems.

Figure 1

(Color online) DUV photolithography system and proposed reaction scheme. Light emitted from the ArF laser has a parabolic profile that is passed through a beam homogenizer to produce a beam with a top-hat intensity profile. Using a wavelength selective mirror, the homogenized light is then reflected onto the sample, which has been placed under a patterned chrome photomask. The exposed regions of the PEGSi monolayer are then photoablated by the incident laser light leaving a substrate suitable for reaction with another silane, in this case DETA.

Figure 2

Contact angle measurements and droplet visualizations for (a) PEGSi control, (b) DETA control, (c) DETA backfilled into unablated PEGSi control, (d) DETA backfilled into ablated PEGSi, and (e) PEGSi backfilled into a DETA control.

Figure 3

Representative high resolution N 1s and C 1s spectra for (a) PEGSi monolayer, (b) photoablated PEGSi, (c) DETA monolayer formed on ablated PEGSi surface, (d) DETA monolayer formed on clean Si, (e) mixed monolayer formed by reacting PEGSi onto a control DETA monolayer, and (f) DETA reacted onto unablated PEGSi monolayer.

Figure 4

(a) Atomic percent of C 1s signal vs ablation time. After 30 s atomic percent of C 1s was comparable to that of clean Si (dashed line). (b) Atomic percent of N 1s signal vs ablation time. Ablated samples were reacted with 0.1% DETA for 30 min to form a monolayer of DETA in the ablated regions. The graph shows nitrogen content (from N 1s high resolution spectra) vs ablation time. The dashed line indicates nitrogen content measured from control DETA samples.

Figure 5

Nitrogen content as incorporated into unablated vs ablated PEGSi monolayers as a function of reaction time. After 30 min reaction time, nitrogen content in the ablated PEGSi samples (solid line) reached values suitable for use in cell culture. Nitrogen content on unablated PEGSi monolayers (dashed line), however, reached a maximum of $\sim 1.9\mathrm{\%}$ at about 10 min, which ensured that longer reaction times could be used to achieve an optimal DETA monolayer while not sacrificing pattern contrast.

Figure 6

Metalized PEGSi-DETA patterns and resulting cell cultures with varying cell types. [(a), (c) and (e)] Metallization results for three different patterns [(a) $100\mu \text{m}$ lines, (b) $30\times 30\mu {\text{m}}^2$ squares, and (c) two-cell circuit pattern]. [(b), (d), and (f)] Rat cells cultured on PEGSi-DETA patterns [(b) embryonic skeletal muscle, (d) embryonic motoneuron, and (f) hippocampal neurons].

Figure 7

Skeletal muscle on patterned PEG-DETA surface remained confined to the DETA regions of the pattern up to 41 days on $100\mu \text{m}$ wide lines. (a) 9 days in culture, (b) 16 days in culture, (c) 26 days in culture, and (d) 41 days in culture. Although many of the myotubes at a later time pulled off the surface due to spontaneous contraction, the remaining myotubes were still confined to the patterns.