The use of three-dimensional cell cultures has been widely used for efficacy and/or toxicity testing of compounds. One of the most promising systems, based on magnetic levitation, is dependent on proper cell magnetization, achieved through adsorption of iron-oxide nanoparticles on cell membranes. These particles must bare not only significant responses to magnetic fields, but also a stable mechanism to attachment to cells. This work proposes a simple, one-pot synthesis method to produce magnetite nanoparticles, using a Fe2+ precursor associated with amino acids under microwave heating, and successive steps to confer positive charges to particles. X-ray diffraction could confirm Fe3O4 composition, and TEM analysis showed cubic-like crystallites with less than 50 nm. Zeta-potential experiments showed that particles remained positively charged (20.98 ± 0.28 mV) in physiological pH, suggesting ability to attach to (negatively charged) cell membranes, observed through optical microscopy. Iron colloid was found to be non-cytotoxic in concentrations up to 8% in cell culture media. Finally, human prostate cancer cells were cultured in 96-well plates using magnetic levitation and could be kept 8 days in culture. The results showed a feasible way to produce spheroids relying on magnetic levitation, using a newly described method of magnetic and cell adherent nanoparticle production.