TIL cell, preparation method thereof and application of TIL cell in cancer treatment
A cell and application technology, applied in the field of TIL cells and their preparation, can solve the problems of not many and not many lung cancer treatments, and achieve the effect of inhibiting proliferation and good effect
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Embodiment 1
[0025] Example 1 Isolation and Proliferation of Lung Cancer Tumor-Infiltrating Lymphocytes
[0026] Soak fresh lung cancer tumor tissue in cold Hanks' solution containing antibiotics for 30 minutes, remove necrosis, hemorrhage and fat tissue, weigh, cut into 1mmxlmmxlmm small pieces, add to the digestive enzyme solution prepared with RPMI1640 containing collagenase type Ⅱ Shake and digest for 12 hours, then add hyaluronidase and DNase, and stir for 4 hours to digest. The completely digested tissue was passed through a 120-mesh copper mesh to obtain a single-cell suspension containing TIL and tumor cells.
[0027]Gently add 3ml of the mixed cell suspension to the interface of 1ml of calf serum, centrifuge at 500r / min for 15s, the bottom layer is the tumor cells, and the upper layer of the cell suspension is subjected to continuous gradient separation to obtain TIL. The viability of the obtained TIL was measured by the trypan blue exclusion method, and the smear was stained wit...
Embodiment 2
[0030] Example 2 Preparation of transgenic TIL cells
[0031] Prepare the transgene according to the construction method in "Construction of Transgenic TNF-α Gene Tumor Infiltrating Lymphocytes and Research on Their Biological Characteristics, Luo Yige et al., Journal of Guang Medical University, 2002, 19(2), p151-154" TIL cells. Specifically, a cell line FIyRUFN14 containing a recombinant retrovirus containing human TNF-α gene was prepared first. The cell line was cultured, the supernatant of the culture solution was taken, and Polybrane was added to a final concentration of 8 mg / L, and the supernatant was added to the TIL cells in the logarithmic growth phase prepared in Example 1. After transfection for 3 days, 400ug / ml G418 was added for screening . After 2 weeks, they were cloned by limiting dilution method, and cultured with culture medium containing 1000u / ml IL-2. Another non-transgenic TIL cells were cultured with the same concentration of G418 as a control.
[003...
Embodiment 3
[0033] Example 3 Preparation of CTLA-4 Monoclonal Antibody
[0034] The antigen is CTLA-4 recombinant protein, Cat. No. JN1568, Biolab. BALB / c mice were injected with 10 mg of immune antigen on the back of the neck, boosted once every two weeks, and blood was collected from the tail 7 to 10 days after three immunizations, and the serum antibody titer was detected by indirect ELISA. The specific steps were as follows: the original mass concentration of the test was 5 μg / mL, 100 μL per well of a 96-well ELISA plate, coated overnight at 4 °C; 0.01 mol / L phosphate buffered saline (PBS) containing 0.1% Tween-20 by volume fraction was used as washing solution, washed 3 times, each Use 300 mL each time, and pat dry (the same below); add a washing solution containing 10% fetal bovine serum to each well to seal, and incubate at 37°C for 2 hours; wash and pat dry; dilute the serum to be tested 1000 times, add 100 μL to each well, And set blank control wells (PBS) and negative control w...
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