This method can help answer key questions, such as how cells behave in a 3D microenvironment, cell-cell and cell-matrix interaction, how they proliferate, migrate, differentiate, and form functional tissues. This technique is advantageous, as it creates a cellular microenvironment closer to physiological conditions. Demonstrating the procedure will be Nausika Betriu, a graduate student from my laboratory.

The implications of the method extends toward therapy of injured tissues, as it may be used to obtain tissue equivalents for reparative or regenerative medicine. The method can also be applied to other systems, such as tumor models, diabetes, or neurodegenerative diseases. To begin, prepare 500 microliters of a 0.5%solution of peptide RAD16-I in 20%weight by volume sucrose in a microcentrifuge tube.

Sonicate the solution in an ultrasonic bath at maximum power for 20 minutes. Next, dilute 300 microliters of the peptide solution prepared previously with 200 microliters of 10%sucrose to obtain 500 microliters of 0.3%solution of peptide RAD16-I. Then, place the microcentrifuge tube containing the peptide solution into an ultrasonic bath at room temperature for 20 minutes.

Next, centrifuge the previously prepared cell suspension. Then, using a Pasteur pipette connected to vacuum line, remove the supernatant. Add two milliliters of 10%sucrose to the cell pellet.

Then, use a manual cell counter to count the cells. After resuspending the cells, centrifuge the cell suspension at room temperature for five minutes. Remove the supernatant, and suspend the cell pellet in the necessary volume of 10%sucrose to obtain the desired concentration of four million cells per milliliter.

Next, sonicate the previously prepared peptide solution in the ultrasonic bath at maximum power for five minutes at room temperature. Place a tissue culture insert in each well of a six-well plate. Next, mix 120 microliters of RAD16-I solution with an equal volume of cell suspension into a microcentrifuge tube.

Mix slowly, avoiding bubble formation, by pipetting up and down. An important factor to take into consideration is mixing cells with peptide solution. It is crucial to be gentle during mixing, slowly, to avoid bubble formation, by pipetting up and down.

Then, using a micropipette, load 80 microliters of the RAD16-I and cell suspension mixture into each insert. Loading 80 microliters of the RAD16-I and cell suspension mixture into each insert should be performed rapidly. Add 500 microliters of media to the bottom of each well to wet the insert membrane.

Allow the peptide to gel for 20 minutes in the laminar flow cabinet. Adding 500 microliters of media to the bottom of each well to wet the insert membrane will promote gel formation and 3D culture equilibration with media. After 20 minutes, add 40 microliters of medium to the insert very slowly, and let it slide down the inner wall to the gel.

Then, incubate the plate at 37 degrees Celsius for 20 minutes. After the incubation is complete, add 60 microliters of medium to the inner wall of the insert, and allow it to flow down the wall. Next, aspirate the sucrose-rich medium out of the well, and add 500 microliters of fresh medium.

Then, add 60 microliters of medium to the insert. Incubate the plate at 37 degrees Celsius for 10 minutes. Next, aspirate the medium out of the well.

Add 2.5 milliliters of fresh medium to the well and 200 microliters of medium into the insert. Incubate the plate in an incubator at 37 degrees Celsius. To perform the cell viability assay, prepare a solution of two micromolar calcein AM and two micromolar ethidium homodimer-1 in PBS in a 15-milliliter tube.

Vortex for 10 seconds, and cover the tube with aluminum foil. Next, using a micropipette, rinse the 3D-SAPS samples with two milliliters of PBS three times. Add the calcein/ethidium homodimer solution over the samples.

Then, cover the samples with aluminum foil, and incubate at room temperature for 40 minutes. After the incubation is complete, rinse the constructs with two milliliters of PBS five times. Finally, visualize the samples under a fluorescent microscope using 20X magnification.

In this protocol, a simple and fast method to culture cells in 3D using SAPS is described. The cell viability assay after 3D construct formation depicts few dead cells, as illustrated by cells stained red after five days of encapsulation. After four weeks of culture, almost no dead cells are observed.

Molecular markers for chondrogenesis and hypertrophy are analyzed by western blot. The collagen type one band is observed in both 2D and 3D systems. However, the lower band representing the processed, mature collagen protein is observed only in the 3D system, indicating differentiation in 3D proceeds in a more physiological way.

Additionally, collagen type two is detected only in 3D constructs. Collagen type 10 is detected in both 2D and 3D constructs, which is indicative of some degree of hypertrophy after four weeks of culture. Once mastered, this technique can be done in two hours if performed properly.

Following this procedure, other methods like collagen type one three-dimensional cell encapsulation can be performed in order to answer additional questions, like cell behavior in a natural degradable scaffold. After watching this video, you should have a good understanding of how to obtain your three-dimensional culture of your desired cell type. Don't forget that working with human cancer cells can be hazardous and precautions, such as the use of biosafety class two facilities and work knowledge of working in sterile conditions, should always be taken while performing this procedure.