This novel methodology allows us to construct three-dimensional assemblies, adapted from desired individual cells, in an aqueous buffer solution containing nonspecific hydrophilic polymer, by establishing stable cell-cell contact. In this video, we will demonstrate how we manipulate desired single cells and construct 3D cellular assemblies without artificial scaffold. Here we will show the experimental procedure in our medium with Dextran as an example.
To begin, maintain NAMRU mouse mammary gland epithelial cells with five milliliters of DMEM, containing 10%FBS and 1%Penicillin-Streptomycin, in a 25 cubic centimeter flask for two to three days. When ready to proceed, use an aspirator to remove the supplemented DMEM and add three to five milliliters of PBS, that is preheated to 37 degrees Celsius to wash the cells. Using an aspirator, remove all of the PBS from the flask.
Add 1.5 milliliters of trypsin that is preheated at 37 degrees Celsius. Incubate in a CO2 incubator at 37 degrees Celsius for at least one to two minutes. After this, add 3.5 milliliters of DMEM containing 10%FBS and 1%Penicillin-Streptomycin.
Pipette up and down to mix. Transfer the cell suspension to a 15 milliliter centrifuge tube and centrifuge it at 417 times G for three minutes at room temperature. Then, aspirate the medium.
Add five millimeters of fresh DMEM containing 10%FBS and 1%Penicillin-Streptomycin. And if required, use a cryopreservation solution to cryopreserve the cells as outlined by the manufacturer's instructions. First mix 10 milliliters of DMEM supplemented with 10%FBS and 1%Penicillin-Streptomycin with 0.8 grams of Dextran, to prepare an 80 milligram per milliliter Dextran solution.
Mix 200 microliters of this Dextran solution with 200 microliters of the previously prepared cell suspension, to create a cell suspension containing 40 milligrams per milliliter Dextran medium. To begin, turn on laser, note that the use of a laser beam with a wavelength in the red to near-infrared region is most effective. Next open the software by double clicking the software icon.
Double click the icon for the camera and note that the corresponding display will pop up. Then double click on the icons for the light emitting diode, the focus adjust, and the moving stage to open up their displays as well. First place two glass spacers onto the bottom cover glass slide.
Transfer 20 microliters, the previously prepared Dextran supplemented cell suspension onto the bottom cover glass slide. Then place the top cover glass slide onto the spacers covering the cell suspension. Place the sample cell onto the lower objective lens with 10 microliters of distilled water for water immersion microscopy.
Attach the upper objective lens at the top of the sample cell using 10 microliters of distilled water. Next, click the on-off button in the microscope illumination window to turn the LED on. Click on the direction buttons in the objective positioning window to adjust the distance between the sample and the lower objective lens until it is in focus.
Set the intensity of each laser beam to 1500 milliwatts in the signal attenuation window. Then, click the two laser icons to irradiate the laser beams at position one and position two. Click on the directional buttons in the sample positioning window to move the sample stage until a cell is trapped at position one.
Click and drag the cursor indicating position two until another cell is trapped at position two. To begin, manipulate a single cell, so that it is in contact with another cell. Maintain this condition for 300 seconds, so that cell is exposed to a laser for 300 seconds.
Record the x-y-axis. Trap another cell and transport it to the first two cells to construct an arbitrary 2D cell assembly. Then move the stage up and down to construct a 3D cellular assembly.
Confirm that the assembly is still stable even after the laser is switched off. In this study soluble polymers are used in the construction of 3D single cell assemblies. An example structure formed using the double-beam optical tweezers is shown here.
If the experiment is successful, these cellular assemblies remain stable even after the laser is switched off. This new methodology are for the construction of 3D cellular assemblies in a aqueous medium with natural hydrophilic polymer. It is highly expected to start construction of such kind of next generation cellular assemblies with some of the powerful tools in the field of regenerative medicine and tissue engineering.
