Monitoring cell fate in 3D organotypic human squamous epithelial cultures

Here, we provide a protocol to model the effects of changes to a small number of cells, such as those arising from a mutation or a virus infection, in stratified epithelia. We describe steps for diluting engineered human keratinocytes into a larger population of unmodified cells and using these cells to grow three-dimensional organotypic cultures. We detail steps to observe effects that are not apparent in homogenous organotypic epithelial cultures by visualizing the localization of modified keratinocytes in epithelial layers. For complete details on the use and execution of this protocol, please refer to Hatterschide et al. (2022).1 [Display omitted] •Modify human keratinocytes to model virus infection or mutation•Generate three-dimensional organotypic cultures with a small number of modified cells•Monitor localization of modified cells in tissue•Determine the effect of virus infection or mutation on epithelial cell identity Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Here, we provide a protocol to model the effects of changes to a small number of cells, such as those arising from a mutation or a virus infection, in stratified epithelia. We describe steps for diluting engineered human keratinocytes into a larger population of unmodified cells and using these cells to grow three-dimensional organotypic cultures. We detail steps to observe effects that are not apparent in homogenous organotypic epithelial cultures by visualizing the localization of modified keratinocytes in epithelial layers.