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Abstract
BackgroundPancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. Mouse pancreatic organoids exhibit unlimited expansion potential, while previously reported human pancreas organoid (hPO) cultures do not expand efficiently long-term in a chemically defined, serum-free medium. We sought to generate a 3D culture system for long-term expansion of human pancreas ductal cells as hPOs to serve as the basis for studies of human pancreas ductal epithelium, exocrine pancreatic diseases and the development of a genomically stable replacement cell therapy for diabetes mellitus.ResultsOur chemically defined, serum-free, human pancreas organoid culture medium supports the generation and expansion of hPOs with high efficiency from both fresh and cryopreserved primary tissue. hPOs can be expanded from a single cell, enabling their genetic manipulation and generation of clonal cultures. hPOs expanded for months in vitro maintain their ductal morphology, biomarker expression and chromosomal integrity. Xenografts of hPOs survive long-term in vivo when transplanted into the pancreas of immunodeficient mice. Notably, mouse orthotopic transplants show no signs of tumorigenicity. Crucially, our medium also supports the establishment and expansion of hPOs in a chemically defined, modifiable and scalable, biomimetic hydrogel.ConclusionshPOs can be expanded long-term, from both fresh and cryopreserved human pancreas tissue in a chemically defined, serum-free medium with no detectable tumorigenicity. hPOs can be clonally expanded, genetically manipulated and are amenable to culture in a chemically defined hydrogel. hPOs therefore represent an abundant source of pancreas ductal cells that retain the characteristics of the tissue-of-origin, which opens up avenues for modelling diseases of the ductal epithelium and increasing understanding of human pancreas exocrine biology as well as for potentially producing insulin-secreting cells for the treatment of diabetes.
Highlights
Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human
The pancreas exhibits dual functions: on the one hand, acinar and ductal cells act as an exocrine organ which aids in digestion, whilst on the other hand, pancreatic β cells, which are located in the islets of Langerhans together with α, δ, ε and PP cells, perform an endocrine function by regulating blood glucose levels through the secretion of insulin [1]
Generation, long-term expansion and clonal derivation of human ductal pancreatic organoids We and others have previously reported culture systems that support human ductal pancreatic organoid growth [21,22,23]. These suffer from several shortcomings in their application for disease modelling and cell therapy: (1) they do not support the long-term expansion required to generate the necessary cell numbers [22], (2) the medium compositions are not chemically defined and require the addition of serum to the medium [21, 23], 3) the extracellular matrix (ECM) used, namely Matrigel, suffers from batch-to-batch effects and is derived from mouse tumours, which makes it difficult to produce under Good Manufacturing Practice (GMP) compliant conditions [21,22,23]
Summary
Pancreatic organoid systems have recently been described for the in vitro culture of pancreatic ductal cells from mouse and human. The pancreas exhibits dual functions: on the one hand, acinar and ductal cells act as an exocrine organ which aids in digestion, whilst on the other hand, pancreatic β cells, which are located in the islets of Langerhans together with α, δ, ε and PP cells, perform an endocrine function by regulating blood glucose levels through the secretion of insulin [1]. Both endocrine and exocrine cells are derived during development from the ventral and dorsal PDX1+ foregut endoderm, which fuse to give rise to the head, body and tail of the pancreas. Human ductal cells could serve as a starting material for modelling pancreas ductal diseases ex vivo as well as for the derivation of glucose-responsive insulin-producing cells, provided they can be efficiently expanded in vitro
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