Transduction of a Humanized Rat Liver Using Adeno-associated Virus Vectors

Abstract

Source: Hiller, T., et al. Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2. J. Vis. Exp., (2016)

This video demonstrates adeno-associated virus-mediated gene delivery in a humanized rat liver scaffold. The vector delivers a transgene encoding a fluorescent reporter and gene-silencing RNA into liver cells via receptor-mediated endocytosis. This model supports efficient transduction in a perfused 3D liver tissue system.

Protocol

Transduction of Liver Model

1. Adjust the adeno-associated virus (AAV) vector solution to a final concentration of 2.7 x 10¹³ vector genomes in 5 ml by adding the respective volume of phosphate-buffered saline (PBS).
2. Disconnect the liver from the media circuit by removing the tubing from the cannula. Connect a 5 ml syringe to the cannula of the portal vein and inject the complete AAV vector solution (5 ml).
3. NOTE: Optionally, add phenol red (5 µg/ml) to follow the distribution of the AAV vector solution throughout the liver.
4. Incubate for 1 hr without pumping. Gradually increase flow rate, starting with 1.25 ml/min for 10 min; 2.5 ml/min for 20 min and 3.75 ml/min for 30 min. Culture the recellularized rat liver over 6 days.
5. NOTE: Replace one third of the medium with fresh medium (50 ml) every other day. Sample the culture medium to measure physiological parameters, such as lactate dehydrogenase activity, the pH of medium samples and the concentration of glucose and lactate.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Incubator	Fraunhofer	/
Peristaltic Pump	Fraunhofer	/
Flange with groove	Duran	2439454
O-Ring Transparent	Duran	2922551
Quick Release Clamp	Duran	2907151
Flat Flange Lid	Duran	2429857
Screw thread Tube	Duran	2483802
Screw thread Tube	Duran	2483602
Silicone sealing Ring	Duran	2862012
Screw Cap	Duran	2924013
Screw Cap	Duran	2924008
Screw Cap with aperture	Duran	2922709
Screw Cap with aperture	Duran	2922705
Filter	Sarstedt	831,826,001
Silicone Tubing	VWR	228-1500
Tube connector	Ismatec	ISM556A
Biocompatible Tubing	Ismatec	SC0736
T175 culture flasks	Greiner bio-one	660 160
RPMI 1640	BioWest SAS (Th. Geyer)	L0501-500
glutamine	BioWest SAS (Th. Geyer)	X0551-100
Trypsin	BioWest SAS (Th. Geyer)	L0940-100
penicillin/streptomycin	BioWest SAS (Th. Geyer)	L0022-100
fetal calf serum	cc pro	S-10-M
HepG2	DSMZ	ACC 180