A method for freezing cells

Abstract

The present invention provides a novel cell freezing method, and the present invention belongs to the field of cell protection technology. The method comprises the following steps: (1) mixing and incubating cells with a DSPE-PEG-TAT solution to obtain a cell suspension; (2) mixing the cell suspension with a dextran 70 solution and freezing. The method of the present invention is safe and efficient, and carries substances that cannot effectively penetrate the cell membrane barrier into the cell through the characteristics of the transmembrane peptide, and replaces the traditional permeable DMSO, glycerol and other cytotoxic components to play the role of intracellular protective agents, which greatly broadens the application methods of traditional protective agents and provides a new idea for realizing efficient cell preservation.

Description

Technical Field

[0001] The present invention relates to the technical field of cell protection, and in particular to a cell freezing method. Background Art

[0002] Cryocell biology is a discipline that studies and applies cryogenic freezing technology to protect cells, so as to achieve the purpose of long-term storage and use of cells. It is an important branch of cryobiology. With the development of the discipline, the research objectives of cryocell biology have spread to many different types of cells such as germ cells, immune cells, stem cells, red blood cells, platelets, etc., especially in the context of the rapid development of the cell therapy industry. Cell cryopreservation technology is one of the important research directions. Cell freezing technology mainly solves the "solute damage" and "ice crystal damage" produced during the cell freezing process. Ice crystal damage is an important factor of cell freezing damage. Ice crystals can easily cause physical damage to cell structures such as organelles, cell membranes, and cytoskeleton.

[0003] In the cell cryopreservation technology, the traditional method is to use a combination of permeable protective agents and non-permeable protective agents for cell cryopreservation protection; permeable protective agents are generally organic solvents such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, etc. Permeable protective agents can easily penetrate the cell membrane and combine with intracellular water, reduce the number of ice crystals generated during the freezing process, and change the shape of ice crystals, thereby reducing the probability of cell membranes and organelle membranes being pierced and damaged by ice crystals to a certain extent; non-permeable protective agents are mainly macromolecules, including hydroxyethyl starch, dextran, polyvinyl pyrrolidone, albumin, polyethylene glycol, polysucrose, etc. Macromolecule protective agents are generally used as cell extracellular membrane protective agents. They do not have the ability to permeate the cell membrane, cannot combine with intracellular free water to reduce freezing ice crystal damage, and cannot play a role in stabilizing the protection of intracellular organelle membranes and cytoskeleton. Damage to organelles and cytoskeleton is a common form of cell damage.

[0004] From the perspective of the safety of cell cryopreservation methods, the biggest problem with traditional protective agents such as dimethyl sulfoxide and glycerol is the toxic side effects on cells and the human body. Even excipient-grade permeable organic solvents also have side effects such as vascular irritation and oral mucosal irritation. When used in cell therapy, permeable organic solvents will increase the metabolic burden on the patient's liver, kidneys and other organs due to impaired organ function.

[0005] On the other hand, the use of traditional protective agents containing high osmotic pressure substances such as dimethyl sulfoxide and glycerol also has great limitations on the large-scale preparation of cell drugs. The survival rate of cells in a high osmotic pressure environment is low, and their functions will also be greatly affected. Secondly, under room temperature conditions, osmotic organic solvents such as dimethyl sulfoxide and glycerol still have great cytotoxicity. Therefore, in the application of cell drug preparation, the lower ambient temperature must be strictly controlled, which increases the difficulty of the preparation process and greatly limits the development of cell drug process. Summary of the invention

[0006] The purpose of the present invention is to provide a cell cryopreservation method. The cell cryopreservation method of the present invention breaks the traditional method of using a permeable organic solvent as an intracellular protective agent in traditional cell cryopreservation technology, and adopts a cell-penetrating peptide to anchor a macromolecular substance as a macromolecular protective agent for transmembrane protection. It does not need to use DMSO, glycerol and other components that are toxic to cells and human bodies, and can safely and efficiently achieve high-quality cell preservation.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a cell freezing method, comprising the following steps:

[0009] (1) Mixing cells with DSPE-PEG-TAT solution and incubating to obtain a cell suspension;

[0010] (2) Mix the cell suspension with dextran 70 solution and freeze the mixture.

[0011] The structural formula of the DSPE-PEG-TAT is:

[0012] .

[0013] Preferably, the cells include mesenchymal stem cells or VERO cells.

[0014] Preferably, the DSPE-PEG-TAT solution uses PB solution as solvent, has a concentration of 0.2-1.5 mM, and a pH of 6.0-7.0.

[0015] Preferably, the density of the cells after mixing with the DSPE-PEG-TAT solution is (4-10)×10 6 cells / mL.

[0016] Preferably, the mixed incubation temperature is 18-30° C., and the mixed incubation time is 30-60 min.

[0017] Preferably, the concentration of the dextran 70 solution is 0.5~2 mM.

[0018] Preferably, the volume ratio of the cell suspension to the dextran 70 solution is 0.8-1.2:1.

[0019] Preferably, during the cryopreservation, the temperature is first lowered and then cryopreserved in liquid nitrogen.

[0020] Preferably, the cooling is to reduce the temperature to -70 to -80°C, and the cooling rate is 1 to 2°C / min.

[0021] Preferably, the freezing temperature is -150 ~ -196°C.

[0022] The cell freezing method provided by the present invention can realize safe and efficient cryopreservation of cells. The method that cell-penetrating peptides carry macromolecular protective agents to realize transmembrane protection of cells breaks the limitation that the traditional freezing method must use dimethyl sulfoxide, glycerol and other toxic components to cells and human bodies, and the extracellular protective effect of dextran 70 is combined to reduce ice crystal damage and solute damage, stabilize cell membranes and organelle membranes. The method of the present invention is simple, the additives are safe, and the limitations of key conditions such as time and temperature for industrial preparation of cell preparations are also solved, which is easier for large-scale production of cell preparations. At the same time, the freezing preservation method of the present invention can effectively protect different types of cells, and efficient preservation is achieved in mesenchymal stem cells and VERO cells.

[0023] Substances such as dimethyl sulfoxide and glycerol have side effects such as irritation of blood vessels and oral mucosa. Especially in cell therapy, the organ functions of tumor patients are generally damaged to varying degrees. Organic solvents such as dimethyl sulfoxide and glycerol will further increase the metabolic burden of the patient's liver, kidney and other organs. The method of the present invention creatively uses cell-penetrating peptides to mediate the protective agent components that cannot penetrate the membrane into the cell, thereby playing a role in cell protection. In addition, due to the extremely high osmotic pressure of traditional cell protectants and high cytotoxicity under normal temperature conditions, the cell preparation conditions, especially the large-scale cell preparation conditions, are demanding, and the preparation temperature, time and other parameters must be strictly controlled. The cell freezing method of the present invention can effectively avoid the above problems. The method of the present application does not require the use of permeable protective agents, and can effectively avoid the effects of high osmotic pressure and cytotoxicity on the preparation efficiency of cell preparations, greatly extending the preparation time of cell preparations. At the same time, the traditional method needs to complete the preparation of cell preparations at low temperatures (2~8°C) because the higher the temperature, the greater the impact on cell activity. The cell freezing method can also effectively avoid the limitation of preparation temperature, making it easier to achieve large-scale cell preparation production. The experimental results show that the method of the present invention is simple to operate and can effectively solve key problems such as cytotoxicity and human toxicity caused by traditional cell cryopreservation methods in production and clinical applications, while maintaining a high cell preservation survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the microfilament structure detection picture of MSCs cells after 3 months of cryopreservation;

[0025] Figure 2 This is a picture of the microfilament structure detection of VERO cells after 3 months of cryopreservation;

[0026] Figure 3 This is a picture of mitochondrial structure detection after MSCs cells were frozen for 3 months;

[0027] Figure 4 This is a picture of mitochondrial structure detection of VERO cells after 3 months of cryopreservation. DETAILED DESCRIPTION

[0028] The present invention provides a cell freezing method, comprising the following steps:

[0029] (1) Mixing cells with DSPE-PEG-TAT solution and incubating to obtain a cell suspension;

[0030] (2) Mix the cell suspension with dextran 70 solution and freeze the mixture.

[0031] In the present invention, cells are mixed with DSPE-PEG-TAT solution for incubation to obtain a cell suspension.

[0032] In the present invention, the cells include mesenchymal stem cells or VERO cells.

[0033] In the present invention, the DSPE-PEG-TAT solution uses PB solution as solvent, the concentration is 0.2-1.5 mM, preferably 0.5-1.2 mM, and more preferably 0.9 mM; the pH is 6.0-7.0, preferably 6.5.

[0034] In the present invention, the DSPE-PEG-TAT solution is prepared from the PB solution and then sterilized by filtration, wherein the filtration sterilization is performed using a 0.22 μm sterile filter.

[0035] In the present invention, the DSPE-PEG-TAT was purchased from Nanjing Peptide Biotechnology Co., Ltd.

[0036] In the present invention, the DSPE-PEG-TAT solution refers to a phospholipid-polyethylene glycol-targeted cell-penetrating peptide TAT solution, wherein the amino acid sequence of the cell-penetrating peptide TAT is YGRKKRRQRRR.

[0037] In the present invention, the structural formula of the DSPE-PEG-TAT is:

[0038] .

[0039] Cell-penetrating peptides are a type of short peptide that can carry macromolecules into cells, and their ability to penetrate the membrane does not rely on classical endocytosis. After TAT-linked polyethylene glycol (PEG) and phospholipids (DSPE) achieve transmembrane transport, polyethylene glycol can bind to intracellular free water and reduce the formation of intracellular ice crystals; phospholipids, as the basic substance of cell structure, can stabilize cell membranes and organelle membranes and activate cells.

[0040] In the present invention, the density of the cells after mixing with the DSPE-PEG-TAT solution is (4-10)×10 6 cells / mL.

[0041] In the present invention, the temperature of the mixed incubation is 18-30° C., preferably 22-26° C., and more preferably 24° C. Under suitable incubation temperature conditions, the fluidity of the cell membrane increases, and the efficiency of transmembrane transport is improved accordingly.

[0042] In the present invention, the mixed incubation time is 30 to 60 min, preferably 40 to 50 min, and more preferably 45 min.

[0043] After the cell suspension is prepared in the present invention, the cell suspension is mixed with a dextran 70 solution and then frozen.

[0044] In the present invention, the concentration of the dextran 70 solution is 0.5-2 mM, preferably 0.8-1.5 mM, and more preferably 1.2 mM.

[0045] In the present invention, the dextran 70 solution is prepared from a PB solution with a pH of 6.0-7.0.

[0046] In the present invention, the volume ratio of the cell suspension to the dextran 70 solution is 0.8-1.2:1, preferably 1:1.

[0047] Dextran 70, as a macromolecular extracellular protective agent, can preferentially bind to extracellular water and reduce the formation of extracellular ice crystals. At the same time, the macromolecular protective agent can also protect the stability of the cell membrane, reduce the electrolyte concentration in the solution, and reduce the impact of solute damage on the cell membrane. The mixing process in the present invention is completed at room temperature, and the cells are frozen after the mixing is completed.

[0048] In the present invention, during the cryopreservation, the temperature is first lowered and then cryopreserved in liquid nitrogen.

[0049] In the present invention, the cooling is to reduce the temperature to -70 to -80°C, more preferably to -80°C.

[0050] In the present invention, the cooling rate is 1-2°C / min, more preferably 1.5°C / min.

[0051] In the present invention, the freezing temperature is -150 ~ -196 ° C, more preferably -196 ° C.

[0052] 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. Example 1

[0053] Use PB solution to prepare DSPE-PEG-TAT solution with a concentration of 0.5 mM, filter (sterile filter pore size 0.22 μm) for sterilization and set aside.

[0054] The concentration of dextran 70 solution was adjusted to 0.8 mM with PB solution and set aside.

[0055] Mesenchymal stem cells and VERO cells were resuspended in DSPE-PEG-TAT solution and incubated at 22°C for 40 min. The two cell suspensions were mixed with dextran 70 solution at a volume ratio of 1:1. The mixture was slowly cooled to -80°C at a rate of 1°C / min and then transferred to liquid nitrogen for long-term storage at -196°C. Example 2

[0056] Use PB solution to prepare DSPE-PEG-TAT solution with a concentration of 0.7 mM, filter (sterile filter pore size 0.22 μm) for sterilization and set aside.

[0057] The concentration of dextran 70 solution was adjusted to 1.0 mM with PB solution and set aside.

[0058] Mesenchymal stem cells and VERO cells were resuspended in DSPE-PEG-TAT solution and incubated at 30°C for 40 min. The two cell suspensions were mixed with dextran 70 solution at a volume ratio of 1:1. The mixture was slowly cooled to -80°C at a rate of 2°C / min and then transferred to liquid nitrogen for long-term storage at -196°C. Example 3

[0059] Use PB solution to prepare DSPE-PEG-TAT solution with a concentration of 0.9 mM, filter (sterile filter pore size 0.22 μm) for sterilization and set aside.

[0060] Adjust the concentration of dextran 70 solution to 1.2 mM with PB solution and set aside.

[0061] Mesenchymal stem cells and VERO cells were resuspended in DSPE-PEG-TAT solution and incubated at 22°C for 50 min. The two cell suspensions were mixed with dextran 70 solution at a volume ratio of 1:1. The mixture was slowly cooled to -80°C at a rate of 1.5°C / min and then transferred to liquid nitrogen for long-term storage at -196°C. Example 4

[0062] Use PB solution to prepare DSPE-PEG-TAT solution with a concentration of 1.2 mM, filter (sterile filter pore size 0.22 μm) for sterilization and set aside.

[0063] Adjust the concentration of dextran 70 solution to 1.5 mM with PB solution and set aside.

[0064] Mesenchymal stem cells and VERO cells were resuspended in DSPE-PEG-TAT solution and incubated at 30°C for 50 min. The two cell suspensions were mixed with dextran 70 solution at a volume ratio of 1:1. The mixture was slowly cooled to -80°C at a rate of 1.5°C / min and then transferred to liquid nitrogen for long-term storage at -196°C.

[0065] Experimental results:

[0066] Using a traditional permeable protective agent plus a non-permeable protective agent combination (1.4 mol / L DMSO + 0.71 mmol / L dextran 70) as a control, the survival rates of the different cells prepared in Examples 1 to 4 after being cryopreserved for 3 months were determined using AO / PI staining.

[0067] The specific results are shown in the table below:

[0068]

[0069]

[0070] It can be seen from Tables 1 and 2 that the cell cryopreservation method of the present invention can effectively protect mesenchymal stem cells and VERO cells, and the effects of different concentration ratios of DSPE-PEG-TAT solution and dextran 70 solution on cell preservation activity in different embodiments are different. Compared with the control group, the effect of Example 3 is the closest, indicating that when the concentration of DSPE-PEG-TAT solution is 0.9 mM and the concentration of dextran 70 solution is 1.2 mM, it is the most suitable working concentration in the present invention, and DSPE-PEG-TAT has the effect of replacing DMSO as an intracellular protective agent.

[0071] The results of cytoskeleton detection after cryopreservation of MSCs and VERO cells are as follows Figure 1-2As shown, the cell freezing method of the present invention plays a good role in maintaining the cytoskeleton structure. Both mesenchymal stem cells and VERO cells have clear microfilament structures, indicating that the combination of DSPE-PEG-TAT and dextran 70 can play a good role in cell stabilization.

[0072] Detection of organelles in MSCs and VERO cells after cryopreservation Figure 3-4 As shown, the cell freezing method of the present invention plays a good role in protecting the organelle structure. The mitochondria of mesenchymal stem cells and VERO cells both show a highly active orange high membrane potential morphology, indicating that the combination of DSPE-PEG-TAT and dextran 70 can also play a good role in protecting the organelle membrane.

[0073] It can be seen from the above examples that the present invention provides a method for using cell-penetrating peptides as non-membrane-permeable substances to protect cells through membranes, which effectively solves the cell and human toxicity risks of permeable organic solvents such as dimethyl sulfoxide and glycerol, as well as the restrictive problems of permeable protective agents on the large-scale production of cell preparations.

[0074] 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 cell freezing method, characterized in that: The following steps are involved: (1) Mixing cells with DSPE-PEG-TAT solution and incubating to obtain a cell suspension; (2) mixing the cell suspension with a dextran 70 solution and freezing the mixture; The cells include mesenchymal stem cells or VERO cells; The DSPE-PEG-TAT solution uses PB solution as solvent, with a concentration of 0.2-1.5 mM and a pH of 6.0-7.0; The structural formula of the DSPE-PEG-TAT is: The concentration of the dextran 70 solution is 0.5-2 mM; The volume ratio of the cell suspension to the dextran 70 solution is 0.8-1.2:1; During the freezing, the temperature is first lowered and then placed in liquid nitrogen for freezing; The cooling is to reduce the temperature to -70 ~ -80 ° C, and the cooling rate is 1 ~ 2 ° C / min; The freezing temperature is -150 ~ -196 ° C.

2. The cell freezing method according to claim 1, characterized in that: The density of the cells after mixing with the DSPE-PEG-TAT solution is (4-10)×10 6 cells / mL.

3. The cell freezing method according to claim 1, characterized in that: The temperature of the mixed incubation is 18-30° C., and the time of the mixed incubation is 30-60 min.

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