Background

Kit (Cat. No.: DNK2308-2 L) Composition

NKT cell serum-free medium

NKT cell activation and expansion reagent

NKT cell culture process (for reference only)

NKT Cell Activation and Expansion Culture Kit (2L System) Reference Operation Procedure

Day 0 (PBMC isolation and seeding)：

1.20-30 mL of fresh anticoagulated whole blood from the donor (usually, 1 mL of blood sample can harvest 1×10 6 mononuclear cells ) , take a small amount of blood sample for sterile streak test, and use lymphocyte separation fluid or lymphocyte separation tube to separate PBMC from the remaining blood sample (see the instructions for use of the corresponding product for details ). After centrifugation, cell stratification can be seen, the bottom layer is red blood cells, the top layer is plasma, and the middle white film layer is the mononuclear cell layer .

2.Preparation of heat-inactivated autologous plasma: Use a sterile pipette to draw the upper plasma (do not touch the buffy coat) into a centrifuge tube, centrifuge at 1000g for 10 minutes at room temperature , take the supernatant and inactivate it at 56℃ for 30 minutes, place it in a 4℃ refrigerator for 30 minutes (or -20 ℃ , 15 minutes) , cool and let it stand, then centrifuge at 1000g for 10 minutes at room temperature, transfer the supernatant to a new tube to obtain the heat-inactivated autologous plasma.;

3. PBMC separation: aspirate the buffy coat layer ( mononuclear cell layer) into a centrifuge tube, add 0.9% saline (containing 1-2% human albumin) and mix well, centrifuge at 250g for 10 minutes at room temperature, wash 1-2 times, resuspend with an appropriate amount of NKT serum-free medium , count the cells and set aside.;

4.  In a T75 culture flask, the final volume is 20 mL , and the PBMC concentration is adjusted to 50 μg/mL using NKT serum-free medium. 1.5 ×10 6 cells/mL , add 1.0 mL of heat inactivated autologous plasma ( 5 % ) . μL III NKT-A Add the factor reagent to the culture bottle, mix well, and place it flat in a 37°C, 5.0 % CO2 incubator for culture.

Note: Take an appropriate amount of culture medium and rewarm it to 37℃ before each rehydration.

Day 1 (complement factor)：

After 24 hours, take a 200 μL tube of III NKT-B factor and add it to a T75 culture flask. After gently mixing, place the flask flat in a 37°C, 5.0 % CO2 incubator for culture.

Note: Do not pipette the cells, just shake gently to mix.

Day 3 (rehydration)：

1. expansion medium I : Take a 10 00 Add μL of III NKT-C factor to the first bottle of NKT serum-free medium and mix well.

2. Add 30 mL of expansion medium I to the culture flask and add 1.5 mL of heat-inactivated autologous plasma (5 %), mix gently, and the final volume is about 50 mL. Place it flat in a 37°C, 5.0 % CO2 incubator for culture.

Note: Do not pipette the cells, just shake gently to mix.

Day 5-Day 9 (rehydration and bag transfer)：

1. On Day 5, observe the cell growth under the microscope. When the cells are clumping (40×) as shown in the figure, add 75 mL of Expansion Medium I into the culture flask, and add 3.5 mL of heat-inactivated autologous plasma (5 %). Gently pipette 2-3 times to mix. The final volume is about 125 mL . Place the cells flat in a 37°C, 5.0 % CO2 incubator for culture.

2. On Day 7, the cell culture medium in the T175 culture flask was gently pipetted 2-3 times and then transferred to the culture bag. The residual cells in the original T175 culture flask were rinsed with about 180 mL of expansion base medium I and transferred to the culture bag. 1-2 % heat-inactivated autologous plasma was added to a final volume of about 300 mL. The flask was placed in a 37°C, 5.0 % CO2 incubator for folding and culture.

3. After the cells are transferred to the culture bag, the liquid can be replenished flexibly by observing the cell growth status and the color of the culture fluid; usually, the liquid is replenished at an interval of 2 Days at a volume of 1.5 times ; if the sample is mixed and counted, the liquid can be replenished at a density of about 1.0×10 6 cells/mL . If the liquid in the cell culture bag is less than 500mL, it needs to be folded and placed. After the bag is loaded, the culture bag can be gently shaken every Day to break up the cell clumps.

Day 11 -Day 15 (rehydration)：

1. expansion medium II : add one 1000 μl of NKT serum-free medium to each bottle. μL of III NKT-D factor was prepared, and after adding expansion medium I, expansion medium II was used.

2. In the later stage of cell expansion, the solution is usually replenished at 0.5 times the volume every 2 Days ; if the sample is mixed and counted, the solution can be replenished at a density of about 1.5×10 6 cells/mL.

Day 1 7 -Day 19 (Harvest)：

1. Sampling inspection (as needed): Take a small amount of cell suspension from the bag for testing of bacteria, fungi, endotoxin, mycoplasma, cell phenotype, etc.

2. Delaying the harvest appropriately according to the cell density and viability can increase the purity and harvest quantity of NKT cells. It is generally recommended to harvest cells at a cell density of 3.0-3.5 × 106 cells/mL.