Three-dimensional (3D) cell culture refers to the co-culture of carriers with different materials of three-dimensional structure and different kinds of cells in vitro, so that the cells can migrate and grow in the three-dimensional spatial structure of the carriers, constituting three-dimensional cell-carrier complexes. According to the different culture modes can be divided into static three-dimensional cell culture and dynamic three-dimensional cell culture, the commonly used three-dimensional cell culture modes include: matrix-covered culture, rotary flask culture, microcarrier culture, pre-positioned scaffold culture and rotary cell culture system, etc. The main technical route is: first of all, carry out the conventional two-dimensional culture of the cells, and when the cells grow up to the full monolayer, trypsin digestion to get the cultured cell suspension, and then A certain amount of cell suspension is added into the holes of artificial basement membrane matrix gel (matrigel) containing type I collagen or rich in laminin, and the cell growth solution is added to make the cells grow in this artificial microenvironment. The traditional flat cell culture method can only make the cell monolayer growth in a two-dimensional environment, can not produce the extracellular matrix barrier in vivo, while the three-dimensional cell culture technology through the simulation of cell growth in vivo physiological micro-environment, the use of a variety of scaffolds or equipment to promote the growth of cells and tissue differentiation, to produce a reasonable morphology and structure and functionality of the tissue cells, with the advantages of intuitive and controllable conditions of cell culture. Three-dimensional culture technology currently has a wide range of application prospects in the fields of stem cell differentiation, tumor research, regenerative medicine, tissue engineering, and high-throughput drug screening.
Principle
The basic principle of three-dimensional cell culture experiment is to have three-dimensional structure of different materials of the carrier and a variety of different types of cells in vitro co-culture, so that the cells can migrate and grow in the three-dimensional spatial structure of the carrier, constituting a three-dimensional cell and carrier complex.
According to the different culture modes can be divided into static three-dimensional cell culture and dynamic three-dimensional cell culture, the commonly used three-dimensional cell culture modes include: matrix-covered culture, rotary flask culture, microcarrier culture, pre-positioned scaffold culture and rotary cell culture system, etc. The main technical route is: first of all, carry out the conventional two-dimensional culture of the cells, and when the cells grow up to the full monolayer, trypsin digestion to get the cultured cell suspension, and then A certain amount of cell suspension is added into the holes of artificial basement membrane matrix gel (matrigel) containing type I collagen or laminin-rich proteins, and the cell growth solution is added to make the cells grow in this artificial microenvironment.
Operation method
Three-dimensional cell culture experiment
Materials and Instruments
Equipment: 48-well plate, thermostat incubator. Move The basic procedure of the 3-D cell culture experiment can be divided into the following steps: Caveat Some companies such as Microtissues have also developed a miniature mold, the 3D Petri dish, which is a natural three-dimensional cell culture environment without scaffolds to maximize cell-cell interactions.More than 40 cell types can now be grown in these three-dimensional dishes, offering a wide range of adaptations, as well as the advantages of autoclavability and reusability, which can save on culture costs. For more product details, please visit Aladdin Scientific website.
Reagents:
① Cell dilution solution (98 ml of complete culture medium and 2 ml of 3-D matrix gel (final concentration of 2%), vortex and mix, incubate at 37℃ for 30 minutes);
A. Routinely culture the cells required for 3-D culture, and then do the following after the number of cells is sufficient for 3-D culture.
B. Thaw the substrate components required for 3-D culture, i.e., 3-D culture medium, e.g., Matrix RGF BME, AlgiMatrix sponge.
C. Add 250μl of substrate to each well of a 48-well plate. Add 250 μl of substrate to each well of the 48-well plate and incubate at 37°C for 30 minutes to promote substrate gelation.
D. Add 98 ml of complete culture medium and 2 ml of 3-D substrate gel (final concentration of 2%) to a sterile vessel, vortex to mix, and incubate at 37°C for 30 minutes to serve as a cell dilution.
E. Harvest the cells and adjust the cell density to 1x104 cells/ml in 24 ml of cell dilution.
F. Add 500 μl of substrate per well of the plate.
F. Add 500 μl of cell suspension to each well of the plate.
G. Incubate the plate overnight at 37°C in a 5% CO2 cell culture incubator.
H. Observe the cell growth and structure formation under the microscope every day. L. Change the medium every 4 days. I. Repeat 3 times. Repeat 3 times.
I. When the cell space structure has grown to the appropriate size, then complete the appropriate cytological analysis.