The overall goal of this procedure is to directly pattern cells in entirely aqueous environments. This is accomplished by first dissolving long chain polymers, such as polyethylene glycol and dextrin in separate tubes containing cell culture medium. The second step is to tryps anize, harvest pellet and resuspend cells at the desired seeding density in one or both polymer solutions.
Next, the polymer solutions are dispensed onto a cell culture dish. The incompatibility of the two polymers allow spatial patterning of the two fully aqueous liquids. Cells can be included in either polymer solution or in both polymer solutions to produce a variety of patterns.
The final step is to remove the polymer solutions once the cells have been allowed to adhere for four hours or longer and replace it with culture medium. Ultimately, brightfield or fluorescence microscopy can be used to confirm cell patterning. The main advantage of this technique over existing methods like micro contact printing, is that cells are patterned in an entirely aqueous environment with simple tools like micro pipe patterns.
This method can be used for tissue engineering to assess cell migration and to investigate cell to cell pericrine and autocrine signaling demonstrating the procedures will be a Abraham, an undergraduate student, and Joshua White, a graduate student from the Takayama lab. Begin by dissolving the two different polymers into the specific medium that will be used in your experiments. In this example, polyethylene glycol, also known as PEG and Dextrin, also known as Dex, have molecular weights of 35 kilodaltons and 500 kilodaltons respectively.
Prepare 16 two milliliter samples in 15 milliliter conical tubes, each with different concentrations of PEG and dex, similar to the chart shown here. To accomplish this, weigh out the appropriate amounts of PEG and dex according to the chart and mix them in the media by gently rocking them until both polymers are fully dissolved. This may take several hours after the polymers are fully dissolved.
The solution should be cloudy. Confirm phase separation by centrifuging the samples at 1000 Gs for five minutes. The denser bottom phase will be dex rich.
The top phase will be peg rich. Next, slowly add 0.1 milliliters of additional medium to the tubes and vortex the samples for 20 seconds. Then centrifuge the samples again to check for phase separation.
The solution is clear after additional volumes of medium have been added, repeat this step adding another 0.1 milliliter of medium mixing and centrifuging the samples until the solution becomes clear and no longer phase separates. This indicates that the critical point threshold has been reached for that specific mixture. Record the final weight of each tube at this point.
Finally, determine the percent weight per weight of the two polymers at the face Separation point. Plot the bin nodal curve, placing percent weight per weight peg on the y axis and percent weight per weight decks on the X axis. This curve is the threshold concentrations for face separation of PEG and dex in this medium.
To begin exclusion patterning of cells prepare separate solutions of PEG and dex at twice their critical point values as determined by the bin nodal threshold. For these experiments, 5%weight per weight PEG and 12.8%weight per weight decks are added to DMEM with 10%FBS and 1%antibiotics. The tubes are placed on a rocker to let them fully dissolve.
Next, harvest the cells to be used for exclusion patterning for this experiment, hela cells at 80%confluence are tryps inized using 0.5%tripsin for three minutes. Once harvested, count the number of cells using a hemo cytometer then pellet and resuspend the cells at 375, 000 cells per milliliter in medium with 5%PEG added. This concentration will produce samples that when cultured will become confluent the following day.
Next, using a micro pipetter dispense 0.5 microliter droplets of the 12.8%deck solution onto a dry 96 well plate for exclusion patterning. Dex droplets can be allowed to dry under sterile conditions for later use. Then dispense 200 microliters of PEG hela cell suspension into the well slowly.
To cover the D droplets, it is important to first cover the D droplet with the PEG solution before filling in the surrounding areas as this prevents it from disrupting the droplet when it spreads across the well. Once all the wells have been patterned and seeded carefully, place the cell culture plate into a humidified incubator. Make sure that the dish is not tilted during handling and that it is placed on a level incubator shelf to avoid disrupting the patterns after the cells have been allowed to adhere for 12 hours.
Remove the PEG solution and wash three times with 200 microliters of culture medium to remove any residual polymers. Then add 200 microliters of fresh culture medium and return the plate to the incubator. Monitor the cultures periodically to observe cell proliferation and migration into the exclusion zones To begin island patterning.
Prepare solutions of 5%weight per weight PEG and 12.8%weight per weight decks in cell culture medium. Also harvest the hela cells to be used for island patterning and again, determine the total number of cells available using a hemo cytometer. Next pellet and resus.
Spin the cells at 5 million cells per milliliter in medium. Continuing 12.8%dex, a higher concentration of cells may be needed to reach confluence in 24 hours if the cells do not adhere as well as heela cells. Next, pipette 200 microliters of PEG solution into each well of a 96 well plate.
Then slowly add a 0.5 microliter droplet of deck cell suspension in each well containing the PEG solution. The droplets will sink to the bottom and contact the culture surface. Then carefully transfer the plate into the incubator, taking care to not tilt the dish at any point, which may disrupt the patterns after 12 hours.
Remove the PEG solution and wash three times with 200 microliters of culture medium. Then add 200 microliters of fresh culture medium and return the plate to the incubator. Monitor the cultures periodically to observe cell proliferation and migration outwards from the islands.
Begin co-culture patterning by preparing solutions of 5%weight per weight PEG and 12.8%weight per weight decks in DMEM with 10%FPS and 1%Antibiotics also grow up the two cell types to be used in the exclusion and island patterning so they are near confluence. At the same time here, we will use Hep G two C3 a hepatocytes islands with NIH three T three fibroblasts surrounding them. Then harvest the two cell types and count them separately using a hemo cytometer.
These two cell types can be easily distinguished using brightfield microscopy pellet, the Hep G two C3 A hepatocytes for island patterning and resus spend in 12.8%decks. Then pellet the fibroblasts for exclusion, patterning and resus suspend in 5%peg. Next dispense 0.5 microliter droplets of D cell suspension onto a dry 96 well plate and cover the dex droplets gently with 200 microliters of peg cell suspension.
Then transfer the plate into an incubator after 12 hours. Remove the media and rinse three times with 200 microliters of culture medium. Then add 200 microliters of fresh culture medium and return the plate to the incubator.
Finally, monitor the co cultures albumin staining intensity can be used to assess the beneficial effects of fibroblast co culture. The polymer concentrations required to form a variety of aqueous two-phase systems are described here by the bin nodal curves at concentrations above the bin nodal curve. The system forms two phases below the bin nodal curve.
The solution is a transparent single phase two-phase systems of at least twice. The concentrations of those found on the binal curve are sufficient for use in the aqueous two-phase systems described in this video. The first example of their use is an exclusion patterning where small areas can be left void of cells.
Island patterning creates the exact opposite of exclusion patterning where small regions can be seated with cells. The combination of these two procedures can be utilized to form a unique co-culture where an island of cells can be surrounded by another cell type as shown here. Hela cells plated using exclusion patterning will proliferate and migrate to fill the void.
Over time. The size of the void can be monitored to provide valuable insights into cell migration and growth under varying conditions. Island patterning, on the other hand, can be used to measure cell outgrowth.
The combination of island and exclusion patterning produces well-defined regions of co-culture. Shown here are islands of hepatocytes surrounded by fibroblast cells. These colonies have been found to maintain their organization for at least four days in culture.
Using this system, multiple islands of cells can be grown on the same plate. When grown as islands alone, hepatocytes produced slightly less albumin than when grown in co-culture with fibroblasts. While attempting this procedure, it's important to remember to use concentrations of polymers at twice the critical concentration to form two-phase solutions.
After watching this video, you should have a good understanding of how to formulate an aqueous two-phase system and use it to pattern cells and define geometries.
