Reusable, reversibly sealable parylene membranes for cell and protein patterning

Abstract

The patterned deposition of cells and biomolecules on surfaces is a potentially useful tool for in vitro diagnostics, high-throughput screening, and tissue engineering. Here, we describe an inexpensive and potentially widely applicable micropatterning technique that uses reversible sealing of microfabricated parylene-C stencils on surfaces to enable surface patterning. Using these stencils it is possible to generate micropatterns and copatterns of proteins and cells, including NIH-3T3 fibroblasts, hepatocytes and embryonic stem cells. After patterning, the stencils can be removed from the surface, plasma treated to remove adsorbed proteins, and reused. A variety of hydrophobic surfaces including PDMS, polystyrene and acrylated glass were patterned using this approach. Furthermore, we demonstrated the reusability and mechanical integrity of the parylene membrane for at least 10 consecutive patterning processes. These parylene-C stencils are potentially scalable commercially and easily accessible for many biological and biomedical applications.

Figure 1

The mechanical properties of parylene membranes. A: Parylene membrane stencils, created using a vapor deposition and etching process, are peeled off individually with tweezers for laboratory applications. B,C: SEM images of the parylene membrane stencil. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 2

Schematic of the patterning process using reversibly sealing, reusable parylene stencils. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

Fluorescent images of protein copatterning on PDMS using parylene. A: Initially, a Texas Red-BSA protein solution is incubated on the parylene membrane for 15–30 min. The protein (red) adsorbs both to the parylene and the exposed regions of the PDMS. B: After the parylene is peeled off, the adsorbed protein pattern remains on the substrate (red), while the unexposed regions are free of protein (black). C: A second protein solution of FITC-BSA (green) is incubated on the first pattern, selectively binding to the protein-free regions and creating a protein copattern. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Fluorescent images of proteins patterned on (A) polystyrene, (B) methacrylated glass, and (C) curved PDMS. The success of these substrates in creating patterns is dependent on the formation of a tight reversible seal between the substrate and the parylene membrane upon contact. Glass slides did not provide a tight seal. We conclude that only hydrophobic surfaces, such as PDMS, polystyrene, and methacrylated glass, can be used for protein patterning with parylene. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 5

Reusability of parylene membranes as a stencil for multiple protein patterning experiments. FITC-BSA was patterned on PDMS (A) and polystyrene (B) using a single parylene membrane for 10 different patterning experiments. The structural integrity of the membrane was easily preserved through the 10 experiments, as evidenced by the nearly identical protein patterns it produced. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 6

Cell patterning on PDMS using parylene stencils. A,B: Phase contrast images of patterned NIH-3T3 fibroblasts after stencil removal. Substrate was initially coated with FN to increase adhesion. C–F: Cell cocultures of AML12 hepatocytes (blue) and NIH-3T3 fibroblasts (red). AML12 hepatocytes are patterned first, and viewed with phase contrast (C) and fluorescence (E) microscopy. Subsequently, NIH-3T3 cells are seeded, allowed to grow to confluence, and viewed with phase contrast (D) and fluorescence (F) microscopy. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 7

Recovery of a parylene membrane by plasma treatment. A parylene membrane used in protein patterning adsorbs an amount of protein, which may inhibit its effectiveness in further experiments. Plasma treatment for 300 sec reduces this adsorbed protein concentration to the original value.