Serum-free culture medium for cryopreserved umbilical cord blood-derived NK (Natural Killer) cells and culture method of serum-free culture medium

Abstract

The invention relates to the technical field of NK cell culture, and discloses a serum-free medium for NK cells from umbilical cord blood after cryopreservation and a culture method thereof.The medium comprises a coating solution, an induction medium and an amplification medium; wherein the coating liquid is prepared from an anti-CD16 monoclonal antibody and DPBS (diphenylphosphonic acid); the induction culture medium comprises a basic culture medium, a human-derived platelet lysate and cell factors IL-15, IL-18 and IL-21; the amplification culture medium comprises a basic culture medium, a human-derived platelet lysate and IL-2. The culture method comprises the following steps: obtaining mononuclear cells from fresh umbilical cord blood, and then cryopreserving and resuscitating; and carrying out multiplication culture on the recovered umbilical cord blood mononuclear cells by using the serum-free culture medium. According to the NK cell culture medium, the NK cells with high purity, high killing activity and high cytotoxic activity can be obtained without autoserum culture.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to the field of NK cell amplification technology, and specifically to a serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood and a culture method thereof. Background Art

[0002] The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.

[0003] Natural killer cells (NK) are a type of innate lymphocytes with strong natural killer activity. They directly recognize and eliminate tumor cells and virus-infected cells in a non-MHC restricted manner, and indirectly improve the cellular immune response mediated by antibodies and T cells. They play a key role in clearing abnormal and damaged cells and senescent cells, killing pathogenic microorganisms, tumor immune monitoring and hematopoietic reconstruction.

[0004] In recent years, immunotherapy has developed rapidly, and NK cells have great potential in tumor immune cell therapy. For some blood system tumors, such as leukemia, lymphoma, etc., NK cell therapy has achieved good results. Due to the limited content of NK cells in the human body, it is impossible to obtain large quantities of high-purity human NK cells from the human body, which limits the application of NK cells in immunotherapy. It is particularly important to develop a safe NK cell culture system that can ensure the efficiency of NK cell proliferation and the killing effect on tumor cells. The in vitro expansion method can obtain a sufficient number of human NK cells with high purity, but it is not suitable for long-term storage, and the cytotoxicity is reduced after low-temperature storage, which is not conducive to later clinical treatment.

[0005] Currently, the sources of NK cell in vitro expansion are peripheral blood and umbilical cord blood. The variability of peripheral blood donors is large, and the total amount of NK cells obtained is unstable. NK cells from umbilical cord blood are not only safe and convenient, but also contain more NK precursor cells, making them a more ideal source for NK cell preparation. By separating, purifying and freezing fresh umbilical cord blood, mononuclear cells are obtained, and then the NK cells in the mononuclear cells are selectively induced and stimulated to expand, which expands the application direction of frozen umbilical cord blood. Autologous plasma is added to the culture process of preparing NK cells from fresh umbilical cord blood, but the autologous plasma of umbilical cord blood is limited. After freezing, the umbilical cord blood mononuclear cells face the problem of insufficient plasma after recovery. Fetal bovine serum is often used instead during the culture process. Fetal bovine serum is an animal-derived component and has certain risks in clinical practice.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide a serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood and a culture method thereof in view of the current situation that plasma is insufficient for culturing NK cells from cryopreserved umbilical cord blood, thereby solving the problems in the prior art of insufficient autologous plasma for culturing NK cells from cryopreserved umbilical cord blood mononuclear cells, complicated operation steps, and low purity of cultured NK cells.

[0008] The technical solution of the present invention is as follows:

[0009] A serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood, the NK cell culture medium comprising a coating solution, an induction culture medium and an expansion culture medium; wherein:

[0010] The coating solution includes anti-CD16 monoclonal antibody and DPBS;

[0011] The induction medium includes a basal medium, human platelet lysate and cytokines IL-15, IL-18 and IL-21;

[0012] The expansion medium comprises a basal medium, human platelet lysate and IL-2.

[0013] According to a preferred embodiment, the concentration of the anti-CD16 monoclonal antibody in the coating solution is 1-5 μg / mL, preferably 2.2-2.8 μg / mL.

[0014] According to a preferred embodiment, the concentration of IL-15 in the induction medium is 40-80 ng / mL, preferably 55-65 ng / mL; the concentration of IL-18 is 40-80 ng / mL, preferably 55-65 ng / mL; the concentration of IL-21 is 40-80 ng / mL, preferably 55-65 ng / mL.

[0015] According to a preferred embodiment, the amount of human platelet lysate in the induction medium is 5%-12% by volume, preferably 10%; the amount of human platelet lysate in the expansion medium is 1%-8% by volume, preferably 5%.

[0016] According to a preferred embodiment, the concentration of IL-2 in the expansion medium is 500-1500 IU / mL, preferably 900-1100 IU / mL.

[0017] According to a preferred embodiment, the basal culture medium is AIM-V culture medium.

[0018] Another aspect of the present invention provides a serum-free culture method for cryopreserved NK cells derived from umbilical cord blood, using the serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood as described above, specifically comprising the following steps:

[0019] Step A, extracting and separating mononuclear cells from fresh umbilical cord blood;

[0020] Step B, freezing the separated umbilical cord blood mononuclear cells;

[0021] Step C, thawing the umbilical cord blood mononuclear cells frozen in step B;

[0022] Step D: inducing, expanding and culturing the cord blood mononuclear cells resuscitated in step C.

[0023] According to a preferred embodiment, the step A specifically includes the following sub-steps:

[0024] Step A1: Collect fresh umbilical cord blood and separate it by gradient centrifugation. Put the fresh umbilical cord blood into the clean bench, use a 50ml syringe to extract the umbilical cord blood from the blood bag into a 50ml centrifuge tube, and centrifuge at 3500r for 20min at room temperature.

[0025] Step A2: After centrifugation, draw the upper plasma into a new 50 ml centrifuge tube, add an appropriate amount of physiological saline to dilute the blood cell pellet in a ratio of 1:1, mix by pipetting, and slowly add the suspension along the wall of the centrifuge tube into the 15 ml centrifuge tube containing the lymphocyte separation solution to ensure clear stratification. Centrifuge at 2500r for 20 minutes at room temperature, with slow rise and slow fall.

[0026] Step A3: After centrifugation, the middle white film layer is aspirated, and the volume is resuspended and mixed with physiological saline to 50 ml, and samples are taken for counting. The obtained 50 ml mononuclear cell suspension is further diluted 5 times to reduce cell centrifugation loss; centrifuged at 1500r for 5 minutes at room temperature, and the supernatant is discarded after centrifugation. The preparation of umbilical cord blood mononuclear cells is completed.

[0027] According to a preferred embodiment, the step B specifically includes the following sub-steps:

[0028] Step B1: Resuspend the umbilical cord blood mononuclear cells with an appropriate amount of serum-free cell freezing solution and adjust the frozen cell density to (1-10)×10 7 cells / ml. Furthermore, the cryopreservation density of umbilical cord blood mononuclear cells is (2-4)×10 7 cells / ml.

[0029] Step B2: Dispense the resuspended cells evenly into cryotubes, with each cryotube dispensing approximately 1.5ml-4ml. Tighten the cryotube caps, label, and transfer to a liquid nitrogen tank for long-term storage after programmed cooling.

[0030] According to a preferred embodiment, the step C specifically includes the following sub-steps:

[0031] Step C1, taking out the frozen umbilical cord blood mononuclear cells from liquid nitrogen and placing them in a 37°C water bath for recovery;

[0032] Step C2: After the ice crystals melt, take out the cells immediately, transfer the thawed mononuclear cells to a sterile centrifuge tube containing an appropriate amount of culture medium, add an appropriate amount of NK cell culture medium, mix gently, take samples and count. Centrifuge at 1500r for 5 minutes at room temperature, remove the supernatant. Furthermore, add NK cell culture medium at a volume ratio of 1:10.

[0033] According to a preferred embodiment, the step D specifically includes the following sub-steps:

[0034] Step D1: Use coating solution to coat T75 bottle at room temperature for 30 minutes in advance.

[0035] Step D2, resuspend the umbilical cord blood mononuclear cells centrifuged in step C2 in an appropriate amount of induction medium, then inoculate them in a coated T75 culture bottle, and place them in a 37°C, 5% CO2 incubator for induction culture. The day of inoculation is recorded as day 0, and the induction culture is 5-6 days. Further, the volume of the NK cell induction medium is 20 ml.

[0036] Furthermore, the inoculation amount of the umbilical cord blood mononuclear cells is 1-2×10 6 cells / mL.

[0037] Step D3, after culturing to the 6th to 7th day, take out the T75 flask, blow the culture flask to disperse the cell clones attached to the bottom of the flask, transfer the cell suspension to a 50 ml centrifuge tube, centrifuge at 1500r for 5 min, remove the supernatant after centrifugation, add 200 ml of expansion medium, transfer the resulting precipitated cells into a cell culture bag, expel excess air and bubbles, and continue culturing in a 37°C, 5% CO2 incubator;

[0038] Step D4: After culturing for 8-11 days, add 800 ml of expansion medium to the cell culture bag and place the bag in a 37° C., 5% CO 2 incubator for further culturing;

[0039] Step D5: Cultivate until the 12th to 21st day to obtain NK cells. During the culture process, the NK cells can be further cultured in separate bags according to their proliferation, and the volume can be expanded to 1500-2000 ml.

[0040] Preferably, the 800 ml expansion medium added in step D4 contains or does not contain human platelet lysate.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] 1. A serum-free culture medium for NK cells derived from umbilical cord blood after cryopreservation, which uses human platelet lysate instead of autologous serum, and the synergistic stimulation of cytokines IL-2, IL-15, IL-18 and IL-21 in the combined induction culture medium to induce cell growth, and CD16 monoclonal antibodies are used to coat the culture bottles to act on mononuclear cells derived from umbilical cord blood, so that serum-free and efficient amplification of NK cells derived from umbilical cord blood after cryopreservation can be achieved, and a large number of NK cells with high purity, high killing activity and high cytotoxic activity can be obtained, and a stable and efficient clinical-grade NK cell preparation method has been established; the problem of insufficient plasma in the later culturing of NK cells when a large number of mononuclear cells are separated from umbilical cord blood is solved, which meets the needs of clinical applications; the clinical risks of fetal bovine serum are avoided; and even NK cells with reduced cell activity after cryopreservation can achieve efficient amplification, and the function of promoting NK cell growth is significant;

[0043] 2. The present invention provides a new idea for adoptive NK cell therapy, which solves the problems of decreased NK cell toxicity and cumbersome operation in the current cryopreservation of large-scale expanded NK cells in vitro. The use of cryopreserved mononuclear cells derived from umbilical cord blood to induce and expand NK cells ensures the same donor source in the subsequent adoptive NK cell therapy, solves the problem of donor differences, is more flexible and convenient in the clinical treatment cycle, and can better meet the cell quantity requirements of different cycles in NK cell therapy. It expands new directions for the application of cryopreserved umbilical cord blood. The use of cryopreserved mononuclear cells derived from umbilical cord blood to induce and expand NK cells does not require the introduction of tumor cells or the addition of animal serum, avoids safety hazards, and has higher safety.

[0044] 3. The NK culture method provided by the present invention solves the problem of low recovery rate of mononuclear cells from umbilical cord blood, effectively utilizes umbilical cord blood resources, provides a stable donor source for later clinical research, and solves the problems of insufficient number and low purity of in vitro expanded NK cells; by optimizing the umbilical cord blood separation technology, the recovery rate of mononuclear cells from umbilical cord blood is improved, so that 20-40 NK cells can be expanded simultaneously from one umbilical cord blood;

[0045] 4. The influence of red blood cells is excluded in the initial stage of culturing umbilical cord blood mononuclear cells after freezing. The purity of NK cells harvested later can reach more than 80%, or even as high as 90%, which has great advantages in the application of allogeneic NK cells;

[0046] 5. It overcomes the technical bias of adding K562 trophoblast cells and fetal bovine serum in the existing NK cell culture process, greatly improving the safety and clinical applicability of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1The cell morphology diagrams of NK cells cultured on the 3rd, 7th and 14th days provided in Example 1 of the present invention;

[0048] Figure 2 This is a flow cytometric graph of NK cells cultured on the 20th day provided in Example 1 of the present invention;

[0049] Figure 3 The NK cell killing activity against K562 provided in Examples 1-3 of the present invention;

[0050] Figure 4 This is a diagram showing the killing of K562 cells by NK cells provided in Example 1 of the present invention;

[0051] Figure 5 The NK cell culture proliferation curve provided in Example 1 of the present invention;

[0052] Figure 6 The flow cytometry diagram of NK cells cultured on the 20th day provided in Comparative Example 2 of the present invention;

[0053] Figure 7 This is a flow cytometry graph of NK cells after cryopreservation provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0054] The specific embodiments listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific embodiments described below. For those skilled in the art, any equivalent modifications and substitutions to the embodiments described below are also within the scope of the present invention. Therefore, the equal transformations and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturers are not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are given in the specific embodiments below. It should be understood by those skilled in the art that the present invention can also be implemented without certain specific details. In other embodiments, the methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the purport of the present invention.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. Unless otherwise specified, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0056] In the embodiments and comparative examples of the present invention:

[0057] Anti-CD16 monoclonal antibody was purchased from Tongli Haiyuan Company;

[0058] IL-15, IL-18, and IL-21 were purchased from Peprotech;

[0059] IL-2 was purchased from Beijing Shuanglu;

[0060] AIM-V medium was purchased from Gibco;

[0061] Lymphocyte separation medium was purchased from Tianjin Haoyang;

[0062] 1-liter NK cell culture bags were purchased from Corning;

[0063] DPBS was purchased from BI;

[0064] K562 tumor cells were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences.

[0065] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0066] Example 1

[0067] This embodiment provides a serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood and a preparation method thereof, comprising the following process:

[0068] 1. Extract and separate mononuclear cells from fresh umbilical cord blood.

[0069] 1. Take 100ml of fresh umbilical cord blood and put it into the clean bench. Use a 50ml syringe to transfer the umbilical cord blood in the blood bag into a 50ml centrifuge tube. Each tube is filled with 25ml. Centrifuge at 3500r for 20min at room temperature.

[0070] 2. After centrifugation, slowly draw the upper plasma into a new 50ml centrifuge tube for later use, and keep a sample for inspection;

[0071] 3. Use physiological saline or PBS solution to dilute the lower red blood cell sediment at a volume ratio of 1:1. After mixing by pipetting, slowly add the suspension into a 15 ml centrifuge tube containing 6 ml of lymphocyte separation solution, and centrifuge at 2500r for 20 min at room temperature.

[0072] 4. After centrifugation, the blood was separated into four layers. The mononuclear cell layer (buffy coat layer) of the second layer was aspirated and processed. The mononuclear cell layer was resuspended to 50 ml with saline solution. Samples were taken for counting and flow cytometry was performed to detect the cell phenotype to obtain the initial number and proportion of NK cells.

[0073] 5. Take another 5 new 50ml centrifuge tubes, dilute the obtained 50ml mononuclear cell suspension 5 times with physiological saline, centrifuge at 1500r for 5 minutes at room temperature, remove the supernatant after centrifugation, and the preparation of umbilical cord blood mononuclear cells is completed.

[0074] 2. Cryopreservation and thawing of cord blood mononuclear cells

[0075] 1. Collect the mononuclear cells from the umbilical cord blood and precipitate them according to the cell counting results. 6 cells / ml freezing density, add appropriate amount of pre-cooled NK cell freezing solution to resuspend the cells, mix well, and dispense the cell suspension into cryopreservation tubes.

[0076] 2. Cryopreserved umbilical cord blood mononuclear cells were cryopreserved in a programmed cooling manner. After being placed in a -80°C refrigerator for 18-24 hours, the cryopreserved cells were taken out and transferred to liquid nitrogen for storage as soon as possible;

[0077] 3. Take out the cells from liquid nitrogen and quickly immerse the cryotube in a 37°C constant temperature water bath to thaw it as soon as possible. After the ice crystals melt, immediately transfer the cell suspension to a 15ml centrifuge tube containing 10ml NK cell culture medium, mix gently, centrifuge at 1500r for 5min at room temperature, and discard the supernatant.

[0078] 3. Induction and Expansion of NK Cells

[0079] 1. Induction of NK cells

[0080] (1) Before culture: Add coating solution to the T75 culture flask and place in a 4°C refrigerator overnight, or leave at room temperature for at least 30 minutes.

[0081] The coating solution is prepared by diluting the anti-CD16 monoclonal antibody to a concentration of 2.5 μg / mL using an appropriate amount of DPBS solution.

[0082] (2) Prepare NK cell serum-free induction medium: add 60 ng / ml IL-15, 60 ng / ml IL-18, 60 ng / ml IL-21 and 2 ml of human platelet lysate to 18 ml AIM-V medium.

[0083] (3) Inoculation of cryopreserved umbilical cord blood mononuclear cells at a density of 1x10 6 After the cord blood mononuclear cells were resuspended in the induction medium, they were inoculated into the coated T75 culture flask. The day of inoculation was recorded as day 0, and the flask was placed in a 37°C, 5% CO2 incubator for 6-7 days.

[0084] 2. Expansion and culture of NK cells

[0085] (1) Prepare NK cell serum-free expansion medium without human platelet lysate: add 1000 IU / ml IL-2 to 1000 ml AIM-V medium,

[0086] (2) On the 7th day of induction culture, take out the T75 flask from the 37°C, 5% CO2 incubator, blow the culture flask to disperse the cell clones attached to the bottom of the flask, and transfer the cell suspension to a 50ml centrifuge tube and centrifuge at 1500r for 5min. After centrifugation, remove the supernatant, add 190ml of NK cell serum-free expansion medium without human platelet lysate, and add 10ml of human platelet lysate to it to prepare NK cell serum-free expansion medium containing 5% human platelet lysate. After blowing and mixing, transfer the cells to the NK cell culture bag, expel excess air and bubbles, and continue to culture in a 37°C, 5% CO2 incubator for 8-11 days.

[0087] (3) On the 11th day of expansion culture, 800 ml of NK cell serum-free expansion medium without human platelet lysate was added to the NK cell culture bag, excess air and bubbles were extracted, and the bag was placed in a 37° C., 5% CO 2 incubator for continued culture.

[0088] (4) Harvest cells on day 12-21 of culture: transfer the cell culture medium to a 250 ml centrifuge tube, centrifuge at 1500 r / min for 5 min, collect cells, take samples and count, calculate the viability, and freeze for storage or future use.

[0089] IV. NK cell quality detection

[0090] (1) Morphology and number of NK cells

[0091] During the culture process, the morphology of NK cells is examined under a microscope. An appropriate amount of cell suspension is pipetted into a T25 bottle, which is then placed under a microscope to observe the cell morphology and take photos for recording. The number and viability of NK cells are counted using a hemacytometer. 500ul of the mixed cell suspension is pipetted into a 1.5ml EP tube. 10ul of the mixed cell suspension is pipetted with a pipette and 10ul of 0.4% trypan blue staining solution is added to mix. Then 10ul of the stained cell suspension is taken for flushing and counting. Under a microscope, dead cells are stained light blue or blue, and live cells are bright particles. The specific cell morphology is as follows: Figure 1 The cell proliferation curve is shown in Figure 5 shown.

[0092] (2) NK cell surface markers (CD3 - CD56 + )Detection

[0093] The umbilical cord blood mononuclear cells before co-culture and the NK cells after culture were collected, centrifuged at 1500r for 5min, resuspended in 1ml DPBS, and the cells were counted. After the cell count, the cell density was adjusted to 1×10 6cells / 100μl, and the corresponding NK cell flow cytometry antibody was added at a ratio of 4μl to each tube of cell suspension, and flow cytometry was used for flow cytometry detection.

[0094] (3) NK cell killing experiment in vitro

[0095] 3.1 Lactate dehydrogenase release method

[0096] Cryopreserved cord blood NK cells were used as effector cells, and K562 tumor cells were used as target cells. Added to 96-well plates at a ratio of 5:1, 10:1, 20:1, and 40:1, with a total volume of 200uL per well. Three replicate wells were set up in each group and placed in a 37°C, 5% CO2 incubator for 4 hours. Operated according to the lactate dehydrogenase cytotoxicity detection kit, the absorbance A value was measured at 490nm. The killing efficiency was calculated according to the formula. Cytotoxicity (%) = (absorbance of effector cell and target cell experimental wells - absorbance of target cell natural release control well) / (absorbance of maximum release control well - absorbance of target cell natural release control well) × 100.

[0097] 3.2 Calcein / PI double staining method

[0098] Target cell preparation: Collect target cells K562, centrifuge at 1200r for 5min at room temperature, resuspend the cells in basal medium, and adjust the target cell density to 1×10 6 Effector cell preparation: Take an appropriate amount of NK cells and collect the cells by centrifugation at room temperature; resuspend the cells in basal medium and adjust the target cell density to 1×10 6 cells / ml. Add the cells to a 96-well plate at a ratio of 5:1, 10:1, 20:1, and 40:1, with a total volume of 200uL per well. Set up 3 replicate wells in each group and culture them in a 37°C, 5% CO2 incubator for 4 hours. Prepare the working solution according to the Calcein-PI cell activity and cytotoxicity detection kit, and incubate the cells in a 37°C incubator in the dark for 30 minutes. After the incubation, take out the cell culture plate and take pictures with a fluorescence microscope. The results are as follows Figure 4 shown.

[0099] In order to verify the technical stability of the present invention, the operation is consistent with the method of Example 1, and Example 2 and Example 3 are added.

[0100] Comparison of NK cell expansion multiples and viability:

[0101] Three cryopreserved umbilical cord blood NK cells were induced and cultured using the patented method of the present invention. After culturing for 25 days, the cells were centrifuged at 1500 r / min, collected, stained with trypan blue, and counted. As shown in Table 1, the total number of cryopreserved umbilical cord blood NK cells amplified and cultured by the present invention was >3×10 at 25 days as the culture time was prolonged. 9 , the survival rate is above 95%.

[0102] Table 1:

[0103]

[0104] Purity comparison of NK cells:

[0105] During the culture process, NK cells on days 0, d7, and d25 were selected for phenotype detection using flow cytometry. The results are shown in Table 2 and Figure 2 As shown, the surface marker of NK cells is CD3 - CD56 + , and CD3 - CD56 + CD16 + The indexes were all above 85%, and the purity was high. The NK cells of Example 1 were frozen for 3 months and then revived for flow cytometry detection. The detection results were as follows: Figure 7 As shown, despite cryopreservation, NK cells’ CD3 - CD56 + CD16 + The indicator is still above 90%.

[0106] Table 2:

[0107]

[0108] Comparison of NK cell killing function:

[0109] Cryopreserved cord blood NK cells were used as effector cells; K562 tumor cells were used as target cells, with effector-target ratios of 5:1, 10:1, 20:1, and 50:1. The killing efficiency was calculated according to the formula: cytotoxicity (%) = (absorbance of effector cell and target cell experimental wells - absorbance of target cell natural release control wells) / (absorbance of maximum release control wells - absorbance of target cell natural release control wells) × 100. The results are shown in Figure 3 shown.

[0110] Comparative Example 1

[0111] The NK cell culture medium of this comparative example is used to induce and expand NK cells.

[0112] The NK cell culture medium provided in this comparative example is different from that in Example 1 in that IL-18 is not added for expansion culture during the NK cell induction stage. The formula of the induction culture medium used in this comparative example 1 is: AIM-V culture medium containing 10% human platelet lysate, 60ng / ml IL-15, and 60ng / ml IL-21 by volume.

[0113] The results showed that without IL-18 induction and stimulation, the number of cells that expanded was very small and almost no expansion was achieved.

[0114] Comparative Example 2

[0115] The NK cell culture medium of this comparative example is used to induce and expand NK cells.

[0116] The NK cell culture medium provided in this comparative example is different from that in Example 1 in that autologous serum is used for expansion culture. The formula of the induction culture medium used in this comparative example is: AIM-V culture medium containing 10% autologous serum by volume, 60ng / ml IL-15, 60ng / ml IL-18, and 60ng / ml IL-21; the formula of the expansion culture medium is: AIM-V culture medium containing 5% autologous serum by volume, and 1000IU / ml IL-2.

[0117] Flow cytometry images on day 21 of culture Figure 6 As shown, CD3 - CD56 + CD16 + The index is 83.86%, and the purity is significantly lower than that of Example 1.

[0118] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood, characterized in that: It includes coating solution, induction medium and expansion medium; wherein, The coating solution includes anti-CD16 monoclonal antibody and DPBS; The induction medium includes a basal medium, human platelet lysate and cytokines IL-15, IL-18 and IL-21; The expansion medium comprises a basal medium, human platelet lysate and IL-2.

2. The serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood according to claim 1, characterized in that: The concentration of the anti-CD16 monoclonal antibody in the coating solution is 1-5 μg / mL, preferably 2.2-2.8 μg / mL.

3. The serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood according to claim 1, characterized in that: The concentration of the IL-15 in the induction medium is 40-80 ng / mL, preferably 55-65 ng / mL; the concentration of the IL-18 is 40-80 ng / mL, preferably 55-65 ng / mL; the concentration of the IL-21 is 40-80 ng / mL, preferably 55-65 ng / mL.

4. The serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood according to claim 1, characterized in that: The amount of human platelet lysate in the induction medium is 5%-12% by volume, preferably 10%; the amount of human platelet lysate in the expansion medium is 1%-8% by volume, preferably 5%.

5. The serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood according to claim 1, characterized in that: The concentration of IL-2 in the expansion medium is 500-1500 IU / mL, preferably 900-1100 IU / mL.

6. The serum-free culture medium for NK cells derived from cryopreserved umbilical cord blood according to claim 1, characterized in that: The basic culture medium is AIM-V culture medium.

7. A serum-free culture method for NK cells derived from umbilical cord blood after cryopreservation, characterized in that: Cultivating the NK cells derived from cryopreserved umbilical cord blood using a serum-free culture medium according to any one of claims 1 to 6 specifically comprises the following steps: Step A, extracting and separating umbilical cord blood mononuclear cells from fresh umbilical cord blood; Step B, freezing the separated umbilical cord blood mononuclear cells; Step C, thawing the umbilical cord blood mononuclear cells frozen in step B; Step D: Inducing and amplifying the cord blood mononuclear cells resuscitated in step C using a serum-free culture medium for NK cells derived from frozen cord blood as described in any one of claims 1 to 6.

8. The serum-free culture method of NK cells derived from cryopreserved umbilical cord blood according to claim 7, characterized in that: The step D specifically includes the following sub-steps: Step D1, coating the T75 bottle with coating solution at room temperature in advance; Step D2, resuspending the cord blood mononuclear cells resuscitated in step C in the induction medium, and then inoculating them in a T75 culture flask coated with the coating solution, the day of inoculation is recorded as day 0, and culturing them in an incubator; Step D3, culturing to the 6th to 7th day, obtaining cells by centrifugation, transferring the obtained precipitated cells into a cell culture bag, adding expansion medium, and placing in an incubator for further culturing; Step D4: On the 8th to 11th day of culturing, add expansion medium to the cell culture bag and place the bag in an incubator for further culturing; Step D5: Culture until the 12th to 21st day to obtain NK cells.

9. The serum-free culture method of NK cells derived from cryopreserved umbilical cord blood according to claim 8, characterized in that: In step D4, the added expansion medium may or may not contain human platelet lysate.

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