A resuscitation medium and resuscitation method for NK cells

Abstract

The present invention relates to the field of immune cell recovery technology, and more particularly to a recovery culture medium and recovery method for NK cells. The NK cell recovery culture medium of the present invention, the following components by weight are added per liter of basal culture medium: mevalonate lactone 0.015-0.05mg, polygonatum spirosterol 0.05-0.2mg, IL-15 0.02-0.1mg, transferrin 2-10mg, insulin 6-17mg, polylysine 0.01-0.03mg, aloin 3-8mg. The present invention adds mevalonate lactone and polygonatum spirosterol components to the recovery culture medium, and the experimental results confirm that the synergistic effect of mevalonate lactone and polygonatum spirosterol can effectively improve the viability of NK cells after cryopreservation recovery, maintain the killing activity of NK cells after cryopreservation recovery, and is of great significance for the clinical application of subsequent NK cells.

Description

Technical Field

[0001] The present invention relates to the technical field of immune cell resuscitation, and in particular to a resuscitation culture medium and a resuscitation method for NK cells. Background Art

[0002] Tumor cell immunotherapy technology is a new type of tumor treatment method. It is a treatment method that uses in vitro amplification technology to expand and culture the patient's autologous or allogeneic immune cells and then re-injects the immune cells into the patient's body. It has the advantages of a wide tumor killing recognition spectrum, strong lethality, and small toxic side effects. Natural killer cells (NK) are one of the cells in tumor immunotherapy technology.

[0003] NK cells are a type of lymphoid stem cells derived from bone marrow and have CD3 - CD56 + Immune effector cells with large granules have dual functions of cytotoxicity and immunoregulation, and can play an important role in the immune clearance of abnormal cells. Unlike other lymphocytes, such as T lymphocytes and B lymphocytes, NK cells are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization. Studies have found that NK cells can release cytotoxic molecules or granzymes, thereby eliminating tumor lesions, inducing tumor cell apoptosis, and inhibiting tumor metastasis.

[0004] NK cells used in clinical practice are usually cells that have been frozen in liquid nitrogen. The applicant team has previously analyzed and summarized a large amount of clinical experimental data and found that NK cells that have been frozen in liquid nitrogen often have technical problems such as low cell viability and poor killing activity, which will affect the subsequent clinical application of NK cells. In view of this, it is urgent to adjust the NK cell recovery medium to improve the viability and killing activity of NK cells after recovery. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, one of the purposes of the present invention is to provide a NK cell recovery medium. The recovery medium of the present invention can effectively improve the viability of NK cells after cryopreservation and recovery, maintain the killing activity of NK cells after cryopreservation and recovery, which is of great significance for the subsequent clinical application of NK cells.

[0006] A second object of the present invention is to provide a method for resuscitating NK cells, which is simple and easy to operate.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A NK cell recovery medium is provided, wherein the following components are added in parts by weight per liter of basic medium: 0.015-0.05 mg of mevalonate lactone, 0.05-0.2 mg of polygonatum spirosterol, 0.02-0.1 mg of IL-15, 2-10 mg of transferrin, 6-17 mg of insulin, 0.01-0.03 mg of polylysine, and 3-8 mg of aloin.

[0009] Preferably, the following components are added in parts by weight per liter of basal culture medium: 0.03 mg of mevalonolactone, 0.07 mg of polygonatum spirosterol, 0.05 mg of IL-15, 7 mg of transferrin, 11 mg of insulin, 0.02 mg of polylysine, and 5 mg of aloin.

[0010] Preferably, the basal culture medium is RPMI 1640 serum-free culture medium.

[0011] The second object of the present invention is achieved by adopting the following technical solution:

[0012] A method for resuscitating NK cells comprises the following steps: thawing a cryopreservation tube of NK cells frozen in liquid nitrogen in a water bath, adding a basal culture medium for dilution and then centrifuging, collecting cells, and inoculating the cells into the above-mentioned resuscitation culture medium for cultivation to complete resuscitation.

[0013] Preferably, the inoculation density of the cells in the recovery medium is 1.5×10 5 -5×10 5 Pieces / mL.

[0014] Preferably, the temperature of the water bath is 37°C-40°C, and the time is 2-3 minutes.

[0015] Preferably, the culture is carried out at 37°C and 5% CO 2 Conditions are carried out.

[0016] Preferably, the culture time is 16-24 hours.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The NK cell recovery medium of the present invention adds mevalonate lactone and polygonatum spirosterol components, and the experimental results also confirm that the synergistic effect of mevalonate lactone and polygonatum spirosterol can effectively improve the viability of NK cells after cryopreservation and recovery, and maintain the killing activity of NK cells after cryopreservation and recovery, which is of great significance for the subsequent clinical application of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the morphology of NK cells;

[0020] Figure 2The activity rate of NK cells after cryopreservation and resuscitation of Example 1-3 and Comparative Example 1-5 is shown;

[0021] Figure 3 This is a diagram showing the expression of CD107a on NK cells after resuscitation in Example 1-3 and Comparative Example 1-5;

[0022] Figure 4 This is a graph showing the killing activity of NK cells after resuscitation in Example 1-3 and Comparative Example 1-5. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0024] The method for obtaining NK cells in the embodiment of the present invention is:

[0025] Peripheral venous blood was collected from healthy volunteers using a blood collection tube supplemented with sodium heparin. Lymphocyte separation solution was then added and centrifuged to obtain a mononuclear cell layer, which was then washed with PBS and incubated at 37°C and 5% CO. 2 The cells were cultured in a saturated humidity incubator for 14 days in a serum-free RPMI-1640 medium supplemented with 500 U / mL interleukin-2 and 5% human AB plasma to obtain NK cells for later use. Figure 1 shown.

[0026] 1. Implementation

[0027] Example 1

[0028] Example 1 provides a NK cell recovery medium, wherein the following components in parts by weight are added to each liter of RPMI 1640 serum-free medium:

[0029] Table 1

[0030]

[0031] A method for resuscitating NK cells, wherein the specific resuscitation process is as follows:

[0032] The cryopreserved tubes containing NK cells (frozen for 1 month) were taken out of liquid nitrogen, thawed in a 37°C water bath for 2 min, diluted with RPMI 1640 serum-free medium, and centrifuged to collect the cells. The cells were inoculated into the above-mentioned recovery medium at a density of 2×10 5 / mL, at 37°C, 5% CO 2 The cells were resuscitated after culturing for 20 h under the given conditions.

[0033] Example 2

[0034] Example 2 provides a NK cell recovery medium, wherein the following components in parts by weight are added per liter of RPMI 1640 serum-free medium:

[0035] Table 2

[0036]

[0037] A method for resuscitating NK cells, wherein the specific resuscitation process is as follows:

[0038] The cryopreserved tubes containing NK cells (frozen for 1 month) were taken out of liquid nitrogen, thawed in a 38°C water bath for 3 min, diluted with RPMI 1640 serum-free medium, and centrifuged to collect the cells. The cells were inoculated into the above-mentioned recovery medium. The inoculation density of the cells in the recovery medium was 1.5×10 5 / mL, at 37°C, 5% CO 2 The cells were resuscitated after culturing for 16 h under the given conditions.

[0039] Example 3

[0040] Example 3 provides a NK cell recovery medium, wherein the following components in parts by weight are added per liter of RPMI 1640 serum-free medium:

[0041] Table 3

[0042]

[0043] A method for resuscitating NK cells, wherein the specific resuscitation process is as follows:

[0044] The cryopreserved tubes containing NK cells (frozen for 1 month) were taken out of liquid nitrogen, thawed in a 40°C water bath for 2 min, diluted with RPMI 1640 serum-free medium, and centrifuged to collect the cells. The cells were inoculated into the above-mentioned recovery medium at a density of 5×10 5 / mL, at 37°C, 5% CO 2 The cells were resuscitated after culturing for 24 h under the given conditions.

[0045] 2. Comparison

[0046] Comparative Example 1

[0047] Comparative Example 1 provides a NK cell recovery medium, wherein the following components in parts by weight are added per liter of RPMI 1640 serum-free medium:

[0048] Table 4

[0049]

[0050] A method for resuscitating NK cells, wherein the specific resuscitation process is as follows:

[0051] The cryopreserved tubes containing NK cells (frozen for 1 month) were taken out of liquid nitrogen, thawed in a 37°C water bath for 2 min, diluted with RPMI 1640 serum-free medium, and centrifuged to collect the cells. The cells were inoculated into the above-mentioned recovery medium at a density of 2×10 5 / mL, at 37°C, 5% CO 2 The cells were resuscitated after culturing for 20 h under the given conditions.

[0052] Comparative Example 2

[0053] Comparative Example 2 provides a NK cell recovery medium, wherein the following components in parts by weight are added per liter of RPMI 1640 serum-free medium:

[0054] Table 5

[0055]

[0056] A method for resuscitating NK cells, wherein the specific resuscitation process is as follows:

[0057] The cryopreserved tubes containing NK cells (frozen for 1 month) were taken out of liquid nitrogen, thawed in a 37°C water bath for 2 min, diluted with RPMI 1640 serum-free medium, and centrifuged to collect the cells. The cells were inoculated into the above-mentioned recovery medium at a density of 2×10 5 / mL, at 37°C, 5% CO 2 The cells were resuscitated after culturing for 20 h under the given conditions.

[0058] Comparative Example 3

[0059] Comparative Example 3 provides a NK cell recovery medium, wherein the following components in parts by weight are added per liter of RPMI 1640 serum-free medium:

[0060] Table 6

[0061]

[0062] A method for resuscitating NK cells, wherein the specific resuscitation process is as follows:

[0063] The cryopreserved tubes containing NK cells (frozen for 1 month) were taken out of liquid nitrogen, thawed in a 37°C water bath for 2 min, diluted with RPMI 1640 serum-free medium, and centrifuged to collect the cells. The cells were inoculated into the above-mentioned recovery medium at a density of 2×10 5 / mL, at 37°C, 5% CO2 The cells were cultured under the same conditions for 20 h and the recovery was completed.

[0064] Comparative Example 4

[0065] Comparative Example 4 provides a NK cell recovery medium, wherein the following components in parts by weight are added per liter of RPMI 1640 serum-free medium:

[0066] Table 7

[0067]

[0068] A method for resuscitating NK cells, wherein the specific resuscitation process is as follows:

[0069] The cryopreserved tubes containing NK cells (frozen for 1 month) were taken out of liquid nitrogen, thawed in a 37°C water bath for 2 min, diluted with RPMI 1640 serum-free medium, and centrifuged to collect the cells. The cells were inoculated into the above-mentioned recovery medium at a density of 2×10 5 / mL, at 37°C, 5% CO 2 The cells were resuscitated after culturing for 20 h under the given conditions.

[0070] Comparative Example 5

[0071] Comparative Example 5 provides a NK cell recovery medium, wherein the following components in parts by weight are added per liter of RPMI 1640 serum-free medium:

[0072] Table 8

[0073]

[0074] A method for resuscitating NK cells, wherein the specific resuscitation process is as follows:

[0075] The cryopreserved tubes containing NK cells (frozen for 1 month) were taken out of liquid nitrogen, thawed in a 37°C water bath for 2 min, diluted with RPMI 1640 serum-free medium, and centrifuged to collect the cells. The cells were inoculated into the above-mentioned recovery medium at a density of 2×10 5 / mL, at 37°C, 5% CO 2 The cells were cultured under the same conditions for 20 h and the recovery was completed.

[0076] 3. Experimental Examples

[0077] Experimental Example 1

[0078] Experimental Example 1 The viability of NK cells after cryopreservation and resuscitation of Examples 1-3 and Comparative Examples 1-5 was investigated. The specific experimental process was as follows:

[0079] The NK cells revived in Examples 1-3 and Comparative Examples 1-5 were mixed with trypan blue (concentration of 0.4%) at a ratio of 9:1. After staining for 2 minutes, the viability of the revived NK cells was counted using a cell counter. Each group of experiments was repeated 3 times. The results are as follows: Figure 2 shown.

[0080] Figure 2 The figure shows the activity of NK cells after cryopreservation and resuscitation in Example 1-3 and Comparative Example 1-5. Figure 2 It can be seen that the NK cell resuscitation activity rates of Examples 1-3 are all higher than those of Comparative Examples 1-5.

[0081] Comparative Examples 1-5 adjust the components of the recovery medium, wherein Comparative Example 1 omits the Polygonatum spirosterol in the recovery medium; Comparative Example 2 omits the Polygonatum spirosterol in the recovery medium and increases the amount of mevalonate lactone; Comparative Example 3 omits the mevalonate lactone in the recovery medium; Comparative Example 4 omits the mevalonate lactone in the recovery medium and increases the amount of Polygonatum spirosterol; Comparative Example 5 omits Polygonatum spirosterol and mevalonate lactone in the recovery medium. It is shown that the two components of Polygonatum spirosterol and mevalonate lactone act synergistically, and can effectively improve the viability of NK cells after cryopreservation recovery. It can be seen from this that the recovery medium of the present invention can effectively improve the recovery viability of NK cells after cryopreservation and increase the number of viable cells after recovery.

[0082] Experimental Example 2

[0083] Experimental Example 2 investigated the CD107a expression rate of NK cells after resuscitation in Examples 1-3 and Comparative Examples 1-5. The specific experimental process was as follows:

[0084] (1) The resuscitated NK cells of Example 1-3 and Comparative Example 1-5 were centrifuged and resuspended in RPMI 1640 serum-free medium to adjust the density of NK cells to 1×10 6 / mL, 100μL per tube;

[0085] (2) Add 5 μL CD107a-PE antibody to each tube and place at 37°C and 5% CO 2 Incubate the cells in a dark incubator for 1 h;

[0086] (3) Add 7 μL of Golgi blocking solution (containing monensin) to each tube and place at 37°C and 5% CO 2 Incubate the cells in a light-proof incubator for 3 h;

[0087] (4) Add CD56-FITC antibody and CD3-PE antibody to each tube and incubate in a 4°C cell culture incubator for 30 min;

[0088] (5) Add PBS to each tube, centrifuge twice (each centrifugation speed is 1200 rpm, and the centrifugation time is 5 min). Discard the supernatant and resuspend the cells with PBS.

[0089] (6) Flow cytometry was used to detect cell phenotypes, and FlowJo 7.6 software was used to analyze CD3 - CD56 + The expression rate of CD107a of NK cells is shown in Table 9. Figure 3 shown.

[0090] Table 9

[0091]

[0092] Table 9 Figure 3 The expression of CD107a in NK cells after resuscitation in Examples 1-3 and Comparative Examples 1-5. Figure 3 It can be seen that the expression rate of the NK cell killing activity marker molecule CD107a after resuscitation in Example 1-3 and Comparative Example 1-5 is higher than that in Comparative Example 1-5. The above results indicate that the resuscitation method of the present invention will not reduce the expression rate of the NK cell killing activity marker molecule CD107a.

[0093] The resuscitation medium of Comparative Examples 1-5 respectively omitted one or two components of mevalonate lactone and Polygonatum spirosterol and adjusted the dosage, which would lead to a decrease in the expression rate of CD107a. This shows that the synergistic effect of the two components of mevalonate lactone and Polygonatum spirosterol can effectively maintain the expression rate of CD107a, a marker molecule for the killing activity of NK cells after resuscitation. It can be seen that the resuscitation medium of the present invention is more conducive to maintaining the killing activity of NK cells after cryopreservation and resuscitation.

[0094] Experimental Example 3

[0095] Experimental Example 3 investigates the killing activity of NK cells after resuscitation in Examples 1-3 and Comparative Examples 1-5. The specific experimental process is as follows:

[0096] NK cells as effector cells: Take the NK cells revived in Example 1-3 and Comparative Example 1-5, centrifuge and remove the supernatant, resuspend the NK cells in RPMI 1640 serum-free medium, and adjust the cell density to 1×10 6 Pieces / mL, for future use;

[0097] K562 cells were used as target cells: K562 cells were human chronic myeloid leukemia cells in the logarithmic growth phase, and the density was adjusted to 5×10 4 Pieces / mL, for future use;

[0098] Killing activity experiment: Effector cells and target cells were inoculated into 96-well plates at a ratio of 20:1, with a total volume of 200 μL per well. Three replicate wells were set up in each group and placed at 37°C and 5% CO. 2 After culturing in the cell culture incubator for 24 h, 20 μL CCK-8 reagent was added to each well and the cells were placed in the incubator for another 4 h. The absorbance was measured at 450 nm and the cell killing activity was counted. The results are as follows: Figure 4 shown.

[0099] Figure 4 The following is a graph showing the killing activity of NK cells after resuscitation in Example 1-3 and Comparative Example 1-5. Figure 4 It can be seen that the killing activity of NK cells in Examples 1-3 is higher than that in Comparative Examples 1-5. Adjusting the components or dosage of the recovery medium in Comparative Examples 1-5 will lead to a decrease in killing activity. That is, the synergistic effect of the two components of mevalonate lactone and Polygonatum spirosterol can effectively maintain the killing activity of NK cells after recovery. This result also directly confirms that the recovery medium of the present invention can maintain the killing activity of NK cells after cryopreservation and recovery.

[0100] In summary, the present invention adds mevalonate lactone and polygonatum spirosterol components to the recovery medium. The experimental results confirm that the synergistic effect of mevalonate lactone and polygonatum spirosterol can effectively improve the recovery activity rate of NK cells after cryopreservation and maintain the killing activity of NK cells after cryopreservation and recovery, which is of great significance for the subsequent clinical application of NK cells.

[0101] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A NK cell recovery medium, characterized in that The following components are added in parts by weight per liter of basal culture medium: 0.015-0.05 mg of mevalonate lactone, 0.05-0.2 mg of polygonatum spirosterol, 0.02-0.1 mg of IL-15, 2-10 mg of transferrin, 6-17 mg of insulin, 0.01-0.03 mg of polylysine and 3-8 mg of aloin; the basal culture medium is RPMI1640 serum-free culture medium.

2. The NK cell recovery medium according to claim 1, characterized in that The following components were added in parts by weight per liter of basal culture medium: 0.03 mg of mevalonolactone, 0.07 mg of polygonatum spirosterol, 0.05 mg of IL-15, 7 mg of transferrin, 11 mg of insulin, 0.02 mg of polylysine and 5 mg of aloin; the basal culture medium was RPMI 1640 serum-free culture medium.

3. A method for resuscitating NK cells, characterized in that: The following steps are involved: The NK cell cryopreservation tube frozen in liquid nitrogen is thawed in a water bath, diluted with basal culture medium and centrifuged to collect the cells, and the cells are inoculated into the recovery medium described in claim 1 or 2 for culture to complete recovery; the basal culture medium is RPMI 1640 serum-free culture medium.

4. The method for resuscitation of NK cells according to claim 3, characterized in that: The cells were seeded at a density of 1.5×10 5 -5×10 5 Pieces / mL.

5. The method for resuscitation of NK cells according to claim 3, characterized in that: The temperature of the water bath is 37°C-40°C, and the time is 2-3 minutes.

6. The method for resuscitation of NK cells according to claim 3, characterized in that: The culture was carried out at 37° C. and 5% CO 2 .

7. The method for resuscitation of NK cells according to claim 3, characterized in that: The culture time is 16-24h.

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