A growing body of evidence suggests that studying cell biology in classical two-dimensional formats, such as cell culture plasticware, results in misleading, non-physiological findings. This paper describes the optimization of a microsphere-based system permitting 3D cell culture incorporating physiological extracellular matrix components. Bio-electrospraying, the most advanced method currently available, is used to produce microspheres containing THP-1 cells as a model cell line. The bio-electrospraying parameters of nozzle size, polymer flow rate, and voltage are systematically investigated in order to allow stable production of size-controlled microspheres containing extracellular matrix material and human cells. The effect of bio-electrospraying parameters, alginate type and cell concentration on cell viability are investigated using trypan blue and propidium iodide staining. Bio-electrospraying has no effect on cell viability nor the ability of cells to proliferate. Cell viability is similarly minimally affected by encapsulation in all types of alginate tested (MVM, MVG, chemical and food-grade). Cell density of 5 x 106 cells mL(-1) within microspheres is the optimum for cell survival and proliferation. The stable generation of microspheres incorporating cells and extracellular matrix for use in a 3D cell culture will benefit study of many diverse diseases and permit investigation of cellular biology within a 3D matrix.