Isolation and long-term culture of human hepatocytes.
Pressure-controlled perfusion of specimens of surgical hepatic resection produced improved yields of human hepatocytes for studies of long-term cultures. The effect of an extracellular matrix configuration on albumin secretion was evaluated by culture on a single layer of collagen or between double layers of collagen gel with Dulbecco's modified Eagle and Williams E media. Hepatocytes from 12 patients were maintained for more than 30 days, and in five of 12 experiments cells were cultured beyond 2 months. In the double gels the cells demonstrated typical polygonal liver cell morphology and higher albumin secretion (p < 0.01) up to 65 days; in contrast, in the single gels cells spread horizontally, and albumin secretion declined rapidly within 3 weeks. A comparison of media formulations showed that maximum albumin secretion occurred 5 days later but was maintained significantly longer with Dulbecco's medium (p < 0.01). The simple addition of a second layer of gelled collagen forming a collagen sandwich significantly stabilizes and supports the long-term culture of human hepatocytes.
Ryan
C M
CM
Surgical Service, Massachusetts General Hospital, Boston 02114.
Carter
E A
EA
Jenkins
R L
RL
Sterling
L M
LM
Yarmush
M L
ML
Malt
R A
RA
Tompkins
R G
RG
D000418:Albumins / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen; D003470:Culture Media; D003584:Cytological Techniques; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors
D000418:Albumins; D003470:Culture Media; D003094:Collagen