Demetris, AJ

Duquesnoy, R

Fung, JJ

Graner, S

Makowka, L

Markus, BH

Starzl, TE

PubMed

D-Scholarship@Pitt

MANCA ET AI. the human system. iable procedures for T hybridomas are reo 3da F: Transplantation ;hi y, et al: J Immunol Primary Cultures of Human Intrahepatic (Biliary) Epithelial Cells A.J. Demetris, B.H. Markus, J.J. Fung, L. Makowka. S. Graner. R. Duquesnoy. and T.E. Starzl THE BILIARY epithelium is a relatively select target in liver allograft rejection.1•2 An ideal approach to study interactions between immune cells and the biliary epithe-lium would be the use of an in vitro system. The goal of this study was to establish primary cultures of bile duct cells. MATERIALS AND METHODS Source of Tissue The tissues used were intact normal human livers (unused donor grafts) or large segments of normal liver obtained from liver transplant and primary resection procedures. Biliary Epithelial Cel/Isolation The liver was placed on a sterile drape under a laminar flow hood with all subsequent steps performed under sterile conditions. The right and left hepatic bile ducts were identified and a silastic cannula (19 x '/a. 12 inches) was advanced into the duct and sutured in place. A segment (I to 2 cm) of the lateral portion of the respective lobe was then resected and the biliary tree flushed with HBSS (calcium and magnesium free Hanks' balanced salt solution. GIBCO Laboratories. Life Technologies Inc. Grand Island, NY). The biliary tree was then filled with a 0.1 % collagenase intravenous (IV) (Worthington. Diagnostics Systems Inc) solution in HBSS, kept at 37°C. and left for 30 minutes to digest off the cells. which were then collected by refiushing the organ with HBSS. The fluid containing the cells was transferred into SO·mL plastic centrifuge tubes, spun at 1.200 RPM for ten minutes, and resuspended in a minimal volume of culture medium and plated at a density of I to lOx \06/m L on 24·well plastic Falcon tissue culture plates previously coated with MATRIGEL (Collaborative Research Inc, Lexington, MA). Tissue Culture Medium and Substrata Basal media used for culturing the epithelial cells (Table I). included CEM 2000 (Scott Laboratories, West Warwick, RI), Williams E with L-glutamine or a 50/50 (vol/vol) mixture of Ham's FI2 and Dulbecco's modified medium (DMEM). Serum supplementation was with 10% heat·inactivated fetal calf serum. Supplements (Ta· bles I and 2) consisted ofSGF-7 (1 mL/loo mL media), SGF·9 (I mL/loo mL media. Scott Laboratories), 100 Ilg/mL endothelial cell growth factor (Collaborative Research), 250 Ilg/mL Fungizone, 56 Ilg/mL Gentamy-cin (G1BCO), and 10 U/mL heparin (O'Neal, Jones, & Feldman, St Louis, MO). The cells were plated on a MATRIGEL (Collaborative Research) substrate. Follow-Up of Primary NPE Cullures The plates were washed free of nonadherent cells after two days and these were centrifuged at 1,200 RPM for five minutes. The pellet was resuspended in medium and replated using the previously described techniques. Half of the NPE culture medium was removed every second day and replaced with fresh complete medium in the previously described formulation. Once the cells were firmly attached (four to five days) the medium was replaced by fresh medium every two to three days. RESULTS Bile Ducl Cell Iso/ales: Identification, Purity and Viability Phase contrast microscopy of eluates obtained after tissue digestion revealed orga-noid clusters of cells intermixed with single small round cells ranging in size from eight to 12 microns in diameter. Immunoperoxidase staining of the organoid clusters for prekera-tin was positive, while factor VIII-related antigen and albumin stains were negative. The organoid clusters and the single cell sus-pensions possessed from 70% to 90% viability based on trypan blue exclusion. Effects of Culture Medium Attempts to establish cell cultures on a MATRIGEL substrate were performed with From the Departments of Pathology and Surgery. Presbyrtrian-University Hospital. Pittsburgh, and the Department of Surgery. University of Rochester. NY. Address reprint requests to A.J. Demetris, Department of Pathology, Presbyterian-University Hospital, DeSoto at O'Hara Streets. Pittsburgh, PA 1521J. C> /988 by Grune d Stratton. Inc. 0041-1345/88/2002-0005$03.00/0 TrBnsplBntBtioo Prrx;eedings. Vol Xx. No 2 (April!. 1988: pp 161-163 161 162 Table 1. Constituents of Serum Supplemented Ba .. 1 Medie Constituent Amount Basal media 90% Fetal calf 10% SGF-7 1mL/100ml-SGF-9 1 mL/ 1 00 ml-Endothelial cell growth factor (ECGF} 100 I'g/mL Fungizone 2501'g/mL Gentamycin 56 I'g/mL Heparin 10 U/mL -See Table 2 for a listing of the constituents and their final concentrations. the following: (I) basal medium alone, (2) basal medium plus SGF-7 and SGF-9, (3) basal medium supplemented with 10% fetal calf serum and, (4) basal medium with serum and growth factor supplements as described in Table 1. Monolayers could be achieved using any of the basal media, but supplementation with fetal calf serum was required. Serum-free medium, on the other hand, despite the addi-tion of fetuin and growth factor supplements, was unable to support the initiation of cul-tures. Morphologic and Phenotypic Characteristic o/Cultures NPE cultures were examined routinely each day using an inverted phase contrast microscope. Cells from the organoid clusters began to spread over the plate within 24 hours and growth to confluence occurred by eight to 12 days. Confluent wells presented as mono-layers with a characteristic cobblestone appearance. The individual cells were mostly small and polygonal with smooth intercellular borders and slightly granular cytoplasm. However, as the cultures aged, the cells became larger and squamoid in appearance and detached from the plates at 6 to 7 weeks. Nearly 100% of the cells comprising the monolayers were uniformly prekeratin posi-tive. Staining for albumin to detect hepato-cytic differentiation, alphafetoprotein, and DEMETRIS ET AL Table 2. Constituents and Final Concentrlltion of Growth Supplements (SGF-7I1nd SGF-9} Growth Factor Supplemented Bllsal Medill Epidermal growth factor (EGF} Transferrin (iron-saturated} Insulin (bovine} Fetuin Hydrocortisone Selenous Acid T3 Progesterone Oleic-BSA and linoleic acid-BSA 2-aminoethanol o-phosphorylethanolamine Concentration 17.5 ng/mL 5.01'g/mL 1.1I'g/mL 500I'g/mL 750 ng/mL 5.6 ng/mL 1.5 ng/ml 1.5 ng/mL 600ng/mL 920 ng/mL 2.1 p.g/mL factor VIII-related antigen to detect endothe-lial cell differentiation were negative. Ultrastructural studies of the monolayers revealed numerous intercellular epithelial-type junctional complexes (tight and desmoso-mal), surface microvilli, and lumenal-like intercellular spaces. The cells contained a few cytoplasmic organelles such as small mito-chondria, a paucity of endoplasmic reticulum, ample glycogen, and cytoplasmic interme-diate filaments that were recognized as pre-keratin by light microscopy immunoperoxi-dase. DISCUSSION Cells isolated by our technique were in all probability derived from biliary ducts. This contention is based on the appearance of his to-logic sections of the liver after dislodging and harvesting the cells (absence of biliary epithe-lium in some ducts), the phenotypic charac-teristics of the isolates immediately after har-vesting, and the routine, immunohistologic, and ultrastructural characteristics of mono-layer cultures. Further experimentation is, however, nec-essary to define more precisely the optimal growth conditions of these cells in culture, and to further characterize the cell type pheno-typically, immunologically, and biochemical-ly. Also, we have to date been unable to establish cell lines from these tissues, but rather have extended primary culture viability HUMAN IN to 6 to 7 date has, playa ro histocom. antigens i \. Deme Patboll18: .. TAIS ET At ation of 3rowth ,,"relion ng/mL ,g/mL ,g/mL ug/mL ~g/mi .g/mL g/mL g/mL )g/mL 19/mL g/mL ;ndothe-10layers ithelial-esmoso-nal-Iike ~d a few !l mito-iculum, 1terme-as pre-lperoxi-e in all .s. This lfhisto-ng and epithe-;harac-er har-~ologicI mono-r, nec-ptimal re, and pheno-mica}-ble to s, but lbility HUMAN INTRAHEPATIC EPITHELIAl CELLS to 6 to 7 weeks. Nevertheless, the success to date has enabled us to investigate factors that playa role in antigenic modulation of major histocompatibility complex (MHC) surface antigens in these cells. 163 ACKNOWLEDGMENT We wish to express our thanks to B. Lombardi for encouragement and helpful discussions during perfor-mance of this work. REFERENCES I. Demetris AJ, Lasky S, Van Thiel DH, et al: Am J Pathol 118:151, 1985 2. Snover DC, Sibley RK, Freese OK, et al: Hepatol-ogy 4:1212,1984 

Primary cultures of human intrahepatic (biliary) epithelial cells

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Primary cultures of human intrahepatic (biliary) epithelial cells

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