Irene Henning1 Paul Williams1 Richard Hague1 Morgan Alexander1

1, University of Nottingham, Nottingham, Nottinghamshire, United Kingdom

To our knowledge, we are the first to demonstrate the multiphoton fabrication of alginate hydrogel structures. The physiochemical properties of hydrogels are well suited to facilitate three-dimensional (3D) cell culture as they mimic the characteristics of a native cellular microenvironment. Alginate is one of the most prevalent biopolymers used for hydrogels due to its biocompatibility, reversible ionic gelation, and natural abundancy. Multiphoton fabrication allows for structures to be fabricated with sub-micron feature size, and therefore on the scale of single cells, while under conditions which living cells can tolerate. Combining the high resolution of multiphoton fabrication with the desirable properties of alginate enables control of the geometrical and mechanical properties of artificial cell microenvironments.
Alginate was functionalized with methacrylate groups in order to make it compatible with multiphoton polymerization. The methacrylation of the alginate was achieved using sodium alginate and 2-aminoethyl methacrylate (AEMA) in combination with the coupling agent 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and 4-methylmorpholine (NMM). The degree of methacrylation could be controlled by varying the amount of reactive compounds and can be used to tune the mechanical properties of the hydrogel. The methacrylated alginate was crosslinked in the presence of a water soluble photoinitiator, P2CK, developed for efficient multiphoton polymerization and biocompatibility. One to two weight percent of methacrylated alginate in water enabled the fabrication of microstructures with a minimum feature size of approximately 200 nm, characterized by scanning electron microscopy. Microstructures with the dimensions 50μm x 50μm x 10μm were fabricated at a laser scanning speed of 20 mm/s, printing one structure in 1-2 min, which is in one of the fastest speeds reported for multiphoton fabrication of hydrogels. We demonstrated that the microstructured alginate was stable under culture conditions with the gram-negative bacteria Pseudomonas aeruginosa over a period of 24 hours at 37°C. The viability of the bacteria cultured in the presence of the microstructures was shown by imaging the fluorescent marker that the bacteria were genetically modified to produce constitutively.
Here we report a method of alginate methacrylation that enables the multiphoton fabrication of biocompatible alginate hydrogels with sub-micron resolution. We predict this approach will enable the engineering of artificial cell microenvironments for a range of cell types and applications.