( Dear customers, please read the following precautions carefully and refer to the operating procedures for the experiment)

1. Sample requirements: 1) When collecting fresh peripheral blood (umbilical cord blood), avoid using EDTA blood collection tubes. 

2) separate mononuclear cells from fresh blood samples within 6 hours. As time goes by, the number and activity of separated mononuclear cells will decrease, affecting the induction and expansion of NK cells. 

3) If the anticoagulant volume in whole blood is greater than 30% of the whole blood volume, it is recommended to use AB serum or serum substitutes instead of autologous plasma. 

4) Umbilical cord blood contains a large amount of red blood cells. It is recommended to separate and culture mononuclear cells after red blood cell sedimentation to reduce the impact of red blood cells. 

5) Cryopreserved mononuclear cells: Depending on the cell cryopreservation conditions, selective resting treatment is performed to restore the cell state or remove cells damaged by cryopreservation. (Brief description of steps: resuspend PBMC or CBMC in serum-free medium containing 10% cryopreserved autologous plasma or serum substitute to a density of 2.0-3.0 × 106 cells /mL, place in a culture flask, and rest in a 37°C incubator for 12-16 hours. Centrifuge at 400g for 5 minutes, resuspend the cells, and inoculate according to the counting density). 

2. Cultivation and treatment: 

1) Inoculation density requirements: Too low density is not conducive to cell proliferation, and too high density affects the purity of NK cells . The recommended seeding density of PBMC is 2.0-2.5 × 10 6 cells/mL . The recommended seeding density for CBMC is 3.0 - 3.5 ×10 6 cells/mL. It is recommended to increase the seeding density of cryopreserved mononuclear cells by 10-20% . 

2) The coating solution needs to be dissolved in DPBS without calcium or magnesium or 0.9% saline, and other lyophilized factors can be dissolved in NK serum-free medium .

3) Initial stage of culture (DAY 0-DAY 5): Do not shake the bottle at will, especially when replenishing the fluid on DAY 3. Slowly inject the prepared culture medium along the side wall of the culture bottle, be gentle and avoid disturbing the cells at the bottom of the bottle. 

4) Before each rehydration, an appropriate amount of NK serum-free medium needs to be taken for rewarming. If the temperature is too low, the cells are prone to form floccules and the viability is reduced. 

5) Fluid replenishment density and timing: The timing of fluid replenishment should be flexibly controlled. It is recommended to replenish fluid when the cell density reaches 1.5-3.5 ×106 cells /mL. After replenishment, the cell density is adjusted to about 0.8-1.2 ×106 cells /mL. When the cell expansion state is not ideal, the fluid replenishment can be appropriately postponed or /and plasma (5%-10%) can be added. If the operator is not sure whether fluid replenishment is needed, please contact our technical staff . 

6) the cells are transferred to the culture bag, it is recommended to tap or shake the culture bag regularly to prevent the cells from clumping too large or becoming too dense and to prevent the formation of flocs. 7) This product should be used within the validity period. After unsealing, the product should be used immediately or stored in aliquots, otherwise the product performance may be affected.

Background

NK cell serum-free medium

NK cell activation and expansion reagent (Cat. No.: CNK2401)

NK cell culture process ( for reference only, fluid can be replenished according to the actual growth of cells )

Day -1 (encapsulation)：

1. Take one tube of lyophilized powder of coating solution and dissolve it with 1 mL of DPBS (or normal saline).Add the dissolved coating solution to 8 mL DPBS and mix well, a total of 9 mL. Tilt the T75 culture flask (TC treatment) to transfer the coating solution to the bottom of the flask, then slowly level it and gently shake the flask to evenly cover the bottom of the flask with the coating solution.3. Place the culture flask flat in a 4°C refrigerator overnight. If time is urgent, you can also incubate it in a 37°C incubator for 2 hours. h ( If not used immediately, it can be stored in a 4°C refrigerator. It is recommended to use within one week ) .

Day 0 (mononuclear cell isolation and seeding) ：

1.30-40 mL of fresh anticoagulated whole blood from the donor (fresh cord blood is recommended to be treated with red blood cell sedimentation), take a small amount of blood sample for sterile streak test, and use lymphocyte separation fluid or lymphocyte separation tube to separate mononuclear cells from the remaining blood sample (see the instructions for use of the corresponding product for details). After centrifugation, cell stratification can be seen, with red blood cells at the bottom and plasma at the top, and mononuclear cells in the middle white film layer . 

2. Preparation of heat-inactivated autologous plasma: Pipette the upper plasma (avoid contact with the buffy coat layer) into a centrifuge tube and Centrifuge at room temperature for 10. The supernatant was transferred to a new centrifuge tube and inactivated at 56°C for 30 min. After the plasma was cooled in a 4°C refrigerator for 30 min (or in a -20°C refrigerator for 15 min), 1500 Centrifuge at room temperature for 10 min, and transfer the supernatant to a new centrifuge tube, which is the heat-inactivated autologous plasma. 

3. mononuclear cells : Pipette the buffy coat layer (mononuclear cells) into a 50 mL centrifuge tube, add DPBS or 0.9% saline (optionally add 1-2% 20% human albumin), mix well, centrifuge at 250g for 10 minutes at room temperature, wash 1-2 times, resuspend with 5-10 mL NK culture medium, count the cells and set aside. 

4. According to the counting results, adjust the PBMC density to 2.0-2.5 ×10 6 cells/mL (CBMC, the density is adjusted to 3.0 - 3.5 ×10 6 cells/mL; for cryopreserved mononuclear cells, it is recommended to increase the seeding density by 10-20%) using NK culture medium. Dissolve the lyophilized powder in 1 mL of NK culture medium and add it to the culture bottle. Add 1.5 mL of heat-inactivated autologous plasma to a final volume of 15 mL. Place the culture bottle in a 37°C, 5% CO 2 incubator and culture it horizontally.Note: The culture medium needs to be rewarmed at 37°C before each use. If the anticoagulant volume in the whole blood is greater than 30% of the whole blood volume, it is recommended to use AB serum or serum substitute..

Day 3 (rehydration, key step)：

1. The cell growth was observed under the microscope, and the cell clumping was as follows ( below, 40×). When the cell clumping rate was >30%, the fluid could be replenished normally (the picture shows the cell status of frozen cord blood D4, delayed for one Day). 

 2. Preparation of culture medium: Take one tube of NK-A freeze-dried powder, dissolve it with 1 mL of NK culture medium, add it to 26 mL of NK culture medium (in a 50 mL centrifuge tube), and add 3 mL of heat-inactivated autologous plasma ( total content is 10%), mix well, and the rehydration volume is 30 mL. 

3. Slowly add the prepared culture medium into the culture flask along the side wall of the culture flask to a final volume of about 45 mL. Mix gently and place flat in a 37°C, 5% CO2 incubator for culture. Note: Do not blow the cells at the bottom, just shake gently. For cryopreserved single nuclei or samples of poor quality, add fluid when the culture flask shows about 30% cell clumping rate. Otherwise, the rehydration can be appropriately delayed..

Day 5 (rehydration and bottle transfer)：

1. Observe the cell growth under the microscope, gently pipette 2-3 times to mix, and then transfer to a T175 culture flask. At this time, count the cells and pay attention to the cell density . 

2. Add 90 mL of NK culture medium (containing 5% heat-inactivated autologous plasma) and use part of the culture medium to rinse the residual cells in the original T75 culture flask and transfer them to a new T175 culture flask. Add one NK-B freeze-dried powder (dissolved in NK culture medium), and the final volume is about 135 mL. Mix gently and place in a 37°C, 5% CO 2 incubator for culture.

Day 7 (bag transfer, counting and rehydration)：

1. Preparation of culture medium: Take 1 tube of NK-C lyophilized powder, dissolve it in 1 mL of NK culture medium, and add it to the remaining NK culture medium in the culture medium bottle (about 865 mL).

2. the cells gently by pipetting, take a small amount of cell suspension for counting, adjust the cell density after rehydration to about 1.0 × 106 cells/mL, and calculate the rehydration volume. 

3. the cell suspension to a cell culture bag, add the prepared NK culture medium and the remaining plasma (about 2% heat-inactivated autologous plasma) according to the calculated volume of rehydration. Rinse the remaining cells in the original T175/T225 culture flask, transfer them to the culture bag, and culture them in a 37°C, 5% CO 2 incubator. Note: If the liquid in the cell culture bag is less than 500 mL, the culture bag needs to be folded and placed (one side of the culture bag needs to be rolled up to half its volume); after putting the bag in place, shake the cell bag as much as possible every Day to break up the cell clumps to prevent the cells from proliferating too quickly and clumping too large.

Day 9/11 / 13 (rehydration)：

1. Preparation of culture medium: Take 1 bottle of NK-C freeze-dried powder, dissolve it in 1 mL of NK culture medium, add it to the remaining 1 bottle of 1000 mL of NK culture medium and set aside. 

2. Before sampling, shake and tap the culture bag to mix the cell suspension (to avoid inaccurate counting), take a small amount of cell suspension for counting, and calculate the volume of rehydration according to the cell density of 1.0 × 10 6 cells/mL. Then, tap the culture bag to evenly distribute the cells and place them in a 37°C, 5% CO 2 incubator for culture (refer to the cell culture process table. Cord blood samples vary greatly, so you can pay attention to the node density. When proliferation slows down, increase the rehydration density in time to maintain 1.0-1.5E6/mL). 

3. DAY13 sampling and testing (as needed): Take a small amount of cell suspension for testing of bacteria, fungi, endotoxins, mycoplasma, cell phenotype, etc. .

Day 15-Day17 (Harvest)：

According to the cell proliferation, it is recommended to harvest the cells when the cell density reaches above 3.0×10 6 cells/mL . NK serum-free medium and NK-C factor can be added to extend the culture time to 20-23 Days to obtain more NK cells.