Cell cryoprotectant and cryopreservation method for in-vitro induced differentiation of porcine bone marrow derived cell macrophages

Abstract

The invention provides a cell cryoprotectant and a cryopreservation method for in-vitro induced differentiation of porcine bone marrow derived cell macrophages, and relates to the technical field of cell cryopreservation. The cell cryoprotectant comprises a permeable cryoprotectant, a non-permeable cryoprotectant and a serum-free basic culture medium, wherein the permeable cryoprotectant comprises dimethyl sulfoxide and ethylene glycol; wherein the impermeable cryoprotectant is prepared from trehalose, dextran 40, hydroxyethyl starch and methyl cellulose. The cell cryoprotectant can be specially used for cryopreserving bone marrow-derived macrophages, and has the advantages of high cryopreservation efficiency, good cryopreservation quality, good activity after cell resuscitation, no change in cell morphology and high survival rate.

Description

Technical Field

[0001] The invention relates to the technical field of cell cryopreservation, and in particular to a cell cryoprotectant solution and a cryopreservation method for in vitro induced differentiation of porcine bone marrow derived cell macrophages. Background Art

[0002] Bone marrow is the most important hematopoietic organ in animals and is also the site of immune cell generation and differentiation. Bone marrow cells are a general term for various cells in the bone marrow. The multipotent stem cells in the bone marrow can differentiate into myeloid stem cells and lymphoid stem cells. Myeloid stem cells further differentiate into erythroid cells, monocytes, granulocytes, and megakaryocytes. The monocytes differentiate into mononuclear macrophages under the action of specific cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), colony stimulating factor (M-CSF), and interferon-γ (IFN-γ).

[0003] Monocytes and macrophages have phagocytic functions, mainly phagocytizing various bacteria and viruses that enter the body. They are widely distributed in organs and tissues throughout the body, so they are also important sites for viral infection and replication. Macrophage extraction and induction culture technology is of great significance to immunology, virology and vaccine-related research. For example, African swine fever virus (ASFV), novel coronavirus pneumonia (NCP) and porcine reproductive and respiratory syndrome virus (PRRSV) all use monocytes and macrophages as target cells for massive replication, destroying the body's immune system.

[0004] At present, the use of freshly prepared bone marrow-derived cells to induce differentiation into static or suspended macrophages for virus isolation and culture is particularly important for the vaccine industry. During the culture process, the operation is cumbersome, time-consuming, labor-intensive, inefficient, and the cost of cell induction production is high. Using the traditional freezing method of 10% DMSO and 90% FBS, it is susceptible to ice crystal damage, the cell viability is low after cell recovery, the number of live cells is insufficient, the number of live cells frozen is greatly reduced, the efficiency is low, and the freezing quality is poor. There is currently no better cell freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, but the induced cells are expensive and not fully utilized, which will cause serious losses, and the workload of repeated cell preparation is increased, so it is very important to obtain a better cell freezing method, which provides great help for scientific researchers engaged in research in related fields.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a cell cryoprotectant and a method for freezing porcine bone marrow-derived macrophages induced to differentiate in vitro. The cell cryoprotectant of the present invention is composed of a specific permeable cryoprotectant and a non-permeable cryoprotectant. The cryoprotectant is used to freeze porcine bone marrow-derived macrophages, which can effectively solve the problem that the existing cryopreservation solution has low cell recovery efficiency and the cells cannot be used after the cells are preserved. The cryopreservation cells of the present invention have a high density of live cells after recovery and good cell viability, and the recovered cells are used for the propagation of porcine reproductive and respiratory syndrome virus NADC30 virus.

[0007] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0008] In a first aspect, the present invention provides a cell cryoprotectant, the cell cryoprotectant comprising: an osmotic cryoprotectant, a non-osmotic cryoprotectant and a serum-free basal culture medium;

[0009] Wherein, the permeable cryoprotectant comprises dimethyl sulfoxide and ethylene glycol;

[0010] Wherein, the non-permeable cryoprotectant includes trehalose, dextran-40, hydroxyethyl starch and methylcellulose.

[0011] Preferably, the serum-free basal culture medium is selected from serum-free DMEM / F-12 culture medium.

[0012] Preferably, the cell cryoprotectant solution comprises the following components:

[0013]

[0014] Further preferably, the cell cryoprotectant solution comprises the following components:

[0015]

[0016] In a second aspect, the present invention provides a use of the cell cryoprotectant solution in preparing a cryoprotectant solution for inducing differentiation of porcine bone marrow-derived macrophages in vitro.

[0017] In a third aspect, the present invention provides a method for freezing and preserving in vitro induced differentiation of porcine bone marrow-derived macrophages, the freezing and preserving method comprising the following steps:

[0018] The porcine bone marrow-derived macrophages are placed in a cell cryoprotectant solution for resuspending, and the porcine bone marrow-derived macrophages are pipetted into single cells and then dispensed into cell cryopreservation tubes for cryopreservation;

[0019] Wherein, the cell cryoprotective solution includes the cell cryoprotective solution as described in the first aspect.

[0020] Preferably, the density of the frozen storage is 0.5×10 7 / mL~50×10 7 / mL, preferably 1×10 7 / mL~3×10 7 Pieces / mL.

[0021] Preferably, the freezing method includes the following method I and / or method II:

[0022] Method I: Cool down to -85--75℃ at a rate of -1.5--0.5℃ / min and store for 6-18h, then transfer to liquid nitrogen for long-term storage;

[0023] Method II: First store at 2-6°C for 10-20 min, then cool to -30-20°C at a cooling rate of -1.5-0.5°C / min, then cool to -140-130°C at a cooling rate of -15-5°C / min, and transfer to liquid nitrogen for long-term storage.

[0024] Preferably, the method for preparing porcine bone marrow-derived macrophages comprises the following steps:

[0025] The bone marrow-derived cells were added to DMEM / F-12 medium containing FBS and GM-CSF for culture to obtain porcine bone marrow-derived macrophages.

[0026] Preferably, the initial cell density of the bone marrow-derived cells is 1.0×10 6 / mL~2.0×10 6 Pieces / mL.

[0027] Preferably, the culture parameters include: temperature of 36-38° C., CO 2 volume percentage of 4-6%, and humidity of 75-85%.

[0028] Preferably, the culture time is 6 to 10 days, preferably 6 to 8 days.

[0029] Preferably, in the DMEM / F-12 culture medium, the content of FBS is 8-12% (v / v); the content of GM-CSF is 5-50 ng / mL.

[0030] Preferably, during the culture process, the medium is replaced every 3 to 5 days, and 5 to 50 ng / mL of M-CSF is added.

[0031] Preferably, the method for preparing porcine bone marrow-derived macrophages further comprises the following post-processing steps:

[0032] The cultured porcine bone marrow-derived macrophages are digested, and then a cell culture medium is added to terminate the digestion. The cells are collected and centrifuged, and the supernatant is discarded.

[0033] Preferably, the digestion treatment comprises: digestion by blowing and / or digestion using Tyrpsin.

[0034] Preferably, the centrifugal speed is 1000-2000 rpm, and the centrifugal time is 5-15 min.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The cell cryopreservation solution of the present invention is suitable for freezing porcine bone marrow-derived macrophages with good effects; and after resuscitation, porcine bone marrow-derived macrophages can be used for virus isolation and virus passage culture; in addition, the cell cryopreservation solution does not contain animal serum, which can avoid the risk of introducing and transmitting pathogens and allergens, and has higher clinical safety than conventional cell cryopreservation solution; secondly, due to the very complex components of serum, differences in sources and production batches, adverse or inconsistent effects may be caused; thirdly, serum is rich in various cytokines, hormones and other active ingredients, which are easy to interfere with and affect the biological characteristics of bone marrow-derived macrophages, reduce the number of cells after resuscitation, low viability, easy aging, low efficiency, and also greatly reduce costs.

[0037] (2) The combination of the permeable protective agent and the non-permeable protective agent of the present invention can produce a synergistic low-temperature protective effect, while reducing their toxic effects, so as to obtain a better protective effect; excessively high concentrations of DMSO are toxic to cells, and the mixed protective agent can effectively reduce the cytotoxicity;

[0038] Among them, methylcellulose, hydroxyethyl starch, dextran-40, and trehalose are all non-permeable cryoprotectants. They cannot penetrate into the cell membrane, but can increase the extracellular osmotic pressure, allowing the water in the cell to quickly seep out during cryopreservation, thereby reducing the formation of ice crystals in the cell, protecting the cell from damage, and maintaining the stability of the cell membrane to protect the cell.

[0039] Among them, DMSO and ethylene glycol are osmotic cryoprotectants. When combined with dimethyl sulfoxide, on the one hand, they combine with water molecules in the extracellular solution to produce hydration, weaken the crystallization process of water, and thus reduce the formation of ice crystals; on the other hand, before the cell suspension is completely frozen and solidified, they penetrate into the cells and produce a certain molar concentration inside and outside the cells, reducing the concentration of electrolytes in the unfrozen solution inside and outside the cells, thereby protecting the cells from damage by high-concentration electrolytes. At the same time, they can prevent excessive extravasation of intracellular water and avoid excessive dehydration and shrinkage of cells.

[0040] (3) The present invention can complete cell cryopreservation by using a programmed cooling box or a traditional cryopreservation method, which is easy to popularize; and the use of this bone marrow-derived macrophage cryopreservation solution to freeze cells has high cryopreservation efficiency, good cryopreservation quality, good cell activity after recovery, no change in cell morphology, and high survival rate.

[0041] (4) The cell activity and characteristics after resuscitation in the present invention remain unchanged, and the virus infectivity remains stable; the frozen porcine bone marrow-derived macrophages can be used for PRRSV virus isolation and subculture, which greatly improves the work of repeated preparation of primary cells, improves efficiency and saves costs, and is safe and effective; the operation is simple, the ingredients are few, the cost is low, the preparation process is simple, and it is easy to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 This is a picture of the porcine bone marrow-derived macrophages provided in Example 1 before freezing (scale bar 50 μm).

[0044] Figure 2 This is a cell recovery diagram of the porcine bone marrow-derived macrophages provided in Example 1 after being cryopreserved for 3 months (scale 50 μm).

[0045] Figure 3 This is a cell recovery diagram of the porcine bone marrow-derived macrophages provided in Example 1 after being cryopreserved for 6 months (scale 50 μm).

[0046] Figure 4 This is a cell recovery diagram of the porcine bone marrow-derived macrophages provided in Example 1 after being cryopreserved for 12 months (scale 50 μm).

[0047] Figure 5 This is a cell recovery diagram of the porcine bone marrow-derived macrophages provided in Example 1 after being cryopreserved for 18 months (scale 50 μm).

[0048] Figure 6 This is a cell recovery diagram of the porcine bone marrow-derived macrophages provided in Example 1 after being cryopreserved for 24 months (scale 50 μm).

[0049] Figure 7 This is a picture of the porcine bone marrow-derived macrophages provided in Example 1 inoculated with PRRSV virus cells before freezing (scale bar 50 μm).

[0050] Figure 8This is a picture of the pig bone marrow-derived macrophages provided in Example 1 being revived and inoculated with PRRSV virus cells after being frozen for 3 months (scale 50 μm).

[0051] Fig. 9 This is a picture of the pig bone marrow-derived macrophages provided in Example 1 being revived and inoculated with PRRSV virus cells after being frozen for 6 months (scale 50 μm).

[0052] Fig.10 This is a picture of the pig bone marrow-derived macrophages provided in Example 1 being revived and inoculated with PRRSV virus cells after being frozen for 12 months (scale 50 μm).

[0053] Fig.11 This is a picture of the pig bone marrow-derived macrophages provided in Example 1 being revived and inoculated with PRRSV virus cells after being frozen for 18 months (scale 50 μm).

[0054] Fig.12 This is a picture of the pig bone marrow-derived macrophages provided in Example 1 being revived and inoculated with PRRSV virus cells after being frozen for 24 months (scale 50 μm).

[0055] Fig.13 Fluorescence image of the pig bone marrow-derived macrophages inoculated with PRRSV virus before freezing provided in Example 1 (scale bar 100 μm).

[0056] Fig.14 This is a fluorescence image of the porcine bone marrow-derived macrophages provided in Example 1 after cell recovery and inoculation with PRRSV virus after being frozen for 3 months (scale 100 μm).

[0057] Fig.15 This is a fluorescence image of the porcine bone marrow-derived macrophages provided in Example 1 after cell recovery and inoculation with PRRSV virus after being frozen for 6 months (scale bar 100 μm).

[0058] Fig.16 This is a fluorescence image of the porcine bone marrow-derived macrophages provided in Example 1 after cell recovery and inoculation with PRRSV virus after being frozen for 12 months (scale 100 μm).

[0059] Fig.17 This is a fluorescence image of the porcine bone marrow-derived macrophages provided in Example 1 after cell recovery and inoculation with PRRSV virus after 18 months of cryopreservation (scale 100 μm).

[0060] Fig.18 This is a fluorescence image of the porcine bone marrow-derived macrophages provided in Example 1 after cell recovery and inoculation with PRRSV virus after 24 months of cryopreservation (scale 100 μm). DETAILED DESCRIPTION

[0061] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0062] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0063] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0064] In a first aspect, the present invention provides a cell cryoprotectant, the cell cryoprotectant comprising a permeable cryoprotectant, a non-permeable cryoprotectant and a serum-free basal culture medium;

[0065] Wherein, the permeable cryoprotectant comprises dimethyl sulfoxide and ethylene glycol;

[0066] Wherein, the non-permeable cryoprotectant includes trehalose, dextran-40, hydroxyethyl starch and methylcellulose.

[0067] In the present invention, a serum-free basal culture medium is selected, and a permeable cryoprotectant and a non-permeable cryoprotectant of a specific composition are added thereto, and the protective solution is used to cryopreserve porcine bone marrow-derived macrophages, which can effectively solve the problem that the existing cryopreservation solution causes low cell recovery efficiency and the cells cannot be used after the cells are preserved. The cryopreserved cells of the present invention have a high live cell density and a good cell viability after recovery, and the recovered cells are used for the propagation of porcine reproductive and respiratory syndrome virus NADC30.

[0068] Among them, the osmotic cryoprotectant is composed of dimethyl sulfoxide and ethylene glycol, which cooperate with each other. On the one hand, they combine with water molecules in the extracellular solution to produce hydration, weaken the crystallization process of water, and thus reduce the formation of ice crystals; on the other hand, before the cell suspension is completely frozen and solidified, they penetrate into the cells and produce a certain molar concentration inside and outside the cells, reducing the concentration of electrolytes in the unfrozen solution inside and outside the cells, thereby protecting the cells from damage by high-concentration electrolytes. At the same time, they can prevent excessive extravasation of water in the cells and avoid excessive dehydration and shrinkage of the cells.

[0069] Among them, the non-permeable cryoprotectant includes trehalose, dextran-40, hydroxyethyl starch and methylcellulose; it itself cannot penetrate into the cell membrane, but can increase the osmotic pressure outside the cell, allowing the water in the cell to quickly seep out during freezing, thereby reducing the formation of ice crystals in the cell, protecting the cell from damage, and maintaining the stability of the cell membrane to protect the cell.

[0070] Among them, the trehalose is a safe and reliable natural sugar, and also a non-permeable protective agent. Appropriate addition is conducive to balancing osmotic pressure, reducing the osmotic damage of cells and the damage to cell structure and skeleton, thereby protecting the cell membrane from rupture. On the other hand, it can wrap the protein molecules of the cell membrane, making it less likely to deform, inhibiting cell membrane fusion, reducing the permeability of the cell membrane, and reducing the formation of ice crystals. When the trehalose concentration is 200mmol / L, immune cells and embryonic stem cells have a higher recovery rate, significantly improving the cell freezing survival rate and vitality.

[0071] Among them, the dextran-40 is a macromolecular compound that can be used as a non-permeable cryoprotectant in the cryopreservation of cells. It forms hypertonicity outside the cells to dehydrate the cells, thereby protecting the cells from damage caused by ice crystal formation and reducing excessive cell expansion caused by osmotic pressure changes during cell recovery. Literature reports that dextran-40 is beneficial to improving the cryopreservation effect of macrophages, with a high viability rate of more than 90% after recovery, and good cell growth status.

[0072] In the present invention, the hydroxyethyl starch is a polysaccharide derivative and a good cryoprotectant. It not only has a good effect but also has low toxicity to the human body and cells. Even if a small amount of HES is injected into the body of experimental animals, no obvious toxic side effects are observed.

[0073] In the present invention, cellulose compounds are selected as stabilizers. They belong to macromolecular sugars and are very stable in aqueous solutions, which are conducive to long-term storage and can be used as stabilizers. Cellulose compounds are also conducive to increasing the viscosity of the solvent and can be used as thickeners. As a preferred embodiment of the present invention, the cellulose non-permeable cryoprotectant is methylcellulose. In the present invention, methylcellulose is preferred, which can better reduce the ice crystals formed during cell freezing and cell damage caused by changes in osmotic pressure. Methylcellulose is widely used in pharmaceuticals, food, and cosmetics, and is generally considered to be non-toxic, harmless, and non-irritating cellulose.

[0074] As an optional embodiment of the present invention, the serum-free basal culture medium is selected from serum-free DMEM / F-12 culture medium.

[0075] As an optional embodiment of the present invention, the cell cryoprotectant solution comprises the following components:

[0076]

[0077]

[0078] Wherein, the content of dimethyl sulfoxide (DMSO) is 5-12% (v / v), for example, it can be 5% (v / v), 5.5% (v / v), 6% (v / v), 6.5% (v / v), 7% (v / v), 7.5% (v / v), 8% (v / v), 8.5% (v / v), 9% (v / v), 9.5% (v / v), 10% (v / v), 10.5% (v / v), 11% (v / v), 11.5% (v / v), 12% (v / v), etc.

[0079] Wherein, the content of ethylene glycol is 2-8% (v / v), for example, it can be 2% (v / v), 2.5% (v / v), 3% (v / v), 3.5% (v / v), 4% (v / v), 4.5% (v / v), 5% (v / v), 5.5% (v / v), 6% (v / v), 6.5% (v / v), 7% (v / v), 7.5% (v / v), 8% (v / v), etc.

[0080] Wherein, the content of trehalose is 100-300mmol / L, for example, it can be 100mmol / L, 110mmol / L, 120mmol / L, 130mmol / L, 140mmol / L, 150mmol / L, 160mmol / L, 170mmol / L, 180mmol / L, 190mmol / L, 200mmol / L, 210mmol / L, 220mmol / L, 230mmol / L, 240mmol / L, 250mmol / L, 260mmol / L, 270mmol / L, 280mmol / L, 300mmol / L, etc.

[0081] Wherein, the content of dextran-40 is 3-7% (v / v), for example, it can be 3% (v / v), 3.5% (v / v), 4% (v / v), 4.5% (v / v), 5% (v / v), 5.5% (v / v), 6% (v / v), 6.5% (v / v), 7% (v / v), etc.

[0082] Wherein, the content of hydroxyethyl starch is 4-8% (w / w), for example, it can be 4% (w / w), 4.5% (w / w), 5% (w / w), 5.5% (w / w), 6% (w / w), 6.5% (w / w), 7% (w / w), 7.5% (w / w), 8% (w / w), etc.

[0083] The content of methyl cellulose is 0.01-0.05 mg / L, for example, 0.01 mg / L, 0.015 mg / L, 0.02 mg / L, 0.025 mg / L, 0.03 mg / L, 0.035 mg / L, 0.04 mg / L, 0.045 mg / L, 0.05 mg / L, etc.

[0084] As a preferred embodiment of the present invention, the cell cryoprotectant solution comprises the following components:

[0085]

[0086] In a second aspect, the present invention provides a use of the cell cryoprotectant solution in preparing a cryoprotectant solution for inducing differentiation of porcine bone marrow-derived macrophages in vitro.

[0087] In a third aspect, the present invention provides a method for freezing and preserving in vitro induced differentiation of porcine bone marrow-derived macrophages, the freezing and preserving method comprising the following steps:

[0088] The porcine bone marrow-derived macrophages are placed in a cell cryoprotectant solution for resuspending, and the porcine bone marrow-derived macrophages are pipetted into single cells and then dispensed into cell cryopreservation tubes for cryopreservation;

[0089] Wherein, the cell cryoprotective solution includes the cell cryoprotective solution as described in the first aspect.

[0090] As an optional embodiment of the present invention, the density of the frozen storage is 0.5×10 7 / mL~50×10 7 / mL, for example, 0.5×10 7 / mL, 1×10 7 / mL, 2×10 7 / mL, 4×10 7 / mL, 6×10 7 / mL, 8×10 7 / mL, 10×10 7 / mL, 12×10 7 / mL, 14×10 7 / mL, 16×10 7 / mL, 18×10 7 / mL, 20×10 7 / mL, 22×10 7 / mL, 24×10 7 / mL, 26×10 7 / mL, 28×10 7 / mL, 30×10 7 / mL, 35×10 7 / mL, 40×10 7 / mL, 45×10 7 / mL, 50×10 7 Pieces / mL, etc.

[0091] As an optional embodiment of the present invention, the density of the frozen storage is 1×10 7 / mL~3×10 7 / mL, for example, 1×10 7 / mL, 1.2×10 7 / mL, 1.4×10 7 / mL, 1.6×10 7 / mL, 1.8×10 7 / mL, 2×10 7 / mL, 2.2×10 7 / mL, 2.4×10 7 / mL, 2.6×10 7 / mL, 2.8×10 7 / mL, 3×10 7 Pieces / mL, etc.

[0092] As an optional embodiment of the present invention, the freezing method includes the following method I and / or method II:

[0093] Method I: Cool down to -85--75℃ at a rate of -1.5--0.5℃ / min and store for 6-18h, then transfer to liquid nitrogen for long-term storage;

[0094] Method II: First store at 2-6°C for 10-20 min, then cool to -30-20°C at a cooling rate of -1.5-0.5°C / min, then cool to -140-130°C at a cooling rate of -15-5°C / min, and transfer to liquid nitrogen for long-term storage.

[0095] In the above method I, the cooling rate is -1.5 to -0.5°C / min, for example, it can be -1.5°C / min, -1.4°C / min, -1.2°C / min, -1°C / min, -0.8°C / min, -0.6°C / min, -0.5°C / min, etc.

[0096] In the above method I, the temperature is lowered to -85 to -75°C, for example, -85°C, -84°C, -82°C, -80°C, -78°C, -76°C, -75°C, etc.

[0097] In the above method I, the storage time is 6 to 18 hours, for example, it can be 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, etc.

[0098] In the above method II, it is stored at 2-6°C (for example, it can be 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, etc.) for 10-20min (for example, it can be 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, etc.).

[0099] In the above method II, the temperature is further reduced to -30 to -20°C (for example, -30°C, -28°C, -26°C, -25°C, -24°C, -22°C, -20°C, etc.) at a cooling rate of -1.5 to -0.5°C / min (for example, it can be -1.5°C / min, -1.4°C / min, -1.2°C / min, -1°C / min, -0.8°C / min, -0.6°C / min, -0.5°C / min, etc.).

[0100] In the above method II, the temperature is then cooled to -140 to -130°C (for example, -140°C, -138°C, -136°C, -135°C, -134°C, -132°C, -130°C, etc.) at a cooling rate of -15 to -5°C / min (for example, it may be -1.5°C / min, -1.4°C / min, -1.2°C / min, -1°C / min, -0.8°C / min, -0.6°C / min, -0.5°C / min, etc.).

[0101] As an optional embodiment of the present invention, the method for preparing porcine bone marrow-derived macrophages comprises the following steps:

[0102] The bone marrow-derived cells were added to DMEM / F-12 medium containing FBS and GM-CSF for culture to obtain porcine bone marrow-derived macrophages.

[0103] As an optional embodiment of the present invention, the initial cell density of the bone marrow-derived cells is 1.0×10 6 / mL~2.0×10 6 / mL, for example, it can be 1.0×10 6 / mL, 1.2×10 6 / mL, 1.4×10 6 / mL, 1.6×10 6 / mL, 1.8×10 6 / mL, 2×10 6 Pieces / mL, etc.

[0104] As an optional embodiment of the present invention, the culture parameters include: a temperature of 36-38°C, for example, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, etc., a CO2 volume percentage of 4-6%, for example, 4%, 4.5%, 5%, 5.5%, 6%, etc., and a humidity of 75-85%, for example, 75%, 76%, 78%, 80%, 82%, 84%, 85%, etc.

[0105] As an optional embodiment of the present invention, the culture time is 6 to 10 days, for example, 6 days, 7 days, 8 days, 9 days, 10 days, etc.

[0106] As a preferred embodiment of the present invention, the culture time is 6 to 8 days.

[0107] In the present invention, bone marrow-derived cells are added to a DMEM / F-12 medium containing FBS and GM-CSF and cultured for 6 to 10 days to obtain porcine bone marrow-derived macrophages that can be cryopreserved; wherein, the best cryopreservation effect is achieved when the culture time is 6 to 8 days.

[0108] As an optional embodiment of the present invention, in the DMEM / F-12 culture medium, the content of FBS is 8-12% (v / v), for example, it can be 8% (v / v), 8.5% (v / v), 9% (v / v), 9.5% (v / v), 10% (v / v), 10.5% (v / v), 11% (v / v), 11.5% (v / v), 12% (v / v), etc.

[0109] As an optional embodiment of the present invention, in the DMEM / F-12 medium, the content of GM-CSF is 5-50 ng / mL, for example, it can be 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL.

[0110] As an optional embodiment of the present invention, the medium is changed every 3 to 5 days of culture, and 5 to 50 ng / mL of M-CSF is added.

[0111] As an optional embodiment of the present invention, the liquid replacement is specifically: half of the culture medium (the culture medium here refers to DMEM / F-12 medium + 8-12% (v / v) FBS + 5-50ng / mL M-CSF) is replaced every 3-5 days of culture.

[0112] As an optional embodiment of the present invention, the method for preparing porcine bone marrow-derived macrophages further comprises the following post-processing steps:

[0113] The cultured porcine bone marrow-derived macrophages are digested, and then a cell culture medium is added to terminate the digestion. The cells are collected and centrifuged, and the supernatant is discarded.

[0114] As an optional embodiment of the present invention, the digestion treatment method includes: digestion by blowing and / or digestion using Tyrpsin.

[0115] It should be noted that after the porcine bone marrow-derived macrophages are cultured, the cells need to be gently blown and / or digested with Tyrpsin. When trypsin digestion is used, the cytoplasm shrinkage is observed under an inverted microscope and the cells are no longer attached to the wall, indicating that the cells are moderately digested at this time, and cell culture medium can be added to terminate the digestion.

[0116] As an optional embodiment of the present invention, the cell culture fluid added during the termination of digestion is specifically: DMEM / F-12 culture medium + 8-12% (v / v) FBS).

[0117] As an optional embodiment of the present invention, the centrifugal rotation speed is 1000-2000rpm, for example, it can be 1000rpm, 1200rpm, 1400rpm, 1600rpm, 1800rpm, 2000rpm, etc.; the centrifugal time is 5-15min, for example, it can be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.

[0118] In a fourth aspect, the present invention provides a cell recovery method of the porcine bone marrow-derived macrophages obtained in the third aspect, the cell recovery method comprising the following steps:

[0119] (A) thawing the porcine bone marrow-derived macrophages frozen in the cell cryoprotectant solution described in the first aspect;

[0120] (B) adding culture medium to the thawed porcine bone marrow-derived macrophages, centrifuging, and discarding the supernatant;

[0121] (C) Adding culture medium again to the porcine bone marrow-derived macrophages after discarding the supernatant, culturing the cells, and completing cell recovery of the porcine bone marrow-derived macrophages.

[0122] As an optional embodiment of the present invention, in step (A), the thawing method adopts a water bath, and the temperature of the water bath is 35-43°C, for example, it can be 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, etc.

[0123] In the present invention, a water bath of suitable temperature is used for gentle and rapid thawing to avoid repeated formation of ice crystals, and the operation of step (B) is performed only after the cells in the tube are completely melted.

[0124] As an optional embodiment of the present invention, in step (B), the centrifugal rotation speed is 1000-2000rpm, for example, it can be 1000rpm, 1200rpm, 1400rpm, 1600rpm, 1800rpm, 2000rpm, etc.; the centrifugal time is 5-15min, for example, it can be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.

[0125] As an optional embodiment of the present invention, in step (C), the culture parameters include: temperature of 36-38°C, for example, 36°C, 36.5°C, 37°C, 37.5°C, 38°C, etc., CO2 volume percentage of 4-6%, for example, 4%, 4.5%, 5%, 5.5%, 6%, etc., humidity of 75-85%, for example, 75%, 76%, 78%, 80%, 82%, 84%, 85%, etc.

[0126] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0127] Example 1

[0128] This embodiment provides a cell cryoprotectant solution, a freezing method for in vitro induced differentiation of porcine bone marrow derived macrophages, and a recovery method;

[0129] Wherein, the cell cryoprotectant solution comprises the following components:

[0130]

[0131] Wherein, the freezing method of porcine bone marrow-derived macrophages comprises the following steps:

[0132] S1. Preparation of differentiation medium for porcine bone marrow-derived macrophages:

[0133] Porcine femur and tibia were used to prepare fresh bone marrow-derived cells. The density of bone marrow-derived cells was adjusted to 1.5×10 6 The cells were added with DMEM / F-12 medium supplemented with 10% (v / v) FBS + 20ng / mL GM-CSF, and cultured in an incubator at 37°C, 5% CO2, and 80% humidity for 8 days. The medium was changed every 4.5 days and 20ng / mL M-CS was added. The induced cells were porcine bone marrow-derived macrophages. The porcine bone marrow-derived macrophages were digested with Tyrpsin, and the cytoplasm was observed to shrink under an inverted microscope using trypsin digestion, and it was no longer attached to the wall, indicating that the cells were properly digested at this time. An equal volume of cell culture medium was added to terminate the digestion, and the cells were collected in a centrifuge tube, centrifuged at 1500rpm for 5min, and the supernatant was discarded.

[0134] S2. Cell cryopreservation:

[0135] Resuspend the cells in the above-mentioned cell cryoprotectant solution, blow the cells into single cells without clumping, and dispense them into cell cryopreservation tubes, 1 mL per tube, with the tube mouth tightly sealed;

[0136] Wherein, the freezing method includes the following method I or method II:

[0137] Method I: Store at -80°C in a programmed cooling box with a freezing rate of -1°C / min (to allow the cells to have enough time to dehydrate and prevent intracellular freezing), store overnight, and then transfer to liquid nitrogen for long-term storage.

[0138] Method ⅠⅠ: The programmed cooling instrument can automatically control the programmed cooling process. The cryotubes were kept at 4°C for 15 min, and then transferred to the programmed cooling instrument (Thermo Fisher Scientific Biochemicals Co., Ltd.). The samples were first cooled to -25°C at 1°C / min; the samples were then cooled to -135°C at 10.0°C / min and transferred to liquid nitrogen for long-term storage.

[0139] The cell density and number of the frozen cells are shown in Table 1 below:

[0140] Table 1

[0141] serial number Cryopreservation density 1# <![CDATA[0.5×10 7 pcs / mL]]> 2# <![CDATA[2×10 7 pcs / mL]]> 3# <![CDATA[3×10 7 pcs / mL]]> 4# <![CDATA[5×10 7 pcs / mL]]>

[0142] Wherein, the resuscitation method comprises the following steps:

[0143] The porcine bone marrow macrophages frozen in the above method Ⅰ or method Ⅱ were thawed gently and quickly in a 40°C water bath to avoid repeated formation of ice crystals until the cells in the tube were completely thawed; the cells were added to the culture medium and centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the culture medium (DMEM / F-12+10% FBS, cell density 1.5×10 6 / mL) and cultured in an incubator at 37°C, 5% CO2, and 80% humidity.

[0144] Among them, the survival status of four batches (1#, 2#, 3#, 4#) of porcine bone marrow-derived macrophages cryopreserved for 3, 6, 12, 18, and 24 months is shown in Table 2, and the cell recovery status cell pictures are shown in Figure 1 to Figure 6 .

[0145] Table 2

[0146]

[0147]

[0148] Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6The cell recovery diagrams of porcine bone marrow-derived macrophages provided in Example 1 before freezing, 3 months later, 6 months later, 12 months later, 18 months later, and 24 months later (cell recovery diagrams of freezing using method I). As shown in the figure, the cell cryoprotectant of the present invention is suitable for freezing porcine bone marrow-derived macrophages. The combination of permeable protective agents and non-permeable protective agents can produce a synergistic low-temperature protective effect, while reducing their toxic effects to obtain a better protective effect. Excessively high concentrations of DMSO are toxic to cells, and mixed protective agents can effectively reduce cytotoxicity.

[0149] Example 2

[0150] This embodiment provides a cell cryoprotective solution, a freezing method (method I) for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which differs from embodiment 1 only in that the contents of the components in the cell cryoprotective solution are different, and the freezing method and the recovery method are consistent with embodiment 1;

[0151]

[0152] Example 3

[0153] This embodiment provides a cell cryoprotective solution, a freezing method (method I) for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which differs from embodiment 1 only in that the contents of the components in the cell cryoprotective solution are different, and the freezing method and the recovery method are consistent with embodiment 1;

[0154] Wherein, the cell cryoprotectant solution comprises the following components:

[0155]

[0156] Example 4

[0157] This embodiment provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differs from Example 1 only in that the freezing method I is specifically: cooling to -75°C at a cooling rate of -1.5°C / min for 18 hours, and then transferring to liquid nitrogen for long-term storage.

[0158] Example 5

[0159] This embodiment provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differs from Example 1 only in that the freezing method I is specifically: cooling to -85°C at a cooling rate of -0.5°C / min for 6 hours, and then transferring to liquid nitrogen for long-term storage.

[0160] Example 6

[0161] This embodiment provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differs from Example 1 only in that the freezing method II is as follows: first store at 2°C for 20 minutes, then cool to -30°C at a cooling rate of -1.5°C / min, then cool to -140°C at a cooling rate of -12°C / min, and transfer to liquid nitrogen for long-term storage.

[0162] Example 7

[0163] This embodiment provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differs from Example 1 only in that the freezing method II is as follows: first store at 6°C for 10 minutes, then cool to -20°C at a cooling rate of -0.5°C / min, then cool to -130°C at a cooling rate of -8°C / min, and transfer to liquid nitrogen for long-term storage.

[0164] Comparative Example 1

[0165] This comparative example provides a cell cryoprotectant, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotectant are different, wherein the cell cryoprotectant comprises: 8% (v / v) dimethyl sulfoxide, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0166] Comparative Example 2

[0167] This comparative example provides a cell cryoprotectant, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotectant are different, wherein the cell cryoprotectant comprises: 5% (v / v) ethylene glycol, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0168] Comparative Example 3

[0169] This comparative example provides a cell cryoprotectant, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which are different from Example 1 only in that the contents of the components in the cell cryoprotectant are different, wherein the cell cryoprotectant comprises: 0.03 mg / L methylcellulose, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with Example 1.

[0170] Comparative Example 4

[0171] This comparative example provides a cell cryoprotectant, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which are different from Example 1 only in that the contents of the components in the cell cryoprotectant are different, wherein the cell cryoprotectant comprises: 200 mmol / L trehalose, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with Example 1.

[0172] Comparative Example 5

[0173] This comparative example provides a cell cryoprotective solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotective solution are different, wherein the cell cryoprotective solution comprises: 5% (v / v) dextran-40, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0174] Comparative Example 6

[0175] This comparative example provides a cell cryoprotective solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotective solution are different, wherein the cell cryoprotective solution comprises: 6% (w / w) hydroxyethyl starch, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with Example 1.

[0176] Comparative Example 7

[0177] This comparative example provides a cell cryoprotectant, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotectant are different. The cell cryoprotectant comprises: 8% (v / v) DMSO, 0.03 mg / L methyl cellulose, 200 mmol / L trehalose, 5% (v / v) dextran-40, 6% (w / w) hydroxyethyl starch, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0178] Comparative Example 8

[0179] This comparative example provides a cell cryoprotectant, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotectant are different. The cell cryoprotectant comprises: 8% (v / v) DMSO, 5% (v / v) ethylene glycol, 200 mmol / L trehalose, 5% (v / v) dextran-40, 6% (w / w) hydroxyethyl starch, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0180] Comparative Example 9

[0181] This comparative example provides a cell cryoprotective solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotective solution are different, wherein the cell cryoprotective solution comprises: 8% (v / v) DMSO, 5% (v / v) ethylene glycol, 0.03 mg / L methyl cellulose, 5% (v / v) dextran-40, 6% (w / w) hydroxyethyl starch, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with Example 1.

[0182] Comparative Example 10

[0183] This comparative example provides a cell cryoprotectant, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotectant are different. The cell cryoprotectant comprises: 8% (v / v) DMSO, 5% (v / v) ethylene glycol, 0.03 mg / L methyl cellulose, 200 mmol / L trehalose, 6% (w / w) hydroxyethyl starch, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0184] Comparative Example 11

[0185] This comparative example provides a cell cryoprotective solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method. The only difference from Example 1 is that the contents of the components in the cell cryoprotective solution are different, wherein the cell cryoprotective solution comprises: 8% (v / v) DMSO, 5% (v / v) ethylene glycol, 0.03 mg / L methyl cellulose, 5% (v / v) dextran-40, and the remainder is serum-free DMEM / F-12 culture medium; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0186] Comparative Example 12

[0187] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that DMSO is replaced with an equal amount of glycerol; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0188] Comparative Example 13

[0189] This comparative example provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that ethylene glycol is replaced with an equal amount of propylene glycol; the freezing method (method I) and the resuscitation method are consistent with those in Example 1.

[0190] Comparative Example 14

[0191] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that trehalose is replaced with an equal amount of sucrose; the freezing method (method I) and the recovery method are consistent with Example 1.

[0192] Comparative Example 15

[0193] This comparative example provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that dextran-40 is replaced with an equal amount of dextran-70; the freezing method (method I) and the resuscitation method are consistent with those in Example 1.

[0194] Comparative Example 16

[0195] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that hydroxyethyl starch is replaced with an equal amount of hydroxypropyl starch; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0196] Comparative Example 17

[0197] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that methyl cellulose is replaced with an equal amount of carboxymethyl cellulose; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0198] Comparative Example 18

[0199] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which are different from Example 1 only in that DMEM / F-12 culture medium is replaced with DMEM culture medium; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0200] Comparative Example 19

[0201] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that dimethyl sulfoxide is no longer added and the content of ethylene glycol is increased to 13% (v / v); the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0202] Comparative Example 20

[0203] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that ethylene glycol is no longer added and the content of dimethyl sulfoxide is increased to 13% (v / v); the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0204] Comparative Example 21

[0205] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which are different from Example 1 only in that trehalose is no longer added and the content of dextran-40 is increased to 10% (v / v); the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0206] Comparative Example 22

[0207] This comparative example provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that dextran-40 is no longer added and the trehalose content is increased to 400 mmol / L; the freezing method (method I) and the resuscitation method are consistent with those in Example 1.

[0208] Comparative Example 23

[0209] This comparative example provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which are different from Example 1 only in that hydroxyethyl starch is no longer added and the content of methyl cellulose is increased to 0.1 mg / L; the freezing method (method I) and the resuscitation method are consistent with those in Example 1.

[0210] Comparative Example 24

[0211] This comparative example provides a cell cryoprotectant solution, a freezing method and a recovery method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which are different from Example 1 only in that methyl cellulose is no longer added and the content of hydroxyethyl starch is increased to 10% (w / w); the freezing method (method I) and the recovery method are consistent with Example 1.

[0212] Comparative Example 25

[0213] This comparative example provides a cell cryoprotectant solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which differs from Example 1 only in that the content of dimethyl sulfoxide is reduced to 3% (v / v) and the content of ethylene glycol is increased to 10% (v / v); the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0214] Comparative Example 26

[0215] This comparative example provides a cell cryoprotectant solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which differs from Example 1 only in that the content of dimethyl sulfoxide is increased to 13% (v / v) and the content of ethylene glycol is reduced to 1% (v / v); the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0216] Comparative Example 27

[0217] This comparative example provides a cell cryoprotectant solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which differs from Example 1 only in that the content of trehalose is reduced to 80 mmol / L, while the content of dextran-40 is increased to 8% (v / v); the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0218] Comparative Example 28

[0219] This comparative example provides a cell cryoprotectant solution, a freezing method and a resuscitation method for in vitro induced differentiation of porcine bone marrow-derived macrophages, which differ from Example 1 only in that the content of trehalose is increased to 320 mmol / L, while the content of dextran-40 is reduced to 2% (v / v); the freezing method (method I) and the resuscitation method are consistent with those in Example 1.

[0220] Comparative Example 29

[0221] This comparative example provides a cell cryoprotectant solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which are different from Example 1 only in that the content of hydroxyethyl starch is increased to 9% (w / w) and the content of methylcellulose is reduced to 0.008 mg / L; the freezing method (method I) and the recovery method are consistent with Example 1.

[0222] Comparative Example 30

[0223] This comparative example provides a cell cryoprotectant solution, a freezing method for in vitro induced differentiation of porcine bone marrow-derived macrophages, and a recovery method, which differs from Example 1 only in that the content of hydroxyethyl starch is reduced to 3% (w / w) and the content of methylcellulose is increased to 0.08 mg / L; the freezing method (method I) and the recovery method are consistent with those in Example 1.

[0224] Test Example 1

[0225] Test samples: resuscitated living cells provided in Examples 1 to 7, and resuscitated living cells provided in Comparative Examples 1 to 30;

[0226] Test method: Different cryopreservation solutions were used to cryopreserve porcine bone marrow-derived macrophages. The cryopreservation density was 2×10 7 The cells were cryopreserved at 100 μg / mL and stored in method I. The survival status of the cells after recovery is shown in Table 3.

[0227] Table 3

[0228]

[0229]

[0230] As shown in Table 3, when cryoprotectants were used alone, the number of live cells and cell viability after resuscitation were low, and the recovery efficiency was less than 51%; 6 groups of cells were cryopreserved with combined cryoprotectants, among which Example 1 used "8% DMSO + 5% ethylene glycol + 0.03 mg / L methyl cellulose + 200 mmol / L trehalose + 5% dextran-40 + 6% hydroxyethyl starch" combined cryoprotectant experimental group with a recovery rate of 96.05%, and the preservation effect was better than other combined cryoprotectant ratios.

[0231] Test Example 2

[0232] Test sample: the resuscitated living cells provided in Example 1;

[0233] Test items: Cultivation of porcine reproductive and respiratory syndrome virus NADC30 strain on resuscitated porcine bone marrow-derived macrophages and determination of virus content;

[0234] Test method:

[0235] The NADC30 viruses prepared by adherent cells were continuously domesticated for three generations in resuscitated porcine bone marrow macrophages, with a dose of 5% to 10% V / V, and culture parameters: temperature 37°C, 5% CO2, 80% RH, and the virus content was not less than 106.0 TCID50 / mL after 48 hours of culture. Porcine reproductive and respiratory syndrome virus has a good sensitivity to macrophages after cryopreservation and resuscitation.

[0236] The virus content of the samples after being docked with the virus using porcine bone marrow macrophages was determined as follows:

[0237] ① Use DMEM / F-12 + 4% FBS culture medium at a rate of 1.0-1.5×10 6 / mL was plated into 96-well cell culture plates, 100 μL / well;

[0238] ② After culturing the cells for 24 hours, the virus samples were diluted 10 times in a serial increment using cell culture medium (the dilution range was from 10 -1 Up to 10 -7 ), each dilution was inoculated in 96-well cell culture plates, 8 wells were inoculated for each dilution multiple, 100 μL / well;

[0239] ③At the same time, virus control and normal cell control were set up, and the 96-well cell culture plate was placed in a 37°C constant temperature incubator for 5 days to observe the cytopathic effect, and the Reed-Muench method was used to calculate the virus half-cell infection dose;

[0240] The results of the virus content determination of porcine bone marrow-derived macrophages after cell recovery and inoculation with PRRSV virus at different freezing times are shown in Table 4:

[0241] Table 4

[0242]

[0243] in, Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 The figures are respectively the porcine bone marrow derived macrophages provided in Example 1 before freezing, 3 months after freezing, 6 months after freezing, 12 months after freezing, 18 months after freezing, and 24 months after freezing, and inoculated with PRRSV virus cells. The results showed that all the virus control groups had cytopathic effects characterized by cell fragmentation and shedding, while the cell control group was normal and had no cytopathic effects.

[0244] Test Example 3

[0245] Test sample: the well plate provided in Test Example 2 that has been tested for virus content;

[0246] Test items: Detection of specific antigen-antibody reaction between virus and porcine reproductive and respiratory syndrome virus monoclonal antibody;

[0247] Test method: Perform immunofluorescence detection on the plate that has been tested for virus content. The steps are as follows:

[0248] ① Fix with 80% pre-cooled acetone for 10 min and wash with PBST three times;

[0249] ② Add porcine reproductive and respiratory syndrome virus monoclonal antibody (1:1000), incubate at 37°C for 2 h, and wash 4 times with PBST;

[0250] ③ Add anti-mouse FITC secondary antibody (1:200), incubate at 37°C for 1 h, wash 4 times with PBST, and finally add 50 μL PBST to each well.

[0251] ④ Using an inverted fluorescence microscope to observe, the virus and porcine reproductive and respiratory syndrome virus monoclonal antibody produced specific antigen-antibody reaction;

[0252] Test results:

[0253] in, Fig.13 , Fig.14 , Fig.15 , Fig.16 , Fig.17 , Fig.18 Fluorescence images of the porcine bone marrow-derived macrophages provided in Example 1 before freezing, 3 months after freezing, 6 months after freezing, 12 months after freezing, 18 months after freezing, and 24 months after freezing were inoculated with PRRSV virus. As shown in the figure, after adding the fluorescent secondary antibody, obvious specific fluorescence appeared in the field of view, and the blank control well had no fluorescence.

[0254] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cell cryoprotectant solution, characterized in that: The cell cryoprotectant solution includes: a permeable cryoprotectant, a non-permeable cryoprotectant and a serum-free basal culture medium; Wherein, the permeable cryoprotectant comprises dimethyl sulfoxide and ethylene glycol; Wherein, the non-permeable cryoprotectant includes trehalose, dextran-40, hydroxyethyl starch and methylcellulose.

2. The cell cryoprotectant according to claim 1, characterized in that: The serum-free basal culture medium is selected from serum-free DMEM / F-12 culture medium.

3. The cell cryoprotectant according to claim 1, characterized in that: The cell cryoprotectant solution comprises the following components: The remainder was serum-free DMEM / F-12 medium.

4. The cell cryoprotective solution according to claim 1 or 3, characterized in that: The cell cryoprotectant solution comprises the following components: The remainder was serum-free DMEM / F-12 medium.

5. Use of the cell cryoprotective solution according to any one of claims 1 to 4 in preparing a cryoprotective solution for inducing in vitro differentiation of porcine bone marrow-derived macrophages.

6. A method for freezing and preserving in vitro differentiated porcine bone marrow-derived macrophages, characterized in that: The freezing method comprises the following steps: The porcine bone marrow-derived macrophages are placed in a cell cryoprotectant solution for resuspending, and the porcine bone marrow-derived macrophages are pipetted into single cells and then dispensed into cell cryopreservation tubes for cryopreservation; Wherein, the cell cryoprotective solution comprises the cell cryoprotective solution as described in any one of claims 1 to 4.

7. The method for freezing and preserving in vitro differentiated porcine bone marrow-derived macrophages according to claim 6, characterized in that: The density of the frozen 7 / mL~50×10 7 / mL, preferably 1×10 7 / mL~3×10 7 Pieces / mL.

8. The method for freezing and preserving in vitro differentiated porcine bone marrow-derived macrophages according to claim 6, characterized in that: The freezing method includes the following method I and / or method II: Method I: Cool down to -85--75℃ at a rate of -1.5--0.5℃ / min and store for 24-48h, then transfer to liquid nitrogen for long-term storage; Method II: First store at 2-6°C for 10-20 minutes, then cool to -30-20°C at a cooling rate of -1.5-0.5°C / min, then cool to -140-130°C at a cooling rate of -12-8°C / min, and transfer to liquid nitrogen for long-term storage.

9. The method for freezing and preserving in vitro differentiated porcine bone marrow-derived macrophages according to claim 6, characterized in that: The method for preparing porcine bone marrow-derived macrophages comprises the following steps: The bone marrow-derived cells were added to DMEM / F-12 medium containing FBS and GM-CSF for culture to obtain porcine bone marrow-derived macrophages.

10. The method for freezing and preserving in vitro differentiated porcine bone marrow-derived macrophages according to claim 9, characterized in that: The initial cell density of the bone marrow-derived cells was 1.0×10 6 / mL~2.0×10 6 Pieces / mL; Preferably, the culture parameters include: temperature of 36-38°C, CO2 volume percentage of 4-6%, humidity of 75-85%; Preferably, the culturing time is 6 to 10 days, preferably 6 to 8 days; Preferably, in the DMEM / F-12 medium, the content of FBS is 8-12% (v / v); the content of GM-CSF is 5-50 ng / mL; Preferably, during the culture process, the medium is changed every 3 to 5 days, and 5 to 50 ng / mL of M-CSF is added; Preferably, the method for preparing porcine bone marrow-derived macrophages further comprises the following post-processing steps: Digesting the cultured porcine bone marrow-derived macrophages, adding a cell culture medium to terminate the digestion, collecting the cells and centrifuging them, and discarding the supernatant; Preferably, the digestion treatment comprises: digestion by blowing and / or digestion using Tyrpsin; Preferably, the centrifugal speed is 1000-2000 rpm, and the centrifugal time is 5-15 min.

Images