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Simple and Quick Method to Obtain a Decellularized, Functional Liver Bioscaffold

  • Matteo Ghiringhelli
  • Alessandro Zenobi
  • Stefano Brizzola
  • Fulvio Gandolfi
  • Valentino Bontempo
  • Sandro Rossi
  • Tiziana A. L. BreviniEmail author
  • Fabio Acocella
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1577)

Abstract

The development of new approaches for organ transplantation has become crucial in the last years. In particular, organ engineering, involving the preparation of acellular matrices that provide a natural habitat for reseeding with an appropriate population of cells, is an attractive although technically demanding approach. We here describe a method that allows for the derivation of functional in vitro hepatic organoids and that does not require a previous selection of all the parenchymal hepatocytes and non-parenchymal cells, namely, Kupffer cells, liver endothelial cells, and hepatic stellate cells. The procedure also replaces the costly standard collagenase perfusion step with a trypsin-based enzymatic digestion that results in high-yield decellularization. A combination of physical and chemical treatments through deep immersion and intraluminal infusion of two different consecutive solutions is used: (1) deionized water (DI) and (2) DI + Triton X 1% + ammonium hydroxide (NH4OH) 0.1%. This ensures the isolation of the hepatic constructs that reliably maintain original architecture and ECM components while completely removing cellular DNA and RNA. The procedure is fast, simple, and cheap and warrants an optimal organoid functionality that may find applications in both toxicological and transplantation studies.

Keywords

Hepatocyte Scaffold Decellularization Organoid Liver bioengineering 

Notes

Acknowledgement

This work was supported by Carraresi Foundation and by Fondazione CMT Cura Mini-invasiva Tumori ONLUS. T.A.L.B., F.G., M.G., and A.Z. are members of the COST Actions CA16119.

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Matteo Ghiringhelli
    • 1
  • Alessandro Zenobi
    • 1
    • 2
  • Stefano Brizzola
    • 1
  • Fulvio Gandolfi
    • 2
    • 3
  • Valentino Bontempo
    • 1
  • Sandro Rossi
    • 4
  • Tiziana A. L. Brevini
    • 1
    • 2
    Email author
  • Fabio Acocella
    • 1
  1. 1.Department of Health, Animal Science and Food SafetyUniversità degli Studi di MilanoMilanItaly
  2. 2.Laboratory of Biomedical Embryology, Centre for Stem Cell ResearchUniversità degli Studi di MilanoMilanItaly
  3. 3.Department of Agricultural and Environmental Science – Production, Landscape, AgroenergyUniversità degli Studi di MilanoMilanItaly
  4. 4.Medicina Interna e Gastroenterologia, Università di Pavia, Fondazione IRCCS Policlinico S. MatteoPaviaItaly

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