Background knowledge

DCIt is an abbreviation for "Dendritic Cells" and its full Chinese name is "Dendritic Cells". It is named after the many dendritic or pseudopod like protrusions that extend when it matures. DC was discovered by Canadian scientist Ralph M. Steinman, who won the Nobel Prize in 2011, in 1973. It is currently the most powerful antigen-presenting cell (APC) discovered. It has been confirmed that DCs are APCs that can significantly stimulate the proliferation of naive T cells (Na ï ve T cells). Mature DCs can present tumor antigens through pathways such as type II histocompatibility antigen (MHC-II), effectively resisting the immune escape mechanism of tumor cells, while other APCs (such as monocytes, B cells, etc.) can only stimulate activated or memory T cells. DC is the initiator of adaptive T cell immune response in the body and plays an extremely important role in tumor immunity.

CIKIt is a heterogeneous cell population obtained by culturing mononuclear cells under the action of CD3 monoclonal antibody and various cytokines (including IFN - γ, IL-2, etc.), with CD3+CD56+cells as the main effector cells. It has both strong anti-tumor activity of T lymphocytes and non MHC (major histocompatibility antigen) limited tumor killing ability of NK cells (natural killer cells).

DC-CIK (Or DC+CIK) [1] refers to CIK cells co cultured with DC cells, or it can be said that the final effector cells are CIK cells activated by DC in vitro. Multiple studies have shown that DC and CIK have a synergistic effect. After co incubation, the expression of co stimulatory molecules and antigen presentation ability on the surface of DC are significantly improved, while the proliferation ability and in vitro and in vivo cytotoxic activity of CIK are also enhanced. Therefore, DC-CIK treatment is more effective than CIK alone. If DC loaded with tumor antigens is co cultured with CIK, it can stimulate the production of tumor antigen-specific T cells. Such DC-CIK therapy has both specific and non-specific dual tumor killing effects, with stronger CIK activity activated by DC stimulation than DC without tumor antigens. It is often used in clinical and scientific research.

CIKCells and DC cells are two important components of cells, and their combination ensures an efficient immune response.

【 Cultivation Principle 】

1Cytokines for DC culture:

1.1 GM-CSF(Granulocyte/macrophage colony-stimulating factor

GM-CSFIt is a hematopoietic growth factor that can stimulate the colony formation of neutrophils and macrophages in vitro, and has the function of promoting the proliferation and development of early red megakaryocytes and eosinophil progenitor cells.

GM-CSFIt is one of the cytokines that has been identified to have an effect on DCs for a long time. The function of GM-CSF in DC culture is to promote the differentiation of monocytes into macrophages, increase the expression of MHC class II molecules on the cell surface, and enhance the antigen presentation function of cells. In addition, GM-CSF can also promote the survival of DCs.

1.2 IL-4 (Interleukin-4)

IL-4The role played in inducing DCs from monocytes is to inhibit the overgrowth of macrophages, thereby guiding monocytes to differentiate towards DCs. If IL-4 is not added to the culture system, monocytes will differentiate into macrophages. Meanwhile, IL-4 also has the ability to reduce the expression of CD14 molecules on the cell surface. The decrease in CD14 expression level is an important marker for monocyte differentiation into DCs.

GM-CSFCombined with IL-4, monocytes can differentiate into immature DCs, which have strong antigen uptake and processing abilities but weak antigen presentation abilities. The cell surface moderately expresses MHC class I, II molecules, and B7 family molecules (CD80, CD86, etc.), but does not express CD14.

1.3 TNF-Alpha (tumor necrosis factor alpha)

TNF-Alpha can downregulate the macropinocytosis and surface Fc receptor expression of immature DCs, causing the disappearance of MHC class II compartments in cells. However, it can upregulate the expression of MHC class I, II molecules, and B7 family molecules (CD80, CD86, etc.) on the cell surface, causing immature DCs to differentiate into mature DCs. At this time, the antigen uptake and processing ability of DCs is significantly weakened, while the antigen presentation ability is significantly enhanced, which can activate T cells.

Cell Preparation

1Collection of peripheral blood mononuclear cells

1.1Collect 80-100mL of peripheral blood mononuclear cells from patients using a blood cell separator;

1.2Further purification of mononuclear cells (PBMCs) using density gradient centrifugation with lymphocyte separation solution;

1.3Wash the serum-free culture medium twice to obtain PBMCs with a purity of over 90%, and the number of cells should reach 1-3x10⁸.

2. DCCell culture and identification

2.1 The PBMCs obtained in step 1 were adjusted to a cell concentration of 2 x 10 ^ 6/ml using serum-free culture medium and placed in a culture bottle;

2.2 37Incubate in a 5% CO2 incubator at ℃ for 2 hours to allow monocytes to adhere to the wall;

2.3 Remove suspended cells, and add the remaining adherent cells (mainly CD14+monocytes) to serum-free culture medium containing recombinant human GM-CSF 500-1000U/ml and recombinant human IL-4 500U/ml. Cultivate in a 37 ℃, 5% CO2 incubator to induce monocyte differentiation into DC cells;

3.2 Change the medium by half every 3 days and replenish cytokines;

3.3On the 6th day of cultivation, recombinant human TNF-a (500U/ml) was added to induce DC cell maturation;

3.4On the 7th or 8th day of cultivation, the number of harvested DC cells should reach 1 × 10 ^ 6 or more;

3.5 DCQuality inspection:

3.5.1 Trypan blue staining is used to detect cell viability: live cells should be above 80%;

3.5.2 Flow cytometry is used to detect the expression of molecules such as HLA-DR, CD83, and CD86 on the surface of DC cells to determine whether the DC is mature.

4.Before harvesting cells, take a small amount of culture for bacterial and fungal cultivation, and test for mycoplasma, chlamydia, and endotoxins (standard: negative pathogen test, endotoxin<5 Eu).

【 * Reagent 】

Product Name	Product Number	Product Specifications	Use concentration
Recombinant human GM-CSF	kx30-GM	50μg/1mg	50-100ng/ml
Recombinant human TNF-a	kx30-T	50μg/1mg	500U/ml
Recombinant human IL-4	kx20-4	50μg/1mg	100ng/ml

[Other related reagents]

Product Name	Product Number	Product Specifications
Recombinant Flt-3 Ligand	kx30-F	50μg /1mg
Recombinant human IL-7	kx20-7	50μg /1mg
Recombinant human IL-15	kx20-15	50μg /1mg
Recombinator SCF	kx30-S	50μg /1mg