phalloidine and cyanine-dye-3

Polymer microarrays as platforms for cell-based assays are presented, offering a unique approach to high-throughput cellular analysis. These high-throughput (HT) platforms are used for the screening of new materials with the purpose of first finding substrates upon which a specific cell line would adhere and second to gain a rapid understanding of the interactions between cells and biomaterials. Arrays presented here are fabricated using pre-synthesised polymers by contact printing via a robotic microarrayer. These large arrays of polymers are then incubated with cell cultures and the results obtained are used to significantly help the design of synthetic biomaterials, implant surfaces and tissue-engineering scaffolds by finding correlations between their chemical structure and their biological performance. The flexibility of polymer microarrays analysis not only greatly refines our knowledge of multitude of cell-biomaterial interactions but could also be used in biocompatibility assessments as novel biomarkers.

Topics: Animals; Benzimidazoles; Biocompatible Materials; Carbocyanines; Cell Adhesion; Cell Culture Techniques; Cell Line; Cells, Cultured; Fluorescein-5-isothiocyanate; Fluorescent Dyes; High-Throughput Screening Assays; Humans; Indoles; K562 Cells; L Cells; Mice; Microarray Analysis; Phalloidine; Polymers; Rhodamines; Substrate Specificity

Mycophenolic acid (MPA) is a potent inhibitor of the inosine monophosphate dehydrogenase and used as an immunosuppressive drug in transplantation. MPA inhibits proliferation of T- and B-lymphocytes by guanosine depletion. Since fibroblasts rely on the de novo synthesis of guanosine nucleotides, it is assumed that MPA interacts with fibroblasts causing an increased frequency of wound healing problems. We show a downregulation of the cytoskeletal proteins vinculin, actin and tubulin in fibroblasts exposed to pharmacological doses of MPA using microarray technology, real-time polymerase chain reaction (PCR) and Western blot. This reduction in RNA and protein content is accompanied by a substantial rearrangement of the cytoskeleton in MPA-treated fibroblasts as documented by immunofluorescence. The dysfunctional fibroblast growth was validated by scratch test documenting impaired migrational capacity. In contrast, cell adhesion was increased in MPA-treated fibroblasts. The results of the cultured human fibroblasts were applied to skin biopsies of renal transplant recipients. Skin biopsies of patients treated with MPA expressed less vinculin, actin and tubulin as compared to control biopsies that could explain potential wound healing problems posttransplantation. The perspective of MPA-induced cytoskeletal dysfunction may go beyond wound healing disturbances and may have beneficial effects on (renal) allografts with respect to scarring.

Topics: Biopsy; Carbocyanines; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Dermatologic Surgical Procedures; Dose-Response Relationship, Drug; Fibroblasts; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; Immunohistochemistry; Immunosuppressive Agents; Indoles; Mycophenolic Acid; Phalloidine; Rhodamines; Skin