This protocol uses a simple method for enhancing the reliability of a 3D cell culture model without the use of any special equipment. The main advantage of this technique is that we can conduct the repeatable 3D culture experiment by controlling the initial culture condition within the cubic device. After preparing a five by five millimeter polycarbonate cubic frame, place the frame on a pre cooled slide and an ice box and use a pipette to add 12 microliters of preheated 1.5%agarose from the top face of the cubic frame to the bottom surface.
Spread the agarose to obtain a flat surface and allow the polymer to cure. Use tweezers to slide the frame to the edge of the glass and rotate the frame so that the open side is facing down before placing the frame back onto the slide. After filling the next two surfaces of the frame with more agarose, as just demonstrated, drop the agarose into a container from an open face to form an agarose wall on the fourth and fifth sides.
To set up an initial cell cluster shape inject an appropriate extracellular matrix for the cell culture of interest into the hybrid gel cube culture space and set a fabricated micro mold onto the cube. Then place the cube in a carbon dioxide incubator for 25 minutes at 37 degrees Celsius. When the extracellular matrix is cured, carefully lift out the micro mold so that the matrix does not deteriorate.
A pocket in the desired mold shape will be fabricated in the extracellular matrix. To load the cells concentrate the experimental cell suspension after centrifugation in the appropriate experimental cell culture medium and inject the cells into the pocket within the extracellular matrix. Place the cube into the carbon dioxide incubator for 20 minutes at 37 degrees Celsius to allow the cells to fall into the extracellular matrix pocket, filling the space created by the micro mold.
At the end of the incubation, place the cube into one well of a 24 well plate and add 100 microliters of the appropriate cell culture medium to the well. Inject additional extracellular matrix to close the pocket and return the cube to the incubator for 25 minutes. When the extracellular matrix is cured drop approximately 10 microliters of 20 degree Celsius agarose onto the top surface to the cube to close the surface and cure the drop for 10 to 20 minutes at 37 degrees Celsius.
Then cover the entire cube with fresh medium to promote osmotic pressure for facilitating the transfer of nutrition to the cells within the cube. For noninvasive 3D shape recognition by multidirectional observation, place the plate onto a microscope stage and obtain images of each side of the cube by bright field or phase contrast microscopy, using tweezers to rotate the cube service to be imaged between captures. For immunofluorescent imaging by multidirectional observation, first aspirate the supernatant from the well containing the cube and fix the cube in 4%paraformaldehyde for 20 minutes at room temperature.
At the end of the fixation, wash the cube two times with PBS for 10 minutes per wash. Impermeabilize the cube with 0.5%triton x-100 and PBS for 10 minutes at four degrees Celsius. Next, wash the cube three times in PBS for 10 minutes per wash, before blocking any nonspecific bonding with goat serum and immunofluorescence buffer for 60 minutes at room temperature.
Then stain the cells with the appropriate primary antibody of interest according to standard antibody staining protocols. Then image all six sides of the cube on a laser or fluorescent microscope as just demonstrated. In this representative experiment multidirectional imaging of hybrid gel cubes, cultured with normal human bronchial epithelial cells, demonstrates the generation of an initial cylindrical or prism shaped cell culture by phase contrast and immunofluorescent imaging of the cultures.
Here immuno staining of normal human epithelial cells initially controlled to a cylindrical shape in the hybrid gel cube, after generation of the bronchial tree is shown. The branches demonstrate the perpendicular to the cylindrical axis, as revealed by multidimensional imaging. It is important to inject a high density of cells into the extracellular matrix pocket, as a low density of cells may result in deterioration of that cell quality after incubation.
This method pattern is the repeatable formation of a 3D cellular pattern with a quantitative measurement by multidirectional imaging to study the mechanism of dish development.
