title: Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models.
abstract: Hepatic/nonhepatic cell cocultures are widely used in studies on the role of homo- and heterotypic interactions in liver physiology and pathophysiology. In this article, for the first time, establishment of the coculture model employing hepatoma C3A cells and human skin fibroblasts, stably expressing fluorescent markers, is described. Suitability of the model in studying coculture conditions using fluorescence microscopy and flow cytometry was examined. C3A cells spontaneously formed island-like growth patterns surrounded by fibroblasts. The "islands" size and resulting intensity of the homo- and heterotypic interactions can easily be tuned by applying various plated cells ratios. We examined the capability of the hepatoma cells to produce albumin in hepatic/nonhepatic cell cocultures. The enzyme-linked immunosorbent assay (ELISA) tests showed that greater number of fibroblasts in coculture, resulting in smaller sizes of hepatoma "islands," and thus, a larger heterotypic interface, promoted higher albumin synthesis. The use of fluorescently labeled cells in flow cytometry measurements enabled us to separately gate two cell populations and to evaluate protein expression only in/on cells of interest. Flow cytometry confirmed ELISA results indicating the highest albumin production in hepatoma cells cocultured with the greatest number of fibroblasts and the inhibited protein synthesis in coculture with osteosarcoma cells.
authors.affiliation: Department of Hybrid Microbiosystem Engineering, Nalecz Institute of Biocybernetics and Biomedical Engineering, PAS, Warsaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Samluk
authors.forename: Karolina Ewa
authors.initials: KE
authors.lastname: Zakrzewska
authors.forename: Krzysztof Dariusz
authors.initials: KD
authors.lastname: Pluta
mesh_terms: D015415:Biomarkers / Q000378:metabolism; D001859:Bone Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D002450:Cell Communication; D045744:Cell Line, Tumor; D002469:Cell Separation / Q000379:methods; D018920:Coculture Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism*; D005434:Flow Cytometry; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D008297:Male; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D012516:Osteosarcoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D012709:Serum Albumin / Q000378:metabolism; D000075462:Serum Albumin, Human; D012867:Skin / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D014162:Transfection; D000097573:Red Fluorescent Protein
chemical_list: D015415:Biomarkers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D012709:Serum Albumin; D000075462:Serum Albumin, Human; D000097573:Red Fluorescent Protein; C422455:ALB protein, human; C485184:enhanced green fluorescent protein
keywords: C3A fluorescent cell line; Flow cytometry; Fluorescently labeled fibroblasts; Lentiviral vectors; Liver coculture model