A stereotypic,transplantable liver tissue-culture system |
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Authors: | Brian A Naughton Benson Sibanda Jory P Weintraub Julia San Román Vafa Kamali |
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Institution: | (1) Hematology Laboratory, Advanced Tissue Sciences Inc., 92037 LaJolla, CA |
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Abstract: | A method of coculturing adult rat hepatic parenchymal cells (PC) and stromal cells in a three-dimensional framework of nylon
filtration screens or biodegradable polymer meshes was developed in our laboratory. Rat liver stroma, which includes vascular
and bile duct endothelial cells, fat-storing cells, fibroblasts, and Kupffer cells, were isolated by gradient centrifugation
afterin situ liver perfusion and expanded in monolayer culture prior to seeding onto nylon screens or bioresorbable polyglycolic acid
(PGA) polymers oriented into a felt-like construct. A second inoculum of freshly isolated PC was applied after the stromal
cells became established. Histological analyses revealed that PC proliferation occurred until all available space for expansion
within the template was exhausted. These cells retained their rounded morphology, and after 4–5 wk 7–9 “layers” of PC filled
the 140-μm deep template. Dioxin-inducible cytochrome P450 activity was detected for up to 58 d in culture, and albumin, fibrinogen,
transferrin, and soluble fibronectin were detected in the medium by enzyme-linked immunosorbent assay (ELISA) for 48 d in
vitro. Immunohistochemical analysis of sections through the cultures confirmed the presence of these proteins as well as cytokeratin
at the cellular level; the extracellular matrix stained for both collagen type III and laminin. Long-term PC proliferation
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 transplantation, co-cultures of hepatic PC and stromal
cells were established on PGA felt constructs instead of nylon screens. After 24 d in vitro, these constructs were grafted
into sites in the mesentery, omentum, and sub-cutaneous tissues of adult Long-Evans rats. The growth of hepatocytes after
30 din situ was evident by histological analysis; grafts of co-cultures regenerated a liver-like architecture consisting of sinusoids
and putative biliary structures. In addition, PC at these extrahepatic graft sites were positive for albumin, transferrin,
and fibrinogen synthesis by immunohistochemistry. Graft survival was enhanced when recipients were subjected to 40% hepatectomy.
Hepatic PC:stromal cell cocultures may prove useful in the restoration of liver function either by direct transplantation
using PGA or similar templates, or as extracorporeal devices, using nylon screens. |
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Keywords: | Albumin cytochrome P450 fibrinogen hepatic parenchymal cells stromal cells three-dimensional culture bioresorbable polymers |
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