Reagent kit and method for cultivating immune cells and application thereof
A technology of immune cells and culture methods, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of lack of anti-tumor cell group synergy, limited effect, etc., to improve systemic immune suppression and prolong survival long-term, good synergistic effect
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Embodiment 1
[0042] A kit for culturing immune cells, which includes culture medium A and coating fluid.
[0043] Among them, the culture medium A is prepared according to the following method: add rhIFN-γ, rhIL-2, rhIL-15 and human transferrin into the serum-free lymphocyte culture medium and mix well, wherein, the serum-free lymphocyte culture medium used Specifically, KBM561 medium (TaKaRa company, Japan); rhIFN-γ (trade name: Clone Gamma), rhIL-2 (Beijing Shuanglu Pharmaceutical Co., Ltd.), rhIL-15 and human transferrin (Sigma company, U.S.A.) The final concentrations in culture solution A were 1000U / mL, 1000U / mL, 5ng / mL, 5ug / ml respectively.
[0044] The coating solution was prepared as follows: OKT3 antibody and Retronectin were added to PBS buffer and mixed evenly. Among them, the final concentrations of OKT3 antibody (Janssen Pharmaceutical Company, Japan) and Retronectin (TaKaRa Company, Japan) in the coating solution were both 100 ng / mL.
Embodiment 2
[0046] A method for culturing immune cells, carried out according to the following steps:
[0047] 1) Mononuclear cells (PBMC) were isolated from the peripheral blood of head and neck cancer patients who had not undergone chemotherapy by Ficoll–Hypaque density gradient centrifugation;
[0048] 2) Inoculate the mononuclear cells in step 1) into the culture flasks coated with the coating solution of Example 1, and culture them with activation medium, at 37°C, 5% CO 2 4 days in the cell culture box; Wherein, the activation medium used is to add the plasma (autologous plasma) of this head and neck tumor patient to the culture medium A of embodiment 1 to make, and this plasma is in this activation medium The mass percentage is 2%;
[0049] 3) Transfer the cells cultured in step 2) to a large culture bottle or a culture bag, and continue culturing with the culture solution A in Example 1. During the culture process, replace the culture solution A every 2 to 3 days.
[0050] 4) The...
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