A Versatile Protein and Cell Patterning Method Suitable for Long-Term Neural Cultures.

Herein, we present an easy-to-use protein and cell patterning method relying solely on pipetting, rinsing steps and illumination with a desktop lamp, which does not require any expensive laboratory equipment, custom-built hardware or delicate chemistry. This method is based on the adhesion promoter poly(allylamine)-grafted perfluorophenyl azide, which allows UV-induced cross-linking with proteins and the antifouling molecule poly(vinylpyrrolidone). Versatility is demonstrated by creating patterns with two different proteins and a polysaccharide directly on plastic well plates and on glass slides, and by subsequently seeding primary neurons and C2C12 myoblasts on the patterns to form islands and mini-networks. Patterning characterization is done via immunohistochemistry, Congo red staining, ellipsometry, and infrared spectroscopy. Using a pragmatic setup, patterning contrasts down to 5 µm and statistically significant long-term stability superior to the gold standard poly(l-lysine)-grafted poly(ethylene glycol) could be obtained. This simple method can be used in any laboratory or even in classrooms and its outstanding stability is especially interesting for long-term cell experiments, e.g., for bottom-up neuroscience, where well-defined microislands and microcircuits of primary neurons are studied over weeks.

Easy to Apply Polyoxazoline-Based Coating for Precise and Long-Term Control of Neural Patterns.

Arranging cultured cells in patterns via surface modification is a tool used by biologists to answer questions in a specific and controlled manner. In the past decade, bottom-up neuroscience emerged as a new application, which aims to get a better understanding of the brain via reverse engineering and analyzing elementary circuitry in vitro. Building well-defined neural networks is the ultimate goal. Antifouling coatings are often used to control neurite outgrowth. Because erroneous connectivity alters the entire topology and functionality of minicircuits, the requirements are demanding. Current state-of-the-art coating solutions such as widely used poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) fail to prevent primary neurons from making undesired connections in long-term cultures. In this study, a new copolymer with greatly enhanced antifouling properties is developed, characterized, and evaluated for its reliability, stability, and versatility. To this end, the following components are grafted to a poly(acrylamide) (PAcrAm) backbone: hexaneamine, to support spontaneous electrostatic adsorption in buffered aqueous solutions, and propyldimethylethoxysilane, to increase the durability via covalent bonding to hydroxylated culture surfaces and antifouling polymer poly(2-methyl-2-oxazoline) (PMOXA). In an assay for neural connectivity control, the new copolymer's ability to effectively prevent unwanted neurite outgrowth is compared to the gold standard, PLL-g-PEG. Additionally, its versatility is evaluated on polystyrene, glass, and poly(dimethylsiloxane) using primary hippocampal and cortical rat neurons as well as C2C12 myoblasts, and human fibroblasts. PAcrAm-g-(PMOXA, NH2, Si) consistently outperforms PLL-g-PEG with all tested culture surfaces and cell types, and it is the first surface coating which reliably prevents arranged nodes of primary neurons from forming undesired connections over the long term. Whereas the presented work focuses on the proof of concept for the new antifouling coating to successfully and sustainably prevent unwanted connectivity, it is an important milestone for in vitro neuroscience, enabling follow-up studies to engineer neurologically relevant networks. Furthermore, because PAcrAm-g-(PMOXA, NH2, Si) can be quickly applied and used with various surfaces and cell types, it is an attractive extension to the toolbox for in vitro biology and biomedical engineering.