Tissue serum-free vitrification cryopreservation liquid and application thereof

Abstract

The invention belongs to the technical field of tissue cryopreservation, discloses a serum-free tissue vitrification cryopreservation solution and application thereof, and particularly discloses a serum-free vitrification cryopreservation solution which comprises a permeable cryoprotectant, a non-permeable cryoprotectant, an energy metabolism regulator, an antioxidant and a culture medium. The tissue serum-free vitrification cryopreservation liquid provided by the invention is invented based on a vitrification preservation principle, is simple in procedure, short in time consumption and low in cost, does not need an expensive programmed cooling instrument to strictly control a cooling step, can effectively avoid damage caused by generation of ice crystals, and has a cryopreservation effect far better than that of slow quick freezing. The serum-free tissue vitrification cryopreservation liquid does not need to add unsafe components such as animal-derived serum, reduces the possibility of animal pathogen pollution, can be used for cryopreservation of tissues such as liver, kidney, skin and tumor, and has a recovery rate of more than 90%.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue cryopreservation, and in particular relates to a serum-free tissue vitrification cryopreservation solution and an application thereof. Background Art

[0002] Living tissues have important clinical and scientific research values. Whether the activity of tissues preserved in vitro can be maintained for a long time and with high quality is the key to realizing these values. It is generally believed that when biological materials are kept at deep low temperatures (less than -130°C), the internal kinetic energy and molecular motion slow down, the chemical and biological reaction rates decrease, and the metabolism, transport, enzyme reaction and diffusion processes are very weak. In theory, they can be stored indefinitely. Cryopreservation technology has been successfully applied to the preservation of single, small-volume biological materials such as cells and sperm. However, for biological tissues, which are composed of multiple layers of cells, their structural complexity will limit the conduction of heat and the penetration of cryoprotectants (CPA), making it difficult to fully preserve their original physiological structure and function.

[0003] In recent years, exploring the long-term high-quality cryopreservation of complex tissues has become a new research hotspot internationally. Existing published patents also involve the field of tissue cryopreservation fluids, mainly including: umbilical cord (patent publication number: CN104472473A, CN113133444A, CN116941606A), placenta (patent publication number: CN116171984A), fat (patent publication number: CN102907416A, CN106465710A, CN110547286A, CN109845728A, CN110973121A, CN111449055A), tumors (patent publication numbers: CN107041362A, CN113475500A, CN114467913A) and ovaries (patent publication numbers: CN111789107A, CN111789106A, CN110074097A, CN110352949A) and other tissues, but the above-mentioned tissue freezing solution has the following shortcomings.

[0004] First of all, the development of most cryopreservation solutions is based on the principle of programmed preservation, which includes precise and complex cooling steps. The cooling speed and time require expensive programmed cooling devices or other instruments to control, and the growth of ice crystals during cooling and preservation can cause fatal mechanical damage to cells and tissues. Therefore, in recent years, some researchers have turned their attention to a new deep-cryopreservation technology - vitrification, which does not require special instruments and can be directly put into liquid nitrogen. The procedure is simple, time-saving and low-cost. More importantly, the essence of vitrification is that the liquid forms a highly viscous state during the freezing and solidification process, and the movement of molecules is highly constrained, thereby avoiding damage caused by ice crystal formation. The effect of vitrification is far better than slow preservation, and it is considered to be the only promising technology for long-term preservation of large tissues and organs.

[0005] Secondly, the existing tissue cryopreservation fluids are only developed for a single tissue, with simple formulas and narrow application ranges. There are only two cryopreservation fluids that can be used for multiple tissues. Among them, Chinese invention patent CN114946836A discloses a microtissue cryopreservation fluid that can be used for breast organoids, kidney cancer organoids, bladder organoids, small intestinal organoids, and lung organoids, but it uses a programmed preservation technology that requires a strict control of the cooling rate to 0.5-3°C / min, and it can only be transferred to liquid nitrogen for long-term storage after 24 hours. Finally, most cryopreservation fluids contain fetal bovine serum, which not only contains xenogeneic immunogens of unknown composition, but also poses potential safety risks during clinical applications.

[0006] The ideal tissue preservation fluid should have the following characteristics: 1) good permeability, able to penetrate multiple layers of cells into the tissue; 2) based on the principle of vitrification preservation, to the greatest extent possible inhibit fatal damage such as ice crystal growth to improve the activity of tissue after recovery, and no expensive program cooling device is required to strictly control the cooling steps; 3) suitable for long-term preservation of various tissues; 4) avoid the introduction of xenogeneic immunogens such as animal serum. Currently, there is no cryopreservation fluid that meets the above characteristics. Therefore, it is urgent to establish a serum-free vitrification cryopreservation fluid suitable for various biological tissues. Summary of the invention

[0007] In order to overcome the defects of the prior art, the present invention proposes a serum-free vitrification cryopreservation solution for tissues. The cryopreservation solution is used for cryopreservation of human and animal tissues, which can effectively maintain the cryopreservation viability of the tissues and the integrity of their structure and function, with a recovery rate of over 90%.

[0008] The purpose of the first aspect of the present invention is to provide a serum-free vitrification cryopreservation solution.

[0009] The purpose of the second aspect of the present invention is to provide an application of the serum-free vitrification cryopreservation solution of the first aspect of the present invention.

[0010] The third aspect of the present invention is to provide a kit.

[0011] The fourth aspect of the present invention aims to provide a method for tissue cryopreservation.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is:

[0013] The first aspect of the present invention provides a serum-free vitrification freezing solution, comprising a permeable cryoprotectant, a non-permeable cryoprotectant, an energy metabolism regulator, an antioxidant and a culture medium.

[0014] The energy metabolism regulator can reduce the damage caused by insufficient synthesis of adenosine triphosphate in tissues at low temperatures, and provide certain nutrients. The antioxidant can reduce the cell damage caused by the large-scale production of reactive oxygen during the freezing process. And the freezing solution does not contain other animal protein components, avoiding the potential risks of introducing unknown pathogens. In addition, the freezing solution is suitable for vitrification freezing, which can effectively avoid ice crystal damage during the freezing process. The use of this freezing solution for freezing human and animal tissues can effectively maintain the freezing activity of tissues and the integrity of tissue structure and function, with a recovery rate of more than 90%.

[0015] In some embodiments of the present invention, the serum-free vitrification solution includes 35-50 v / v% osmotic cryoprotectant, 1000-1100 mmol / L non-osmotic cryoprotectant, 1900-2100 mmol / L energy metabolism regulator, 5-20 mmol / L antioxidant and culture medium.

[0016] In some preferred embodiments of the present invention, the serum-free vitrification solution includes 40-45 v / v% osmotic cryoprotectant, 1000-1050 mmol / L non-osmotic cryoprotectant, 2000-2100 mmol / L energy metabolism regulator, 5-15 mmol / L antioxidant and culture medium.

[0017] In some more preferred embodiments of the present invention, the serum-free vitrification solution includes 40-45 v / v% osmotic cryoprotectant, 1000-1020 mmol / L non-osmotic cryoprotectant, 2000-2020 mmol / L energy metabolism regulator, 6-10 mmol / L antioxidant and culture medium.

[0018] In some embodiments of the present invention, the osmotic protectant includes dimethyl sulfoxide and polyol.

[0019] In some embodiments of the present invention, the polyol includes one or more of ethylene glycol, propylene glycol, and glycerol.

[0020] In some preferred embodiments of the present invention, the osmotic protectant includes dimethyl sulfoxide and ethylene glycol.

[0021] Dimethyl sulfoxide and ethylene glycol are permeable cryoprotectants. They are small molecules that can penetrate the plasma membrane and form hydrogen bonds with water molecules to lower the freezing point and prevent ice formation inside and outside the cell. It is generally believed that ethylene glycol combined with dimethyl sulfoxide is the first choice for vitrification cryoprotectants. Compared with other cryoprotectants, dimethyl sulfoxide has strong permeability, low relative molecular weight, and can form a stable vitrified state at a low concentration. It is a basic cryoprotectant for cell cryopreservation. However, dimethyl sulfoxide has strong biochemical toxicity to tissue cells, and reducing its concentration is beneficial to improving the biological activity of tissue cells after vitrification. Ethylene glycol has strong permeability and low toxicity, but its vitrification formation ability is slightly poor. Therefore, the combined use of the two can complement each other, and a large number of studies have shown that the use of ethylene glycol combined with dimethyl sulfoxide is better than the use of dimethyl sulfoxide or ethylene glycol alone in the cryopreservation of neural tissue, ovarian tissue, embryos, oocytes, etc. In addition, the combination of non-permeable (sucrose) and permeable cryoprotectants can further reduce the cytotoxic effects of permeable cryoprotectants and improve the results of vitrification.

[0022] In some embodiments of the present invention, the non-permeable protective agent includes human serum albumin and water-soluble sugar.

[0023] In some embodiments of the present invention, the water-soluble sugar includes one or more of sucrose, trehalose, hydroxyethyl starch, and dextran.

[0024] In some embodiments of the present invention, the non-permeable protective agent includes human serum albumin and sucrose.

[0025] Human serum albumin can not only reduce side effects, but also stabilize proteins. Sucrose is a natural non-reducing sugar and the most effective cryoprotectant among all disaccharides. As a non-permeable protective agent, it will not penetrate the plasma membrane during the cooling process, but will concentrate between the plasma membranes to form a buffer layer, or adsorb on the surface of the plasma membrane to form a sticky layer to prevent the formation of ice crystals. In addition, it increases the viscosity of the vitrified solution and the glass transition temperature required for the vitrified solution, making the glassy system stable and less prone to physical and chemical changes.

[0026] In some embodiments of the present invention, the energy metabolism regulator includes at least one of hydrocortisone, insulin, glutamine, and adenosine triphosphate.

[0027] Hydrocortisone, insulin, glutamine, and adenosine triphosphate are good nutrients that can maintain the energy required for cell metabolism and prevent ATP depletion. Under low temperatures, the ATP produced by mitochondria is significantly reduced, but energy is still needed to maintain cell metabolism. ATP depletion will inhibit Na + / K +The activity of ATPase leads to cell membrane depolarization and destruction of lipid bilayer integrity and cytoskeletal structure. The damaged cell membrane loses control over ion flow, leading to ion homeostasis disorder, further disrupting cell metabolism, and ultimately leading to cell necrosis. Previous literature has shown that adding ATP to hair follicle preservation solution can prolong the in vitro preservation time of hair follicles and better maintain the activity of hair follicles.

[0028] In some preferred embodiments of the present invention, the energy metabolism regulators include hydrocortisone, insulin, glutamine and adenosine triphosphate.

[0029] In some embodiments of the present invention, the antioxidant includes at least one of melatonin, epigallocatechin-3-ol, epigallocatechin-3-ol gallate, and resveratrol.

[0030] In some embodiments of the present invention, the antioxidants include melatonin, epigallocatechin gallate and resveratrol.

[0031] The decrease in enzyme activity at low temperatures will lead to excessive production of ROS in cells. ROS will destroy essential macromolecules such as proteins, lipids and nucleic acids, and further aggravate ATP depletion, ultimately leading to cell necrosis and apoptosis. Antioxidants can provide a good reducing environment for frozen tissues, effectively preventing the accumulation of ROS in cells and avoiding damage to cells. Melatonin has the effects of a free radical scavenger and a DNA repair inducer. Epigallocatechin gallate (EGCG) has very strong antioxidant activity, which is at least 100 times that of vitamin C and 25 times that of vitamin E. The pharmacological benefits of resveratrol include anti-tumor, antioxidant, anti-inflammatory and anti-aging.

[0032] In some embodiments of the present invention, the serum-free vitrification solution comprises dimethyl sulfoxide, ethylene glycol, human serum albumin, sucrose, hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate, resveratrol and culture medium.

[0033] In some embodiments of the present invention, the serum-free vitrification solution comprises dimethyl sulfoxide, ethylene glycol, human serum albumin, sucrose, hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate, resveratrol and culture medium.

[0034] In some embodiments of the present invention, the serum-free vitrification solution includes 15-25 v / v% dimethyl sulfoxide, 15-25 v / v% ethylene glycol, 2-3 mmol / L human serum albumin, 0.5-2.5 mol / L sucrose, 0.01-1 mmol / L hydrocortisone, 0.2-20 mol / L insulin, 1-4 mmol / L glutamine, 0.1-20 mmol / L adenosine triphosphate, 2-8 mmol / L melatonin, 0.4-10 mmol / L epigallocatechin gallate, 0.4-10 mmol / L resveratrol and culture medium.

[0035] In some preferred embodiments of the present invention, the serum-free vitrification solution includes 18-20 v / v% dimethyl sulfoxide, 18-20 v / v% ethylene glycol, 2.1-2.7 mmol / L human serum albumin, 0.5-2 mol / L sucrose, 0.05-0.1 mmol / L hydrocortisone, 1-2 mol / L insulin, 1-3 mmol / L glutamine, 1-5 mmol / L adenosine triphosphate, 5-8 mmol / L melatonin, 1-5 mmol / L epigallocatechin gallate, 1-5 mmol / L resveratrol and culture medium.

[0036] In some embodiments of the present invention, the culture medium is Walliam's E medium.

[0037] William's E medium contains a variety of inorganic salts, amino acids and vitamins. Compared with other culture media such as DMEM, its ingredients are more suitable for tissue growth needs. In addition, energy metabolism regulators and antioxidants can reduce cell damage caused by metabolic and redox imbalances caused by insufficient synthesis of adenosine triphosphate (ATP) and the massive production of reactive oxygen species (ROS) during the freezing and thawing process of tissues, and provide certain nutrients to maintain the cryopreservation viability of tissues.

[0038] In some embodiments of the present invention, the method for preparing the serum-free vitrification solution comprises:

[0039] (1) dissolving hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate, resveratrol and other powders according to dosage in a culture medium to prepare a basic solution;

[0040] (2) dissolving human serum albumin and sucrose in a base solution according to dosage to prepare an initial freezing solution;

[0041] (3) Dimethyl sulfoxide, ethylene glycol and the initial freezing solution are mixed in corresponding volume ratios to prepare a freezing solution.

[0042] The second aspect of the present invention provides the use of the serum-free vitrification cryopreservation solution of the first aspect of the present invention in (1) or (2):

[0043] (1) Cryopreservation of tissues and / or cells;

[0044] (2) Preparation of tissue and / or cell cryopreservation products.

[0045] In some embodiments of the present invention, the tissues include but are not limited to liver, kidney, skin, tumor, hair follicle and other tissues.

[0046] The third aspect of the present invention provides a kit comprising the serum-free vitrification solution of the first aspect of the present invention.

[0047] In some embodiments of the present invention, the base solution includes hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate and resveratrol.

[0048] In some embodiments of the present invention, the basic solution includes 0.01-1 mmol / L of hydrocortisone, 0.2-20 mol / L of insulin, 1-4 mmol / L of glutamine, 0.1-20 mmol / L of adenosine triphosphate, 2-8 mmol / L of melatonin, 0.4-10 mmol / L of epigallocatechin gallate, 0.4-10 mmol / L of resveratrol and culture medium.

[0049] In some preferred embodiments of the present invention, the basic solution includes 0.05-0.1 mmol / L of hydrocortisone, 1-2 mol / L of insulin, 1-3 mmol / L of glutamine, 1-5 mmol / L of adenosine triphosphate, 5-8 mmol / L of melatonin, 1-5 mmol / L of epigallocatechin gallate, 1-5 mmol / L of resveratrol and culture medium.

[0050] In some embodiments of the present invention, the culture medium is Walliam's E medium.

[0051] The fourth aspect of the present invention provides a tissue cryopreservation method, comprising the step of treating the tissue using the serum-free vitrification cryopreservation solution of the first aspect of the present invention or the kit of the third aspect of the present invention.

[0052] In some embodiments of the present invention, the tissues include but are not limited to liver, kidney, skin, tumor, hair follicle and other tissues.

[0053] In some embodiments of the present invention, when the kit is used to process tissue, the tissue cryopreservation method specifically comprises the following steps:

[0054] Preparation of balancing solution: Mix the base solution in the kit with the freezing solution at a volume ratio of 2-3:1, 1:1, and 1:2-3 to obtain balancing solutions A, B, and C;

[0055] Immerse the tissue in balancing solutions A, B, and C in sequence for 5 to 20 minutes;

[0056] The tissue in the balancing solution was taken out, immersed in the freezing solution for 5 to 10 minutes, and stored in liquid nitrogen.

[0057] The beneficial effects of the present invention are:

[0058] The serum-free vitrification cryopreservation solution provided by the present invention is invented based on the principle of vitrification preservation. It has a simple procedure, short time consumption, low cost, and does not require an expensive program cooling device to strictly control the cooling steps. It can effectively avoid the damage caused by ice crystal formation, and the freezing effect is far better than slow freezing. The serum-free vitrification cryopreservation solution does not need to add unsafe ingredients such as animal-derived serum, thereby reducing the possibility of animal pathogen contamination. Among them, the combined use of non-permeable and permeable cryoprotectants can inhibit ice crystal formation and reduce the cytotoxic effect of permeable cryoprotectants. Energy metabolism regulators and antioxidants can reduce the cell damage caused by metabolism and redox imbalance caused by insufficient ATP synthesis and the large-scale production of reactive oxygen ROS during the freezing and thawing process of tissues. The serum-free vitrification cryopreservation solution is used for freezing tissues (such as liver, kidney, skin, tumor and other tissues), and the survival rate after tissue recovery is more than 90%, which has good clinical application prospects and scientific research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a HE staining comparison of the mouse back skin frozen using the serum-free vitrification solution of Example 1 and its fresh tissue.

[0060] Figure 2 This is a comparison chart of the cell viability of mouse liver and kidney tissues cryopreserved using the serum-free vitrification solution of Example 1 and their fresh tissues. In the figure, ns means P>0.05.

[0061] Figure 3 This is a comparison chart of cell survival rates of human breast tumor and lung tumor tissues cryopreserved using the serum-free vitrification solution of Example 1 and fresh tissues. In the figure, ns means P>0.05. DETAILED DESCRIPTION

[0062] The present invention is further described in detail below through specific examples.

[0063] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0064] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0065] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0066] Example 1

[0067] A serum-free tissue vitrification cryopreservation solution comprises 20 v / v% dimethyl sulfoxide, 20 v / v% ethylene glycol, 2.4 mmol / L human serum albumin, 1 mol / L sucrose, 0.1 mmol / L hydrocortisone, 2 mol / L insulin, 2 mmol / L glutamine, 1.7 mmol / L adenosine triphosphate, 5 mmol / L melatonin, 2 mmol / L epigallocatechin gallate (CAS No. 989-51-5), 2 mmol / L resveratrol, and Walliam's E medium (Gibco TM A1217601, the same below).

[0068] The method for preparing the above-mentioned freezing solution comprises the following steps:

[0069] (1) dissolving hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate, resveratrol and other powders according to dosage in Walliam's E medium to prepare a basal solution;

[0070] (2) dissolving human serum albumin and sucrose in a base solution according to dosage to prepare an initial freezing solution;

[0071] (3) Dimethyl sulfoxide, ethylene glycol and the initial freezing solution are mixed in corresponding volume ratios to prepare a freezing solution.

[0072] Example 2

[0073] A serum-free tissue vitrification freezing solution comprises 18 v / v% dimethyl sulfoxide, 18 v / v% ethylene glycol, 2.1 mmol / L human serum albumin, 0.5 mol / L sucrose, 0.01 mmol / L hydrocortisone, 0.2 mol / L insulin, 1 mmol / L glutamine, 0.17 mmol / L adenosine triphosphate, 2 mmol / L melatonin, 0.4 mmol / L epigallocatechin gallate, 0.4 mmol / L resveratrol and Walliam's E medium.

[0074] Example 3

[0075] A serum-free tissue vitrification cryopreservation solution comprises 22 v / v% dimethyl sulfoxide, 22 v / v% ethylene glycol, 2.7 mmol / L human serum albumin, 2 mol / L sucrose, 1 mmol / L hydrocortisone, 20 mol / L insulin, 3 mmol / L glutamine, 17 mmol / L adenosine triphosphate, 8 mmol / L melatonin; 10 mmol / L epigallocatechin gallate; 10 mmol / L resveratrol and Walliam's E culture medium.

[0076] Comparative Example 1

[0077] The conventional freezing solution on the market is composed of 90% fetal bovine serum and 10% dimethyl sulfoxide.

[0078] Comparative Example 2

[0079] A freezing solution, which is different from Example 1 only in that no osmotic protective agent (dimethyl sulfoxide and ethylene glycol) is added.

[0080] Comparative Example 3

[0081] A freezing solution, which is different from Example 1 only in that no non-permeable protective agent (human serum albumin and sucrose) is added.

[0082] Comparative Example 4

[0083] A freezing solution, which is different from Example 1 only in that: no energy metabolism regulator (hydrocortisone, insulin, glutamine and adenosine triphosphate) and antioxidant (melatonin, epigallocatechin gallate and resveratrol) are added.

[0084] Comparative Example 5

[0085] A freezing solution, which is different from Example 1 only in that no energy metabolism regulator (hydrocortisone, insulin, glutamine and adenosine triphosphate) is added.

[0086] Comparative Example 6

[0087] A freezing solution, which is different from Example 1 only in that no antioxidant (melatonin, epigallocatechin gallate and resveratrol) is added.

[0088] Example 4

[0089] A method for cryopreservation of tissues, comprising the following steps:

[0090] (1) Preparation of equilibrium solution: Figure 1 The base solution was mixed evenly with the freezing solution of the corresponding example at a volume ratio of 3:1, 1:1 and 1:3 to prepare balanced solutions A, B and C.

[0091] (2) Obtain tissue and cut it into tissue blocks no larger than 1 cm × 1 cm × 1 cm. Place it in Walliam's E medium at 4°C and wash it twice.

[0092] (3) Place the washed tissue blocks in 4°C equilibrated solutions A, B, and C in sequence for 5 to 20 minutes.

[0093] (4) Add 1 mL of cryopreservation solution to the cryopreservation tube, pick up the tissue block from the equilibrium solution C, and soak it in the cryopreservation tube for 5 to 10 minutes.

[0094] (5) Cryotubes are placed directly into liquid nitrogen for long-term storage.

[0095] Table 1 Basic solution and freezing solution formula

[0096]

[0097] Effect Example

[0098] 1. Cryopreservation of Mouse Back Skin Tissue

[0099] C57 mice were taken, shaved and depilated with a depilatory cream, the back skin was separated and unfolded, and first divided into left and right parts. The left part was used as fresh tissue to directly make paraffin sections, and the right part was first divided into 1 cm × 1 cm tissue blocks, and cryopreserved using the method of Example 4 (the cryopreservation solution here is the tissue serum-free vitrification cryopreservation solution of Example 1). After 30 days, they were thawed and made into paraffin sections, HE staining was performed, and pictures were collected using a slide scanner.

[0100] The results are as follows Figure 1 As shown, the skin tissue after being cryopreserved for 30 days using the tissue serum-free vitrification cryopreservation solution of Example 1 according to the method of Example 4 is the same as the fresh skin tissue, the tissue structure is intact, and the skin appendages such as hair follicles and sebaceous glands are intact, indicating that the tissue serum-free vitrification cryopreservation solution of Example 1 can be used for the cryopreservation of skin tissue.

[0101] 2. Cell survival rate of liver and kidney tissues of mice

[0102] The liver and kidney tissues of C57 mice were taken and divided into two equal parts, one of which was used as fresh tissue to directly prepare a single cell suspension (as a control example); the other was divided into tissue blocks with a volume of no more than 1 cm×1 cm×1 cm, and cryopreserved using the method of Example 4 (using the cryopreservation solutions of Examples 1 to 3 and Comparative Examples 1 to 6, respectively), thawed after 30 days, and prepared into a single cell suspension; the single cell suspension was stained for live and dead cells, and counted using a fluorescent cell counter.

[0103] The results are shown in Tables 2 and 3. Figure 2As shown, when the serum-free vitrification cryopreservation solution of Example 1 is used to cryopreserve liver tissue and kidney tissue, the cell survival rate of the liver tissue can reach 92%, and the survival rate of the kidney tissue can reach 90%, which is not significantly different from the survival rate of cells in fresh tissue (single cell suspension is directly prepared from fresh liver / kidney tissue), indicating that the serum-free vitrification cryopreservation solution of Example 1 can be used for the cryopreservation of liver and kidney tissues, and has no obvious effect on the cells in these tissues.

[0104] Table 2 Survival rate of hepatocytes in mouse liver tissue and fresh liver tissue

[0105]

[0106] Table 3 Survival rate of renal cells in mouse liver tissue and fresh liver tissue

[0107]

[0108] 3. Cell survival rate of human breast cancer and lung cancer tissues

[0109] Human breast cancer and lung cancer tissues were taken and divided into two equal parts, one of which was used as fresh tissue to directly prepare a single cell suspension; the other was divided into tissue blocks with a volume of no more than 1 cm×1 cm×1 cm, and cryopreserved using the method of Example 4 (the cryopreservation solution here is the serum-free vitrification cryopreservation solution of Example 1), thawed after 30 days, and prepared into a single cell suspension; the single cell suspension was stained for live and dead cells, and counted using a fluorescent cell counter.

[0110] The results are as follows Figure 3 As shown, the cell survival rate of breast cancer tissue cryopreserved using the serum-free vitrification cryopreservation solution of Example 1 can reach 91%, and the cell survival rate of lung cancer tissue can reach 95%, which is not significantly different from the survival rate of cells in fresh tissue, indicating that the serum-free vitrification cryopreservation solution of Example 1 can be used for the cryopreservation of tumor tissue.

[0111] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A serum-free vitrification freezing solution, comprising a permeable cryoprotectant, a non-permeable cryoprotectant, an energy metabolism regulator, an antioxidant and a culture medium.

2. The serum-free vitrification cryopreservation solution according to claim 1, characterized in that: The serum-free vitrification freezing solution comprises 35-50 v / v% of a permeable cryoprotectant, 1000-1100 mmol / L of a non-permeable cryoprotectant, 1900-2100 mmol / L of an energy metabolism regulator, 5-20 mmol / L of an antioxidant and a culture medium.

3. The serum-free vitrification cryopreservation solution according to claim 1 or 2, characterized in that: The permeability protective agent includes dimethyl sulfoxide and polyol; and / or, the non-permeability protective agent includes human serum albumin and water-soluble sugar.

4. The serum-free vitrification solution according to claim 3, characterized in that: The polyol includes one or more of ethylene glycol, propylene glycol, and glycerol; and / or the water-soluble sugar includes one or more of sucrose, trehalose, hydroxyethyl starch, and dextran.

5. The serum-free vitrification solution according to claim 4, characterized in that: The energy metabolism regulator includes at least one of hydrocortisone, insulin, glutamine, and adenosine triphosphate; and / or the antioxidant includes at least one of melatonin, epigallocatechin gallate, and resveratrol.

6. The serum-free vitrification cryopreservation solution according to claim 5, characterized in that: The serum-free vitrification freezing solution comprises dimethyl sulfoxide, ethylene glycol, human serum albumin, sucrose, hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate, resveratrol and culture medium.

7. Use of the serum-free vitrification solution according to any one of claims 1 to 6 in (1) or (2): (1) Cryopreservation of tissues and / or cells; (2) Preparation of tissue and / or cell cryopreservation products.

8. A kit comprising the serum-free vitrification solution according to any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that The kit further comprises a base solution; preferably, the base solution comprises hydrocortisone, insulin, glutamine, adenosine triphosphate, melatonin, epigallocatechin gallate and resveratrol.

10. A tissue cryopreservation method, comprising the step of treating tissue using the serum-free vitrification cryopreservation solution according to any one of claims 1 to 6 or the kit according to claim 8 or 9.

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