Electrohydrodynamic spraying (EHS) has recently gained popularity for microencapsulation of cells for applications in cell delivery and tissue engineering. Some of the polymers compatible with EHS are alginate, chitosan, and other similar natural polymers, which are subject to ionotropic or physical gelation. It is desirable to further extend the use of the EHS technique beyond such polymers for wider biofabrication applications. Here, building upon our previous work of making PEG microspheres via EHS, we utilized the principles of EHS to fabricate cell-laden polyethylene glycol (PEG) hydrogel microspheres. The gelation of PEG hydrogel microspheres was achieved by forming covalent crosslinks between multiarm PEG acrylate and dithiol crosslinkers via Michael-type addition. We conducted a detailed investigation of the critical parameters of EHS, such as the applied voltage, inner needle diameter (i.d. needle), and flow rate, to obtain PEG microspheres with high cell viability and tightly-controlled diameters in the range of 70–300 μ m. The polydispersity of cell-laden PEG hydrogel microspheres as measured by % coefficient of variation was between 6% and 23% for all conditions tested. We established that our method was compatible with different cell types and that all tested cell types could be encapsulated at high densities of 10 ⁶ –10 ⁹ and ≥90% encapsulation efficiency. We observed cell aggregation within the hydrogel microspheres at applied voltage >5 kV. Since PEG is a synthetic polymer devoid of cell attachment sites, we could overcome this limitation by tethering Arg-Gly-Asp-Ser (RGDS) peptide to the PEG hydrogel microspheres; upon RGDS tethering, we observed uniform cell dispersion. The microencapsulated cells could be cultured in the PEG hydrogel microspheres of different sizes for up to one week without significant loss in cell viability. In conclusion, the EHS technique developed here could be used to generate cell-laden PEG hydrogel microspheres of controlled sizes for potential applications in cell delivery and organoid cultures.

Tuning hydrogel properties through minor modifications of the crosslinker structure is a beneficial approach for hydrogel design that could result in hydrogels with wide range of properties to match a desired application. In this study, we analyzed the relationship between the dithiol crosslinker chemical and physical structure and the resulting properties of polyethylene glycol (PEG) hydrogels formed via Michael-type addition reaction. Specifically, the dithiol crosslinker properties and chemical structure were correlated with gelation time, hydrolytic degradation rate, reaction rate constant, crosslink density and storage modulus of PEG hydrogels. By changing the properties and structure of the crosslinker, hydrogels with controlled degradation ranging from 10 h to 22 d were obtained. It was also established that hydrogel gelation times correlated closely with degradation times. By extensive characterization of the dithiol crosslinker chemical structure and physical properties, we identified two sets of conditions which yielded fast-gelling, fast-degrading hydrogels and slow-gelling, slow-degrading hydrogels. Uniquely, the hydrogel storage moduli could be controlled by the dithiol crosslinker chemical identity independent of the degradation time of the hydrogel or the mesh size.