Immune cell cryopreservation liquid and immune cell cryopreservation method

Abstract

The invention discloses an immune cell cryopreservation liquid, which comprises the following components: 0.02 to 0.06 mg / mL of dimethyl sulfoxide, 1 to 6 mg / mL of cucurbitacine, 0.01 to 0.03 mL / mL of gleditsia sinensis polysaccharide, 1 to 2 mg / mL of lentinan, 1 to 5 [mu]g / mL of polyglycerol fatty acid ester, 0.01 to 0.03 mg / mL of polyethylene glycol, 10 to 16 mg / mL of non-essential amino acid and 12 to 20 mg / mL of human serum albumin. The invention discloses an immune cell cryopreservation method which comprises the following steps: uniformly mixing peripheral blood mononuclear cells with the immune cell cryopreservation liquid to obtain a cell suspension, transferring the cell suspension into a sterile cryopreservation tube, then putting the cell suspension into a cryopreservation box, carrying out programmed cooling to-70 DEG C, carrying out cryopreservation for 10-15 hours, and then transferring the cell suspension into liquid nitrogen for cryopreservation, wherein the concentration of the cells in the cell suspension is (2-4) * 10<7> cells / mL.

Description

technical field

[0001] The invention relates to the technical field of cell cryopreservation, in particular to an immune cell cryopreservation solution and an immune cell cryopreservation method. Background technique

[0002] Biological immune cell therapy can induce autologous antiviral immune response through in vitro culture, proliferation, activation, and reinfusion into the body. Once the human antiviral immunity is activated, it will continuously produce antiviral substances to kill the virus. After activation, most of the T cells that can kill tumor cells become memory cells and store in lymphoid tissues, providing long-term protection for the complete elimination of tumor cells and the prevention and treatment of metastasis and recurrence.

[0003] With the rapid development of immune cell biology and immune molecular biology, somatic cell immunotherapy has become one of the important means of adjuvant therapy after radiotherapy and chemotherapy for tumor patients. ...

Examples

Embodiment 1

[0031] An immune cell cryopreservation solution, the components of which include:

[0032] Dimethyl sulfoxide 0.02mg / mL,

[0033] Cucurbitacin 1mg / mL,

[0034] Acacia polysaccharide 0.01mL / mL,

[0035] Lentinan 1mg / mL,

[0036] Polyglycerol fatty acid ester 1 μg / mL,

[0037] Polyethylene glycol 0.01mg / mL,

[0038] Non-essential amino acids 10mg / mL,

[0039] Human serum albumin 12mg / mL.

[0040] A method for cryopreserving immune cells, comprising the steps of:

[0041] S1. Take peripheral blood in a centrifuge tube, centrifuge for 10 minutes, remove the upper layer of plasma, add an equal volume of PBS buffer to the lower layer of cells and mix evenly, then add Ficoll separation solution to make the layers clear, absorb the peripheral blood mononuclear cell layer after centrifugation, add PBS buffer was mixed and centrifuged to obtain peripheral blood mononuclear cells;

[0042] S2. Mix the peripheral blood mononuclear cells with the above-mentioned immune cell cryopre...

Embodiment 2

[0045] An immune cell cryopreservation solution, the components of which include:

[0046] Dimethyl sulfoxide 0.06mg / mL,

[0047] Cucurbitacin 6mg / mL,

[0048] Acacia polysaccharide 0.03mL / mL,

[0049] Lentinan 2mg / mL,

[0050] Polyglycerol fatty acid ester 5μg / mL,

[0051] Polyethylene glycol 0.03mg / mL,

[0052] Non-essential amino acids 16mg / mL,

[0053] Human serum albumin 20mg / mL.

[0054] A method for cryopreserving immune cells, comprising the steps of:

[0055] S1. Take peripheral blood in a centrifuge tube, centrifuge for 20 minutes, remove the upper layer of plasma, add an equal volume of PBS buffer to the lower layer of cells and mix evenly, then add Ficoll separation solution to make the layers clear, absorb the peripheral blood mononuclear cell layer after centrifugation, add PBS buffer was mixed and centrifuged to obtain peripheral blood mononuclear cells;

[0056] S2. Mix the peripheral blood mononuclear cells with the above-mentioned immune cell cryopres...

Embodiment 3

[0059] An immune cell cryopreservation solution, the components of which include:

[0060] Dimethyl sulfoxide 0.03mg / mL,

[0061] Cucurbitacin 5mg / mL,

[0062]Acacia polysaccharide 0.015mL / mL,

[0063] Lentinan 1.7mg / mL,

[0064] Polyglycerol fatty acid ester 2μg / mL,

[0065] Polyethylene glycol 0.025mg / mL,

[0066] Non-essential amino acids 12mg / mL,

[0067] Human serum albumin 18mg / mL.

[0068] A method for cryopreserving immune cells, comprising the steps of:

[0069] S1. Take peripheral blood in a centrifuge tube, centrifuge for 13 minutes, remove the upper layer of plasma, add an equal volume of PBS buffer to the lower layer of cells and mix evenly, then add Ficoll separation solution to make the layers clear, absorb the peripheral blood mononuclear cell layer after centrifugation, add PBS buffer was mixed and centrifuged to obtain peripheral blood mononuclear cells;

[0070] S2. Mix the peripheral blood mononuclear cells with the above-mentioned immune cell cryop...

technical field

[0001] The invention relates to the technical field of cell cryopreservation, in particular to an immune cell cryopreservation solution and an immune cell cryopreservation method. Background technique

[0002] Biological immune cell therapy can induce autologous antiviral immune response through in vitro culture, proliferation, activation, and reinfusion into the body. Once the human antiviral immunity is activated, it will continuously produce antiviral substances to kill the virus. After activation, most of the T cells that can kill tumor cells become memory cells and store in lymphoid tissues, providing long-term protection for the complete elimination of tumor cells and the prevention and treatment of metastasis and recurrence.

[0003] With the rapid development of immune cell biology and immune molecular biology, somatic cell immunotherapy has become one of the important means of adjuvant therapy after radiotherapy and chemotherapy for tumor patients. ...

Examples

Embodiment 1

[0031] An immune cell cryopreservation solution, the components of which include:

[0032] Dimethyl sulfoxide 0.02mg / mL,

[0033] Cucurbitacin 1mg / mL,

[0034] Acacia polysaccharide 0.01mL / mL,

[0035] Lentinan 1mg / mL,

[0036] Polyglycerol fatty acid ester 1 μg / mL,

[0037] Polyethylene glycol 0.01mg / mL,

[0038] Non-essential amino acids 10mg / mL,

[0039] Human serum albumin 12mg / mL.

[0040] A method for cryopreserving immune cells, comprising the steps of:

[0041] S1. Take peripheral blood in a centrifuge tube, centrifuge for 10 minutes, remove the upper layer of plasma, add an equal volume of PBS buffer to the lower layer of cells and mix evenly, then add Ficoll separation solution to make the layers clear, absorb the peripheral blood mononuclear cell layer after centrifugation, add PBS buffer was mixed and centrifuged to obtain peripheral blood mononuclear cells;

[0042] S2. Mix the peripheral blood mononuclear cells with the above-mentioned immune cell cryopre...

Embodiment 2

[0045] An immune cell cryopreservation solution, the components of which include:

[0046] Dimethyl sulfoxide 0.06mg / mL,

[0047] Cucurbitacin 6mg / mL,

[0048] Acacia polysaccharide 0.03mL / mL,

[0049] Lentinan 2mg / mL,

[0050] Polyglycerol fatty acid ester 5μg / mL,

[0051] Polyethylene glycol 0.03mg / mL,

[0052] Non-essential amino acids 16mg / mL,

[0053] Human serum albumin 20mg / mL.

[0054] A method for cryopreserving immune cells, comprising the steps of:

[0055] S1. Take peripheral blood in a centrifuge tube, centrifuge for 20 minutes, remove the upper layer of plasma, add an equal volume of PBS buffer to the lower layer of cells and mix evenly, then add Ficoll separation solution to make the layers clear, absorb the peripheral blood mononuclear cell layer after centrifugation, add PBS buffer was mixed and centrifuged to obtain peripheral blood mononuclear cells;

[0056] S2. Mix the peripheral blood mononuclear cells with the above-mentioned immune cell cryopres...

Embodiment 3

[0059] An immune cell cryopreservation solution, the components of which include:

[0060] Dimethyl sulfoxide 0.03mg / mL,

[0061] Cucurbitacin 5mg / mL,

[0062]Acacia polysaccharide 0.015mL / mL,

[0063] Lentinan 1.7mg / mL,

[0064] Polyglycerol fatty acid ester 2μg / mL,

[0065] Polyethylene glycol 0.025mg / mL,

[0066] Non-essential amino acids 12mg / mL,

[0067] Human serum albumin 18mg / mL.

[0068] A method for cryopreserving immune cells, comprising the steps of:

[0069] S1. Take peripheral blood in a centrifuge tube, centrifuge for 13 minutes, remove the upper layer of plasma, add an equal volume of PBS buffer to the lower layer of cells and mix evenly, then add Ficoll separation solution to make the layers clear, absorb the peripheral blood mononuclear cell layer after centrifugation, add PBS buffer was mixed and centrifuged to obtain peripheral blood mononuclear cells;

[0070] S2. Mix the peripheral blood mononuclear cells with the above-mentioned immune cell cryop...

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