The overall goal of the falling experiment is to deliver macromolecules vector free to target cells using mechanical disruption. This is achieved by first placing the target cell type in suspension with the desired delivery material and loading into the reservoir of a specially designed microfluidic device. As a second step, the microfluidic device is pressurized and the cells are driven through an array of microfluidic channels that squeeze the cells to facilitate transient membrane disruption.
Next, the cells are allowed to incubate in the presence of the target delivery material to facilitate diffusive transport of the material into the cell cytoplasm. While the cell membrane is repaired. Results are obtained that show effective delivery of macromolecules through the use of flow cytometry.
The main advantage of this technique over existing methods like ation and lipo perfection, is that it's capable of addressing challenging cell types and materials that cannot be effectively delivered by other methods. This method can answer key questions in the biological field, such as how to improve cell re programming. The implications of this technique extend towards the therapy and diagnosis of cancer because the system enables manipulation of immune cells to enhance their anti-tumor efficacy.
To begin, fabricate or purchase the microfluidic device, device holder, plastic fluid reservoirs and O-rings shown here and listed with details in the accompanying text protocol, then fill three sealable containers partway with 70%ethanol. Place the devices in the first container, the reservoirs and O-rings in the second container and holders in the third. Then fill the containers the rest of the way with 70%ethanol.
Next place, the container containing the reservoirs and O-rings as well as the one containing the holders in an ultrasound bath for five to 10 minutes before use. This will help to remove any contaminating particles from previous experiments while the component sonicate wipe down the workspace in a biosafety cabinet with 70%ethanol. Then sterilize all the required materials in including the three containers and a pair of tweezers by wiping them down with 70%ethanol and place them inside the biosafety cabinet to begin assembly.
First, set down two to three low lint wipes in the work area. Then remove the plastic reservoirs from the ethanol container with tweezers and set them on the wipes to facilitate wicking and evaporation of ethanol from it inner surface. Gently tap the reservoirs to assist in the removal of the ethanol solution.
If necessary, use pressurized gas to fully purge the reservoir. Once the reservoirs have dried, insert the O-rings into the appropriate slots on the reservoirs. Then use tweezers to place the chip face up in the device holder.
Make sure the chip is lying flat in the holder and adjust if necessary using the tweezers. If the device does not fit properly in its holder, there is a risk that it will break during the subsequent steps. Next, gently place the reservoirs on the holder and align them with the clips.
Make sure the O-rings don't fall outta their slots. During this process, gently press down on the reservoirs until they click into place. Ensure that both sides of the reservoirs are secure and that the chip appears to be in the correct position.
Do not press down on the reservoirs with excessive force. As the pressure could break the chips for primary or adherence. Cell lines, place cells one to two days prior to the experiment so that they are no more than 80%confluent.
On the day of the experiment. Immediately before the experiment, harvest the cells and resus, suspend them in media or other desired buffer at between one and 10 million cells per milliliter. Then carefully pass the cell suspension through a 40 micron cell strainer mesh to prevent clumps of cells and matrix from clogging the device.
Once strained, mixed 300 microliters of the cell suspension with the desired concentration of delivery material in a separate tube. For this demonstration, two microliters of dextrin at five milligrams per milliliter and two microliters of Alexa. 4 88 isotope control antibody are added per 50 microliters of cell suspension.
Next, gently mix 100 microliters of the cells by pipetting. Then using gel loading tips, pipette the cells and delivery material into either one of the reservoirs, attach pressure tubing to the filled reservoir and finger tighten the knot to ensure proper ceiling. Then adjust the air pressure to 70 PSI on the regulator to control the speed at which cells travel through the device.
The pressure should be optimized for the specific cell and microfluidic chip combination used in each experiment. With everything prepared, raise the device to eye level and orient it so that the liquid in the reservoir is easily visible. Then press the push button valve to pressurize the reservoir and begin cell flow.
Watch the speed at which the fluid is flowing from the reservoir. If the fluid slows substantially relative to its initial flow rate, the mounted device is likely clogged and needs to be exchanged. The increased shear caused by the blockage will cause greater than normal cell death.
When the liquid level is approximately two millimeters from the bottom of the reservoir, quickly turn the regulator to zero PSI to stop the flow. It is important to stop the flow of air before the reservoir is emptied, or the sample may be ejected from the collection reservoir. Then collect the treated cells from the outflow reservoir and place them into a micro centrifuge tube.
At room temperature, the rated cells will continue to uptake material for up to 10 minutes. After 10 minutes, plate the cells with additional media or continue on to use the cells according to experimental needs. Repeat these steps to collect as many treated cells as are needed and exchange the chips as needed if they clog and discard the C clogged devices for each subsequent sample.
Change the flow direction to minimize clogging effects. When finished collecting cells, gently disconnect the reservoirs from the main holder by pushing aside the clip arms. Then place each part to be reused in the appropriate storage container and fill them with fresh 70%ethanol.
Finally place all the used chips in a separate container for proper disposal. The The delivery efficiency and cell viability of hela cells were measured. Following a series of experiments, three different microfluidic chips were tested in a range of speeds up to 600 millimeters per second.
These tests showed that increased flow speeds improved delivery of fluorescently conjugated, three kilodalton dextrin into the cells for each of the chips tested. Additionally, increased speeds also caused the cell viability to drop by about 20%in the tested devices. This amount of cell death is relatively low and is often much higher in inappropriately designed devices.
The uptake of Pacific blue conjugated dextrin into the control cell population shown on the left is the result of limited surface binding and endocytic effects. As the cells are exposed to the delivery material for the same amount of time as the treated cells. On the right is the population of live cells that pass through the microfluidic device while exposed to the same concentration of dextrin as the control cells.
The cells in the shaded region are considered undelivered, and the cells to the right of the line are defined as delivered. Once mastered, this technique can be completed in under one hour if it is performed properly. Following this procedure, other methods, life post optometry can be performed in order to answer additional questions like delivery efficiency and material functionality After its development.
This technique paved the way for researchers in the field of regenerative medicine to explore cell programming in adult primary cells using direct protein delivery. After watching this video, you should have a good understanding of how to set up and operate the self squeezing system for your desired application.
