Gel preparation for cryopreservation of CIK (cytokine-induced killer) cells as well as preparation method and application of gel preparation

Abstract

The invention relates to the technical field of CIK cell cryopreservation, in particular to a gel preparation for CIK cell cryopreservation and a preparation method and application thereof. The skin care solution is specifically prepared from sodium hyaluronate, sodium carboxymethyl cellulose, guaiacol, human serum albumin, ornithine, L-proline, low-molecular dextran, dimethyl sulfoxide, 1-aniline, isomaltose oligosaccharide, glutathione, propylene glycol alginate tobramycin, povidone and the balance of water. The skin care solution is prepared from the following raw materials: sodium hyaluronate, sodium carboxymethylcellulose, guaiacol, human serum albumin, ornithine, L-proline, low-molecular dextran, dimethyl sulfoxide, 1-aniline, isomaltose oligosaccharide, glutathione, propylene glycol alginate tobramycin, povidone and the like. The gel preparation provided by the invention can be used for long-time cryopreservation of CIK cells, and the survival rate of immune cells can still reach 96.4% after the CIK cells are preserved for 12 months.

Description

Technical Field

[0001] The present invention relates to the technical field of CIK cell cryopreservation, and in particular to a gel preparation for CIK cell cryopreservation, and a preparation method and application thereof. Background Art

[0002] Cytokine-induced killer cells (CIK cells) are a group of heterogeneous cells obtained by culturing mononuclear cells of bone marrow, human peripheral blood or umbilical cord blood, or in vitro with cytokines for a period of time. According to existing research records, CIK cell therapy can improve patients' disease-free survival and overall survival, and enhance the body's anti-tumor immune function, thereby improving patients' quality of life.

[0003] Since CIK cells express two membrane protein molecules, CD3 and CD56, they are also called NK cell-like T lymphocytes. They have both the powerful anti-tumor activity of T lymphocytes and the non-major histocompatibility complex (MHC)-restricted tumor-killing advantages of NK cells.

[0004] CD3+CD56+ double positive cells are the main effector cells in CIK cells, which have the advantages of fast proliferation, high tumor killing activity, non-major histocompatibility complex (MHC) restriction, broad tumor killing spectrum, and slight impact on normal bone marrow hematopoiesis. In particular, they play an important role in clearing micro-metastatic lesions after surgery, preventing the spread and recurrence of cancer, and improving the patient's own anti-tumor immunity. They can also play a positive role in improving the quality of life and prolonging the life of patients with advanced tumors who cannot undergo surgery or tolerate chemotherapy.

[0005] However, the CIK cell preparation technology in the prior art still has the disadvantages of poor CIK cell proliferation and low cytotoxic activity. In addition, the induction period of CIK cells is very long, and multiple blood sampling and induction are required in clinical practice, which causes great pain to patients. If the induced and cultured CIK cells are not used in time, they are easy to die, which wastes a lot of medical costs. Therefore, those skilled in the art are committed to developing a CIK cell preservation method to facilitate the preservation of CIK cells and the clinical application of related treatment therapies.

[0006] Gel preparations are a new drug dosage form that has emerged in China in recent years. Gel preparations refer to thick liquid or semi-solid preparations in the form of solutions, suspensions or emulsions made from drugs and excipients that can form gels. The commonly used matrix for preparing gel preparations is generally composed of water, glycerol or propylene glycol and polymer materials such as cellulose derivatives, carbomer and alginate. This type of gel is a hydrogel. Although it is non-greasy and easy to apply and remove, it has a slow release rate and is prone to water loss and mildew, which seriously affects the quality and various properties of the gel preparation.

[0007] Therefore, how to provide a new type of gel preparation to solve the technical problem that CIK cells are difficult to preserve in the prior art is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0008] The purpose of the present invention is to provide a gel preparation for CIK cell cryopreservation and a preparation method and application thereof.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The invention provides a gel preparation for cryopreservation of CIK cells. Each 100 mL of the gel preparation comprises: 0.2-0.4 g of sodium hyaluronate, 0.1-0.2 g of sodium carboxymethyl cellulose, 2-3 g of guaiac gum, 50-70 mg of human serum albumin, 0.3-0.4 g of ornithine, 0.1-0.2 g of L-proline, 0.6-0.8 g of low molecular weight dextran, 4-6 mL of dimethyl sulfoxide, 2-4 mL of 1-aniline, 2-4 mL of isomaltose oligosaccharide, 0.3-0.5 g of glutathione, 2-3 g of propylene glycol alginate, 0.06-0.08 g of tobramycin, 0.1-0.2 g of povidone iodine, and the rest is water.

[0011] Preferably, the water is deionized water or physiological saline injection.

[0012] The present invention also provides a method for preparing the gel preparation for CIK cell cryopreservation, comprising the following steps:

[0013] (1) mixing sodium hyaluronate, sodium carboxymethyl cellulose, guaiac gum and water, and letting stand to obtain material 1;

[0014] (2) mixing the material 1, human serum albumin, ornithine, L-proline, low molecular weight dextran, dimethyl sulfoxide, 1-aniline, propylene glycol alginate, isomaltose oligosaccharide and glutathione to obtain material 2;

[0015] (3) The material 2, tobramycin and povidone-iodine are mixed and stirred to obtain a gel preparation for CIK cell cryopreservation.

[0016] Preferably, the stirring speed in step (1) is 300 to 500 rpm and the stirring time is 10 to 14 min.

[0017] Preferably, the stirring speed in step (2) is 100 to 200 rpm and the stirring time is 5 to 7 minutes.

[0018] Preferably, the stirring speed in step (3) is 100 to 200 rpm and the stirring time is 3 to 5 min.

[0019] Preferably, the standing time in step (1) is 15 to 21 hours.

[0020] The present invention also provides a CIK cell cryopreservation method, wherein the CIK cells are mixed with the gel preparation and then cryopreserved.

[0021] Preferably, the freezing temperature is -160 to -196°C.

[0022] Preferably, each 100 mL of the gel preparation contains 5 to 7×10 12 CIK cells.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The low-molecular-weight dextran added in the present invention has the function of maintaining the balance of colloidal osmotic pressure, and dimethyl sulfoxide, 1-aniline and isomaltose oligosaccharide as cryoprotectants can improve the permeability of cell membrane to water, delay the freezing process, and thus reduce the damage of ice crystals to cells.

[0025] The glutathione added in the present invention can protect cells from oxidative stress and help form a favorable redox environment inside and outside the cells to avoid damage to the activity of CIK cells. Ornithine and L-proline can hydrogen bond with intracellular water to inhibit ice crystal formation at low temperatures, and can also bind with intracellular water to reduce intracellular water loss.

[0026] The addition of propylene glycol alginate and guaiac gum added in the present invention can improve the stability of the gel matrix and can be used to preserve immune cells for a long time. The tobramycin and povidone-iodine added in the present invention have antibacterial effects and can effectively play an antiseptic and sterilizing effect.

[0027] The gel preparation provided by the present invention can be used for long-term cryopreservation of CIK cells. After being stored for 12 months, the survival rate of CIK cells can still reach 96.4%. DETAILED DESCRIPTION

[0028] The invention provides a gel preparation for CIK cell cryopreservation. Each 100 mL of the gel preparation comprises: 0.2-0.4 g of sodium hyaluronate, 0.1-0.2 g of sodium carboxymethyl cellulose, 2-3 g of guaiac gum, 50-70 mg of human serum albumin, 0.3-0.4 g of ornithine, 0.1-0.2 g of L-proline, 0.6-0.8 g of low molecular weight dextran, 4-6 mL of dimethyl sulfoxide, 2-4 mL of 1-aniline, 2-4 mL of isomaltose oligosaccharide, 0.3-0.5 g of glutathione, 2-3 g of propylene glycol alginate, 0.06-0.08 g of tobramycin, 0.1-0.2 g of povidone iodine, and the rest is water.

[0029] Preferably, each 100 mL of the gel preparation comprises: 0.3 g of sodium hyaluronate, 0.15 g of sodium carboxymethylcellulose, 2.5 g of guaiac gum, 54-66 mg of human serum albumin, 0.35 g of ornithine, 0.15 g of L-proline, 0.7 g of low molecular weight dextran, 5 mL of dimethyl sulfoxide, 3 mL of 1-aniline, 3 mL of isomaltose oligosaccharide, 0.4 g of glutathione, 2.5 g of propylene glycol alginate, 0.07 g of tobramycin, 0.15 g of povidone iodine, and the remainder is water;

[0030] It is further preferred that each 100 mL of the gel preparation comprises: 0.3 g of sodium hyaluronate, 0.15 g of sodium carboxymethyl cellulose, 2.5 g of guaiac gum, 60 mg of human serum albumin, 0.35 g of ornithine, 0.15 g of L-proline, 0.7 g of low molecular weight dextran, 5 mL of dimethyl sulfoxide, 3 mL of 1-aniline, 3 mL of isomaltose oligosaccharide, 0.4 g of glutathione, 2.5 g of propylene glycol alginate, 0.07 g of tobramycin, 0.15 g of povidone iodine, and the remainder is water.

[0031] In the present invention, the water is deionized water or physiological saline injection; preferably physiological saline injection.

[0032] The present invention also provides a method for preparing the gel preparation for CIK cell cryopreservation, comprising the following steps:

[0033] (1) mixing sodium hyaluronate, sodium carboxymethyl cellulose, guaiac gum and water, and letting stand to obtain material 1;

[0034] (2) mixing the material 1, human serum albumin, ornithine, L-proline, low molecular weight dextran, dimethyl sulfoxide, 1-aniline, propylene glycol alginate, isomaltose oligosaccharide and glutathione to obtain material 2;

[0035] (3) The material 2, tobramycin and povidone-iodine are mixed and stirred to obtain a gel preparation for CIK cell cryopreservation.

[0036] In the present invention, the stirring speed in step (1) is 300-500 rpm, and the time is 10-14 min; preferably, the speed is 340-460 rpm, and the time is 11-13 min; further preferably, the speed is 380-420 rpm, and the time is 12 min; more preferably, the speed is 400 rpm, and the time is 12 min.

[0037] In the present invention, the stirring speed in step (2) is 100-200 rpm, and the time is 5-7 min; preferably, the speed is 120-180 rpm, and the time is 6 min; further preferably, the speed is 140-160 rpm, and the time is 6 min; more preferably, the speed is 150 rpm, and the time is 6 min.

[0038] In the present invention, the stirring speed in step (3) is 100-200 rpm, and the time is 3-5 min; preferably, the speed is 120-180 rpm, and the time is 4 min; further preferably, the speed is 140-160 rpm, and the time is 4 min; more preferably, the speed is 150 rpm, and the time is 4 min.

[0039] In the present invention, the standing time in step (1) is 15 to 21 hours, preferably 16 to 20 hours, more preferably 17 to 19 hours, and more preferably 18 hours.

[0040] The present invention also provides application of the gel preparation for CIK cell cryopreservation in CIK cell cryopreservation, wherein CIK cells are mixed with the gel preparation and then cryopreserved.

[0041] In the present invention, the freezing temperature is -160 to -196°C; preferably -170 to -186°C; more preferably -180°C.

[0042] In the present invention, each 100 mL of the gel preparation contains 5 to 7 × 10 12 CIK cells; preferably 6×10 12 CIK cells.

[0043] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0044] Example 1

[0045] A gel preparation for CIK cell cryopreservation, wherein each 100 mL of the gel preparation comprises: 0.2 g of sodium hyaluronate, 0.1 g of sodium carboxymethyl cellulose, 2 g of guaiac gum, 50 mg of human serum albumin, 0.3 g of ornithine, 0.1 g of L-proline, 0.6 g of low molecular weight dextran, 4 mL of dimethyl sulfoxide, 2 mL of 1-aniline, 2 mL of isomaltose oligosaccharide, 0.3 g of glutathione, 2 g of propylene glycol alginate, 0.06 g of tobramycin, 0.1 g of povidone iodine, and the remainder is water.

[0046] The preparation method of the gel preparation for CIK cell cryopreservation comprises the following steps:

[0047] (1) Sodium hyaluronate, sodium carboxymethyl cellulose, guaiac gum and water are mixed and stirred, and allowed to stand for 15 hours to obtain material 1; the rotation speed is 300 rpm, and the time is 10 minutes;

[0048] (2) mixing the material 1, human albumin, ornithine, L-proline, low molecular weight dextran, dimethyl sulfoxide, 1-aniline, propylene glycol alginate, isomaltose oligosaccharide and glutathione to obtain material 2; the stirring speed is 100 rpm, and the stirring time is 5 min;

[0049] (3) The material 2, tobramycin and povidone-iodine are mixed and stirred to obtain a gel preparation for CIK cell cryopreservation; the stirring speed is 100 rpm and the stirring time is 3 min.

[0050] Example 2

[0051] A gel preparation for CIK cell cryopreservation, wherein each 100 mL of the gel preparation comprises: 0.4 g of sodium hyaluronate, 0.2 g of sodium carboxymethyl cellulose, 3 g of guaiac gum, 70 mg of human serum albumin, 0.4 g of ornithine, 0.2 g of L-proline, 0.8 g of low molecular weight dextran, 6 mL of dimethyl sulfoxide, 4 mL of 1-aniline, 4 mL of isomaltose oligosaccharide, 0.5 g of glutathione, 3 g of propylene glycol alginate, 0.08 g of tobramycin, 0.2 g of povidone iodine, and the rest is normal saline injection.

[0052] The preparation method of the gel preparation for CIK cell cryopreservation comprises the following steps:

[0053] (1) Sodium hyaluronate, sodium carboxymethyl cellulose, guaiac gum and water were mixed and stirred, and allowed to stand for 21 hours to obtain material 1; the rotation speed was 500 rpm, and the time was 14 minutes;

[0054] (2) mixing the material 1, human albumin, ornithine, L-proline, low molecular weight dextran, dimethyl sulfoxide, 1-aniline, propylene glycol alginate, isomaltose oligosaccharide and glutathione to obtain material 2; the stirring speed is 200 rpm, and the stirring time is 7 minutes;

[0055] (3) The material 2, tobramycin and povidone-iodine are mixed and stirred to obtain a gel preparation for CIK cell cryopreservation; the stirring speed is 200 rpm and the stirring time is 5 min.

[0056] Example 3

[0057] A gel preparation for CIK cell cryopreservation, wherein each 100 mL of the gel preparation comprises: 0.3 g of sodium hyaluronate, 0.15 g of sodium carboxymethyl cellulose, 2.5 g of guaiac gum, 60 mg of human serum albumin, 0.35 g of ornithine, 0.15 g of L-proline, 0.7 g of low molecular weight dextran, 5 mL of dimethyl sulfoxide, 3 mL of 1-aniline, 3 mL of isomaltose oligosaccharide, 0.4 g of glutathione, 2.5 g of propylene glycol alginate, 0.07 g of tobramycin, 0.15 g of povidone iodine, and the rest is normal saline injection.

[0058] The preparation method of the gel preparation for CIK cell cryopreservation comprises the following steps:

[0059] (1) Sodium hyaluronate, sodium carboxymethyl cellulose, guaiac gum and water were mixed and stirred, and allowed to stand for 18 hours to obtain material 1; the rotation speed was 400 rpm, and the time was 12 minutes;

[0060] (2) mixing the material 1, human albumin, ornithine, L-proline, low molecular weight dextran, dimethyl sulfoxide, 1-aniline, propylene glycol alginate, isomaltose oligosaccharide and glutathione to obtain material 2; the stirring speed is 150 rpm, and the stirring time is 6 minutes;

[0061] (3) Material 2, tobramycin and povidone-iodine are mixed and stirred to obtain a gel preparation for CIK cell cryopreservation; the stirring speed is 150 rpm and the time is 4 minutes.

[0062] Example 4

[0063] A method for freezing CIK cells, comprising mixing CIK cells with the gel preparation obtained in Example 1 and freezing the mixture at a temperature of -160°C; each 100 mL of the gel preparation contains 5×10 12 CIK cells.

[0064] Example 5

[0065] A method for freezing CIK cells, comprising mixing CIK cells with the gel preparation obtained in Example 2 and freezing the mixture at a temperature of -180°C; each 100 mL of the gel preparation contains 7×10 12 CIK cells.

[0066] Example 6

[0067] A method for freezing CIK cells, comprising mixing CIK cells with the gel preparation obtained in Example 3 and freezing the mixture at a temperature of -196°C; each 100 mL of the gel preparation contains 6×10 12 CIK cells.

[0068] Control group 1: other methods were the same as those in Example 6, except that ornithine and L-proline were not added to the gel preparation of Example 3.

[0069] Control group 2: other methods were the same as those in Example 6, except that dimethyl sulfoxide, 1-aniline and isomaltose oligosaccharide were not added to the gel preparation of Example 3.

[0070] Control group 3: other methods were the same as those in Example 6, except that glutathione was not added to the gel preparation of Example 3.

[0071] Experimental Example 1

[0072] The cells were cryopreserved according to the methods of Example 6 and Control Groups 1 to 3, and taken out at 1 month, 3 months, 6 months and 12 months, respectively, and revived in a 37°C water bath for 2 min. The cells were counted by trypan blue staining, and the survival rate was calculated. The average value of the measurements was taken 3 times. The results are shown in Table 1.

[0073] Table 1 Cell survival rate statistics

[0074] Group 1 month 3 months 6 months 12 months Example 6 99.9％ 99.3％ 98.5％ 96.4％ Control group 1 99.3％ 96.0％ 92.1％ 84.7％ Control group 2 98.5％ 94.6％ 88.9％ 80.5％ Control group 3 99.2％ 97.2％ 93.8％ 86.1％

[0075] It can be seen from the contents recorded in Table 1 that the gel preparation prepared by the method of the present invention can be used for long-term cryopreservation of CIK cells. After being stored for 12 months, the survival rate of immune cells can still reach 96.4%.

[0076] It can be seen from the data of the control group that only by using the complete formulation of the gel preparation of the present invention can an excellent cryopreservation effect be achieved. If one or more components thereof are discarded, the survival rate of the cells will be significantly affected.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A gel preparation for CIK cell cryopreservation, characterized in that: Each 100 mL of gel preparation includes: 0.2-0.4 g of sodium hyaluronate, 0.1-0.2 g of sodium carboxymethyl cellulose, 2-3 g of guaiac gum, 50-70 mg of human serum albumin, 0.3-0.4 g of ornithine, 0.1-0.2 g of L-proline, 0.6-0.8 g of low molecular weight dextran, 4-6 mL of dimethyl sulfoxide, 2-4 mL of 1-aniline, 2-4 mL of isomaltose oligosaccharide, 0.3-0.5 g of glutathione, 2-3 g of propylene glycol alginate, 0.06-0.08 g of tobramycin, 0.1-0.2 g of povidone iodine, and the rest is water.

2. A gel preparation for CIK cell cryopreservation according to claim 1, characterized in that: The water is deionized water or physiological saline injection.

3. The method for preparing a gel preparation for CIK cell cryopreservation according to claim 1 or 2, characterized in that: The steps include: (1) mixing sodium hyaluronate, sodium carboxymethyl cellulose, guaiac gum and water, and letting stand to obtain material 1; (2) mixing the material 1, human serum albumin, ornithine, L-proline, low molecular weight dextran, dimethyl sulfoxide, 1-aniline, propylene glycol alginate, isomaltose oligosaccharide and glutathione to obtain material 2; (3) The material 2, tobramycin and povidone-iodine are mixed and stirred to obtain a gel preparation for CIK cell cryopreservation.

4. The method for preparing a gel preparation for CIK cell cryopreservation according to claim 3, characterized in that: The stirring speed in step (1) is 300 to 500 rpm, and the stirring time is 10 to 14 minutes.

5. The method for preparing a gel preparation for CIK cell cryopreservation according to claim 3, characterized in that: The stirring speed in step (2) is 100-200 rpm and the stirring time is 5-7 min.

6. The method for preparing a gel preparation for CIK cell cryopreservation according to claim 3, characterized in that: The stirring speed in step (3) is 100-200 rpm and the stirring time is 3-5 min.

7. The method for preparing a gel preparation for CIK cell cryopreservation according to claim 3, characterized in that: The standing time in step (1) is 15 to 21 hours.

8. Use of the gel preparation for CIK cell cryopreservation according to claim 1 or 2 in CIK cell cryopreservation, characterized in that: CIK cells are mixed with the gel preparation according to claim 1 or 2 and then frozen.

9. The use according to claim 8, characterized in that: The freezing temperature is -160 to -196°C.

10. The use according to claim 8, characterized in that: Each 100 mL of the gel preparation contains 5 to 7 × 10 12 CIK cells.