The aim of this study was to determine the optimal conditions for culturing coelomocytes (leukocytelike cells) from the annelid Eisenia hortensis. It was of particular interest to determine if CO2 could be omitted to permit more wide-spread use of earthworms in cell biology curricula using standard incubators. Two different types of media, DMEM and SFX-Insect Media, were used at varying conditions including: temperature, serum concentration, antimycotic concentration, CO2, and time. Cell viability was measured using propidium iodide and flow cytometry in addition to analysis of forward and side light scatter properties. It was found that the coelomocytes of E. hortensis exhibit the highest level of cell viability when cultured with DMEM supplemented with 10% newborn calf serum at 25 °C. Longer incubations showed lower cell death when CO2 was provided, but CO2 could be omitted for shorter periods of culture without significant loss of cell viability providing 10 mM HEPES was included in the culture medium. It was also observed that SFX-Insect Medium was a suitable alternative to DMEM and was used without the need for 5% CO2, but a minimum of 5% serum needed to be included. The toxicity of amphotericin B was tested and 0.875 μg/ml in DMEM and SFX-Insect Medium did not compromise cell viability. This information shows that earthworms can be cultured easily without the need for a CO2 incubator, thus simplifying laboratory conditions and minimizing costs associated with using earthworms for cell biology curricula and research purposes.