CMOD Pack allows you to pattern cells with very high precision and culture them in 2d or 3d. This opens up opportunities to study cell-cell interactions and the morphogenesis of engine new tissues. The main advantage of this technique is that it's easy for other labs to adopt, it doesn't require a clean room, specialized equipment, or custom synthesized reagents.
Begin by dropping small drops of the positive photo resist onto the aldehyde slide, using a disposable pipette. Spin the slide at 3000 RPM for 30 seconds using the spin coder, then place it on a 100 degrees Celsius hot plate for 1.5 minutes to cross-link the photo resist. Remove the slide from the hot plate and place a photo mask with the desired features on top of the slide, weigh it down with a piece of glass and cover the entire setup in an opaque box.
Expose the setup with a UV lamp for two minutes, develop the slide by immersing it in the developer solution for three to five minutes, then rinse away excess developer solution with water, dry it under a stream of air or nitrogen. Observe this light under a microscope to confirm the success of photo lithography and store it in the dark. Add a droplet of 20 micromolar amine modified oligos solution onto each photo patterned region of the slide and spread the droplet gently across the entire region, using a pipette tip, taking care, not to scratch the slide, bake the slide in a 65 degrees Celsius oven until the DNA solution has fully dried, perform reductive amination by placing the baked slide and a 15 centimeter cell culture dish in a fume hood on top of a shaker.
Gently mix 100 milligrams of sodium boro hydride in 40 milliliters of PBS and add it to the dish. Then turn on the shaker for 15 minutes. After the reaction, wash this slide twice with 0.1%sodium dodecyl sulfate to remove unreacted DNA.
Then wash the slide three times with water. Drive this slide under the stream of nitrogen or air. Finally rinse it with acetone to remove the remaining photo resist.
Prepare a four micromolar universal anchor and adapter solution as described in the text manuscript and prepare a 20 micromolar universal co-anchor solution in PBS. Prepare the cell suspension by resuspending the cell pellet in one milliliter of ice cold PBS or serum free media and transfer one to 3 million cells to a 1.5 milliliter micro centrifuge tube centrifuge. Centrifuge at 160 times G for four minutes.
Resuspend the obtained cell pellet in 75 microliters of ice cold PBS or serum free media and add 75 microliters of the prepared for micromolar universal anchor and adapter solution. Mix thoroughly and incubate for five minutes on ice, add 15 microliters of the universal co-anchor solution to the tube and mix thoroughly, then incubate the sample for five minutes on ice. To remove excess oligos from the cell suspension, add one milliliter of ice cold PBS or serum free media to the tube and mix with a pipette.
Pellet the cells by centrifuging at 160 times G for four minutes at four degrees Celsius, then discard the supernatant. Repeat this step two more times. Resuspend the cells in ice col PBS or serum free media to create a cell dense solution of at least 25 million cells per milliliter, slightly tilt the pattern slide, then add 25 microliters of this cell suspension to the inlet of each flow cell.
Remove the PBS and 1%BSA solution from the outlet, allowing the cell suspension to fill the PDMs flow cell. Incubate on ice or at room temperature for 30 seconds, aspirate five microliters of the cell suspension from the outlet of the slide and add it back into the inlet. Repeat this 10 times per flow cell.
Gently pipette PBS or serum free media into the inlet of each flow cell to wash out the excess cells and collect the cell suspension from the outlet. Repeat this two to four times until no excess cells remain on the slide. For 3d culture, prepare a hydro gel precursor solution containing 2%DNAs and add 50 microliters of it to the inlet of each flow cell.
Aspirate the excess fluid from the outlet, driving the hydrogel solution into the flow cell. Incubate the slide at 37 degrees Celsius for 30 to 45 minutes to allow the hydrogels to set and to cleave the DNA based adhesion between the cells and the surface. Add 50 microliters of hydro gel precursor to a well of a two well chamber slide or a six well plate.
Pipette 10 microliters of PBS on either side of each flow cell. Distribute it along the full length of the flow cell using a razorblade or find point tweezers and gently lift the sides of the flow cells so that the PBS rushes under the hydro gel. Using a razorblade, move the flow cell to the edge of the slide by inverting the slide and nudge the flow cell off the slide so that it lands on top of the razorblade.
Pick the flow cell off the razorblade using curved forceps. Invert the flow cells so that the cells are on the bottom and place them on top of the droplet of hydro gel precursor solution. Incubate for at least 30 minutes so that the hydrogel containing the pattern cells can bind to the hydrogel underlay resulting in the full embedding of the pattern cells.
Remove the flow cell and immerse it completely in media. Gently nudge the flow cell using curved forceps until it pops off and floats into the media, then discard it. The quantification of the DNA spot adhesion to CMO labeled cells, which increases.
as a function of CMO concentration is represented as the mean and standard deviation from three experiments. The DNA patterns are shown in magenta and the adhered CMO labeled cells in cyan at different concentrations of CMO. A comparison of CMO labeled huvecs and LMO labeled huvecs adhered to a linear DNA pattern is shown here.
Single MDCKs pattern VSC MOD pack, and transferred into maitre gel were able to proliferate and polarize after five days of culture. Multilayered multicellular aggregates were created by alternating layers of cells labeled with complimentary CMOs. Multiple unique cell populations can be patterned together with high precision and without cross-contamination.
When attempting this protocol, it is critical to have a dense cell suspension while adding the cells to the slide in order to maximize opportunities for the cells to stick to DNA spots.
