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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol presents an optimized two-step collagenase liver perfusion technique in a rat model and shows the use of isolated hepatocytes for in vitro long-term culture of 3D organoids.

Abstract

Primary hepatocytes are a commonly used tool for in vitro liver-related studies. However, the maintenance of these cells has always been a challenge due to the rapid loss of morphology, viability, and functionality in culture. A recent approach to long-term culture is the generation of three-dimensional (3D) organoids, an in vitro tool that can recapitulate tissues in a dish based on the marvelous ability of the liver to regenerate itself. Published protocols have been designed to obtain long-term functional 3D organoids from primary adult hepatocytes (Hep-Orgs). The 3D organoid cutting-edge tool requires the ability to isolate cells from adult tissue, and this initial step is crucial for a high-quality final result. The two-step collagenase perfusion, introduced in the 1970s, is still a valid procedure to obtain single hepatocytes. The present article aims to describe all the crucial steps of the surgical procedure, thereby optimizing the primary hepatocytes isolation procedure in the rat model. Moreover, particular attention is paid to the PREPARE guidelines to increase the likelihood of successful procedures and ensure high-quality results. A detailed protocol allows researchers to speed up and optimize the downstream work to establish 3D organoids from primary adult rat hepatocytes. Compared to 2D hepatocytes, Hep-Orgs were still viable and in active proliferation at Day 15, demonstrating a long-term potential.

Introduction

Primary hepatocytes are an important and widely used tool for in vitro liver-related studies. However, their expansion and maintenance have been historically challenging, as they lose morphology and functionality after a few days in the culture1. 2D culture is a limiting condition, in particular, for hepatocytes that have a polygonal shape and polarized structure with differentiated apical and basolateral membranes. In fact, hepatocyte adhesion to the plate interferes with their normal activity because it leads to a flat cytoskeleton with limited interaction among cells and between cells and extracellular matrix (ECM), reducing the pol....

Protocol

All procedures and animal housing were conducted according to the guidelines of the Italian Law and European Community directive. The experimental protocol was approved by the local Animal Care Committee and by the Italian Health Ministry (permit n° 321/2022-PR) according to art.31 of decree 26/2014.

1. Preparation for the animal procedure

NOTE: Please refer to Table 1 for the medium and buffer composition and to the Tabl.......

Representative Results

At the end of the set-up procedures (step 6.13), we obtained a cell yield of up to 1 x 108 cells per isolation from the liver of about 300 g of a rat. Cell viability between 78% and 97% was established by Trypan blue counting.

As already described in previous studies1,18,19, primary hepatocytes in culture lose their morphology, liver-specific functions, and die within a few days.

Discussion

3D-organoids are a frontier for personalized medicine and allow a long-term hepatocyte culture. The quality of this innovative technique requires a good yield of viable primary hepatocytes and well-performed liver perfusion and hepatocytes isolation. This old procedure is still widely used; however, it comprises different steps that can be challenging. Approaching the procedure, we experienced critical issues such as bacterial contamination, low liver digestion efficiency, low primary hepatocyte yield, and low hepatocyte.......

Acknowledgements

We thank Dr. Davide Selvestrel and prof. Giovanni Sorrentino of the SorrentinoLab at the University of Trieste for helping us perform the EdU proliferation assay. The work was supported by a Banca d'Italia ad hoc grant and intramural FIF grants.

....

Materials

NameCompanyCatalog NumberComments
A83-01- ALK5 Inhibitor IVTwin Helix T3031
B27Thermofisher Scientific0080085SA
CFX Connect Real-Time PCR Detection System  Bio-Rad
CHIR99021Twin HelixT2310
Click EdU Alexa 488 imaging kitThermofisher ScientificC10499
Collagen, Type I, solution from rat tailMerckC3867-1VL
Dexamethasone MerckD4902
EGFMerck E9644
Fetal bovine serum (FBS)EurocloneECS0180L
GELTREX LDEV FREE RGF BMEThermofisher ScientificA1413202
Heparin Sodium 25000 IU/5 mlB. Braun Melsungen AGB01AB01
HGFPeprotech 100-39H 
Insulin-Transferrin-Selenium solution 100x Thermofisher Scientific41400045
L-Glutamine solutionEurocloneECB3000D
Liver Digest Medium Thermofisher Scientific 17703-034
Liver Perfusion MediumThermofisher Scientific17701038
N2 supplementThermofisher Scientific17502048
N-acetylcysteineMerckA9165
NicotinamideMerck N-0636
Non-Essential Amino AcidsMerckM7145
NormocinAurogeneant-nr-1
PBS buffer 1XPanReac AppliChemA0964,9050
Penicillin-streptomycin solution 100xEurocloneECB3001D
PercollSanta Cruzsc-296039A
Peristaltic pumpIsmatec™ MS-4/12 Reglo Digital Pump
TNFaPeprotech 300-01A
TRI ReagentMerckT9424
Tubing Ismatec™  ID.2,79mm
Williams' E Medium, no glutamineThermofisher Scientific31415029
Y27632Twin HelixT1725

References

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