Approaching xeno-free cultivation of pulmonary pathogens in vitro

In biomedical research, the cultivation of the bacterial pathogen Streptococcus pneumoniae for in vitro and in vivo experiments requires products based on animal derived substances. Here, we aim to replace or dramatically reduce these products in order to improve experimental reproducibility and animal welfare. To establish alternative cultivation protocols, we selected xeno-reduced agar plates (Standard Nutrient Agar I) and xeno-free agar plates (CASO-Bouillon vegetal, Lysogeny Broth Agar vegetal, Standard Nutrient Agar I vegetal), as well as liquid growth media alternatives (containing CASO-Bouillon media vegetal, Lysogeny Broth media vegetal, human platelet lysate). To evaluate the applicability of xeno-free and xeno-reduced products we compare bacterial growth behavior, colony morphology and virulence to traditionally cultivated bacteria.

Preliminary results of alternatively cultivated bacteria show that Streptococcus pneumoniae serotypes 2 and 3 can be cultivated with xeno-reduced and xeno-free products, albeit at the expense of longer incubation times and an increased number of agar plates utilized. The high similarity between alternatively and traditionally grown bacteria regarding growth behavior and morphology, indicates a promising favor towards xeno-reduced and xeno-free growth media containing CASO-Bouillon media vegetal, human platelet lysate and heparin. Furthermore, alternative agar plates achieve characteristic growth curves deviating in a delayed start of the exponential growth phase.

Although similar growth characteristics and morphology can be achieved when cultivating Streptococcus pneumoniae using xeno-free products, more detailed conclusions will be drawn after further in vitro experiments on human cell lines (A549 cells) in which bacteria grown in xeno-free or xeno-reduced conditions will be evaluated with regards to their virulence and infectivity.

Publication History

Article published online:
09 March 2023