Medium for culturing immune cells isolated from umbilical cord blood and its culturing method
A technology of immune cells and culture medium, applied in the biological field, can solve the problems of high cost, cell growth rate, cell activity, cell killing ability, etc., and achieve the effects of avoiding quality changes, reducing costs, and strong killing ability
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0044] The formulation of the GR medium for cultivating immune cells isolated from umbilical cord blood provided in this example is:
[0045] The basal medium is CTS AIM-V SFM medium containing 20% volume concentration of KSR (Knockout serum replacement) serum substitute, in which the following five cytokines are added:
[0046] The mass ratio of OKT-3 to medium is 1:5000;
[0047] IL-2 1000 IU / mL;
[0048] IL-1α 1000 IU / mL;
[0049] IL-15 1000 IU / mL;
[0050] IFN-γ 1000 IU / mL.
Embodiment 2
[0064] This embodiment provides a method for culturing immune cells induced by cytokines, so as to obtain a new type of immunocompetent cells. Specifically, it is cultivated and obtained by the following methods:
[0065] (1) Isolation of cord blood mononuclear cells:
[0066] ①In a 15 mL centrifuge tube pre-added with 3 mL Ficoll lymphocyte separation medium, combine a 1 mL syringe needle with a 10 mL syringe, slowly add 5 mL of cord blood into the above centrifuge tube (inject along the wall, keep stratification) 400 g, L Speed 9, speed down 0, centrifuge for 30 minutes; use a 3 mL sterile dropper to remove the upper layer of plasma from the centrifuge, and transfer it to a 50 mL centrifuge tube (all plasma is combined into the same centrifuge tube);
[0067] ②Use a 3 mL sterile dropper to take the buffy coat and its upper layer of residual plasma and a small amount of Ficoll lymphocyte separation fluid in the lower layer, and transfer them to a clean 50 mL centrifuge tub...
Embodiment 3
[0071] Embodiment 3 Comparison of three kinds of medium culture effects
[0072] (1) Comparison of cell growth
[0073] Use GR, G, and 505 medium to culture immune cells according to the method in Example 2, and do three repeated experiments with three cord blood to verify whether the effect of the three mediums is reproducible, and exclude other conditions. interference. The trypan blue activity was determined when each experiment was carried out to the 13th day, and the data of the second experiment were taken as an example. The three culture media were operated in parallel, and the initial cell concentration was 1×10 6 / mL.
[0074] Immune cell activity detection method: Take an appropriate amount of immune cell fluid cultured to d13, blow and beat the agglomerate, take 100 μL, mix well with 100 μL trypan blue dye, place it on a glass slide, and examine it under a microscope (×100 ) to observe, take pictures, and estimate cell viability.
[0075] The cells were counted...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More