ProductSpecifications

Table 2 Immun-Eximmune cell-based medium product specifications

Table 3 Immun-ExImmun Cell Culture Medium Product specifications

Scope of Application

Onlyfor scientific and industrial use, cannot be used for human body.

Storage

Table 4. Immun-Exserum-free immune cell medium storage conditions

0°C-30°C environment for transportation.

VI. Experimental Materials

6.1Instruments and Equipment: Biological safety cabinet, CO2 incubator, invertedmicroscope, water bath, centrifuge, cell counter, and related equipment.

6.2Testing: DPBS, 0.2% of the blue or AO/PI chromosome, Immun-Ex serum-free immunecell medium, proteine median 2, CD3 antibodies, CD28 antibodies, L-glutamine,self-plasma or blood replacement, nutrient layer cells and so on.

6.3Consumer materials and container: cell cultivation bottles, cardiac tubes,transmissive tubes, etc.

Immunic Cell GeneralMedium

7.1Immun-Ex immune Cell Culture Medium additives, distributed to them according tothe actual dose, after distributed at ≤-20°C. The total freezing number of immun-Eximmun Cell Culture Medium additives should not exceed 2 times.

7.2Reference Table 5 describes the combined immune cell general medium, which canbe saved at 2-8°Cfor 2 weeks.

Table 5Immuno-cell General Medium Preparation Instructions

VIII. NK Cell Expansion (2 LCulture Example)

8.1NK cells fully medium preparation: add 5% self-plasma or blood replacement,IL-2 (200IU/mL) to the immune cells general medium, adding antibiotics asneeded. NK cells are fully medium and need to be presently attached.

8.2NK cells are fully medium for extending NK cells from fresh or frozenPBMC/UBMC. Table 6 is a proposal for expansion.

Table 6 NKExtension Programs

8.3Cell vaccination and activation (Day 0): with the frozen UBMC for example, acell (2-3 x 107), re-clean with PBS, and off-hearted cleansing. The NK cellsare fully medium resuspend the cells, adding to T75. Similarly restore anutrient layer cell, with NK cells completely medium hanging, adding to thecultivation bottle, mixed uniformly. The medium total is about 30 mL, the UBMCseeding density is about 1.0×106 Cells/mL, placed at 37°C,5% CO2incubator.

8.4Medium Exchange (Day 3): Collect the cells by centrifugation, resuspend them inNK Cell Complete Medium, and return them to the original culture flask. Adjustthe total culture volume to approximately 30 mL.

8.5Extension (4-6 days): Daily observation and supplementation of fresh medium,according to the medium color or cell density supplementation NK cells fullymedium. The supplement is 50%-100% of the original volume.

8.6reactivate (day 7): adding NK cells fully medium, making cell density at0.8-1.0 x 106 Cells/mL, and recovering a nutritious layer cell, mixed withmedium, transferred to cultivating bag.

8.7Extension (8-13 days): daily counting and adding to the cultivation bag freshNK cells completely medium, making cell density at 1.0-2.0×106 Cells/mL. Extending the last2-3 days, cells can grow at a higher density, but it is not recommended toexceed 3×106 Cells/mL.

8.8Crop: Depending on the required number of cells, NK cell purity and so onjudging the time of crop, generally grown up to the 12-14 day cells can becroped.

Use to expand T cells.

9.1T cells fully medium preparation: add 5% self plasma or blood replacement, IL-2(300IU/mL), L-glutamine (2mM) to the immune cells general medium, addantibiotics as needed. The T cell is fully medium and needs to be presentlyattached.

9.2package is prepared for work liquid: with DPBS, CD3 and CD28 antibodies arediluted to work concentration (1-20µg/mL), mixing two antibodies in the packagefor work liquid, available. The package required for different cultivationcontainers is the work liquid volume in Table 7, which can be made up accordingto the experimental plan and the package required to be prepared by the workliquid.

Table 7 Package ofdifferent cultivation containers is volume

9.2container package is: Package is added to the cell cultivation panel by workliquid, adding a circle of DPBS to prevent the marginal effect. The rollingplant plaque makes the package fully covered by liquid, and the back of themembrane is placed at 2-8°Covernight.

9.3Cell vaccination: with a frozen PBMC for example, recovering a PBMC, washingwith DPBS once again, separating the heart, with adequate T cells completelymedium hanging, counting. The package in the cell cultivation board is absorbedby work liquid, washed 2 times with DPBS and added PBMC cell suspension. Theseeding density is 1.0 x 106 cells/ml.

9.4Extended cultivation: when the living cell density reaches 2-3 x 106 cells/mL,it can be transmitted, the transmitted density is 0.5-0.8 x 106 cell/mL and 2-3days. The height of the cultivation fluid during the process of cultivationremains at 1-1.2 cm. The culture volume of different containers see Table 8.

Table 8 T cellcultivation system reference table