Ready-to-use serum-free DMSO-free cell freezing medium as well as preparation method and application thereof

Abstract

The invention relates to a ready-to-use serum-free DMSO-free cell cryopreservation solution, a preparation method and application, and relates to the field of cell cryopreservation, and the cell cryopreservation solution comprises the following components: sodium lactobionate, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, potassium bicarbonate, potassium chloride, sucrose, mannose, trehalose, hydroxyethyl starch, 4-hydroxyethyl piperazine ethanesulfonic acid, water-soluble vitamin E and ethylene glycol. The cell cryopreservation liquid provided by the invention has the advantages of high efficiency, no foreign protein, easiness in use and the like, cells resuscitated after cryopreservation are high in activity, and the cell characteristics can be kept after resuscitation; the cell freezing medium does not contain serum and other foreign proteins, so that the influence of endotoxin, latent virus and inter-batch difference is avoided; the cell freezing medium disclosed by the invention does not contain DMSO, so that the toxic effect on cells is further reduced, and the cell characteristics are favorably maintained; the cell freezing medium can be directly stored in a refrigerator at the temperature of-80 DEG C without programmed cooling, and is quick and convenient to use.

Description

Technical Field

[0001] The invention relates to the field of cell cryopreservation, and in particular to a ready-to-use serum-free and DMSO-free cell cryopreservation solution, a preparation method and an application thereof. Background Art

[0002] During the cell culture process, primary cells need to be preserved in time to prevent the biological characteristics of cells from changing with the number of passages. In addition, during the cell procurement and transportation process, some cells also need to be preserved in the form of cryopreservation. Therefore, timely and convenient cryopreservation of cells is particularly important in practical work.

[0003] In the research on cell cryopreservation, there are two mainstream views to explain the damage caused by cryopreservation to cells: (1) ice crystals mechanically destroy the cell membrane, so it is impossible to obtain structurally intact cells after thawing; (2) when ice crystals form in cells during cooling, the concentration of remaining liquid solutes increases fatally. In order to mitigate these effects, appropriate protective measures must be taken: use of cryoprotectants and selection of appropriate cooling and thawing rates.

[0004] Cryoprotectants used in cell cryopreservation are mainly divided into osmotic protectants and non-osmotic protectants. Osmotic protectants can enter the interior of cells and protect cells from mechanical damage caused by ice crystals and the osmotic effect of solutes during freezing. Osmotic protectants are highly water-soluble at low temperatures, can easily pass through the cell membrane and have relatively low cytotoxicity. Non-osmotic protectants cannot enter the interior of cells and work outside the cells, usually with a larger molecular weight. Osmotic protectants and non-osmotic protectants work synergistically to protect cells from damage caused by freezing.

[0005] Traditional cell freezing solutions are mainly prepared with fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO). Fetal bovine serum may carry potential unknown viruses or endotoxins, and there are batch differences, which may damage the cells in cryopreservation. Dimethyl sulfoxide has been shown to be slightly toxic to cells and may cause changes in the biological properties of cells. In addition, traditional cell freezing solutions adopt a slow freezing and fast thawing method. During the freezing process, the freezing temperature needs to be manually adjusted or a program cooling box needs to be used, which is cumbersome and time-consuming. In view of this, the present invention provides a ready-to-use serum-free and DMSO-free cell freezing solution, a preparation method and an application. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a ready-to-use serum-free and DMSO-free cell freezing solution, a preparation method and an application thereof. The purpose is to provide a serum-free, DMSO-free, exogenous protein-free cell freezing solution that has high cell activity after cryopreservation and can maintain cell characteristics after recovery.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] In the first aspect, a ready-to-use serum-free and DMSO-free cell freezing solution is provided, wherein each 100 mL of the cell freezing solution comprises the following components: 2 g to 6 g of sodium lactobionate, 0.0003 g to 0.0008 g of calcium chloride, 0.03 g to 0.08 g of magnesium chloride, 0.08 g to 0.2 g of potassium dihydrogen phosphate, 0.03 g to 0.08 g of potassium bicarbonate, 0.1 g to 0.4 g of potassium chloride, 0.5 g to 2 g of sucrose, 0.2 g to 0.6 g of mannose, 1 g to 4 g of trehalose, 0.1 g to 1 g of hydroxyethyl starch, 0.3 g to 1 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 0.0005 g to 0.003 g of water-soluble vitamin E, and 2 mL to 10 mL of ethylene glycol.

[0009] Based on the above technical solution, the present invention can also be improved as follows.

[0010] Furthermore, each 100 mL of the cell freezing solution includes the following components: 3 g to 5 g of sodium lactobionate, 0.0004 g to 0.0007 g of calcium chloride, 0.04 g to 0.07 g of magnesium chloride, 0.09 g to 0.15 g of potassium dihydrogen phosphate, 0.04 g to 0.07 g of potassium bicarbonate, 0.2 g to 0.3 g of potassium chloride, 0.6 g to 0.8 g of sucrose, 0.3 g to 0.5 g of mannose, 1 g to 3 g of trehalose, 0.2 g to 0.5 g of hydroxyethyl starch, 0.4 g to 0.8 g of 4-hydroxyethylpiperazineethanesulfonic acid, 0.001 g to 0.002 g of water-soluble vitamin E, and 3 mL to 8 mL of ethylene glycol.

[0011] Furthermore, each 100 mL of the cell freezing solution includes the following components: 5 g of sodium lactobionate, 0.0005 g of calcium chloride, 0.05 g of magnesium chloride, 0.15 g of potassium dihydrogen phosphate, 0.05 g of potassium bicarbonate, 0.2 g of potassium chloride, 0.7 g of sucrose, 0.4 g of mannose, 2 g of trehalose, 0.4 g of hydroxyethyl starch, 0.6 g of 4-hydroxyethylpiperazineethanesulfonic acid, 0.001 g of water-soluble vitamin E, and 6 mL of ethylene glycol.

[0012] In a second aspect, a ready-to-use serum-free and DMSO-free cell freezing kit comprises the components described above.

[0013] In a third aspect, a method for preparing a ready-to-use serum-free and DMSO-free cell freezing solution comprises the following steps:

[0014] (1) In double distilled water, add sodium lactobionate, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, potassium bicarbonate, potassium chloride, and 4-hydroxyethylpiperazineethanesulfonic acid and dissolve them; continue to add sucrose, mannose, trehalose, and hydroxyethyl starch and dissolve them; continue to add ethylene glycol and mix well; continue to add water-soluble vitamin E and dissolve it to obtain a mixed solution;

[0015] (2) The pH of the mixed solution is adjusted to 7.2, sterilized, and fixed to volume to obtain a cell freezing solution.

[0016] Furthermore, the sterilization in step (2) is specifically performed by filtering and sterilizing with a 0.22 um pore size filter membrane.

[0017] Furthermore, the step (2) of adjusting the volume specifically includes: adjusting the volume using a volumetric flask.

[0018] In a fourth aspect, a ready-to-use serum-free and DMSO-free cell freezing solution is used for cell freezing.

[0019] In a fifth aspect, a cell freezing method comprises the following steps: using the cell freezing solution to dilute the cells to be frozen into a cell suspension; and then transferring the cell suspension to a cryopreservation tube for freezing.

[0020] Furthermore, the concentration of cells in the cell suspension is 1×10 6 cells / ml~1×10 7 cells / ml.

[0021] In the cell freezing solution provided by the present invention, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, potassium bicarbonate, potassium chloride and 4-hydroxyethylpiperazineethanesulfonic acid provide a suitable buffer system and ionic strength, sodium lactobionate, sucrose, trehalose and hydroxyethyl starch are non-permeable protective agents, ethylene glycol is a permeable protective agent, and mannose and water-soluble vitamin E are reducing agents, which reduce the damage of free radicals to cells.

[0022] The beneficial effects of the present invention are:

[0023] (1) The cell freezing solution provided by the present invention has the advantages of high efficiency, no exogenous protein, and easy use. The cells revived after freezing have high activity and can maintain their cell characteristics after resuscitation;

[0024] (2) The cell freezing solution provided by the present invention does not contain serum and other exogenous proteins, thus avoiding the influence of endotoxins, latent viruses and batch differences;

[0025] (3) The cell freezing solution provided by the present invention does not contain DMSO, which further reduces the toxicity to cells and helps maintain cell characteristics;

[0026] (4) The cell freezing solution provided by the present invention does not need to be cooled through a programmed cooling process and can be directly stored in a -80°C refrigerator, which is quick and convenient to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a diagram of the state of HELA cells after thawing using the cryopreservation solution of Example 1 of the present invention;

[0028] Figure 2 This is a diagram of the state of HELA cells after thawing using the traditional cryopreservation solution of Comparative Example 1;

[0029] Figure 3 This is a diagram of the state of THP-1 cells after thawing using the cryopreservation solution of Example 1 of the present invention;

[0030] Figure 4 This is a diagram of the THP-1 cell status after thawing using the traditional freezing solution of Control Example 1. DETAILED DESCRIPTION

[0031] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0032] Sources of materials and reagents:

[0033] Sodium lactobionate (Aladdin, catalog number: S192464); mannose (Merck, catalog number: M6020); hydroxyethyl starch (Aladdin, H498478); water-soluble vitamin E (Merck, catalog number: 238813); HELA cells (Boster Biological, catalog number: CX0003); THP-1 cells (Boster Biological, catalog number: CX0042).

[0034] Example 1

[0035] A method for preparing a ready-to-use serum-free and DMSO-free cell freezing solution, the preparation of 1L usage volume comprises the following steps:

[0036] (1) In a 1000 ml beaker, add 800 ml of double distilled water, slowly add 50 g of sodium lactobionate, 5 mg of calcium chloride, 0.5 g of magnesium chloride, 1.5 g of potassium dihydrogen phosphate, 0.5 g of potassium bicarbonate, 2 g of potassium chloride, and 6 g of 4-hydroxyethylpiperazineethanesulfonic acid, stir and fully dissolve; gradually add 7 g of sucrose, 4 g of mannose, 20 g of trehalose, and 2 g of hydroxyethyl starch, stir and fully dissolve; continue to add 60 ml of ethylene glycol, stir and fully dissolve; continue to add 10 mg of water-soluble vitamin E, stir and fully dissolve;

[0037] (2) Adjust the pH to 7.2, make up to 1000 ml with double distilled water, filter and sterilize with a 0.22 μm pore size filter membrane, and store at 2-8°C.

[0038] The method for using the cryopreservation solution prepared in this embodiment comprises the following steps:

[0039] S1. Digestion: Take cells in the logarithmic growth phase, remove the old culture medium in the culture dish, wash twice with PBS; digest with trypsin, centrifuge and discard the waste liquid, retain the precipitate; add freezing solution and resuspend to obtain cell suspension, the cell suspension concentration is about 1×10 6 Pieces / mL.

[0040] S2. Cryopreservation: the cell suspension using the cryopreservation solution of the present invention is directly placed at -80°C;

[0041] S3. Recovery: Thaw the frozen cells at 37±0.5℃, centrifuge, retain the precipitate, add complete culture medium to resuspend, inoculate, and culture.

[0042] Example 2

[0043] Compared with Example 1, except for the different amounts of components in each 1000 mL of the cell freezing solution: 50 g of sodium lactobionate, 5 mg of calcium chloride, 0.5 g of magnesium chloride, 1.5 g of potassium dihydrogen phosphate, 0.5 g of potassium bicarbonate, 2 g of potassium chloride, 7 g of sucrose, 4 g of mannose, 20 g of trehalose, 2 g of hydroxyethyl starch, 6 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), and 60 mL of ethylene glycol; the rest are the same as Example 1.

[0044] Example 3

[0045] Compared with Example 1, except for the different amounts of components in each 1000 mL of the cell freezing solution: 50 g of sodium lactobionate, 5 mg of calcium chloride, 0.5 g of magnesium chloride, 1.5 g of potassium dihydrogen phosphate, 0.5 g of potassium bicarbonate, 2 g of potassium chloride, 7 g of sucrose, 4 g of mannose, 20 g of trehalose, 2 g of hydroxyethyl starch, 6 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 20 mg of water-soluble vitamin E, and 60 mL of ethylene glycol; the rest are the same as Example 1.

[0046] Example 4

[0047] Compared with Example 1, except for the different amounts of components in each 1000 mL of the cell freezing solution: 20 g of sodium lactobionate, 5 mg of calcium chloride, 0.5 g of magnesium chloride, 1.5 g of potassium dihydrogen phosphate, 0.5 g of potassium bicarbonate, 2 g of potassium chloride, 5 g of sucrose, 4 g of mannose, 10 g of trehalose, 1 g of hydroxyethyl starch, 6 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 10 mg of water-soluble vitamin E, and 60 mL of ethylene glycol; the rest are the same as Example 1.

[0048] Example 5

[0049] Compared with Example 1, except for the different amounts of components in each 1000 mL of the cell freezing solution: 60 g of sodium lactobionate, 5 mg of calcium chloride, 0.5 g of magnesium chloride, 1.5 g of potassium dihydrogen phosphate, 0.5 g of potassium bicarbonate, 2 g of potassium chloride, 20 g of sucrose, 4 g of mannose, 40 g of trehalose, 10 g of hydroxyethyl starch, 6 g of 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), 10 mg of water-soluble vitamin E, and 60 mL of ethylene glycol; the rest are the same as Example 1.

[0050] Comparative Example 1

[0051] The preparation of traditional 1L cryopreservation solution includes the following steps:

[0052] (1) Take 900 ml of fetal bovine serum and add 100 ml of dimethyl sulfoxide;

[0053] (2) After thorough mixing, filter and sterilize using a 0.22 μm filter membrane and store at 2-8°C.

[0054] The method for using the freezing solution in this reference example comprises the following steps:

[0055] S1. Digestion: Take cells in the logarithmic growth phase, remove the old culture medium in the culture dish, wash twice with PBS; digest with trypsin, centrifuge and discard the waste liquid, retain the precipitate; add freezing solution and resuspend to obtain cell suspension, the cell suspension concentration is about 1×10 6 Pieces / mL.

[0056] S2. Cryopreservation: The cell suspension obtained by traditional cryopreservation solution is placed in a programmed cooling box and stored at -80°C, or placed in a -80°C box after manual programmed cooling.

[0057] S3. Recovery: Thaw the frozen cells at 37±0.5℃, centrifuge, retain the precipitate, add complete culture medium to resuspend, inoculate, and culture.

[0058] Test example

[0059] (1) Cryopreservation of HELA cells

[0060] According to the method of using the cryopreservation solution, HELA cells (from Boster Biotech) were directly cryopreserved using the cryopreservation solution of the embodiment of the present invention and HELA cells were cryopreserved using the cryopreservation solution of the control example using a programmed cooling box. After 1 month of cryopreservation, the cells were revived, the cell status was checked, and the cell viability was counted. Figure 1 The state of HELA cells after thawing using the cryopreservation solution of Example 1 of the present invention, Figure 2 This is the state of HELA cells after thawing using the traditional cryopreservation solution of Comparative Example 1. The statistics of HELA cell viability are shown in Table 1:

[0061] Table 1

[0062] Cryopreservation medium HeLa cell viability Example 1 98％ Example 2 87％ Example 3 92％ Example 4 83％ Example 5 91％ Comparative Example 1 97％

[0063] (2) Cryopreservation of THP-1 cells

[0064] According to the method of using the freezing solution, THP-1 cells (from Boster Biotech) were directly frozen using the freezing solution of the embodiment of the present invention and the storage solution of the control example was used to freeze THP-1 cells in a programmed cooling box. After one month of freezing, the cells were revived, the cell status was checked, and the cell viability was counted. Figure 3 The state of THP-1 cells after thawing using the cryopreservation solution of Example 1 of the present invention, Figure 4 This is the state of THP-1 cells after thawing using the traditional cryopreservation solution of Control Example 1. The statistics of THP-1 cell viability are shown in Table 2:

[0065] Table 2

[0066]

[0067]

[0068] It can be seen from Application Example 1 that after the cryopreservation solution of Example 1 of the present invention was frozen and revived HELA cells, the cell state and viability of HELA were comparable to those of the traditional cryopreservation solution. It can be seen from Application Example 2 that after the cryopreservation solution of Example 1 of the present invention was frozen and revived THP-1 cells, the cell viability was significantly improved compared with THP-1 after cryopreservation and revivification with the traditional cryopreservation solution, the cell state was better, and fewer dead cells appeared. It can be seen from the comparison of the experimental results of Example 1, Example 2, and Example 3 that the addition of water-soluble vitamin E to the cell freezing solution can improve the cell recovery survival rate after freezing, and the addition of water-soluble vitamin E at a suitable concentration can obtain a higher cell recovery survival rate. It can be seen from the comparison of the experimental results of Example 1, Example 4, and Example 5 that non-permeable protective agents such as sodium lactobionate, sucrose, trehalose, and hydroxyethyl starch in the cell freezing solution can maintain a higher viability after cell freezing at a suitable concentration, and too low or too high non-permeable protective agent concentrations are not conducive to cell recovery and survival after freezing.

[0069] In summary, the cell freezing solution provided by the present invention has the advantages of high efficiency, no exogenous protein and easy to use. The cells revived after freezing have high activity and can maintain their cell characteristics after resuscitation. The cell freezing solution provided by the present invention does not contain serum and other exogenous proteins, avoiding the influence of endotoxins, latent viruses and batch differences. The cell freezing solution provided by the present invention does not contain DMSO, and the toxic effect on cells is further reduced, which helps to maintain cell characteristics. The cell freezing solution provided by the present invention does not need to be cooled by a program, and can be directly placed in a -80°C refrigerator for storage, which is quick and convenient to use.

[0070] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A ready-to-use serum-free and DMSO-free cell freezing solution, characterized in that: Each 100 mL of the cell freezing solution includes the following components: 2 g to 6 g of sodium lactobionate, 0.0003 g to 0.0008 g of calcium chloride, 0.03 g to 0.08 g of magnesium chloride, 0.08 g to 0.2 g of potassium dihydrogen phosphate, 0.03 g to 0.08 g of potassium bicarbonate, 0.1 g to 0.4 g of potassium chloride, 0.5 g to 2 g of sucrose, 0.2 g to 0.6 g of mannose, 1 g to 4 g of trehalose, 0.1 g to 1 g of hydroxyethyl starch, 0.3 g to 1 g of 4-hydroxyethylpiperazineethanesulfonic acid, 0.0005 g to 0.003 g of water-soluble vitamin E, and 2 mL to 10 mL of ethylene glycol.

2. A ready-to-use serum-free and DMSO-free cell freezing solution according to claim 1, characterized in that: Each 100 mL of the cell freezing solution includes the following components: 3 g to 5 g of sodium lactobionate, 0.0004 g to 0.0007 g of calcium chloride, 0.04 g to 0.07 g of magnesium chloride, 0.09 g to 0.15 g of potassium dihydrogen phosphate, 0.04 g to 0.07 g of potassium bicarbonate, 0.2 g to 0.3 g of potassium chloride, 0.6 g to 0.8 g of sucrose, 0.3 g to 0.5 g of mannose, 1 g to 3 g of trehalose, 0.2 g to 0.5 g of hydroxyethyl starch, 0.4 g to 0.8 g of 4-hydroxyethylpiperazineethanesulfonic acid, 0.001 g to 0.002 g of water-soluble vitamin E, and 3 mL to 8 mL of ethylene glycol.

3. A ready-to-use serum-free and DMSO-free cell freezing solution according to claim 1, characterized in that: Each 100 mL of the cell freezing solution includes the following components: 5 g of sodium lactobionate, 0.0005 g of calcium chloride, 0.05 g of magnesium chloride, 0.15 g of potassium dihydrogen phosphate, 0.05 g of potassium bicarbonate, 0.2 g of potassium chloride, 0.7 g of sucrose, 0.4 g of mannose, 2 g of trehalose, 0.4 g of hydroxyethyl starch, 0.6 g of 4-hydroxyethylpiperazineethanesulfonic acid, 0.001 g of water-soluble vitamin E, and 6 mL of ethylene glycol.

4. A ready-to-use serum-free and DMSO-free cell freezing kit, characterized in that: The composition comprises the components according to any one of claims 1 to 3.

5. A method for preparing a ready-to-use serum-free and DMSO-free cell freezing solution according to any one of claims 1 to 3, characterized in that: The steps include: (1) In double distilled water, add sodium lactobionate, calcium chloride, magnesium chloride, potassium dihydrogen phosphate, potassium bicarbonate, potassium chloride, and 4-hydroxyethylpiperazineethanesulfonic acid and dissolve them; continue to add sucrose, mannose, trehalose, and hydroxyethyl starch and dissolve them; continue to add ethylene glycol and mix well; continue to add water-soluble vitamin E and dissolve it to obtain a mixed solution; (2) The pH of the mixed solution is adjusted to 7.2, sterilized, and fixed to volume to obtain a cell freezing solution.

6. The method for preparing a ready-to-use serum-free and DMSO-free cell freezing solution according to claim 5, characterized in that: The sterilization in step (2) is specifically as follows: sterilization by filtering with a 0.22 um pore size filter membrane.

7. The method for preparing a ready-to-use serum-free and DMSO-free cell freezing solution according to claim 5, characterized in that: The specific method of determining the volume in step (2) is: using a volumetric flask to determine the volume.

8. A use of a ready-to-use serum-free and DMSO-free cell freezing solution, characterized in that: The cell freezing solution according to any one of claims 1 to 3 is used for freezing cells.

9. A cell freezing method, characterized in that: The method comprises the following steps: using the cell freezing solution according to any one of claims 1 to 3 to dilute the cells to be frozen into a cell suspension; and then transferring the cell suspension to a cryopreservation tube for freezing.

10. A cell freezing method according to claim 9, characterized in that: The cell concentration in the cell suspension is 1×10 6 cells / ml~1×10 7 cells / ml.

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