Method of extracorporeal induction, proliferation and cryopreservation of immune cells
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An immune cell and cryopreservation technology, applied in the biological field, can solve the problems of culture system safety risks, natural killer cell culture and cryopreservation methods need to be improved, expansion multiples and poor tumor killing effect, etc.
Active Publication Date: 2017-04-26
广州沙艾生物科技有限公司
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However, there are already NK cell therapy products on the market, but the effect of cryopreservation and recovery is poor, the culture system used is complicated, the amplification multiple and tu
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[0076] Example 1:
[0077] Using the method for inducing expansion of immune cells in the embodiment of the present invention, the isolated mononuclear cells are induced and expanded into immune cells, and the activity of the immune cells is detected.
[0080] 1.1 Add 5 mL of 2.5 μg / mL anti-human CD16 monoclonal antibody dissolved in medical normal saline into a sterile culture bottle, shake the culture bottle gently to make the antibody cover the culture surface, and overnight at 4°C in the dark.
[0081] 1.2 Recover the antibody coating solution before use, wash the culture bottle once with 5 mL of normal saline, and then use 5 mL of T cell expansion medium (OpTmizer TM CTS TM T-cell expansion SFM) and washed once.
[0082] 2. Collect peripheral blood, separate peripheral blood plasma and mononuclear cells
[0083] 2.1 Collect about 100ml of human peripheral blood with a sterile blood c...
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[0125] Example 2
[0126] In this example, 6 kinds of cryopreservation solutions without plasma and 6 kinds of cryopreservation solutions containing plasma were used respectively to freeze the NK cells induced and expanded in vitro in Example 1, and compare the cryopreservation effects so as to observe the effect of plasma on Effects of cryopreservation of NK cells, and the possibility of reducing DMSO concentrations to reduce cytotoxicity. details as follows:
[0127] 1. Use plasma-free cryopreservation medium to freeze NK cells
[0128] 1.1. NK cell cryopreservation and recovery methods:
[0129] The NK cells obtained on the 10th day of in vitro induction and expansion in Example 1 were cryopreserved in six cryopreservation solutions containing HSA, and the components of the six cryopreservation solutions were as follows:
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Abstract
The invention discloses a method of extracorporeal induction, proliferation and cryopreservation of immune cells. The method comprises the steps of utilizing a CD16 antibody to cover a culture vessel to obtain the culture vessel after covering; utilizing the immune cells to activate a culture medium, and placing single cell nucleus in the culture vessel after covering to activate the culture medium through the immune cells to obtain preliminarily induced and proliferated immune cells; utilizing a immune cell proliferation culture medium to conduct second induction and proliferation culture on the preliminarily induced and proliferated immune cells to obtain differentiated immune cells; utilizing immune cell scale proliferation culture to conduct third induction proliferation culture on the differentiated immune cells to obtain large-scale immune cells with activated functions; utilizing immune cell cryopreservation liquid to cryopreserve the large-scale immune cells with the activated functions. The method of extracorporeal induction, proliferation and cryopreservation of the immune cells has the advantages of being high in induction efficiency, high in proliferation speed, high in safety, low in cost and the like. Moreover, the cryopreserved large-scale immune cells have long effective storing time and high cell recovery rate.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to a method for inducing expansion and cryopreservation of immune cells in vitro. Background technique [0002] In recent years, biological therapy has become the fifth largest treatment mode after surgery, radiotherapy and chemotherapy, and endocrine therapy, and has gradually received attention. Adoptive cellular immunotherapy (ACI) is one of the methods of cell biological therapy. It refers to the infusion of immune cells with anti-tumor activity to tumor patients, directly killing tumor cells or stimulating the body's immune response to kill tumor cells, so as to achieve the goal of treating tumors. Purpose. Currently, adoptive immune cells in clinical use include DC-CIK cells, TIL cells, LAK cells and NK cells, among which CIK cells, LAK cells and A-NK cells are all anti-cancer systems with natural killer cells as the main body. [0003] Natural killer cells (natural killer cells...
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