Biosynthetic microspheres have the potential to address some of the limitations in cell microencapsulation; however, the generation of biosynthetic hydrogel microspheres has not been investigated or applied to cell encapsulation. Droplet microfluidics has the potential to produce more uniform microspheres under conditions compatible with cell encapsulation. Therefore, the aim of this study was to understand the effect of process parameters on biosynthetic microsphere formation, size, and morphology with a co-flow microfluidic method. Poly(vinyl alcohol) (PVA), a synthetic hydrogel and heparin, a glycosaminoglycan were chosen as the hydrogels for this study. A capillary-based microfluidic droplet generation device was used, and by varying the flow rates of both the polymer and oil phases, the viscosity of the continuous oil phase, and the interfacial surface tension, monodisperse spheres were produced from $200 to 800 lm. The size and morphology were unaffected by the addition of heparin. The modulus of spheres was 397 and 335 kPa for PVA and PVA/heparin, respectively, and this was not different from the bulk gel modulus (312 and 365 for PVA and PVA/heparin, respectively). Mammalian cells encapsulated in the spheres had over 90% viability after 24 h in both PVA and PVA/heparin microspheres. After 28 days, viability was still over 90% for PVA-heparin spheres and was significantly higher than in PVA only spheres. The use of biosynthetic hydrogels with microfluidic and UV polymerisation methods offers an improved approach to long-term cell encapsulation. V C 2013 AIP Publishing LLC. [http://dx