Preparation method and application of mesenchymal stem cell preparation

Abstract

The invention provides a preparation method and application of a mesenchymal stem cell preparation. The preparation method of the mesenchymal stem cell preparation comprises the following steps: S1, taking and separating mesenchymal stem cells; s2, pretreatment of the mesenchymal stem cells; s3, purification and amplification of the mesenchymal stem cells; s4, carrying out induced differentiation on the mesenchymal stem cells to pancreas islet beta-like cells; and S5, preparation preparation. According to the preparation method of the mesenchymal stem cell preparation, the placenta is used as a mesenchymal stem cell source; carrying out pretreatment on the mesenchymal stem cells; sorting the mesenchymal stem cells through immunomagnetic beads; adding a plurality of growth factors and an inducer into an induced differentiation culture medium; and adopting a compound cell protective agent. According to the preparation method disclosed by the invention, the mesenchymal stem cell preparation which is high in purity, strong in activity, good in stability and capable of effectively treating diabetes mellitus can be obtained.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell preparations, and more particularly relates to a preparation method and application of a mesenchymal stem cell preparation. BACKGROUND

[0002] Diabetes is a metabolic disease characterized by high blood sugar, and long-term high blood sugar can cause chronic damage and dysfunction of various tissues and organs such as eyes, kidneys, hearts, blood vessels and nerves, which seriously threatens human health. At present, the treatment of diabetes mainly relies on insulin injection, oral hypoglycemic drugs and other methods. Although these methods can control blood sugar to a certain extent, they cannot fundamentally repair damaged islet tissue and are difficult to avoid the occurrence and development of complications.

[0003] Mesenchymal stem cells (MSCs) have shown broad application prospects in the field of diabetes treatment due to their multi-directional differentiation potential, immunoregulatory function and paracrine effect. In terms of differentiation potential, MSCs can be induced to differentiate into insulin-secreting beta-like cells in vitro and in vivo; in terms of immunoregulatory function, MSCs can inhibit T cell proliferation, regulate macrophage polarization and reduce immune rejection; in terms of paracrine effect, MSCs can secrete various growth factors and cytokines to promote tissue repair.

[0004] However, there are still many deficiencies in the preparation method of mesenchymal stem cell preparations in the prior art: firstly, the stem cells are mostly derived from bone marrow, umbilical cord and the like, and the activity of mesenchymal stem cells derived from bone marrow decreases with the increase of donor age, and the mesenchymal stem cells derived from umbilical cord are limited in quantity and controversial in ethics; secondly, the stem cells have low purity and poor activity in the preparation process, and the efficiency of differentiation into islet beta-like cells is not high, which affects the treatment effect; thirdly, the preparation has poor stability and is prone to cell apoptosis during storage and transportation. Therefore, it has become a problem to be solved in the field to develop a mesenchymal stem cell preparation method which is convenient to obtain, has high purity, strong activity, good stability and can effectively treat diabetes. SUMMARY

[0005] To achieve the above-mentioned purpose, the technical solution adopted by the present application is to provide a preparation method of a mesenchymal stem cell preparation, comprising the following steps: S1. Obtaining and separating mesenchymal stem cells: selecting placental tissue, adding a mixed digestive solution containing collagenase IV and trypsin after fine processing, then adding a high-sugar culture medium containing human platelet lysate to terminate digestion, and obtaining a mesenchymal stem cell initial extract after filtration and centrifugation; S2. Pretreatment of mesenchymal stem cells: the mesenchymal stem cell primary extract is inoculated into a culture medium containing a pretreatment factor, and cultured in a low-oxygen environment; the pretreatment factor is composed of 5-10 ng / mL fibroblast growth factor, 2-5 ng / mL insulin-like growth factor, and 1-3 mmol / L β-hydroxybutyric acid; S3. Purification and expansion of mesenchymal stem cells: mesenchymal stem cells with a purity of ≥98% are obtained by immunomagnetic bead sorting; the purified mesenchymal stem cells are cultured for expansion and passaged for 3-5 generations; S4. Induced differentiation of mesenchymal stem cells into pancreatic beta-like cells: the expanded mesenchymal stem cells are inoculated into an induced differentiation culture medium and cultured for 14-21 days; the induced differentiation culture medium is based on a high-glucose culture medium, and 15-20 ng / mL nerve growth factor, 10-15 ng / mL hepatocyte growth factor, 50-100 nmol / L FLI-06, 10-15 mmol / L nicotinamide (NAM), 1-3 μmol / L retinoic acid, and 3-5 μg / mL insulin are added; S5. Preparation of preparation: undifferentiated placental mesenchymal stem cells and induced pancreatic beta-like cells are mixed with a cell protection agent at a volume ratio of 1:2:2-1:2:3 to prepare a preparation, and the preparation is cryopreserved, the cell protection agent including 5%-8% trehalose by mass fraction, 2%-3% human blood albumin by mass fraction, 0.1-0.3 mmol / L vitamin E, 0.5%-1% hydroxyethyl starch with a molecular weight of 200 kDa by mass fraction, and the balance physiological saline.

[0006] In an embodiment, the placenta in step S1 is derived from a healthy full-term woman with a gestational age of 37-40 weeks.

[0007] In an embodiment, in step S1, the mixed digestive solution further contains 2 mmol / L EDTA (ethylenediaminetetraacetic acid), and the digestion condition is 37°C shaking at a speed of 150-200 rpm for 30-45 minutes, and then the tissue digestion is observed under a microscope.

[0008] In an embodiment, in step S3, the immunomagnetic bead sorting is based on at least three of the following surface markers of mesenchymal stem cells: CD29, CD44, CD90, and CD105.

[0009] In an embodiment, the specific operation of immunomagnetic bead sorting in step S3 includes: washing the pretreated cells with PBS buffer for 2-3 times; adding 5-10 μL of anti-human CD44 magnetic beads per 5×10 6 -1×10 7The cells are mixed with 100-200 μL of a magnetic bead suspension coated with any one of CD29, CD44, CD90, and CD105 antibodies, and incubated at 4°C for 15-30 minutes, and then subjected to positive cell screening using a magnetic bead sorting column; the positive cells obtained by screening are subjected to stepwise sorting using magnetic beads coated with another surface marker antibody of CD29, CD44, CD90, and CD105 to obtain positive cells, and the stepwise sorting is repeated until positive cells with the target surface marker combination are obtained.

[0010] In an embodiment, the specific steps for inducing differentiation in step S4 are as follows: Stage 1: culture for 1-7 days in a high-sugar medium containing 15-20 ng / mL nerve growth factor and 10-15 ng / mL hepatocyte growth factor; Stage 2: culture for 8-14 days by adding 50-100 nmol / L of FLI-06 and 10-15 mmol / L of nicotinamide to the medium of stage 1; Stage 3: culture for 15-21 days by adding 1-3 μmol / L of retinoic acid and 3-5 μg / mL of insulin to the medium of stage 2.

[0011] In an embodiment, in step S5, the cryopreservation is performed using a programmed cooling method, and the specific cooling program is as follows: 4°C for 30 minutes, cooling to -20°C at a rate of 1°C / min and maintaining for 1 hour, then cooling to -80°C at a rate of 5°C / min and maintaining for at least 12 h, and finally transferring into liquid nitrogen for long-term storage.

[0012] The application also provides a mesenchymal stem cell preparation obtained by the preparation method of the mesenchymal stem cell preparation, characterized in that it comprises placental mesenchymal stem cells, pancreatic beta-like cells differentiated from the placental mesenchymal stem cells, and a cell protective agent, wherein the cell protective agent comprises trehalose with a mass fraction of 5%-8%, human blood albumin with a mass fraction of 2%-3%, 0.1-0.3 mmol / L vitamin E, hydroxyethyl starch with a mass fraction of 0.5%-1% and a molecular weight of 200 kDa, and the rest is physiological saline.

[0013] The application also provides the use of the mesenchymal stem cell preparation of the application in the preparation of a drug for treating diabetes, wherein the diabetes includes type 1 diabetes, type 2 diabetes, and diabetes-related complications, and the diabetes-related complications include diabetic retinopathy and diabetic nephropathy.

[0014] In an embodiment, the mesenchymal stem cell preparation is administered by intravenous injection.

[0015] Compared with the prior art, the preparation method of the mesenchymal stem cell preparation provided in the application selects placenta as the source of mesenchymal stem cells, the placenta is easy to obtain and belongs to postpartum waste, and there is no ethical controversy, and the mesenchymal stem cells derived from the placenta are higher in activity and stronger in proliferation capacity than the traditional bone marrow and umbilical cord derived stem cells; the purity and activity of the mesenchymal stem cells can be improved by pretreating the mesenchymal stem cells by using a pretreatment factor combination composed of fibroblast growth factor, insulin-like growth factor and hydroxybutyric acid; the mesenchymal stem cells can be efficiently induced to differentiate into pancreatic beta-like cells with insulin secretion function by adding various growth factors and inducers in the induction differentiation culture medium; the apoptosis of the mesenchymal stem cell preparation can be effectively reduced and the cell survival rate can be improved during the cryopreservation by adding a composite protective agent composed of trehalose, human blood albumin, vitamin E and hydroxyethyl starch in the preparation. Therefore, the preparation method can obtain the mesenchymal stem cell preparation which is high in purity, strong in activity, good in stability and can effectively treat diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0017] Figure 1 The insulin secretion amount of different mesenchymal stem cell preparation pancreatic beta-like cells in low sugar / high sugar environment is shown; Figure 2 The blood glucose change trend of different mesenchymal stem cell preparations after administration to type 1 diabetic rats for 1-8 weeks is shown. DETAILED DESCRIPTION

[0018] In order to make the technical problems to be solved by the application, the technical solutions and the beneficial effects more clearly, the following will further describe the application in combination with the drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the application and not to limit the application.

[0019] It should be noted that when an element is referred to as being "fixed to" or "disposed on" another element, it can be directly on the other element or indirectly on the other element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element.

[0020] It should be understood that the terms "length", "width", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and cannot be understood as indicating or implying that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0021] In addition, the terms "first", "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined as "first", "second" can explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "multiple" is two or more, unless otherwise explicitly specified.

[0022] Please refer to Figure 1 and Figure 2 , a preparation method of a mesenchymal stem cell preparation provided by the embodiments of the present application will be described. The preparation method of the mesenchymal stem cell preparation comprises the following steps: S1. Tissue selection and separation of mesenchymal stem cells: selecting placental tissue for fine processing, adding a mixed digestive solution containing collagenase IV and trypsin for digestion, then adding a high-sugar culture medium containing human platelet lysate to terminate digestion, and obtaining a mesenchymal stem cell initial extract after filtration and centrifugation; S2. Pretreatment of mesenchymal stem cells: inoculating the mesenchymal stem cell initial extract into a culture medium containing a pretreatment factor and culturing in a low-oxygen environment; the pretreatment factor is composed of 5-10 ng / mL fibroblast growth factor, 2-5 ng / mL insulin-like growth factor and 1-3 mmol / L β-hydroxybutyric acid; S3. Purification and expansion of mesenchymal stem cells: obtaining mesenchymal stem cells with a purity of ≥98% by immunomagnetic bead sorting; and subculturing the purified mesenchymal stem cells for 3-5 generations; S4. Induced differentiation of mesenchymal stem cells into pancreatic beta-like cells: inoculating the expanded mesenchymal stem cells into an induced differentiation culture medium and culturing for 14-21 days; the induced differentiation culture medium is based on a high-sugar culture medium, and 15-20 ng / mL nerve growth factor, 10-15 ng / mL hepatocyte growth factor, 50-100 nmol / L FLI-06, 10-15 mmol / L nicotinamide (NAM), 1-3 μmol / L retinoic acid and 3-5 μg / mL insulin are added; S5. Preparation of the preparation: the undifferentiated placental mesenchymal stem cells and the induced differentiated pancreatic beta-like cells are mixed with the cell protective agent in a volume ratio of 1:2:2-1:2:3 to prepare the preparation, and the cell protective agent includes 5%-8% trehalose by mass fraction, 2%-3% human blood albumin by mass fraction, 0.1%-0.3 mmol / L vitamin E, 0.5%-1% hydroxyethyl starch with a molecular weight of 200 kDa by mass fraction, and the rest is physiological saline.

[0023] Now the step S1 is described in detail: compared with traditional bone marrow and umbilical cord-derived mesenchymal stem cells, placenta-derived mesenchymal stem cells have the advantages of no ethical controversy, large cell reserves and high activity stability. Specifically, the placenta is a postpartum waste, and does not need additional invasive collection; a single placenta can obtain 1×10 8 -5×10 8 initial mesenchymal stem cells, far more than 1×10 7 -5×10 7 of a single umbilical cord; the placental tissue has a low degree of development, and the differentiation potential and proliferation capacity of stem cells are significantly better than those of bone marrow-derived stem cells, and are not affected by donor age, and are more widely applicable. For the detailed processing of the placenta, surgical scissors can be used for cutting and crushing, or a tissue dissociator can be used, and the size of the fragments should be as small as possible to facilitate subsequent digestion of the tissue. Collagenase IV can specifically degrade collagen fibers in the placental tissue, avoiding damage to the surface receptors of stem cells. Trypsin can gently decompose intercellular adhesion proteins to promote cell dispersion and avoid cell rupture caused by excessive digestion. Human platelet lysate contains rich growth factors, which can quickly repair cell surface damage during digestion, improve the activity of initial cells, and at the same time avoid the "xenoprotein pollution risk" of fetal bovine serum, reducing the probability of immune rejection in subsequent clinical applications. High-sugar medium contains rich glucose, which can promote the proliferation of mesenchymal stem cells. After termination of digestion, filtration and centrifugation are performed to remove undigested tissue blocks, vascular fragments, and enzyme degradation products, dead cell fragments and other impurities in the supernatant, to obtain mesenchymal stem cells with high purity and activity.

[0024] Now step S2 is described in detail: hypoxic culture, i.e. oxygen concentration of 1%-5%, can reduce the generation of reactive oxygen species (ROS) of mesenchymal stem cells, reduce cell oxidative damage; can also induce the expression of hypoxia-inducible factor (HIF-1), and then promote the expression of vascular endothelial growth factor (VEGF), improve cell proliferation ability. Fibroblast growth factor can activate the MAPK / ERK pathway, promote stem cell proliferation and early lineage orientation. Insulin-like growth factor can activate the PI3K / Akt pathway, enhance the cell anti-apoptosis ability, and at the same time up-regulate the basic expression of key genes for islet development. β-hydroxybutyric acid is a ketone metabolite, which can simulate the metabolic microenvironment in the fasting state, activate the PPARγ signaling pathway, promote the differentiation of stem cells into insulin-secreting cells, and improve the differentiation efficiency of mesenchymal stem cells.

[0025] Now step S3 is described in detail: immunomagnetic bead sorting is a high-efficiency cell separation and purification method, which is based on the characteristics that cell surface antigens can be combined with specific antibodies connected with magnetic beads. In an external magnetic field, cells connected with magnetic beads through antibodies are adsorbed and retained in the magnetic field, while cells without such surface antigens cannot be combined with specific antibodies connected with magnetic beads and have no magnetism, so they do not stay in the magnetic field to achieve the purpose of cell separation. The equipment required for magnetic bead cell sorting is simple, and the technical personnel are not high. The sensitivity, purity, cell activity and recovery rate of the sorted cells are good, and the influence on downstream application is small, so it has a wide application prospect. Mesenchymal stem cells are passaged for 3-5 generations to balance the number and activity of cells. If the passage number is too high, the cells are prone to show signs of aging, such as slow proliferation rate and decreased differentiation potential.

[0026] Now step S4 is described in detail: nerve growth factor and hepatocyte growth factor promote the differentiation of stem cells into endoderm, up-regulate the expression of endoderm markers such as Sox17 and FoxA2, and orient the subsequent islet lineage differentiation. FLI-06 is a Notch signaling pathway inhibitor that can block the undifferentiated state of stem cells and promote the differentiation of cells into islet precursor cells. Nicotinamide is a PARP inhibitor that can inhibit apoptosis of islet precursor cells and up-regulate the expression of the key transcription factor PDX-1 for islet development. Retinoic acid is a vitamin A derivative that can activate the RAR / RXR signaling pathway, promote the differentiation of islet precursor cells into β cell lineage, and avoid differentiation into glucagon-secreting α cells. Insulin as an autocrine or paracrine signal can simulate the in vivo insulin feedback regulation to promote the maturation of islet β-like cells. The differentiation of islet β-like cells is a gradual maturation process, so a certain culture time is needed.

[0027] The step S5 is described in detail as follows: the undifferentiated placental mesenchymal stem cells and the induced pancreatic beta-like cells are mixed with the cell protective agent at a volume ratio of 1:2:2-1:2:3, so that the cells are sufficiently protected and the cell concentration is not too low. The cell protective agent can improve the storage stability of the cells in the preparation and avoid excessive decrease in cell activity during transportation. Trehalose can form a glassy protective layer on the cell surface to avoid damage to the cell membrane caused by ice crystal formation during freezing and maintain the stability of the cell osmotic pressure. Human blood albumin can bind to the cell surface to reduce cell aggregation and provide nutrients to avoid apoptosis caused by nutrient deficiency. Vitamin E as an antioxidant can scavenge reactive oxygen species generated during storage, reduce cell oxidative damage, and avoid the decrease in insulin secretion capacity caused by oxidative stress. Hydroxyethyl starch has high colloid osmotic pressure and can maintain the stability of the cell suspension to avoid cell sedimentation. Meanwhile, the molecular weight of hydroxyethyl starch is 200 kDa, which can avoid entering the interstitial space of tissues through capillaries and reduce the risk of adverse reactions.

[0028] Compared with the prior art, the preparation method of the mesenchymal stem cell preparation provided in the present application selects placenta as the source of mesenchymal stem cells. The placenta is easy to obtain and belongs to postpartum waste, which has no ethical controversy. Meanwhile, the placenta-derived mesenchymal stem cells have high activity and strong proliferation capacity, which are superior to traditional bone marrow-derived and umbilical cord-derived stem cells. The combination of the pretreatment factors composed of fibroblast growth factor, insulin-like growth factor and hydroxybutyric acid is used to pretreat the mesenchymal stem cells, which can improve the purity and activity of the mesenchymal stem cells. The addition of various growth factors and inducers in the induction differentiation medium can efficiently induce the mesenchymal stem cells to differentiate into pancreatic beta-like cells with insulin secretion function. The addition of the composite protective agent composed of trehalose, human blood albumin, vitamin E and hydroxyethyl starch in the preparation can effectively reduce the apoptosis of the mesenchymal stem cell preparation during cryopreservation and improve the cell survival rate. Therefore, the preparation method of the present application can obtain mesenchymal stem cell preparations with high purity, high activity, good stability and effective treatment of diabetes.

[0029] In an embodiment, the placenta in step S1 is derived from a healthy full-term pregnant woman with a gestational age of 37-40 weeks.

[0030] Specifically, the placenta with a gestational age of 37-40 weeks has been developed and matured, and the number of stem cells in the tissue reaches a peak value and the activity is stable. The placenta of a healthy full-term pregnant woman has a low probability of being affected by factors such as pathogen infection and developmental abnormalities, which can greatly reduce the risk of contamination or potential defects of the stem cells and is conducive to the subsequent preparation of mesenchymal stem cell preparations with high purity and high activity.

[0031] In an embodiment, in step S1, the mixed digestive solution further comprises 2 mmol / L EDTA (ethylenediaminetetraacetic acid), and the digestion condition is that the tissue is shaken at 37°C with a shaking speed of 150-200 rpm for 30-45 minutes, and then the digestion of the tissue is observed under a microscope. Specifically, EDTA is a calcium ion chelator, which can destroy the calcium ion-dependent adhesion junction between cells, and assist trypsin in more efficiently decomposing the intercellular matrix. In addition, EDTA can inhibit the autodegradation of part of the protease, prolong the maintenance time of enzyme activity, and avoid the problem of incomplete digestion caused by enzyme activity attenuation. In addition, collagenase, dispersion enzyme, deoxyribonuclease, etc. can also be added to the mixed digestive solution.

[0032] The mixed digestive solution contains active enzyme components of collagenase IV and trypsin, and 37°C is the physiological temperature of the human body, which is the optimum temperature for enzyme activity. A shaking speed of 150-200 rpm can ensure that the digestive solution and the tissue block are in full contact, while avoiding mechanical damage to the cells caused by too high a shaking speed. Because the composition of the tissue is complex, too short a digestion time will lead to incomplete decomposition of the tissue block and a decrease in cell yield, but too long a digestion time will cause excessive enzyme digestion and damage to the cell membrane, resulting in a decrease in cell viability. After digestion, the digestion of the tissue is observed under a microscope, which can ensure that the tissue block is fully digested and the quality of the mesenchymal stem cells is controlled.

[0033] In an embodiment, in step S3, the immunomagnetic bead sorting is based on at least three of the following surface markers of mesenchymal stem cells: CD29, CD44, CD90, and CD105.

[0034] Specifically, CD29, CD44, CD90, and CD105 are all internationally recognized specific surface markers of human mesenchymal stem cells. Among them, CD29 is also known as integrin β1, which is involved in cell adhesion and matrix interaction and is stably expressed in mesenchymal stem cells, but not expressed in hematopoietic stem cells or immune cells. CD44 is a hyaluronic acid receptor that is involved in the regulation of proliferation and migration of mesenchymal stem cells. CD90, also known as Thy-1, is a glycoprotein molecule that is highly expressed in undifferentiated mesenchymal stem cells and gradually down-regulated as the cells differentiate, and can be used as a marker for the undifferentiated state of mesenchymal stem cells. CD105 is an endothelial glycoprotein that is involved in the regulation of TGF-β signaling pathway and is closely related to the immune regulation function of mesenchymal stem cells.

[0035] In addition to mesenchymal stem cells, placental tissue also contains other cells, and a single marker screening can have cross-reactions, for example, CD44 is also lowly expressed in some hematopoietic cells. Therefore, using a combination of at least three markers for immunomagnetic bead sorting can help improve the purity of mesenchymal stem cells, remove mixed hematopoietic cells, fibroblasts and other impurities, and avoid interference with subsequent induction and differentiation.

[0036] In an embodiment, the specific operation of immunomagnetic bead sorting in step S3 includes: washing the pretreated cells with PBS buffer for 2-3 times; adding 100-200 μL of magnetic bead suspension coated with any one of CD29, CD44, CD90 and CD105 antibodies to mix with 5x10 6 -1x10 7 cells, 4°C incubation for 15-30 minutes, and then using a magnetic bead sorting column to screen positive cells; the positive cells obtained by screening are further subjected to stepwise sorting using magnetic beads coated with another surface marker antibody of CD29, CD44, CD90 and CD105 to obtain positive cells, and the stepwise sorting is repeated until positive cells with the target surface marker combination are finally obtained.

[0037] Specifically, washing the cells with PBS buffer for 2-3 times can effectively remove the human albumin component and residual factors in the pretreatment medium, avoid interference with the specific binding of the antibody to the cell surface marker, and affect the antibody binding efficiency. The ratio of 5x10 6 -1x10 7 cells to 100-200 μL of magnetic bead suspension containing surface marker antibodies can ensure sufficient contact between the cells and the sorting magnetic beads, and avoid non-specific binding caused by excessive antibodies.

[0038] The magnetic bead sorting column is an important consumable in immunomagnetic bead sorting technology, and is filled with magnetic particles inside. When the sorting column is placed in a strong magnetic field, a high-strength, gradient magnetic field is formed inside the column. When the mixture containing labeled magnetic beads and cells flows through the sorting column, the target cells labeled with magnetic beads are adsorbed on the magnetic particles in the sorting column by the magnetic field, i.e. positive cells, while the unlabeled cells are not affected by the magnetic field and can directly flow through the sorting column, i.e. negative cells. After separation, the positive cells adsorbed on the sorting column can be eluted to obtain high-purity target cells. Immunomagnetic bead sorting cannot recognize cells with multiple markers, so stepwise sorting is required.

[0039] In an embodiment, the specific steps of inducing differentiation in step S4 are: Stage 1: culture for 1-7 days, and the culture medium is a high-sugar medium containing 15-20 ng / mL nerve growth factor and 10-15 ng / mL hepatocyte growth factor; Stage 2: culture for 8-14 days, add 50-100 nmol / L FLI-06 and 10-15 mmol / L nicotinamide to the medium of stage 1; Stage 3: culture for 15-21 days, add 1-3 μmol / L retinoic acid and 3-5 μg / mL insulin to the medium of stage 2.

[0040] Specifically, the differentiation of pancreatic beta-like cells is a gradual maturation process, so the stepwise addition of different induction differentiation substances is based on the biological law of cell differentiation to scientifically regulate the differentiation process of mesenchymal stem cells. Among them, stage 1 induces endoderm differentiation, and the origin layer of islet cells is endoderm; stage 2 induces the formation of islet precursor cells; and stage 3 induces the maturation of islet beta-like cells, which have the ability to secrete insulin.

[0041] In an embodiment, in step S5, the cryopreservation adopts a programmed cooling method, and the specific cooling program is as follows: 4°C for 30 minutes, cooling to -20°C at a rate of 1°C / min and keeping for 1 hour, then cooling to -80°C at a rate of 5°C / min and keeping for at least 12 hours, and finally transferring into liquid nitrogen for long-term storage.

[0042] Specifically, attention should be paid to avoid the mechanical damage of ice crystal formation to the cell membrane and organelles during cell cryopreservation, that is, when the cell is rapidly cooled, the water inside and outside the cell is easy to form sharp ice crystals, which pierces the cell membrane and leads to cell death; while slow cooling will cause excessive dehydration of the cell, which destroys the osmotic pressure balance. Therefore, the cooling rate of the cell should be reasonably controlled to make the water in the cell orderly exude and form a glassy state outside the cell, thereby maximizing the preservation of cell activity.

[0043] As the initial stage of cooling, setting the temperature to 4°C helps the cells gradually adapt to the low-temperature environment and reduces the stress response caused by sudden temperature drop; 30 minutes of pre-cooling time is sufficient to reduce the cell metabolic rate and avoid cell shock caused by direct entry into low temperature. Slow cooling at a rate of 1°C / min helps to prevent the formation of ice crystals in the cell, because the water outside the cell freezes first, and the osmotic pressure gradient formed promotes the slow exudation of water in the cell. Keeping at -20°C for 1 hour can make the ice crystals outside the cell fully form and stabilize, and at the same time make the remaining water in the cell further balance through osmotic action. When the temperature drops below -20°C, the liquid outside the cell has been completely solidified, at which time the cooling rate can be increased to 5°C / min to promote the remaining water in the cell to form an amorphous glass state. Keeping at -80°C for at least 12 hours can completely balance the temperature inside and outside the cell, and at the same time verify the preliminary stability of the cell in ultra-low temperature, avoiding the temperature shock caused by direct transfer into liquid nitrogen. The ultra-low temperature environment of liquid nitrogen can completely stop the cell metabolism, prevent cell aging, apoptosis or functional degradation, and greatly improve the preservation time of the cell.

[0044] The application further provides a mesenchymal stem cell preparation, which is obtained by the preparation method of the mesenchymal stem cell preparation provided by the application, and comprises placental mesenchymal stem cells, islet beta-like cells differentiated from the placental mesenchymal stem cells, and a cell protective agent, wherein the cell protective agent comprises trehalose with a mass fraction of 5-8%, human blood albumin with a mass fraction of 2-3%, 0.1-0.3 mmol / L vitamin E, hydroxyethyl starch with a mass fraction of 0.5-1% and a molecular weight of 200 kDa, and the rest is physiological saline.

[0045] Specifically, the mesenchymal stem cell preparation of the application has islet beta-like cells differentiated from placental mesenchymal stem cells and high insulin secretion; and the cell activity under long-term storage conditions is improved by improving the formula of the cell protective agent.

[0046] The application further provides an application of the mesenchymal stem cell preparation of the application in the preparation of a drug for treating diabetes, wherein the diabetes includes type 1 diabetes, type 2 diabetes and diabetes-related complications, and the diabetes-related complications include diabetic retinopathy and diabetic nephropathy.

[0047] Specifically, type 1 diabetes is mainly caused by autoimmune abnormalities, resulting in the destruction of islet beta cells and the absolute lack of insulin. The islet beta-like cells in the mesenchymal stem cell preparation of the application can directly supplement insulin-secreting cells, and the immunoregulatory function of the placental mesenchymal stem cells can reduce autoimmune attack and delay further damage to islet beta cells. Type 2 diabetes is mainly caused by insulin resistance and the decline of islet beta cell function. The mesenchymal stem cell preparation of the application can secrete insulin through islet beta-like cells, and can improve the insulin sensitivity of tissues such as liver and muscle and reduce insulin resistance through the cytokines secreted by the placental mesenchymal stem cells.

[0048] Diabetes-related complications are complications caused by long-term high blood sugar damage to multiple tissues and organs in the body. Among them, diabetic retinopathy is caused by high blood sugar, resulting in damage to retinal microvessels, ischemia and hypoxia. The mesenchymal stem cell preparation of the application can promote vascular repair through the paracrine effect of placental mesenchymal stem cells, and can inhibit abnormal proliferation of retinal neovascularization. Diabetic nephropathy is caused by long-term high blood sugar, resulting in mesangial proliferation and decreased kidney function. The placental mesenchymal stem cells can reduce the levels of inflammatory factors such as TNF-α and IL-6, inhibit the inflammatory response of the kidney, and reduce the deposition of mesangial matrix.

[0049] In an embodiment, the mesenchymal stem cell preparation is administered by intravenous injection.

[0050] Specifically, intravenous injection can make the pancreatic beta-like cells in the preparation circulate with the blood to the target organs throughout the body. Compared with local injection, intravenous injection is simple to operate, less traumatic, and has high patient compliance, and can act on multiple lesion sites at the same time. Intravenous injection is a routine clinical administration method, and does not require complex surgical equipment, and can be carried out in primary hospitals.

[0051] The application is further described below through specific examples: Example 1 The preparation method of the mesenchymal stem cell preparation in this example includes the following steps: S1. Obtaining and separating mesenchymal stem cells: select a placenta from a healthy full-term woman with a gestational age of 37 weeks, peel off the chorion of the placenta under sterile operation, remove the amnion and blood vessels, rinse with 0.9% normal saline for 5 times until no blood color, and cut into about 2mm 3 tissue fragments; place the tissue fragments in a centrifuge tube, add a mixed digestion solution containing 0.15% collagenase IV, 0.25% trypsin and 2mmol / L EDTA for digestion, the digestion conditions are 37℃ shaking bed with 150rpm rotation speed for 45 minutes, then observe the tissue digestion under a microscope; add DMEM / F12 high-sugar medium containing 10% human platelet lysate to terminate the digestion; filter through a 200 mesh sieve, centrifuge at 1100rpm for 9min, discard the supernatant, resuspend the precipitate with PBS (phosphate buffer), and obtain the mesenchymal stem cell primary extract.

[0052] S2. Pretreatment of mesenchymal stem cells: inoculate the mesenchymal stem cell primary extract at 5×10 4 cells / cm² on a low adsorption culture dish, the culture medium contains pretreatment factors, the pretreatment factors are composed of 5ng / mL fibroblast growth factor, 5ng / mL insulin-like growth factor and 3mmol / L β-hydroxybutyric acid; place the culture dish in a culture box and culture in a low-oxygen environment at 37℃, 3%O2 and 5%CO2.

[0053] S3. Purification and expansion of mesenchymal stem cells: obtain mesenchymal stem cells with a purity of ≥98% by immunomagnetic bead sorting; culture the purified mesenchymal stem cells for expansion, and subculture for 3 generations. The immunomagnetic bead sorting is based on the surface markers of mesenchymal stem cells, CD29, CD44, CD90 and CD105, and the specific operation is as follows: Wash the pretreated cells with PBS buffer for 3 times; add 200μL of magnetic bead suspension coated with CD29 antibody to mix per 1×10 7 cells, incubate at 4℃ for 30 minutes, and then use MACS sorting column for positive cell screening; screen the CD29 +positive cells, and then the positive cells were obtained by stepwise sorting using magnetic beads coated with CD90 antibody; the CD29 + CD44 + positive cells, and then the positive cells were obtained by stepwise sorting using magnetic beads coated with CD90 antibody; the CD29 + CD44 + CD90 + positive cells, and then the positive cells were obtained by stepwise sorting using magnetic beads coated with CD105 antibody; the CD29 + CD44 + CD90 + CD105 + positive cells.

[0054] S4. Inducing differentiation of mesenchymal stem cells into pancreatic beta-like cells: the expanded mesenchymal stem cells were inoculated in an inducing differentiation medium and cultured for 21 days; the specific steps of inducing differentiation were as follows: Stage 1: cultured for 1-7 days in a DMEM / F12 high-sugar medium containing 15 ng / mL nerve growth factor and 10 ng / mL hepatocyte growth factor; Stage 2: cultured for 8-14 days by adding 50 nmol / L of FLI-06 and 10 mmol / L of nicotinamide to the medium of stage 1; Stage 3: cultured for 15-21 days by adding 1 μmol / L of retinoic acid and 3 μg / mL of insulin to the medium of stage 2.

[0055] S5. Preparation of preparation: the undifferentiated placental mesenchymal stem cells and the pancreatic beta-like cells obtained by inducing differentiation were mixed with a cell protective agent at a volume ratio of 1:2:2 to prepare a preparation, and the preparation was cryopreserved by using a programmed cooling method; the specific cooling program was as follows: kept at 4°C for 30 minutes, cooled to -20°C at a rate of 1°C / min and kept for 1 hour, then cooled to -80°C at a rate of 5°C / min and kept for at least 12 h, and finally transferred into liquid nitrogen for long-term storage.

[0056] The cell protective agent comprises 5% trehalose by mass fraction, 2% human blood albumin by mass fraction, 0.1 mmol / L vitamin E, 1% hydroxyethyl starch with a molecular weight of 200 kDa by mass fraction, and the balance of physiological saline.

[0057] The mesenchymal stem cell preparation of the embodiment was obtained by using the preparation method of the mesenchymal stem cell preparation of the embodiment.

[0058] The mesenchymal stem cell preparation of the embodiment was applied to type 1 diabetes, and was administered by intravenous injection.

[0059] Example 2 Based on Example 1, the difference is that the placenta is derived from a healthy but preterm woman of 35 weeks of gestation.

[0060] Example 3 Based on Example 1, the difference is that the mesenchymal stem cells are induced to differentiate into pancreatic beta-like cells without staging in step S4, i.e. the expanded mesenchymal stem cells are seeded in the induction differentiation medium and cultured for 14-21 days; the induction differentiation medium is based on high-glucose medium, and 15 ng / mL nerve growth factor, 10 ng / mL hepatocyte growth factor, 50 nmol / L FLI-06, 10 mmol / L nicotinamide, 1 μmol / L retinoic acid and 3 μg / mL insulin are added.

[0061] Comparative Example 1 Based on Example 1, the difference is that the mesenchymal stem cells are extracted from umbilical cord tissue.

[0062] Comparative Example 2 Based on Example 1, the difference is that the mesenchymal stem cells in step S2 are cultured in a normoxic environment at 37°C, 21% O2 and 5% CO2.

[0063] Based on Example 1, the difference is that the cell protective agent in step S5 is composed of 10% DMSO (dimethyl sulfoxide) and 20% fetal bovine serum by mass fraction.

[0064] Experimental Example 1 The insulin secretion function of the pancreatic beta-like cells induced to differentiate from the mesenchymal stem cells is detected by enzyme-linked immunosorbent assay (ELISA).

[0065] In this experiment, a human insulin (ultra-sensitive) enzyme-linked immunosorbent assay kit is used, which is purchased from Biyun Tian Biotechnology, with the item number PI608. According to the kit instructions, the specific steps are as follows: (1) Group setting: for each example and comparative example, set up a low-sugar group and a high-sugar group, each with 3 replicates; set up a blank control group with 3 replicates; set up 3 background correction holes, i.e. blank holes, which only add TMB solution and stop solution.

[0066] (2) Take the pancreatic beta-like cells obtained by induction differentiation of each example and comparative example, remove the induction differentiation medium, wash 3 times with 1xPBS, and discard the PBS. Perform the following settings: Low-sugar group: add 10 mL of DMEM / F12 medium containing 2.8 mM glucose; High-sugar group: add 10 mL of DMEM / F12 medium containing 16.7 mM glucose; Blank control group: take a new culture dish, add 10 mL of DMEM / F12 medium.

[0067] (3) Then incubate each group of culture dishes in the cell incubator for 2 hours. Collect the supernatant of each group, centrifuge the supernatant, 500g, 5 minutes, and take the supernatant as the sample to be tested.

[0068] (4) Preparation before detection: after taking the kit out of the refrigerator, place it at room temperature (25-28°C) for 20 minutes, dilute the washing solution (20X) to 1X with double distilled water or deionized water; add the standard sample diluent to 1 bottle of standard sample according to the volume marked on the standard sample label, and incubate at room temperature for 15 minutes; take 5 clean 1.5 mL centrifuge tubes, and add 250 μL of standard sample diluent to each tube in advance, then perform a dilution of the standard sample by a factor of 2 to obtain 200, 100, 50, 25, 12.5, 6.25 mIU / L, a total of six standard sample concentrations, and finally add the diluted standard sample to the pre-coated plate holes in turn, and add the standard sample diluent directly as the 0 mIU / L concentration, a total of seven standard sample concentrations.

[0069] (5) Sample addition: take out the pre-coated plate strips required for the experiment and place them in the 96-well frame; add the sample to be tested or different concentrations of standard samples to the corresponding wells according to 25 μL / well; immediately add the horseradish peroxidase-labeled human insulin antibody according to 100 μL / well; cover the reaction wells with a sealing film (transparent) and incubate at room temperature for 120 minutes; wash the plate 5 times and the last time is placed on thick absorbent paper to dry; add the color developing agent TMB solution according to 100 μL / well, cover the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 10 minutes; (6) Detection: add the stop solution according to 50 μL / well, mix well, and then immediately measure the A450 absorbance value with an enzyme marker.

[0070] (7) Data processing: the absorbance value of each standard sample or sample to be tested should be subtracted from the absorbance value of the background correction hole. Use the detection data of the standard sample to make a standard curve, and then convert the insulin secretion amount of the sample to be tested according to the standard curve.

[0071] (8) Result analysis: Figure 1 The insulin secretion amount of different mesenchymal stem cell preparations in a low sugar / high sugar environment is shown in Table 1. Figure 1 As shown in Table 1, the insulin secretion amount of the mesenchymal stem cell preparations of Examples 1-3 is significantly higher than that of Comparative Examples 1-3, indicating that the mesenchymal stem cell preparations of the present application have the potential to treat diabetes; the insulin secretion amount of the mesenchymal stem cell preparations of Example 1 is significantly higher than that of Examples 2-3, indicating that the placenta is derived from a healthy full-term pregnant woman with a gestational age of 37-40 weeks or the induction differentiation step is performed in stages, which can further improve the insulin secretion function of the mesenchymal stem cell preparations.

[0072] Experimental Example 2 The effect of mesenchymal stem cell preparation in treating diabetes was verified by using a type 1 diabetes rat model.

[0073] In this experiment, 6-8 week old SPF male SD rats weighing 200-250 g were used, and streptozotocin (STZ, dissolved in 0.1 mol / L citric acid buffer) was used for modeling; the specific steps are as follows: (1) Modeling: fasting for 12 hours before administration, but not water. In the fasting state of rats, intraperitoneal injection was performed. The model group, each example and the comparative example group were injected with STZ solution at a dose of 60 mg / kg based on the body weight of the rats; the control group was injected with the same amount of 0.1 mol / L citric acid buffer; after injection, the rats were returned to the cage for normal feeding, and sufficient food, water and activity space were ensured.

[0074] (2) 72 hours later, tail vein blood was taken (fasting for 6 hours before blood collection), and fasting blood glucose was detected by blood glucose meter. Two consecutive blood glucose values ≥16.7 mmol / L indicated that the diabetes model was successful and could be included in the experiment.

[0075] (3) Group setting: set the control group, model group, each example and comparative example group, and randomly divide the successfully modeled rats into each group, 10 rats per group. The rats in the control group and model group were injected with 0.2 mL of 0.9% normal saline through the tail vein; the mesenchymal stem cell preparation was injected in each example and comparative example group, and the cell concentration was 5×10 6 cells / mL, and the dose was 0.2 mL. Note that the mesenchymal stem cell preparation is balanced to room temperature before administration, and gently mixed to avoid cell aggregation.

[0076] (4) Blood sampling was performed at 1 week, 2 weeks, 4 weeks and 8 weeks after administration, respectively. The rats were fasted for 6 hours before sampling, and 50 μL of blood was taken from the tail vein, which was detected by a portable blood glucose meter, and the blood glucose value (unit: mmol / L) was recorded.

[0077] (5) Result analysis: Figure 2 The blood glucose change trend of type 1 diabetes rats after administration of different mesenchymal stem cell preparations for 1-8 weeks is shown in the following table. For example, Figure 2As shown, the blood glucose of the control group was always maintained in the normal range (5.1-5.3 mmol / L), the blood glucose of the model group continued to rise over time, verifying the stability of the diabetes model; the blood glucose of the rats in Example 1-3 at each time point was significantly lower than that of the control group, and after 8 weeks of treatment, it was close to the control group, which embodied the excellent effect of the application in treating type 1 diabetes; in the examples, the treatment effect of Example 1 was better than that of Examples 2-3, which corresponded to the results of Experimental Example 1, indicating that the placenta was derived from a healthy full-term puerpera of 37-40 weeks of gestation or the induced differentiation step was carried out in stages, which could further improve the treatment effect of the mesenchymal stem cell preparation on type 1 diabetes.

[0078] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a mesenchymal stem cell preparation, characterized by, The method comprises the following steps: S1. Taking and separating mesenchymal stem cells: selecting placental tissue, adding a mixed digestive solution containing collagenase IV and trypsin for digestion after fine processing, then adding a high-sugar culture medium containing human platelet lysate to terminate the digestion, and obtaining a mesenchymal stem cell primary extract after filtration and centrifugation; S2. Pre-treating the mesenchymal stem cells: inoculating the mesenchymal stem cell primary extract into a culture medium containing a pre-treatment factor and culturing in a low-oxygen environment; the pre-treatment factor is composed of 5-10 ng / mL fibroblast growth factor, 2-5 ng / mL insulin-like growth factor and 1-3 mmol / L beta-hydroxybutyric acid; S3. Purifying and amplifying the mesenchymal stem cells: obtaining mesenchymal stem cells with a purity of ≥98% by immunomagnetic bead sorting; and subculturing the purified mesenchymal stem cells for 3-5 generations; S4. Inducing differentiation of the mesenchymal stem cells into islet beta-like cells: inoculating the amplified mesenchymal stem cells into an induction and differentiation culture medium and culturing for 14-21 days; the induction and differentiation culture medium is based on a high-sugar culture medium and is added with 15-20 ng / mL nerve growth factor, 10-15 ng / mL hepatocyte growth factor, 50-100 nmol / L FLI-06, 10-15 mmol / L nicotinamide, 1-3 μmol / L retinoic acid and 3-5 μg / mL insulin; S5. Preparing a preparation: mixing undifferentiated placental mesenchymal stem cells and islet beta-like cells obtained by induction and differentiation with a cell protective agent at a volume ratio of 1:2:2-1:2:3 to prepare a preparation, and freezing and storing the preparation; the cell protective agent comprises 5%-8% trehalose by mass fraction, 2%-3% human blood albumin by mass fraction, 0.1-0.3 mmol / L vitamin E, 0.5%-1% hydroxyethyl starch with a molecular weight of 200 kDa by mass fraction, and the balance of physiological saline.

2. The method for preparing a mesenchymal stem cell preparation according to claim 1, wherein The placenta in step S1 is derived from a healthy full-term woman with a gestational age of 37-40 weeks.

3. The method for preparing a mesenchymal stem cell preparation according to claim 1, wherein In step S1, the mixed digestive solution further contains 2 mmol / L ethylenediaminetetraacetic acid, and the digestion condition is 37°C shaking at a speed of 150-200 rpm for 30-45 minutes, and then the digestion of the tissue is observed under a microscope.

4. The method for preparing a mesenchymal stem cell preparation according to claim 1, wherein In step S3, the immunomagnetic bead sorting is based on surface markers of the mesenchymal stem cells, and the markers include at least three of CD29, CD44, CD90 and CD105.

5. The method for preparing a mesenchymal stem cell preparation according to claim 4, wherein The specific operation of the immunomagnetic bead sorting in step S3 includes: washing the pretreated cells with PBS buffer for 2-3 times; adding 100-200 μL of magnetic bead suspension coated with antibodies of any one of CD29, CD44, CD90 and CD105 to 5x10 6 -1x10 7 cells, mixing, incubating at 4°C for 15-30 minutes, and then performing positive cell sorting by using a magnetic bead sorting column; using magnetic beads coated with antibodies of another surface marker of CD29, CD44, CD90 and CD105 to perform step-by-step sorting on the obtained positive cells, to obtain positive cells, and then repeating the step-by-step sorting until the positive cells with the target surface marker combination are finally obtained.

6. The method for preparing a mesenchymal stem cell preparation according to claim 1, wherein The specific steps of induction and differentiation in step S4 are as follows: Stage 1: culturing for 1-7 days in a high-sugar culture medium containing 15-20 ng / mL nerve growth factor and 10-15 ng / mL hepatocyte growth factor; Stage 2: culturing for 8-14 days by adding 50-100 nmol / L FLI-06 and 10-15 mmol / L nicotinamide to the medium in stage 1; Stage 3: culturing for 15-21 days by adding 1-3 μmol / L retinoic acid and 3-5 μg / mL insulin to the medium in stage 2.

7. The method of claim 1, wherein the mesenchymal stem cell preparation is prepared by the method comprising: In step S5, the cryopreservation is performed by using a programmed cooling method, and the specific cooling program is as follows: 4 DEG C for 30 minutes, cooling to-20 DEG C at a rate of 1 DEG C / min and keeping for 1 hour, then cooling to-80 DEG C at a rate of 5 DEG C / min and keeping for at least 12 hours, and finally transferring into liquid nitrogen for long-term storage. ​ 8. A mesenchymal stem cell preparation obtained by the method of any one of claims 1 to 7, characterized in that, The placental mesenchymal stem cells, the islet beta-like cells differentiated from the placental mesenchymal stem cells and the cell protective agent are used in the preparation of a medicament for treating diabetes.

9. Use of the mesenchymal stem cell preparation according to claim 8 for the manufacture of a medicament for the treatment of diabetes, characterized in that, The diabetes includes type 1 diabetes, type 2 diabetes and diabetes-related complications, and the diabetes-related complications include diabetic retinopathy and diabetic nephropathy.

10. The use of the mesenchymal stem cell preparation according to claim 9 in the manufacture of a medicament for treating diabetes, characterized in that, The mesenchymal stem cell preparation is administered by intravenous injection.

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