Injectable stem cell freezing medium as well as preparation method and application thereof

Abstract

The invention relates to the technical field of stem cells, in particular to an injectable stem cell freezing medium which comprises human serum albumin, dimethyl sulfoxide, 1, 2-propylene glycol, trehalose, hydroxyethyl starch and methyl cellulose. According to the embodiment of the invention, the human serum albumin with a specific content and the permeable and impermeable protective agents with specific types and dosages are compounded, so that the stem cell cryopreservation liquid which is high in safety and good in stability and can be directly used for injection is obtained, and the stem cell cryopreservation liquid does not contain animal-derived components, has low-content dimethyl sulfoxide, and is suitable for clinical application. According to the present invention, the formula is formed by using the pharmaceutical grade components, can be directly used as the clinical injection liquid, can be opened for use after the cryopreservation recovery, can be stored for a long time at-80 DEG C or in liquid nitrogen, and can ensure the cell activity to be more than 85%;

Description

Technical Field

[0001] The present application relates to the field of stem cell technology, and in particular to an injectable stem cell cryopreservation solution and a preparation method and application thereof. Background Art

[0002] Stem cell therapy has always been one of the most important areas in the forefront of life sciences in various countries. It has become a frontier hotspot, supported by government policies and promoted by new research fields and methods. Among the many types of stem cells, mesenchymal stem cells (MSCs) derived from tissues such as umbilical cord, placenta, and bone marrow have become the most advantageous clinical application-level stem cells due to their powerful self-renewal and immune regulation capabilities. Clinical research on mesenchymal stem cells covers a variety of diseases such as osteoarthritis, spinal cord injury, lower limb ischemia, diabetes, diabetic complications, cerebral infarction, Parkinson's disease, cardiovascular disease, stroke, liver disease and tumors, refractory lung disease and infertility, and is expected to provide a new treatment approach for these diseases.

[0003] However, most clinical studies on stem cells at home and abroad are still in the clinical phase I and II, and many clinical studies have failed or terminated due to the quality stability of stem cell drugs. The challenges faced by stem cell therapy include how to ensure a stable cell source and sufficient transplanted cells, how to obtain cell preservation technology to maintain cell activity, how to ensure the safety of stem cell transplantation therapy, and how to effectively improve the effect of stem cell therapy. Among them, how to achieve a stem cell drug formula that maintains a highly active cell population and is easy to use in clinical applications is the first problem to be solved.

[0004] Since the active ingredients of stem cell drugs are living cells, the storage conditions require extremely high standards. When stem cells are frozen using conventional cell freezing fluid, the conventional cell freezing fluid contains at least 10% dimethyl sulfoxide. Direct infusion of stem cell suspensions containing high concentrations of dimethyl sulfoxide into the human body can cause serious side effects. In general, stem cells frozen using high concentrations of dimethyl sulfoxide need to be thawed, cleaned to remove dimethyl sulfoxide, and then resuspended in physiological saline or other solutions to make a cell suspension before clinical use to ensure clinical safety. It must be used within a short time (24h-48h), and the operation is relatively complicated. For the convenience of clinical use, it is particularly important to develop ready-to-use stem cell freezing fluids as stem cell drug preparations.

[0005] To ensure the activity of stem cells, they need to be placed in a liquid nitrogen environment after being frozen using conventional cell freezing fluid. However, for hospital applications, it is difficult to equip gaseous or liquid nitrogen tanks and their management and maintenance. Therefore, stem cell drugs face the problem of inconvenient storage in hospitals after they are launched on the market. In order to solve the problems of clinical storage and use, it is urgent to develop stem cell drug preparations that can be stored for a long time under non-liquid nitrogen conditions (such as -80°C). Summary of the invention

[0006] The present application solves at least one of the problems of the related art from the following aspects.

[0007] The first aspect of the present application provides an injectable stem cell freezing solution, comprising: human serum albumin, dimethyl sulfoxide, 1,2-propylene glycol, trehalose, hydroxyethyl starch and methyl cellulose.

[0008] In some embodiments, the injectable stem cell freezing solution further comprises an electrolyte solution, wherein the electrolyte solution comprises sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.

[0009] In some embodiments, the electrolyte solution comprises 5.26 g / L sodium chloride, 5.02 g / L sodium gluconate, 3.68 g / L sodium acetate, 0.37 g / L potassium chloride and 0.3 g / L magnesium chloride, with the remainder being water.

[0010] In some embodiments, based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises:

[0011] 3 to 5 wt% human albumin,

[0012] 2 to 8 wt% dimethyl sulfoxide,

[0013] 2 to 8 wt% 1,2-propylene glycol,

[0014] 0.05 to 0.17 wt% trehalose,

[0015] 1×10 -5 Up to 5×10 -5 wt% hydroxyethyl starch, and

[0016] 1×10 -5 Up to 10×10 -5 wt% methylcellulose.

[0017] In some embodiments, based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises:

[0018] 3.5 to 4.5wt% human albumin,

[0019] 4 to 6 wt% dimethyl sulfoxide,

[0020] 4 to 6 wt% 1,2-propylene glycol,

[0021] 0.06 to 0.1 wt% trehalose,

[0022] 1×10 -5 Up to 2×10 -5wt% hydroxyethyl starch, and

[0023] 4×10 -5 Up to 6×10 -5 wt% methylcellulose.

[0024] In some embodiments, based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises:

[0025] 4wt% human albumin,

[0026] 5wt% dimethyl sulfoxide,

[0027] 5wt% 1,2-propylene glycol,

[0028] 0.083wt% trehalose,

[0029] 1×10 -5 wt% Hydroxyethyl starch,

[0030] 5×10 -5 wt% methylcellulose,

[0031] The balance is the electrolyte solution.

[0032] In some embodiments, the stem cells are mesenchymal stem cells.

[0033] In some embodiments, the stem cells are mesenchymal stem cells derived from humans.

[0034] In some embodiments, the mesenchymal stem cells are selected from the group consisting of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, amniotic fluid mesenchymal stem cells, menstrual blood mesenchymal stem cells, peripheral blood mesenchymal stem cells, dental pulp mesenchymal stem cells and skin mesenchymal stem cells.

[0035] The second aspect of the present application provides a method for preparing an injectable stem cell cryopreservation solution according to any one of the first aspects, comprising:

[0036] Dissolving trehalose, hydroxyethyl starch and methyl cellulose in electrolyte solution respectively and sterilizing the solution to obtain trehalose solution, hydroxyethyl starch solution and methyl cellulose solution;

[0037] The trehalose solution, the hydroxyethyl starch solution, the methylcellulose solution, human serum albumin, 1,2-propylene glycol and dimethyl sulfoxide are added to the electrolyte solution, wherein the human serum albumin is added to the electrolyte solution first and the dimethyl sulfoxide is added to the electrolyte solution last.

[0038] A third aspect of the present application provides an injectable stem cell system, comprising the injectable stem cell cryopreservation solution and mesenchymal stem cells according to any one of the first aspects.

[0039] In some embodiments, the injectable stem cell system comprises 0.2×10 7 Up to 5×10 7 The mesenchymal stem cells contain cells / mL.

[0040] The embodiments of the present application achieve the following beneficial effects:

[0041] The embodiments of the present application provide an innovative stem cell cryopreservation solution formula, which does not contain animal-derived ingredients, reduces the content of dimethyl sulfoxide, and uses pharmaceutical-grade components to form the formula. It can be directly used as a clinical injection solution, and can be used immediately after cryopreservation and resuscitation. It can be stored for a long time at -80°C or in liquid nitrogen, and the cell activity is guaranteed to be above 85%, thereby obtaining the expected therapeutic effect.

[0042] The present application provides an injectable stem cell cryopreservation solution, a preparation method thereof, and an injectable stem cell system. By using a specific content of human albumin and a specific type and amount of permeability and non-permeability protective agent, a stem cell cryopreservation solution with high safety, good stability, and direct injection is obtained, which can be stored at -80°C or in liquid nitrogen for a long time, significantly improving the efficiency of stem cell recovery after long-term storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0044] Figure 1 The total number of viable cells and cell viability of MSC001 cells in Example 2 recovered after being frozen at -80°C for 2 weeks;

[0045] Figure 2 The total number of live cells and cell viability of MSC001 cells in Example 2 after being frozen at -80°C for 2 weeks and then revived and cultured for 72 hours;

[0046] Figure 3 The total number of viable cells and cell viability of MSC001 cells in Example 2 recovered after being frozen at -80°C for one month;

[0047] Figure 4 The total number of live cells and cell viability of MSC001 cells in Example 2 after being frozen at -80°C for one month and then revived and cultured for 72 hours;

[0048] Figure 5 The total number of viable cells and cell viability of MSC001 cells in Example 3 recovered after being frozen in liquid nitrogen for 2 weeks;

[0049] Figure 6 The total number of live cells and cell viability of MSC001 cells in Example 3 after being frozen in liquid nitrogen for 2 weeks and then revived and cultured for 72 hours;

[0050] Figure 7 The total number of viable cells and cell viability of MSC001 cells in Example 3 recovered after being frozen in liquid nitrogen for one month;

[0051] Figure 8 The total number of live cells and cell viability of MSC001 cells in Example 3 after being frozen in liquid nitrogen for one month and then revived and cultured for 72 hours;

[0052] Fig. 9 The total number of viable cells and cell viability of MSC006 in Example 4 were recovered by freezing at -80°C for 2 weeks and 1 month using F-11 / F-16 / F-24 cryopreservation solution;

[0053] Fig.10 The total number of live cells and cell viability of MSC006 in Example 4 after being frozen at -80°C for 2 weeks and 1 month using F-11 / F-16 / F-24 cryopreservation solution and then cultured for 72 hours;

[0054] Fig.11 The total number of viable cells and cell viability of MSC-006 in Example 4 were recovered by freezing in liquid nitrogen for 2 weeks and 1 month using F-11 / F-16 / F-24 cryopreservation solution;

[0055] Fig.12 The total number of live cells and cell viability of MS-C006 in Example 4 were cryopreserved in liquid nitrogen for 2 weeks and 1 month and then revived and cultured for 72 hours using F-11 / F-16 / F-24 cryopreservation solution. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art and are not intended to limit the present invention in any way.

[0057] This application is made based on the following knowledge of the inventor:

[0058] When designing the ingredients of stem cell cryopreservation injections, on the one hand, it is necessary to provide the best cryopreservation environment for stem cells during the storage process to ensure the vitality and biological properties of stem cells, and on the other hand, it is necessary to be as non-toxic and non-side effects on the patient's human tissues and organs as possible during use to avoid adverse reactions. Therefore, it is necessary to study the applicability and concentration of the cryopreservation solution formula in order to obtain a cryopreservation solution formula that can ensure the activity and biological properties of stem cells for as long as possible and has low toxicity, so as to be used as a stem cell drug.

[0059] The first aspect of the present application provides an injectable stem cell freezing solution, comprising: human serum albumin, dimethyl sulfoxide, 1,2-propylene glycol, trehalose, hydroxyethyl starch and methyl cellulose.

[0060] The present invention has developed an innovative stem cell cryopreservation solution formula, which does not contain animal-derived ingredients and reduces the content of dimethyl sulfoxide. The formula is composed of pharmaceutical-grade components and can be directly used as a clinical injection solution. It can be used immediately after cryopreservation and resuscitation, and can be stored for a long time at -80°C or in liquid nitrogen, and the cell activity is guaranteed to be above 85%, thereby obtaining the expected therapeutic effect.

[0061] In some embodiments, the injectable stem cell freezing solution further comprises an electrolyte solution, wherein the electrolyte solution comprises sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride.

[0062] In some embodiments, the electrolyte solution comprises 5.26 g / L sodium chloride, 5.02 g / L sodium gluconate, 3.68 g / L sodium acetate, 0.37 g / L potassium chloride and 0.3 g / L magnesium chloride, with the remainder being water.

[0063] In some embodiments, based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises:

[0064] 3 to 5 wt% (e.g., 3.25 wt%, 3.5 wt%, 3.75 wt%, 4.0 wt%, 4.25 wt%, 4.5 wt%, 4.75 wt%) human albumin,

[0065] 2 to 8 wt% (e.g. 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%) of dimethyl sulfoxide,

[0066] 2 to 8 wt% (e.g., 3 wt%, 4 wt%, 5 wt%, 6 wt%, 7 wt%) 1,2-propylene glycol,

[0067] 0.05 to 0.17 wt% (e.g., 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.10 wt%, 0.11 wt%, 0.12 wt%, 0.13 wt%, 0.14 wt%, 0.15 wt%, 0.16 wt%) trehalose,

[0068] 1×10 -5 Up to 5×10 -5 wt% (e.g. 2×10 -5 wt%,3×10 -5 wt%、4×10 -5 wt %) hydroxyethyl starch, and

[0069] 1×10 -5 Up to 10×10 -5 wt% (e.g. 2×10 -5 wt%,3×10 -5 wt%、4×10 -5 wt%,5×10 - 5 wt%、6×10 -5 wt%、7×10 -5 wt%、8×10 -5 wt%、9×10 -5 wt %) methyl cellulose.

[0070] It is understandable that the human albumin contained in the injectable stem cell cryopreservation solution can be added in the form of an albumin preparation. As an example, a preparation containing 20wt% human albumin produced by Shanghai Laishi Blood Products Co., Ltd. can be used to prepare the injectable stem cell cryopreservation solution, so that the final net content of human albumin therein is 3 to 5wt% of the total mass of the injectable stem cell cryopreservation solution.

[0071] In some embodiments, based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises:

[0072] 3.5 to 4.5wt% human albumin,

[0073] 4 to 6 wt% dimethyl sulfoxide,

[0074] 4 to 6 wt% 1,2-propylene glycol,

[0075] 0.06 to 0.1 wt% trehalose,

[0076] 1×10 -5 Up to 2×10 -5 wt% hydroxyethyl starch, and

[0077] 4×10-5 Up to 6×10 -5 wt% methylcellulose.

[0078] In some embodiments, based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises:

[0079] 4wt% human albumin,

[0080] 5wt% dimethyl sulfoxide,

[0081] 5wt% 1,2-propylene glycol,

[0082] 0.083wt% trehalose,

[0083] 1×10 -5 wt% Hydroxyethyl starch,

[0084] 5×10 -5 wt% methylcellulose,

[0085] The balance is the electrolyte solution.

[0086] In some embodiments, the stem cells are mesenchymal stem cells.

[0087] In some embodiments, the stem cells are mesenchymal stem cells derived from humans.

[0088] In some embodiments, the mesenchymal stem cells are selected from the group consisting of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, amniotic fluid mesenchymal stem cells, menstrual blood mesenchymal stem cells, peripheral blood mesenchymal stem cells, dental pulp mesenchymal stem cells and skin mesenchymal stem cells.

[0089] The second aspect of the present application provides a method for preparing an injectable stem cell cryopreservation solution according to any one of the first aspects, comprising:

[0090] Dissolving trehalose, hydroxyethyl starch and methyl cellulose in electrolyte solution respectively and sterilizing the solution to obtain trehalose solution, hydroxyethyl starch solution and methyl cellulose solution;

[0091] The trehalose solution, the hydroxyethyl starch solution, the methylcellulose solution, human serum albumin, 1,2-propylene glycol and dimethyl sulfoxide are added to the electrolyte solution, wherein the human serum albumin is added to the electrolyte solution first and the dimethyl sulfoxide is added to the electrolyte solution last.

[0092] A third aspect of the present application provides an injectable stem cell system, comprising the injectable stem cell cryopreservation solution and mesenchymal stem cells according to any one of the first aspects.

[0093] In some embodiments, the injectable stem cell system comprises 0.2×10 7 Up to 5×10 7 cells / mL (e.g. 0.5×10 7 cells / mL, 1×10 7 cells / mL, 2×10 7 cells / mL, 3×10 7 cells / mL, 4×10 7 cells / mL) of the mesenchymal stem cells.

[0094] In this application, the term "comprising" is an open expression, that is, including the contents specified in the present invention, but not excluding other contents.

[0095] The stem cell freezing solution in the embodiment of the present invention comprises 15-25wt% human albumin preparation (Shanghai Laishi Blood Products Co., Ltd., the specification is a preparation containing 20wt% human albumin), 2-8wt% dimethyl sulfoxide, 2-8wt% 1,2-propylene glycol, 0.3-1wt% trehalose mother solution (16.67% concentration), 0.01-0.05wt% hydroxyethyl starch mother solution (0.1% concentration), 0.001-0.01wt% methyl cellulose mother solution (1% concentration), and the remainder is a compound electrolyte solution (Shijiazhuang Siyao Co., Ltd. National Medicine Standard No. H20133245, each 500ml contains 2.63g sodium chloride, 2.51g sodium gluconate, 1.84g sodium acetate, 0.185g potassium chloride, and 0.15g magnesium chloride.).

[0096] The method involved in the embodiments of the present invention is as follows:

[0097] Preparation method of stem cell cryopreservation solution

[0098] S1. Prepare reagents:

[0099] Prepare trehalose, hydroxyethyl starch and methyl cellulose separately, dissolve them in the composite electrolyte solution and sterilize them (sterilize at 115°C for 30 minutes, or sterilize by filtering with a 0.22 μm filter membrane);

[0100] Propylene glycol and dimethyl sulfoxide were sterilized by filtering through a 0.22 μm filter membrane before use.

[0101] S2. Preparation steps:

[0102] Take an appropriate amount of compound electrolyte solution, first add human albumin preparation (Shanghai Laishi Blood Products Co., Ltd., the specification is a preparation containing 20wt% human albumin), then add trehalose, methylcellulose, hydroxyethyl starch, propylene glycol (in no particular order), and finally add dimethyl sulfoxide, and make up the rest with compound electrolyte solution. Please refer to the specific examples for the content of each component in the prepared stem cell freezing solution.

[0103] Weigh 4 g of trehalose powder into a 50 mL centrifuge tube, add the compound electrolyte solution to make up to 20 mL, sterilize at 115°C for 30 minutes after fully dissolving, or filter with a 0.22 μm filter membrane for sterilization and set aside to obtain a trehalose mother solution with a mass concentration of 16.67%;

[0104] Weigh 0.02 g of hydroxyethyl starch into a 50 mL centrifuge tube, add the compound electrolyte solution to make up to 20 mL, sterilize at 115° C. for 30 minutes after fully dissolving, or filter through a 0.22 μm filter membrane for sterilization and set aside to obtain a hydroxyethyl starch mother solution with a mass concentration of 0.1%;

[0105] Take 100 mL of the composite electrolyte solution and heat it to 80°C, weigh 1 g of methyl cellulose, dissolve it at 4°C for 30 minutes, stir it for 30 minutes until it is completely dissolved, filter it with a 0.22 μm filter membrane for sterilization and set it aside to obtain a methyl cellulose mother solution with a mass concentration of 1%.

[0106] 1,2-Propanediol was sterilized by filtration through a 0.22 μm filter membrane and set aside.

[0107] The formula of stem cell freezing solution contains 15-25wt% human albumin preparation (Shanghai Laishi Blood Products Co., Ltd., the specification is a preparation containing 20wt% human albumin), 2-8wt% DMSO, 2-8wt% 1,2-propylene glycol, 0.3-1wt% trehalose mother solution (16.67% concentration), 0.01-0.05wt% hydroxyethyl starch mother solution (0.1% concentration), 0.001-0.01wt% methyl cellulose mother solution (1% concentration), and the rest is made up by compound electrolyte solution according to the preparation system.

[0108] Human albumin preparation, trehalose mother solution, methylcellulose mother solution, hydroxyethyl starch mother solution, propylene glycol and dimethyl sulfoxide are sequentially added to the composite electrolyte solution, and each one is fully mixed. Among them, human albumin is added first, and dimethyl sulfoxide is added last.

[0109] Human Mesenchymal Stem Cell Resuscitation

[0110] Human mesenchymal stem cells were taken for cell recovery and inoculated into a cell culture flask. After shaking, they were placed in a 37°C, 5% CO 2 Culture in an incubator.

[0111] Human Mesenchymal Stem Cell Passaging

[0112] When the cell confluence reaches 80-90%, subculture is performed. First, discard the cell culture supernatant, wash the cells with an appropriate amount of DPBS, add TryPLE (brand and item number: GIBCO, 12563029) and digest at room temperature for 2-3 minutes. After the cells fall off, add an appropriate amount of complete cell culture medium (brand and item number: Youkang, NC0106) to reduce the digestion effect. Transfer the cell suspension to a centrifuge tube, discard the supernatant after centrifugation, inoculate in a cell culture bottle, shake well and place in 37°C, 5% CO 2 Culture in an incubator.

[0113] Human Mesenchymal Stem Cell Harvest and Cryopreservation

[0114] Resuspend the cell pellet after centrifugation in the pre-prepared stem cell freezing solution, place it in a programmed cooling box after aliquoting, store it at -80℃ overnight, and transfer it to liquid nitrogen for long-term storage the next day, or store it at -80℃ for long-term storage.

[0115] The following examples are used to further illustrate the advantages and features of the present method, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0116] The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. Unless otherwise specified, the quantitative analysis experiments in the following examples were repeated three times, and the results were averaged.

[0117] Example 1: Preparation of cryopreservation solutions with different components and different component concentrations, and measurement of their osmotic pressure and pH. 1.1 Screening of cryopreservation solution components and concentration ranges

[0118] Table 1. Screening component combinations and concentrations

[0119]

[0120] Table 2. Orthogonal permutation and combination method configuration table

[0121]

[0122]

[0123] After preparation, the osmotic pressure and pH value of each dose were measured. The freezing solution that met the osmotic pressure of 1800-2300mosm / kg and pH of 6.8-7.5 was the optimal freezing solution formula in the initial screening.

[0124] Table 3.34 Results of osmotic pressure and pH value of freezing solution

[0125]

[0126]

[0127] From the results in Table 3, it can be seen that F-1 / F-10 / F-11 / F-13 / F-16 / F-17 / F-20 / F-21 / F-24 / F-27 / F-31 / F-32 / F-33 / F-34 are the formula components that basically meet the osmotic pressure: 1800-2300mosm / kg, pH: 6.8-7.5. According to the data in Table 3, after preliminary screening of the osmotic pressure and pH of the mixed solution of 34 cryopreservation solution formula components, the F-1 / F-11 / F-16 / F-24 / F-27 / F-31 / F-32 / F-33 / F-34 cryopreservation solution formula was finally selected for further comparison and screening.

[0128] Example 2: Comparative example of the recovery of human umbilical cord mesenchymal stem cells frozen at -80°C for 2 weeks and 1 month using the cryopreservation solution formula F-1 / F-11 / F-16 / F-24 / F-27 / F-31 / F-32 / F-33 / F-34

[0129] 2.1 Cell recovery: Human umbilical cord mesenchymal stem cells MSC001 were recovered and inoculated into cell culture bottles. After shaking, they were placed in a 37°C, 5% CO 2 Culture in an incubator.

[0130] 2.2 MSC001 cell passaging: When the cell confluence reaches 80-90%, subculture is performed. First, discard the cell culture supernatant, wash the cells with an appropriate amount of DPBS, add TryPLE to each cell culture bottle and digest at room temperature for 2-3 minutes. After the cells fall off, add an appropriate amount of complete cell culture medium to reduce the digestion effect, blow 3-4 times to make the cells into a single cell state, and transfer the cell suspension to a centrifuge tube; take an appropriate amount of DPBS to wash the bottom of the bottle, transfer it to the same centrifuge tube, put it into a centrifuge at 500g for 5 minutes, discard the supernatant after centrifugation, inoculate it into a cell culture bottle, shake it well, and put it into 37℃, 5% CO 2 Culture in an incubator.

[0131] 2.3MSC001 cell harvesting and freezing: observe the cells about 3 days after inoculation, and when the cell fusion degree reaches 80%-90%, put the culture flask into the biosafety cabinet, aspirate the cell culture supernatant, gently wash it with an appropriate amount of DPBS, discard the DPBS, add an appropriate amount of TryPLE digestion solution and digest for 2-4 minutes, observe the cell digestion under a microscope, and when the adherent cells become round and bright, tap them to make them fall off. After the cells fall off, add an appropriate amount of complete cell culture medium to reduce the digestion effect, blow and beat 3-4 times to make the cells into a single cell state, transfer the cell suspension to a centrifuge tube, take an appropriate amount of DPBS to wash the bottom of the flask twice, collect them in the same centrifuge tube, and centrifuge them in a centrifuge at 500g for 5 minutes at room temperature.

[0132] 2.4 MSC001 cell cryopreservation: discard the supernatant immediately after centrifugation, carefully flick off the precipitate, add an appropriate volume of culture medium into the centrifuge tube and gently blow to resuspend the cells, count the cells, calculate the cell viability and the total number of live cells harvested based on the cell counting results, centrifuge the remaining cell suspension at 500g for 5 minutes to obtain the cell precipitate, use different self-prepared cryopreservation solutions (F-1, F-11, F-16, F-24, F-27, F-31, F-32, F-33, F-34) and D10 cryopreservation solution (commercial cell cryopreservation solution, BI, catalog number 05-714-1A) to resuspend the cells according to the cell amount and the required cryopreservation quantity, adjust the cell density to 1E7 / mL, cryopreserve in cryopreservation tubes, each tube is 0.5mL, set up at least 6 replicate tubes for each dose and D10 group, store in a programmed cooling box at -80℃ overnight, and store at -80℃ for a long time.

[0133] 2.5 Recovery test: Recover three samples of cryopreserved solutions with different storage conditions 2 weeks and 1 month after cryopreservation for cell counting. Groups with cell viability below 80% do not need to be inoculated and cultured. Groups with cell viability above 80% should be counted at 10,000 ± 2,000 cells / cm 2 Inoculate one T75 and culture for 72 hours.

[0134] 2.6 Results of cryopreservation and thawing of MSC001 using different formulas of stem cell cryopreservation solutions Figure 1-4 shown.

[0135] according to Figure 1 and Figure 2 , when MSC001 was frozen at -80℃ for 2 weeks, the total number of viable cells after revival in each cryopreservation solution group was above 3.00E6, and the cell viability was above 90% except for F-34; when MSC001F was frozen for 2 weeks and then revived and cultured for 72 hours, the total number of viable cells and cell viability after revival in the cryopreservation solution formulas F-1 / F-11 / F-16 / F-24 / F-32 were relatively high;

[0136] according to Figure 3 and Figure 4 When MSC001 was frozen at -80℃ for one month, the total number of viable cells after revival in each cryopreservation solution group was above 3.00E6, and the cell viability was above 90% except for F-34; when MSC001F was frozen for one month and then revive and cultured for 72 hours, the total number of viable cells and cell viability after revival in F-1 / F-11 / F-16 / F-24 cryopreservation solution formulas were relatively high.

[0137] Example 3: Comparative example of the recovery of human umbilical cord mesenchymal stem cells frozen in liquid nitrogen for 2 weeks and 1 month using the cryopreservation solution formula F-1 / F-11 / F-16 / F-27 / F-31 / F-32 / F-33 / F-34

[0138] 3.1 Cell recovery: Human umbilical cord mesenchymal stem cells MSC001 were recovered and inoculated into cell culture bottles. After shaking, they were placed in a 37°C, 5% CO 2 Culture in an incubator.

[0139] 3.2 MSC001 cell passaging: When the cell confluence reaches 80-90%, subculture is performed. First, discard the cell culture supernatant, wash the cells with an appropriate amount of DPBS, add TryPLE to each cell culture bottle and digest at room temperature for 2-3 minutes. After the cells fall off, add an appropriate amount of complete cell culture medium to reduce the digestion effect, blow 3-4 times to make the cells into a single cell state, and transfer the cell suspension to a centrifuge tube; take an appropriate amount of DPBS to wash the bottom of the bottle, transfer it to the same centrifuge tube, put it into a centrifuge at 500g for 5 minutes, discard the supernatant after centrifugation, inoculate it into a cell culture bottle, shake it well, and put it into 37℃, 5% CO 2 Culture in an incubator.

[0140] 3.3 MSC001 cell harvesting and freezing: observe the cells about 3 days after inoculation, and when the cell fusion degree reaches 80%-90%, put the culture flask into the biosafety cabinet, aspirate the cell culture supernatant, gently wash it with an appropriate amount of DPBS, discard the DPBS, add an appropriate amount of TryPLE digestion solution and digest for 2-4 minutes, observe the cell digestion under a microscope, and when the adherent cells become round and bright, tap them to make them fall off. After the cells fall off, add an appropriate amount of complete cell culture medium to reduce the digestion effect, blow and beat 3-4 times to make the cells into a single cell state, transfer the cell suspension to a centrifuge tube, take an appropriate amount of DPBS to wash the bottom of the flask twice, collect them in the same centrifuge tube, and centrifuge them in a centrifuge at 500g for 5 minutes at room temperature.

[0141] 3.4 MSC001 cell cryopreservation: Immediately discard the supernatant after centrifugation, carefully flick off the precipitate, add an appropriate volume of culture medium into the centrifuge tube and gently blow to resuspend the cells, perform cell counting, calculate the cell viability and the total number of live cells harvested based on the cell counting results, centrifuge the remaining cell suspension at 500g for 5 minutes to obtain a cell pellet, resuspend the cells in different cryopreservation solutions (F-1, F-11, F-16, F-27, F-31, F-32, F-33, F-34) and D10 cryopreservation solution (commercial cell cryopreservation solution, BI, catalog number 05-714-1A) according to the cell amount and the required cryopreservation quantity, adjust the cell density to 1E7 / mL, and cryopreserve in cryopreservation tubes, each with 0.5mL. Set up at least 6 replicate tubes for each dose and D10 group, store in a programmed cooling box at -80℃ overnight, and store in liquid nitrogen for a long time.

[0142] 3.5 Recovery test: Recover cells from three different cryopreservation solutions at 2 weeks and 1 month after cryopreservation and count the cells. Groups with cell viability below 80% do not need to be cultured. Groups with cell viability above 80% should be cultured at 12,000 cells / cm 2 Inoculate one T75 and culture for 72 hours.

[0143] 3.6 The results of long-term storage and resuscitation of MSC001 in liquid nitrogen after cryopreservation using different cryopreservation solutions are as follows Figure 5-8 shown.

[0144] according to Figure 5 and Figure 6 After MSC001 was frozen in liquid nitrogen for 2 weeks, the total number of living cells of MS-C001 revived after being frozen in each cryopreservation solution group with F-1 / F-11 / F-16 / F-24 / F-27 / F-32 cryopreservation solution formula was above 3.0E6, and the cell viability was above 90%; after 72 hours of culture, the total number of living cells in each cryopreservation solution formula group was higher than 4.0E6, and the cell viability was higher than 95% except for F-31.

[0145] according to Figure 7 and Figure 8 When MSC001 was frozen in liquid nitrogen for one month, the total number of viable cells in each cryopreservation solution group after thawing was above 3.00E6, and the cell viability was above 90% except for F-34; after thawing for 72 hours, the viability of MSC001 cells thawing after thawing in F-1 / F-11 / F-16 / F-27 / F-32 / F-33 cryopreservation solution formulas was higher than 80%, and the total number of viable cells was higher than that of other groups.

[0146] Example 4: Comparative example of human umbilical cord mesenchymal stem cells frozen in liquid nitrogen and -80°C for 2 weeks and 1 month after recovery. Based on the above Examples 2-3 and the components of the cryopreservation solution formula, the F-11, F-16, and F-24 cryopreservation solution formula groups and D10 cryopreservation solution (commercial cell cryopreservation solution, BI, product number 05-714-1A) were selected for further comparative screening under liquid nitrogen and -80°C conditions, and recovered after 2 weeks and 1 month.

[0147] 4.1 Cell recovery: Human umbilical cord mesenchymal stem cells MSC006 were recovered and inoculated into a cell culture bottle. After shaking, they were placed in a 37°C, 5% CO 2 Culture in an incubator.

[0148] 4.2 MSC006 cell passaging: When the cell confluence reaches 80-90%, subculture is performed. First, discard the cell culture supernatant, wash the cells with an appropriate amount of DPBS, add TryPLE to each cell culture bottle and digest at room temperature for 2-3 minutes. After the cells fall off, add an appropriate amount of complete cell culture medium to reduce the digestion effect, blow 3-4 times to make the cells into a single cell state, and transfer the cell suspension to a centrifuge tube; take an appropriate amount of DPBS to wash the bottom of the bottle, transfer it to the same centrifuge tube, put it into a centrifuge at 500g for 5 minutes, discard the supernatant after centrifugation, inoculate it into a cell culture bottle, shake it well, and put it in 37℃, 5% CO 2 Culture in an incubator.

[0149] 4.3 Harvesting and freezing of MSC006 cells: observe the cells about 3 days after inoculation, and when the cell fusion degree reaches 80%-90%, put the culture flask into the biosafety cabinet, aspirate and discard the cell culture supernatant, gently wash it with an appropriate amount of DPBS, discard the DPBS, add an appropriate amount of TryPLE digestion solution and digest for 2-4 minutes, observe the cell digestion under a microscope, and when the adherent cells become round and bright, tap them to make them fall off. After the cells fall off, add an appropriate amount of complete cell culture medium to reduce the digestion effect, blow and beat 3-4 times to make the cells into a single cell state, transfer the cell suspension to a centrifuge tube, take an appropriate amount of DPBS to wash the bottom of the flask twice, collect them in the same centrifuge tube, and centrifuge them in a centrifuge at 500g for 5 minutes at room temperature.

[0150] 4.4 MSC006 cell cryopreservation: discard the supernatant immediately after centrifugation, carefully flick off the precipitate, add an appropriate volume of culture medium into the centrifuge tube and gently blow to resuspend the cells, count the cells, calculate the cell viability and the total number of live cells harvested based on the cell counting results, centrifuge the remaining cell suspension at 500g for 5 minutes to obtain the cell precipitate, resuspend the cells in different cryopreservation solutions (F-11, F-16, F-24) and D10 cryopreservation solution (commercial cell cryopreservation solution, BI, catalog number 05-714-1A) according to the cell amount and the required cryopreservation quantity, adjust the cell density to 1E7 / mL, and cryopreserve in cryopreservation tubes, each with 0.5mL. Set up at least 6 replicate tubes for each dose and D10 group, store in a programmed cooling box at -80℃ overnight, and store in liquid nitrogen for a long time.

[0151] 4.5 Recovery test: Recover cells from three different cryopreservation solutions at 2 weeks and 1 month after cryopreservation and count the cells. Groups with cell viability below 80% do not need to be cultured. Groups with cell viability above 80% should be cultured at 12,000 cells / cm 2 Inoculate one T75 and culture for 72 hours.

[0152] 4.6 Results of MSC006 cryopreservation with different cryopreservation solutions Figure 9-12 shown.

[0153] from Fig. 9 It can be seen that the total number of live cells and cell viability of MSC006 cells frozen in F-16 cryopreservation solution (MSC006F-16 for short) were the highest when they were frozen in -80℃ for 2 weeks; the total number of live cells and cell viability of MSC006 cells frozen in F-24 cryopreservation solution (MSC006F-24 for short) were the highest when they were frozen in -80℃ for 1 month. Fig.10 It can be seen that the total number of live cells and cell viability of MSC006F-16 cells after being frozen at -80℃ for 2 weeks and 1 month and then thawed for 72 hours were the highest. Fig.11 It can be seen that the total number of live cells and cell viability of MSC006F-16 cells were the highest when they were frozen in liquid nitrogen for 2 weeks; when they were frozen in liquid nitrogen for 1 month, the total number of live cells of F-24 cells was the highest and the cell viability of F-16 cells was the highest. Fig.12 It can be seen that the total number of viable cells of MSC006F-24 cells was the highest when they were frozen in liquid nitrogen for 2 weeks, followed by F-16; the total number of viable cells of MSC006F-16 cells was the highest when they were frozen in liquid nitrogen for 1 month;

[0154] MSC006F-24 cells had the highest cell viability.

[0155] Therefore, based on the overall results, F-16 is the best freezing solution formula.

[0156] Example 5: Human umbilical cord mesenchymal stem cells MSC001 were cryopreserved at -80°C for 2 weeks using stem cell cryopreservation solution and then revived

[0157] The following is intended to further illustrate the present invention in conjunction with specific embodiments:

[0158] 5.1 MSC001F-16 cells were frozen at -80°C for 2 weeks and then revived to detect cell viability, total number of living cells, cell apoptosis and sterility (the method is the same as in Example 2)

[0159] Table 4

[0160]

[0161] 5.2 After being frozen at -80℃ for 2 weeks, MSC001 cells were revived and their phenotype and purity were tested (as shown in the table below). After being frozen at -80℃ for 2 weeks, the expression rates of cell phenotype surface markers CD73 / CD90 / CD105 were not less than 95%, and the expression rates of cell purity phenotype markers HLA-DR+CD19+CD31+CD34+CD45+CD11b and CD33 / CD133 / CD14 were not higher than 2%, which met the requirements of the standard for the characteristics of stem cell surface markers.

[0162] Table 5

[0163]

[0164] 5.3 MSC001F-16 cells were cryopreserved at -80°C for 2 weeks and then revived and cultured for 72 hours.

[0165] Table 6

[0166]

[0167] Example 6: Human umbilical cord mesenchymal stem cells MSC001 were cryopreserved at -80°C for 3 months using stem cell cryopreservation solution and then revived

[0168] 6.1 MSC001 was cryopreserved at -80°C using the stem cell cryopreservation solution of dosage number F-16 for 3 months and then tested for cell viability, cell apoptosis and sterility (the method is the same as in Example 2)

[0169] Table 7

[0170]

[0171]

[0172] 6.2 The phenotype and purity of MSC001 cells frozen in F-16 cryopreservation solution at -80°C were tested 3 months after freezing (as shown in the following table). The expression rates of high expression CD73 / CD90 / CD105 were not less than 95%, and the expression rates of low expression HLA-DR+CD19+CD31+CD34+CD45+CD11b and CD33 / CD133 / CD14, cell purity phenotype markers, were not higher than 2%, which met the requirements of the standard for stem cell surface marker characteristics.

[0173] Table 8

[0174]

[0175] 6.3 MSC001 was cryopreserved at -80℃ using F-16 stem cell cryopreservation solution for 3 months and then revived and cultured for 72 hours

[0176] Table 9

[0177]

[0178] 6.4 MSC002 was frozen at -80℃ using F-16 and D10 cryopreservation solutions and then revived for 3 months to test its immune regulation ability. As shown in the table below, the results show that the same batch of cells frozen at -80℃ using F-16 stem cell cryopreservation solution were significantly better than the commercial cryopreservation solution D10 in terms of Th1 cell proliferation inhibition rate, Th17 cell proliferation inhibition rate, Treg cell proliferation promotion rate and inhibition of TNF-α secretion by mononuclear cells. This indicates that F-16 stem cell cryopreservation solution is suitable for freezing cells at -80℃ and can significantly enhance the immune regulation ability of cells.

[0179] Table 10

[0180]

[0181]

[0182] Example 7: Human umbilical cord mesenchymal stem cells MSC001 were cryopreserved in liquid nitrogen using stem cell cryopreservation solution for 2 weeks and then revived

[0183] 7.1 MSC001 cells were cryopreserved in liquid nitrogen using F-16 cryopreservation solution for 2 weeks and then tested for cell viability, apoptosis and sterility (the method is the same as in Example 3)

[0184] Table 11

[0185]

[0186] 7.2 After MSC001 cells were cryopreserved in F-16 stem cell cryopreservation medium under liquid nitrogen conditions for 2 weeks, their phenotype and purity were detected (as shown in the following table). The expression rates of high expression of CD73 / CD90 / CD105 were not less than 95%, and the expression rates of low expression of cell purity phenotype markers HLA-DR+CD19+CD31+CD34+CD45+CD11b and CD33 / CD133 / CD14 were not higher than 2%, which met the requirements of the standard for the characteristics of stem cell surface markers.

[0187] Table 12

[0188]

[0189] 7.3 MSC001 was cryopreserved in liquid nitrogen using F-16 stem cell cryopreservation solution for 2 weeks and then revived and cultured for 72 hours

[0190] Table 13

[0191]

[0192]

[0193] Example 8: Human umbilical cord mesenchymal stem cells MSC001 were cryopreserved in liquid nitrogen using stem cell cryopreservation solution for 3 months and then revived (the method is the same as that of Example 3)

[0194] 8.1 MSC001 was cryopreserved in liquid nitrogen using F-16 stem cell cryopreservation solution for one month before testing cell viability, apoptosis and sterility

[0195] Table 14

[0196]

[0197] 8.2 After MSC001 was cryopreserved in liquid nitrogen using F-16 stem cell cryopreservation solution for 3 months, its phenotype and purity were tested (as shown in the following table). The expression rates of high expression of CD73 / CD90 / CD105 were not less than 95%, and the expression rates of low expression of cell purity phenotype markers HLA-DR+CD19+CD31+CD34+CD45+CD11b and CD33 / CD133 / CD14 were not higher than 2%, which met the requirements of the standard for the characteristics of stem cell surface markers.

[0198] Table 15

[0199]

[0200] 8.3 MSC001 was cryopreserved in liquid nitrogen using F-16 stem cell cryopreservation solution for 3 months and then revived and cultured for 72 hours

[0201] Table 16

[0202]

[0203]

[0204] 8.4 MSC001 was frozen in liquid nitrogen conditions using F-16 and D10 cryopreservation solutions and then revived for 3 months to test its immune regulation ability. As shown in the table below, the results show that the same batch of cells frozen in liquid nitrogen using F-16 stem cell cryopreservation solution was significantly better than the commercial cryopreservation solution D10 in terms of Th17 cell proliferation inhibition rate, Treg cell proliferation promotion rate and inhibition of TNF-α secretion by mononuclear cells, indicating that F-16 stem cell cryopreservation solution is also suitable for conventional liquid nitrogen conditions to freeze cells and can ensure the immune regulation ability of cells.

[0205] Table 17

[0206]

[0207] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0208] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. An injectable stem cell cryopreservation solution, characterized in that: Contains: human albumin, dimethyl sulfoxide, 1,2-propylene glycol, trehalose, hydroxyethyl starch and methylcellulose.

2. The injectable stem cell cryopreservation solution according to claim 1, characterized in that: The invention also comprises an electrolyte solution, wherein the electrolyte solution comprises sodium chloride, sodium gluconate, sodium acetate, potassium chloride and magnesium chloride. Preferably, the electrolyte solution comprises 5.26 g / L sodium chloride, 5.02 g / L sodium gluconate, 3.68 g / L sodium acetate, 0.37 g / L potassium chloride and 0.3 g / L magnesium chloride, with the remainder being water.

3. The injectable stem cell cryopreservation solution according to claim 1, characterized in that: Based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises: 3 to 5 wt% human albumin, 2 to 8 wt% dimethyl sulfoxide, 2 to 8 wt% 1,2-propylene glycol, 0.05 to 0.17 wt% trehalose, 1×10 -5 Up to 5×10 -5 wt% hydroxyethyl starch, and 1×10 -5 Up to 10×10 -5 wt% methylcellulose.

4. The injectable stem cell cryopreservation solution according to claim 1, characterized in that: Based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises: 3.5 to 4.5wt% human albumin, 4 to 6 wt% dimethyl sulfoxide, 4 to 6 wt% 1,2-propylene glycol, 0.06 to 0.1 wt% trehalose, 1×10 -5 Up to 2×10 -5 wt% hydroxyethyl starch, and 4×10 -5 Up to 6×10 -5 wt% methylcellulose.

5. The injectable stem cell cryopreservation solution according to claim 2, characterized in that: Based on the total mass of the injectable stem cell cryopreservation solution, the injectable stem cell cryopreservation solution comprises: 4wt% human albumin, 5wt% dimethyl sulfoxide, 5wt% 1,2-propylene glycol, 0.083wt% trehalose, 1×10 -5 wt% Hydroxyethyl starch, 5×10 -5 wt% methylcellulose, The balance is the electrolyte solution.

6. The injectable stem cell cryopreservation solution according to any one of claims 1 to 5, characterized in that: The stem cells are mesenchymal stem cells; preferably, the stem cells are mesenchymal stem cells derived from humans.

7. The injectable stem cell cryopreservation solution according to claim 6, characterized in that: The mesenchymal stem cells are selected from the group consisting of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells, placental mesenchymal stem cells, amniotic membrane mesenchymal stem cells, amniotic fluid mesenchymal stem cells, menstrual blood mesenchymal stem cells, peripheral blood mesenchymal stem cells, dental pulp mesenchymal stem cells and skin mesenchymal stem cells.

8. The method for preparing an injectable stem cell cryopreservation solution according to any one of claims 1 to 7, characterized in that: include: Dissolving trehalose, hydroxyethyl starch and methyl cellulose in electrolyte solution respectively and sterilizing the solution to obtain trehalose solution, hydroxyethyl starch solution and methyl cellulose solution; The trehalose solution, the hydroxyethyl starch solution, the methylcellulose solution, human serum albumin, 1,2-propylene glycol and dimethyl sulfoxide are added to the electrolyte solution, wherein the human serum albumin is added to the electrolyte solution first and the dimethyl sulfoxide is added to the electrolyte solution last.

9. An injectable stem cell system, characterized in that: The invention comprises the injectable stem cell cryopreservation solution according to any one of claims 1 to 7 and mesenchymal stem cells.

10. The injectable stem cell system according to claim 9, characterized in that: The injectable stem cell system contains 0.2×10 7 Up to 5×10 7 The mesenchymal stem cells contain cells / mL.

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