# Long-term feeder cell-free cat intestinal organoid cultures to study Toxoplasma gondii’s sexual development

## TL;DR

Researchers developed a new lab model using feline intestinal organoids to study how Toxoplasma gondii develops sexually, which only happens in cats.

## Contribution

A feeder cell-free feline intestinal organoid system was established to study T. gondii sexual development in vitro.

## Key findings

- Feline intestinal organoids supported elevated levels of T. gondii sexual stage-specific transcripts.
- The model allows systematic investigation of host factors influencing parasite sexual development.
- Advanced sexual stages were not observed, but the system is reproducible and controlled.

## Abstract

Toxoplasma gondii is a protozoan parasite able to infect and survive in diverse host environments. However, its sexual reproduction, culminating in infectious oocysts, occurs exclusively in feline intestines. Recent studies identified the transcription factors AP2XII-1 and AP2XI-2 as crucial for pre-sexual development. Their depletion enabled merozoite formation in human fibroblasts, but progression to sexual stages appeared to require additional cues. Host-specific factors governing this process are suspected but remain elusive. Here, we describe a robust continuous feline intestinal organoid culture system without feeder cells to investigate whether the feline cellular and metabolic environment promotes sexual development of in vitro-generated merozoites. Using ultrastructural and transcriptional analyses, we found elevated levels of sexual stage-specific transcripts. While advanced sexual stage formation could not yet be observed, our feline intestinal organoid model provides a controlled and reproducible experimental system to systematically uncover the feline host factors and molecular mechanisms of T. gondii’s sexual development.

Establishment of continuously expanding feline intestinal organoids in feeder cell-free cultures provides a robust in vitro model for the study of Toxoplasma gondi’s sexual stages and other intestinal pathogens.