11. Place each plate (500 total) at 37 °C, 5% C02- 
Dav 8 (or when plates are subconfluent) 
1. Aspirate off media using a pasteur pipette. 
2. Add 10 ml of fresh complete medium to each plate using a 10 ml pipette. 
3. Place each plate at 37 °C, 5% CO2 
Day 9 
1. Take off supernatant using a 10 ml pipette and filter through a 0.45|im one liter 
filter unit. Pool 100 plates into each filter unit. 
2. Repeat the above step for the other 400 plates. 
3. Pool all the supernatants by pouring the supernatants together until thoroughly 
mixed. 
4. From the 5 one liter filter bottles, remove 50 ml using a 25 ml pipette and transfer 
to a 50 ml conical test tube. (See section F for assays to be performed on the 
supernatants.) 
5. Aliquot the 5000 ml of supernatant into 200 ml storage bottles. 
6. Store the 200 ml aliquots at -70 °C. 
6. Characterization of the LDL receptor viral supernatant 
Aliquots of the pooled supernatants will be analyzed for the following tests and certified 
by the FDA prior to use in the human experiments: 
Performed Bv 
H.A.L. 1 
OL2 
OL 
OL 
1 Human Applications Laboratory, University of Michigan 
2 Outside Laboratory 
Test 
1 . Identity 
A. Fluorescent LDL assay 
B. Southern blot analysis 
2. Sterility 
4. General Safety 
5. Extended S+L- Focus 
D. Harvest of Liver 
Autologous hepatocytes will be harvested from liver tissue obtained via a partial 
hepatectomy of the recipient. Through a bilateral subcostal incision, attachments to the left 
Recombinant DNA Research, Volume 15 
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