The overall goal of the following experiment is to investigate the deform ability of different cell types using a simple microfluidics based assay. This is achieved by fabricating a poly dimethyl suboxane or PDMS microfluidic device to probe the timescale of cell transit through a sequence of micron scale Constrictions pressure driven flow is then used to drive cells through their microfluidic channels, which enables the deformation and time dependent relaxation of individual cells to be assayed. Next, the automated image analysis program is run in order to process the videos and obtain a histogram of the transit time data.
The results show that different cell types exhibit differences in cell deform ability based on their transit time through a series of constrictions. The main advantage of this technique over existing techniques such as atomic force microscopy or micro pipet aspiration is that microfluidic devices can successfully operate with high throughput capacities. While other microfluidic devices can also be used to assay cell de formability.
We design our device so that cells passage through sequential constrictions, a geometry common in physiological contexts such as the pulmonary capillary bed. To begin, design the microfluidic device by selecting the width of constriction array channels to be approximately 30 to 50%of the average cell diameter and the channel height to be at least 50%of the cell diameter. Include a filter at the entry ports to remove debris and disaggregate cell clusters.
Before commencing the experiment, fabricate the device master using standard techniques for lithographic micro machining. Verify the height of the pattern features using a profilometer. Next, repair the air source for pressure driven flow using a tank of compressed air and a sequence of air regulators and fittings.
First set up an air supply tank and manual regulator. Then set up an an electronically controlled pressure regulator in line with the manual regulator. Use the simple code written in LabVIEW to input the desired pressure.
The electro pneumatic converter uses an internal feedback loop to adjust the valve outlet pressure to match the specified pressure across the microfluidic device. Set up a pressurized chamber to drive flow of the cell suspension by assembling a cell suspension chamber out of a standard flow cytometer tube and a machined cap that creates a pressure tight seal on the tube. The pressure chamber cap contains two orifices, an inlet that connects to the compressed air tank and an outlet through which cells flow from the pressurized chamber into the device.
Next, set up an inverted microscope fitted with a camera that has an acquisition rate of at least 100 frames per second. To capture images of the cells flowing through the constriction array, then prepare a stock surfactant solution in phosphate buffered saline or PBS as adding a small amount of surfactant to the cell media solution will help to minimize cell adhesion to the PDMS walls. To fabricate the PDMS block with microfluidic channels, add one gram of curing agent to 10 grams of PDMS base and mix thoroughly.
Pour the mixture over the device. Master Degas in a bell jar with applied vacuum until the entrapped bubbles disappear, or for about 10 to 20 minutes. After this time, there may still be bubbles at the AIR PDMS interface, which is normal.
These will typically dissipate during baking. Then bake the Degas device at 65 degrees Celsius for four hours after the device has cooled. Remove the microfluidic devices from the master mold.
Start by gently lifting off one corner of the device. Slow and gentle peeling as opposed to lifting The PD DMS block straight up reduces the stress on both the PDMS and the master Cut individual microfluidic devices out of the PDMS block with a razor blade and remove the device from the master Punch holes in the PDMS device to create connection ports for access between tubing and micro channels. Create holes from the channel side through to the exterior side of the device using a biopsy punch.
Rinse the PDMS device with isopropanol to remove dust and lodge chunks of PDMS. Make sure the punched holes are clear of debris by directing a steady stream of pure isopropanol from a squeeze bottle through the holes. Blow dry with filtered air.
Clean the glass substrate by rinsing with methanol. Then blow dry with filtered air and place on a 200 degree Celsius hot plate for five to 10 minutes to ensure the glass is completely clean and dry before plasma treatment. As detailed in the text protocol, inspect the microfluidic device under a microscope.
Ensure that the channels are not collapsed or broken, and that both inlet and outlet holes connect directly into the device channels by using a low power objective. Place cell suspension in a flow cytometer tube and connect to the pressure cap before connecting the tubing to the microfluidic device, adjust the pressure to about 14 to 21 kilopascals and flush until the cell suspension emerges from the tip of the tubing. Next, place the device on a flat surface to insert the tubing into the device inlet.
As the glass cover slip bonded to the underside of the PDMS device is fragile. Insert the tip of the tubing containing the cell suspension into the device inlet. Then insert a piece of tubing into the exit port and route it into an empty tube for waste collection, such as an empty Falcon tube taped to the side of the microscope stage.
Gently ramp up the pressure to about 28 kilopascals or until cells flow through the channels. Position the device so that multiple channels are in the field of view and are perpendicular to the bottom of the screen. To begin data analysis, open the master script m file and run the program.
Select the first video to be analyzed from the Windows Explorer window that appears. Specify the frame rate for the selected video and press enter. A figure will appear that prompts the user to select a rectangular cropping window.
For the first video, select a window that encompasses all the channels from left to right and intersects the channels just above the first bulb of the array of channels. At the top and bottom, select the constriction regions from the cropped image. The algorithm displays the segmentation of cells for the first 50 frames of each video.
Monitor the top left image closely to determine if the algorithm is accurately locating cells. A ized image is superimposed on the source video. To demonstrate the location of identified cells, select the next video to be processed from the Windows Explorer window.
The algorithm will repeat. For each video selected, select cancel. Once all desired videos have been added via the Windows Explorer window to instruct the function that the video list is complete representative results for the transit time of HL 60 and neutrophil type HL 60 cells show the timescale for a single cell to transit through a series of constrictions transit.
Time is measured for a population of individual cells at each seven micrometer constriction in a series of seven constrictions. At a driving pressure of 28 Kilopascals HL 60 cells temporarily occlude the first constriction for a median time of 9.3 milliseconds before packaging through the subsequent constrictions. By contrast, neutrophil type HL 60 cells occlude the first constriction for only 4.3 milliseconds before packaging.
Once through the first constriction cells transit more quickly through the remaining constrictions from two to seven, with a median transit time of 4.0 milliseconds for the HL 60 cells and 3.3 milliseconds for the neutrophil type cells. By comparing transit time among cell populations, differences in cell de informability can be revealed. After watching this video, you should have a good understanding of how to measure the deform ability of different cell types using the simple microfluidic assay.
Here, pressure driven flow is used to drive cells through microfluidic channels, which enables the deformation and relaxation of individual cells to be assay.
