The overall goal of the following experiment is to quantitatively measure ation of circulating cells toward a chemokine gradient. Under conditions of physiological sheer stress, this is achieved by first culturing, a monolayer of endothelial cells onto collagen coated microporous inserts, and then assembling them into a flow chamber where they cover a separate static compartment containing chemokines. Then shear stress is applied to the cells in order to mimic the physiological conditions in the vasculature.
As a second step test cells of interest such as leukocytes stem cells or tumor cells are allowed to circulate in the 3D system during circulation. The contents in the static compartment influence the efficiency of interactions between circulating cells and endothelial cells, and subsequently the rate of transmigration of the test cells across the endothelial layer. Next, the 3D system is disassembled and trans migrated test cells are collected from the lower static compartment in order to quantitatively measure cell ization using various state-of-the-art methods, results are obtained that show the ability of circulating test cells to interact with endothelial cells under conditions of physiological flow and exit the circulation based on the concentration of chemokines in the static compartment.
The main advantage of this technique over existing methods like the use of parallel lamina flow chambers and static transfer assays is that it combines both she stress in the upper compartment and kines in the lower static compartment. This allows researchers to quantitatively measure externalization of circulating cells to the kin gradient and the conditions of physiological flow. This method can also be used to study the effect of a crosstalk between the cells of local microenvironment and endothelial cells on extravasation of circulating cells.
By aiding an additional layer of cells on a separate insert below the endothelial cells demonstrating the procedure will be Valentina against rova, a technician from my laboratory. To begin, calculate the number of inserts needed for the experiment and place each insert in a separate sterile Petri dish. Sterilize them by gamma irradiation until the total dose reaches 11 gray based on individual setups.
Then dilute the stock collagen. Type one solution to 50 micrograms per milliliter using 0.01 molar hydrochloric acid stored at four degrees Celsius and prepare 154 microliter aliquots of the final solution for each insert. Next place several drops from one Eloqua onto the surface of the insert in a circular pattern.
Then add the rest of the aliquot to form one large drop on the insert surface. Ensure that the pipette tip does not touch the insert at any point and that the collagen covers the entire surface of the insert, but does not drain away. Once the insert is covered, place the lid on the Petri dish and carefully place it on a flat surface.
Incubate the inserts for one hour at room temperature following incubation, aspirate the collagen solution and wash the membrane with 160 microliters of PBS. Then aspirate the PBS. Replace the lid and wrap the dish with paraform.
If the inserts will be used on the same day, place the Petri dish in a dry place at room temperature. Otherwise, place the dish at four degrees Celsius and use the insert within seven days. Harvest cultured human umbilical vein endothelial cells right before use and re suspend them in the desired culture medium at 2.5 million cells per milliliter.
When counting the cells, ensure that the cell viability is greater than 98%using trian blue, then add 160 microliters of the cell suspension onto each insert. By first placing several small drops onto the surface of the collagen coated insert in a circle, and then carefully adding the rest of the cell suspension to form one large drop. When finished, replace the lids and carefully place the dishes into an incubator with 5%carbon dioxide at 37 degrees Celsius and incubate for 30 minutes to allow the cells to attach to the membrane.
After 30 minutes, remove the plates from the incubator and gently add five milliliters of prewarm culture medium over each insert. Taking care to ensure that the insert remains on the bottom of the dish and is completely covered by the medium. Then replace the lids and carefully put the dishes back into the incubator culture.
The cells at 37 degrees Celsius overnight. To assemble the flow chamber first, screw together the upper and lower plates. Then connect the plates to the gas exchange unit using 1.42 millimeter bore PVC tubing with 0.8 millimeter thick walls.
Next, place the assembled device into a clean plastic bag and sterilize it by gamma irradiation until the total dose reaches 11 gray. The next step is to remove an incubator tray, place it into a sterile tissue culture hood and sterilize it with 70%ethanol. Then cover the tray with a large sterile napkin.
Next, remove the device from the plastic bag and place it on the sterile incubator tray. Then connect the device to the peristaltic pump and program the pump to run at a speed of 0.2 milliliters per minute. Pipette five to seven milliliters of the desired culture medium into a sterile 15 milliliter tube to be used for the inlet.
Then disconnect the inlet tubing from the outlet by pulling the metal needle from the tubing using small sterile napkins to maintain sterility of the tubing, place the metal inlet needle into the 15 milliliter tube with the media and place the outlet tube into the empty 15 milliliter tube. Next, turn on the pump, so the flow is counterclockwise. Allow the negative pressure to draw the medium from the 15 milliliter inlet tube into the tubing and stop the pump when the medium reaches the end of the tubing just before it enters the device.
Then open the device by unscrewing the tube plates and carefully remove the upper plate pipette 1500 to 1, 550 microliters of medium into the lower wells, making sure the wells contain sufficient medium to form a hemisphere that reaches one to two millimeters above the lower plate. Next, transfer the prepared inserts from the petri dishes to the wells of the lower plate. Using a sterile forceps, make sure that no bubbles are trapped beneath the inserts and aspirate any medium that appears on the surface of the lower plate.
Then place the upper plate back onto the lower plate and reconnect them. Using the screws immediately turn on the peristaltic pump and allow medium to flow through the 3D device. Elevate the outlet end of the device and maintain the chamber in this position to prevent formation of bubbles within the space between the plates.
Continue to fill the tubing connecting the chamber to the gas exchange unit with medium until it reaches the gas exchange unit. Next place three milliliters of medium into the gas exchange unit. Turn on the pump and allow the air bubbles to escape through it.
Elevate the unit to allow the medium to fill the tubing that exits it and stop the pump when the medium is two to three centimeters before the end. Once fully primed, connect the inlet needle to the outlet tube using small sterile napkins. Then reverse the pump so the medium flows clockwise towards the gas exchange unit and ensure any remaining air bubbles are removed.
With the pump again flowing counterclockwise. Add 100 microliters of the test cell suspension to the gas exchange unit. Then close the lid of the respirator.
Place the tray back into the incubator and allow the test cells to circulate in the device at 37 degrees Celsius. Once the desired time has passed, remove the tray with the working system from the incubator and place it into the hood. Stop the pump, disconnect the inlet and outlet and place the ends into empty sterile 15 milliliter tubes.
Then turn on the pump and collect the cell suspension from the system. Place the cell suspension on ice until use. Next, disassemble the chamber by removing the screws and lifting off the upper plate.
Remove the inserts from the lower plate and transfer them to petri dishes for staining or cell collection. Finally, remove cell suspension from the lower wells and place it into 15 milliliter tubes. Rinse each well twice with 1.5 milliliters of medium and add it to its respective tube.
Then centrifuge the tubes for five minutes at 200 Gs and process the cells according to specific experimental goals such as those listed in the accompanying text protocol shown. Here is an example of one of the many tests that can be performed using this system. The setup consists of mirroring bone marrow derived endothelial cells grown on inserts with five micron pores below the inserts stromal cell derived factor one or SDF one was added at concentrations of zero five or 50 nanograms per milliliter.
Bone marrow cells were allowed to circulate in the 3D system. Cells that migrated through the insert were collected and then cultured on plates with Methylcellulose media. In the presence of hematopoietic growth factors causing progenitor cells to form colonies, the number of colonies were scored 14 days later to determine the number of progenitors in each well and to reveal their ability to migrate toward sdf.
One under conditions of physiological flow. With the addition of stromal fibroblasts grown on the lower insert in the static chamber, more hematopoietic cells migrated from the circulation into the chamber with the addition of sdf, one at 50 nanograms per milliliter than with just SDF one alone After its development. This technique paved the way for researchers in different fields of medical and biological sciences to model tissue specific vascular beds in vitro and to explore cellular and molecular mechanisms, mediating migration of circulating cells toward normal tissues versus diseased.
