Retroviral particles

There have already been clinical trials of porcine hepatocyte-based bioartificial livers [5, 6]. However, we believe these systems to represent temporary and short-lived approaches. Compelling evidence from recent experiments show that primary porcine liver cells express and release endogenous retroviral particles that are able to infect human cells. However, long term in vivo investigations of patients previously exposed to porcine tissues over a period of 12 year did not show any porcine endogenous retrovirus (PERV) viremia [7]. Therefore, we consider the further pursuit of porcine bioartificial livers the only solution at present with regard to the cell source. However, as an intermediate term alternative human cell sources are in development [8]. Expansion technologies for human fetal cells may contribute to resolve these limitations in the future. 

Good, high-level titre stocks of replication-incompetent retroviral particles can be produced. 

The next day, infect the HT-29 cells with luciferase or mock retroviral particles. A 2.5 ml solution containing retroviral particles is either prepared fresh or obtained from a frozen aliquot (see above). Prepare polybrene as 100 x stock solution (0.8 mg/ml) in 1 x PBS (can be stored at —20°C). Add polybrene to the tubes containing the retroviral particles at a final concentration of 8 pg/ml. Aspirate the medium from the HT-29 cells in the two T25 flasks. Initiate the infection by adding 2.5 ml of the solutions containing the luciferase or mock retroviral particles with polybrene to both flasks. 

Before moving the HT-29-luciferase cells from the BSL-2 lab back to a regular tissue culture lab, verify that the cells do not secrete infectious retroviral particles ... 

When working with infectious retroviral particles, consider the following safety points ... 

To generate retroviral particles, murine leukemia virus (MLV) pseudotypes are produced by calcium phosphate-mediated transfection of HEK-293 T cells with a 3 1 ratio of receptor plasmid to pCGP, which encodes the MLV gag andpol genes. When a specific chemokine receptor must be evaluated, HEK-293 T cells transfected transiendy or stably with the receptor should be used. At 4 h posttransfection, fresh medium supplemented with 10 mM -butyric acid is added to increase protein expression supernatant... 

Production of Supernatant Containing Infectious Retroviral Particles... 

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