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Patterning Cell Arrays Using a Versatile Polymer Template

Christine P. Tan**, Daniel J. Brooks**, Harold Craighead*, and Claudia Fischbach**

*School of Applied and Engineering Physics, Cornell University

**Department of Biological and Environmental Engineering, Cornell University

Abstract

Cell-cell interactions play a critical role in tissue homeostasis, and dysregulation of this interplay may contribute to cancer initiation and progression. The goal of this study is to create tailored, cell-patterned surfaces to study the angiogenic capability of tumor cells in the absence and presence of cell-cell interactions. To this end, we fabricated a versatile polymer template on a glass cover slip that enables the patterning individual cells and clusters in a well-defined manner. Our polymer template offers several advantages over the widely popular polydimethylsiloxane (PDMS) used in micro-patterning.

Research Summary

Alterations in cell signaling contribute to tumor vascularization, a hallmark of cancer [1]. By comparing the angiogenic behavior of individual cancer cells and cell clusters containing varying number of cancer cells, one may be able to identify signaling pathways leading towards enhanced tumor vascularization. Knowledge of these pathways is important in designing more efficacious anti-cancer treatments, as well as identifying biomarkers for cancer diagnosis and patient prognosis. Cell arrays have previously been patterned by micro-contact printing (mCP) using PDMS stamps, to study cell-cell interactions, to measure cellular forces and to introduce time-variant stimuli to cells [2].

In this work, we describe a versatile polymer (Parylene C) template to create tailored surfaces for patterning cell arrays of individual and clustered cells, for studying the angiogenic behavior of tumor cells as a function of cell-cell interactions. Parylene is chemically inert, pinhole free and resist swelling in aqueous medium. Our Parylene templates overcome the problem of sagging and swelling associated with the PDMS in mCP, thus leading to more reproducible feature patterns. In addition, we can fabricate parylene templates with features, ranging from nanometers to micrometers. We have reported the use of Parylene templates to pattern cells [3], DNA [4] and lipid bilayers[5].

Figure 1: Fabrication process flow

Briefly, the fabrication process is shown in Figure 1. Using standard photolithography technique and oxygen plasma etching, we fabricated Parylene templates with micrometer-sized features on glass cover slips (Figure 2). The Parylene film served as a template for coating defined areas of fibronectin onto the underlying cover slip, and afterwards this template was peeled off. Laying down the fibronectin was necessary for cell adhesion in later steps. Figure 3 shows fibronectin coated cover slips, as visualized by immunostaining. The cover slip surface was treated with a blocking agent (e.g. bovine serum albumin, polyethylene-glycol) to prevent non-specific cell adhesion. Figure 3 also shows the cell array patterned using this method. We quantified the number of adherent cells based on fluorescent DAPI-staining of the nuclei. The surface area of the features determined the number of cells adhered and thus, able to interact with each other.

We are currently using these Parylene template cover slips to pattern oral squamous cancer cells and study their ability to up-regulate angiogenic factor secretion as a function of cell-cell interactions.

Figure 2: Parylene templates with micrometer-sized features on a glass cover slip (left to right: 25x25 microns squares, 20x40 microns rectangles, 40x40 microns squares)

Figure 3: Microscopy of surfaces patterned with 25x25 micron squares of fibronectin (Left to right: Fibronectin pattern visualized using fluorescent immunostaining, phase contrast image of cells patterned in an array, and fluorescent microscopy of DAPI-stained cell nuclei for the quantification of the number of cell adhered to each feature).

References

  1. Hanahan, D. and Weinberg, R.A., Cell (2000) 100:57-70
  2. Raghava, S. and Chen, C.S., Adv. Mat. (2004) 16:1303-1313
  3. Ilic, B. and Craighead, H.G., Biomed. Devices (2000) 2, 317-322
  4. Moran-Mirabal, J.M., et al, Anal. Chem. (2007) 79, 1109-1114
  5. Moran-Mirabal, J.M., et al, Biophy. J. (2005) 89, 296-305