The Evaluation of Carbon Dioxide-Independent Media for Toxoplasma gondii Growth

Toxoplasma gondii is a human pathogen that plays a role as the causative agent of toxoplasmosis, a disease that produces many deleterious effects for immunocompromised individuals and infants. Current research involving this intracellular parasite requires the use and continues maintenance of costly cell incubators attached to bulky carbon dioxide canisters. To reduce laboratory costs and space requirements, we, therefore, propose a novel procedure by which human foreskin fibroblasts, as well as Toxoplasma gondii tachyzoites, may be grown in a commercially available carbon dioxide-independent media. Growth assays utilizing resazurin dye or relative fluorescence from parasites modified with a red fluorescent protein (dTomato) were performed over 10 days and revealed that human foreskin fibroblasts and type I RH-strain Toxoplasma gondii parasites are fully capable of proliferating in carbon dioxide-independent media supplemented with 10% bovine serum albumin. Further, type II ME49-strain tachyzoites were shown by manual cell counts to be able to grow in such an environment, though the addition of growth factors from carbon dioxide-independent RH tachyzoites was shown to significantly increase growth such that the proliferation exceeded that of the parasites grown in D10 media (Gibco® DMEM supplemented with 20% Gibco® Medium 199, 10% bovine serum albumin, and antibiotics) over a 36-hour period. The viability of this inexpensive procedure suggests an alternative means by which parasites may be grown in a carbon dioxide-independent environment, thus offering a potential to reduce both the amount of space required in the laboratory and the project costs for the continuation of critical Toxoplasma gondii research