High-throughput microcontact printing of proteins in microwell cell culture plates

Abstract

Microcontact printing (MCP) is used to pattern a surface with a specific compound, allowing the spatially restricted response of cells to be assayed as they encounter a molecule of interest. MCP is a relatively low-cost and accessible technique that uses commercially available reagents and common cell culture equipment. However, it can be technically challenging, slow, and incompatible with microwell cell culture plates that are widely used for screening and other applications. Here, we describe a novel protocol using medical biopsy punches to transfer patterns into standard 96-well plates via polydimethylsiloxane (PDMS) cutouts. We demonstrate that this method can be used to deposit patterns of poly-D-lysine (PDL) into the microwells of glass-bottom plates. As a proof-of-concept, we show that cultured rodent glial cells preferentially grow and extend processes on the pattern. This method will allow larger scale MCP experiments in which different patterns, proteins, or other factors can be assayed in parallel.•Biopsy punches enable both cutting out small circular stamps and plunging them into tissue culture microwells to transfer proteins.•Compared to standard MCP, this method offers a more rapid workflow to pattern proteins onto substrates, and allows use of microwell plates that permits larger-scale experiments.

Keywords: Cell assay; High-content; High-throughput microwell microcontact printing; Micropatterning.

Graphical abstract

Fig. 1

Schematic of the microwell MCP workflow.

Fig. 2

A) Representative well in a 96-well plate stamped with 10 µg/mL PDL mixed with 0.5 µg/mL Alexa488-conjugated IgG (green) in a honeycomb pattern with 5 µm circular holes, with a 50% PDL-50% PEG backfilled reference surface. Neonatal rat glia were cultured for 7 days and labeled with Alexa546 conjugated Phalloidin to visualize F-actin along cellular processes (magenta). B) Magnified image of a glial cell corresponding to the box in A. Some cellular processes can be seen growing over the holes (blue arrows), while most grow around on the more adhesive PDL pattern (yellow arrows). C) A glial cell from a different plate printed with 10 µm-thick stripe of PDL with a 50% PDL-50% PEG reference surface. Instead of growing outward, once encountering the stripe, processes deflect to extend along the edge of the comparatively more adhesive PDL stripe substrate (yellow arrows).