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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 operator-depending 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 well-defined 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.

SUBMITTER: Dossena M

PROVIDER: S-EPMC7055108 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Standardized GMP-compliant scalable production of human pancreas organoids.

Dossena Marta M Piras Roberta R Cherubini Alessandro A Barilani Mario M Dugnani Erica E Salanitro Francesca F Moreth Till T Pampaloni Francesco F Piemonti Lorenzo L Lazzari Lorenza L

Stem cell research & therapy 20200304 1

<h4>Background</h4>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.<h4>Methods</h4>Starting from discarded pancreatic tissu ...[more]

PMID: 32127043

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Standardized GMP-compliant scalable production of human pancreas organoids.

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Standardized GMP-compliant scalable production of human pancreas organoids.

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