A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

The aim of this experiment is to generate 3D tissue like steroids. This is accomplished by first harvesting cultured cells and then generating a single cell suspension. Next drops of the cell suspension, a deposited onto the underside of the inverted lid from a tissue culture dish.

Then the lid is inverted onto the hydration chamber. The final step is to incubate the hanging drops for 24 to 48 hours to allow aggregates to form. These can then be transferred to a shaking water bath and incubated for up to five more days.

Ultimately, results can be obtained that show differences in aggregate cohesion or cell sorting through compaction assays tissue surface 10 symmetry or epi fluorescence microscopy. The main advantage of this technique over existing methods like conventional 2D tissue culture, for example, is that cells grown in 3D more closely mimic cellular interactions experienced by cells in a tissue environment. I'll be demonstrating this method to show you that it's so simple that even a PI can do it.

To begin this experiment, harvest adherence cells at 90%co fluency. First, aspirate the culture media and rinse the cell sheet twice with PBS. Then aspirate the PBS and pipette two milliliters of 0.05%trips in EDTA solution onto the monolayer layer.

Place the plate into the 37 degree Celsius tissue culture incubator and incubate until cells detach. Next, remove the plate from the incubator and halt ization. Adding two milliliters of complete medium.

Use a five milliliter pipette to tritrate the mixture until the cells are fully in suspension. Then transfer the cell suspension into a 15 milliliter conical tube. Pipette 40 microliters of a 10 milligrams per milliliter, DNA solution into the cell suspension, and incubate it room temperature for five minutes following incubation.

Vortex the cell suspension briefly and then centrifuge at 200 times gravity for five minutes. Once the centrifugation is complete, aspirate the S supernatant and discard, then resuspend the palette in one milliliter of complete medium to wash. Repeat the centrifugation and Reese from the pellet in one milliliter of complete medium.

Next, perform a cell count using an automated cell counter or hemo cytometer. Adjust the cell suspension with media to a final concentration of 2.5 times 10 to the six cells per milliliter. Then proceed to the formation of hanging drops.

Prepare a hydration chamber by removing the lid from a 60 millimeter tissue culture dish and pipetting five milliliters of PBS into the bottom of the dish. Invert the lid of the plate and pipette up to 2010 microliter drops of cell suspension onto the underside of the lid. Ensure that the drops are placed sufficiently apart so as not to touch.

Gently replace the lid containing the drop cultures back onto the bottom of the PBS containing dish. Place the culture plate into a tissue culture incubator. Set at 37 degrees Celsius, 5%carbon dioxide and 95%humidity and incubate overnight.

Monitor the drops daily and continue the incubation until either cell sheets or aggregates have formed. Once cell sheets or aggregates have formed, transfer them into round bottom glass shaker flasks containing three milliliters of complete medium. Incubate the flasks containing the cell sheets or aggregates in a shaking water bath.

Maintained at 37 degrees Celsius and 5%carbon dioxide for around 24 hours until the formation of steroid can be visualized under a dissecting microscope. Once steroids have been formed, the effect of experimental manipulations on steroid formation may be quantified according to the procedure outlined in the written protocol. Also, the membrane intercalation of any previously included fluorescent dyes may now be visualized using fluorescence microscopy.

This brightfield microscope image demonstrates the aggregation of rat prostate cancer MLL cells after 18 hours of incubation using the hanging drop method. This image shows a second aggregate of MLL cells formed after 18 hours of culture using the hanging drop method. These cells were cultured in the presence of 25 micromolar of the MEK inhibitor PD 9 8 0 5 9 resulting in a substantially more compact aggregate of cells.

Here, the difference between MEK inhibitor treated MLL hanging drop culture, aggregate size and untreated control aggregate size is demonstrated. The asterisk represents a statistically significant difference of P less than 0.0001. As determined by students T-test between untreated and MEK inhibitor treated aggregates and suggests that MEK inhibitor treatment results in significantly smaller and more compact aggregates.

This image was captured using a Nikon Smz 800 stereo microscope and illustrates a steroid formed by incubating chick embryonic liver cells in hanging drop culture for 18 hours. Then in a 37 degrees Celsius shaking water bath for 48 hours. This confocal microscope image demonstrates the sorting out behavior that occurs when certain cell populations are co cultured using the hanging drop method before proceeding to hanging drop culture chick embryonic liver cells were stained with pkh two membrane inter coating dye and chick embryonic.

Heart cells were stained with PKH 26. The cell suspensions were then mixed and hanging drop. Culture was performed for 18 hours followed by 48 hours of incubation in the 37 degrees Celsius shaking water bath.

It can be seen from the optical section that the heart cells colored yellow are enveloped by liver cells. Pseudo colored blue here, two nardi and transfected L cell clones expressing NCA at their surfaces. At a ratio of 2.4 to one, were stained with the fluorescent membrane into collating dyes, PKH two and PK KH 26 mixed in equal proportions and cultured as hanging drops as before.

In this example, the high-end cadian expressing cells, which are pseudo colored green are enveloped by those expressing lower levels of en cadian, which are pseudo colored red. Once mastered, this technique can be done in 30 minutes or less if it's performed properly.