Derivation, expansion and cryopreservation of primary fetal organoids from second and third trimester human amniotic fluid cells

Human primary fetal stem cell-derived organoids are used to model developing tissues in vitro. However, ethical and legislative constraints restrict fresh fetal tissue collection in several countries. Amniotic fluid (AF) is easily accessible with minimal ethical and regulatory constraints for collection. Our team recently showed that tissue-specific stem/progenitor cells can be isolated from fetal fluids collected during pregnancy through clinically indicated minimally invasive procedures conducted during the second and third trimesters. These samples consistently generate fetal lung, kidney tubule and gastrointestinal epithelial organoids autologous to the developing fetus. AF-derived organoids (AFOs) allow the investigation of fetal epithelia at developmentally relevant stages. Moreover, AFOs allow research to be conducted on late gestational stages, hardly accessible with other methods. Here, we provide a detailed protocol to establish, characterize and cryopreserve AFOs from viable AF cells. This includes the processing of patient-derived AF samples, viable cell sorting, seeding, establishment of clonal AFO lines, tissue phenotyping, expansion and cryopreservation. Additionally, we describe a straightforward immunofluorescence-based approach to pinpoint the tissue identity of the AFOs in a quick and cost-effective manner. In our hands, the protocol enabled the generation of primary fetal AFOs from 85.71% of samples (62.5% ascribed to the fetal lung, 59.4% to the kidney tubule and 6.2% to the small intestine). It takes 4–6 weeks to implement, requiring only standard equipment and expertise commonly available in cell biology laboratories