CN116083360A - Efficient amplification method for autologous CIK cells - Google Patents

Efficient amplification method for autologous CIK cells Download PDF

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CN116083360A
CN116083360A CN202310190552.2A CN202310190552A CN116083360A CN 116083360 A CN116083360 A CN 116083360A CN 202310190552 A CN202310190552 A CN 202310190552A CN 116083360 A CN116083360 A CN 116083360A
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culture
platelets
platelet aggregation
cik cells
autologous cik
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徐子恩
弗莱德·范德里希
胡建杰
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Vandrich Shanghai Biotechnology Co ltd
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Abstract

The invention discloses an efficient amplification method of autologous CIK cells, which comprises the following steps of S1, preparing a culture bottle: and adding an activating agent into the culture flask until the bottom of the flask is covered, and incubating to obtain an activated culture flask, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution. S2, separation of peripheral blood mononuclear cells: peripheral blood mononuclear cells are separated from the blood of a patient and suspended for later use. S3, preparation of platelet aggregation release materials: separating to obtain platelets and re-suspending the platelets by using phosphate buffer solution to obtain re-suspended platelets; adenosine diphosphate is added into the resuspended blood platelets, the culture is incubated in an incubator for 2-4 hours, 1800-2000 g is centrifuged for 20-25 min, and the supernatant component is harvested and directly used as a solution containing the platelet aggregation releaser, or the platelet aggregation releaser is obtained through freeze-drying treatment.

Description

Efficient amplification method for autologous CIK cells
Technical Field
The invention relates to the technical field of cell culture, in particular to an efficient amplification method of autologous CIK cells.
Background
CIK cells, i.e., cytokine-induced killer cells (cytokine-induced killercells), are a population of heterogeneous cells obtained by co-inducing human peripheral blood mononuclear cells with a plurality of cytokines in vitro. Is a new generation of cytokine activated lymphocytes developed on the basis of LAK cells (Lymphokine-activated killer cells). LAK cells are first reported by Grimm et al in 1982, and various cytokines are added into peripheral blood mononuclear cells for in vitro culture for 4-6 days, so that the LAK cells can kill various tumor cells by inducing a nonspecific killer cell. In 1984, the Rosenberg study group used IL-2 and LAK for the first time to treat 25 cases of renal cell carcinoma, melanoma, lung cancer, colon cancer and other tumor patients, wherein 11 cases of tumors were reduced by 50% and 1 case of melanoma was completely resolved.
The study group summarized 222 tumor patients treated in conjunction in 1988, in which 16 patients had complete tumor metastasis regressions and 26 patients had tumor regressions of 50% or more, the treatment was remarkable in metastatic renal cell carcinoma, melanoma, colon cancer and non-hodgkin's lymphoma.
Autologous CIK cell culture has obvious advantages in clinical treatment, and because the risk of cross infection of diseases is greatly reduced due to the culture of tissues taken from a patient, and the safety is greatly ensured, an efficient autologous CIK cell expansion method is needed.
Disclosure of Invention
In view of the problems existing in the prior art, the invention discloses an efficient amplification method of autologous CIK cells, which adopts the following technical scheme:
s1, preparing a culture bottle: and adding an activating agent into the culture flask until the bottom of the flask is covered, and incubating to obtain an activated culture flask, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution.
S2, separation of peripheral blood mononuclear cells: peripheral blood mononuclear cells are separated from the blood of a patient and suspended for later use.
S3, preparation of platelet aggregation release materials: separating to obtain platelets and re-suspending the platelets by using phosphate buffer solution to obtain re-suspended platelets; adenosine diphosphate is added into the resuspended blood platelets, the culture is incubated in an incubator for 2-4 hours, 1800-2000 g is centrifuged for 20-25 min, and the supernatant component is harvested and directly used as a solution containing the platelet aggregation releaser, or the platelet aggregation releaser is obtained through freeze-drying treatment.
S4, preparing an activation culture medium: to 49mL of basal medium containing plasma, 1mL of CIK cell activator was added to obtain an activated medium.
S5, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture medium for culture, supplementing 5% -20% of serum-free culture medium containing the platelet aggregation release substances during culture, and adding cytokines IL2, IL1a and anti-CD3 for induction until the autologous CIK cells are harvested.
As a preferable technical scheme of the efficient amplification method of the autologous CIK cells, the concentration of the CD3 monoclonal antibody in the activator in the S1 is 50 mug/mL, and the concentration of IFN-gamma is 2000U/mL.
As a preferable technical scheme of the efficient amplification method of the autologous CIK cells, the incubation condition in the S1 is 4-37 ℃ and the incubation time is 1-18h.
As a preferable technical scheme of the high-efficiency expansion method of the autologous CIK cells, the S2 is used for separating peripheral blood mononuclear cells by using a density gradient centrifugation method.
As a preferable technical scheme of the efficient amplification method of the autologous CIK cells, the phosphate buffer solution used in the S3 is a phosphate buffer solution containing Ca2+ and Mg2+.
As a preferable technical scheme of the efficient amplification method of the autologous CIK cells, the adenosine diphosphate in the S3 is 80-150 mu mol/L.
As an optimal technical scheme of the efficient amplification method of the autologous CIK cells, the CIK cell activator in the S4 is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution.
As a preferable technical scheme of the efficient amplification method of the autologous CIK cells, the contents of platelet-derived growth factors, transforming growth factors, insulin-like growth factors and epidermal growth factors in the platelet aggregation release are all more than 150ng/g.
The invention has the beneficial effects that: the invention passes through.
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FIG. 1 is a schematic flow chart of the present invention.
Detailed Description
Example 1
As shown in fig. 1, the invention discloses an efficient amplification method of autologous CIK cells, which adopts the following technical scheme:
s1, preparing a culture bottle: and adding an activating agent into the culture flask until the bottom of the flask is covered, and incubating to obtain an activated culture flask, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution, the concentration of the CD3 monoclonal antibody in the activating agent is 50 mug/mL, the concentration of the IFN-gamma is 2000U/mL, the incubation condition is 4 ℃, and the incubation time is 1h.
S2, separation of peripheral blood mononuclear cells: peripheral blood mononuclear cells are separated from the blood of a patient, suspended for later use and separated by using a density gradient centrifugation method.
S3, preparation of platelet aggregation release materials: separating to obtain platelets and re-suspending the platelets by using phosphate buffer solution to obtain re-suspended platelets; adding adenosine diphosphate into the resuspended blood platelets, incubating and culturing for 2 hours in an incubator, centrifuging for 20 minutes at 1800g, and collecting supernatant components, wherein the supernatant components are directly used as a solution containing the blood platelet aggregation release substances, or freeze-drying to obtain the blood platelet aggregation release substances, wherein the adenosine diphosphate is 80 mu mol/L, and the contents of blood platelet derived growth factors, transforming growth factors, insulin-like growth factors and epidermal growth factors in the blood platelet aggregation release substances are all more than 150ng/g.
S4, preparing an activation culture medium: adding 1mL of CIK cell activator into 49mL of basal medium containing plasma to obtain an activated medium, wherein the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution.
S5, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture medium for culture, supplementing 5 percent of serum-free culture medium containing the platelet aggregation release substances during culture, and adding cytokines IL2, IL1a and anti-CD3 for induction until the autologous CIK cells are harvested.
Example 2
As shown in fig. 1, the invention discloses an efficient amplification method of autologous CIK cells, which adopts the following technical scheme:
s1, preparing a culture bottle: and adding an activating agent into the culture flask until the bottom of the flask is covered, and incubating to obtain an activated culture flask, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution, the concentration of the CD3 monoclonal antibody in the activating agent is 50 mug/mL, the concentration of the IFN-gamma is 2000U/mL, the incubation condition is 10 ℃, and the incubation time is 6 hours.
S2, separation of peripheral blood mononuclear cells: peripheral blood mononuclear cells are separated from the blood of a patient, suspended for later use and separated by using a density gradient centrifugation method.
S3, preparation of platelet aggregation release materials: separating to obtain platelets and re-suspending the platelets by using phosphate buffer solution to obtain re-suspended platelets; adding adenosine diphosphate into the resuspended blood platelets, incubating and culturing for 3 hours in an incubator, centrifuging for 23 minutes 1850g, and collecting supernatant components, wherein the supernatant components are directly used as a solution containing the blood platelet aggregation release substances, or freeze-drying to obtain the blood platelet aggregation release substances, wherein the adenosine diphosphate is 100 mu mol/L, and the contents of blood platelet derived growth factors, transforming growth factors, insulin-like growth factors and epidermal growth factors in the blood platelet aggregation release substances are all more than 150ng/g.
S4, preparing an activation culture medium: adding 1mL of CIK cell activator into 49mL of basal medium containing plasma to obtain an activated medium, wherein the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution.
S5, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture medium for culture, supplementing 10% of serum-free culture medium containing the platelet aggregation release substances during culture, and adding cytokines IL2, IL1a and anti-CD3 for induction until the autologous CIK cells are harvested.
Example 3
As shown in fig. 1, the invention discloses an efficient amplification method of autologous CIK cells, which adopts the following technical scheme:
s1, preparing a culture bottle: and adding an activating agent into the culture flask until the bottom of the flask is covered, and incubating to obtain an activated culture flask, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution, the concentration of the CD3 monoclonal antibody in the activating agent is 50 mug/mL, the concentration of the IFN-gamma is 2000U/mL, the incubation condition is 30 ℃, and the incubation time is 12 hours.
S2, separation of peripheral blood mononuclear cells: peripheral blood mononuclear cells are separated from the blood of a patient, suspended for later use and separated by using a density gradient centrifugation method.
S3, preparation of platelet aggregation release materials: separating to obtain platelets and re-suspending the platelets by using phosphate buffer solution to obtain re-suspended platelets; adding adenosine diphosphate into the resuspended blood platelets, incubating and culturing for 3 hours in an incubator, centrifuging for 24 minutes at 1900g, and collecting supernatant components, wherein the supernatant components are directly used as a solution containing the blood platelet aggregation release substances, or freeze-drying to obtain the blood platelet aggregation release substances, wherein the adenosine diphosphate is 1200 mu mol/L, and the contents of blood platelet derived growth factors, transforming growth factors, insulin-like growth factors and epidermal growth factors in the blood platelet aggregation release substances are all more than 150ng/g.
S4, preparing an activation culture medium: adding 1mL of CIK cell activator into 49mL of basal medium containing plasma to obtain an activated medium, wherein the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution.
S5, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture medium for culture, supplementing 15% of serum-free culture medium containing the platelet aggregation release substances during culture, and adding cytokines IL2, IL1a and anti-CD3 for induction until the autologous CIK cells are harvested.
Example 4
As shown in fig. 1, the invention discloses an efficient amplification method of autologous CIK cells, which adopts the following technical scheme:
s1, preparing a culture bottle: and adding an activating agent into the culture flask until the bottom of the flask is covered, and incubating to obtain an activated culture flask, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution, the concentration of the CD3 monoclonal antibody in the activating agent is 50 mug/mL, the concentration of the IFN-gamma is 2000U/mL, the incubation condition is 37 ℃, and the incubation time is 18 hours.
S2, separation of peripheral blood mononuclear cells: peripheral blood mononuclear cells are separated from the blood of a patient, suspended for later use and separated by using a density gradient centrifugation method.
S3, preparation of platelet aggregation release materials: separating to obtain platelets and re-suspending the platelets by using phosphate buffer solution to obtain re-suspended platelets; adding adenosine diphosphate into the resuspended blood platelets, incubating and culturing for 4 hours in an incubator, centrifuging for 25 minutes at 2000g, and collecting supernatant components, wherein the supernatant components are directly used as a solution containing the blood platelet aggregation release substances, or freeze-drying to obtain the blood platelet aggregation release substances, wherein the adenosine diphosphate is 150 mu mol/L, and the blood platelet derivative growth factors, transforming growth factors, insulin-like growth factors and epidermal growth factors in the blood platelet aggregation release substances are all more than 150ng/g.
S4, preparing an activation culture medium: adding 1mL of CIK cell activator into 49mL of basal medium containing plasma to obtain an activated medium, wherein the CIK cell activator is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution.
S5, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture medium for culture, supplementing 20% of serum-free culture medium containing the platelet aggregation release substances during culture, and adding cytokines IL2, IL1a and anti-CD3 for induction until the autologous CIK cells are harvested.
The components not described in detail herein are prior art.
Although the specific embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes and modifications without inventive labor may be made within the scope of the present invention without departing from the spirit of the present invention, which is within the scope of the present invention.

Claims (8)

1. The high-efficiency amplification method of the autologous CIK cells is characterized by comprising the following steps of:
s1, preparing a culture bottle: and adding an activating agent into the culture flask until the bottom of the flask is covered, and incubating to obtain an activated culture flask, wherein the activating agent is prepared by dissolving a CD3 monoclonal antibody and IFN-gamma in a PBS buffer solution.
S2, separation of peripheral blood mononuclear cells: peripheral blood mononuclear cells are separated from the blood of a patient and suspended for later use.
S3, preparation of platelet aggregation release materials: separating to obtain platelets and re-suspending the platelets by using phosphate buffer solution to obtain re-suspended platelets; adenosine diphosphate is added into the resuspended blood platelets, the culture is incubated in an incubator for 2-4 hours, 1800-2000 g is centrifuged for 20-25 min, and the supernatant component is harvested and directly used as a solution containing the platelet aggregation releaser, or the platelet aggregation releaser is obtained through freeze-drying treatment.
S4, preparing an activation culture medium: to 49mL of basal medium containing plasma, 1mL of CIK cell activator was added to obtain an activated medium.
S5, culturing autologous CIK cells: carefully paving the peripheral blood mononuclear cells into a culture medium for culture, supplementing 5% -20% of serum-free culture medium containing the platelet aggregation release substances during culture, and adding cytokines IL2, IL1a and anti-CD3 for induction until the autologous CIK cells are harvested.
2. The method for efficiently expanding autologous CIK cells according to claim 1, wherein: the concentration of the CD3 monoclonal antibody in the activator in the S1 is 50 mug/mL, and the concentration of IFN-gamma is 2000U/mL.
3. The method for efficiently expanding autologous CIK cells according to claim 1, wherein: the incubation condition in the S1 is 4-37 ℃ and the incubation time is 1-18h.
4. The method for efficiently expanding autologous CIK cells according to claim 1, wherein: the peripheral blood mononuclear cells are separated in the S2 by using a density gradient centrifugation method.
5. The method for efficiently expanding autologous CIK cells according to claim 1, wherein: the phosphate buffer used in S3 is ca2+ and mg2+ containing phosphate buffer.
6. The method for efficiently expanding autologous CIK cells according to claim 1, wherein: the adenosine diphosphate in the S3 is 80-150 mu mol/L.
7. The method for efficiently expanding autologous CIK cells according to claim 1, wherein: the CIK cell activator in the S4 is prepared by dissolving IL-2, IL-1 alpha and IFN-gamma in PBS buffer solution.
8. The method for efficiently expanding autologous CIK cells according to claim 1, wherein: the platelet derived growth factor, transforming growth factor, insulin-like growth factor and epidermal growth factor content in the platelet aggregation release are all greater than 150ng/g.
CN202310190552.2A 2023-03-02 2023-03-02 Efficient amplification method for autologous CIK cells Pending CN116083360A (en)

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