The next generation of low adhesion
INTRODUCTION
Cell based in vitro assays are becoming more important in the basic research but also in applied research in pharmacology and toxicology. All these methods are generating important information and finally help to understand the human (patho-) physiology. In order to improve the predictivity of these assays, traditional two-dimensional cell culture models evolve into three-dimensional models (spheroids and organoids) and microfluidic systems (e.g. organ on a chip)1. Both trends require a robust and reproducible coating of the used hardware to generate valid data2.
Low attachment U-bottom shaped plates for spheroid generation are very convenient for the generation and usage of such spheroids and most of the major laboratory suppliers provide such plates in a variety of formats. However, this first wave of “low-binding plates” differs strongly in quality and a range of flaws. For example, multiple spheroids or irregular aggregates can be observed leading to reduced uniformity and reproducibility of assay results. Furthermore, plates are available in a limited number of formats, restricting applications severely to the standard microtiter surfaces and dishes. “Self-made systems” to coat other surfaces, e.g. based on agarose, come with increased variability and challenges in handling. More standardized 3D cell culture procedures could therefore further reduce data variability and enhance biological relevance of in vitro assays3.
To address these shortcomings, we have developed a polymeric coating solution. The BIOFLOAT™ FLEX coating solution generates a surface with excellent protein and cellrepellent properties, leading to a biologically inert surface on different kinds of lab consumables. Owing to its highly anti-adhesive properties, cell-to-cell interactions are favored, leading to the formation of highly uniform spheroids which float in the medium without interaction with the surface. The coating solution allows to treat different formats of plates or microfluidic devices, which are amenable for 3D spheroid screening approaches or organoid models in cancer research or toxicology.
The BIOFLOAT™ FLEX coating solution passivates plastic and glass surfaces
The aim was to develop a polymer solution that i) rapidly and strongly binds to the surface without any complex pretreatment of the surface, that (ii) is highly protein and cell-repellent, better than previously available systems and that iii) is stable under cell culture conditions.
The QCM detects mass changes of a sensor surface due to the binding of protein. Such protein adsorption is found in all materials tested including plastic and glass materials. In contrast, after coating the surface using BIOFLOAT FLEX coating solution, protein binding is significantly reduced.
After optimizing the polymer chemistry and composition, the physical properties of the coated surfaces were initially characterized using a Quartz-Crystal Microbalance (QCM), which detects the kinetics of mass adsorption and allows to precisely monitor the coating process. Hereby, a strong adsorption of the BIOFLOAT™ FLEX coating solution to polystyrene was detected already seconds after passing the solution through the flow cell. The adsorbed polymer layer of about 600 ng / cm2 corresponds to a monolayer of polymer molecule. Accordingly, the polymer-coated surface will not affect the geometry of the device. This is of importance, in particular for microfluidic devices since the diameter and flow rate of the microfluidic channels will not be changed by the deposition of such a layer.
The protein repellency of the BIOFLOAT™ FLEX coating solution was analyzed on different commonly used substrate materials such as polystyrene, quartz glass, polycarbonate and polyethylene. To this end, a casein solution was introduced into the QCM flow cell as a model protein to monitor the protein loading on the surfaces. The control experiments clearly showed that casein adsorbs to all materials tested in the absence of any other coating. However, once the surfaces were passivated with the BIOFLOAT™ FLEX coating solution, protein adsorption was fully prevented as can be seen from the minimal mass loadings in the QCM study.
