A method of coculturing adult rat hepatic parenchymal cells (PC) and s
tromal cells in a three-dimensional framework of nylon filtration scre
ens or biodegradable polymer meshes was developed in our laboratory. R
at liver stroma, which includes vascular and bile duct endothelial cel
ls, fat-storing cells, fibroblasts, and Kupffer cells, were isolated b
y gradient centrifugation after in situ liver perfusion and expanded i
n monolayer culture prior to seeding onto nylon screens or bioresorbab
le polyglycolic acid (PGA) polymers oriented into a felt-like construc
t. A second inoculum of freshly isolated PC was applied after the stro
mal cells became established. Histological analyses revealed that PC p
roliferation occurred until all available space for expansion within t
he template was exhausted. These cells retained their rounded morpholo
gy, and after 4-5 wk 7-9 ''layers'' of PC filled the 140-mu m deep tem
plate. Dioxin-inducible cytochrome P450 activity was detected for up t
o 58 d in culture, and albumin, fibrinogen, transferrin, and soluble f
ibronectin were detected in the medium by enzyme-linked immunosorbent
assay (ELISA) for 48 d in vitro. Immunohistochemical analysis of secti
ons through the cultures confirmed the presence of these proteins as w
ell as cytokeratin at the cellular level; the extracellular matrix sta
ined for both collagen type III and laminin. Long-term PC proliferatio
n and function were enhanced by the presence of stromal cells as well
as by a meshwork template whose geometry allows the interaction of PC
with stroma and matrix on several different planes. To permit transpla
ntation, cocultures of hepatic PC and stromal cells were established o
n PGA felt constructs instead of nylon screens. After similar to 24 d
in vitro, these constructs were grafted into sites in the mesentery, o
mentum, and subcutaneous tissues of adult Long-Evans rats. The growth
of hepatocytes after 30 d in situ was evident by histological analysis
; grafts of cocultures regenerated a liver-like architecture consistin
g of sinusoids and putative biliary structures. In addition, PC at the
se extrahepatic graft sites were positive for albumin, transferrin, an
d fibrinogen synthesis by immunohistochemistry. Graft survival was enh
anced when recipients were subjected to similar to 40% hepatectomy. He
patic PC:stromal cell cocultures may prove useful in the restoration o
f liver function either by direct transplantation using PGA or similar
templates, or as extracorporeal devices, using nylon screens.