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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.
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.
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....
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.......
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.
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.......
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.
....Name | Company | Catalog Number | Comments |
A83-01- ALK5 Inhibitor IV | Twin Helix | T3031 | |
B27 | Thermofisher Scientific | 0080085SA | |
CFX Connect Real-Time PCR Detection System | Bio-Rad | ||
CHIR99021 | Twin Helix | T2310 | |
Click EdU Alexa 488 imaging kit | Thermofisher Scientific | C10499 | |
Collagen, Type I, solution from rat tail | Merck | C3867-1VL | |
Dexamethasone | Merck | D4902 | |
EGF | Merck | E9644 | |
Fetal bovine serum (FBS) | Euroclone | ECS0180L | |
GELTREX LDEV FREE RGF BME | Thermofisher Scientific | A1413202 | |
Heparin Sodium 25000 IU/5 ml | B. Braun Melsungen AG | B01AB01 | |
HGF | Peprotech | 100-39H | |
Insulin-Transferrin-Selenium solution 100x | Thermofisher Scientific | 41400045 | |
L-Glutamine solution | Euroclone | ECB3000D | |
Liver Digest Medium | Thermofisher Scientific | 17703-034 | |
Liver Perfusion Medium | Thermofisher Scientific | 17701038 | |
N2 supplement | Thermofisher Scientific | 17502048 | |
N-acetylcysteine | Merck | A9165 | |
Nicotinamide | Merck | N-0636 | |
Non-Essential Amino Acids | Merck | M7145 | |
Normocin | Aurogene | ant-nr-1 | |
PBS buffer 1X | PanReac AppliChem | A0964,9050 | |
Penicillin-streptomycin solution 100x | Euroclone | ECB3001D | |
Percoll | Santa Cruz | sc-296039A | |
Peristaltic pump | Ismatec™ | MS-4/12 Reglo Digital Pump | |
TNFa | Peprotech | 300-01A | |
TRI Reagent | Merck | T9424 | |
Tubing | Ismatec™ | ID.2,79mm | |
Williams' E Medium, no glutamine | Thermofisher Scientific | 31415029 | |
Y27632 | Twin Helix | T1725 |
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