Digestive juice for enzyme digestion method of mesenchymal stem cells and application of digestive juice

Abstract

The invention discloses digestive juice for a mesenchymal stem cell enzyme digestion method, which is prepared from the following components in percentage by mass on the basis of the total mass of the digestive juice: 0.1-0.5% of digestive enzyme, 0.1-0.5% of vitamin C, 0.1-0.5% of vitamin E, 0.1-0.5% of vitamin E, 0.1-0.5% of vitamin E, 0.1-0.5% of vitamin E, 0.1-0.5% of vitamin E, 0.1-0.5% of vitamin E and the balance of water. 1%-3% of human serum albumin; the content of the heparin sodium is 0.008%-0.02%; the kukoamine A accounts for 0.01 to 0.03 percent; the balance is 1 * HBSS. The invention also discloses an application of the digestive juice in a process of separating and culturing to obtain mesenchymal stem cells by an enzyme digestion method. The invention also discloses an application of the digestive juice in preparation of a medicine for separating and culturing mesenchymal stem cells by an enzyme digestion method. The invention also discloses application of the digestive juice in preparation of drugs for breeding and amplifying mesenchymal stem cells.

Description

Technical Field

[0001] The invention relates to the field of cell biology, and in particular to a digestion solution for a mesenchymal stem cell enzyme digestion method and an application thereof. Background Art

[0002] Mesenchymal stem cells are a type of adult stem cell with multidirectional differentiation potential, and have broad application prospects in tissue repair and regenerative medicine. Mesenchymal stem cells have all the common characteristics of stem cells, namely self-renewal and multidirectional differentiation capabilities. They are also the most widely used in clinical applications. When used in combination with hematopoietic stem cells, they can improve the success rate of transplantation and accelerate hematopoietic reconstruction. When patients receive high-dose chemotherapy, the infusion of mesenchymal stem cells together with hematopoietic stem cells can significantly accelerate the recovery time of patients' blood cells, and is safe and has no adverse reactions.

[0003] Mesenchymal stem cells exist not only in the bone marrow, but also in skeletal muscle, periosteum and trabecular bone. Because they can differentiate into a wide range of tissue types, they are of great clinical value.

[0004] Enzyme digestion is a commonly used method in the isolation and culture of mesenchymal stem cells. However, the enzyme digestion solutions used in the traditional mesenchymal stem cell enzymatic digestion method mainly include trypsin, collagenase, etc. These enzyme digestion solutions may have the following shortcomings during the digestion process:

[0005] (1) Excessive digestion of cell surface proteins, leading to decreased cell viability;

[0006] (2) Mechanical stress generated during digestion may also cause damage to cells;

[0007] (3) Chemical components in digestive juices may have toxic effects on cells, further affecting cell viability and function.

[0008] Therefore, developing a digestive fluid that can protect mesenchymal stem cells from damage during enzymatic digestion has become a technical problem that needs to be solved urgently. Summary of the invention

[0009] Purpose of the invention: In view of the above-mentioned deficiencies of the prior art, the present invention discloses a digestion solution for mesenchymal stem cell enzymatic digestion and its application. The digestion solution of the present invention can effectively digest cells while minimizing damage to mesenchymal stem cells and improving the activity and function of mesenchymal stem cells.

[0010] Technical solution: A digestion solution for mesenchymal stem cell enzymatic digestion method, which is composed of the following components in terms of mass percentage based on the total mass of the digestion solution for mesenchymal stem cell enzymatic digestion method:

[0011] Digestive enzymes account for 0.1% to 0.5%;

[0012] Human serum albumin accounts for 1% to 3%;

[0013] Heparin sodium accounts for 0.008% to 0.02%;

[0014] Lycopodiol accounts for 0.01% to 0.03%;

[0015] The remainder was 1× HBSS.

[0016] Furthermore, the enzyme activity of the digestion solution used for the mesenchymal stem cell enzymatic digestion method is at least 190U.

[0017] Furthermore, the enzyme activity of the digestion solution used for the mesenchymal stem cell enzymatic digestion method is 190U-220U.

[0018] Furthermore, the digestive enzyme consists of the following components in terms of mass percentage:

[0019] Type I collagenase accounts for 40-50%;

[0020] Type II collagenase accounts for 30-40%;

[0021] The remainder is type IV collagenase.

[0022] Furthermore, the enzyme activity of the heparin sodium is at least 80U.

[0023] Furthermore, the enzyme activity of the heparin sodium is 80U-100U.

[0024] The digestion solution described in any one of the above items is used as a digestion solution in the process of isolating and culturing mesenchymal stem cells by enzyme digestion.

[0025] Use of any of the above digestive solutions in the preparation of a drug for separating and culturing mesenchymal stem cells by enzyme digestion.

[0026] Use of any of the above-mentioned digestive fluids in the preparation of a drug for propagating and expanding mesenchymal stem cells.

[0027] The components used in the present invention are described below:

[0028] 1. Digestive enzymes are all mild enzyme ingredients: collagenase type I, collagenase type II and collagenase type IV are mainly used to decompose the collagen components in the extracellular matrix. Different types of collagenase have specific effects on different types of collagen, which can separate mesenchymal stem cells from the surrounding matrix, making it easier to separate and collect mesenchymal stem cells. By controlling the ratio of different types of collagenase, the extracellular matrix in a specific tissue or cell culture system can be digested more effectively, while reducing damage to cells.

[0029] 2. Human serum albumin: It can protect mesenchymal stem cells and form a protective barrier during the digestion process to prevent digestive enzymes from excessively damaging mesenchymal stem cells. It can also maintain osmotic pressure balance, provide a relatively stable osmotic pressure environment for mesenchymal stem cells, and prevent mesenchymal stem cells from swelling or shrinking. It can also bind and transport small molecules, regulate the concentration of some important substances in the microenvironment outside mesenchymal stem cells, and maintain normal metabolism and signal transduction of mesenchymal stem cells.

[0030] 3. Sodium heparin: It has an anticoagulant effect, preventing the digestive fluid from causing blood coagulation or thrombus formation during contact with mesenchymal stem cells, ensuring the smooth progress of the digestion process. During the culture of mesenchymal stem cells, especially in the stage of re-seeding after digestion of mesenchymal stem cells, cells are prone to aggregation. Sodium heparin can cover the surface of mesenchymal stem cells, producing a charge repulsion or steric hindrance effect, preventing mesenchymal stem cells from aggregating together due to charge attraction or protein-protein interaction. This effect helps to maintain the uniform distribution of mesenchymal stem cells in the culture system, effectively reduce the adhesion between mesenchymal stem cells, make the mesenchymal stem cells more evenly dispersed, and improve the quality and repeatability of mesenchymal stem cell culture.

[0031] 4. Lycopodiella citrate: It has antioxidant effects, removes reactive oxygen species produced during cell digestion, and reduces oxidative damage; it has anti-inflammatory effects, inhibits the activation of inflammatory signal molecules and the release of inflammatory factors; it also has the possibility of regulating the metabolism of mesenchymal stem cells, helping mesenchymal stem cells adapt to changes in energy metabolism after digestion; it can also protect mesenchymal stem cells, interact with mesenchymal stem cell receptors to activate protective signal pathways, reduce mesenchymal stem cell apoptosis, and increase cell survival rate after digestion, providing healthy cells for subsequent mesenchymal stem cell experiments or applications.

[0032] 5.1×HBSS: Provide a suitable ion environment for mesenchymal stem cells. Calcium and magnesium ions in 1×HBSS participate in physiological processes such as cell signal transduction and enzyme activity regulation. At the same time, it can maintain the physiological state of mesenchymal stem cells. As a buffer system, it ensures that mesenchymal stem cells are in a suitable environment, which is conducive to mesenchymal stem cells maintaining normal morphology and function, preventing mesenchymal stem cells from being damaged by changes in the external environment, and ensuring the stability of the environment around the cells during cell digestion, providing a good foundation for the separation and subsequent culture of mesenchymal stem cells.

[0033] Beneficial effects: The digestion solution for mesenchymal stem cell enzymatic digestion disclosed in the present invention and its application have the following beneficial effects:

[0034] 1. The combination of type I collagenase, type II collagenase and type IV collagenase can reduce the damage to mesenchymal stem cells while improving the vitality and function of mesenchymal stem cells. By controlling the ratio of different types of collagenase, specific tissues can be digested more effectively.

[0035] 2. Add protective ingredients, such as human serum albumin, sodium heparin, and lycopodiol to provide nutritional support for mesenchymal stem cells and reduce the damage to mesenchymal stem cells caused by oxidative stress and mechanical stress. In this digestive solution formula, the role played by sodium heparin and lycopodiol is unique. Sodium heparin mainly plays an anticoagulant role in the digestive solution. It binds to antithrombin III to enhance the inhibitory effect of antithrombin III on thrombin and other coagulation factors (such as factors Xa, IXa, XIa, etc.) to prevent blood coagulation. At the same time, it may also have a certain promoting effect on the adhesion and growth of mesenchymal stem cells, and can bind and regulate some growth factors to affect the proliferation and differentiation of mesenchymal stem cells; lycopodiol has antioxidant and anti-inflammatory effects, which can reduce the oxidative stress and inflammatory damage to mesenchymal stem cells during the digestion process. It may also have a regulatory effect on the metabolism and survival of mesenchymal stem cells, which helps to maintain the healthy state of mesenchymal stem cells. It is difficult for these two ingredients to be completely replaced by other equivalent substances.

[0036] 3. A good buffer system maintains appropriate pH and ion concentration to ensure enzyme activity and normal metabolism of mesenchymal stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a microscopic view of human mesenchymal stem cells after digesting a placental tissue sample using the digestion solution for the mesenchymal stem cell enzymatic digestion method of Example 3.

[0038] Figure 2 A microscope image of human mesenchymal stem cells after digestion of placental tissue using trypsin.

[0039] Figure 3 This is a schematic diagram of the proliferation of human mesenchymal stem cells under the action of different digestive fluids in Example 5.

[0040] Figure 4 Schematic diagram of the expression of surface markers of human mesenchymal stem cells cultured after the embryonic tissue sample was digested with the digestion solution for the mesenchymal stem cell enzyme digestion method in Example 3.

[0041] Figure 5A This is a graph showing the osteogenic induction staining result of human mesenchymal stem cells cultured after the embryonic tissue sample was digested with the digestion solution for the mesenchymal stem cell enzyme digestion method of Example 3.

[0042] Figure 5B This is the result of osteogenic induction staining of human mesenchymal stem cells after placental tissue samples were digested with trypsin digestion solution.

[0043] Fig. 6A This is a diagram showing the adipogenic induction staining result of human mesenchymal stem cells cultured after the embryonic tissue sample was digested with the digestion solution for the mesenchymal stem cell enzyme digestion method in Example 3.

[0044] Figure 6B This is the result of adipogenic induction staining of human mesenchymal stem cells after placental tissue samples were digested with trypsin digestion solution.

[0045] Fig. 7A This is a diagram showing the chondrogenic induction staining result of human mesenchymal stem cells cultured after the embryonic tissue sample was digested with the digestion solution for the mesenchymal stem cell enzyme digestion method in Example 3.

[0046] Figure 7B This is the result of chondrogenic induction staining of human mesenchymal stem cells after placental tissue samples were digested with trypsin digestion solution. DETAILED DESCRIPTION

[0047] The specific embodiments of the present invention are described in detail below.

[0048] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 10 to 50 is listed for a specific parameter, it is understood that the range of 10 to 40 and 20 to 50 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In this application, unless otherwise specified, the range of values ​​"a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the range of values ​​"0 to 5" means that all real numbers between "0 to 5" have been fully listed in this article, and "0 to 5" is only an abbreviation of these numerical combinations.

[0049] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0050] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0051] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0052] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0053] Unless otherwise specified, the reaction is carried out at room temperature and pressure.

[0054] Unless otherwise specified, all parts or percentages are by weight.

[0055] In the present invention, all substances used are known substances and can be purchased or synthesized by known methods.

[0056] In the present invention, the devices or equipment used are all conventional devices or equipment known in the art and are commercially available.

[0057] In the present invention, unless otherwise specified, the working concentration of the trypsin digestion solution used is: 0.25% trypsin and 0.02% EDTA dissolved in PBS buffer.

[0058] The digestion solution for the enzymatic digestion method of mesenchymal stem cells used was the digestion solution for the enzymatic digestion method of mesenchymal stem cells in Example 3.

[0059] Example 1

[0060] A digestion solution for mesenchymal stem cell enzymatic digestion method, which is composed of the following components in terms of mass percentage based on the total mass of the digestion solution for mesenchymal stem cell enzymatic digestion method:

[0061] Digestive enzymes account for 0.5%;

[0062] Human serum albumin accounts for 3%;

[0063] Heparin sodium accounts for 0.02%;

[0064] Lycopodiol accounts for 0.03%;

[0065] The remainder was 1× HBSS.

[0066] Furthermore, the enzyme activity of the digestion solution used for the mesenchymal stem cell enzymatic digestion method is 300U.

[0067] Furthermore, the digestive enzyme consists of the following components in terms of mass percentage:

[0068] Type I collagenase accounts for 40%;

[0069] Type II collagenase accounts for 30%;

[0070] The remainder is type IV collagenase.

[0071] Furthermore, the enzyme activity of the heparin sodium is 150 U. In other embodiments, the enzyme activity of the heparin sodium is 100 U.

[0072] The digestion solution described in any one of the above items is used as a digestion solution in the process of isolating and culturing mesenchymal stem cells by enzyme digestion.

[0073] Example 2

[0074] A digestion solution for mesenchymal stem cell enzymatic digestion method, which is composed of the following components in terms of mass percentage based on the total mass of the digestion solution for mesenchymal stem cell enzymatic digestion method:

[0075] Digestive enzymes account for 0.125%;

[0076] Human serum albumin 1%;

[0077] Heparin sodium accounts for 0.008%;

[0078] Lycopodiol accounts for 0.015%;

[0079] The remainder was 1× HBSS.

[0080] Furthermore, the enzyme activity of the digestion solution used for the mesenchymal stem cell enzymatic digestion method is 220U.

[0081] Furthermore, the digestive enzyme consists of the following components in terms of mass percentage:

[0082] Type I collagenase accounts for 45%;

[0083] Type II collagenase accounts for 35%;

[0084] The remainder is type IV collagenase.

[0085] Furthermore, the enzyme activity of the heparin sodium is 120 U. In other embodiments, the enzyme activity of the heparin sodium is 90 U.

[0086] The digestion solution described in any one of the above items is used as a digestion solution in the process of isolating and culturing mesenchymal stem cells by enzyme digestion.

[0087] Example 3

[0088] A digestion solution for mesenchymal stem cell enzymatic digestion method, which is composed of the following components in terms of mass percentage based on the total mass of the digestion solution for mesenchymal stem cell enzymatic digestion method:

[0089] Digestive enzymes account for 0.1%;

[0090] Human serum albumin accounts for 2%;

[0091] Heparin sodium accounts for 0.01%;

[0092] Lycopodiol accounts for 0.01%;

[0093] The remainder was 1× HBSS.

[0094] Further, the enzyme activity of the digestion solution used for the enzymatic digestion of mesenchymal stem cells is 190 U. In other embodiments, the enzyme activity of the digestion solution used for the enzymatic digestion of mesenchymal stem cells is 200 U.

[0095] Furthermore, the digestive enzyme consists of the following components in terms of mass percentage:

[0096] Type I collagenase accounts for 50%;

[0097] Type II collagenase accounts for 40%;

[0098] The remainder is type IV collagenase.

[0099] Furthermore, the enzyme activity of the heparin sodium is 100 U. In other embodiments, the enzyme activity of the heparin sodium is 80 U.

[0100] The digestion solution described in any one of the above items is used as a digestion solution in the process of isolating and culturing mesenchymal stem cells by enzyme digestion.

[0101] In the present invention, the enzymatic digestion method is used to separate and culture mesenchymal stem cells, and the specific steps are as follows:

[0102] 1. Processing of tissue samples:

[0103] (11) Select fresh tissue samples, place them in a sterile container, and rinse them with saline to remove blood and impurities.

[0104] (12) Cut the tissue sample into small pieces of 1–2 mm in size. 3 ;

[0105] 2. Preparation of digestive juice:

[0106] Prepare digestive juice according to the proportions;

[0107] 3. Tissue Digestion:

[0108] (31) Place the processed tissue sample into a sterile container containing tissue separation and digestion solution, ensuring that the tissue is completely immersed in the digestion solution;

[0109] (32) Place the container in a 37°C constant temperature incubator for digestion. During the digestion process, gently shake the container to promote full contact between the tissue and the digestive fluid;

[0110] (33) Take out a small amount of digestion fluid at regular intervals and observe the digestion of the tissue under a microscope. When most of the tissue is digested and mesenchymal stem cells begin to be released, the digestion time can be appropriately extended, but care should be taken to avoid damage to the mesenchymal stem cells due to over-digestion.

[0111] 4. Cell collection:

[0112] (41) After digestion is complete, the digestion solution is filtered through a filter to remove undigested tissue fragments. The pore size of the filter should be appropriately selected to ensure that larger tissue fragments can be filtered out without losing too many cells.

[0113] (42) The filtered cell suspension was transferred to a centrifuge tube and centrifuged at 1000-1500 rpm / min for 5-10 min. After centrifugation, the supernatant was removed and the cell pellet was retained;

[0114] (43) Resuspend the cell pellet with an appropriate amount of cell culture medium to obtain a mesenchymal stem cell suspension.

[0115] (44) Count and detect the activity of the mesenchymal stem cell suspension to determine the quantity and quality of the mesenchymal stem cells.

[0116] Example 4

[0117] Digestion efficiency experiment:

[0118] 1. Experimental materials: Select several placental tissue samples. The samples should come from the same human tissue to ensure the comparability of the experiment;

[0119] 2. Experimental methods:

[0120] (1) The placental tissue samples were divided into two groups. One group of placental tissue samples was digested using the digestion solution for mesenchymal stem cell enzyme digestion method in Example 3 using the above method, and the other group of placental tissue samples was digested using the above method using trypsin digestion solution.

[0121] (2) Under the same experimental conditions, the digestion time of the two groups of placental tissue samples was recorded. The digestion time was defined as the time from the addition of the digestion solution to the time when most of the tissue was digested and mesenchymal stem cells began to be released.

[0122] (3) After digestion, human mesenchymal stem cells (P0 cells) were cultured in complete culture medium for 1 to 2 weeks, and the number of cells in the two groups was counted using a cell counter, and the cells were observed and photographed under a 40X microscope.

[0123] 3. Experimental results:

[0124] like Figure 1 , Figure 2, within the same microscope field of view, the human mesenchymal stem cells cultured after digestion with the digestion solution for mesenchymal stem cell enzyme digestion method of Example 3 are evenly distributed and significantly more numerous, indicating that the digestion solution of the present invention has a promoting effect on cell proliferation and can provide more sufficient cell resources for subsequent experiments and applications. At the same time, the number of floating cells in the field of view is extremely small and almost negligible, which means that the cells are more stable during the culture process and are not easy to fall off from the surface of the culture vessel, greatly reducing the risk of cell damage or death due to detachment.

[0125] Compared with the cells treated with trypsin, the number of cells in the field of view is relatively small, the distance between cells is more obvious, and the overall growth trend is not as good as the cells treated with the digestion solution of the present invention. At the same time, a certain number of floating cells can be seen, indicating that the cells treated with trypsin have certain shortcomings in maintaining cell adhesion growth, which easily leads to poor growth or even apoptosis of some cells.

[0126] The average digestion time of the placenta tissue using the digestion solution group for mesenchymal stem cell enzyme digestion method of Example 3 was 60 to 90 minutes, and the average digestion time using the trypsin digestion solution group was 120 to 150 minutes. Relatively speaking, the digestion time of the present invention was shortened by 40 to 50%.

[0127] For placental tissue, the number of P0 cells released by the digestion solution used for the mesenchymal stem cell enzyme digestion method in Example 3 of the present invention is 1500-2000W, and the number of P0 cells released by the trypsin digestion solution group is 1000-1300W, and the cell number of the present invention is increased by 50-53%.

[0128] Example 5

[0129] Cell viability assay:

[0130] 1. Experimental materials:

[0131] In Example 4: human mesenchymal stem cells cultured after digesting embryonic tissue samples with the digestion solution for mesenchymal stem cell enzyme digestion method in Example 3;

[0132] In Example 4: Human mesenchymal stem cells were cultured after the embryonic tissue sample was digested with trypsin solution.

[0133] 2. Experimental methods:

[0134] The cell proliferation ability was detected using CCK-8 kit.

[0135] The cells were seeded in a 96-well plate, CCK-8 reagent was added, and the absorbance value was measured with an enzyme marker at different time points (such as 24 hours, 48 ​​hours, 72 hours, etc.) to reflect the proliferation of the cells.

[0136] 3. Experimental results:

[0137] like Figure 3 As shown, in the cell proliferation experiment, the absorbance values ​​of human mesenchymal stem cells cultured after digesting the embryonic tissue sample with the digestion solution for the mesenchymal stem cell enzymatic digestion method in Example 3 at different time points were higher than those of human mesenchymal stem cells cultured after digesting the embryonic tissue sample with the trypsin digestion solution, indicating that the proliferation ability of human mesenchymal stem cells cultured after digesting the embryonic tissue sample with the digestion solution for the mesenchymal stem cell enzymatic digestion method in Example 3 was significantly enhanced.

[0138] Example 6

[0139] Cell phenotyping experiments:

[0140] 1. Experimental materials:

[0141] In Example 4: human mesenchymal stem cells cultured after digesting embryonic tissue samples with the digestion solution for mesenchymal stem cell enzyme digestion method in Example 3;

[0142] 2. Experimental methods:

[0143] Flow cytometry was used to detect the expression of surface markers of human mesenchymal stem cells cultured after the embryonic tissue sample was digested with the digestion solution for the mesenchymal stem cell enzyme digestion method in Example 3. The cells were labeled with specific antibodies, and then the expression levels of the markers were analyzed by flow cytometry.

[0144] 3. Experimental results:

[0145] like Figure 4 As shown, the surface markers of human mesenchymal stem cells cultured after the embryonic tissue sample was digested with the digestion solution for the mesenchymal stem cell enzyme digestion method of Example 3 were stably expressed, indicating that the digestion process did not change the stem cell characteristics of the cells.

[0146] Example 7

[0147] Differentiation potential assay:

[0148] 1. Experimental materials:

[0149] In Example 4: human mesenchymal stem cells cultured after digesting embryonic tissue samples with the digestion solution for mesenchymal stem cell enzyme digestion method in Example 3;

[0150] In Example 4: Human mesenchymal stem cells were cultured after the embryonic tissue sample was digested with trypsin solution.

[0151] 2. Experimental methods:

[0152] (1) Cell inoculation and preliminary preparation

[0153] Take an appropriate amount of digested human mesenchymal stem cells and use a blood cell counting plate or an automatic cell counter to accurately count them. According to the experimental design and subsequent testing requirements, count the cells at an appropriate cell density (e.g., 1×10 per square centimeter). 4 ~5×10 4 The cells are inoculated into a cell culture dish or culture plate that has been pre-coated with poly-lysine or the like to ensure that the cells can grow evenly by adhering to the wall.

[0154] Place the culture vessel inoculated with cells in a cell culture incubator at 37°C and 5% CO2 to allow the cells to adhere to the wall and return to normal physiological state. The incubation time is about 2 to 4 hours, and the cell adhesion is observed regularly during this period.

[0155] (2) Multidirectional differentiation induction operation

[0156] (2a) Osteogenesis induction: When the cells are well attached to the wall and reach a confluence of about 70% to 80%, carefully discard the original culture medium and replace it with a specially prepared osteogenic induction medium. The culture medium is mainly composed of a basal culture medium (DMEM low-glucose culture medium) and a final concentration of 10 -8 ~10 -7 M dexamethasone, 10mM sodium β-glycerophosphate, and 50μg / mL vitamin C. Gently shake the culture vessel to evenly distribute the culture medium, and then place it in a 37°C, 5% CO2 incubator for culture. The induction period is usually 14 to 21 days. During this period, replace fresh osteogenic induction medium every 2 to 3 days to maintain the stability of the induction environment and nutrient supply.

[0157] (2b) Adipogenesis induction: When the cells adhere to the wall to 70% to 80% confluence, the original culture medium is discarded and replaced with adipogenesis induction medium. The base medium is DMEM high-glucose medium, with insulin at a final concentration of 10 μg / mL and 10 -6 ~10 -5 M of dexamethasone and 100 μM of indomethacin. For other operations such as osteogenic induction, the culture vessel was returned to the incubator, the induction time was set to 10 to 14 days, and the culture medium was changed every 2 to 3 days to ensure that the cells were in a continuous and effective induction environment.

[0158] (2c) Chondrogenic induction: When the cells adhered to the appropriate density (60% to 70% confluence), the chondrogenic induction medium was replaced with DMEM / F12 medium, and a final concentration of 10 ng / mL of transforming growth factor-β, 10 -8 ~10 - 7 M of dexamethasone and 50 μg / mL of vitamin C. Subsequent operations were the same as before, the incubator environment remained unchanged, the induction time was 14 to 21 days, and the medium was changed every 2 to 3 days to ensure stable induction conditions.

[0159] (3) Monitoring and analysis of the induction process

[0160] During the entire induction process, the morphological changes of cells were observed every day or every other day using an inverted microscope. The key features of the cell morphology gradually changing from the initial state to the direction of osteogenic, adipogenic, and chondrogenic were recorded, such as the gradual aggregation of cells, the appearance of multi-layered growth, and the formation of calcium nodules on the edges during osteogenic induction; the gradual appearance of round and translucent lipid droplets in cells under adipogenic induction; and the aggregation of cells into cartilage-like nodules and the decrease of intercellular spaces during chondrogenic induction.

[0161] (3a) Alizarin red staining after osteogenic induction:

[0162] After induction, carefully discard the culture medium and gently wash the cells 2 to 3 times with PBS buffer for 5 minutes each time to remove residual culture medium components and impurities.

[0163] Add 4% paraformaldehyde solution to fix the cells for 15 to 20 minutes, and then wash them three times with PBS, each time for 5 minutes.

[0164] Prepare fresh alizarin red staining solution according to the instructions of the alizarin red staining kit, immerse the fixed cells in the staining solution, and stain for 30 to 60 minutes at room temperature in the dark.

[0165] After staining, wash the cells thoroughly with PBS to remove excess stain and observe under a microscope. The calcium nodules appear red and the formation of calcium nodules is quantitatively analyzed by counting or area measurement.

[0166] (3b) Oil red O staining after adipogenic induction

[0167] After induction, discard the culture medium and wash the cells with PBS as above.

[0168] The cells were fixed with 4% paraformaldehyde for 10-15 minutes and washed with PBS three times, 5 minutes each time.

[0169] Prepare the staining solution according to the requirements of the Oil Red O staining kit, immerse the cells in the staining solution, and stain at room temperature for 15 to 30 minutes, taking care to avoid light.

[0170] After staining, wash thoroughly with PBS and observe under a microscope. The lipid droplets are stained red, and the size and number of lipid droplets can be statistically analyzed using image analysis software.

[0171] (3c) Alcian blue staining after chondrogenic induction

[0172] At the end of the induction period, the cells were treated as before, i.e., the culture medium was discarded, the cells were washed with PBS, and they were fixed (the fixative and time were the same as those for adipogenic induction).

[0173] Prepare the dye solution according to the instructions of the Alcian blue staining kit, immerse the cells in the dye solution, and stain at 4°C in the dark for 1 to 2 hours.

[0174] Wash the cells several times with PBS to remove excess dye and observe under a microscope. The cartilage matrix appears blue and relevant quantitative analysis can be performed on the blue area, such as the proportion of the visual field area.

[0175] 3. Experimental results:

[0176] like Figure 5A , Figure 5B , Fig. 6A , Figure 6B , Fig. 7A and Figure 7B As shown, the human mesenchymal stem cell group cultured after digesting the embryonic tissue sample with the digestion solution for the mesenchymal stem cell enzyme digestion method in Example 3 showed good differentiation ability after induction of osteogenic, adipogenic and chondrogenic differentiation, which was comparable to the control group of human mesenchymal stem cells cultured after digesting the embryonic tissue sample with trypsin digestion solution.

[0177] The above describes the embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A digestion solution for mesenchymal stem cell enzymatic digestion, characterized in that: Measured in mass percentage, based on the total mass of the digestion solution used for the mesenchymal stem cell enzymatic digestion method, the composition is composed of the following components: Digestive enzymes account for 0.1% to 0.5%; Human serum albumin accounts for 1% to 3%; Heparin sodium accounts for 0.008% to 0.02%; Lycopodiol accounts for 0.01% to 0.03%; The remainder was 1× HBSS.

2. A digestion solution for mesenchymal stem cell enzymatic digestion as claimed in claim 1, characterized in that: The enzyme activity of the digestion solution used for the mesenchymal stem cell enzymatic digestion method is at least 190U.

3. A digestion solution for mesenchymal stem cell enzymatic digestion as claimed in claim 2, characterized in that: The enzyme activity of the digestion solution used for the mesenchymal stem cell enzyme digestion method is 190U-220U.

4. The digestion solution for mesenchymal stem cell enzymatic digestion as claimed in claim 1, characterized in that: The digestive enzyme consists of the following components in terms of mass percentage: Type I collagenase accounts for 40-50%; Type II collagenase accounts for 30-40%; The remainder is type IV collagenase.

5. The digestion solution for mesenchymal stem cell enzymatic digestion as claimed in claim 1, characterized in that: The enzyme activity of the heparin sodium is at least 80U.

6. The digestion solution for mesenchymal stem cell enzymatic digestion as claimed in claim 5, characterized in that: The enzyme activity of the heparin sodium is 80U-100U.

7. Use of the digestive solution according to any one of claims 1 to 6 as a digestive solution in the process of isolating and culturing mesenchymal stem cells by enzymatic digestion.

8. Use of the digestive fluid according to any one of claims 1 to 6 in the preparation of a medicament for separating and culturing mesenchymal stem cells by enzyme digestion.

9. Use of the digestive juice according to any one of claims 1 to 6 in the preparation of a medicament for propagating and expanding mesenchymal stem cells.

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