The overall goal of this protocol is to demonstrate the fabrication and operation of a microfluidic chip, which allows for high-efficiency single-cell isolation and culture. This method provides a useful tool for any area that would benefit from single-cell isolation and culture. The main advantage of this technique is that it is highly efficient at loading single cells into large microwell arrays.
Additionally, only gentle flow and the gravitational force are used to operate the device, which minimizes the cell damage. The implication of this technique is turned towards the end diagnosis of human disease such as cancer, in which cellular heterogeneity affects the response of cell, so We came up with the idea of developing this method when we were establishing microwell cell line, because limit dilution method was very time-consuming and inefficient. Now this method can provide insight into individual cell analysis.
It can also be applied to other applications such as the establishment of and time-ness of observation of individual cell course and behavior. To begin, fabricate silanized master molds for both the single cell isolation layer and the microwell culture layer, using standard photolithography as described in the accompanying text protocol. For each master mold, combine 16 grams of PDMS base with 1.6 grams of a curing agent in a disposable cup, and stir the mixtures until combined.
Then, place the master molds into 150 millimeter petri dishes, and pour the PDMS on top of the molds. Place the petri dishes into a desiccator and apply a vacuum for one hour to remove air bubbles from the PDMS. After one hour, remove the dishes from the desiccator and place them in a conventional oven at 65 degrees Celsius for three to six hours to cure the PDMS.
Then, remove the dishes from the oven and let them cool down to room temperature. Once cool, peel the cured PDMS capture-well array and microwell culture array from the master molds and cut out the devices using a razor blade. Next, use a 0.75 millimeter punch to create one hole at each end of the microchannel on the capture-well array layer.
Use tape to clean what will become the inner surface of the device, and then place the individual layers with the inner surfaces facing up into a plasma cleaner for a brief oxygen plasma treatment. When finished, remove the PDMS layers from the plasma cleaner and use a stereo microscope to align and connect the top and bottom layers of the device. Place the aligned device in an oven at 65 degrees Celsius for 24 hours to create a permanent bond between the PDMS layers.
Next, place the bonded PDMS device in deionized water and place the container in a desiccator under vacuum for 15 minutes, in order to remove the air from the microchannel of the device. With all of the air now removed from the device, place the PDMS device in a tissue culture hood and use UV light to sterilize the surface of the device for 30 minutes. Next, replace the deionized water in the device with 5%bovine serum albumin in PBS and incubate the device at 37 degrees Celsius for 30 minutes.
This will block the surface and improve the transfer efficiency of isolated cells from capture-wells to the culture-wells. After 30 minutes, replace the blocking solution in the device with sterile PBS. Prepare a single-cell suspension with 2.5 million cells per milliliter and load a pipette with 50 microliters of the suspension.
Then, transfer the cells into the device through the device's outlet hole. Load another 50 microliters of the cell suspension and transfer it into the device through the inlet hole to evenly fill up the entire microchannel with cells. Once loaded, use a sterile plug made from one millimeter diameter nylon fishing line to seal the outlet hole of the device.
Then, fill a one milliliter sterile syringe with culture medium, eject any residual air bubbles, and set it into the syringe pump. Connect a 23 gauge blunt needle to the syringe and use polytetrafluoroethylene tubing to connect the needle to the inlet of the device. After connecting the tube, remove the plug from the outlet hole and wait two minutes while the cells settle into the single cell capture-wells by gravitational force.
Next, set the syringe pump to 600 microliters per minute. Remove the outlet plug and wash away the uncaptured cells for 30 seconds. Wait two minutes for the pressure to stabilize.
Then remove the tube from the inlet of the device and seal both the inlet and outlet holes with plugs to form a closed culture system. Now flip the device by hand to transfer the captured single cells to the culture microwells on the other side of the device. Then, place the device in a 100 millimeter tissue culture dish, add 10 milliliters of sterilized PBS around the device, and place the dish into an incubator.
To replace the culture medium, first place two droplets of culture medium on top of the PDMS device near the inlet and outlet to avoid introducing air bubbles into the microchannel. Then, use a 0.75 millimeter biopsy puncher to punch two holes from the top of the PDMS device near the ends of the microchannel. Be sure to only punch through the top layer of the device.
Next, fill a one milliliter plastic syringe containing fresh prewarmed medium, and use a 23 gauge blunt needle and PTFE tubing to connect it to the newly created inlet port. Slowly flow approximately 120 microliters of fresh medium into the device over five minutes to replace the old medium. When finished, seal the inlet and outlet ports using two plugs, and return the device to the cell culture incubator.
The number of cells that end up in the capture-wells ranges from about 68%to 85%depending on the type of cell. In addition, most of the capture-wells contain only a single cell, for all three of the cell types. Following the transfer of captured KT98 cells to the culture-wells, about 77%of the wells had only a single cell, 16%had no cells, 6%had two cells, and less than 1%of the wells had three or more cells.
Over the course of seven days, isolated single cells exhibited heterogeneous growth patterns. While some of the cells multiplied, others did not. Once mastered, this technique can be done in approximately 20 minutes if it is performed properly.
After watching this video, you should have a good understanding of how to use this high-throughput single cell isolation endocrinal culture platform to boost the productivity of your single cell experiments. While attempting this procedure, it's important to remember to avoid air bubbles getting into the microchannel and to prevent cells from aggregating together as time goes on.
