DC-CIK co-culture method

Abstract

The invention discloses a DC-CIK co-culture method which comprises the following steps: S1, collecting blood of a tumor patient, and separating PBMC cells in the blood of the tumor patient by adopting a Ficoll separation method; s2, resuspending the separated cells by using a culture solution, then transferring the cells into a T75 bottle, and culturing the T75 bottle in a carbon dioxide incubator; s3, culturing for 2.5-3.5 hours in a carbon dioxide incubator, pouring supernatant into a new T175 bottle, adding a CIK factor 1 to culture CIK cells, and adding a DC starting factor into a T75 bottle to culture DC cells; s4, continuing to culture for 20-26 hours, and adding a CIK factor 2 into the T175 bottle; s5, continuing to culture for 110-130 hours, and adding a DC maturation factor into the T75 bottle; s6, continuing to culture for 160-170 hours, harvesting the matured DC cells, and transferring the DC cells and the CIK cells into a culture bag for culture; and S7, culturing for 330-340 hours, harvesting, and testing the DCCIK flow phenotype. The DC-CIK cell solves the problems of low purity, poor cell killing function and long tumor killing period of the existing DC-CIK cell.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a DC-CIK co-culture method. Background Art

[0002] DC cells are the most powerful professional antigen-presenting cells in the body, which can efficiently take up, process and present antigens; CIK cells have the powerful anti-tumor activity of T lymphocytes and the non-MHC-restricted tumor-killing advantages of NK cells.

[0003] After DC cells are co-cultured with CIK cells, the proliferation rate and killing activity of cells are increased, and the killing effect on tumor cells is more specific. The existing DC-CIK cells have low purity, weak cell killing function, and long tumor killing cycle. Summary of the invention

[0004] In view of the problems that the existing DC-CIK cells have low purity, poor cell killing function and long tumor killing cycle, the present invention provides a DC-CIK co-culture method.

[0005] In order to achieve the above technical objectives, the technical solution adopted by the present invention is as follows:

[0006] A DC-CIK co-culture method comprises the following steps:

[0007] S1. Collect blood from tumor patients and separate PBMC cells from the blood of tumor patients using Ficoll separation method;

[0008] S2, resuspend the separated cells in culture medium, then transfer them to a T75 bottle and culture them in a carbon dioxide incubator;

[0009] S3. Culture in a carbon dioxide incubator for 2.5 to 3.5 hours, pour the supernatant into a new T175 bottle, add CIK factor 1 to culture CIK cells, and add DC priming factor to the T75 bottle to culture DC cells;

[0010] S4, continue culturing for 20 to 26 hours, and add CIK factor 2 into the T175 bottle;

[0011] S5. Continue culturing for 110 to 130 hours and add DC maturation factors to the T75 flask;

[0012] S6. Continue culturing for 160 to 170 hours until the DC cells mature and are harvested, and the DC cells and CIK cells are transferred to a culture bag for culture;

[0013] S7. After another 330 to 340 hours of culture, the cells were harvested and the DCCIK flow cytometry phenotype was tested.

[0014] Furthermore, the optimal time for incubation in the carbon dioxide incubator in step S3 is 3 hours.

[0015] Furthermore, the optimal time for continuing the culture before adding CIK factor 2 to the T175 bottle in step S4 is 24 hours.

[0016] Furthermore, the optimal time for continuing the culture before adding DC maturation factors to the T75 bottle in step S5 is 120 hours.

[0017] Furthermore, in step S6, the optimal culture time for culturing DC cells until they are mature and harvested is 168 hours.

[0018] Furthermore, in step S7, the DC cells and CIK cells were harvested after being cultured in the culture bag for an optimal time of 336 hours.

[0019] Furthermore, CIK factor 1 is IFN-γ, and DC activation factors are GM-CSF and IL-4.

[0020] Further, CIK factor 2 is CD3, CD28, IL-1a and IL-2.

[0021] Furthermore, DC maturation factors are TNF-a and CD40L.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention is simple to operate. The cells are separated and induced to mature stably at an early stage and then combined for culture, so that both DC and CIK can grow stably and ensure that they can stably exert their efficacy.

[0024] Through the above scheme, the antigens in the patient's plasma are used for antigen presentation by DCs, giving CIK memory so that it can quickly identify and proliferate in the patient's body and accurately attack cancer cells in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an overall flow chart of a DC-CIK co-culture method in an embodiment of the present invention;

[0026] Figure 2 Schematic diagram of the results of testing the DCCIK flow table in an embodiment of the present invention. DETAILED DESCRIPTION

[0027] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments and drawings. The contents mentioned in the implementation modes are not intended to limit the present invention.

[0028] like Figure 1 As shown, this embodiment provides a DC-CIK co-culture method, comprising the steps of:

[0029] S1. Collect blood from tumor patients and separate PBMC cells from the blood of tumor patients using Ficoll separation method;

[0030] S2. Resuspend the separated cells in culture medium (GT-581), then transfer them to a T75 flask and culture them in a carbon dioxide incubator;

[0031] S3. Culture in a carbon dioxide incubator for 2.5 to 3.5 hours, pour the supernatant (containing cells) into a new T175 bottle, add CIK factor 1 (IFN-γ) to culture CIK cells, and add DC activating factors (GM-CSF, IL-4) to culture DC cells in the T75 bottle;

[0032] S4, continue culturing for 20 to 26 hours, and add CIK factor 2 (CD3, CD28, IL-1a, IL-2) into the T175 flask;

[0033] S5, continue culturing for 110 to 130 hours, and add DC maturation factors (TNF-a and CD40L) into the T75 flask;

[0034] S6. Continue culturing for 160 to 170 hours until the DC cells mature and are harvested, and the DC cells and CIK cells are transferred to a culture bag for culture;

[0035] like Figure 2 As shown, S7 and cultured for 330 to 340 hours, and the cells were harvested and tested for flow cytometry phenotype of DCCIK.

[0036] The optimal time for incubation in the carbon dioxide incubator in step S3 is 3 hours.

[0037] The optimal time for continued culture before adding CIK factor 2 to the T175 bottle in step S4 is 24 hours.

[0038] The optimal culture time before adding DC maturation factors to the T75 bottle in step S5 is 120 hours.

[0039] The optimal culture time for culturing DC cells to maturity and harvesting in step S6 is 168 hours.

[0040] In step S7, the optimal culture time of DC cells and CIK cells in the culture bag is 336 hours before harvesting.

[0041] CIK factor 1 is IFN-γ, and DC initiation factors are GM-CSF and IL-4.

[0042] CIK factors 2 are CD3, CD28, IL-1a and IL-2.

[0043] DC maturation factors are TNF-a and CD40L.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The present invention is simple to operate. The cells are separated and induced to mature stably at an early stage and then combined for culture, so that both DC and CIK can grow stably and ensure that they can stably exert their efficacy.

[0046] Through the above scheme, the antigens in the patient's plasma are used for antigen presentation by DCs, giving CIK memory so that it can quickly identify and proliferate in the patient's body and accurately attack cancer cells in the body.

[0047] The above is a detailed introduction to a DC-CIK co-culture method provided by the present application. The description of the specific embodiment is only used to help understand the method and its core idea of ​​the present application. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A DC-CIK co-culture method, characterized in that: Includes steps: S1. Collect blood from tumor patients and separate PBMC cells from the blood of tumor patients using Ficoll separation method; S2. Resuspend the separated PBMC cells in culture medium, then transfer them to a T75 flask and culture them in a carbon dioxide incubator; S3. Culture in a carbon dioxide incubator for 2.5 to 3.5 hours, pour the supernatant into a new T175 bottle, add CIK factor 1 to culture CIK cells, and add DC priming factor to the T75 bottle to culture DC cells; S4, continue culturing for 20 to 26 hours, and add CIK factor 2 into the T175 bottle; S5. Continue culturing for 110 to 130 hours and add DC maturation factors to the T75 flask; S6. Continue culturing for 160 to 170 hours until the DC cells mature and are harvested, and the DC cells and CIK cells are transferred to a culture bag for culture; S7. After another 330 to 340 hours of culture, the cells were harvested and the DCCIK flow cytometry phenotype was tested.

2. A DC-CIK co-culture method according to claim 1, characterized in that: The optimal time for incubation in the carbon dioxide incubator in step S3 is 3 hours.

3. A DC-CIK co-culture method according to claim 2, characterized in that: The optimal time for continued culture before adding CIK factor 2 to the T175 bottle in step S4 is 24 hours.

4. A DC-CIK co-culture method according to claim 3, characterized in that: The optimal culture time before adding DC maturation factors to the T75 bottle in step S5 is 120 hours.

5. A DC-CIK co-culture method according to claim 4, characterized in that: The optimal culture time for culturing DC cells to maturity and harvesting in step S6 is 168 hours.

6. A DC-CIK co-culture method according to claim 5, characterized in that: In step S7, the optimal culture time of DC cells and CIK cells in the culture bag is 336 hours before harvesting.

7. A DC-CIK co-culture method according to claim 6, characterized in that: CIK factor 1 is IFN-γ, and DC initiation factors are GM-CSF and IL-4.

8. A DC-CIK co-culture method according to claim 7, characterized in that: CIK factors 2 are CD3, CD28, IL-1a and IL-2.

9. A DC-CIK co-culture method according to claim 8, characterized in that: DC maturation factors are TNF-a and CD40L.

Images