Protocol to study regeneration in human pancreatic slices using viral tracing, extended culture, dissociation, and functional Ca2+ influx techniques

Human pancreatic slices provide an organotypic platform that preserves native cell interactions for functional studies under near-physiological conditions. We present a protocol to regenerating human pancreatic slices, including lentiviral and adenoviral transduction for lineage tracing and a dual-reporter HIP-Cre system to label neogenic β-cells. The workflow includes tissue dissociation for single-cell analysis and calcium influx measurements to assess glucose responsiveness, enabling identification, tracking, and functional evaluation of newly formed β-cells. For complete details on the use and execution of this protocol, please refer to Doke et al. 1 • Prepare and maintain long-term cultures of human pancreatic slices • Perform dual lentiviral and adenoviral transduction for lineage tracing • Acquire daily fluorescence imaging to monitor β-cell lineage conversion • Dissociate pancreatic slices to obtain viable single cells for scRNA-seq Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Human pancreatic slices provide an organotypic platform that preserves native cell interactions for functional studies under near-physiological conditions. We present a protocol to regenerating human pancreatic slices, including lentiviral and adenoviral transduction for lineage tracing and a dual-reporter HIP-Cre system to label neogenic β-cells. The workflow includes tissue dissociation for single-cell analysis and calcium influx measurements to assess glucose responsiveness, enabling identification, tracking, and functional evaluation of newly formed β-cells.