Standardized GMP-compliant scalable production of human pancreas organoids: Stem Cell Research and Therapy

M. Dossena, R. Piras, A. Cherubini, M. Barilani, E. Dugnani, F. Salanitro, T. Moreth, F. Pampaloni, L. Piemonti, L. Lazzari

Research output: Contribution to journalArticlepeer-review

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. © 2020 The Author(s).
Original languageEnglish
Article number94
JournalStem Cell Res. Ther.
Volume11
Issue number1
DOIs
Publication statusPublished - 2020

Keywords

  • adult
  • Article
  • automation
  • biobank
  • controlled study
  • digestion
  • donor
  • flow cytometry
  • gene expression
  • genetic analysis
  • good manufacturing practice
  • human
  • human tissue
  • insulin dependent diabetes mellitus
  • organoid
  • pancreas organoid
  • priority journal
  • standardization
  • tissue culture
  • tissue expansion
  • tissue growth

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