Table 3. Characterization of cellular input into bioreactors: hepatocytes were isolated by a modified two-step collagenase perfusion process described earlier [117] and isolates with viability > 90% were consistently used.
NPC fractions were purified from supernatants based on published methods [119] and stained with different immunostains (SE-1 for sinusoidal endothelial cells, ED-2 for Kupffer cells, GFAP for stellate cells) before quantifying by flow cytometry. NPC isolates were consistently shown to have a viability of > 95%. Based on the proportions, defined numbers of each cell type were added back into reactors at the time of seeding.
| Cell type | Function | Reference in vivo values |
Seeding numbers/reactor well |
|
|---|---|---|---|---|
| Monocultures | Co-cultures | |||
| Hepatocytes | Main metabolic cells | 60% | 800,000 (95%) | 400,000 (40%) |
| Endothelial cells | Filtration, secrete cytokines | 20% | (1 –2%) | 400,000 (40%) |
| Kupffer cells | Macrophages, inflammatory response | 15% | (1 –2%) | 150,000 (15%) |
| Stellate cells | Fibroblasts, store fat, secrete ECM | 5% | (< 1 %) | 50,000 (5%) |
NPC: Non-parenchymal cell.