YY/T 1465.1-2016Immunogenic evaluation method of medical devices - Part 1: T Lymphocyte transformation test in vitro (English PDF)

5.1 General

All animal tests shall be carried out in the laboratories approved by the national accreditation agency and in compliance with all applicable regulations for laboratory animal welfare; and shall also comply with the requirements of GB/T 16886.2.

5.2 Animal species and requirements

The commonly used experimental animal is a mouse. This Part recommended to use the healthy Grade-SPF, 6-8 weeks of age BALB/c mice that have not been tested. If other strains of mice are used, the suitability shall be explained.

7.1 Principle

MTT is a pale-yellow cell dye; it enters into the living cells and forms water-insoluble blue-purple crystalline formazan particles in mitochondria under the role of succinate dehydrogenase and deposits in the cells;

7.2 Lymphocyte preparation

The BALB/c mice were sacrificed by taking the cervical vertebrae off; the single cell suspension was prepared by grinding; collect the cell suspension; take 400g to centrifuge for 5min; discharge the supernatant; add 5mL of red blood cell lysate; mix up; incubate at 4°C for 5min; add RPMI1640 nutrient solution containing 10% FCS to stop the reaction; take 400g to centrifuge for 5min; discharge the supernatant; add RPMI1640 nutrient solution containing 10% FCS to adjust the cell concentration into 2 x 10^6 cells/mL.

7.4 Test procedures

Add the cell suspension into a 96-well plate; 200µL per well; set six replicate wells.

Add ConA to positive control group till the final concentration is 5µg/mL.

7.5 Data analysis

Record the absorbance values, A, for each group; use appropriate statistical method to deal with the test results. Use mitogen to confirm whether the test system is working properly; meanwhile, it can compare the immune response from the cells to antigen and mitogen.

8.1 Principle

Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a living fluorescent dye that can penetrate the living cell membrane and irreversible binds to intracellular proteins.

When the cells divide, CFSE-labeled fluorescence is evenly distributed into the two daughter cells; its fluorescence intensity is half of that of the parental cells.

8.2 Test procedures

8.2.1 Aseptically prepare the mouse spleen lymphocyte suspension according to 7.2; wash at least 2 times by PBS; discharge the supernatant; resuspend the cells by 2mL of PBS containing 0.1% BSA; separately add CFSE till the final concentration is 5µM; mix up. Incubate at 37°C for 15min to 30min in the dark.

8.2.2 Add 3mL of pre-cooled RPMI1640 nutrient solution containing 2% FCS to stop the staining reaction; mix gently; after placing on the ice for 5min; take 400g to centrifuge for 5min; discharge the supernatant; add 2mL of RPMI1640 nutrient solution containing 10% FCS; mix up.

8.3 Data analysis

The test results shall be recorded by using the mean fluorescence intensity combined with the percentage of proliferating cells; appropriate statistical methods shall be used to deal with the test results.