Detailed Analysis of Pancreatic Tumor Cell Attachment on Gradient PDEGMA Brushes.

Poly(di(ethylene glycol) methyl ether methacrylate) (PDEGMA) brushes show interesting thermoresponsive behavior that can be applied for cell release surfaces. Here it is shown that PDEGMA thickness gradients, which are synthesized by surface-initiated atom transfer radical polymerization, allow the systematic and precise analysis of the attachment of PaTu 8988 cells. By pumping the polymerization solution into the reactor with vertically fixed initiator samples, PDEGMA gradients with linearly increasing dry ellipsometric thickness with typical slopes of 2.5 nm cm-1 are obtained. A very narrow transition of PaTu 8988t cell attachment is observed that starts for a thickness larger than 7.1 ± 0.2 nm. For PDEGMA layers thicker than 8.7 ± 0.2 nm no attached cells are found. This very narrow transition in brush properties within a thickness difference of <2 nm from cell-adherent to cell-nonadherent can be determined in much greater detail than before owing to the thickness gradients with shallow slope.

Long-Term Stable Poly(acrylamide) Brush Modified Transparent Microwells for Cell Attachment Studies in 3D.

The fabrication of 3D cell microenvironments exploiting versatile, long-term stable passivating poly(acryl amide) brushes in a microwell format and the study of the behavior of fibroblast and pancreatic tumor cells in wells of systematically varied shape and size is reported. The microwells, which are obtained by combining micromolding in capillaries with microcontact printing of initiator monolayers for subsequent surface-initiated polymerization of acrylamide and controlled functionalization with fibronectin (FN), expose cell adhesive areas inside the wells and protein and cell resistant brushes on the topside plateaus. NIH 3T3 fibroblast and pancreatic tumor (Patu 8988T) cells adhere and remain viable in the FN coated microwells for more than 1 week. Compared to 2D patterns, both cell lines are observed to attach to the bottom as well as the sidewalls of the microwells. The cytoskeleton alignment is found to be less pronounced compared to 2D patterned substrates, independent of microwell size and geometry.

Block Copolymer Brushes for Completely Decoupled Control of Determinants of Cell-Surface Interactions.

The known determinants for cell-surface interactions, comprising biochemical cues, patterns, passivating functionality, and control of tether mechanical properties, are fully decoupled in tailored block copolymer brushes synthesized by surface-initiated atom transfer radical polymerization. Exploiting sequential polymerization of a passivating underlying polyacrylamide (PAAm) block with defined cross-linking followed by a second poly(acrylic acid) block, which can be conjugated with a selective adhesion peptide, hierarchically structured brushes that can be micro-patterned by soft lithography were obtained. The interaction of NIH 3T3 fibroblasts and PaTu 8988t pancreatic tumor cells with brushes that differed only in the stiffness of the hidden PAAm block or only in the peptide ligand, while keeping all other parameters constant, revealing profound differences in cell adhesion and morphology. In particular, cells could only attach well to stiff RGD presenting brushes.

The Effect of Size and Geometry of Poly(acrylamide) Brush-Based Micropatterns on the Behavior of Cells.

In this study, the fabrication, detailed characterization, and application of long-term stable micropatterned bio-interfaces of passivating poly(acrylamide) (PAAm) brushes on transparent gold for application in the study of cell-surface interactions is reported. The micropatterns were fabricated by microcontact printing of an initiator for surface-initiated atom transfer radical polymerization (SI-ATRP), SI-ATRP of acrylamide, and subsequently backfilling of the unfunctionalized areas of 400-2500 µm(2) size and systematically altered number of corners with octadecanethiol. As verified by surface plasmon resonance spectroscopy, the physisorption of fibronectin (FN) was restricted to the adhesive areas. Exploiting this platform, the effect of micropattern geometry and size of cell-adhesive FN areas surrounded by passivating PAAm brushes on transparent gold substrates on the attachment of cells and cytoskeleton alignment was investigated at the single-cell level. Exceptional long-term stability of the patterned PAAm brushes and arrays of adhesive areas, in which human pancreatic tumor cells (Patu 8988T) and fibroblast cells (NIH 3T3) were confined for more than one week, was observed. Adhesive areas of 1600 µm(2) or less constrained the cell shape and caused focal adhesions to accumulate in the corners of the pattern. These changes were most obvious for the PatuT cells in adhesive areas of ∼900 µm(2), in which the actin filaments were aligned, following the boundary of the pattern, and merged in the focal adhesions concentrated in the corners of the pattern. NIH 3T3 cells possessed a larger cell area, which led to an optimal cytoskeleton alignment in adhesive patterns of ∼1600 µm(2). The alignment of the cytoskeleton was found to be less pronounced in cells on larger adhesive areas, where the PatuT cells spread similarly to cells on unpatterned substrates. By contrast, the NIH 3T3 cells were found to stretch even on larger adhesive areas, spanning from one corner to the other. The long-term stability under cell culture conditions of the patterns introduced here will also be useful for long-term studies of single and multiple cells, cell motility in toxicity assays, and stem cell differentiation.

Control of Cell Attachment and Spreading on Poly(acrylamide) Brushes with Varied Grafting Density.

To achieve spatial control of fibroblast cell attachment and spreading on a biocompatible polymer coating, the effect of poly(acrylamide) (PAAm) brushes with varied grafting density was investigated. The synthesis of the brushes was performed by surface-initiated atom transfer radical polymerization (SI-ATRP). Gold substrates were modified with binary self-assembled monolayers (SAMs) of an initiator and 16-mercaptohexadecanoic acid (MHDA) as an "inert" thiol to initiate the ATRP of AAm. By using different mixtures for the binary SAMs, a series of polymer brushes with varied grafting densities were prepared. The fractional coverage of surface bound initiator was determined by grazing incidence Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and contact angle measurements. A linear relationship between the Br/S ratio determined by XPS and ToF-SIMS versus the fraction of initiator on the surface determined by water contact angle measurements was observed. The varied initiation concentration on the gold substrates yielded PAAm brushes with different thicknesses, indicating a transition from mushroom to brush regimes with increasing grafting density. Thereby we achieved exquisite control of the degree of cell adhesion. Cell attachment experiments with NIH 3T3 fibroblast cells revealed cell spreading on PAAm brushes with low grafting densities (initiator fractional coverage <0.2) as well as a complete passivation by polymer brushes with higher grafting densities.