Background: The most important tool for the study of the malaria parasite is probably the in vitro culture that was established in 1976 by Trager and Jensen. However, the methodology is technically complex, expensive, and unsuitable for use in laboratories and regions with limited resources. Human factors such as hormones and the immune system are frequently ignored, and the similarity between the in vitro culture conditions and physiologic-like environment has been questioned. One of the most controversial factors about Plasmodium culture is the gaseous composition employed in vitro, which consists of a mixture poor in O2 (1%–5%) and rich in CO2 (approximately 5%).Results: The requirement of employing commercial gaseous mixtures and their suitability for use in reusable glass bottles under agitation for culturing were assessed. The methodology presented here is suitable for small- and large-scale culturing in cell culture flasks or hermetic and sterile glass bottles in agitation and provides an easier and cheaper Plasmodium culture alternative, which dispenses several sophisticated technical requirements of the classical methodology. The parasite viability, free oxygen, and drug-screening assays under these conditions were compared with those in the classic conditions. The cultivation under atmospheric air did not substantially affect the free oxygen levels and parasitic proliferation for periods longer than two years. However, it changed atovaquone, artesunate, and chloroquine efficacy in a different ways across the different isolates. Oxygraphic experiments demonstrated that the free oxygen concentration contained in the media was slightly superior compared to both the human blood and the media employed the classical methodology.Conclusions: The results indicate that the described culture conditions are suitable for parasite maintenance and can be used to obtain high yields of parasite. Although parasites proliferate under various culture conditions, it is clear that it is necessary to evaluate the different gaseous conditions to validate an antimalarial candidate. With this, it will be possible to detect an oxygen level that encompasses the physiological oxygen levels of human blood. Since oxygen decreases through media layers, future studies should further explore the use of oxygen-rich mixtures as a strategy to expose static cultures to more physiologic-like conditions.
