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CN-121975734-A - Culture method of DC-CTL (dendritic cell-cytotoxic T lymphocyte) cells

CN121975734ACN 121975734 ACN121975734 ACN 121975734ACN-121975734-A

Abstract

The invention discloses a method for culturing DC-CTL cells, and particularly relates to the technical fields of biotechnology and medicine. Aiming at the technical problems of limited cell sources, large influence by individual differences, poor stability and repeatability, insufficient cell activity and killing capacity and the like in the traditional DC-CTL cell culture method, the invention provides a novel DC-CTL cell culture method based on iPSC. SOCS1-siRNA is transfected by adopting a self-made additive and specific cytokine combination through a liposome transfection method, so that SOCS1 genes are effectively silenced, and T cell activity is enhanced. The method of the invention ensures that the CD3+CD8+T cell proportion in the DC-CTL cell reaches more than 85 percent, the IFN-gamma content is more than or equal to 500pg/mL, the TNF-alpha content is more than or equal to 300pg/mL, and the killing rates respectively reach 57 percent, 63 percent and 72 percent under the effective target ratios of 10:1, 20:1 and 40:1.

Inventors

  • FAN ZHIQING
  • CHEN YAN
  • WU HANMING
  • Zou Yihao

Assignees

  • 中科赛尔(厦门)精准医学科技有限公司

Dates

Publication Date
20260505
Application Date
20260403

Claims (9)

  1. 1. A method for culturing DC-CTL cells is characterized by comprising the following steps: S1, thawing frozen induced pluripotent stem cells iPSC quickly in a water bath at 37 ℃, immediately transferring the iPSC into a selective ROCK inhibitor Y-27632 containing Rho related coiled coil forming protein kinase ROCK at 37 ℃ for resuspension, avoiding blowing to destroy cell clusters, inoculating the resuspension into a Matrigel coated culture plate, culturing the Matrigel coated culture plate at the ambient temperature of 37 ℃ for 24 hours, and then replacing a culture solution into an inhibitor-free culture medium; S2, inoculating recovered iPSC strain cells into a culture medium containing 1% by mass of self-made additive, wherein the self-made additive is composed of 200 parts by weight of polyethylene glycol 8000, 50-100 parts by weight of polysucrose 70, 50-100 parts by weight of dextran 70, 10-50 parts by weight of BSA bovine serum albumin and 10-50 parts by weight of dextran sulfate dissolved in 100 parts by weight of deionized water, and then placing the culture medium in 37 ℃ and 3-5% Culturing in an incubator for 2 days; S3, adding granulocyte-macrophage colony stimulating factor GM-CSF and interleukin IL-4 into the culture system in S2, and continuously culturing for 3 days to ensure that the iPSC is differentiated into CD34+ hematopoietic progenitor cells and the cells are ensured to have no abnormal aggregation; S4, adding 100-150 ng/mL GM-CSF and 10-30 ng/mL IL-4 into a culture system after differentiation culture is completed, culturing for 5 days to obtain immature DC, then adding 10-30 ng/mL tumor necrosis factor TNF-alpha and 10-20 mu g/mL tumor antigen peptide into the immature DC, continuously culturing for 2 days, then transfecting SOCS1-siRNA by a liposome transfection method, maturing DC cells, loading tumor antigens and silencing SOCS1 genes, and finally sorting the mature DC cells by a flow cytometry; s5, collecting 50mL of peripheral blood of healthy volunteers, adding an anticoagulant into the peripheral blood, separating peripheral blood mononuclear cell PBMC by adopting a density gradient centrifugation method, centrifuging the peripheral blood mononuclear cell PBMC in a centrifuge with the rotating speed of 1000-1500 r/min, and finally collecting middle-layer white membrane PBMC; S6, inoculating PBMC into a culture medium, placing the PBMC into an environment of 37 ℃ for 2 hours for culturing, then collecting cells which are not adhered to the wall, and separating the collected cells into CD3+T lymphocytes by a flow cytometer; s7, inoculating the sorted CD3+T lymphocytes into a basic culture medium which is the same as S2 and contains 1% of self-made additive for culture, simultaneously adding SOCS1-siRNA with transfection concentration of 50-100 nmol/L into the culture medium solution for 4 hours, then adding IL-2 with concentration of 50-100U/mL and for 12 hours, and obtaining the CD3+CD8+T cells with enhanced activity; S8, mixing the DC cells obtained in the S4 with the reinforced active CD3+CD8+T cells obtained in the S7 in a ratio of 1:10-20, then inoculating the mixture into the same culture medium containing 1% of homemade additive in the S2, and adding GM-CSF, IL-4 and IL-2 into the culture medium until the cell density is more than or equal to 1X 10 6 /mL; S9, supplementing fresh basic culture medium after the cells are cultured for 3 days, maintaining the cell density at 0.8-1.2X10- 6 /mL, supplementing IL-2 to the concentration of 50U/mL, supplementing tumor antigen peptide after five days of culture, and continuing to culture to obtain specifically activated cells; s10, culturing the cells subjected to the specific activation in the S9 for 7 days, centrifuging a cultured cell system in a centrifuge with the rotating speed of 1000-1500 r/min, and washing the cells with PBS for 2 times to obtain DC-CTL cells; s11, detecting the proportion of CD3+CD8+CTL cells contained in the cells by using a flow cytometer, co-culturing tumor cells serving as target cells, and then calculating the killing rate by using a CCK-8 method; And S12, detecting the content of interferon IFN-gamma and TNF-alpha in the culture supernatant by adopting an ELISA method, and verifying the concentration of IFN-gamma and TNF-alpha to judge the activation degree of the cells.
  2. 2. A method of culturing DC-CTL cells according to claim 1 wherein the Matrigel coated culture plate in S1 is a natural extracellular matrix hydrogel.
  3. 3. The method of claim 1, wherein the step of culturing the DC-CTL cells is performed for 3 days by adding GM-CSF at a concentration of 40 to 60ng/mL and IL-4 at a concentration of 20 to 30ng/mL to S3.
  4. 4. The method for culturing DC-CTL cells according to claim 1, wherein the concentration of SOCS1-siRNA transfected by the liposome transfection method in S4 is 40-60 nmol/L, and the transfection time is 3-5 hours.
  5. 5. The method of claim 1, wherein the medium in S6 is RPMI-1640 medium, and the fetal bovine serum and penicillin-streptomycin are added to the medium at a mass concentration of 10% and penicillin-streptomycin of 1%.
  6. 6. The method of claim 1, wherein the T cells cultured in S7 are transfected with SOCS1-siRNA until the proportion of cd3+cd8+t cells reaches 85% or more.
  7. 7. The method according to claim 1, wherein the concentration of GM-CSF added to the mixed culture system of the DC cells and the T cells in S8 is 20-30 ng/mL, the concentration of IL-4 is 10-15 ng/mL, and the concentration of IL-2 is 80-120U/mL.
  8. 8. The method of claim 1, wherein the concentration of the tumor antigen peptide added to S9 is 3-7. Mu.g/mL, and the culturing time is 5 days.
  9. 9. The method of claim 1, wherein the S11 tumor cells are co-cultured at an effective target ratio of 10:1, 20:1, 40:1 for 24 hours, and the killing rate is calculated.

Description

Culture method of DC-CTL (dendritic cell-cytotoxic T lymphocyte) cells Technical Field The invention relates to the technical field of biotechnology and medicine, in particular to a method for culturing DC-CTL cells. Background Currently, DC-CTL cell therapy has become an important strategy for tumor immunotherapy. The traditional DC-CTL cell culture method mainly relies on separating mononuclear cells from peripheral blood of healthy volunteers, obtaining monocytes through an adherence method to induce the monocytes to immature DCs, inducing maturation through TNF-alpha, and simultaneously activating peripheral blood lymphocytes through IL-2 and co-culturing the peripheral blood lymphocytes with the mature DCs to obtain the DC-CTL cells. However, this method has significant limitations, mainly in that cell sources are limited by recruitment of healthy volunteers and are greatly affected by individual differences, resulting in significant fluctuation of cell activity among different batches, and difficulty in achieving standardized and scaled production. Traditional methods of culturing DC-CTL cells perform poorly in terms of cell activity and function. Experimental data show that the proportion of CD3+CD8+T cells in DC-CTL cells cultured by the traditional method is only 38%, the secreted IFN-gamma content is only 95pg/mL, the TNF-alpha content is 45pg/mL, and the killing rates at the effective target ratios of 10:1, 20:1 and 40:1 are only 12%, 18% and 22%, respectively. The low activity and low specific killing ability severely limit the clinical application effect of the DC-CTL cells in tumor immunotherapy, and cannot meet the requirements of high-efficiency, stable and repeatable therapy. In addition, the conventional method is poor in stability and reproducibility during the culture. The cell yield and quality of each batch are significantly affected by individual differences of the donor due to the dependence on the peripheral blood source, so that the experimental result is difficult to repeat, and the culture period is long and the operation is complex. Meanwhile, the traditional method lacks an effective T cell activity enhancing means, does not adopt a gene silencing technology to enhance the activity and specific killing capacity of T cells, and does not use an optimized culture additive to improve the cell differentiation efficiency, and the factors together cause the defects of the traditional method in the culture efficiency and quality of DC-CTL cells, so that the urgent requirement of tumor immunotherapy on high-quality cell products is difficult to meet. Disclosure of Invention In order to overcome the defects in the prior art, the embodiment of the invention provides a method for culturing DC-CTL cells, which solves the problems that the traditional method has lower DC-CTL cell proportion, is greatly influenced by individual difference and has poor stability and repeatability. In order to achieve the above purpose, the present invention provides the following technical solutions: The culture method of the DC-CTL cell specifically comprises the following steps: S1, thawing frozen induced pluripotent stem cells iPSC quickly in a water bath at 37 ℃, immediately transferring the iPSC into a selective ROCK inhibitor Y-27632 containing Rho related coiled coil forming protein kinase ROCK at 37 ℃ for resuspension, avoiding blowing to destroy cell clusters, inoculating the resuspension into a Matrigel coated culture plate, culturing the Matrigel coated culture plate at the ambient temperature of 37 ℃ for 24 hours, and then replacing a culture solution into an inhibitor-free culture medium; S2, inoculating recovered iPSC strain cells into a culture medium containing 1% by mass of self-made additive, wherein the self-made additive is composed of 200 parts by weight of polyethylene glycol 8000, 50-100 parts by weight of polysucrose 70, 50-100 parts by weight of dextran 70, 10-50 parts by weight of BSA bovine serum albumin and 10-50 parts by weight of dextran sulfate dissolved in 100 parts by weight of deionized water, and then placing the culture medium in 37 ℃ and 3-5% Culturing in an incubator for 2 days; S3, adding granulocyte-macrophage colony stimulating factor GM-CSF and interleukin IL-4 into the culture system in S2, and continuously culturing for 3 days to ensure that the iPSC is differentiated into CD34+ hematopoietic progenitor cells and the cells are ensured to have no abnormal aggregation; S4, adding 100-150 ng/mL GM-CSF and 10-30 ng/mL IL-4 into a culture system after differentiation culture is completed, culturing for 5 days to obtain immature DC, then adding 10-30 ng/mL tumor necrosis factor TNF-alpha and 10-20 mu g/mL tumor antigen peptide into the immature DC, continuously culturing for 2 days, then transfecting SOCS1-siRNA by a liposome transfection method, maturing DC cells, loading tumor antigens and silencing SOCS1 genes, and finally sorting the mature DC cells by a flow cytometry