title: Analysis of local tissue-specific gene expression in cellular micropatterns.
abstract: While cellular micropatterning approaches are employed extensively in cell biology and tissue engineering, only a limited number of methods for analysis of local function in the context of a complex, microfabricated environment are currently available. The present study develops a novel strategy for analysis of local tissue-specific function in cellular micropatterns. Model hepatocytes (HepG2 cells) were seeded onto silane-modified glass slides containing robotically printed arrays of collagen type I. These model hepatocytes formed cell arrays with individual cell cluster dimensions (150 or 500 microm) corresponding in size to the printed collagen spots. Non-parenchymal cells (3T3 fibroblasts) were added to hepatocellular micropatterns to create heterotypic cocultures. Expression of hepatic phenotype in HepG2 cells was first verified by traditional techniques including intracellular staining and ELISA for albumin. In order to evaluate local liver function in the cellular microarray, individual array members composed of approximately 400 hepatocytes were retrieved using laser capture microdissection and analyzed with real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). Hepatic function was assessed based on expression of four genes associated with differentiated liver phenotype: albumin, transferrin, alpha-fetoprotein, and alpha1-antitrypsin. "Titration" experiments, carried out to identify the smallest population of HepG2 cells yielding detectable mRNA levels and RT-PCR signals, showed that extraction area of 12,500 microm2 (corresponding to approximately 70 cells) provided detectable gene expression signals. All four liver-specific genes were routinely evaluated after extraction of approximately 400 HepG2 from the micropatterned surfaces. Significantly, selective retrieval and subsequent analysis of tissue-specific function was demonstrated for hepatic cells micropatterned alone and in coculture with non-parenchymal cells. In the future, methods described in this study will offer the possibility to investigate dynamic and reciprocal interactions between two or more cell types positioned on a microfabricated cell culture surface. We also envision the proposed approaches to be ideally suited for cell analysis in the context of combinatorial microenvironment.
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, Davis, California 95616, USA.
authors.forename: Ji Youn
authors.initials: JY
authors.lastname: Lee
authors.forename: Caroline
authors.initials: C
authors.lastname: Jones
authors.forename: Mark A
authors.initials: MA
authors.lastname: Zern
authors.forename: Alexander
authors.initials: A
authors.lastname: Revzin
mesh_terms: D016475:3T3 Cells; D000418:Albumins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000378:metabolism*; D020869:Gene Expression Profiling; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D051379:Mice; D036103:Nanotechnology; D009928:Organ Specificity; D010641:Phenotype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000235:genetics / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000418:Albumins; D014168:Transferrin; D000515:alpha 1-Antitrypsin; D000509:alpha-Fetoproteins