The overall goal of this procedure is to avoid distracting physical effects during in vitro shockwave treatment in order to achieve reproducible results. This is accomplished by first filling a cell culture flask completely with culture medium, and then placing the flask into a heated water bath. The second step is to use a generous amount of ultrasound gel when coupling the applicator to the water bath.
Next, the applicator is placed against the water bath's membrane in a horizontal manner. The final step is to ensure that the shockwave source is in line with the center of the cell culture flask in order to have the cells positioned within the wave focus. Ultimately, analysis of your cells can be performed as an now example with real-time PCR and a cytokine array to show an alteration in the genes and cytokines of interest.
We first had the idea for this model when we saw that existing methods of applying short waves directly to cell cultures do not consider the effect of wave reflection at the transition from culture medium to the ambient air. Demonstrating the procedure will be Dr.Chante PhD student from my research group. To begin heat 3.5 liters of tap water to 37 degrees Celsius.
Then transfer the heated water to the water bath. The water level should be three centimeters below the top of the water bath and should cover the membrane completely. Next, connect the temperature sensor to a power supply and place the temperature sensor in the fitting on the back wall of the water bath.
Then while stirring the water, continuously connect the heater to a power supply and heat the water to a stable temperature of 37 degrees Celsius. Under sterile conditions. Seat cells in T 25 culture flasks.
So they will reach 90%con fluency following overnight incubation. Then stand flasks vertically and fill with culture medium to the bottom of the neck of the flask to avoid contamination. Do not allow medium to come in contact with the neck of the flask or the closing cap.
Next close flasks with a solid cap without a filter to prevent contamination from the water in the bath. Then seal the caps with paraform. After the flasks are sealed, clamp them into a stand.
Then insert the fixed flasks into the water bath and align the middle of the cell culture flask. With the center of the shock shockwave applicator's membrane, apply ample amounts of ultrasound transmission gel on the shockwave applicator and the membrane of the water bath. Then connect the applicator to the membrane and to make sure no air bubbles are between them.
Next, hold the shockwave applicator in the center of the membrane and ensure that it is lined horizontally and vertically with the cell culture flask to guarantee treatment of the entire cell growth area. Then conduct a pilot experiment to determine the ideal distance between the shockwave applicator and the culture flask, as well as for defining the correct treatment parameters. Make sure to maintain the culture flask and the applicator in a stable position during activation of the shockwave device.
Once an ideal distance is determined, perform the experiment under the desired shockwave treatment parameters. After shockwave treatment is completed, remove the cell culture flask from the water bath, dry with paper towels, and disinfect the outside surface of the flask. Next, transfer the medium from the flask into a centrifuge tube.
Pellet the cells under normal centrifuge parameters. Resus suspend the cell pellet in two milliliters of cell culture medium, then transferred the cell suspension back into the flask containing five milliliters of cell culture medium, and grow the cells to the required co fluency for cell analysis experiments. Real time PCR analysis of human umbilical vein endothelial cells revealed an upregulation in tie two mRNA levels after shockwave treatment.
This suggests shockwave treatment, increased endothelial cell proliferation and angiogenesis. The cytokine levels in the umbilical vein. Endothelial cells were quantified using a cytokine array.
The two inflammatory cytokines IL six and IL eight were also found to be significantly increased, both of which are involved in inflammation accompanying the angiogenic process After its development. This technique paved the way for numerous researchers in the field of shockwave basic science to explore effects on a single cell type in vitro experiments. We hope to convince even more researchers to use this model in order to gain reproducible and comparable results in shockwave science.
