Method for culturing autologous peripheral blood lymphocytes

Abstract

The invention relates to a method for culturing autologous peripheral blood lymphocytes, comprising the following steps of: (1) separating a PBMC (Peripheral Blood Mononuclear Cell) from peripheral blood, then heavily suspending in a serum-free culture medium, and statically culturing to prepare a primary culture solution; (2) replenishing the serum-free culture medium to the primary culture solution, simultaneously adding IL (Interleukin)-2, and statically culturing to prepare a secondary culture solution; (3) replenishing the serum-free culture medium to the secondary culture solution, simultaneously adding IL-2, and statically culturing to prepare a culture solution; and (4) uniformly dividing the culture solution into two parts, adding the serum-free culture medium to complement a sufficient volume, adding IL-2, statically culturing, and repeating the step (4) to prepare the autologous peripheral blood lymphocytes. As multiple monoclonal antibodies and cell factors are added to the serum-free culture medium, the activation efficiency and the amplification efficiency of effector cell masses are improved. Through detection, the activation efficiency and the amplification efficiency are obviously improved as compared with the existing method, and a large amount of cell masses occur after the effector cell masses are activated about 3 days.

Description

technical field

[0001] The invention relates to a method for culturing autologous peripheral blood lymphocytes (Autologous immunotherapy lymphocytes, AIL), which belongs to the technical field of in vitro culture of immune cells. Background technique

[0002] Adoptive immunotherapy is to infuse sensitized lymphocytes (with specific immunity) or their products to people with low cellular immunity (such as tumor patients) to obtain anti-tumor immunity. It is an immune system used to treat tumors. therapy. Based on the close relationship between the occurrence, development and prognosis of tumors and the immune function of the body, especially the cellular immune function, in 1985, the National Cancer Research Committee of the United States established cancer immunotherapy as the fourth treatment after surgery, radiotherapy and chemotherapy. therapy. AIL cells are a member of adoptive immunotherapy. Compared with traditional methods, AIL cells are widely used clinically due ...

Examples

Embodiment 1

[0072] A method for cultivating autologous peripheral blood lymphocytes, comprising the steps of:

[0073] (1) After separating PBMC from 50-100mL peripheral blood, resuspend in 50mL serum-free medium, the cell concentration of PBMC is 2×10 6 cells / mL, placed at 37°C, 7.5% CO by volume 2 , 100% relative humidity of CO 2 Static culture in the incubator for 72 hours to obtain the primary culture solution;

[0074] (2) Add serum-free medium to 200 mL of the initial culture medium prepared in step (1), and add IL-2 at the same time, so that the concentration of IL-2 is 1×10 3 U / mL, placed at 37°C, 7.5% CO by volume 2 , 100% relative humidity of CO 2 Stand and cultivate in the incubator for 96 hours to obtain the secondary culture solution;

[0075] (3) Transfer the secondary culture solution prepared in step (2) to a 1.8L cell culture bag, add serum-free medium to 1000mL, and add IL-2 at the same time, so that the concentration of IL-2 is 1×10 3 U / mL, placed at 37°C, 7.5% CO...

Embodiment 2

[0093] The method described in Example 1, the difference is that every milliliter of serum-free medium contains the following components:

[0094] IFN-γ 1500U, PHA-p 750ng, IL-1α 150U, CD3mAb 75ng, CD28mAb 75ng.

[0095] Experimental results

[0096] The detection process is as described in Example 1. After testing,

[0097] 1. Determination of cell proliferation multiple

[0098] The results showed that the expansion factor of AIL cells gradually increased over time from 1 to 21 days of culture, basically showed a linear trend from 1 to 5 days, and the expansion efficiency remained constant. The reasons for the amplification factor basically showed a linear increase, and the amplification efficiency increased on the 18th to 21st day, and reached the maximum value on the 21st day of cultivation. Compared with Example 1, the maximum amplification factor was increased by 1 times, and the results were as follows: figure 2 shown.

[0099] 2. Cytotoxicity experiment

[0100]...

Embodiment 3

[0104] The method described in Example 1, the difference is that every milliliter of serum-free medium contains the following components:

[0105] IFN-γ 2000U, PHA-p 1000ng, IL-1α 200U, CD3mAb 100ng, CD28mAb 100ng.

[0106] Experimental results

[0107] The detection process is as described in Example 1. After testing,

[0108] 1. Determination of cell proliferation multiple

[0109] The results showed that the amplification factor of AIL gradually increased over time from 1 to 21 days of culture, basically showed a linear trend on 1 to 5 days, and the expansion efficiency remained constant. The reason is that the amplification factor basically rises linearly, and the amplification efficiency increases linearly from 18 to 21 days, and reaches the maximum value on the 21st day of cultivation. Compared with Example 2, the maximum amplification factor increases by about 1 / 3, The result is as image 3 shown.

[0110] 2. Cytotoxicity experiment

[0111] The results showed th...

technical field

[0001] The invention relates to a method for culturing autologous peripheral blood lymphocytes (Autologous immunotherapy lymphocytes, AIL), which belongs to the technical field of in vitro culture of immune cells. Background technique

[0002] Adoptive immunotherapy is to infuse sensitized lymphocytes (with specific immunity) or their products to people with low cellular immunity (such as tumor patients) to obtain anti-tumor immunity. It is an immune system used to treat tumors. therapy. Based on the close relationship between the occurrence, development and prognosis of tumors and the immune function of the body, especially the cellular immune function, in 1985, the National Cancer Research Committee of the United States established cancer immunotherapy as the fourth treatment after surgery, radiotherapy and chemotherapy. therapy. AIL cells are a member of adoptive immunotherapy. Compared with traditional methods, AIL cells are widely used clinically due ...

Examples

Embodiment 1

[0072] A method for cultivating autologous peripheral blood lymphocytes, comprising the steps of:

[0073] (1) After separating PBMC from 50-100mL peripheral blood, resuspend in 50mL serum-free medium, the cell concentration of PBMC is 2×10 6 cells / mL, placed at 37°C, 7.5% CO by volume 2 , 100% relative humidity of CO 2 Static culture in the incubator for 72 hours to obtain the primary culture solution;

[0074] (2) Add serum-free medium to 200 mL of the initial culture medium prepared in step (1), and add IL-2 at the same time, so that the concentration of IL-2 is 1×10 3 U / mL, placed at 37°C, 7.5% CO by volume 2 , 100% relative humidity of CO 2 Stand and cultivate in the incubator for 96 hours to obtain the secondary culture solution;

[0075] (3) Transfer the secondary culture solution prepared in step (2) to a 1.8L cell culture bag, add serum-free medium to 1000mL, and add IL-2 at the same time, so that the concentration of IL-2 is 1×10 3 U / mL, placed at 37°C, 7.5% CO...

Embodiment 2

[0093] The method described in Example 1, the difference is that every milliliter of serum-free medium contains the following components:

[0094] IFN-γ 1500U, PHA-p 750ng, IL-1α 150U, CD3mAb 75ng, CD28mAb 75ng.

[0095] Experimental results

[0096] The detection process is as described in Example 1. After testing,

[0097] 1. Determination of cell proliferation multiple

[0098] The results showed that the expansion factor of AIL cells gradually increased over time from 1 to 21 days of culture, basically showed a linear trend from 1 to 5 days, and the expansion efficiency remained constant. The reasons for the amplification factor basically showed a linear increase, and the amplification efficiency increased on the 18th to 21st day, and reached the maximum value on the 21st day of cultivation. Compared with Example 1, the maximum amplification factor was increased by 1 times, and the results were as follows: figure 2 shown.

[0099] 2. Cytotoxicity experiment

[0100]...

Embodiment 3

[0104] The method described in Example 1, the difference is that every milliliter of serum-free medium contains the following components:

[0105] IFN-γ 2000U, PHA-p 1000ng, IL-1α 200U, CD3mAb 100ng, CD28mAb 100ng.

[0106] Experimental results

[0107] The detection process is as described in Example 1. After testing,

[0108] 1. Determination of cell proliferation multiple

[0109] The results showed that the amplification factor of AIL gradually increased over time from 1 to 21 days of culture, basically showed a linear trend on 1 to 5 days, and the expansion efficiency remained constant. The reason is that the amplification factor basically rises linearly, and the amplification efficiency increases linearly from 18 to 21 days, and reaches the maximum value on the 21st day of cultivation. Compared with Example 2, the maximum amplification factor increases by about 1 / 3, The result is as image 3 shown.

[0110] 2. Cytotoxicity experiment

[0111] The results showed th...

Images