Cell hybridization and selection culture - Database & Sql Blog Articles

(1) Fusion: Before cell hybridization, spleen B cell suspension and mouse myeloma cells (such as SP2/0-Agl4 cell line) should be prepared separately. The spleen of the immunized mouse was disrupted under aseptic conditions, and the B cells were suspended in a serum-free medium (usually prepared using RPMI1640 commercial product), and the serum of the mice was removed 3 times. SP2/0 cells were cultured with 10% fetal bovine or calf serum, and fresh medium was replaced every day to make cells that grew vigorously during the logarithmic division. The cells were washed 2-3 times with RPMIl640, and the two cells were combined in the same tube, and 50% polyethylene glycol (relative molecular mass 1000-1500) was used as a fusion agent, and fused at 37 ° C for 1-2 min. . The cells were then slowly diluted with 1640 medium, then the PEG was removed and the cells were dispersed into HAT selection plates. The electrofusion method can also be used for the preparation of monoclonal antibodies. Although the fusion rate is high, the number of cells in one fusion is small, and special equipment is required, which limits its widespread use. The ratio of spleen cells to myeloma cells at the time of fusion was satisfactory at 5:1 to 10:1, and the number of cells per fusion was 10-10. The fused cells were cultured in HAT medium (RPMI1640 containing 10% to 20% fetal bovine or calf serum and HAT) on a 40- or 96-well plate at 37 ° C under 5% CO 2 . Only the hybridoma cells formed by splenocytes and myeloma cells in the cell suspension after fusion can grow in HAT medium, and other forms of fused cells can not grow. Unfused cells also failed to survive in HAT medium.

In the cell culture process after fusion, a feeder cell contributes to the growth of hybridoma cells. The feeder cells can be used as peritoneal cells or thoracic and abdominal cells of the same species. Phagocytes in the peritoneal cells remove dead cell debris. Make the background cleaner and "clean." At the same time, the cytokines or active substances secreted by the feeder cells contribute to the growth of the hybridoma cells. The existing commercial "hybridoma growth factor" can be used to replace feeder cells.

(2) Screening of positive hybridoma cells and single-gram degradation: After hybrid cells are cultured for about 10-14 days, available cell colonies (clone) are formed. The antibody activity was measured after several changes of the culture solution (HT medium). Commonly used screening methods are ELISA and agglutination tests. The former is often used for soluble antigens, and the latter is suitable for surface antigens such as cells and bacteria. In addition, Dot-ELISA, immunoblotting and immunofluorescence assays can be used for the screening of hybridoma cells.

The process of separating cells from which many cell clones are mixed and grown into individual cell clones. The most common monoclonalization method for colonization is limited dilution, that is, the mixed cells are diluted and then dispensed on a culture plate. So that only one cell appears in most of the wells of the plate. To ensure that the antibody secreting cells are derived from a single cell, the cloning process can be repeated, referred to as subclonization. In addition to the limiting dilution method, fluorescence activated cell sorting (FACS) is also used for the cloning process of hybridoma cells.