Adhesion, proliferation, and detachment of various cell types on thermoresponsive microgel coatings.

Cutting-edge biomedical applications require increasingly complex and fastidious cell systems, for example, various classes of primary or stem cells. Their cultivation, however, still differs little from 30 years ago. This especially applies to the use of indiscriminative proteases for nonspecific cell detachment. A far more gentle alternative changes the adhesive properties of the cell culture substrates through coatings based on thermoresponsive polymers. Such polymers mediate cell adhesion at 37°C, but become repulsive upon a cell-compatible temperature drop to, for example, 32°C. While the high functionality of this method has already been well proven, it must also be easy and reproducible to apply. Here, we emphasize the potential of standard cell culture materials coated by spraying with thermoresponsive microgels for routine cultivation and beyond. On these surfaces, we successfully cultivated and detached various cell types, including induced pluripotent stem cells and cells in serum-free culture. In addition, we evaluated the compatibility of the microgel-sprayed surfaces with adhesion-promoting proteins, which are essential for, for example, stem cells or neuronal cells. Finally, we demonstrate that the microgel surfaces do not impair proliferation and show their long-term stability. We conclude that for cell detachment, thermoresponsive cell culture substrates can fully substitute proteases, like trypsin, by employing a comparably straightforward protocol that is compatible with many industrial processing lines.

Patterned Thermoresponsive Microgel Coatings for Noninvasive Processing of Adherent Cells.

Cultivation of adherently growing cells in artificial environments is of utmost importance in medicine and biotechnology to accomplish in vitro drug screening or to investigate disease mechanisms. Precise cell manipulation, like localized control over adhesion, is required to expand cells, to establish cell models for novel therapies and to perform noninvasive cell experiments. To this end, we developed a method of gentle, local lift-off of mammalian cells using polymer surfaces, which are reversibly and repeatedly switchable between a cell-attractive and a cell-repellent state. This property was introduced through micropatterned thermoresponsive polymer coatings formed from colloidal microgels. Patterning was obtained through automated nanodispensing or microcontact printing, making use of unspecific electrostatic interactions between microgels and substrates. This process is much more robust against ambient conditions than covalent coupling, thus lending itself to up-scaling. As an example, wound healing assays were accomplished at 37 °C with highly increased precision in microfluidic environments.

Microporous polymeric 3D scaffolds templated by the layer-by-layer self-assembly.

Polymeric scaffolds serve as valuable supports for biological cells since they offer essential features for guiding cellular organization and tissue development. The main challenges for scaffold fabrication are i) to tune an internal structure and ii) to load bio-molecules such as growth factors and control their local concentration and distribution. Here, a new approach for the design of hollow polymeric scaffolds using porous CaCO3 particles (cores) as templates is presented. The cores packed into a microfluidic channel are coated with polymers employing the layer-by-layer (LbL) technique. Subsequent core elimination at mild conditions results in formation of the scaffold composed of interconnected hollow polymer microspheres. The size of the cores determines the feature dimensions and, as a consequence, governs cellular adhesion: for 3T3 fibroblasts an optimal microsphere size is 12 µm. By making use of the carrier properties of the porous CaCO3 cores, the microspheres are loaded with BSA as a model protein. The scaffolds developed here may also be well suited for the localized release of bio-molecules using external triggers such as IR-light.

Control of cell adhesion by mechanical reinforcement of soft polyelectrolyte films with nanoparticles.

Chemical cross-linking is the standard approach to tune the mechanical properties of polymer coatings for cell culture applications. Here we show that the elastic modulus of highly swollen polyelectrolyte films composed of poly(L-lysine) (PLL) and hyaluronic acid (HA) can be changed by more than 1 order of magnitude by addition of gold nanoparticles (AuNPs) in a one-step procedure. This hydrogel-nanoparticle architecture has great potential as a platform for advanced cell engineering application, for example remote release of drugs. As a first step toward utilization of such films for biomedical applications we identify the most favorable polymer/nanoparticle composition for optimized cell adhesion on the films. Using atomic force microscopy (AFM) we determine the following surface parameters that are relevant for cell adhesion, i.e., stiffness, roughness, and protein interactions. Optimized cell adhesion is observed for films with an elastic modulus of about 1 MPa and a surface roughness on the order of 30 nm. The analysis further shows that AuNPs are not incorporated in the HA/PLL bulk but form clusters on the film surface. Combined studies of the elastic modulus and surface topography indicate a cluster percolation threshold at a critical surface coverage above which the film stiffness drastically increases. In this context we also discuss changes in film thickness, material density and swelling ratio due to nanoparticle treatment.

Thermoresponsive PEG-based polymer layers: surface characterization with AFM force measurements.

Thermoresponsive polymer-coated surfaces based on poly(2-(2-methoxyethoxy)ethyl methacrylate-co-oligo(ethylene glycol) methacrylate) [P(MEO(2)MA-co-OEGMA)] allow switching between cell attachment and detachment. Here, we investigate the temperature-dependent surface interactions between the polymer coating and a colloidal probe in an aqueous medium by means of atomic force microscopy (AFM) force-distance measurements. The analysis of the adhesion forces from AFM retraction curves identifies two kinds of regimes for the copolymer at temperatures below and above the lower critical solution temperature (LCST). Whereas at 25 degrees C the surface interactions with the polymer in the swollen state are dominated by repulsive forces, at 37 degrees C the surface interactions switch to attractive forces and a stronger adhesion is detected by AFM. Running several heating/cooling cycles repeatedly shows that switching the surface properties provides reproducible adhesion force values. Time-dependent measurements give insight into the switching kinetics, demonstrating that the cell response is coupled to the polymer kinetics but probably limited by the cellular rearrangements.

Tuning the thickness of polymer brushes grafted from nonlinearly growing multilayer assemblies.

A new versatile method for tuning the thickness of surface-tethered polymer brushes is introduced. It is based on the combination of polyelectrolyte multilayer deposition and surface-initiated atom transfer radical polymerization. To control the thickness of the brushes, the nonlinear growth of certain polyelectrolyte multilayer systems is exploited. The method is demonstrated to work with different polyelectrolytes and different monomers. The relevance for applications is demonstrated by cell adhesion experiments on grafted thermoresponsive polymer layers with varying thickness.