Specification	Details
Design specifications for bioprinting of cell-laden bioinks	A key challenge is that properties required for cell survival and tissue deposition are often in conflict with the needs for printability of high fidelity constructs (Cooke and Rosenzweig, 2021).
Phototoxicity in light-based manipulation of cell-laden hydrogel properties	In the presence of cells, irradiation can cause cell death through phototoxicity or radical generation. Care must be taken to limit the prolonged exposure to low wavelength light, particularly in the ultraviolet range. (Glass et al., 2018).
Ligand and protein bioactivity can be affected by light irradiation	Radical termination can disrupt protein folding, compromising bioactivity of bioactive ligands. The effect of radicals can be mitigated by optimizing the wavelength and intensity of light to reduce the local radical concentration or by including chemical species that terminate or stabilize radicals.
Stability of hydrogels with dynamic covalent crosslinks	Dynamic gels will eventually erode over time with media changes and cell remodeling, which could potentially limit the duration of experiments. To overcome this, hydrogels have sometimes involved stabilization with a secondary crosslinking mechanism.
Off-target effects of functional groups in dynamic hydrogels	Dynamic chemistries can have off target effects. Aldehyde groups that make up one side of the hydrazone bond can crosslink to a free amine group found in soluble or cell derived proteins to form a weak imine bond. Cyclodextrins used in dynamic guest-host bonds can absorb cholesterol molecules from cell membranes and alter membrane fluidity. Additionally, disruption of ionic bonds in alginate gels could locally change the concentration of calcium, which could impact cell signaling.
Imaging cells in 3D hydrogels	Hydrogels can interfere with standard imaging, staining, and immunolabeling methods. Hydrogels may attenuate light, which must be considered when designing experiments. As cells contract fibrous gels, they may densify the material and reduce light penetration to 10s of microns.