**2.6 Technical challenges**

The classical nature of 3D organoid models was closed round structures embedded in Matrigel, challenging to infect with viruses as receptors needed for infection are always located deep inside. This shortcoming was overcome in HAE cultures where cells are grown on a Transwell. Therefore, round gut organoids can be easily transformed into an open organoid model where they are accessible from the upper and lower sides simultaneously to establish the desired infection [28, 57, 58]. This model system is technically advantageous for infectious disease studies and drugtesting in antimicrobial therapy.

The next significant challenge worth consideration is readouts used for analysis after infection. Due to the release of viral particles in a nonlytic manner, virus cultures in primary cellular models do not result in plaque-like cytopathic effect (CPE) most of the time, for example, in the case of enterovirus A71. Huang et al. have shown using human intestinal organoids that are infected with enterovirus A71 that viral release happens through exosomes instead of a lytic process characteristic of a classical RD cell line [59]. This production is quantified through back titration or plaque assays using cell lines. The aforementioned protocol of measurement of the number of viral particles is a matter of concern in the case of primary cultures, which calls for more suitable evaluation methods.
