Nuclear translocation of myocardin-related transcription factor-A during transforming growth factor beta-induced epithelial to mesenchymal transition of lens epithelial cells.

PURPOSE: Transforming growth factor beta (TGFß) is a known inducer of epithelial to mesenchymal transition (EMT), and studies in other systems have shown that nuclear localization of the myocardin-related transcription factor (MRTF) is downstream of TGFß. In the following study, we investigated whether nuclear translocation of MRTF-A or MRTF-B is involved in TGFß-induced EMT of lens epithelial cells (LECs). We further investigated the relationship between matrix metalloproteinase-2 and -9 (MMP-2/9) and MRTF in the EMT of LECs. METHODS: Rat lens explant cultures were used as the model system. Explants were treated with TGFß, an MMP-2/9 inhibitor, or actin binding drugs and immunostained for alpha smooth muscle actin (αSMA), MRTF-A, and MRTF-B. Cytoplasmic and nuclear intensities of cells were measured using ImageJ. Production of αSMA was measured using western blot analysis and ImageJ. RESULTS: Untreated explant cells exhibited little αSMA expression, and MRTF-A and B were found to reside primarily in the cytosol. However, when stimulated with TGFß, a significantly greater number of cells exhibited nuclear expression of MRTF-A, accompanied by an increase in αSMA expression. However, MRTF-B remained in the cytoplasm following TGFß treatment. Cotreatment with an MMP-2/9 inhibitor and TGFß resulted in reduced MRTF-A nuclear localization and αSMA expression compared to cells treated with TGFß alone. CONCLUSIONS: Our results are the first to demonstrate the expression of MRTF-A in LECs and that its nuclear translocation can be stimulated by TGFß. Our data further suggest that MMP-2 and -9 are involved in the translocation of MRTF-A in LECs during TGFß-induced EMT.

Alginate-based microfluidic system for tumor spheroid formation and anticancer agent screening.

We demonstrate a microfluidic system for long-term tumor cell culture and drug testing. Three-dimensional cell culture is critical in characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Breast tumor cells were encapsulated within alginate which was gelled in situ within the microchannels. Tumor spheroid formation was observed several days after cell seeding, and various concentrations of doxorubicin were applied to the encapsulated cell aggregates. Drug effects on cell viability and proliferation were measured. In future, hydrogel-based microfluidic devices can comprise part of systems which replace labor intensive screening platforms currently implemented in the laboratory, and they address a need for improving preclinical testing of cancer cell sensitivity to anti-cancer drugs.

Effects of surfactant and gentle agitation on inkjet dispensing of living cells.

Inkjet dispensing is a promising method for patterning cells and biomaterials for tissue engineering applications. In a novel approach, this work uses a biocompatible surfactant to improve the reliability of droplet formation in piezoelectric drop-on-demand inkjet printing of Hep G2 hepatocytes onto hydrogels. During a long printing process, cell aggregation and sedimentation within the inkjet reservoir can lead to inconsistent printing results. In order to improve repeatability, the effects of gentle agitation on cell sedimentation and aggregation within the inkjet reservoir were also investigated. Cell viability and proliferation when printed onto prepared collagen substrates were assessed using live/dead staining and the Alamar Blue metabolic assay. The addition of 0.05% Pluronic as a surfactant did not reduce cell viability, which remained above 95% 2 days after printing. The surfactant improved the reliability of droplet formation. Although gentle stirring of the inkjet reservoir was sufficient to maintain a cell suspension and reduce sedimentation, aggregation within the suspension continued to affect printing performance over a 180 min printing period.

Hydrogel-based microfluidic systems for co-culture of cells.

Currently, in vivo cellular microenvironments are not well modeled using traditional cell culture methods. Microfluidic technology provides the tools to mimic in vivo environments for cell culture. We use alginate hydrogels to reversibly trap and release cells for incubation. The porous nature of alginate gels resembles the natural extracellular matrix and allows the transport of nutrients and waste. This property makes it an ideal cell trapping and culturing material. Here, we present reversible immobilization of human fetal lung fibroblasts (HFL1) and human hepatocellular liver carcinoma cell (Hep G2) inside microfluidic channels and demonstrate the possibility of co-culturing these two cell types within different alginate gel layers.