Confirming Viral Transduction in Recellularized Rat Liver Using Immunohistochemical Staining

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 immunohistochemical staining to evaluate viral gene transfer in a recellularized rat liver model. Reporter expression, confirmed by antibody detection and co-localization with nuclear staining, indicates successful viral transduction in the cells.

Protocol

1. Transduction of the Recellularized Rat Liver

1. Large-Scale Production of viral vector derived from adeno-associated viruses (AAV vector)
   1. Produce, purify, and quantify AAV vectors:
      1. Briefly, produce AAV vectors in roller bottles and purify them by iodixanol gradient centrifugation. Remove residual iodixanol by filtration over PD10 gel filtration columns. Determine the AAV vector concentration by quantitative polymerase chain reaction (qPCR) using genomic AAV DNA as a standard.
         NOTE: The self-complementary, pseudotyped AAV2/6 vector used in the present study encoded emerald green fluorescent reporter protein, EmGFP, as a reporter to demonstrate transduction efficiency and a short hairpin RNA (shRNA) expression cassette for the knockdown of an endogenously expressed gene (human cyclophilin B (hCycB), Figure 1). Ensure a sufficient amount of pseudotyped scAAV vectors of serotype 6 (2.7 x 10¹³ AAV vectors per liver model).
2. Transduction of Liver Model
   1. Adjust the 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).
      NOTE: Optionally, add phenol red (5 µg/ml) to follow the distribution of the AAV vector solution throughout the liver.
   3. 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.

2. Assessment of the Recellularized Transduced Rat Liver

1. Hematoxylin and eosin (HE) staining and Immunohistochemical Analysis
   1. Take samples (0.5 x 0.5 x 1.5-2 cm) from each liver lobe using a scalpel.
   2. Incubate samples in 4% paraformaldehyde (PFA) + 4% sucrose solution for 1.5 hr at 4 °C. (Caution: PFA is toxic and carcinogenic. Always keep PFA within a fume hood and wear appropriate protective clothing.) Wash three times with PBS (1 min per wash step) and incubate in 8% sucrose overnight at 4 °C.
   3. Pour the fixing medium into plastic cryomolds, and place the samples into the fixing medium, free of air bubbles. Add fixing medium until the sample is well covered. Store embedded samples at -80 °C until further use.
   4. Prepare cryo-sections (10 µm) with a cryotome. Assess recellularization by hematoxylin + eosin staining. Assess transduction efficiency by immunohistochemical staining for the gene-of-interest (here: EmGFP).

Results

Figure 1. Map of the self-complementary, pseudotyped AAV2/6 vector used in the present study. The genome consists of the inverted terminal repeats (ITR) of AAV2 and encodes EmGFP under control of the CMV promoter as well as an shRNA under control of a U6 promoter.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tissue-Tek O.C.T.	Weckert-Labortechnik	600001
Cryomold 15 x 15 x 5 mm	Sakura	4566
Incubator	Fraunhofer	/