Multiprotein microcontact printing with micrometer resolution

Abstract

Depositing multiple proteins on the same substrate in positions similar to the natural cellular environment is essential to tissue engineering and regenerative medicine. In this study, the development and verification of a multiprotein microcontact printing (μCP) technique is described. It is shown that patterns of multiple proteins can be created by the sequential printing of proteins with micrometer precision in registration using an inverted microscope. Soft polymeric stamps were fabricated and mounted on a microscope stage while the substrate to be stamped was placed on a microscope objective and kept at its focal distance. This geometry allowed for visualization of patterns during the multiple stamping events and facilitated the alignment of multiple stamped patterns. Astrocytes were cultured over stamped lane patterns and were seen to interact and align with the underlying protein patterns.

Figure 1

Essential steps of creating multiple protein patterns. (A) A high resolution image of astrocyte expressed laminin (stained green), chondroitin sulfate proteoglycan (stained red) and cell nucleus (stained blue) (courtesy of F. Meng, Tresco’s lab, University of Utah). (B) A smaller region of astrocyte membrane is used to create μCP templates for two proteins. (C) These templates are used to prepare two μCP stamps with the same repeating patterns. (D) The stamps are used to print two proteins in registration.

Figure 2

Schematic of the multi-protein μCP device. A light microscope was modified to accept stamps and substrates while providing real-time imaging capabilities.

Figure 3

Schematic of the multi-protein μCP process. A stamp is inked and dried (1–2) then loaded and leveled using the leveling objective. The substrate stage is advanced with the microscope turret and the stamp is aligned with a previous pattern (3). The stamp is then brought into contact with the surface and the protein pattern is transferred (4–5). This process is repeated to deposit multiple patterns of proteins.

Figure 4

Examples of in situ microscopy available in multi-protein μCP process. (A) Reflectance interference contrast microscopy (RICM) image of a gradient patterned stamp in contact with a substrate. (B) Fluorescence microscopy image of a transferred laminin (AlexaFluor 488-labeled) protein pattern from a gradient stamp.

Figure 5

The registration capability of multi-protein μCP process. (A) A subsection of a random 20% coverage pattern. (B) Bright field image of a randomized 20% coverage stamp. (C) A subsection of the complementary 80% coverage pattern. (D) Bright field image of a complementary 80% coverage stamp. (E) Overlay of the two stamps as seen by operator during the alignment step. False color was added for contrast.

Figure 6

Lane patterns printed with laminin (AlexaFluor 488-labeled) and aggrecan (AlexaFluor 592-labeled). (A) Intended lane spacing of 0 μm. (B) Intended lane spacing of 5 μm.

Figure 7

(A), (B) Fluorescent images produced from the sequential stamping of laminin (green, AlexaFluor 488-labeled) and aggrecan (red, AlexaFluor 592-labeled) using two lane stamps with bright field overlay of cultured astrocytes. Astrocytes were seen to interact with the underlying laminin pattern but not the underlying aggrecan pattern. (C) Histogram showing the alignment of astrocytes cultured on aggrecan lanes relative to underlying pattern (n = 97). (D) Histogram showing the alignment of astrocytes cultured on laminin and aggrecan lanes relative to underlying pattern (n = 179).