Matthew Pan1 Shengyang Chen1 Tae-Sik Jang1 Juha Song1

1, Nanyang Technological University, Singapore, , Singapore

Biofabrication technologies have endowed us with the capability to fabricate complex biological constructs at exceptional resolution and accuracy. Photocrosslinking is commonly coupled with biofabrication to produce stable cell-laden hydrogels due to precise control in the polymerization process, short crosslinking times, and minimal heat production. However, there are three major drawbacks: 1) deleterious effects of UV irradiation such as DNA damage or the cancerization of cells, 2) radicals generated during irradiation which react with cells either via direct contact or the formation of reactive oxygen species, and 3) cytotoxicity from unreacted double bonds of hydrogels functionalized with photoreactive groups such as acrylates and methacrylates. Herein, we implemented a cell protection strategy against harmful external stressors present during biofabrication, which involves the encapsulation of mammalian cells within cytoprotective polyphenol-alginate compartments before embedding them in photocurable bioinks. Polyphenolics (PP) are especially suitable for encapsulation of cells due to the mild coating conditions required. Cell-laden alginate particles are generated followed by PP loading performed at various concentrations. The actual PP loading was quantified via UV-Vis absorbance spectrometry. The cytocompatibility and UV shielding effect of PP-alginate particles were thoroughly investigated with quantitative cell proliferation and LIVE/DEAD cell assays. Through our study, we discovered that even under prolonged UV irradiation (2.5-10 mins @ 18-19 mW/cm2), the viability of mammalian cells was preserved with the encapsulation of cells in PP-alginate particles. Lastly, in order to evaluate the applicability of our cytoprotective PP-alginate particles in biofabrication, we tested them with three photocrosslinkable hydrogels, Poly(ethylene glycol) diacrylate (PEGDA; 10, 15%), gelatin methacryloyl (GelMA ; 20%) and glycidyl methacrylate hyaluronic acid (GMHA; 4%). Hydrogel structures in the form of a thin substrate and a 3D printed scaffold were prepared at a particle to gel mass ratio of 2:3 and 1:3, respectively. The results clearly indicate that PP-alginate encapsulated cells exhibited higher viability under more stressful biofabrication conditions (extrusion-based 3D printing) and in more cytotoxic hydrogels such as PEGDA with high density of photoreactive side groups. The PP-alginate cell compartment acts not only as UV shield but also as diffusion barrier against harmful small molecules (photoinitiators) and larger macromolecules (polymer chains) as well as mechanical barrier against any external shear stresses. We envision this to be a technological breakthrough in biofabrication, maximizing mechanical stability of cell-laden scaffolds whilst minimizing damage to cells for any bioprinting or bioassembly processes.