Peripheral blood mononuclear cell cryopreservation liquid, preparation method and application thereof, and cryopreservation method of peripheral blood mononuclear cells

Abstract

The invention provides a peripheral blood mononuclear cell cryopreservation solution, a preparation method and application of the peripheral blood mononuclear cell cryopreservation solution and a peripheral blood mononuclear cell cryopreservation method, and relates to the technical field of cells. The peripheral blood mononuclear cell cryopreservation liquid comprises the following components: a permeable protective agent, a non-permeable protective agent, human serum albumin, a 1640 basal culture medium, glutathione, autologous plasma, vitamin C and heparin sodium, wherein the autologous plasma is plasma of the peripheral blood mononuclear cells. According to the technical scheme, the permeable protective agent and the non-permeable protective agent are combined, and meanwhile, the human serum albumin, the 1640 basic culture medium, the heparin sodium, the glutathione, the autologous plasma and the vitamin C are added, so that a metabolic environment in the cell cryopreservation process is provided, and the motility rate and the proliferation activity of the recovered cells are ensured.

Description

Technical Field

[0001] The invention relates to the field of cell technology, and in particular to a peripheral blood mononuclear cell freezing solution and a preparation method and application thereof, and a peripheral blood mononuclear cell freezing method. Background Art

[0002] Peripheral blood mononuclear cells (PBMC) are cells with a single nucleus in peripheral blood. Mononuclear cells include two types of cells, one is lymphocytes (lymphocytes) and the other is monocytes (monocytes). Clinically, PBMC is a key component of the immune system, and therefore is widely used as the most basic experimental raw material in the fields of immunology, infectious diseases, malignant tumors, vaccine development, transplantation therapy, personalized medicine and toxicology. At the same time, due to the importance of PBMC in the body's immunity, PBMC has become the focus of attention and research for many scientific researchers.

[0003] Cell cryopreservation is one of the main means of cell preservation. This technology can be used to store cells in liquid nitrogen at -196°C. The cells will temporarily leave the growth state and preserve the cell characteristics. The cells can be revived and used when needed. When cells are cryopreserved, permeable protective agents or non-permeable protective agents can be added to the culture medium. Under slow freezing conditions, the freezing point of the solution can be lowered, and the water in the cells can be released, thereby reducing the formation of ice crystals and avoiding cell damage.

[0004] At present, most methods for freezing mononuclear cells use dimethyl sulfoxide (DMSO) combined with other ingredients to freeze mononuclear cells, but DMSO is toxic to cells. If it is not removed in time after recovery, it will damage the cells and reduce the activity of the cells. At the same time, long-term contact or high concentration will have adverse effects on the cells. Of course, there are also freezing solutions that do not contain DMSO and culture medium, but they are expensive and not suitable for mass production. Summary of the invention

[0005] The main purpose of the present invention is to provide a peripheral blood mononuclear cell freezing solution and its preparation method and application, and a peripheral blood mononuclear cell freezing method, aiming to reduce the degree of damage of the freezing solution to the mononuclear cells.

[0006] To achieve the above object, the present invention provides a peripheral blood mononuclear cell cryopreservation solution, which is used for cryopreserving peripheral blood mononuclear cells, and comprises the following components: an osmotic protective agent, a non-osmotic protective agent, human albumin, 1640 basal culture medium, glutathione, autologous plasma, vitamin C and heparin sodium;

[0007] Wherein, the autologous plasma is the plasma of the peripheral blood mononuclear cells.

[0008] In one embodiment, the peripheral blood mononuclear cell freezing solution comprises the following components by weight:

[0009] 8-10 parts of osmotic protective agent, 14-16 parts of non-osmotic protective agent, 2-4 parts of human serum albumin, 50-70 parts of 1640 basal medium, 0.5-1 part of glutathione, 14-16 parts of autologous plasma and 0.1-0.5 part of vitamin C;

[0010] In the peripheral blood mononuclear cell freezing solution, the concentration of heparin sodium is 4-6 IU / mL.

[0011] In one embodiment, the osmotic protectant comprises at least one of glycerol and acetamide.

[0012] In one embodiment, the permeability protective agent includes glycerol and acetamide, and the mass ratio of glycerol to acetamide is (5-8): (1-5).

[0013] In one embodiment, the non-permeable protective agent includes at least one of polyethylene glycol, trehalose, glucose and dextran.

[0014] In one embodiment, the non-permeable protective agent includes polyethylene glycol, trehalose, glucose and dextran, and the mass ratio of polyethylene glycol, trehalose, glucose and dextran is (2-4):5:2:2:3.

[0015] The present invention also provides a method for preparing a peripheral blood mononuclear cell cryopreservation solution, comprising:

[0016] S10. Obtain a peripheral blood sample and a human lymph separation fluid, spread the peripheral blood on the human lymph separation fluid and centrifuge, take the supernatant and centrifuge to obtain autologous plasma;

[0017] S20. Mix the permeable protective agent, the non-permeable protective agent, human serum albumin, 1640 basal culture medium, glutathione, autologous plasma, vitamin C and sodium heparin to obtain a peripheral blood mononuclear cell freezing solution.

[0018] The present invention also provides an application of a peripheral blood mononuclear cell freezing solution in freezing peripheral blood mononuclear cells. The peripheral blood mononuclear cell freezing solution includes the peripheral blood mononuclear cell freezing solution as described above, or the peripheral blood mononuclear cell freezing solution prepared by the preparation method as described above.

[0019] The present invention also provides a method for freezing peripheral blood mononuclear cells, comprising:

[0020] S100. Obtaining peripheral blood mononuclear cells to be frozen, and resuspending the peripheral blood mononuclear cells with physiological saline to obtain a cell suspension to be frozen;

[0021] S200. Mix the cell suspension to be frozen with a peripheral blood mononuclear cell freezing solution, pre-freeze at -80°C, and then store in liquid nitrogen at -200 to -190°C, wherein the peripheral blood mononuclear cell freezing solution includes the peripheral blood mononuclear cell freezing solution as described above.

[0022] In one embodiment, in step S200, after mixing, the density of peripheral blood mononuclear cells is 1.0-2.0×10 7 cells / ml.

[0023] In the technical scheme of the present invention, by combining permeable protective agents and non-permeable protective agents, and adding human albumin, 1640 basic culture medium, sodium heparin, glutathione, autologous plasma and vitamin C, a metabolic environment is provided during cell cryopreservation and the cell viability and proliferation activity after recovery are guaranteed; wherein, the permeable protective agent can enter the cell and combine with water molecules to reduce the formation of ice crystals, and at the same time, it can also reduce the excessive extravasation of water in the cell, and avoid the cell from shrinking due to excessive dehydration, thereby protecting the cell; the non-permeable protective agent can increase the viscosity and freezing point of the solvent, thereby reducing the formation of ice crystals, and when the extracellular fluid freezes, the volume will expand. The non-permeable cryoprotectant can fill the gaps around the cell, reduce the spatial changes caused by the freezing of water, thereby reducing the pressure on the cell membrane and the mechanical damage caused thereby, and the non-permeable protective agent can maintain the stability of the extracellular environment, and at the same time, it can prevent the fusion and denaturation of proteins during the freezing and thawing process, thereby protecting the protein components inside and outside the cell. DETAILED DESCRIPTION

[0024] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0025] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0026] At present, most methods for freezing mononuclear cells use DMSO combined with other ingredients to freeze mononuclear cells, but DMSO is toxic to cells. If it is not removed in time after recovery, it will damage the cells and reduce the activity of the cells. At the same time, long-term contact or high concentration will have adverse effects on the cells. Of course, there are also freezing solutions that do not contain DMSO and culture medium, but they are expensive and not suitable for mass production.

[0027] In view of this, the present invention provides a peripheral blood mononuclear cell freezing solution, which is used for freezing peripheral blood mononuclear cells, and the peripheral blood mononuclear cell freezing solution comprises the following components: an osmotic protective agent, a non-osmotic protective agent, human albumin, 1640 basal culture medium, glutathione, autologous plasma, vitamin C and heparin sodium; wherein the autologous plasma is the plasma of the peripheral blood mononuclear cells.

[0028] In the technical solution of the present invention, by combining a permeable protective agent with a non-permeable protective agent, and adding human albumin, 1640 basal culture medium, sodium heparin, glutathione, autologous plasma and vitamin C, a metabolic environment is provided during cell cryopreservation and the viability and proliferation activity of cells after recovery are ensured;

[0029] Among them, permeable protective agents can enter cells and combine with water molecules to reduce the formation of ice crystals. At the same time, they can also reduce excessive extravasation of water in cells, preventing cells from shrinking due to excessive dehydration, thereby protecting cells; non-permeable protective agents can increase the viscosity and freezing point of the solvent, thereby reducing the formation of ice crystals. When the extracellular fluid freezes, the volume will expand;

[0030] Non-permeable cryoprotectants can fill the gaps around cells and reduce the spatial changes caused by water freezing, thereby alleviating the pressure on the cell membrane and the resulting mechanical damage. Non-permeable cryoprotectants can maintain the stability of the extracellular environment and prevent protein fusion and denaturation during the freezing and thawing process, thereby protecting the protein components inside and outside the cells.

[0031] Heparin sodium is an anticoagulant drug that exerts its anticoagulant effect by activating antithrombin III (ATIII). In cryopreservation fluid, heparin sodium can prevent cells from being damaged by the formation of ice crystals and the concentration of intracellular fluid during the freezing process. In addition, heparin sodium can also prevent cells from swelling and rupture due to changes in osmotic pressure during the thawing process. Using heparin sodium as a component of cryopreservation fluid can improve cell survival rate and maintain the stability of its function.

[0032] Glutathione is a small molecule peptide with strong antioxidant capacity, which can protect cells from oxidative damage. Adding glutathione to cell freezing solution helps maintain the redox balance in cells and improve the recovery effect of cells after freezing;

[0033] Vitamin C is a powerful antioxidant that can remove free radicals and reduce the damage to cells caused by oxidative stress. During the cryopreservation process, cells are exposed to a low-temperature environment, which may produce more free radicals, leading to oxidative damage to cell membranes, proteins, and DNA. Vitamin C neutralizes the free radicals produced by cells in a low-temperature environment by providing electrons, thereby protecting cells from oxidative damage.

[0034] Glutathione and vitamin C can work together as antioxidants to resist reactive oxygen species (ROS) produced during the freezing process, thereby reducing the damage to cells caused by oxidative stress; heparin sodium is an anticoagulant that can prevent cells in blood samples from being damaged by coagulation;

[0035] Autologous plasma is rich in growth factors and other nutrients. For certain types of cells (such as stem cells), the use of freezing fluid containing autologous plasma can help maintain cell viability, support the basic metabolic needs of cells in a frozen state, protect cells from damage and environmental stress, and promote the recovery process after thawing.

[0036] In one embodiment, the peripheral blood mononuclear cell freezing solution includes the following components, measured by mass: 8 to 10 parts of permeability protective agent, 14 to 16 parts of non-permeability protective agent, 2 to 4 parts of human albumin, 50 to 70 parts of 1640 basal culture medium, 0.5 to 1 part of glutathione, 14 to 16 parts of autologous plasma and 0.1 to 0.5 parts of vitamin C; in the peripheral blood mononuclear cell freezing solution, the concentration of heparin sodium is 4 to 6 IU / mL.

[0037] In the technical scheme of the present invention, by adjusting the mass fraction of each component, the damage of the peripheral blood mononuclear cell freezing solution to the peripheral blood mononuclear cells can be further reduced; by adjusting the concentration of sodium heparin, as an anticoagulant, the coagulation of peripheral blood mononuclear cells can be prevented. If the concentration of sodium heparin is too low, blood coagulation cannot be effectively prevented, thereby causing cells to be damaged due to clot formation. If the concentration of sodium heparin is too high, the stability of the cell membrane may be affected or cell metabolism may be interfered with.

[0038] In one embodiment, the osmotic protectant comprises at least one of glycerol and acetamide.

[0039] Glycerol is a permeable protective agent that can penetrate the cell membrane into the cell, combine with water molecules, reduce the formation of ice crystals, and thus reduce the damage of ice crystals to the cell membrane; during the freezing process, it increases the solute concentration in the cell and reduces the osmotic pressure difference between the inside and outside of the cell, thereby reducing cell shrinkage; at the same time, it reduces the metabolic activity of the cell during low-temperature storage, reduces energy consumption, and helps the cell to resume normal function after recovery; glycerol has antioxidant properties, which can neutralize the free radicals generated during the freezing process and protect the cells from oxidative stress damage; it also helps the cells to quickly restore normal volume and function after thawing, thereby improving cell viability.

[0040] Acetamide can penetrate into the cells and reduce the electrolyte concentration in the unfrozen solution inside and outside the cells, thereby protecting the cells from damage by high concentrations of electrolytes. At the same time, acetamide can also reduce excessive extravasation of water in the cells and prevent the cells from shrinking due to excessive dehydration. Moreover, acetamide can combine with water molecules to produce hydration, weaken the crystallization process of water, increase the viscosity of the solution, and thus reduce the formation of ice crystals.

[0041] In the technical solution of the present invention, by using at least one of glycerol and acetamide as an osmotic protective agent, the crystallization process of water can be weakened, thereby reducing the damage of ice crystals in cells to cell membranes; wherein, glycerol can help stabilize the cell membrane structure by replacing the position of water molecules, prevent cell membrane rupture caused by water freezing, and, during freezing and thawing, glycerol helps to maintain the osmotic pressure balance inside and outside the cell, reducing the osmotic pressure shock caused by rapid temperature changes; acetamide can reduce the solute effect by diluting the solute in the cell, reducing the toxicity of the solute effect on the cell; the technical solution of the present invention uses glycerol and acetamide to avoid the toxicity of DMSO to peripheral blood mononuclear cells. Specifically, in one embodiment of the present invention, the osmotic protective agent includes glycerol and acetamide.

[0042] In one embodiment, the permeability protective agent includes glycerol and acetamide, and the mass ratio of glycerol to acetamide is (5-8): (1-5). In the technical solution of the present invention, by adjusting the mass ratio of glycerol to acetamide to (5-8): (1-5), the appropriate ratio can optimize the effect of cell freezing and help improve the cell survival rate and functional recovery after thawing.

[0043] In one embodiment, the non-permeable protective agent includes at least one of polyethylene glycol, trehalose, glucose and dextran.

[0044] Polyethylene glycol can preferentially bind to water molecules in the solution, reducing the content of extracellular free water, lowering the freezing point and thus reducing the formation of ice crystals. At the same time, due to its large molecular weight, polyethylene glycol can reduce the electrolyte concentration in the solution and reduce solute damage.

[0045] Trehalose helps maintain the fluidity of cell membranes by replacing some water molecules, reduces mechanical damage to cell membranes caused by ice crystal formation, stabilizes the structure of proteins and other biomolecules, and reduces cell dehydration damage caused by water removal during the freezing process.

[0046] Glucose helps maintain an appropriate osmotic pressure environment and avoid osmotic pressure shock caused by rapid temperature changes. As one of the main energy sources for cells, it can provide the necessary energy to support the metabolic needs of cells during the cryopreservation process.

[0047] Dextran is a polysaccharide that increases the viscosity of the solution, thereby slowing down the cooling rate, helping to distribute the protective agent more evenly, and reducing the formation of ice crystals. At the same time, dextran can protect the cell membrane from ice crystal damage to a certain extent through physical isolation.

[0048] In the technical solution of the present invention, by using at least one of polyethylene glycol, trehalose, glucose and dextran as a non-permeable protective agent, the formation of ice crystals can be reduced, thereby preventing ice crystals from damaging cells; specifically, in an embodiment of the present invention, polyethylene glycol, trehalose, glucose and dextran are used as non-permeable protective agents, among which trehalose and glucose both have antioxidant properties, which can reduce the damage caused to cells by oxidative stress.

[0049] In one embodiment, the non-permeable protective agent includes polyethylene glycol, trehalose, glucose and dextran, and the mass ratio of polyethylene glycol, trehalose, glucose and dextran is (2-4): 5: 2: 2: 3. In the technical scheme of the present invention, by adjusting the mass ratio of polyethylene glycol, trehalose, glucose and dextran, trehalose is used as the main non-permeable protective agent, and polyethylene glycol, glucose and dextran are used to assist trehalose, so as to achieve the best protective effect.

[0050] The present invention also provides a method for preparing a peripheral blood mononuclear cell cryopreservation solution, comprising:

[0051] S10. Obtain a peripheral blood sample and a human lymph separation fluid, spread the peripheral blood on the human lymph separation fluid and centrifuge, take the supernatant and centrifuge to obtain autologous plasma;

[0052] S20. Mix the permeable protective agent, the non-permeable protective agent, human serum albumin, 1640 basal culture medium, glutathione, autologous plasma, vitamin C and sodium heparin to obtain a peripheral blood mononuclear cell freezing solution.

[0053] In the technical solution of the present invention, autologous plasma of peripheral blood mononuclear cells is separated while isolating peripheral blood mononuclear cells. The autologous plasma contains nutrients and related growth factors required for the growth of mononuclear cells, which can help maintain cell viability and promote the recovery process after thawing, thereby improving the survival rate of cells after recovery from cryopreservation.

[0054] The present invention also provides an application of a peripheral blood mononuclear cell freezing solution in freezing peripheral blood mononuclear cells. The peripheral blood mononuclear cell freezing solution includes the peripheral blood mononuclear cell freezing solution as described above, or the peripheral blood mononuclear cell freezing solution prepared by the preparation method as described above.

[0055] Since the present application adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0056] The present invention also provides a method for freezing peripheral blood mononuclear cells, comprising:

[0057] S100. Obtaining peripheral blood mononuclear cells to be frozen, and resuspending the peripheral blood mononuclear cells with physiological saline to obtain a cell suspension to be frozen;

[0058] S200. Mix the cell suspension to be frozen with a peripheral blood mononuclear cell freezing solution, pre-freeze at -80°C, and then store in liquid nitrogen at -200 to -190°C, wherein the peripheral blood mononuclear cell freezing solution includes the peripheral blood mononuclear cell freezing solution as described above.

[0059] In the technical scheme of the present invention, peripheral blood mononuclear cells are separated and then dispersed and mixed with peripheral blood mononuclear cell freezing solution to maintain the external environment of the peripheral blood mononuclear cells and improve the survival rate of the peripheral blood mononuclear cells; by pre-freezing and then storing in liquid nitrogen, the cooling rate of the cells can be slowed down to avoid excessive ice crystal formation due to too fast a cooling rate and damage to the cells.

[0060] In one embodiment, in step S200, after mixing, the density of peripheral blood mononuclear cells is 1.0-2.0×10 7 In the technical solution of the present invention, by adjusting the density of peripheral blood mononuclear cells, the survival rate of peripheral blood mononuclear cells after recovery can be improved, and the biological activity of peripheral blood mononuclear cells can be enhanced.

[0061] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0062] Experimental Materials

[0063] Unless otherwise specified, the reagents and materials used in the examples of the present invention are commercially available.

[0064] CIK culture: Purchase CIK immune cell kit from Zhongjiajia Beiying and culture according to the instructions to obtain CIK cells;

[0065] NK culture: Purchase the NK immune cell kit from Zhongjiajia Beiying and culture it according to the instructions to obtain NK cells;

[0066] 1640 culture medium: purchased from Gibco, catalog number 11875119.

[0067] Example 1

[0068] This embodiment provides a method for freezing and thawing peripheral blood mononuclear cells, and the specific steps are as follows:

[0069] (1) Collect 90 ml of peripheral blood from three healthy volunteers of appropriate age, add 15 ml of human lymph separation fluid to the bottom of a 50 ml centrifuge tube, spread the peripheral blood on the human lymph separation fluid in a ratio of (1-2):1, preferably 2:1, tighten the centrifuge tube cap, place in a centrifuge, balance, and centrifuge at room temperature at 460g for 30 min with fast ascent and slow descent.

[0070] (2) After centrifugation, remove the centrifuge tube and transfer the top plasma layer to a new 50 ml centrifuge tube. Place the plasma in a 56°C water bath for 30 min and then place it in a -20°C refrigerator for 10 min for inactivation. Centrifuge the inactivated plasma at 4°C, 1400 g, for 10 min. Transfer the supernatant to a new 50 ml centrifuge tube, label it, and store it in a 4°C refrigerator for later use.

[0071] (3) Use a pipette to aspirate the cells in the buffy coat layer, taking as little of the separation liquid layer as possible, and make up the volume to 50 ml with physiological saline. Centrifuge at room temperature at 600 g for 10 min with rapid rise and fall.

[0072] (4) After centrifugation, discard the supernatant, resuspend the cell pellet with physiological saline and mix it evenly, and adjust the volume to 50 ml. Take samples for counting and viability detection, and centrifuge at room temperature at 500g for 10 minutes. Prepare the freezing solution according to the counting results, and control the freezing density at 1.0-2.0×10 7 cells / ml.

[0073] (5) Cryopreservation solution was prepared according to the counting results. The components and proportions of the cryopreservation solution were glycerol: acetamide: polyethylene glycol: trehalose: glucose: dextran: human serum albumin: 1640 basal culture medium: glutathione: autologous plasma: vitamin C = 8%: 2%: 2%: 5%: 2%: 2%: 3%: 60%: 0.8%: 15%: 0.2% plus 5 IU / mL heparin sodium. After preparation, the cryopreservation solution was placed in a 4°C refrigerator for precooling for half an hour.

[0074] (6) After the cell centrifugation is completed, the supernatant is discarded to obtain a cell pellet rich in mononuclear cells. The 4°C pre-cooled cryopreservation solution is added to the cell pellet, mixed and dispensed into 1.8 ml cryopreservation tubes, 1 ml per tube, and then placed in a programmed cooling box, placed in a -80°C refrigerator overnight, and finally transferred to -196°C liquid nitrogen for storage.

[0075] (7) After three months of cryopreservation, peripheral blood mononuclear cells were removed and revived in a 37°C water bath. The revived cells were quickly transferred to pre-cooled saline and the volume was adjusted to 50 ml. The cells were centrifuged at 500 g for 10 min.

[0076] (8) After centrifugation, discard the supernatant, collect the mononuclear cell pellet, add pre-cooled saline to resuspend and adjust the volume to 50 ml, and centrifuge at 500g for 10 min.

[0077] (9) After centrifugation, discard the supernatant and collect the mononuclear cell pellet.

[0078] (10) Resuspend the mononuclear cells in 30 ml of basal medium without any added factors and take samples for counting and viability.

[0079] (11) CIK and NK mononuclear cells were cultured for 14 days, and the seeding density was controlled at 1.0-2.0×10 7 cells / ml, preferably 1.5-10 7 cells / ml.

[0080] (12) The remaining PBMCs were frozen for six, nine, and twelve months and then removed and revived in a 37°C water bath. The revived cells were transferred to pre-cooled saline and the volume was adjusted to 50 ml. The cells were then centrifuged at 500 g for 10 min.

[0081] (13) After centrifugation, discard the supernatant, collect the mononuclear cell pellet, add pre-cooled saline to resuspend and adjust the volume to 50 ml, and centrifuge at 500g for 10 min.

[0082] (14) After centrifugation, discard the supernatant and collect the mononuclear cell pellet.

[0083] (15) Resuspend the mononuclear cells in 30 ml of basal medium without any added factors and take samples to calculate the viability for stability testing.

[0084] Example 2

[0085] This embodiment provides a method for freezing and thawing peripheral blood mononuclear cells. The specific steps are basically the same as those in Example 1, except that in (5), the volume ratio of the freezing solution is glycerol: acetamide: polyethylene glycol: trehalose: glucose: dextran: human albumin: 1640 basal culture medium: glutathione: autologous plasma: vitamin C = 8%: 1%: 3%: 5%: 2%: 2%: 3%: 60%: 0.8%: 15%: 0.2% plus 5 IU / mL heparin sodium.

[0086] Example 3

[0087] This embodiment provides a method for freezing and thawing peripheral blood mononuclear cells. The specific steps are basically the same as those in Example 1, except that in (5), the volume ratio of the freezing solution is glycerol: acetamide: polyethylene glycol: trehalose: glucose: dextran: human albumin: 1640 basal culture medium: glutathione: autologous plasma: vitamin C = 5%: 3%: 4%: 5%: 2%: 2%: 3%: 60%: 0.8%: 15%: 0.2% plus 5 IU / mL heparin sodium.

[0088] Example 4

[0089] This embodiment provides a method for freezing and thawing peripheral blood mononuclear cells. The specific steps are basically the same as those in Example 1, except that in (5), the volume ratio of the freezing solution is glycerol: acetamide: polyethylene glycol: trehalose: glucose: dextran: human albumin: 1640 basal culture medium: glutathione: autologous plasma: vitamin C = 5%: 5%: 2%: 5%: 2%: 2%: 3%: 60%: 0.8%: 15%: 0.2% plus 5 IU / mL heparin sodium.

[0090] Example 5

[0091] This embodiment provides a method for freezing and thawing peripheral blood mononuclear cells. The specific steps are basically the same as those in Example 1, except that in (5), the volume ratio of the freezing solution is glycerol: acetamide: polyethylene glycol: trehalose: glucose: dextran: human serum albumin: 1640 basal culture medium: glutathione: autologous plasma: vitamin C = 6%: 3%: 3%: 5%: 2%: 2%: 3%: 60%: 0.8%: 15%: 0.2% plus 5 IU / mL heparin sodium.

[0092] Comparative Example 1

[0093] This comparative example provides a method for freezing and thawing peripheral blood mononuclear cells. The specific steps are basically the same as those in Example 1, except that in (5), the freezing solution is FBS:DMSO=9:1.

[0094] Comparative Example 2

[0095] This comparative example provides a method for freezing and resuscitating peripheral blood mononuclear cells. The specific steps are basically the same as those in Example 1, except that in (5), the freezing solution is autologous plasma:DMSO=9:1.

[0096] Comparative Example 3

[0097] This comparative example provides a method for freezing and thawing peripheral blood mononuclear cells. The specific steps are basically the same as those in Example 1, except that in (5), the volume ratio of the freezing solution is glycerol: acetamide: polyethylene glycol: trehalose: glucose: dextran: human albumin: 1640 basal culture medium: glutathione: autologous plasma: vitamin C = 15%: 15%: 10%: 4%: 3%: 3%: 4%: 35%: 0.5%: 10%: 0.5% plus 5 IU / mL heparin sodium.

[0098] Comparative Example 4

[0099] This comparative example provides a freshly prepared peripheral blood mononuclear cells, and the specific steps are as follows:

[0100] (1) Collect 90 ml of peripheral blood from three healthy volunteers of appropriate age, add 15 ml of human lymph separation fluid to the bottom of a 50 ml centrifuge tube, spread the peripheral blood on the human lymph separation fluid in a ratio of (1-2):1, preferably 2:1, tighten the centrifuge tube cap, place in a centrifuge, balance, and centrifuge at room temperature at 460g for 30 min with fast ascent and slow descent.

[0101] (2) After centrifugation, remove the centrifuge tube and transfer the top plasma layer to a new 50 ml centrifuge tube. Place the plasma in a 56°C water bath for 30 min and then place it in a -20°C refrigerator for 10 min for inactivation. Centrifuge the inactivated plasma at 4°C, 1400 g, for 10 min. Transfer the supernatant to a new 50 ml centrifuge tube, label it, and store it in a 4°C refrigerator for later use.

[0102] (3) Aspirate the cells from the buffy coat layer and aspirate as little of the separation liquid layer as possible. Make up the volume to 50 ml with physiological saline and centrifuge at 600 g at room temperature for 10 min.

[0103] (4) After centrifugation, discard the supernatant, mix the cell pellet with physiological saline and make up to 50 ml, take samples for counting and viability testing, and centrifuge at room temperature at 500g for 10 min with rapid rise and fall.

[0104] (5) Resuspend the mononuclear cells in 30 ml of basal culture medium without any added factors and take samples for counting and viability.

[0105] (6) CIK and NK mononuclear cells were cultured for 14 days, and the seeding density was controlled at 1.0-2.0×10 7 cells / ml, preferably 1.5-10 7 cells / ml.

[0106] Performance Testing

[0107] The viability of the resuspended cells in step (10) of Examples 1 to 5 and Comparative Examples 1 to 3 was detected by AO / PI staining, and the detection results are shown in Table 1. As can be seen from Table 1, the viability of Examples 1 to 5 is higher than that of Comparative Examples 1 to 3, and the components of the cryopreservation solution in Examples 1 to 5 greatly improve the survival rate of the cells.

[0108] Table 1 The viability of mononuclear cells recovered after 3 months of cryopreservation

[0109] 1 group 2 groups 3 groups average value Average Deviation Example 1 98.56％ 97.85％ 98.66％ 98.36％ 0.29％ Example 2 96.13％ 96.89％ 97.00％ 96.67％ 0.36％ Example 3 94.25％ 93.17％ 91.88％ 93.1％ 0.81％ Example 4 97.20％ 92.31％ 96.53％ 95.35％ 2.02％ Example 5 93.49％ 91.24％ 91.81％ 92.18％ 0.87％ Comparative Example 1 82.36％ 85.13％ 85.29％ 84.26％ 1.27％ Comparative Example 2 90.72％ 88.90％ 87.85％ 89.16％ 1.04％ Comparative Example 3 80.01％ 79.25％ 76.51％ 78.59％ 1.39％

[0110] After 14 days of CIK culture, the cells of PBMCs in Examples 1 to 5 and Comparative Examples 1 to 4 were counted and the expansion efficiency was calculated. The results are shown in Table 2.

[0111] Table 2 CIK expansion after 14 days of culture

[0112] 1 group 2 groups 3 groups average value Average Deviation Example 1 160.21 158.60 159.81 159.54 0.63 Example 2 155.89 157.52 155.44 156.28 0.82 Example 3 148.10 153.45 151.34 150.96 1.91 Example 4 155.86 157.90 154.12 155.96 1.29 Example 5 149.20 151.55 153.84 151.53 1.55 Comparative Example 1 148.03 145.39 143.30 145.57 1.64 Comparative Example 2 140.91 147.28 145.19 144.46 2.37 Comparative Example 3 140.95 139.30 139.54 139.93 0.68 Comparative Example 4 158.52 158.36 159.27 158.72 0.36

[0113] It can be seen from Table 2 that the CIK amplification rates of Examples 1 to 5 are slightly greater than those of Comparative Examples 1 to 4.

[0114] The PBMCs in Examples 1 to 5 and Comparative Example 4 were subjected to a tumor cell HL60 killing experiment after being cultured with CIK for 14 days. The experimental results are shown in Table 3.

[0115] Table 3 Killing rate of CIK on tumor HL60 after 14 days of culture

[0116]

[0117]

[0118] As can be seen from Table 3, the killing rates of Examples 1 to 5 on tumor cells HL60 are good and comparable to that of Comparative Example 4.

[0119] The PBMCs in Examples 1 to 5 and Comparative Examples 1 to 3 were subjected to a tumor cell K562 killing experiment after NK culture for 14 days. The experimental results are shown in Table 4.

[0120] Table 4 Killing rate of NK cells against tumor cells K562 after 14 days of culture

[0121] 1 group 2 groups 3 groups average value Example 1 43.6％ 43.9％ 43.7％ 43.7％ Example 2 43.2％ 43.2％ 43.0％ 43.1％ Example 3 43.0％ 42.6％ 42.8％ 42.8％ Example 4 42.2％ 43.6％ 43.1％ 43.0％ Example 5 43.1％ 43.0％ 42.6％ 42.9％ Comparative Example 1 31.5％ 31.6％ 31.9％ 31.7％ Comparative Example 2 34.1％ 30.2％ 32.5％ 32.3％ Comparative Example 3 31.5％ 30.6％ 29.8％ 30.6％ Comparative Example 4 43.2％ 43.5％ 42.8％ 43.2％

[0122] As can be seen from Table 4, the killing effects of Examples 1 to 5 and Comparative Example 4 on K562 are more significant than those of Comparative Examples 1 to 3.

[0123] The viability of the resuspended cells in step (15) of Examples 1 to 5 and Comparative Examples 1 to 3 was detected by AO / PI staining. The detection results are shown in Table 5.

[0124] Table 5. Survival rate of PBMCs after cryopreservation for six, nine and twelve months

[0125]

[0126]

[0127] As can be seen from Table 5, the survival rates of Examples 1 to 5 and Comparative Examples 1 to 3 after being frozen for six months, nine months, and twelve months, respectively, showed a downward trend, but the survival rates of Examples 1 to 5 were still above 90%, and the survival rates were higher than those of Comparative Examples 1 to 3.

[0128] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A peripheral blood mononuclear cell freezing solution, characterized in that: The peripheral blood mononuclear cell freezing solution is used for freezing peripheral blood mononuclear cells, and the peripheral blood mononuclear cell freezing solution comprises the following components: an osmotic protective agent, a non-osmotic protective agent, human albumin, 1640 basic culture medium, glutathione, autologous plasma, vitamin C and heparin sodium; Wherein, the autologous plasma is the plasma of the peripheral blood mononuclear cells.

2. The peripheral blood mononuclear cell freezing solution according to claim 1, characterized in that The peripheral blood mononuclear cell freezing solution includes the following components by mass: 8-10 parts of osmotic protective agent, 14-16 parts of non-osmotic protective agent, 2-4 parts of human serum albumin, 50-70 parts of 1640 basal medium, 0.5-1 part of glutathione, 14-16 parts of autologous plasma and 0.1-0.5 part of vitamin C; In the peripheral blood mononuclear cell freezing solution, the concentration of heparin sodium is 4-6 IU / mL.

3. The peripheral blood mononuclear cell freezing solution according to claim 1, characterized in that: The osmotic protectant includes at least one of glycerol and acetamide.

4. The peripheral blood mononuclear cell freezing solution according to claim 3, characterized in that: The permeability protective agent comprises glycerol and acetamide, and the mass ratio of glycerol to acetamide is (5-8): (1-5).

5. The peripheral blood mononuclear cell freezing solution according to claim 1, characterized in that: The non-permeable protective agent includes at least one of polyethylene glycol, trehalose, glucose and dextran.

6. The peripheral blood mononuclear cell freezing solution according to claim 4, characterized in that: The non-permeable protective agent comprises polyethylene glycol, trehalose, glucose and dextran, and the mass ratio of polyethylene glycol, trehalose, glucose and dextran is (2-4):5:2:2:

3.

7. A method for preparing a peripheral blood mononuclear cell cryopreservation solution, characterized in that: include: S10. Obtain a peripheral blood sample and a human lymph separation fluid, spread the peripheral blood on the human lymph separation fluid and centrifuge, take the supernatant and centrifuge to obtain autologous plasma; S20. Mix the permeable protective agent, the non-permeable protective agent, human serum albumin, 1640 basal culture medium, glutathione, autologous plasma, vitamin C and sodium heparin to obtain a peripheral blood mononuclear cell freezing solution.

8. A use of a peripheral blood mononuclear cell freezing solution in freezing peripheral blood mononuclear cells, characterized in that: The peripheral blood mononuclear cell freezing solution includes the peripheral blood mononuclear cell freezing solution as described in any one of claims 1 to 6, or the peripheral blood mononuclear cell freezing solution prepared by the preparation method as described in claim 7.

9. A method for freezing peripheral blood mononuclear cells, characterized in that: include: S100. Obtaining peripheral blood mononuclear cells to be frozen, and resuspending the peripheral blood mononuclear cells with physiological saline to obtain a cell suspension to be frozen; S200. Mix the cell suspension to be frozen with the peripheral blood mononuclear cell freezing solution, pre-freeze at -80°C, and then store in liquid nitrogen at -200 to -190°C, wherein the peripheral blood mononuclear cell freezing solution includes the peripheral blood mononuclear cell freezing solution as described in any one of claims 1 to 6.

10. The method for freezing peripheral blood mononuclear cells according to claim 9, characterized in that: In step S200, after mixing, the density of peripheral blood mononuclear cells is 1.0-2.0×10 7 cells / ml.

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