Biological Sample Printing

Microarray spotters equipped with solenoid valves are capable of printing a wide range of biological samples, including reagents, growth media, compounds, hydrogels, genes, proteins, viruses, and cells, for biochemical and cell-based assays. Solenoid valve-driven bioprinting has clear advantages over other printing technologies in terms of controlling sample volume dispensed and flexibility in the type of samples printed. Unlike robotic liquid dispensers that are more robust due to relatively large orifice sizes and dispensing volumes, microarray spotters must take precautions to prevent the clogging of solenoid valves and ceramic tips due to the extremely small sample volume printed. Therefore, it is essential to avoid dust and large precipitates (e.g., compound precipitates and filamentous microbes), test the viscosity of the material printed, and understand the mechanism of gelation for hydrogels used for cell encapsulation. In general, temperature-sensitive hydrogels such as Matrigel have a high risk of forming a gel spontaneously inside tubes, ceramic tips, and solenoid valves with a slight change in temperature. Therefore, a lower temperature must be maintained constantly across the chip-loading deck and the dispensing head. The clogging may result in replacement of tubes, solenoid valves, and ceramic tips, which can be an expensive affair. Gelation of hydrogels used for 3D cell culture has to be occurred in two steps to avoid clogging. Typically, crosslinkers/initiators are printed first on the micropillar/microwell chip platform and then hydrogels containing cells are dispensed on the top to form a gel. Therefore, ionic crosslinking (e.g., alginate with CaCl2, PuraMatrix with salts), affinity/covalent bonding (e.g., functionalized polymers with streptavidin and biotin), photopolymerization (e.g., methacrylated alginate with photoinitiators), and biocatalysis (e.g., fibrinogen with thrombin) are favorable mechanisms of gelation. Developing proper surface chemistry to attach cell spots in hydrogels robustly on the surface of the micropillar/microwell chip is also critical. Covalent bonding (e.g., poly(maleic anhydride-alt-1-octadecene) and poly-l-lysine), affinity (e.g., streptavidin and biotin), and ionic interaction (poly-l-lysine and negatively charged alginate) are commonly introduced to enhance spot attachment on the chip. Viscosity of the material printed is another important factor that affects the performance of printing, and valve/tip clogging and rinsing. In general, biological samples with low viscosity such as 10–30 centipoise (equivalent to 1 % or lower alginate in distilled water) are preferred. Samples with high viscosity cannot be printed with solenoid valves and are difficult to remove from tubes, solenoid valves, and tips by rinsing with water. Therefore, it has to be diluted properly with either solvents or cell culture media to ensure reproducible printing. In case of cell printing and encapsulation for 3D culture, maintaining cell suspension in hydrogels while printing is important to avoid solenoid valves and tips clogging. In general, over crowded cells (typically seeding density higher than 10 million cells/mL) can result in clogging issues. In addition, cells tend to settle down quicker in a low viscosity solution. Finally, mechanical strength of hydrogels over time needs to be considered to support long-term cell culture. Peptide-based hydrogels (e.g., fibrinogen, Matrigel, and PuraMatrix) tend to lose their strength over time due to degradation by matrix metalloproteinases (MMPs) secreted by many mammalian cells. These MMPs are responsible for hydrogel degradation and eventual spot detachment. To sustain cell spots for a longer time and minimize spot detachment due to degradation, peptide-based hydrogels can be mixed with more stable hydrogels such as alginate. When mixed, the resulting hydrogel has to be transparent with minimal background fluorescence and should not interfere high-content imaging (HCI) assays. The protocols provided in this chapter will give researchers a guidance towards multiplex, 3D-cell based assays on the micropillar/microwell chip platform.