A cryopreservation solution for cryopreserving NK cells at low temperatures and a method of using the same

Abstract

The present invention belongs to the technical field of cell cryopreservation, and specifically relates to a cryopreservation solution for low-temperature freezing of NK cells and a method for using the same. The NK cell cryopreservation solution provided by the present invention has simple ingredients, and by adding hydroxyethyl starch, cedarwood glycol, and butyl ferulate to the culture medium, NK cells can be stored in liquid nitrogen for a long time. And the experimental results show that NK cells frozen for 15 months using the cryopreservation solution provided by the present invention still have a high cell viability and killing activity, which can provide an effective way to provide sufficient NK cells for clinical use, and can effectively reduce the storage cost of NK cells and improve the convenience of using NK cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell cryopreservation, and in particular relates to a cryopreservation solution for cryopreserving NK cells at low temperatures and a method for using the cryopreservation solution. Background Art

[0002] With the rapid development of immune cell biology, immunotherapy has become one of the important means of tumor treatment. Cellular immunotherapy, also known as adoptive immunotherapy, activates immune cells and infuses them back into the patient's body to directly kill tumors or stimulate the body's anti-tumor immunity. It has the characteristics of low immune rejection, good treatment sensitivity, reduced recurrence and metastasis, and is conducive to the recovery of the body's immune system. The immune cells used in cell immunotherapy are mainly natural killer cells (NK), natural killer T cells, etc.

[0003] NK cells originate from bone marrow lymphoid stem cells. Their differentiation and development depend on the bone marrow and thymus microenvironment. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs and lymph nodes. NK cells are different from T and B cells. They are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization. They are also involved in the occurrence of hypersensitivity reactions and autoimmune diseases and can recognize target cells and killing mediators.

[0004] In order to meet the direct clinical demand for NK cells, it is necessary to cryopreserve the cells to achieve cell storage, so that sufficient NK cells with good activity can be provided to the clinic in a timely manner when needed. Patent CN115644166A discloses a method for cryopreserving NK cells, including pre-treating the NK cells and mixing the pre-treated cells with a cell freezing solution. However, 7v / v%~13v / v% DMSO and 5v / v%~15v / v% albumin are added to the freezing solution of the patent. Among them, DMSO has certain cytotoxicity, and human albumin is a kind of blood product. Due to its limited source, it is expensive. Therefore, it is necessary to modify the NK cell freezing solution to improve its safety and ensure the killing activity of NK cells after freezing. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, one of the objectives of the present invention is to provide a cryopreservation solution for cryopreserving NK cells at low temperatures, which has simple components and can effectively ensure the killing activity of NK cells after cryopreservation.

[0006] A second object of the present invention is to provide a method for using a cryopreservation solution for cryopreserving NK cells at low temperatures, 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 cryopreservation solution for cryopreserving NK cells at low temperatures comprises a culture medium and hydroxyethyl starch, cedardiol and butyl ferulate added to the culture medium; the addition amounts of the hydroxyethyl starch, cedardiol and butyl ferulate in the culture medium are: 10-20 μg / mL of hydroxyethyl starch, 3-10 μg / mL of cedardiol and 1-5 μg / mL of butyl ferulate.

[0009] Furthermore, the addition amounts of hydroxyethyl starch, cedarwood glycol and butyl ferulate in the culture medium are: hydroxyethyl starch 13-17 μg / mL, cedarwood glycol 5-8 μg / mL and butyl ferulate 2-4 μg / mL.

[0010] Furthermore, the addition amounts of hydroxyethyl starch, cedarwood glycol and butyl ferulate in the culture medium are: 15 μg / mL hydroxyethyl starch, 6 μg / mL cedarwood glycol and 3 μg / mL butyl ferulate.

[0011] Furthermore, the culture medium is X-VIVO serum-free culture medium.

[0012] The second object of the present invention is achieved by adopting the following technical solution:

[0013] A method for using a cryopreservation solution for cryopreserving NK cells at low temperatures, wherein the cryopreservation is performed using the cryopreservation solution.

[0014] Furthermore, the method comprises the following steps: mixing the NK cells to be frozen with the above-mentioned freezing solution to obtain a cell suspension, pre-freezing the cell suspension in a cryopreservation tube by programmed cooling, and after the pre-freezing is completed, transferring the cells to liquid nitrogen for storage.

[0015] Furthermore, the concentration of cells in the cell suspension is 1×10 6 ~5×10 7 Pieces / mL.

[0016] Furthermore, the programmed cooling pre-freezing process is: cooling to -20°C at 1-4°C / min for pre-freezing for 2-3h, and cooling to -80°C at 8-12°C / min for pre-freezing for 4-6h.

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

[0018] The NK cell freezing solution provided by the present invention has simple ingredients, and by adding hydroxyethyl starch, cedarwood glycol, and butyl ferulate to the culture medium, NK cells can be stored in liquid nitrogen for a long time. And the experimental results show that NK cells frozen for 15 months using the freezing solution provided by the present invention still have a high cell viability and killing activity, which can provide an effective way to provide sufficient NK cells for clinical use, and can effectively reduce the storage cost of NK cells and improve the convenience of using NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a morphological diagram of NK cells of the present invention;

[0020] Figure 2 This is a graph showing the killing rate of NK cells against K562 cells after cryopreservation in Example 1 and Comparative Examples 1 to 6;

[0021] Figure 3 This is a graph showing the killing rate of HepG2 cells by NK cells after cryopreservation in Example 1 and Comparative Examples 1 to 6. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with 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.

[0023] The culture process of NK cells of the present invention is as follows:

[0024] Low molecular weight heparin sodium was added to the peripheral venous blood of healthy blood donors for anticoagulation, and lymphocyte separation solution was added. The blood was centrifuged at 2200 rpm / min for 12 min, and the mononuclear cell layer was aspirated and washed three times with PBS. 310 U / mL interleukin-2 and 8% human AB plasma X-VIVO serum-free medium were added and incubated at 37°C and 5% CO 2 Culture in a saturated humidity incubator for 14 days (replace half of the culture medium every 2 days) to obtain NK cells for future use. See the morphology of NK cells for details. Figure 1 shown.

[0025] 1. Implementation

[0026] Example 1

[0027] Example 1 provides a cryopreservation solution for low-temperature freezing of NK cells, which is composed of X-VIVO serum-free culture medium and hydroxyethyl starch, cedarwood glycol, and butyl ferulate added to the culture medium, wherein the added amounts of hydroxyethyl starch, cedarwood glycol, and butyl ferulate in the culture medium are: 15 μg / mL hydroxyethyl starch, 6 μg / mL cedarwood glycol, and 3 μg / mL butyl ferulate.

[0028] A method for using a cryopreservation solution for cryopreserving NK cells at low temperatures, comprising mixing the NK cells to be cryopreserved with the cryopreservation solution to obtain a cell suspension (with a concentration of 2×10 6 / mL), cool the cell suspension in a cryopreservation tube to -20°C at 2°C / min for 2 h, cool it to -80°C at 10°C / min for 5 h, and transfer the cells to liquid nitrogen for storage.

[0029] Example 2

[0030] Example 2 provides a cryopreservation solution for low-temperature freezing of NK cells, which is composed of X-VIVO serum-free culture medium and hydroxyethyl starch, cedarwood glycol, and butyl ferulate added to the culture medium, wherein the added amounts of hydroxyethyl starch, cedarwood glycol, and butyl ferulate in the culture medium are: 10 μg / mL hydroxyethyl starch, 3 μg / mL cedarwood glycol, and 1 μg / mL butyl ferulate.

[0031] A method for using a cryopreservation solution for cryopreserving NK cells at low temperatures, comprising mixing the NK cells to be cryopreserved with the cryopreservation solution to obtain a cell suspension (with a concentration of 1×10 6 / mL), cool the cell suspension in a cryopreservation tube to -20°C at 2°C / min for 3 h, cool it to -80°C at 10°C / min for 4 h, and transfer the cells to liquid nitrogen for storage.

[0032] Example 3

[0033] Example 3 provides a cryopreservation solution for low-temperature freezing of NK cells, which is composed of X-VIVO serum-free culture medium and hydroxyethyl starch, cedarwood glycol, and butyl ferulate added to the culture medium, wherein the added amounts of hydroxyethyl starch, cedarwood glycol, and butyl ferulate in the culture medium are: 20 μg / mL hydroxyethyl starch, 10 μg / mL cedarwood glycol, and 5 μg / mL butyl ferulate.

[0034] A method for using a cryopreservation solution for cryopreserving NK cells at low temperatures, comprising mixing the NK cells to be cryopreserved with the cryopreservation solution to obtain a cell suspension (with a concentration of 5×10 7 / mL), cool the cell suspension in a cryopreservation tube to -20°C at 2°C / min for 2.5 h, cool it to -80°C at 10°C / min for 6 h, and transfer the cells to liquid nitrogen for storage.

[0035] 2. Comparison

[0036] Comparative Example 1

[0037] Comparative Example 1 provides a freezing solution for NK cells, which is different from Example 1 in that ferulic acid is used to replace butyl ferulate, and the rest is the same as Example 1.

[0038] Comparative Example 2

[0039] Comparative Example 2 provides a freezing solution for NK cells, which is different from Example 1 in that cedardiol is omitted and the rest is the same as Example 1.

[0040] Comparative Example 3

[0041] Comparative Example 3 provides a NK cell freezing solution, which is different from Example 1 in that butyl ferulate is omitted and the rest is the same as Example 1.

[0042] Comparative Example 4

[0043] Comparative Example 4 provides a freezing solution for NK cells, which is different from Example 1 in that hydroxyethyl starch and butyl ferulate are omitted, the addition amount of cedarwood diol is increased to 24 μg / mL, and the rest is the same as Example 1.

[0044] Comparative Example 5

[0045] Comparative Example 5 provides a freezing solution for NK cells, which is different from Example 1 in that hydroxyethyl starch and cedarwood glycol are omitted, the addition amount of butyl ferulate is increased to 24 μg / mL, and the rest is the same as Example 1.

[0046] Comparative Example 6

[0047] Comparative Example 6 provides a freezing solution for NK cells, which is different from Example 1 in that cedardiol and butyl ferulate are omitted, the amount of hydroxyethyl starch added is increased to 24 μg / mL, and the rest is the same as Example 1.

[0048] 3. Test Examples

[0049] Test Example 1

[0050] Experimental Example 1 The viability of NK cells after being cryopreserved for 1 month, 3 months, 9 months, and 15 months using the cryopreservation solutions provided in Examples 1 to 3 and Comparative Examples 1 to 6 was investigated. The specific steps are as follows:

[0051] Take out 1 mL cryovials from liquid nitrogen, thaw them in a 37°C constant temperature water bath, and resuspend them in X-VIVO serum-free medium (cell concentration 1×10 4 The cell suspension was mixed with 0.4% trypan blue at a ratio of 9:1, and the cell viability was detected using a Countstar automatic cell counter after staining for 2 minutes. This was repeated 3 times.

[0052] Table 1

[0053]

[0054] As can be seen from Table 1, compared with the freezing solutions provided in Examples 1 to 3, the NK cells frozen in the freezing solutions provided by Comparative Example 1 (replacing butyl ferulate with ferulic acid), Comparative Example 2 (omitting cedardiol), Comparative Example 3 (omitting butyl ferulate), Comparative Example 4 (omitting hydroxyethyl starch and butyl ferulate, and increasing the amount of cedardiol added), Comparative Example 5 (omitting hydroxyethyl starch and cedardiol, and increasing the amount of butyl ferulate added), and Comparative Example 6 (omitting cedardiol and butyl ferulate, and increasing the amount of hydroxyethyl starch added) have significantly reduced NK cell viability after resuscitation, which indicates that the synergistic effect of hydroxyethyl starch, cedardiol, and butyl ferulate can better protect the viability of NK cells.

[0055] Test Example 2

[0056] Experimental Example 2 investigates the killing rate of NK cells revived by freezing the cryopreservation solutions of Example 1 and Comparative Examples 1 to 6 for 15 months. The specific process is as follows:

[0057] The NK cells frozen for 15 months and revived in Example 1 and Comparative Examples 1 to 6 were used as effector cells. The supernatant was discarded after centrifugation, and the cells were resuspended in X-VIVO serum-free medium and the cell density was adjusted. K562 and HepG2 cells in the logarithmic growth phase were used as target cells, and the cell density was controlled to 5×10 4 / mL. 100 μL of target cells and effector cells were inoculated into 96-well plates according to the effector-target ratio of 5:1, 10:1, 20:1, and 40:1. Three replicate wells were set up for each group and the plates were incubated at 37°C and 5% CO 2 The cells were cultured in a saturated humidity incubator for 24 h. After the culture was completed, 10 μL of CCK-8 reagent was added to each well and incubated at 37°C and 5% CO 2 The cells were incubated in a saturated humidity incubator for 4 h, and then the absorbance OD value at 450 nm was detected using an ELISA reader. The killing rate was calculated based on the absorbance value. The results are shown in Tables 2-3. Figure 2~3 shown.

[0058] Table 2 Killing rate of NK cells against K562 cells after cryopreservation in Example 1 and Comparative Examples 1 to 6

[0059]

[0060] Table 3 Killing rate of NK cells on HepG2 cells after cryopreservation in Example 1 and Comparative Examples 1 to 6

[0061]

[0062] From Table 2~3, Figure 2~3 It can be seen that the killing rate of NK cells against K562 cells and HepG2 cells after being frozen for 15 months using the freezing solution provided in Example 1 is significantly higher than that of NK cells after being frozen for 15 months using the freezing solution provided in Comparative Examples 1 to 6, that is, the freezing solution provided in Example 1 has a better effect of maintaining killing activity. This also shows that the freezing solution of the present invention is more conducive to maintaining killing activity and is suitable for long-term storage of NK cells in liquid nitrogen.

[0063] In summary, the NK cell freezing solution provided by the present invention has simple ingredients, and by adding hydroxyethyl starch, cedarwood glycol, and butyl ferulate to the culture medium, NK cells can be stored in liquid nitrogen for a long time. And the experimental results show that NK cells frozen for 15 months using the freezing solution provided by the present invention still have a high cell viability and killing activity, which can provide an effective way to provide sufficient NK cells for clinical use, and can effectively reduce the storage cost of NK cells and improve the convenience of using NK cells.

[0064] 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 cryopreservation solution for cryopreserving NK cells, characterized in that: The method comprises a culture medium and hydroxyethyl starch, cedarwood glycol and butyl ferulate added to the culture medium; the addition amounts of the hydroxyethyl starch, cedarwood glycol and butyl ferulate in the culture medium are: 10-20 μg / mL of hydroxyethyl starch, 3-10 μg / mL of cedarwood glycol and 1-5 μg / mL of butyl ferulate.

2. The cryopreservation solution for cryopreserving NK cells according to claim 1, characterized in that: The addition amounts of hydroxyethyl starch, cedarwood glycol and butyl ferulate in the culture medium are: 13-17 μg / mL hydroxyethyl starch, 5-8 μg / mL cedarwood glycol and 2-4 μg / mL butyl ferulate.

3. The cryopreservation solution for cryopreserving NK cells according to claim 1, characterized in that: The amounts of hydroxyethyl starch, cedarwood glycol and butyl ferulate added to the culture medium are: 15 μg / mL hydroxyethyl starch, 6 μg / mL cedarwood glycol and 3 μg / mL butyl ferulate.

4. The cryopreservation solution for cryopreserving NK cells according to claim 1, characterized in that: The culture medium is X-VIVO serum-free culture medium.

5. A method for using a cryopreservation solution for cryopreserving NK cells, characterized in that: The cryopreservation is performed using the cryopreservation solution according to any one of claims 1 to 4.

6. The method for using the freezing solution according to claim 5, characterized in that: The following steps are involved: The NK cells to be frozen are mixed with the freezing solution to obtain a cell suspension, the cell suspension is pre-frozen in a cryopreservation tube by programmed cooling, and after the pre-freezing is completed, the cells are transferred to liquid nitrogen for storage.

7. The method for using the freezing solution according to claim 6, characterized in that: The cell concentration in the cell suspension is 1×10 6 ~5×10 7 Pieces / mL.

8. The method for using the freezing solution according to claim 6, characterized in that: The process of programmed cooling pre-freezing is: cooling to -20°C at 1-4°C / min for 2-3h, and cooling to -80°C at 8-12°C / min for 4-6h.

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