Standardized GMP-compliant scalable production of human pancreas organoids

Abstract

Background: Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach. Methods: Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis.Results: This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operatordepending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a welldefined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three-and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%). Conclusion: This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

Advances in Gold Nanoparticle-Based Combined Cancer Therapy

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Long-term live imaging and multiscale analysis identify heterogeneity and core principles of epithelial organoid morphogenesis

“…Starting from cell clusters, the cell survival and therefore the multiplication rate of cell material is higher. The production of large amounts of material for a potential clinical application is therefore ensured [ 37 ]. However, starting from clusters, the heterogeneity of the culture dynamics increases.…”

Long-term live imaging of epithelial organoids and corresponding multiscale analysis reveal high heterogeneity and identify core regulatory principles

“…Starting from cell clusters, the cell survival and therefore the multiplication-rate of cell material is higher. The production of large amounts of material for a potential clinical application is therefore ensured (Dossena et al, 2020). However, starting from clusters, the heterogeneity of the cultures dynamics increases.…”