Fabrication and biological evaluation of uniform extracellular matrix coatings on discontinuous photolithography generated micropallet arrays

A driving force for the development of methodologies for the investigation of rare adherent cell populations is the increasing appreciation of cellular heterogeneity at the tissue level, including the recognition of mesenchymal stem cell and other progenitor cell compartments. The relative proportion of specific cellular subsets has been associated with specific biological behaviors 1-14 suggesting that the study of rare cellular subsets within tissues may provide additional capacity over genomic signatures to direct individualized therapeutic strategies, so called “individualized” or “personalized” medicine 2-4,15. Ideally, these studies would be conducted on primary tissue samples at the single cell level to potentially enable molecular and genomic analysis of individual cells. Primary tissues contain various broad cellular compartments including: stromal (e.g., fibroblasts), glandular or lining structures (e.g., epithelial and endothelial cells), along with cells of the hematologic and immune systems (e.g., macrophages and lymphocytes). Although technologies for the study of rare cell populations of hematopoietic and lymphoid cells has existed for some time 16, the currently available technologies for selection and isolation of single adherent cells from large heterogeneous populations have significant drawbacks, including requirements for large tissue samples and poor viability of recovered cells. Micropallet arrays are a recently developed technology designed to allow the identification, selection, and recovery of a single adherent cell within a given cell population, Fig. 1(a), 17-23. Micropallet arrays consist of tens of thousands of microscale pedestals, or “micropallets”, arrayed on a glass microscope slide and each designed to hold a single adherent cell. The micropallets are made from a transparent polymeric material that can be patterned using photolithographic microfabrication techniques. The cells are confined to the tops of the micropallets by chemical modification of the array such that their surfaces, i.e., the micropallet top surfaces, sidewalls, and underlying glass substrate, are highly hydrophobic. When an aqueous solution is applied to the arrays, air is trapped in the voids between micropallets creating an air network of “virtual walls” that sequesters cells to the top surfaces of individual micropallets, Fig. 1(b). The proof of concept for this technology was established by the use of particularly robust adherent cell lines with little or no specific requirements for optimal adherence, HeLa & RBL 18,19,22. Prior tissue culture experience and knowledge of fundamental biology suggests that the successful application of micropallet array technology to the examination of primary adherent and semi-adherent cells is likely to be critically dependent on the ability to effectively coat micropallet arrays with extracellular matrix (ECM) components. Extracellular matrix components that are commonly used for growth of adherent cells are generally proteins or glycoproteins capable of adhering to standard tissue culture surfaces and/or glass. The unique physical nature of micropallet arrays places additional constraints on the use of ECM components in order to maintain the full functional capacity of the micropallet array. Such considerations are not necessary when utilizing ECM proteins coatings for cell culture on traditional ex vivo culture technologies that employ flat, continuous growth surfaces. Figure 1 Micropallet array and virtual walls. Micropallet arrays are vast arrays of microscale polymer pedestals uniformly arranged on a glass microscope slide. (a) Scanning electron microscope (SEM) micrographs of a micropallet array consisting of micropallets ... The functionality of an ECM coating on micropallet arrays is determined by three critical parameters: the uniformity and stability of adherence of the ECM coatings on individual micropallets, restriction of the ECM to the top micropallet surfaces, and the affinity for the ECM proteins of the specific cell type being investigated. The growth surface of a micropallet array is in reality a vast number of uniformly spaced microscale growth surfaces, arrayed to present a pseudo-solid surface at the macroscale, but very non-uniform and discontinuous at the micro and cellular scale. This drives the need to develop specialized coating protocols to achieve proper and efficacious application of ECM components to the micropallet arrays. We report studies to define conditions and methodologies to generate functional ECM coatings of various types (fibronectin, EHS basement membrane extract (BME), collagen, and laminin-5) on micropallet arrays and the effectiveness of these coatings in capturing cells from different cell lines representing the range of cellular compartments, noted above, that constitute specific tissues.