We propose a novel platform for investigating the effects of cell shape on gene expression, using methods for growing and sorting adherences with different morphologies at the single cell level. The main advantage of this technique is that it facilitates the high throughput study of cell shape in vitro as comparing cells with different shapes in vivo is technically demanding. To set up a patterned cardiomyocyte culture, transfer a cardiomyocyte suspension at the aspect ratio of interest to a 15 milliliter tube for counting and dilute the cells to a one times 10 to the five cells per milliliter of appropriate plating medium concentration.
Add two milliliters of cells onto a fibronectin coated micropatterned chip submerged in two milliliters of warm plating medium inside a 35 milliliter Grenier Petri dish and place the dish in a 37 degrees Celsius and 5%carbon dioxide incubator for 18 hours. The next day, check the chip by light microscopy to confirm that most of the cells have attached. Remove the medium from the dish to remove any debris and or dead cells, and starting from the center and moving towards the sides in a drop-wise fashion, gently add PBS to the cells.
After the third wash, replace the PBS with four milliliter maintenance medium and return the cells to the cell culture incubator. After 72 hours, gently flush the chip surface two times with two milliliters of warm Dulbecco's PBS per wash as demonstrated and use forceps to immediately transfer the washed chip to a new sterile 35 milliliter Grenier Petri dish. Quickly add 1.5 microliters of vibrant dicycle green diluted 1000 fold in DPBS to the chip.
Fix a chamber over the chip and place the dish onto the dish holder of the cell picker stage. Insert the magnetic cap and locate the crosshair in the live view window. Focus on the crosshair and, in the scanning and sorting window, select the calibration for automated injection and calibrate.
Replace the dish supernatant with 1.5 milliliters of a one-to-one triple E in DPBS solution to loosen the cells from the fibronectin and open the scanning tab. To scan the entire chip, focus on the top left corner of the chip in the field of view and click get current microscope position. Next, focus on the bottom right corner of the chip and click get current microscope position.
In the popup window, click set sharpest plane and click go to the top right and go to the bottom left corner buttons. Then, click finish to start scanning. When the scanning is complete, open the analyzing tab and click show map to select the single cells that pass the study criteria.
Center the glass microcapillary in the middle of the microscope live view and open the pump tab. To create a vacuum, retract the plunger from a 50 milliliter number one syringe four milliliters and open the sorting tab. To allow a single cell to be picked, set the valve two to be opened for 120 milliseconds and valve one to be opened for 20 milliseconds after a time-lapse of 200 milliseconds.
To allow the picked cell to be delivered successfully to its PCR tube containing lysis buffer, set the valve one to be opened for 20 milliseconds and valve two to be opened for 10 milliseconds after a time-lapse of 10 milliseconds. When the valve settings have been adjusted, click compute the path. The software will compute the fastest path from cell to cell for picking up and injecting the selected cells throughout the chip.
Then, focus the microscope on a pattern on the chip surface. Use the joystick to move the microcapillary down carefully so that the sharpest image of the tip of the microcapillary can be obtained without touching the chip surface and click set. A new window will open, showing the microcapillary cross-section.
To have the software record the tip offset of the capillary in the X, Y, and Z coordinates, click on the exact center of the capillary. Then, click start sorting to launch the sorting. In this representative analysis of pre-amplified cDNA from a picked single cell, a clear band in the gel like densitometry plot was observed corresponding to the peak at 1, 852 base pairs in the electropherogram.
The average size of fragments was 1, 588 base pairs with a small number of fragments that were shorter than 300 base pairs, indicating the generation of a good cDNA library. Immunofluorescent staining and analysis can also be performed to evaluate the sarcomere structure within the patterned cardiomyocytes. This platform paves the way for high throughput study and drug screening of different types of heart failure.
