# Protocol to study ductal progenitor-like cells from the adult human pancreas using 3D suspension and methylcellulose-based culture systems

## TL;DR

This paper provides a detailed protocol for culturing human pancreatic ductal progenitor-like cells in 3D systems to potentially generate insulin-producing cells for diabetes treatment.

## Contribution

A GMP-compatible 3D culture protocol for human pancreatic ductal progenitor-like cells is introduced.

## Key findings

- A 3D suspension culture system and methylcellulose-based colony assay are described for culturing human pancreatic cells.
- Steps for dissociation, cryopreservation, and downstream analysis of ductal spheroids are detailed.

## Abstract

Primary human ductal progenitor-like cells derived from donated pancreas have the potential to serve as a source of therapeutic insulin-producing beta cells for the treatment of diabetes. Here, we present a protocol for studying ductal progenitor-like cells using a good manufacturing practice (GMP)-compatible 3D suspension culture system and a methylcellulose- and Matrigel-based 3D colony assay. We describe steps for dispersing and cryopreserving human pancreatic cells and initiating and maintaining cultures. We then detail how to prepare ductal spheroids or colonies for downstream applications.

For complete details on the use and execution of this protocol, please refer to Zook et al.1 and Quijano et al.2

•Stepwise protocol to dissociate and cryopreserve primary human exocrine tissues•Instructions to maintain human ductal cells/spheroids using 3D suspension culture•Steps to prepare and use 3D methylcellulose-based colony assay to generate colonies•Steps for analysis and processing of spheroids and colonies

Stepwise protocol to dissociate and cryopreserve primary human exocrine tissues

Instructions to maintain human ductal cells/spheroids using 3D suspension culture

Steps to prepare and use 3D methylcellulose-based colony assay to generate colonies

Steps for analysis and processing of spheroids and colonies

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Primary human ductal progenitor-like cells derived from donated pancreas have the potential to serve as a source of therapeutic insulin-producing beta cells for the treatment of diabetes. Here, we present a protocol for studying ductal progenitor-like cells using a good manufacturing practice (GMP)-compatible 3D suspension culture system and a methylcellulose- and Matrigel-based 3D colony assay. We describe steps for dispersing and cryopreserving human pancreatic cells and initiating and maintaining cultures. We then detail how to prepare ductal spheroids or colonies for downstream applications.