Introduction

Controlled cell adhesion to the extracellular matrix (ECM) is essential for a coordinated morphogenesis and growth of functional tissue during embryonic development, tissue differentiation, and regeneration (1,2,3). Deregulation of cell adhesion occurs in many diseases and can contribute to the malfunction of tissues. For example, the abundance of integrins, a major family of ECM receptors, changes during tumor transformation. This is believed to contribute to the progression from a noninvasive to an invasive tumor stage (4). Therefore, binding specificities between cells and ECM components as well as changes in binding strengths and molecular mechanisms underlying these changes have been studied for many years. At the same time, the continuing discovery of novel ECM proteins, receptors, and related signal transduction pathways results in increasing numbers of cell-substrate interactions being investigated (5,6,7,8). In addition, there is growing interest in characterizing cell attachment to synthetic materials, such as peptides, polymers, or recombinant ECM protein fragments for the purpose of designing matrices for tissue engineering (9,10,11,12). Modulation of cell-substrate interactions by environmental influences, such as growth factors, or intrinsic effects, such as altered gene expression, add additional complexity to the function of cell-substrate interactions being investigated (13). Given the multitude of cell-substrate interactions, systematic screenings of cell attachment to large panels of substrates accelerate a comprehensive characterization and understanding of cell adhesion.

Most in vitro studies on cell-ECM interactions are performed by coating multiwell plates with ECM proteins or other adhesion molecules. This approach requires considerable quantities of substrate and cells. Because most ECM proteins are purified from tissues, they can be expensive and limited in quantity, especially when extracted from human sources. Another limiting factor is cell availability, especially when stem cells or cells from biopsied material are to be tested against a whole panel of ECM proteins or other substrates. The miniaturization of cell adhesion assays helps to facilitate higher throughput for such studies, and large panels of substrates can be examined rapidly while only small amounts of cells are needed. For this reason, microarrays of diverse cell binding substrates have been developed (14,15,16,17,18,19,20,21). These studies were mainly focused on the preparation of arrays containing various types of substrates and their effects on cells, but little attention has been given to the development of methods for accurate comparative measurements of cell adhesion in this miniaturized format. Here we describe an advanced cell microarray system that provides both reproducible immobilization of protein and a better control of cell culture conditions for differential cell adhesion measurements while using small amounts of cells.

Materials and Methods

Proteins

Type I collagen from human placenta, human plasma-derived fibro-nectin, human tenascin, and laminin from human placenta were purchased from Chemicon (Temecula, CA, USA). Type III collagen from human placenta, type IV collagen from basement membrane of Engelbreth-Holm-Swarm (EHS) murine Sarcoma, type V collagen from human placenta, human vitro-nectin, human thrombospondin, EHS laminin, EHS heparan sulfate proteoglycan (HSPG), and poly-L-lysine were purchased from Sigma-Aldrich (Taufkirchen, Germany). Human cellular fibronectin was obtained from Upstate Biotechnology (Lake Placid, NY, USA), type IV collagen from human placenta was purchased from BD Biosciences (Bedford, MA, USA), human placenta-derived type VI collagen was purchased from Acris (Hiddenhausen, Germany), and bovine serum albumin (BSA) was acquired from Roth (Karlsruhe, Germany). BSA was labeled with TAMRA (Fluka Chemie AG, Deisenhofen, Germany), and the labeled protein was purified using a PD10 column (Pharmacia AB, Uppsala, Sweden) according to the manufacturer's instructions.

Preparation of Microarrays

Proteins were microspotted on nitrocellulose-coated glass microslides (NMI Technologietransfer GmbH, Reutlingen, Germany). Protein solutions were prepared in 0.5% (w/v) trehalose in phosphate-buffered saline (PBS) containing 0.0003% (v/v) of the nonionic detergent IGEPAL® CA-630 [(octylphenoxy)polyethoxye thanol; Sigma-Aldrich] for collagens or 0.005% (v/v) IGEPAL CA-630 for all other proteins. For the chondrocyte adhesion assays, type III collagen was spotted in 0.4% (w/v) trehalose, 0.001% IGEPAL CA-630, and 5% glycerol. In general, we used the lowest protein coating concentration of a given protein at which the highest number of cells attached to a microspot in an adhesion assay. Furthermore, economic considerations were taken into account when expensive proteins were used as well as technical feasibility of printing these protein solutions with the ink-jet arrayer. Unless otherwise indicated, proteins were spotted at the following concentrations: 200 µg/mL poly-L-lysine, type III collagen, type V collagen, type VI collagen, fibronectin (human cellular), laminin (human placenta), and laminin (EHS); 400 µg/mL BSA; 50 µg/mL type I collagen (human placenta) and tenascin; 100 µg/ mL type IV collagen (human placenta), type IV collagen (EHS), fibronectin (human plasma), thrombospondin, and HSPG; 250 µg/mL vitronectin.