The Effects of Inhibiting Mechanotransductive Cell Signaling Pathways in Human Bone Marrow Stromal Cells in 2D vs. 3D Culture
Paulamäki, Jannika (2017)
2017
Biotekniikka
Teknis-luonnontieteellinen tiedekunta - Faculty of Natural Sciences
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Hyväksymispäivämäärä
2017-10-04
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tty-201709221950
https://urn.fi/URN:NBN:fi:tty-201709221950
Tiivistelmä
Mechanotransduction is the process of mechanical information transfer between a cell and its surroundings into biochemical responses. Involved in mechanotransduction are several components: extracellular matrix (ECM), transmembrane integrin receptors, focal adhesion protein complexes, and actin stress fibers. Through these complex interactions, cells can exert contractile forces and migrate, proliferate, change morphology and differentiate in the case of stem cells. It has been suggested in the literature that mechanotransductive effects vary between two-dimensional (2D) and three-dimensional environments (3D).
Objectives: In this study, four inhibitors were tested to determine the critical points (concentrations) affecting mechanotransduction. The used inhibitors were PF-562271, a focal adhesion kinase (FAK) inhibitor, PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, Y-27632, a Rho-associated protein kinase (ROCK) inhibitor and (-)-Blebbistatin, a non-muscle myosin II (NMMII) inhibitor. Although all four inhibitors have previously been studied, critical concentrations at which these inhibitors affect human bone marrow stromal cell (hBMSC) survival is unclear. In addition, the effects on morphology and viability are poorly reported, both in 2D and 3D.
Methods: The critical points of four inhibitors were assessed based on two aspects: viability and morphology. Various concentrations of the inhibitors were applied to cell cultures for 24 h or 48 h. After the exposure time in 2D and 3D models, cells were imaged for morphological changes (n=2) and tested for their viability (n=5 and n=3, respectively). Tissue culture plastic/glass and rat collagen I hydrogel were used as a 2D and 3D models, respectively.
Results: IC50 was successfully calculated for FAK inhibitor in both 2D and 3D model (3.5 µM and 5.2 µM, respectively) and for Blebbistatin in 2D (28.5 µM). ROCK inhibitor had little effect on viability, but based on morphological changes IC50 of 6 µM was estimated. MEK inhibitor had no clear effect on viability or morphology of the cells.
Conclusion: Based on the results achieved in the present study, new information was obtained concerning all four inhibitors. In addition, the results correlate the hypotheses of the present study and the mechanotransductive signaling pathway hierarchy.
Objectives: In this study, four inhibitors were tested to determine the critical points (concentrations) affecting mechanotransduction. The used inhibitors were PF-562271, a focal adhesion kinase (FAK) inhibitor, PD98059, a mitogen-activated protein kinase kinase (MEK) inhibitor, Y-27632, a Rho-associated protein kinase (ROCK) inhibitor and (-)-Blebbistatin, a non-muscle myosin II (NMMII) inhibitor. Although all four inhibitors have previously been studied, critical concentrations at which these inhibitors affect human bone marrow stromal cell (hBMSC) survival is unclear. In addition, the effects on morphology and viability are poorly reported, both in 2D and 3D.
Methods: The critical points of four inhibitors were assessed based on two aspects: viability and morphology. Various concentrations of the inhibitors were applied to cell cultures for 24 h or 48 h. After the exposure time in 2D and 3D models, cells were imaged for morphological changes (n=2) and tested for their viability (n=5 and n=3, respectively). Tissue culture plastic/glass and rat collagen I hydrogel were used as a 2D and 3D models, respectively.
Results: IC50 was successfully calculated for FAK inhibitor in both 2D and 3D model (3.5 µM and 5.2 µM, respectively) and for Blebbistatin in 2D (28.5 µM). ROCK inhibitor had little effect on viability, but based on morphological changes IC50 of 6 µM was estimated. MEK inhibitor had no clear effect on viability or morphology of the cells.
Conclusion: Based on the results achieved in the present study, new information was obtained concerning all four inhibitors. In addition, the results correlate the hypotheses of the present study and the mechanotransductive signaling pathway hierarchy.