Axenic Long-Term Cultivation of .

, a fungus causing severe pneumonia (PCP) in humans, has long been described as non-culturable. Only isolated short-term experiments with and a small number of experiments involving animal-derived species have been published to date. However, culture conditions may differ significantly from those of animal-derived , as there are major genotypic and phenotypic differences between them. Establishing a well-performing cultivation is crucial to understanding PCP and its pathophysiological processes. The aim of this study, therefore, was to develop an axenic culture for . To identify promising approaches for cultivation, a literature survey encompassing animal-derived cultures was carried out. The variables identified, such as incubation time, pH value, vitamins, amino acids, and other components, were trialed and adjusted to find the optimum conditions for culture. This allowed us to develop a medium that produced a 42.6-fold increase in qPCR copy numbers after a 48-day culture. Growth was confirmed microscopically by the increasing number and size of actively growing clusters in the final medium, DMEM-O3. doubling time was 8.9 days (range 6.9 to 13.6 days). In conclusion, we successfully cultivated under optimized cell-free conditions in a 70-day long-term culture for the first time. However, further optimization of the culture conditions for this slow grower is indispensable.