Ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration and its application

Abstract

The present invention discloses a ready-to-use preparation of umbilical cord mesenchymal stem cells for topical medication and its application, and belongs to the field of biomedical technology. The biological safety and microbiological safety of the effective ingredient in the preparation, umbilical cord mesenchymal stem cells, are strictly controlled; the auxiliary material components such as trehalose, human serum albumin, and compound electrolytes are all injection-grade auxiliary materials (DMF) approved by the State Food and Drug Administration, and do not contain toxic components such as DMSO, and the ready-to-use preparation can be directly used in clinical practice; in addition, the human serum albumin, sodium octanoate, and sodium gluconate in the auxiliary materials have certain synergistic effects on the immunomodulatory mechanism of mesenchymal stem cells (effective ingredients) and the inhibition of inflammation, etc., in addition to maintaining the stability of the stem cell preparation, the topical medication for osteoarthritis and diabetic foot is all helpful. After testing, the ready-to-use preparation provided by the present invention does not need to be frozen, and the stable period can reach 14 days under the conditions of 2-8°C, which is convenient for product production, storage and transportation, and clinical use.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration and application thereof. Background Art

[0002] Stem cells are a type of multipotent cells with the ability to self-replicate. They have the potential to proliferate and differentiate. They can regenerate various tissues and organs and produce highly differentiated functional cells. The medical community calls them "universal cells."

[0003] Stem cell preparation technology and clinical application programs are diverse, complex and specific. Stem cells are different from other drugs. It is difficult for them to survive for a long time in vitro, and the viability and activity of stem cells will affect the patient's medical safety and efficacy. In actual clinical applications, the prepared stem cell preparations are often not used immediately. Therefore, the appropriate storage solution and placement time have a great impact on cell activity and cell viability, which is also an important factor that limits the clinical application of stem cells.

[0004] There are two types of stem cell preservation technologies that are relatively mature in clinical applications: one is cryopreservation, including liquid nitrogen -196℃ preservation and -80℃ refrigerator preservation. Cryopreserved stem cells are safe and reliable and can be stored for a long time under liquid nitrogen -196℃ conditions. However, cryopreserved stem cells cannot be used directly in clinical practice and need to be thawed and resuspended in cell infusion solution for use. The cell freezing and thawing process requires very professional operations, involving the addition of cryoprotectants and packaging, cooling, storage, melting, cleaning, and reinfusion. Each link in this process may cause cell damage and death. In addition, before using cryopreserved cells, it is necessary to add cleaning and final product testing in a sterile clean environment. It is difficult for general clinical institutions to achieve the above operations, which limits the clinical use of stem cells. Another type is refrigerated storage, that is, non-frozen storage at 4℃. Stem cells are refrigerated at 4℃ and do not need to be thawed before reinfusion. If the components contained in the refrigerated storage solution meet the requirements for clinical use, the cells can be directly reinfused after rewarming. The operation method is relatively simple and convenient for clinical use. However, the current methods of cryopreservation of cells have a limited storage time of less than 7 days (as described in Reference 1), which greatly limits clinical applications. Although some cells can be stored at 4°C for 3 weeks (as described in Reference 2), they cannot be directly used for human transfusion or injection because the protective solution used contains cell culture medium, that is, they cannot be used clinically. Some use dimethyl sulfoxide (DMSO, a cell cryoprotectant component) (as described in Reference 3). Although the amount of DMSO used is within the clinical safety range, the possibility of adverse reactions to the human body cannot be ruled out, which also limits clinical applications (as described in Reference 4).

[0005] Reference 1: A mesenchymal stem cell cryopreservation solution and preservation method, 202210026512.X.

[0006] Reference 2: Preparation of trehalose 4℃ cold storage protection solution for hUC-MSCs and evaluation of its preservation effect, Di Guohu, Wang Jiexi, Liu Minxia, ​​Proceedings of the 11th Annual Academic Conference of Beijing Refrigeration Society, 2012, 147-156.

[0007] Reference 3: Umbilical cord mesenchymal stem cell preparations, preparation methods and their applications in the treatment of knee osteoarthritis, 202310737902.2.

[0008] Document 4: Neurotoxicity associated with dimethylsulfoxide–preservedhematopoietic progenitor cell infusion. AM Júnior, CA Arrais, R Saboya, BoneMarrow Transplant. 2008Jan; 41(1):95-6.doi:10.1038 / sj.bmt.1705883. Summary of the invention

[0009] In order to solve the problems existing in the prior art, the present invention provides the following technical solutions.

[0010] The first aspect of the present invention provides a ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration, the ready-to-use preparation comprising: human umbilical cord mesenchymal stem cells, trehalose or mannitol, medical human serum albumin or bovine serum albumin and medical compound electrolyte injection.

[0011] Preferably, each 2 mL of the ready-to-use preparation comprises: 2.0×10 7 pcs, trehalose or mannitol 0.010-0.018g, medical human albumin or bovine serum albumin 0.12mL-0.2mL and medical compound electrolyte injection 1.6mL-1.8mL.

[0012] Preferably, 2 mL of the ready-to-use preparation includes: 2.0×10 human umbilical cord mesenchymal stem cells 7 pcs, trehalose 0.015g, medical human serum albumin 0.16mL and medical compound electrolyte injection 1.7mL.

[0013] The second aspect of the present invention provides a method for preparing the ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration as described in the first aspect, comprising:

[0014] Obtain the umbilical cord and control the microbial safety of the umbilical cord to meet the preset requirements;

[0015] Prepare umbilical cord mesenchymal stem cells, and perform quality control on the prepared umbilical cord mesenchymal stem cells to ensure that they meet preset requirements;

[0016] A ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration was prepared, and the test results showed that the prepared ready-to-use preparation could be stored at 2-8° C. for 14 days.

[0017] The third aspect of the present invention provides the use of the ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration as described in the first aspect for immunomodulation in the treatment of osteoarthritis.

[0018] The fourth aspect of the present invention provides the use of the ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration as described in the first aspect in the preparation of an immunomodulatory drug for treating osteoarthritis.

[0019] The fifth aspect of the present invention provides the use of the ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration as described in the first aspect for immunomodulation in the treatment of diabetic foot.

[0020] The sixth aspect of the present invention provides the use of the ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration as described in the first aspect in the preparation of an immunomodulatory drug for treating diabetic foot.

[0021] The beneficial effects of the present invention are as follows: the instant preparation of umbilical cord mesenchymal stem cells for local medication provided by the present invention and its application, the biological safety and microbiological safety of the effective ingredient umbilical cord mesenchymal stem cells in the preparation are strictly controlled; the auxiliary material components such as trehalose, human serum albumin, compound electrolytes are all injection-grade auxiliary materials (DMF) approved by the State Food and Drug Administration, and do not contain toxic components such as DMSO, so the instant preparation can be directly used in clinical practice; in addition, the human serum albumin, sodium octanoate and sodium gluconate in the auxiliary materials have certain synergistic effects on the immunomodulatory mechanism and inhibition of inflammation of mesenchymal stem cells (effective ingredients), in addition to maintaining the stability of the stem cell preparation, it is also helpful for local medication for osteoarthritis and diabetic foot. After testing, the instant preparation provided by the present invention does not need to be frozen, and the stable period can reach 14 days at 2-8°C, which is convenient for product production, storage, transportation and clinical use. In 2mL of the instant preparation, when 2.0×10 7Under the conditions of 0.5 g of trehalose, 0.015 g of trehalose, 0.16 mL of medical human albumin, and 1.7 mL of medical compound electrolyte injection, the preparation has better stability, and the osmotic pressure (290-300 mOsmol / kg) is closer to the reference value of human body fluids (280-310 m Osmol / kg). After storage at 2-8°C for 21 days, the cell viability remains above 90%, and the inhibitory effect on Th1 lymphocytes is the best (up to >60%). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the wound healing of mice according to Example 5 of the present invention;

[0023] Figure 2 This is a schematic diagram of the wound healing rate of mice according to Example 5 of the present invention;

[0024] Figure 3 This is a schematic diagram of the histopathological observation results of the mouse wound surface according to Example 5 of the present invention;

[0025] Figure 4 This is a schematic diagram of the changes in SDF-1α protein levels at different stages after wound formation in mice according to Example 5 of the present invention. DETAILED DESCRIPTION

[0026] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0027] The invention provides a ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration. The ready-to-use preparation comprises: human umbilical cord mesenchymal stem cells, trehalose or mannitol, medical human serum albumin or bovine serum albumin and medical compound electrolyte injection; wherein, per 1000 ml of the medical compound electrolyte injection comprises 5.26 g of sodium chloride, 5.02 g of sodium gluconate, 3.68 g of sodium acetate, 0.37 g of potassium chloride and 0.30 g of magnesium chloride; and the medical human serum albumin comprises: 20 g / 100 ml of human serum albumin, 16 mmol / L of sodium N-acetyltryptophanate, 16 mmol / L of sodium zincate and 140 mmol / L of sodium chloride.

[0028] Tests show that the ready-to-use preparation provided by the present invention has a stable period of up to 14 days at 2-8°C, and has good stability. Trehalose or mannitol has a good effect on improving the stability of the ready-to-use preparation, and there is a certain synergistic effect between the components, which can enhance the stability of the ready-to-use preparation.

[0029] Trehalose is a non-reducing disaccharide composed of two glucose molecules and is the most stable type of natural disaccharides. Because it is not reducing, it has very good stability to heat and acid and alkali. When coexisting with amino acids and proteins, the Maillard reaction will not occur even if heated. It can form a unique protective film on the cell surface under harsh environmental conditions, effectively protecting the biological molecular structure from being destroyed, and has a non-specific protective effect on biological macromolecules and organisms. As a stabilizer for biological products, it can not only be stored at room temperature, but also prevent contamination, thereby ensuring the storage, transportation and safety of biological products. As one of the most commonly used non-permeable protective agents for low-temperature preservation, trehalose can increase the solute concentration of the extracellular solution, not only can it form hypertonicity outside the cell and reduce the formation of intracellular ice, but also has a high glass transition temperature and is easy to form a glassy state. On the one hand, this avoids the damage of low temperature to cell membrane proteins and lipids, and on the other hand, it wraps up the protein molecular support of the cell membrane, making it difficult to deform, inhibiting cell membrane fusion, and reducing the permeability of the cell membrane. To successfully achieve low-temperature preservation of organisms, protective agents such as trehalose are indispensable. In addition, trehalose is also a good choice for lyophilized protective agents for liposome injections. Experiments show that the freeze-dried liposomes with trehalose as a protective agent have the smallest change in particle size and the best protective effect; the particle size of glucose changes the most and the worst protective effect. For trehalose, a concentration of 10% has the best protective effect on freeze-dried liposomes. As a protein protective agent for monoclonal antibody injection, trehalose also plays an important role in stabilizing and protecting antibody proteins. Bevacizumab injection (Avastin), a humanized monoclonal antibody against vascular endothelial growth factor (VEGF) developed by Roche, adalimumab (Humira), the "king of medicine" for the treatment of rheumatoid arthritis, and AstraZeneca's PD-1 / PD-L1 inhibitor (durvalumab, Imfinzi) all use ultra-low endotoxin, sterile, high-quality pharmaceutical-grade trehalose (concentration range 50-100 mg / mL). In the present invention, trehalose is the main component for maintaining the stability of the preparation. Furthermore, considering the characteristics of umbilical cord mesenchymal stem cells, the trehalose of the present invention is selected in a lower concentration range (5-9 mg / mL) than cell cryopreservation and monoclonal antibody injection protein protection, and after optimization, its concentration range is controlled within a reasonable range. The preferred concentration is 7.5 mg / mL.

[0030] The chemical formula of mannitol is C6H 14 O6 is a sugar alcohol and an isomer of sorbitol. Mannitol is a good diuretic in medicine, which can reduce intracranial pressure, intraocular pressure, and treat kidney medicine, dehydration medicine, sugar substitute, and is also used as an excipient for tablets and a diluent for solids and liquids. As a solvent and stabilizer, it can increase the stability and solubility of drugs, thereby improving the absorption and bioavailability of drugs. When selecting, it is necessary to pay attention to selecting pharmaceutical-grade raw materials that can be directly used in the human body.

[0031] Human albumin is the main component of human plasma protein. Its important physiological functions are its blood osmotic pressure maintenance and its carrying function for substances with low relative molecular mass. It plays a stabilizing and regulating role in circulating blood volume. It is also a transporter of substances such as hormones, enzymes, drugs and toxins. It has a good effect on maintaining the morphology and function of cells. The main functions and effects include anti-inflammatory, immunomodulatory, and maintaining plasma colloidal osmotic pressure. Considering that human albumin may bring certain exogenous irritation when applied topically to the human body, the present invention reduces the use concentration range of human albumin. The present invention preferably uses medical human albumin (20g / 100ml, i.e. 20%), which contains 0.08mmol sodium octanoate / gram albumin and 0.08mmol sodium acetyltryptophan / gram albumin as a protein stabilizer. The preferred concentration of medical human albumin (20%) is 8%.

[0032] Bovine serum albumin is often used as a stabilizer in the storage solution and reaction solution of biological products or enzymes. It can prevent enzyme decomposition and non-specific adsorption, reduce the denaturation of some enzymes, reduce the denaturation caused by adverse environmental factors such as heating, surface tension and chemical factors, and help cell survival. However, bovine serum albumin is currently mainly used as a scientific research reagent.

[0033] The main function of compound electrolyte injection is to supplement body fluids, and it can also regulate acid-base balance and electrolyte disorders. The ingredients of compound electrolyte injection include sodium chloride, potassium chloride, calcium chloride, potassium-free hydrochlorothiazide, sodium bicarbonate, disodium phosphate, calcium gluconate, sodium lactate, dipotassium sodium phosphate, potassium chloride, and sodium chloride. The neutral environment and stable osmotic pressure of the preparation can be maintained to protect the vitality of cells. The ingredients of compound electrolyte injections from different manufacturers will have certain differences. The present invention preferably produces Sichuan Kelun Pharmaceutical Co., Ltd., and the components are: 5.26g sodium chloride, 5.02g sodium gluconate, 3.68g sodium acetate, 0.37g potassium chloride, and 0.30g magnesium chloride per 1000ml. The preferred volume proportion of compound electrolyte injection is 85%.

[0034] The medical human albumin injection used in the above preparations contains a certain concentration of sodium zincate, and the compound electrolyte injection contains a certain concentration of sodium gluconate. These have a certain synergistic effect on the immunomodulatory and anti-inflammatory effects of mesenchymal stem cells. The effects and uses of sodium gluconate in medicine include regulating acid-base balance and promoting nerve recovery. Sodium gluconate can also be used as a joint lubricant. After many joint surgeries, sodium gluconate will be injected into the joints, which can protect the joints and reduce friction between joints, thereby promoting trauma recovery. In short, the medical human albumin and compound electrolyte injection in the preparations can not only maintain the stability of the stem cell preparations, but also help with local medication for osteoarthritis and diabetic foot.

[0035] Compared with the prior art (such as documents 1 and 3 in the background technology) (as shown in Table 1), the types of components of the instant preparation provided by the present invention are few, and only human umbilical cord mesenchymal stem cells, trehalose or mannitol, medical human albumin or bovine serum albumin and medical compound electrolyte injection are included. Low molecular weight heparin calcium injection, dextran 40 sodium chloride injection and DMSO are not included. In addition, the present invention reduces the amount of human albumin, better controls the cost of the preparation, and makes clinical accessibility higher. Although DMSO is a commonly used cell protectant, it has a certain toxicity risk. In various clinical preparations, its use concentration must be strictly controlled. In order to ensure the clinical safety of the preparation, the present invention does not use DMSO at all.

[0036] The ready-to-use preparation of the present invention has a stable period of more than 14 days at 2-8° C., which is greatly prolonged compared with 4 days in Document 1 and 18 hours in Document 3, making clinical use more convenient and significantly improving clinical accessibility.

[0037] Table 1

[0038]

[0039]

[0040] Example 1: Microbial safety control of umbilical cord, the starting material of umbilical cord mesenchymal stem cell preparation.

[0041] 1. Select a regular clinical institution, pass ethical review, and have the donor sign an informed consent.

[0042] 2. Screening of umbilical cord donors.

[0043] (1) The mother is under 35 years old and has normal development and nutrition;

[0044] (2) Pregnancy is between 36 and 42 weeks, prenatal examination is normal, delivery is normal, and the delivery process is smooth;

[0045] (3) No serious complications during pregnancy;

[0046] (4) No familial genetic disease;

[0047] (5) Prenatal tests for infectious indicators such as hepatitis B, HCV, HIV, EBV, HTLV, CMV, and Treponema pallidum were normal (see Table 2);

[0048] (6) The amniotic fluid is uncontaminated;

[0049] (7) Fetal weight ≥2500 g;

[0050] (8) The fetus is born without congenital malformations.

[0051] Table 2

[0052]

[0053]

[0054] 3. Umbilical cord collection and transportation management.

[0055] 1) The time out of body should not exceed 12 hours.

[0056] 2) The umbilical cord length is greater than 20cm.

[0057] 3) Transported under cold chain at 2-8℃, arrive at the laboratory and be disinfected within 24 hours.

[0058] Example 2: Preparation and quality control of umbilical cord mesenchymal stem cells (UC-MSCs).

[0059] 1. Isolate and culture umbilical cord mesenchymal stem cells in a B+A grade laboratory.

[0060] 2. Strictly control exogenous microorganisms during the production process.

[0061] 3. The umbilical cord tissue in Example 1 can be used to separate and subculture umbilical cord mesenchymal stem cells and to establish a seed cell bank and a working cell bank.

[0062] 4. Take the cells from the working cell bank for resuscitation culture, harvest the P6 generation cells, and conduct tests such as basic biological properties, cell effectiveness, biological safety, and microbiological safety as shown in Table 3.

[0063] Table 3

[0064]

[0065]

[0066] Example 3: Stability of the ready-to-use preparation of umbilical cord mesenchymal stem cells.

[0067] 1. Prepare a ready-to-use preparation of umbilical cord mesenchymal stem cells (medium dose group).

[0068] 1. Preparation

[0069] Freshly harvested P6 generation human umbilical cord mesenchymal stem cells were tested to meet the requirements according to Example 2, and the preparation was prepared according to Table 4. After 0.22 um filtration, they were packaged according to the specification of 2 ml / bottle and stored in a refrigerator at 2-8° C. for future use.

[0070] Table 4

[0071] Components content Human umbilical cord mesenchymal stem cells <![CDATA[2.0×10 7 pcs / 2.0ml]]> Trehalose 0.75%(w / v) Human serum albumin (20%) 8.0%(v / v) Compound Electrolyte Injection Remaining volume (about 1.7mL)

[0072] 2. Stability observation

[0073] The samples were stored in a refrigerator at 2-8°C, and samples were taken on the 0th, 3rd, 5th, 7th, 10th and 14th days for observation and testing of appearance, visible foreign matter, pH, osmotic pressure molarity, cell density, cell viability, sterility, bacterial endotoxins, mycoplasma, inhibition of Th1 lymphocyte subset proliferation, and cell phenotype.

[0074] 3. The results are as follows:

[0075] (1) Appearance: The cell suspension should be uniform when observed with the naked eye and should not change with storage time. The specific results are shown in Table 5.

[0076] (2) Visible foreign matter: The cell suspension must be free of visible foreign matter and must not change over time. The specific results are shown in Table 5.

[0077] (3) pH value: The pH value of the preparation was tested using a calibrated pH meter, and the pH value was required to be between 6.8 and 7.4, which was within the acceptable range over the storage time. The specific results are shown in Table 5.

[0078] (4) Osmolality: The osmolality of the preparation is required to be between 275 and 325 mOsmol / kg, with no significant change over storage time. The specific results are shown in Table 5.

[0079] (5) Cell density: The cell density of the preparation is required to be 9.0x10 6 / ml~1.1x10 7 / ml, and there was no significant change with storage time. The specific results are shown in Table 5.

[0080] (6) Cell viability: The cell viability of the preparation is required to be >80%, with no significant change over storage time. The specific results are shown in Table 5.

[0081] Table 5

[0082]

[0083] (7) Sterility: The sterility test result of the preparation is required to be negative and have no significant change with storage time. The specific results are shown in Table 6.

[0084] (8) Bacterial endotoxin: The bacterial endotoxin test result of the preparation is required to be less than 0.5EU / ml, and there is no significant change with storage time. The specific results are shown in Table 6.

[0085] (9) Mycoplasma: The mycoplasma test result of the preparation is required to be negative and have no significant change with storage time. The specific results are shown in Table 6.

[0086] (10) Th1 lymphocyte subset proliferation: Flow cytometry detection is required, and the inhibition rate is >40%, with no significant change over storage time. The specific results are shown in Table 6.

[0087] (11) Cell phenotype: The preparations are required to undergo cell phenotype testing, with the positive rates of CD73, CD90, and CD105 being no less than 95%; the positive rates of CD34, CD45, CD19, CD11b, and HLA-DR being no more than 2%, with no significant changes over storage time. The specific results are shown in Table 6.

[0088] Table 6

[0089]

[0090]

[0091] (11) The above results show that after storage at 2-8°C for 21 days, the appearance, visible foreign matter, pH, osmotic pressure molar concentration, sterility, bacterial endotoxins, mycoplasma, and cell phenotype of the preparations did not change significantly with the storage time. At the same time, the cell density was 9.8x10 6 / ml~1.1x10 7 / ml, cell viability>95%, and inhibition rate of Th1 lymphocyte subset proliferation above 60%, indicating that the stability of the preparation can reach 21 days and the preparation prescription is stable.

[0092] 2. Prepare a ready-to-use preparation of umbilical cord mesenchymal stem cells (low-dose group).

[0093] 1. Preparation

[0094] Freshly harvested P6 generation human umbilical cord mesenchymal stem cells were tested to meet the requirements according to Example 2, and the preparation was prepared according to Table 7. After 0.22 um filtration, the preparation was packaged according to the specification of 2 ml / bottle and stored in a refrigerator at 2-8° C. for future use.

[0095] Table 7

[0096] Components content Human umbilical cord mesenchymal stem cells <![CDATA[1.6×10 7 pcs / 2.0ml]]> Trehalose 0.50%(w / v) Human serum albumin (20%) 6.0%(v / v) Compound Electrolyte Injection Remaining volume (about 1.8 mL)

[0097] 2. Stability observation

[0098] The samples were stored in a refrigerator at 2-8°C, and samples were taken on the 0th, 3rd, 5th, 7th, 10th and 14th days for observation and testing of appearance, visible foreign matter, pH, osmotic pressure molarity, cell density, cell viability, sterility, bacterial endotoxins, mycoplasma, inhibition of Th1 lymphocyte subset proliferation, and cell phenotype.

[0099] 3. The results are as follows:

[0100] (1) Appearance

[0101] The cell suspension was required to be a uniform suspension observed by naked eyes and not change with the storage time. The specific results are shown in Table 8.

[0102] (2) Visible foreign matter

[0103] It is required that there is no visible foreign matter in the cell suspension by naked eye observation and no change with the storage time. The specific results are shown in Table 8.

[0104] (3) pH value

[0105] The pH value of the preparation was tested using a calibrated pH meter, and the pH value was required to be between 6.8 and 7.4, which was within the qualified range over the storage time. The specific results are shown in Table 8.

[0106] (4) Osmolality

[0107] The osmotic pressure molar concentration of the preparation is required to be between 275 and 325 mOsmol / kg, and there is no significant change with storage time. The specific results are shown in Table 8.

[0108] (5) Cell density

[0109] The cell density of the preparation is required to be 8.0x10 6 / ml~1.0x10 7 / ml, and there was no significant change with storage time. The specific results are shown in Table 8.

[0110] (6) Cell viability

[0111] The cell viability of the preparation was required to be >80%, with no significant change over storage time. The specific results are shown in Table 8.

[0112] Table 8

[0113]

[0114] (7) Sterility

[0115] The sterility test result of the preparation is required to be negative and have no significant change with storage time. The specific results are shown in Table 9.

[0116] (8) Bacterial endotoxins

[0117] The bacterial endotoxin test result of the preparation is required to be less than 0.5EU / ml, and there is no significant change with the storage time. The specific results are shown in Table 9.

[0118] (9) Mycoplasma

[0119] The mycoplasma test results of the preparations are required to be negative and have no significant changes with storage time. The specific results are shown in Table 9.

[0120] (10) Th1 lymphocyte subset proliferation: Flow cytometry detection is required, and the inhibition rate is >40%, with no significant change over storage time. The specific results are shown in Table 9.

[0121] (11) Cell phenotype

[0122] The preparations are required to carry out cell phenotype test results, with the positive rates of CD73, CD90, and CD105 being no less than 95%; the positive rates of CD34, CD45, CD19, CD11b, and HLA-DR being no more than 2%, and having no significant changes with storage time. The specific results are shown in Table 9.

[0123] Table 9

[0124]

[0125] (11) The above results show that after storage at 2-8°C for 14 days, the appearance, visible foreign matter, pH, osmotic pressure molar concentration, cell density, cell viability, sterility, bacterial endotoxins, mycoplasma, and cell phenotype indicators of the preparation did not change significantly with the storage time, indicating that the stability of the preparation can reach 14 days and the preparation prescription is stable.

[0126] 3. Prepare a ready-to-use preparation of umbilical cord mesenchymal stem cells (high-dose group).

[0127] 1. Preparation

[0128] Freshly harvested P6 generation human umbilical cord mesenchymal stem cells were tested to meet the requirements according to Example 2, and the preparation was prepared according to Table 10. After 0.22 um filtration, the preparation was packaged according to the specification of 2 ml / bottle and stored in a refrigerator at 2-8° C. for future use.

[0129] Table 10

[0130] Components content Human umbilical cord mesenchymal stem cells <![CDATA[2.4×10 7 pcs / 2.0ml]]> Trehalose 0.90%(w / v) Human serum albumin (20%) 10.0%(v / v) Compound Electrolyte Injection Remaining volume (about 1.6mL)

[0131] 2. Stability observation

[0132] The samples were stored in a refrigerator at 2-8°C, and samples were taken on the 0th, 3rd, 5th, 7th, 10th and 14th days for observation and testing of appearance, visible foreign matter, pH, osmotic pressure molarity, cell density, cell viability, sterility, bacterial endotoxins, mycoplasma, inhibition of Th1 lymphocyte subset proliferation, and cell phenotype.

[0133] 3. The results are as follows:

[0134] (1) Appearance

[0135] The cell suspension was required to be a uniform suspension observed by naked eyes and not change with the storage time. The specific results are shown in Table 11.

[0136] (2) Visible foreign matter

[0137] It is required that there is no visible foreign matter in the cell suspension by naked eye observation and no change with the storage time. The specific results are shown in Table 11.

[0138] (3) pH value

[0139] The pH value of the preparation was tested using a calibrated pH meter, and the pH value was required to be between 6.8 and 7.4, which was within the qualified range over the storage time. The specific results are shown in Table 11.

[0140] (4) Osmolality

[0141] The osmotic pressure molar concentration of the preparation is required to be between 275 and 325 mOsmol / kg, and there is no significant change with storage time. The specific results are shown in Table 11.

[0142] (5) Cell density

[0143] The cell density of the preparation is required to be 1.0×10 7 / ml~1.2×10 7 / ml, and there was no significant change with storage time. The specific results are shown in Table 11.

[0144] (6) Cell viability

[0145] The cell viability of the preparation was required to be >80%, with no significant change over storage time. The specific results are shown in Table 11.

[0146] Table 11

[0147]

[0148] (7) Sterility

[0149] The sterility test result of the preparation is required to be negative and have no significant change with storage time. The specific results are shown in Table 12.

[0150] (8) Bacterial endotoxins

[0151] The bacterial endotoxin test result of the preparation is required to be less than 0.5EU / ml, and there is no significant change with the storage time. The specific results are shown in Table 12.

[0152] (9) Mycoplasma

[0153] The mycoplasma test results of the preparations are required to be negative and have no significant changes with storage time. The specific results are shown in Table 12.

[0154] (10) Th1 lymphocyte subset proliferation: Flow cytometry detection is required, and the inhibition rate is >40%, with no significant change over storage time. The specific results are shown in Table 12.

[0155] (11) Cell phenotype

[0156] The preparations are required to carry out cell phenotype test results, with the positive rates of CD73, CD90, and CD105 being no less than 95%; the positive rates of CD34, CD45, CD19, CD11b, and HLA-DR being no more than 2%, and having no significant changes with storage time. The specific results are shown in Table 12.

[0157] Table 12

[0158]

[0159]

[0160] (11) The above results show that after storage at 2-8°C for 14 days, the appearance, visible foreign matter, pH, osmotic pressure molar concentration, cell density, cell viability, sterility, bacterial endotoxins, mycoplasma, inhibition of Th1 lymphocyte subset proliferation, and cell phenotype indicators of the preparation did not change significantly with the storage time, indicating that the stability of the preparation can reach 14 days and the preparation prescription is stable.

[0161] Example 4: Effect of the ready-to-use preparation on inflammatory factor analysis and pain threshold of osteoarthritis.

[0162] Osteoarthritis is a degenerative and chronic joint disease characterized by clinical symptoms and deformation of joint tissues. It mainly damages tissues such as articular cartilage, subchondral bone and synovium, leading to pain, swelling and stiffness around the joints. The global prevalence is 3.8%. The knee joint is the largest synovial joint in humans. Compared with other joints, the knee joint has a higher incidence rate, with a lifetime risk of about 45%. Traditional pharmacological intervention focuses on improving symptoms. Currently, the drugs used for intra-articular injection are mainly sodium hyaluronate and glucocorticoid drugs, but their effects are limited and have a series of side effects. In the induced osteoarthritis animal model, mesenchymal stem cells (MSCs) can prevent and treat cartilage degeneration and improve joint pain.

[0163] 1. For the P6 generation of subculture, a stem cell suspension (ready-to-use preparation) was prepared according to the component ratio of the medium-dose preparation in Example 3. There were three dose groups, each with 2 mL. Except for the different cell concentrations, the proportions of other components remained unchanged. The low-dose ready-to-use preparation group had a cell concentration of 3.0×10 6 cells / mL; medium-dose ready-to-use preparation group: cell concentration 1.0×10 7 cells / mL; high-dose ready-to-use preparation group: cell concentration 3.0×10 7 cells / mL.

[0164] 2. The rat knee osteoarthritis model was established by surgical induction, and the above preparation was locally administered into the knee joint cavity: 50uL / joint cavity. The experimental animals were divided into 3 groups of ready-to-use preparations with high, medium and low doses, normal control, model control, etc., with 10 in each group. The serum inflammatory indicators IL-1β, IL-6, and TNF-α of each group were detected by ELISA method (the results are shown in Table 13). The test probe of the VonFrey analgesic instrument was used to tactilely stimulate the sole of the affected side of the rat, and the intensity was gradually increased from small to large, and the mechanical pain threshold when the rat withdrew its foot was recorded (the results are shown in Table 14).

[0165] 3. Compared with the normal control group, the levels of IL-1β, IL-6 and TNF-α in the serum of the model control group were significantly increased (P≤0.05). Compared with the model control group, the low-concentration ready-to-use preparation group significantly reduced the level of IL-1β in the serum (P≤0.05), and the high- and medium-dose ready-to-use preparation groups significantly reduced the levels of IL-1β and IL-6 in the serum (P≤0.01).

[0166] Table 13 Effects of stem cell ready-to-use preparations on serum inflammatory factors in rats (Mean±SEM, n=10)

[0167]

[0168] Note: 1. Compared with the normal control group, △△ P≤0.01; compared with the model control group, * P≤0.05, ** P≤0.01; 2. n represents the number of animals; 3. Mean±SEM represents the mean plus or minus standard error.

[0169] 4. Compared with the normal control group, the pain threshold of the model control group was significantly increased (P≤0.05~0.01). Compared with the model control group, the high and medium dose ready-to-use preparation groups could significantly increase the pain threshold of rats after administration for 2 and 3 times (P≤0.05~0.01), which was dose-dependent. See Table 14 for details.

[0170] Table 14 Effects of stem cell ready-to-use preparations on pain threshold of rats (Mean±SEM, n=10)

[0171]

[0172] Note: 1. Compared with the normal control group, △ P≤0.05, △△ P≤0.01; compared with the model control group, * P≤0.05, ** P≤0.01; 2. n: number of animals; 3. The number of administrations is based on the number of stem cell administrations; 4. Mean±SEM represents the mean plus or minus standard error.

[0173] Example 5. Effect of the ready-to-use preparation on diabetic foot.

[0174] Diabetic foot ulcer (DFU) is a serious complication of diabetes. It is a disease of blood vessels and nerves in the foot, leading to varying degrees of ulceration and infection symptoms in the foot. Because it is difficult to heal, it will infiltrate muscles and bones in the later stage, leading to tissue necrosis. It is also a common type of chronic difficult-to-heal wound. Due to the long-term unsatisfactory control of blood sugar in diabetic foot patients, the basement membrane of the lower limb blood vessels thickens, vascular endothelial cells are damaged, lipid adhesion, plaque formation, lumen stenosis or vascular obstruction, resulting in terminal ischemia, hypoxia, and necrosis. Diabetic autonomic neuropathy can cause a large number of microcirculatory arteriovenous shunts to open, resulting in reduced capillary blood flow, further aggravating tissue hypoxia, ischemia and nutritional disorders. At the same time, the phagocytic function of leukocytes in diabetic patients is reduced, making DFU gangrene infection difficult to control, and ulcer wounds are often difficult to heal. In severe cases, amputation is often required in the later stage, which poses a severe challenge to patients and clinicians. The self-renewal and directional differentiation potential of stem cells has opened up a new treatment path for repairing damaged tissues, anti-inflammation, regeneration, and restoring tissue cell function to promote wound healing.

[0175] 1. At the P6 generation of subculture, 2 mL of stem cell suspension (ready-to-use preparation) was prepared according to the component proportions of the medium-dose preparation in Example 3. The cell concentration was 1.0×10 7 cells / mL.

[0176] 2. A diabetic mouse model was established by intraperitoneal injection of streptozotocin (STZ), and a full-thickness skin defect wound was made on the back of the mouse. The skin wound healing rate was compared with that of normal mice to establish a standard delayed diabetic wound healing model. A circular wound with a diameter of 6 mm was made on both sides of the back of the mouse, and the experimental animals were divided into 3 experimental groups: ① NC group: normal control group; ② DC group: diabetic control group; ③ Ready-to-use preparation (MSC) group: the above preparation (80uL) was injected at the edge of the diabetic wound; 15 mice in each group. The wound healing of each group of mice was tracked and detected on the day after wound formation (0 day), 3rd day, 7th day, 11th day, and 14th day after surgery. The wound healing time of the mice was recorded and photos were taken. The results are as follows Figure 1 shown.

[0177] 3. Wound healing of mice in each group: The wound healing of mice in each group was tracked and tested on the day, 3rd day, 7th day, 11th day and 14th day after wound formation. Compared with the diabetic control group, wound injection of the ready-to-use preparation was helpful for wound healing, and the wound healing speed and healing rate were significantly better than those of the diabetic control group. Figure 2 shown.

[0178] 4. Compared with the diabetic control group, the injection of the ready-to-use preparation into the wound surface can improve the wound surface microenvironment, reduce the infiltration time of inflammatory cells and excessive inflammatory response, promote the proliferation of fibroblasts, the formation of granulation tissue and new blood vessels, and the increase of collagen production in the wound surface, and finally accelerate wound healing. In the embodiment of the present invention, the wound surface histopathological observation was carried out: the changes of inflammatory cells in the wound surface were observed by HE staining and Masson staining. The results are as follows: Figure 3 As shown. SDF-1 (stromal cell derived factor-1) is a homeostatic chemokine, a chemical attractant for T lymphocytes and monocytes, and also has inflammatory activity. It plays a classic chemokine role in immune response by attracting activated CXCR4+T cells and monocytes to the inflammatory site. The present invention measured the SDF-1α protein level in the wound of mice (the results are shown in Figure 4 (shown in Figure 2): On the day of wound formation, the content of SDF-1α in the wound of the NC group was significantly higher than that of the other two groups, with significant differences (P < 0.05). On the 7th day, the SDF-1α in the wounds of the three groups increased to varying degrees, with the increase in the MSC group being more significant (P < 0.01). On the 14th day after wound formation, the SDF-1α content in the normal control group returned to normal levels, but the other three groups were still higher than the levels on the day of wound formation (P < 0.05).

[0179] In summary, the ready-to-use preparation of umbilical cord mesenchymal stem cells for topical administration provided by the present invention has the following beneficial effects:

[0180] 1. The ready-to-use preparation does not need to be frozen. At 2-8°C, the preparation is stable for up to 14 days. It is convenient for product production, storage, transportation and clinical use. At the same time, the selected components are all clinical drug-grade raw materials and can be used directly in clinical practice.

[0181] 2. In addition to maintaining the stability of stem cell preparations, human albumin and compound electrolytes in the ready-to-use preparations also have immunomodulatory mechanisms and anti-inflammatory effects. They have a certain synergistic effect with mesenchymal stem cells and are helpful for local medication of osteoarthritis and diabetic foot.

[0182] 3. The ready-to-use preparation has an immunomodulatory effect in osteoarthritis model animals. Compared with the normal control group, the levels of IL-1β, IL-6, and TNF-α in the serum of the osteoarthritis model control group were significantly increased (P≤0.05). Compared with the model control group, the low-dose ready-to-use preparation group significantly reduced the IL-1β content in the serum (P≤0.05). The high- and medium-dose stem cell ready-to-use preparation groups significantly reduced the IL-1β and IL-6 content in the serum (P≤0.01).

[0183] 4. The ready-to-use preparation has the effect of regulating the pain threshold in osteoarthritis model animals. Compared with the normal control group, the pain threshold of the osteoarthritis model control group was significantly increased (P≤0.05-0.01). Compared with the model control group, the high and medium doses of stem cell ready-to-use preparation groups can significantly increase the pain threshold of rats after 2 and 3 administrations (P≤0.05-0.01), which is dose-dependent.

[0184] 5. The ready-to-use preparation can regulate immunity and accelerate wound healing in the diabetic foot animal model. Compared with the diabetic control group, the injection of the ready-to-use preparation of stem cells into the wound can improve the wound microenvironment, reduce the infiltration time of inflammatory cells and excessive inflammatory response, promote the proliferation of fibroblasts, granulation tissue and new blood vessels, and increase collagen production, ultimately accelerating wound healing.

[0185] Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention. Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A ready-to-use preparation of umbilical cord mesenchymal stem cells for topical administration, characterized in that: Each 2 mL of the ready-to-use preparation consists of the following substances: 2.0×10 human umbilical cord mesenchymal stem cells 7 , 0.015g trehalose, 0.16mL medical human albumin and 1.7mL medical compound electrolyte injection; wherein, each 1000ml of the medical compound electrolyte injection includes 5.26g sodium chloride, 5.02g sodium gluconate, 3.68g sodium acetate, 0.37g potassium chloride and 0.30g magnesium chloride.

2. The method for preparing the ready-to-use preparation of umbilical cord mesenchymal stem cells for topical administration according to claim 1, characterized in that: include: Obtain the umbilical cord and control the microbial safety of the umbilical cord to meet the preset requirements; Prepare umbilical cord mesenchymal stem cells, and perform quality control on the prepared umbilical cord mesenchymal stem cells to ensure that they meet preset requirements; A ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration was prepared, and the test results showed that the prepared ready-to-use preparation could be stored at 2-8° C. for 14 days.

3. Use of the ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration as claimed in claim 1 in the preparation of an immunomodulatory drug for treating osteoarthritis.

4. Use of the ready-to-use preparation of umbilical cord mesenchymal stem cells for local administration as claimed in claim 1 in the preparation of an immunomodulatory drug for treating diabetic foot.

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