C004988c 2771..2777

Microfabrication techniques and microfluidics are increasingly being used in single-cell research. Surface patterning of cell adhesive and non-fouling patches, for instance, can confine cells to arbitrary shapes on two-dimensional surfaces, whereas laminar flow can be used to deliver pharmacological agents to well-defined parts of cells. Together with the powerful tools available through molecular biology, the control of the cellular environment at a size scale equal to or smaller than the cells is currently revealing intriguing details about the cytoskeleton mechanics, transduction schemes, apoptosis, motility and differentiation. The natural environment of mammalian cells, however, is three-dimensional rather than two-dimensional, and usually much softer than the silicon, glass or plastic surfaces typically used for cell culture experiments. For this reason, a strong research effort has gone into the development of artificial three-dimensional analogs of extracellular matrix, and into techniques to pattern them. Major approaches are the use of photolithographic structuring of polyethylene glycol (PEG) hydrogels, and fluidic layer-by-layer deposition using the natural contraction of collagen. The use of UV radiation in the case of PEG hydrogels in the presence of cells, and the fixed layer height given by the contraction fraction of the collagen are the major drawbacks of these two approaches. In addition, it is difficult to precisely engineer the single cell environment using these techniques. We propose on-chip layer-by-layer deposition of chemically modified alginates to achieve three-dimensional environments on the single cell scale. In the past, we have demonstrated layer-by-layer deposition of alginates with different particle loads, here we extend the technique to chemically modified alginates, enabling structuring of the environment around single cells. We provide a proof of principle of this concept by depositing alternatively fluorescently labeled and non-fluorescent alginate under biocompatible conditions; we investigate the maximum resolution achievable and show that is