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

Begin with a bioreactor system containing a decellularized rat liver scaffold repopulated with human liver cells.

The scaffold is maintained in a flow system that circulates media to supply nutrients.

Load a syringe with a buffer containing a recombinant adeno-associated virus vector.

The vector carries self-complementary DNA with a transgene cassette encoding a fluorescent reporter and a gene-silencing RNA.

Stop the media flow and disconnect the inflow tubing.

Inject the vector through the main blood vessel supplying the scaffold.

Incubate to allow vector–cell interaction.

The vector enters the liver cells via receptor-mediated endocytosis.

Once inside, the vector escapes the endosome and releases its self-complementary DNA into the nucleus.

Self-complementary DNA allows direct transgene expression, producing a fluorescent protein and gene-silencing RNA.

Gradually resume the media flow to restore circulation through the scaffold.

The transduced liver model is now suitable for downstream analysis.

Now transduce the liver model. First, get 27 trillion vectors in five milliliters of PBS loaded into a syringe. Phenol red may be added at 5 micrograms per milliliter.

Then disconnect the liver from the media circuit and inject the vectors into the system. Once injected, incubate the system for one hour without pumping. Then gradually increase the flow as previously described.

Later, as the transduced liver is incubated over six days, change 50 milliliters of the medium every other day.