阅读说明：本技术 一种人脐带间充质干细胞注射液冻存液及其制备方法 (Human umbilical cord mesenchymal stem cell injection frozen stock solution and preparation method thereof ) 是由 朱灏 于 2021-01-28 设计创作，主要内容包括：本发明涉及冻存液技术领域,具体涉及一种人脐带间充质干细胞注射液冻存液及其制备方法。一种人脐带间充质干细胞注射液冻存液,其制备原料至少包括以下组分：人血白蛋白、二甲基亚砜、复方电解质注射液、氯化钠注射液、葡萄糖注射液。本发明提供的人脐带间充质干细胞注射液冻存液,取材方便,不违反伦理问题,对细胞的毒副作用小适合临床使用,明显提高细胞回输对人体的安全性和生物相容性,可以在降低DMSO用量的前提下,提高脐带间充质干细胞活率和增殖、分化能力,且复苏后直接回输,无需进行离心洗涤,减少对细胞的损伤。(The invention relates to the technical field of frozen stock solutions, and particularly relates to a human umbilical cord mesenchymal stem cell injection frozen stock solution and a preparation method thereof. A frozen stock solution of human umbilical cord mesenchymal stem cell injection is prepared from at least the following components: human serum albumin, dimethyl sulfoxide, compound electrolyte injection, sodium chloride injection and glucose injection. The human umbilical cord mesenchymal stem cell injection cryopreservation solution provided by the invention is convenient to obtain materials, does not violate ethical problems, has small toxic and side effects on cells, is suitable for clinical use, obviously improves the safety and biocompatibility of cell reinfusion on a human body, can improve the survival rate and the proliferation and differentiation capacities of umbilical cord mesenchymal stem cells on the premise of reducing the dosage of DMSO (dimethyl sulfoxide), is directly reinfused after recovery, does not need centrifugal washing, and reduces the damage to the cells.)

1. The frozen stock solution of the human umbilical cord mesenchymal stem cell injection is characterized by comprising the following raw materials in parts by weight: human serum albumin, dimethyl sulfoxide, compound electrolyte injection, sodium chloride injection and glucose injection.

2. The human umbilical cord mesenchymal stem cell injection cryopreservation liquid as claimed in claim 1, wherein the raw materials for preparing the cryopreservation liquid at least comprise 20-50% of human serum albumin, 2-5% of dimethyl sulfoxide, 40-60% of compound electrolyte injection, 1-10% of sodium chloride injection and 1-10% of glucose injection by volume percentage.

3. The human umbilical cord mesenchymal stem cell injection cryopreservation liquid as claimed in claim 2, further comprising 5-15% dextran.

4. The human umbilical cord mesenchymal stem cell injection cryopreservation solution as claimed in claim 3, wherein the dextran is dextran-40 and/or dextran-70.

5. The human umbilical cord mesenchymal stem cell injection cryopreservation solution as claimed in claim 4, wherein the dextran-40 is dextran-40 glucose injection.

6. The human umbilical cord mesenchymal stem cell injection cryopreservation liquid as claimed in claim 3, wherein the volume ratio of the dimethyl sulfoxide to the dextran is 1: (1-3).

7. The human umbilical cord mesenchymal stem cell injection cryopreservation liquid as claimed in claim 1, wherein the mass concentration of the glucose injection is 10%; the mass concentration of the sodium chloride injection is 0.9%.

8. The human umbilical cord mesenchymal stem cell injection cryopreservation liquid as claimed in claim 7, wherein the volume ratio of the glucose injection to the dextran is 1: (1-10).

9. The preparation method of the human umbilical cord mesenchymal stem cell injection cryopreservation liquid as claimed in claim 1, comprising the following steps: adding pre-cooled glucose injection, sodium chloride injection and compound electrolyte injection into dimethyl sulfoxide, and mixing uniformly; and then adding pre-cooled human serum albumin, and uniformly mixing to obtain the product.

10. The method for preparing the human umbilical cord mesenchymal stem cell injection cryopreservation solution as claimed in claim 9, wherein the pre-cooling is performed at 2-8 ℃ for at least 30 min.

Technical Field

The invention relates to the technical field of frozen stock solutions, and particularly relates to a human umbilical cord mesenchymal stem cell injection frozen stock solution and a preparation method thereof.

Background

The human umbilical cord mesenchymal stem cell is a pluripotent stem cell separated from an umbilical cord, has obvious clinical application advantages compared with mesenchymal stem cells from other sources, such as easy material taking, easy collection, freezing storage, relative purity and the like, has higher differentiation potential, can be differentiated in multiple directions, such as osteoblasts, chondrocytes, heparinoid cells, insulin-like cells, nerve cells, myocardial cells and the like, and has no ethical problem. At present, human umbilical cord mesenchymal stem cells have been applied in the clinical field and have achieved encouraging therapeutic effects.

The seed bank of the human umbilical cord mesenchymal stem cells is widely applied clinically, but the seed bank has the problem that the seed bank cannot be applied in a large scale due to the limitation of freezing conditions, no exogenous pollution is generated in the freezing process of the stem cells, which is the key for the safety and reliability of the human umbilical cord mesenchymal stem cells in clinical treatment application, on one hand, the fetal bovine serum in the conventional freezing solution contains various undefined cell growth factors and can interfere the differentiation of the cells, on the other hand, the stem cells cultured by animal serum are clinically applied and can cause the cross infection of pathogens, so the human umbilical cord mesenchymal stem cell serum-free freezing solution with clear components needs to be developed.

The long-term activity of the human umbilical cord mesenchymal stem cell seed bank cells is closely related to the freezing environment, the microenvironment of the freezing solution has important influence on the human umbilical cord mesenchymal stem cells, and the existing freezing solution has larger toxic effect on the cells. Therefore, there is an urgent need for a cryopreservation solution that can prevent cell damage during cryopreservation and maintain high viability and functionality of cells after recovery.

Disclosure of Invention

In order to solve the technical problems, the invention provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection in a first aspect, and the frozen stock solution is prepared from at least the following components: human serum albumin, dimethyl sulfoxide, compound electrolyte injection, sodium chloride injection and glucose injection.

As a preferred technical scheme of the invention, the preparation raw materials of the frozen stock solution at least comprise 20-50% of human serum albumin, 2-5% of dimethyl sulfoxide, 40-60% of compound electrolyte injection, 1-10% of sodium chloride injection and 1-10% of glucose injection by volume percentage.

As a preferable technical scheme of the invention, the dextran also comprises 5-15 percent of dextran.

As a preferable technical scheme of the invention, the dextran is dextran-40 and/or dextran-70.

As a preferable technical scheme of the invention, the dextran-40 is dextran-40 glucose injection.

As a preferable technical scheme of the invention, the volume ratio of the dimethyl sulfoxide to the dextran is 1: (1-3).

As a preferable technical scheme of the invention, the mass concentration of the glucose injection is 10 percent; the mass concentration of the sodium chloride injection is 0.9%.

As a preferable technical scheme of the invention, the volume ratio of the glucose injection to the dextran is 1: (1-10).

The invention provides a preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution in a second aspect, which comprises the following steps: adding pre-cooled glucose injection, sodium chloride injection and compound electrolyte injection into dimethyl sulfoxide, and mixing uniformly; and then adding pre-cooled human serum albumin, and uniformly mixing to obtain the product.

As a preferable technical scheme of the invention, the precooling is carried out for at least 30min at the temperature of 2-8 ℃.

Advantageous effects

The invention provides a human umbilical cord mesenchymal stem cell injection cryopreservation solution which is convenient to obtain materials, does not violate ethical problems, has small toxic and side effects on human umbilical cord mesenchymal stem cells, can be directly clinically used, obviously improves the safety and biocompatibility of human umbilical cord mesenchymal stem cell reinfusion on human bodies, can improve the survival rate, proliferation and differentiation capacity of human umbilical cord mesenchymal stem cells on the premise of reducing the dosage of DMSO (dimethyl sulfoxide), directly reinfusion after recovery, does not need centrifugal washing, and reduces the damage to cells.

Detailed Description

The disclosure may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the examples included therein. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

The term "prepared from …" as used herein is synonymous with "comprising". The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

The conjunction "consisting of …" excludes any unspecified elements, steps or components. If used in a claim, the phrase is intended to claim as closed, meaning that it does not contain materials other than those described, except for the conventional impurities associated therewith. When the phrase "consisting of …" appears in a clause of the subject matter of the claims rather than immediately after the subject matter, it defines only the elements described in the clause; other elements are not excluded from the claims as a whole.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "optional" or "any" means that the subsequently described event or events may or may not occur, and that the description includes instances where the event occurs and instances where it does not.

Approximating language, as used herein throughout the specification and claims, is intended to modify a quantity, such that the invention is not limited to the specific quantity, but includes portions that are literally received for modification without substantial change in the basic function to which the invention is related. Accordingly, the use of "about" to modify a numerical value means that the invention is not limited to the precise value. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. In the present description and claims, range limitations may be combined and/or interchanged, including all sub-ranges contained therein if not otherwise stated.

The invention provides a human umbilical cord mesenchymal stem cell injection frozen stock solution in a first aspect, wherein the frozen stock solution at least comprises the following components in parts by weight: human serum albumin, dimethyl sulfoxide, compound electrolyte injection, sodium chloride injection and glucose injection.

In a preferred embodiment, the preparation raw materials of the frozen stock solution at least comprise 20-50% of human serum albumin, 2-5% of dimethyl sulfoxide, 40-60% of compound electrolyte injection, 1-10% of sodium chloride injection and 1-10% of glucose injection by volume percentage.

In a more preferred embodiment, the raw material for preparing the frozen stock solution further comprises 5-15% of dextran.

Human serum albumin

In the present invention, the volume percentage of the human serum albumin in the frozen stock solution is 20-50%, and 20%, 25%, 30%, 35%, 40%, 45%, 50%, etc. can be mentioned.

In the invention, the human serum albumin is 20% human serum albumin.

The inventor believes that the addition of a proper amount of human serum albumin in the system does not cause anaphylactic reaction as non-human components such as bovine serum (FBS), has a strong protective effect on cells, can promote the growth of the cells, prolong the survival time of the cells, increase blood volume and maintain osmotic pressure, adjust the dynamic balance of water between tissues and blood vessels, maintain relatively stable osmotic pressure of the cells, ensure the safety of human umbilical cord mesenchymal stem cells in the cryopreservation process, and can be used as a nitrogen source to provide nutrition for the tissues. However, the content of human albumin cannot exceed the range of 20-50%, otherwise, the recovery of human umbilical cord mesenchymal stem cells from the frozen state is not facilitated.

Dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) is a sulfur-containing organic compound with molecular formula of C₂H₆OS, a colorless and odorless transparent liquid at room temperatureIt is a hygroscopic combustible liquid. Has the characteristics of high polarity, high boiling point, good thermal stability, non-proton and water miscibility, can be dissolved in most organic substances such as ethanol, propanol, benzene, chloroform and the like, and is known as an 'universal solvent'.

In the invention, the volume percentage of the dimethyl sulfoxide in the frozen stock solution is 2-5%.

In a preferred embodiment, the volume percentage of the dimethyl sulfoxide in the frozen stock solution is 5%.

The technical scheme of the invention is carried out under the condition of 5 percent of dimethyl sulfoxide.

The inventor believes that in the cryopreservation solution provided by the invention, DMSO easily penetrates cells, so that the freezing point can be lowered in the process of cell cryopreservation, the chance of forming ice crystals in the cells is reduced, and the damage of the ice crystals to the cells is reduced. However, the commonly used dimethyl sulfoxide has certain toxicity to cells, and researches show that the dimethyl sulfoxide can be combined with proteins to cause protein denaturation, thereby causing damage. Therefore, the invention obtains a new frozen stock solution after research on the basis, and the content of dimethyl sulfoxide can be greatly reduced. The preferred human albumin can replace serum to further protect cells and reduce the toxicity of dimethyl sulfoxide to the cells, and can also provide certain nutrition to ensure that the cells still maintain better activity after fast melting. In addition, the risk of virus infection caused by the addition of serum is avoided, the most preferable human albumin is 20% human albumin, and the safety and biocompatibility of cell reinfusion to human bodies can be obviously improved.

Compound electrolyte injection

The product is compatible with blood and blood components, and can be administered by infusion (i.e., as a priming solution) either before or after transfusion using the same administration set, either added to the blood component being infused, or as a diluent for the blood cells.

In the invention, the volume percentage of the compound electrolyte injection in the frozen stock solution is 40-60%, and 40%, 45%, 50%, 55%, 60% and the like can be mentioned.

The inventor thinks that the system has the double advantages of the cell cryopreservation protective solution and the electrolyte injection, can protect cells in the low-temperature cryopreservation process of below 130 ℃ below zero, can be directly used as the electrolyte injection to be infused back to a human body after being thawed, and does not need to pass through an additional treatment process, thereby reducing the operation flow and the pollution probability; meanwhile, the method can reduce the problem of electrolyte disturbance during and after the return transfusion, and can be used as a supplement source of water and electrolyte and an alkalizer.

Sodium chloride injection

Sodium chloride injection, water loss due to various causes including hypotonic, isotonic, and hypertonic water loss; hypertonic nonketotic diabetic coma, use isotonic or hypotonic sodium chloride can correct the state of water loss and hypertonicity; low chloride metabolic alkalosis; washing eyes, wounds and the like with physiological saline; it is also used for induced labor of water bag in obstetrical department.

In the invention, the volume percentage of the sodium chloride injection in the frozen stock solution is 1-10%, and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% and the like can be mentioned.

In a preferred embodiment, the mass fraction of the sodium chloride injection is 0.9%.

The inventor finds that the system generates a synergistic effect after the compound electrolyte injection and the sodium chloride injection are compounded, can further maintain the system balance of the ionic osmotic pressure and the colloid osmotic pressure, forms an environmental condition beneficial to the survival of the human umbilical cord mesenchymal stem cells, can be used for the long-term storage of the human umbilical cord mesenchymal stem cells, can be used for human body reinfusion after cryopreservation, and is more convenient and flexible to use.

Glucose injection

Glucose injection, with indication 1, energy and body fluid supplement; can be used for treating anorexia or large amount of body fluid loss (such as emesis, diarrhea, etc.), intravenous nutrition, and hunger ketosis. 2. Hypoglycemia disease; 3. hyperkalemia; 4. hypertonic solutions are used as tissue dehydrating agents; 5. preparing peritoneal dialysis solution; 6. a pharmaceutical diluent; 7. an intravenous glucose tolerance test; 8. for preparing GIK (polarizing solution).

In the present invention, the volume percentage of the glucose injection in the frozen stock solution is 1-10%, and 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. can be mentioned.

In a preferred embodiment, the glucose injection has a mass fraction of 10%.

The inventor finds that the glucose can be used as a freezing protective agent to reduce the formation of ice crystals and the osmotic contraction of cells during freezing, the glucose can provide parenteral nutrition and supplement energy during reinfusion, and the sodium chloride can supplement electrolytes; after the dextran glucose injection is compounded with the dextran glucose injection for use, microcirculation can be improved during reinfusion, the dextran glucose injection has the effect of expanding blood volume, and the problems of erythrocyte aggregation, thrombosis and the like in blood vessels can be prevented or eliminated.

Dextran

In the invention, the dextran accounts for 5-15% by volume in the frozen stock solution, and 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% and the like can be mentioned.

In a preferred embodiment, the dextran is dextran-40 and/or dextran-70.

In a more preferred embodiment, the dextran-40 is a dextran-40 glucose injection solution.

In a more preferred embodiment, the dextran 40 has a weight average molecular weight of between 32000 and 42000.

In the invention, the volume ratio of the dimethyl sulfoxide to the dextran is 1: (1-3).

In the system, in order to reduce the damage of temperature reduction of stem cells during cryopreservation, the adopted method is mainly to increase the percentage content of dextran 40 glucose injection; on one hand, from the view of the action mode of the cell cryopreservation protective agent, DMSO is used as a permeable reagent and can permeate into cells to enable a large amount of water to permeate out; the dextran 40 glucose injection is used as an impermeable reagent to form hypertonicity outside cells so as to dehydrate the cells, thereby protecting the cells from being damaged by the formation of ice crystals; the system adopts a mode of compounding the two, particularly when the weight average molecular weight of the adopted dextran 40 is 32000-: (1-3), osmotic pressure and pH can be maintained, an environment suitable for survival of the human umbilical cord mesenchymal stem cells is provided, a better cryopreservation effect is obtained, functional indexes and biological functions of the human umbilical cord mesenchymal stem cells are maintained, and the survival rate, proliferation and differentiation capacity of the umbilical cord mesenchymal stem cells are improved; on the other hand, because DMSO has toxicity to tissue cells at normal temperature, the DMSO is reduced, so that the damage to the cells at normal temperature is reduced, the cell viability is improved, and clinical reinfusion is facilitated.

In the present invention, the source of the raw material for preparing the frozen stock solution is not particularly limited, and commercially available frozen stock solutions are suitable for use in the present invention. For example, the human serum albumin is from Octagema AB; the dimethyl sulfoxide is clinical-grade dimethyl sulfoxide and is sourced from WAK-Chemie company; the compound electrolyte injection is from Hebei Tian Cheng pharmaceutical industry GmbH; the dextran 40 is from Sichuan Koran pharmaceutical industry Co., Ltd; the 10% glucose injection is from the national health management corporation; the sodium chloride injection is from Tsukamur Guangdong pharmaceutical Co.

The invention provides a preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution in a second aspect, which comprises the following steps: adding pre-cooled glucose injection, sodium chloride injection and compound electrolyte injection into dimethyl sulfoxide, and mixing uniformly; and then adding pre-cooled human serum albumin, and uniformly mixing to obtain the product.

In a preferred embodiment, after the preparation of the frozen stock solution is finished, a pre-cooling step is further included.

In a preferred embodiment, the precooling is performed at 2-8 ℃ for at least 30 min.

In a most preferred embodiment, the preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding pre-cooled glucose injection, sodium chloride injection and compound electrolyte injection into dimethyl sulfoxide, and mixing uniformly; and then adding pre-cooled human serum albumin, uniformly mixing, and pre-cooling to obtain the product.

The inventor believes that the survival rate of the human umbilical cord mesenchymal stem cells can be improved after precooling other preparation raw materials except DMSO in the frozen stock solution. The inventors believe that a possible reason is that DMSO is severely cytotoxic at normal temperatures; at a deep low temperature, DMSO is a permeable protective agent, and can lower the cell freezing point, reduce the formation of ice crystals and reduce the damage to cells in the freezing storage process. In addition, although the DMSO content of the system is greatly reduced in the preparation process of the frozen stock solution, the pre-cooling of the frozen stock solution can reduce the cytotoxicity of DMSO to a certain extent and improve the survival rate of cells. Meanwhile, DMSO releases heat when meeting water, and precooling can reduce the influence of the reaction on the frozen stock solution after the preparation is finished.

The present invention will be specifically described below by way of examples. It should be noted that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention, and that the insubstantial modifications and adaptations of the present invention by those skilled in the art based on the above disclosure are still within the scope of the present invention.

In addition, the starting materials used are all commercially available, unless otherwise specified.

Examples

Example 1

The embodiment 1 provides a frozen stock solution of a human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials in percentage by volume: 5% of dimethyl sulfoxide, 56% of compound electrolyte injection, 8.5% of dextran-40 glucose injection, 3% of 0.9% sodium chloride injection, 2.5% of 10% glucose injection and 25% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 0.9% sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 2

Embodiment 2 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 40% of compound electrolyte injection, 5% of dextran-40 glucose injection, 1% of 0.9% sodium chloride injection, 1% of 10% glucose injection and 48% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 0.9% sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 3

Embodiment 3 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 60% of compound electrolyte injection, 10% of dextran-40 glucose injection, 2.5% of 0.9% sodium chloride injection, 2.5% of 10% glucose injection and 20% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 0.9% sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 4

Embodiment 4 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 56% of compound electrolyte injection, 8.5% of dextran-40 glucose injection, 3% of 0.9% sodium chloride injection, 2.5% of 10% glucose injection and 25% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 0.9% sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 5

Embodiment 5 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 10% of dimethyl sulfoxide, 56% of compound electrolyte injection, 8.5% of dextran-40 glucose injection, 3% of 0.9% sodium chloride injection, 2.5% of 10% glucose injection and 20% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 0.9% sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 6

Embodiment 6 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 56% of compound electrolyte injection, 3% of 0.9% sodium chloride injection, 2.5% of 10% glucose injection and 33.5% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 0.9% sodium chloride injection and compound electrolyte injection, and shaking while adding until uniformly mixed; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 7

Embodiment 7 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 56% of compound electrolyte injection, 8.5% of dextran-40 glucose injection, 2.5% of 10% glucose injection and 25% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 8

Embodiment 8 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 56% of compound electrolyte injection, 8.5% of dextran-40 glucose injection, 3% of 0.9% sodium chloride injection and 27.5% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding pre-cooled 0.9% sodium chloride injection, dextran-40 glucose injection, and compound electrolyte injection, and shaking while adding until mixing; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 9

Embodiment 9 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 56% of compound electrolyte injection, 8.5% of dextran-40 glucose injection, 3% of 0.9% sodium chloride injection, 2.5% of 50% glucose injection and 25% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 50% glucose injection, 0.9 sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 10

Embodiment 10 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 56% of compound electrolyte injection, 8.5% of dextran-40 glucose injection, 3% of 5% sodium chloride injection, 2.5% of 10% glucose injection and 25% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 5% sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 11

Embodiment 11 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 5% of dimethyl sulfoxide, 56% of compound electrolyte injection, 8.5% of dextran-40 glucose injection, 3% of 0.9% sodium chloride injection, 2.5% of 10% glucose injection and 25% of 20% human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding 10% glucose injection, 0.9 sodium chloride injection, dextran-40 glucose injection, and compound electrolyte injection, and shaking while adding until mixing; then slowly adding the precooled 20% human serum albumin, and evenly mixing the mixture by turning upside down to obtain the product.

Example 12

Embodiment 12 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 2.5 percent of dimethyl sulfoxide, 58.5 percent of compound electrolyte injection, 8.5 percent of dextran-40 glucose injection, 3 percent of 0.9 percent sodium chloride injection, 2.5 percent of 10 percent glucose injection and 25 percent of 20 percent human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 0.9% sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Example 13

Embodiment 13 provides a frozen stock solution of human umbilical cord mesenchymal stem cell injection, which comprises the following raw materials by volume percent: 7.5 percent of dimethyl sulfoxide, 53.5 percent of compound electrolyte injection, 8.5 percent of dextran-40 glucose injection, 3 percent of 0.9 percent sodium chloride injection, 2.5 percent of 10 percent glucose injection and 25 percent of 20 percent human serum albumin.

The preparation method of the human umbilical cord mesenchymal stem cell injection frozen stock solution comprises the following steps: adding DMSO into a 500mL receiver, slowly adding precooled 10% glucose injection, 0.9% sodium chloride injection, dextran-40 glucose injection and compound electrolyte injection, and shaking while adding until mixing uniformly; slowly adding pre-cooled 20% human serum albumin, turning upside down and uniformly mixing, and pre-cooling to obtain the product; the precooling is carried out for 50 minutes in a medical refrigerator at the temperature of 4 ℃.

Performance testing

1. Cell viability assay: taking a proper amount of cell suspension sample, adding AO/PI staining solution according to the proportion of 1:1, mixing uniformly, adding 20 mu l of the cell suspension sample into a Countstar cell counting plate, and carrying out cell counting by selecting an 'AO/PI' mode on a cell counting instrument. The same samples were counted in duplicate. And calculating the average cell viability rate according to the cell viability rate displayed by the counter.

The test results are shown in Table 1.

TABLE 1 results of the Performance test of examples

The foregoing examples are merely illustrative and serve to explain some of the features of the method of the present invention. The appended claims are intended to claim as broad a scope as is contemplated, and the examples presented herein are merely illustrative of selected implementations in accordance with all possible combinations of examples. Accordingly, it is applicants' intention that the appended claims are not to be limited by the choice of examples illustrating features of the invention. Also, where numerical ranges are used in the claims, subranges therein are included, and variations in these ranges are also to be construed as possible being covered by the appended claims.