Screening for Cell Adhesion Properties (PDF)

Figure 1. Top) Brightfield image of multiplexed pattern of eight different ECM proteins: bovine serum albumin (BSA), fibronectin (FN), laminin (LM), vitronectin (VIT), collagen I (Col-I), collagen IV (Col-IV), human extracellular matrix mixture (ECM), and matrigel (MAT). White scale bar is 500 µm. Bottom) Fluorescence image of same pattern, labeling both fibronectin (red) and laminin (green).

Figure 2. Brightfield images of 3T3 fibroblasts and C2C12 myoblasts adhered to DPN-patterned features of bovine serum albumin (BSA), fibronectin (FN), laminin (LM) and matrigel (MAT).

Screening for Cell Adhesion Properties

Abstract

NanoInk's desktop nanolithography platform, the NLP 2000 System, was used to create sub-cellular scaled arrays of multiple extracellular matrix (ECM) proteins on a glass slide. The multiplexed arrays were used to determine the cell binding efficiencies of various cell types, and binding efficiency data was used to identify the optimal ECM and array geometry for each cell type being studied. Using this technique, it was possible to establish a cell's adhesion propensities for multiple ECM proteins over the course of a single screening experiment.

Keywords

Desktop Nanolithography, Lithography, Cell Culture, Cell Adhesion, Cell Function

Introduction

Cell adhesion plays an important functional role in most human cell types; it is known to contribute to many tissue maintenance processes, including cell migration, wound healing and cellular differentiation. To study the impact of cell adhesion on these and other cellular functions, an even more fundamental cell adhesion issue must first be addressed: those cells being studied must be affixed to a suitable culture substrate. Thus, characterization of a cell population's ability to adhere to various types of cell adhesive molecules is a primary component of many cell function experiments. Also, the success of a cell culture experiment often depends on the ability to identify a suitable cell adhesive molecule(s) in order to study downstream cellular functions.

Dip Pen Nanolithography® (DPN®) is an established method of nanofabrication in which materials are deposited onto a surface using a sharp tip. DPN enables controlled deposition of a wide variety of materials onto various substrates with nanoscale registry, all under biocompatible conditions. Using DPN, multiple materials (e.g. cell adhesive molecules) can be patterned over small areas, with the resultant capacity to assess cell binding with very few cells. The ability to use small numbers of cells to determine adhesive response is important for experiments involving very rare or expensive cells or for studies where passage number or time in culture might influence cell function.

Using DPN, researchers can simultaneously pattern several cell adhesive molecules onto a surface and then study cell adhesion. For example, ECM proteins such as fibronectin, laminin or collagen can be deposited and cells subsequently adhered via integrin binding. DPN is also capable of depositing antibodies specific to cell membrane surface antigens for attachment of cells that over express cell membrane receptor proteins.

In previous Application Notes, NanoInk has described direct deposition of proteins onto a substrate followed by patterning of cells onto the printed protein domains. Here we describe the utility of using the DPN platform to screen for cell binding strength using considerably fewer cells than is typically necessary with conventional microtiter plate screening techniques.

Cell Binding Efficiencies

NanoInk's NLP 2000 System was employed to fabricate sub-cellular scaled protein arrays using the process described in the company's Multiplex Protein Arrays Application Note. The NLP 2000 System is a desktop nanolithography instrument capable of printing arbitrary protein patterns at sub-cellular scales. The size and shape of the cell adhesive protein pattern can be tailored to the size and shape of the cell being studied. In this case, seven different ECM proteins and bovine serum albumin (BSA) - a negative control - were simultaneously patterned into 3x3 squares. A representative image is depicted in Figure 1.

The ECM patterned surface was coated with BSA as a blocking agent to render the non-patterned areas less adhesive to cells and the blocked slide was then incubated with cells (at densities varying from 5x10⁴ to 1x10⁶ cells/cm²) in a plating media. Slides were incubated for approximately 30 minutes, and then washed to remove cells not bound to the patterned ECM features. Slides were imaged with an inverted phase contrast microscope to quantify adhesion.

In experiments that require the use of single cell patterns, small features of DPN-deposited cell adhesive proteins and high densities of cells work best. Lower concentration of cells will result in a lower percentage of patterns having bound cells but a higher percentage of bound cells being single cells.

Multiplexed Cell Adhesion Screening

We demonstrated the DPN platform's ability to screen for adherence of various cell types to multiple ECM proteins by depositing fibronectin, laminin, and matrigel (as well as BSA as a negative control) in 2x2 patterns as shown in Figure 2. 3T3 fibroblasts subsequently added to patterned slides bound strongly to fibronectin but not to the other two ECM proteins. Conversely, C2C12 myoblasts adhered strongly to fibronectin and only moderately to both laminin and matrigel. As illustrated in Figure 2, this experiment involved ten binding areas for each of the patterned proteins; it is possible to increase the number of binding areas in order to increase experimental statistical relevance.

Conclusion

This work demonstrates that substrates pattered with various ECM proteins can be used to assess cell binding properties. In addition to ECM proteins, DPN can be used to pattern other types of cell adhesion molecules, such as antibodies and receptor ligands. The DPN platform can also simultaneously deposit multiple concentrations of cell adhesion molecules to enable multiplexed cell adhesion analysis. This screening technique provides the flexibility needed to investigate cell adhesion using minimal amounts of user-determined proteins and cells.

Reference

Collins, J.M. and Nettikadan, S. Sub-cellular scaled multiplexed protein patterns for single cell co-cultures. Analytical Biochemistry. 419(2):339-41, 2011.

NanoInk Products Used

NLP 2000 System
DPN® Pen Arrays: Type M
DPN® Inkwell Arrays: Type 66x12

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