The overall goal of this procedure is to evaluate the contractility of mesenchymal cells that have undergone in vitro inflamatory cytokine induced epithelial mesenchymal transition, or EMT. This mesenchymal is a variation of basic cell function after EMT induced by inflammatory immune responses such as during idiopathic pulmonary fibrosis or, airway remodeling in severe asthma. The main advantages of this technique are that it does not require specific devices and that it demonstrates a high content Demontrating the procedure will be Hideyuki Takeshima and Kosuke Makita graduate students from my laboratory.
Begin by washing a culture of A-549 human lung epithelial cells with five to ten millileters of PBS. Next, detach the adherent cells with two millileters of trypsin-EDTA at 37 degrees Celsius and five percent carbon dioxide. After three minutes, transfer the cell suspension into conical tubes containing cell culture medium and spin down the cells in a centrifuge.
Resuspend the pellets in two millileters of cell culture medium, reserving a small eloquate for counting. Then seed 0.5 to 1 times 10 to the 6th cells in ten millileters of medium per ten centimeter polysterene dish and incubate the cell cultures for 24 hours. For EMT induction, the next day, treat the cells with 10 microliters of TGF beta 1 and TNF alpha, and return the plates to the incubator for another 48 hours.
On day four, wash the emt induced cultures with five to ten millileters of pbs, then detach cells with trypsin-EDTA as just demonstrated. Next, spin down the cell suspensions in DMEM supplemented with tryspsin inhibitor and resuspend the pellets in 500 microliters of pbs. Then count the cells, and add freshley prepared gell medium to a density of 3 times 10 to the 5th cells per well, gently but quickly mixing the solution by pipette before gelation.
Promptly, but carefully, dispense 0.5 millileters of cells into each well of a non-treated 24 well plate in neat, cylindrical forms. Then place the plate in a cell culture incubator for 15 minutes. When the gel has completely solidified, move a sterilized spatuala in a circle around the outside of each gel in one direction, to detach the gels from the plates without breaking.
When removing the gel, be careful not to move the spatula back and forth in the well. Then use the spatula to gently transfer the gels to individual 16 millimeter tissue culture dishes containing five millileters of DMEM supplemented with one percentFBS, with or without TGF beta 1 and TNF alpha. Finally, gently shake the dishes to ensure that the gels are floating on the medium, and return the gels to the cell culture incubator.
Untreated a541 cells display a cobblestone like and triangle shaped appearance, characteristic of epithelial cells. After their stimulation with TGF beta 1 and TNF alpha however, the cells demonstrate a long spindle shape, similar to that observed for mesynchemal cells. QRTCPR of the epithelial and mesynchemal marker expression before and after EMT reveals that A541 cells treated with inflammatory cytokines for 48 hours exhibit a significantly reduced expression of CDH1 and significantly increased expressions of VIAM and ACTA2.
Further, stimulation with these cytokines attenuates E-cadherin expression as determined by western blood analysis, whereas the expressions of vimentin N-cadherin, and alpha smooth muscle actin are induced. In a gel contraction assay, after 48 hours, the gels containing TGF beta 1 and TNF alpha treated cells are smaller than the controlled gels containing cells treated with PBS. Indeed, after 72 hours, the size of the gels containing the cytokine treated gels is significantly reduced, compared to the controlled gels as measured by a gel analyzer.
As observed, the gel contraction steps can be completed in three hours if they are performed properly. While attempting this procedure, it's important to remember to confirm that the EMT has been successfully induced before performing the gel contracting assay. After observement, this technique provides a way for a variating the EMT process during inflammatory state such as fibrosis or lung remodeling.
After watching this video, you should have a good understanding of how to emit cells that have EMT, into a type of cuagenter and to froth them in medium.
