The overall goal of this procedure is to print three T three fibroblasts onto a serum coated surface. This is accomplished by first pre adsorb serum onto a tissue tissue culture surface. The second step is to print a suspension of fibroblasts onto the surface using a continuous flow micro spotter, followed by incubation of the cells at 37 degrees Celsius for two hours.
Next, printed cells as well as cells seeded through standard cell culture are stained with propidium iodide and visualized under an inverted microscope for comparison. Ultimately, fluorescence microscopy is used to assess cell viability, density, and morphology at relevant time points. The main advantage of this technique over existing methods like pin printing, is that the printhead forms a seal over the surface, allowing cells to be printed while submerged in cell media.
While the submerged printing of cells is useful for screening new drug candidates for safety and efficacy, submerged printing in general is also useful for other applications. For example, we can analyze new drug candidates against tissue slices, or we can use it as a model for organ systems. For example, a liver profusion model.
Generally, individuals new to this method will struggle because during printer priming and cell printing, air cannot enter the lines or it can cause cell death. To begin thaw NIH three T three cells for two to three minutes in a shaking water bath at 37 degrees Celsius. Re suspend the cells in five milliliters of complete media and centrifuge at 1500 Gs for three minutes.
Remove the cell supernatant without disturbing the cell pellet. Then re suspend the cells in five milliliters of media and transfer 10 microliters of the cell suspension to a 0.5 milliliter micro centrifuge tube. Next, add 10 microliters of triam blue to the cell suspension and stir with the tip of the pipette pipette 10 microliters of the stained cells into a hemo cytometer chamber.
Before counting the cells at 10 x magnification, count the live cells which resemble small white balls for four of the nine large squares. Only count the live cells that do not appear dark blue from the trippen blue stain. Calculate the average number of cells per large square, resulting in the number of cells times 10, 000 cells per milliliter in the original cell suspension.
Then seed cells at a density of 100, 000 cells per milliliter with a total of five milliliters in each T 25 flask. Culture the cells to between 70 and 80%co fluency before beginning experiments. To prepare the printing surface, use a marker to mark a rectangle the size of the print head on the bottom of a tissue culture treated polystyrene 12.
Well plate pipette 50 microliters of fetal bovine serum into the rectangular area and spread it over the entire region. With the tip, leave the well plate in a sterile biosafety hood overnight to allow the serum spot to dry completely. To begin submerged printing, rinse the print head out with distilled water.
Then fill a 60 millimeter Petri dish with distilled water and dock the print head onto the surface flow distilled water through each of the lines for two minutes at 150 microliters per minute using a pneumatic pump. Then discard the water from the Petri dish and fill it with clean water. Next, move the print head up and down in the water three times.
Dock the print head center over the serum coated spot in the pre-prepared 12 well plate. Then prime the print head with Prewarm. Complete media at 300 microliters per channel.
If performing multiple prints, add a bleach rinse in addition to the water rinse between prints. Proceed to print the suspended cells at a concentration of 50, 000 cells per milliliter onto the surface, and at 60 microliters per minute. 100 microliters per channel.
After cell printing, place the print head left docked against the surface and the manifold in a cell culture incubator. Set to 5%carbon dioxide and 37 to degrees Celsius for two hours. After the two hour incubation, remove the print head and place the lid on the culture dish.
The time at which the print head is removed is considered the zero hour time point. For comparison, standard cell culture is also performed. Add one milliliter of prewarm media to one of the serum spotted 12 well plates, then take one milliliter of the remaining cell suspension and pipette it into a single well of the 12 well plate.
This will produce a culture containing 50, 000 cells in the dish. Place the cell culture plate in an incubator at 37 degrees Celsius for two hours. After the two hour incubation, begin timing for consistency between printed and seeding samples.
This is considered the zero hour time point. After 0 2, 24 and 48 hours, prepare cells from each of the two methods for imaging by staining with propidium iodide, which is only incorporated into the nucleus of dead cells. First, gently rinse the cells three times with one milliliter of phosphate puffed saline with calcium and magnesium to remove any debris.
Next, add one milliliter of the prewarm culture media to each culture vessel, and then one microliter of propidium iodide. Stock solution incubate the cells stained with propidium iodide at 37 degrees Celsius for 10 minutes before imaging the cells using an inverted microscope at 10 x and 40 x magnification. Finally discard the cells in an appropriate biohazard container.
The fibroblast cell line, NIH three T three cells were printed or seeded onto a submerged surface. After printing and seeding, the cells were visualized to assess density and morphology at relevant time points. The images showed that the cells possess almost identical phenotypes at each time point and at each magnification.
Based on these figures, the effective printing was determined to be minimal. The cell viability was assessed using a propidium iodide stain, and revealed that the viability for the printed cells was not significantly different than the seated cells for each time point. The major difference between the two methods of attaching the cells to the surface is the density of the cells.
Printed cell density decreases at the two hour time point by over 50%in both seated and printed cells. Further study is warranted to determine the cause of this decrease. However, overall printed density ratios when compared to seeded remain significantly higher.
The printed cell density in the flow cell was approximately 10 times as dense at each time point as the seeded cells considering cells were printed at the same density as seeding, but in a smaller area. At the final time point, the cells are at the same density, which is likely due to cell motility and resource consumption limitations. Since its development, this technique has paved the way for researchers in the areas of drug discovery to study the effects of drug toxicity and efficacy on cells and cancer aids, heart disease, and other high demand fields.
By using submerged cell printing in conjunction with other technologies such as adherence, programmable cell adhesion, we can extend the reach of the technique to suspension cells for expanded drug discovery and development screening. After watching this video, you should have a solid understanding of how to print cells using our submerged printing technique.
