Generation and Genetic Manipulation of Human Cervical Organoids

Cervical cancer is the fourth most common cancer in women and most frequently affects the uterine ectocervix. The uterine ectocervix is lined by stratified squamous epithelium, comprising basal cells and differentiating parabasal and suprabasal cells. The regeneration observed after cervical conization suggests the presence of robust stem cell activity. However, our understanding of the identity and regulatory mechanisms of cervical stem cells and their malignant transformation process has been limited due to the restricted access to human cervical tissues and the lack of optimal systems to assess stem cell activity in the uterine ectocervix. Recently established human cervical organoids can help overcome these limitations and offer new opportunities to study cervical physiology and pathology. However, no standardized method exists for isolating and establishing human cervical organoids. Here, we describe a method for establishing human ectocervical organoids. Specifically, we present a protocol to isolate single cells from the human ectocervical epithelium. By incubating cervical tissue in an optimized enzyme solution, we peel off the cervical epithelium to maximize cell yield while minimizing fibroblast contamination. We further outline the conditions for culturing human cervical organoids using an optimized medium formulation. In addition, we detail the procedures for passaging and genetically manipulating cervical organoids. This protocol provides a highly efficient approach for establishing human cervical organoids and utilizing them to study cervical stem cells and diseases.