Separation and efficient amplification culture method for antigen specific T lymphocyte

Abstract

The invention relates to a separation and efficient amplification culture method for an antigen specific T lymphocyte. By means of the immunomagnetic bead technology and the character of an active T cell expression CD137, the antigen specific T lymphocyte is separated from a complex cell mass; then an antigen specific T lymphocyte with high tumor killing activity is obtained through in-vitro efficient amplification culture. CD137L (CD137-ligand), IL-7, IL-15 and IL-2 are added in the culturing process, so that the phenomenon that activated and induced cells are liable to apoptosis in the culturing process is avoided, the amplification time is increased, and the killing activity of the antigen specific T lymphocyte is improved.

Description

technical field

[0001] The invention belongs to the technical field of cell culture, and relates to a method for separating and efficiently expanding and culturing antigen-specific T lymphocytes. Background technique

[0002] Dendritic cells (dendritic cells, DC) are the professional antigen-presenting cells (antigen process cells, APCs) with the strongest function known so far, and they play an important role in inducing the body's immune response. Immunotherapy using DCs loaded with tumor antigens as a vaccine can stimulate the body to generate a specific cytotoxic T cell immune response (cytotoxic T lymphocyte, CTL) against tumor antigens. Tumor vaccines can be divided into gene-modified dendritic cell vaccines, dendritic cell vaccines loaded with tumor antigen polypeptides, dendritic cell vaccines transfected with tumor antigen mRNA or RNA, and dendritic cell vaccines sensitized with complete tumor cell antigens. Currently, antigen-loaded DC vaccines have achieved good ...

Examples

Embodiment 1

[0050] Isolation and Efficient Expansion of Antigen-specific T Lymphocytes Induced by Tumor Antigen (Tumor Cell Lysate)

[0051] Step 1: Preparation of autologous tumor cell lysates

[0052] 1.1 Take the tumor tissue resected after the operation of the melanoma patient, remove the normal tissue on the tumor tissue, and rinse it with normal saline until there is no blood and no fat residue.

[0053] 1.2 Soak with 0.1% chlorhexidine (chlorhexidine) for 30 minutes, rinse with sterile saline 3 to 4 times.

[0054] 1.3 Shred the tumor tissue with sterile surgical scissors, add an appropriate amount of saline for grinding, and collect the single cell suspension after filtering through a 200-mesh mesh.

[0055] 1.4 Put the single-cell suspension into a centrifuge tube, freeze at -86°C, thaw at room temperature, repeat the freeze-thaw 3 times, and observe under the microscope until no cells survive.

[0056] 1.5 Centrifuge the single cell suspension at 3000rpm for 30min, filter the ...

Embodiment 2

[0097] Isolation and Efficient Expansion of Antigen-specific T Lymphocytes Induced by Tumor Antigen (Protein)

[0098] Step 1: The operation is the same as Step 2 of Example 1, except that the antigen added in 2.4 is not the tumor cell lysate, but the full-length protein of the tumor antigen MART-1.

[0099] Step 2: Same as Step 3 of Example 1.

[0100] Step 3: Same as Step 4 of Example 1.

[0101] Step 4: Same as Step 5 of Example 1.

[0102] Step 5: Same as Step 6 of Example 1.

Embodiment 3

[0104] Isolation and Efficient Expansion of Antigen-specific T Lymphocytes Induced by Tumor Antigen (Polypeptide)

[0105] Step 1: DC cell culture and antigen peptide loading

[0106] 1.1 Peripheral blood mononuclear cells were collected with an apheresis machine. After Ficoll separation, the cells in the middle layer were taken and washed twice with normal saline to obtain PBMCs (peripheral blood mononuclear cells).

[0107] 1.2 Take PBMC, add RPMI 1640 medium to make 5×10 6 / ml cell suspension, inoculate 3ml per well into a 6-well plate, and put it into an incubator.

[0108] After 1.32 hours, take it out, shake it to remove the supernatant cells, and leave the adherent mononuclear cells (slightly larger than other cells), add 3ml DC medium (IMP serum-free medium, containing 2% autologous plasma, 1000IU) to each well / ml GM-CSF, 1000IU / ml IL-4).

[0109] 1.4 On day 5, add DC maturation agent (final concentration 10ng / ml LPS, 50IU / ml IFN-r).

[0110] 1.5 On day 7, add MAR...

technical field

[0001] The invention belongs to the technical field of cell culture, and relates to a method for separating and efficiently expanding and culturing antigen-specific T lymphocytes. Background technique

[0002] Dendritic cells (dendritic cells, DC) are the professional antigen-presenting cells (antigen process cells, APCs) with the strongest function known so far, and they play an important role in inducing the body's immune response. Immunotherapy using DCs loaded with tumor antigens as a vaccine can stimulate the body to generate a specific cytotoxic T cell immune response (cytotoxic T lymphocyte, CTL) against tumor antigens. Tumor vaccines can be divided into gene-modified dendritic cell vaccines, dendritic cell vaccines loaded with tumor antigen polypeptides, dendritic cell vaccines transfected with tumor antigen mRNA or RNA, and dendritic cell vaccines sensitized with complete tumor cell antigens. Currently, antigen-loaded DC vaccines have achieved good ...

Examples

Embodiment 1

[0050] Isolation and Efficient Expansion of Antigen-specific T Lymphocytes Induced by Tumor Antigen (Tumor Cell Lysate)

[0051] Step 1: Preparation of autologous tumor cell lysates

[0052] 1.1 Take the tumor tissue resected after the operation of the melanoma patient, remove the normal tissue on the tumor tissue, and rinse it with normal saline until there is no blood and no fat residue.

[0053] 1.2 Soak with 0.1% chlorhexidine (chlorhexidine) for 30 minutes, rinse with sterile saline 3 to 4 times.

[0054] 1.3 Shred the tumor tissue with sterile surgical scissors, add an appropriate amount of saline for grinding, and collect the single cell suspension after filtering through a 200-mesh mesh.

[0055] 1.4 Put the single-cell suspension into a centrifuge tube, freeze at -86°C, thaw at room temperature, repeat the freeze-thaw 3 times, and observe under the microscope until no cells survive.

[0056] 1.5 Centrifuge the single cell suspension at 3000rpm for 30min, filter the ...

Embodiment 2

[0097] Isolation and Efficient Expansion of Antigen-specific T Lymphocytes Induced by Tumor Antigen (Protein)

[0098] Step 1: The operation is the same as Step 2 of Example 1, except that the antigen added in 2.4 is not the tumor cell lysate, but the full-length protein of the tumor antigen MART-1.

[0099] Step 2: Same as Step 3 of Example 1.

[0100] Step 3: Same as Step 4 of Example 1.

[0101] Step 4: Same as Step 5 of Example 1.

[0102] Step 5: Same as Step 6 of Example 1.

Embodiment 3

[0104] Isolation and Efficient Expansion of Antigen-specific T Lymphocytes Induced by Tumor Antigen (Polypeptide)

[0105] Step 1: DC cell culture and antigen peptide loading

[0106] 1.1 Peripheral blood mononuclear cells were collected with an apheresis machine. After Ficoll separation, the cells in the middle layer were taken and washed twice with normal saline to obtain PBMCs (peripheral blood mononuclear cells).

[0107] 1.2 Take PBMC, add RPMI 1640 medium to make 5×10 6 / ml cell suspension, inoculate 3ml per well into a 6-well plate, and put it into an incubator.

[0108] After 1.32 hours, take it out, shake it to remove the supernatant cells, and leave the adherent mononuclear cells (slightly larger than other cells), add 3ml DC medium (IMP serum-free medium, containing 2% autologous plasma, 1000IU) to each well / ml GM-CSF, 1000IU / ml IL-4).

[0109] 1.4 On day 5, add DC maturation agent (final concentration 10ng / ml LPS, 50IU / ml IFN-r).

[0110] 1.5 On day 7, add MAR...

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