Gamma delta T-like alpha beta T cell and preparation method and application thereof
A cell and tumor tissue technology, applied in the field of biomedicine, can solve problems such as failure to achieve results
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[0034] The present invention provides a method for preparing γδT-like αβT cells, comprising the following steps: using tumor tissue antigens to induce γδT cells, using the γδT cells as antigen-presenting cells to activate αβT cells, and obtaining bispecific γδT cells induced by tumor tissue antigens Cell stimulation activates γδT-like αβ T cells.
[0035] The present invention does not specifically limit the types of tumor tissue antigens, preferably including lung cancer, breast cancer, liver cancer, rectal cancer and gastric cancer, and the preparation methods of different types of tumor tissue antigens are the same. The preparation method of the tumor tissue antigen according to the present invention preferably comprises: (1) mixing and digesting the tumor tissue piece washed with PBS and the compound enzyme liquid; the volume of the compound enzyme liquid is twice the volume of the tumor tissue block ; Include trypsin, collagenase I and collagenase IV in the compound enzym...
Embodiment 1
[0062] 1. Preparation of tumor tissue antigens from lung cancer, breast cancer, liver cancer, rectal cancer and gastric cancer
[0063] 1.1 Place the fresh tumor tissue in a culture dish, add an appropriate amount of PBS, remove the area where other tissues retain tumor cells, and wash it with PBS.
[0064] 1.2 Put the tumor tissue into a new culture dish, add PBS, and use ophthalmic scissors to cut the tissue into about 1-2mm 3 sized pieces. Transfer to a 50ml centrifuge tube and rinse with PBS. After the tissue pieces sink automatically, remove the PBS.
[0065] 1.3 Add twice the volume of 0.25% trypsin: 2% collagenase I: 2% collagenase IV mixture, shake and digest in a 37°C water bath for 30-60 minutes.
[0066] 1.4 After the digestion of the tissue block is completed, suck out the upper liquid, filter it with a cell mesh with a pore size of 70 μm, and transfer it to a centrifuge tube containing an equal volume of complete medium to terminate the digestion.
[0067] 1.5 ...
Embodiment 2
[0093] Lung cancer antigens are extracted, loaded with γδT cells, and co-cultured with αβT cells, and the obtained γδT-like αβT cells have a specific killing effect on lung cancer cells.
[0094] 1. Count lung cancer cells, add CFSE to make the final concentration of CFSE reach 2μM, and treat at 37°C for 30min.
[0095] 2. After taking it out, centrifuge at 1000rpm for 5 minutes, you can faintly see the cells stained yellow, wash with PBS three times.
[0096] 3. Mixed culture with γδT-like αβT cells, 37°C, 5% CO 2 , saturated humidity, incubated for 24h.
[0097] 4. Add 1 μg / ml PI working solution, keep away from light for 30 minutes, and then flow up.
[0098] 5. CFSE, PI double-positive cells are the killed lung cancer cells, divided by the total number of lung cancer cells, is the killing rate.
[0099] According to the above method to test, the results are as follows Figure 4 As shown, when the effect-to-target ratio of γδT-like αβT cells to lung cancer cells is 1:1,...
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Abstract
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