Evaluating the performance of fibrillar collagen films formed at polystyrene surfaces as cell culture substrates.

While it is well-appreciated that the extracellular matrix plays a critical role in influencing cell responses, well-defined and reproducible presentation of extracellular matrix proteins poses a challenge for in vitro experiments. Films of type 1 collagen fibrils assembled on alkanethiolate monolayers formed at gold-coated surfaces have been shown to elicit a cellular response comparable to collagen gels, but with the advantages of excellent optical properties, and high reproducibility and robustness. To make this collagen matrix more accessible to laboratories that do not have access to gold film deposition the authors have examined the use of untreated polystyrene as a substrate for forming fibrillar collagen films.

Tools for quantitative and validated measurements of cells.

In this chapter, we describe the preparation of thin films of collagen that can serve as reference materials for assuring reproducible and predictable cell responses. Subtle differences in the molecular-scale characteristics of extracellular matrix proteins, including the supramolecular structure of type 1 collagen, can have tremendous influences on cell state and cell-signaling pathways; therefore the careful control and analysis of the culture surface is critical to assure a relevant and consistent response in cell-based assays. We also describe how cell-phenotypic parameters such as morphology, proliferation, and green fluorescent protein expression can be unambiguously quantified in adherent cells by automated fluorescence microscopy or high content screening.

Gradient substrate assembly for quantifying cellular response to biomaterials.

Using quantitative fluorescence microscopy in conjunction with a method of gradient substrate assembly established in their group, the authors were able to introduce and measure reproducible changes in cellular morphology and cell density by manipulating polymer grafting density. The mechanism behind this change in cellular behavior was explained by a semiempirical, geometric model that describes the effect of the spatial distribution of the polymer on protein attachment. A 10-fold increase in graft density of poly(2-hydroxyethyl methacrylate) [PHEMA] along the surface of a gradient sample, preexposed to bovine fibronectin, caused a change in the size of fibroblasts on the surface (i.

Thin films of Type 1 collagen for cell by cell analysis of morphology and tenascin-C promoter activity.

Background: The use of highly reproducible and spatiallyhomogeneous thin film matrices permits automated microscopy and quantitative determination of the response of hundreds of cells in a population. Using thin films of extracellular matrix proteins, we have quantified, on a cell-by-cell basis, phenotypic parameters of cells on different extracellular matrices. We have quantitatively examined the relationship between fibroblast morphology and activation of the promoter for the extracellular matrix protein tenascin-C using a tenascin-C promoter-based GFP reporter construct.

Tuning cell adhesion on gradient poly(2-hydroxyethyl methacrylate)-grafted surfaces.

A simple yet versatile method was developed to prepare a low-density polymerization initiator gradient, which was combined with surface-initiated atom transfer radical polymerization (ATRP) to produce a well-defined poly(2-hydroxyethyl methacrylate) (HEMA) gradient substrate. A smooth variation in film thickness was measured across the gradient, ranging from 20 A to over 80 A, but we observed a nonmonotonic variation in water contact angle. Fits of X-ray reflectivity profiles suggested that at the low graft density end, the polymer chain structure was in a "mushroom" regime, while the polymer chains at high graft density were in a "brush" regime.

Vascular smooth muscle cell response on thin films of collagen.

Vascular smooth muscle cells (vSMC) cultured on gels of fibrillar type I collagen or denatured collagen (gelatin) comprise a model system that has been widely used for studying the role of the extracellular matrix in vascular diseases such as hypertension, restenosis and athrosclerosis. Despite the wide use of this model system, there are several disadvantages to using collagen gels for cellular studies. These include poor optical characteristics for microscopy, difficulty in verifying that the properties of the preparations are identical from experiment to experiment, heterogeneity within the gels, and difficulty in handling the gels because they are fragile.