The protocols presented here have been optimized for 3D culture and allow for efficient and affordable investigation of processes associated with cancer cell growth, proliferation, and death in a 3D environment. The main advantages of using 3D culture is that they are a unique possibility for recapitulating key aspects of the tumor microenvironment in vitro. 3D spheroid's an important tool for cancer drug screening, which is increasingly being used to improve translation between in vitro and in vivo conditions in cancer therapy research.
Some of the protocols require a certain amount of practice and skill. This is in particular true for producing spheroids by the hand drop method, and for embedding spheroids for immunohistochemistry. As key elements of these procedures are difficult to describe in writing, such as how to insert spheroids into a drop of agarose, visual demonstration will help researchers perform these procedures.
Dilute the prepared cell suspension in a 15 milliliter tube to obtain the optimal cell density. Transfer the diluted cell suspension to a sterile reservoir, and use a multi-channel pipette to dispense 200 microliters into the previously prepared plate. Incubate at 37 degrees Celsius with 5%carbon dioxide and 95%humidity.
Every two to three days, acquire light microscopic images of the spheroids. After acquiring the images, remove 100 microliters of the spent medium from each well, and replace it with 100 microliters of fresh medium. First, thaw reconstituted basement membrane on ice.
Keep any individually-wrapped plates and reservoirs on ice before use. Next, dilute the prepared cell suspension in a 15-milliliter tube to obtain the optimal cell density. Place the tube containing the diluted cell suspension on ice.
Fill the plastic containers with ice and transfer them into the hood. Transfer the chilled plates and reservoirs to a cell culture hood. Place the plates and reservoirs back on ice.
Resuspend the rBM gently to ensure a homogenous gel. Add the optimal concentration of the rBM to the chilled cell suspensions. Invert the tube to ensure that the rBM and cell suspension are properly mixed.
Then, transfer this mixture to a sterile reservoir, and use a multichannel pipette to dispense 200 microliters to each well of a chilled, ultra-low-attachment, 96-well plate. Centrifuge the plate at 750 times g for 15 minutes to ensure that the cells are clustered together when the rBM hardens. Use the centrifuge soft descent or minimal braking function.
First, add six milliliters of PBS to a cell culture dish. Dilute the prepared cell suspension to obtain a suitable dilution. A practical dilution is 50, 000 cells per milliliter.
Pour the cell suspension into a sterile reservoir and use a multichannel pipette to carefully place up to 30 drops of suspension into the lid of the cell culture dish, resulting in a concentration of 2000 cells per drop. In a quick but controlled movement, invert the lid and place it on top of the cell culture dish containing PBS. Incubate at 37 degrees Celsius with 5%carbon dioxide and 95%humidity for four to six days.
If the spheroids are to be used for protein lysates or embedding, pull them by removing the lid, tilting it, and then washing down the drops with one milliliter of heated media. Transfer the resulting spheroid-containing medium to a 1.5 milliliter tube. Let the spheroid settle to the bottom of the tube before proceeding with protein lysates or embedding.
Cut the end of a P200 pipette tip to more easily capture the spheroids without disturbing their structure. For each condition, pull a minimum of 12 but ideally between 18 and 24 spheroids in a 1.5 milliliter tube. Place the tubes on ice and allow the spheroids to settle at the bottom.
Next, move from the sterile cell laboratory to the regular laboratory. Wash the spheroids twice in one milliliter of ice-cold one X PBS, making sure to let the spheroids settle before removing the PBS between each washing step. Then, aspirate as much PBS as possible without disturbing or removing the spheroids.
Add five microliters of heated lysis buffer with phosphatase and protease inhibitors per spheroid. Vortex the spheroids for 30 seconds and then perform a quick centrifugation for 10 seconds. Repeat these intervals of vortexing and centrifugation for five to 10 minutes, or until the spheroids are dissolved.
After preparing the PI solution, remove 100 microliters of medium from each well in the 96-well plate, making sure to not remove the spheroids. Add 100 microliters of heated one X PBS to each well, followed by removing 100 microliters of liquid from the wells. Repeat this washing step three times to wash out any remaining medium.
Next, add 100 microliters of the PI solution to each well. Cover the plate in aluminum foil and incubate for 37 degrees Celsius with 5%carbon dioxide and 95%humidity for 10 to 15 minutes. After this, repeat the washing process with heated one X PBS three times, as previously described, to wash out the PI solution and diminish the background signal when imaging.
Using an epifluorescence microscope, image the spheroids. Within the imaging software, open the multi-channel menu. Set the exposure time for the relevant channels, and press Read Settings after adjusting each channel.
Open the Z-stack menu and define the start and end by adjusting the image focus. Initially have the spheroid out of focus, then move the focal point through the entire spheroid until the image becomes blurry once again. Then adjust the step size to be between 18 to 35 micrometers, and press Start.
To begin, fix the spheroids in a fume hood on day one as outlined in the text protocol. On day two, place the agarose gel into a water-filled beaker in a microwave oven to heat it and keep the gel warm on a bench-top heating plate set to 60 degrees Celsius, until needed. Wash the spheroids twice in the fume hood using one milliliter of ice-cold one X PBS per wash.
Then, aspirate most of the PBS. Prepare a 20 microliter pipette tip by cutting it at an incline to obtain a pointier tip with a larger hole. After this, make an agarose gel drop on a microscope slide.
Place the slide on a warm heating block to prevent the agarose from solidifying. Using the modified pipette tip, catch as many spheroids as possible in a volume of 15 to 20 microliters. Carefully inject the spheroids into the center of the agarose gel drop, making sure to not touch the microscope slide.
Incubate at room temperature or at four degrees for five to 10 minutes to let the agarose gel drop harden. When the gel drop has solidified somewhat, but is still rather soft, use a scalpel to carefully push it from the microscope slide into a plastic tissue cassette. Transfer the tissue cassette to a beaker filled with ethanol and store at room temperature.
In this study, a series of methods are presented for the analysis of anti-cancer treatment-induced changes in cancer cell viability and death in 3D culture. The concentration of rBM added can profoundly affect the morphology of the spheroids. The addition of up to 2.5%rBM allows spheroid formation in SKBr-3 breast cancer cells, with no further effect at higher concentrations.
In contrast, BxPC-3 pancreatic cancer cells spontaneously form small compact spheroids. In this cell type, increasing the rBM to 1.5%or above elicits a distinct morphological change from spheroid to more convoluted structures with protrusions and invaginations. An example of the use of spheroid cultures for screening of chemotherapy efficacy is shown here.
Light microscope images are acquired every two to three days during a dose response experiment performed to determine the dose necessary for 50%reduced viability in MDA-MB-231 breast cancer cells. The spatial arrangement of dead cells upon an increasing concentration of an inhibitor can be visualized using PI staining. As seen, control spheroids show a limited necrotic, late apoptotic core, whereas the dead cells are distributed throughout the spheroid as the concentration of inhibitor is increased.
The spheroid technique shown here can be applied to many other assays, such as 3D invasion, migration, or microphoretic analysis. It is also applicable for co-cultures with fibroblast, adipocytes, or immune cells, which can provide important information about cellular interactions in the tumor microenvironment. The development of this technique has been very important in elucidating the role of the tumor microenvironment in numerous aspects of cancer development, including gene expression, invasion, and treatment assistance.
