FS
Frank Seeber
  • Research scientist, Robert Koch Institute Berlin
Research fields
  • Microbiology
Generation of Mature Toxoplasma gondii Bradyzoites in Human Immortalized Myogenic KD3 Cells

Toxoplasma gondii is a zoonotic protozoan parasite and one of the most successful foodborne pathogens. Upon infection and dissemination, the parasites convert into the persisting, chronic form called bradyzoites, which reside within cysts in muscle and brain tissue. Despite their importance, bradyzoites remain difficult to investigate directly, owing to limited in vitro models. In addition, the need for new drugs targeting the chronic stage, which is underlined by the lack of eradicating treatment options, remains difficult to address since in vitro access to drug-tolerant bradyzoites remains limited. We recently published the use of a human myotube-based bradyzoite cell culture system and demonstrated its applicability to investigate the biology of T. gondii bradyzoites. Encysted parasites can be functionally matured during long-term cultivation in these immortalized cells and possess many in vivo–like features, including pepsin resistance, oral infectivity, and antifolate resistance. In addition, the system is scalable, enabling experimental approaches that rely on large numbers, such as metabolomics. In short, we detail the cultivation of terminally differentiated human myotubes and their subsequent infection with tachyzoites, which then mature to encysted bradyzoites within four weeks at ambient CO2 levels. We also discuss critical aspects of the procedure and suggest improvements.


Key features

• This protocol describes a scalable human myotube-based in vitro system capable of generating encysted bradyzoites featuring in vivo hallmarks.

• Bradyzoite differentiation is facilitated through CO2 depletion but without additional artificial stress factors like alkaline pH.

• Functional maturation occurs over four weeks.


Graphical overview


From 3D to 2D: Harmonization of Protocols for Two-dimensional Cultures on Cell Culture Inserts of Intestinal Organoids from Various Species

In the expanding field of intestinal organoid research, various protocols for three- and two-dimensional organoid-derived cell cultures exist. Two-dimensional organoid-derived monolayers are used to overcome some limitations of three-dimensional organoid cultures. They are increasingly used also in infection research, to study physiological processes and tissue barrier functions, where easy experimental access of pathogens to the luminal and/or basolateral cell surface is required. This has resulted in an increasing number of publications reporting different protocols and media compositions for organoid manipulation, precluding direct comparisons of research outcomes in some cases. With this in mind, here we describe a protocol aimed at the harmonization of seeding conditions for three-dimensional intestinal organoids of four commonly used research species onto cell culture inserts, to create organoid-derived monolayers that form electrophysiologically tight epithelial barriers. We give an in-depth description of media compositions and culture conditions for creating these monolayers, enabling also the less experienced researchers to obtain reproducible results within a short period of time, and which should simplify the comparison of future studies between labs, but also encourage others to consider these systems as alternative cell culture models in their research.


Graphic abstract:



Schematic workflow of organoid-derived monolayer generation from intestinal spheroid cultures. ECM, extracellular matrix; ODM, organoid-derived monolayer.


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