Standardized GMP-compliant scalable production of human pancreas organoids

  • Stem Cell Res Ther. 2020 Mar 4;11(1):94. doi: 10.1186/s13287-020-1585-2.
Marta Dossena  1 ,  Roberta Piras  1 ,  Alessandro Cherubini  1 ,  Mario Barilani  1  2 ,  Erica Dugnani  3 ,  Francesca Salanitro  1 ,  Till Moreth  4 ,  Francesco Pampaloni  4 ,  Lorenzo Piemonti  3  5 ,  Lorenza Lazzari  6
Affiliations
  • 1. Laboratory of Regenerative Medicine - Cell Factory, Department of Transfusion Medicine and Hematology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy.
  • 2. Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy.
  • 3. IRCCS Ospedale San Raffaele, San Raffaele Diabetes Research Institute, Milan, Italy.
  • 4. Physical Biology Group, Buchmann Institute for Molecular Life Sciences (BMLS), Goethe Universität Frankfurt am Main, Frankfurt am Main, Germany.
  • 5. Vita-Salute San Raffaele University, Milan, Italy.
  • 6. Laboratory of Regenerative Medicine - Cell Factory, Department of Transfusion Medicine and Hematology, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Via F. Sforza 35, 20122, Milan, Italy. [email protected].
PMID: 32127043 DOI: 10.1186/s13287-020-1585-2
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.

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