Preparation method and application of mesenchymal stem cell preparation

Abstract

The application provides a preparation method and application of a mesenchymal stem cell preparation, wherein the preparation method of the mesenchymal stem cell preparation comprises the following steps: S1. sampling and separation of mesenchymal stem cells; S2. pretreatment of the mesenchymal stem cells; S3. purification and expansion of the mesenchymal stem cells; S4. induced differentiation of the mesenchymal stem cells into pancreatic beta-like cells; and S5. preparation of the preparation. The preparation method of the mesenchymal stem cell preparation provided by the application uses a placenta as a source of mesenchymal stem cells; the mesenchymal stem cells are pretreated; the mesenchymal stem cells are sorted by immunomagnetic beads; a plurality of growth factors and inducers are added to an induced differentiation culture medium; and a composite cell protective agent is used. The preparation method of the application can obtain a mesenchymal stem cell preparation which has high purity, high activity, good stability and can effectively treat diabetes.

Description

Technical Field

[0001] This application belongs to the field of cell preparation technology, and more specifically, relates to a method for preparing and applying a mesenchymal stem cell preparation. Background Technology

[0002] Diabetes is a metabolic disease characterized by hyperglycemia. Long-term hyperglycemia can lead to chronic damage and dysfunction of various tissues and organs, including the eyes, kidneys, heart, blood vessels, and nerves, seriously threatening human health. Currently, the treatment of diabetes mainly relies on insulin injections and oral hypoglycemic drugs. While these methods can control blood sugar to some extent, they cannot fundamentally repair damaged pancreatic tissue and are difficult to prevent the occurrence and development of complications.

[0003] Mesenchymal stem cells (MSCs) have shown great promise in the treatment of diabetes due to their multi-lineage differentiation potential, immunomodulatory function, and paracrine function. Regarding differentiation potential, MSCs can be induced to differentiate into pancreatic β-like cells with insulin-secreting function in vitro and in vivo. In terms of immunomodulatory function, MSCs can inhibit T cell proliferation, regulate macrophage polarization, and reduce immune rejection. Regarding paracrine function, MSCs can secrete various growth factors and cytokines, promoting tissue repair.

[0004] However, existing methods for preparing mesenchymal stem cell preparations have several shortcomings: First, stem cells are mostly obtained from bone marrow, umbilical cord, etc. Bone marrow-derived mesenchymal stem cells show decreased activity with increasing donor age, while umbilical cord-derived stem cells are subject to ethical controversies and are limited in quantity. Second, the stem cells have low purity and poor activity during preparation, resulting in low differentiation efficiency into pancreatic β-like cells, affecting therapeutic efficacy. Third, the preparations have poor stability and are prone to apoptosis during storage and transportation. Therefore, developing a method for preparing mesenchymal stem cell preparations that is convenient to obtain, highly pure, highly active, stable, and effective in treating diabetes has become an urgent problem to be solved in this field. Summary of the Invention

[0005] To achieve the above objectives, the technical solution adopted in this application is: to provide a method for preparing a mesenchymal stem cell preparation, comprising the following steps: S1. Collection and isolation of mesenchymal stem cells: Placental tissue was selected, refined, and then digested with a mixed digestive solution containing collagenase IV and trypsin. The digestion was then terminated by adding a high-glucose culture medium containing human platelet lysate. After filtration and centrifugation, the initial extract of mesenchymal stem cells was obtained. S2. Pretreatment of mesenchymal stem cells: The primary extract of mesenchymal stem cells is inoculated into a culture medium containing pretreatment factors and cultured under hypoxic conditions; the pretreatment factors consist 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% were obtained by immunomagnetic bead sorting; the purified mesenchymal stem cells were expanded and cultured, and passaged for 3-5 generations; S4. Induction of mesenchymal stem cells into pancreatic β-like cells: Expanded mesenchymal stem cells were seeded into an induction differentiation medium and cultured for 14-21 days; the induction differentiation medium was based on a high-glucose medium supplemented 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 (NAM), 1-3 μmol / L retinoic acid and 3-5 μg / mL insulin; S5. Formulation preparation: Undifferentiated placental mesenchymal stem cells and induced pancreatic β-like cells are mixed with a cell protectant at a volume ratio of 1:2:2-1:2:3 to prepare a formulation, which is then frozen and stored. The cell protectant includes 5%-8% trehalose, 2%-3% human serum albumin, 0.1-0.3 mmol / L vitamin E, 0.5%-1% hydroxyethyl starch with a molecular weight of 200 kDa, and the remainder being physiological saline.

[0006] In one embodiment, the placenta described in step S1 is derived from a healthy full-term mother at 37-40 weeks of gestation.

[0007] In one embodiment, in step S1, the mixed digestion solution further contains 2 mmol / L EDTA (ethylenediaminetetraacetic acid), and the digestion conditions are 37°C, shaker at 150-200 rpm for 30-45 minutes, followed by microscopic observation of the tissue digestion.

[0008] In one embodiment, in step S3, the immunomagnetic bead sorting is based on mesenchymal stem cell surface markers, which include at least three of CD29, CD44, CD90, and CD105.

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

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

[0011] In one embodiment, in step S5, the cryopreservation adopts a programmed cooling method. The specific cooling program is as follows: maintain 4°C for 30 minutes, cool down to -20°C at a rate of 1°C / min and maintain for 1 hour, then cool down to -80°C at a rate of 5°C / min and maintain for at least 12 hours, and finally transfer to liquid nitrogen for long-term preservation.

[0012] This application also provides a mesenchymal stem cell preparation, obtained using the preparation method provided in this application. Its characteristics include placental mesenchymal stem cells, pancreatic β-like cells induced from placental mesenchymal stem cells, and a cell protectant. The cell protectant comprises 5%-8% trehalose, 2%-3% human serum albumin, 0.1-0.3 mmol / L vitamin E, 0.5%-1% hydroxyethyl starch with a molecular weight of 200 kDa, and the balance being physiological saline.

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

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

[0015] The method for preparing mesenchymal stem cell (MSC) preparations provided in this application, compared with existing technologies, utilizes the placenta as the source of MSCs. The placenta is readily available, is postpartum waste, and therefore avoids ethical controversies. Furthermore, placental-derived MSCs exhibit high activity and strong proliferative capacity, superior to stem cells derived from traditional bone marrow and umbilical cord. Pretreatment of MSCs with a combination of pretreatment factors consisting of fibroblast growth factor, insulin-like growth factor, and hydroxybutyric acid (HbA1c) enhances their purity and activity. Adding multiple growth factors and inducers to the differentiation-inducing medium efficiently induces MSCs to differentiate into insulin-secreting pancreatic β-like cells. The addition of a composite protectant composed of trehalose, human serum albumin, vitamin E, and hydroxyethyl starch effectively reduces apoptosis and improves cell survival during cryopreservation. Therefore, the preparation method of this application yields a MSC preparation with high purity, strong activity, good stability, and effective treatment for diabetes. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This indicates the amount of insulin secreted by pancreatic β-like cells under low / high glucose conditions using different mesenchymal stem cell preparations; Figure 2 This indicates the trend of blood glucose changes in type 1 diabetic rats 1-8 weeks after administration of different mesenchymal stem cell preparations. Detailed Implementation

[0018] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and are not intended to limit the scope of this application.

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

[0020] It should be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0021] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, "multiple" means two or more, unless otherwise explicitly specified.

[0022] Please refer to the following: Figure 1 and Figure 2 The preparation method of a mesenchymal stem cell preparation provided in this application embodiment will now be described. The preparation method of the mesenchymal stem cell preparation includes the following steps: S1. Collection and isolation of mesenchymal stem cells: Placental tissue was selected and refined, digested with a mixed digestive solution containing collagenase IV and trypsin, and then digested with a high-glucose culture medium containing human platelet lysate to terminate the digestion. After filtration and centrifugation, the initial extract of mesenchymal stem cells was obtained. S2. Pretreatment of mesenchymal stem cells: The primary extract of the mesenchymal stem cells was inoculated into a culture medium containing pretreatment factors and cultured under hypoxic conditions; the pretreatment factors consisted of 5-10 ng / mL fibroblast growth factor, 2-5 ng / mL insulin-like growth factor and 1-3 mmol / L β-hydroxybutyrate; S3. Purification and expansion of mesenchymal stem cells: Mesenchymal stem cells with a purity of ≥98% were obtained by immunomagnetic bead sorting; the purified mesenchymal stem cells were expanded and cultured, and passaged for 3-5 generations; S4. Induction of mesenchymal stem cells into pancreatic β-like cells: Expanded mesenchymal stem cells were seeded into an induction differentiation medium and cultured for 14-21 days; the induction differentiation medium was based on a high-glucose medium supplemented 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 (NAM), 1-3 μmol / L retinoic acid and 3-5 μg / mL insulin; S5. Formulation preparation: Undifferentiated placental mesenchymal stem cells and induced differentiated pancreatic β-like cells are mixed with a cell protectant at a volume ratio of 1:2:2-1:2:3 to prepare a formulation, which is then frozen and stored. The cell protectant includes 5%-8% trehalose, 2%-3% human serum albumin, 0.1%-0.3 mmol / L vitamin E, 0.5%-1% hydroxyethyl starch with a molecular weight of 200 kDa, and the remainder being physiological saline.

[0023] Step S1 will now be described in detail: Compared to traditional bone marrow and umbilical cord-derived mesenchymal stem cells, placental-derived mesenchymal stem cells have the advantages of being ethically uncontroversial, having a large cell reserve, and exhibiting high activity and stability. Specifically, the placenta is a postpartum waste product and does not require additional invasive collection; a single placenta can yield 1×102 8 -5×10 8 Each initial mesenchymal stem cell, far exceeding the 1×10⁻⁶ cells from a single umbilical cord. 7 -5×10 7 Placental tissue has a low degree of development, and its stem cell differentiation potential and proliferation capacity are significantly better than those from bone marrow, and it is not affected by the donor's age, making it more widely applicable. For placental fragmentation, surgical scissors or a tissue dissociator can be used, ensuring the fragments are as small as possible to facilitate thorough digestion. Collagenase IV can specifically degrade collagen fibers in placental tissue, avoiding damage to stem cell surface receptors. Trypsin can gently break down intercellular adhesion proteins, promoting cell dispersion and preventing cell rupture due to over-digestion. Human platelet lysate contains abundant growth factors, which can rapidly repair cell surface damage during digestion, enhance initial cell activity, and avoid the "foreign protein contamination risk" of fetal bovine serum, reducing the probability of immune rejection in subsequent clinical applications. High-glucose culture medium contains abundant glucose, which can promote the proliferation of mesenchymal stem cells. After digestion is terminated, filtration and centrifugation are performed to remove incompletely digested tissue blocks, vascular fragments, and impurities such as enzymatic hydrolysates and dead cell fragments in the supernatant, obtaining a high-purity and highly active initial mesenchymal stem cell extract.

[0024] Step S2 is explained in detail below: Hypoxic culture, with an oxygen concentration of 1%-5%, can reduce the generation of reactive oxygen species (ROS) in mesenchymal stem cells, thereby reducing oxidative damage to cells; it can also induce the expression of hypoxia-inducible factor-1 (HIF-1), which in turn promotes the expression of vascular endothelial growth factor (VEGF) and enhances cell proliferation. Fibroblast growth factor can activate the MAPK / ERK pathway, promoting stem cell proliferation and early lineage orientation. Insulin-like growth factor can activate the PI3K / Akt pathway, enhancing the cell's anti-apoptotic ability, while upregulating the basal expression of key genes for pancreatic islet development. β-hydroxybutyrate (β-hydroxybutyrate), a ketone body metabolite, can mimic the metabolic microenvironment under fasting conditions, 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] Step S3 is described in detail below: Immunomagnetic bead sorting is a highly efficient method for cell separation and purification. It is based on the property that cell surface antigens can bind to specific antibodies linked to magnetic beads. In an external magnetic field, cells linked to the magnetic beads by antibodies are adsorbed and retained in the magnetic field, while cells without the surface antigens cannot bind to the specific antibodies linked to the magnetic beads and are therefore non-magnetic, thus not remaining in the magnetic field, achieving cell separation. Magnetic bead cell sorting requires simple equipment, does not demand highly skilled technicians, and yields cells with high sensitivity, good purity, and good cell viability and recovery rate. It has minimal impact on downstream applications and has broad application prospects. Mesenchymal stem cells should be passaged 3-5 times to achieve a balance between cell number and viability. If the passage number is too high, cells are prone to senescence, manifested as slower proliferation and decreased differentiation potential.

[0026] Step S4 is explained in detail below: Nerve growth factor and hepatocyte growth factor promote the differentiation of stem cells into the endoderm and upregulate the expression of endoderm markers such as Sox17 and FoxA2, ​​thus directing subsequent islet lineage differentiation. FLI-06 is an inhibitor of the Notch signaling pathway, which can block the undifferentiated state of stem cells and promote the differentiation of cells into islet precursor cells. Nicotinamide is a PARP inhibitor, which can inhibit islet precursor cell apoptosis and upregulate the expression of PDX-1, a key transcription factor 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 the β-cell lineage, and prevent differentiation into α-cells that secrete glucagon. Insulin, as an autocrine or paracrine signal, can mimic the in vivo insulin feedback regulation and promote the maturation of islet β-like cells. The differentiation of islet β-like cells is a gradual maturation process, therefore requiring a certain culture time.

[0027] Step S5 is described in detail below: Undifferentiated placental mesenchymal stem cells and induced pancreatic β-like cells are mixed with a cell protectant at a volume ratio of 1:2:2 to 1:2:3 to ensure sufficient cell protection without excessively low cell concentration. The cell protectant improves the storage stability of cells in the formulation and prevents excessive decline in cell viability during transportation. Trehalose forms a glassy protective layer on the cell surface, preventing ice crystal formation and damage to the cell membrane during freezing and maintaining stable cell osmotic pressure. Human serum albumin binds to the cell surface, reducing cell aggregation and providing nutrients to prevent apoptosis due to malnutrition. Vitamin E, as an antioxidant, scavenge reactive oxygen species generated during storage, reducing oxidative damage to cells and preventing functional decline such as insulin secretion caused by oxidative stress. Hydroxyethyl starch has high colloidal osmotic pressure, maintaining the stability of cell suspensions and preventing cell precipitation. Its molecular weight of 200 kDa also prevents it from entering the interstitial space through capillaries, reducing the risk of adverse reactions.

[0028] The method for preparing mesenchymal stem cell (MSC) preparations provided in this application, compared with existing technologies, utilizes the placenta as the source of MSCs. The placenta is readily available, is postpartum waste, and therefore avoids ethical controversies. Furthermore, placental-derived MSCs exhibit high activity and strong proliferative capacity, superior to stem cells derived from traditional bone marrow and umbilical cord. Pretreatment of MSCs with a combination of pretreatment factors consisting of fibroblast growth factor, insulin-like growth factor, and hydroxybutyric acid (HbA1c) enhances their purity and activity. Adding multiple growth factors and inducers to the differentiation-inducing medium efficiently induces MSCs to differentiate into insulin-secreting pancreatic β-like cells. The addition of a composite protectant composed of trehalose, human serum albumin, vitamin E, and hydroxyethyl starch effectively reduces apoptosis and improves cell survival during cryopreservation. Therefore, the preparation method of this application yields a MSC preparation with high purity, strong activity, good stability, and effective treatment for diabetes.

[0029] In one embodiment, the placenta described in step S1 is derived from a healthy full-term mother at 37-40 weeks of gestation.

[0030] Specifically, the placenta is fully developed between 37 and 40 weeks of gestation, and the number of stem cells in the tissue reaches its peak and their activity is stable. The placenta of healthy full-term mothers is less likely to be affected by pathogen infection, developmental abnormalities, or other factors, which greatly reduces the risk of stem cell contamination or potential defects, and is beneficial for the subsequent preparation of high-purity, high-activity mesenchymal stem cell preparations.

[0031] In one embodiment, in step S1, the mixed digestion solution further contains 2 mmol / L EDTA (ethylenediaminetetraacetic acid), and the digestion conditions are 37°C, shaker at 150-200 rpm for 30-45 minutes, followed by microscopic observation of the tissue digestion. Specifically, EDTA is a calcium ion chelating agent that can disrupt calcium-dependent adhesion junctions between cells, assisting trypsin in more efficiently breaking down the intercellular matrix. Simultaneously, EDTA can inhibit the autodegradation of some proteases, prolonging the duration of enzyme activity and preventing incomplete digestion due to enzyme activity decay. In addition, collagenase, dispersinase, and deoxyribonuclease can also be added to the mixed digestion solution.

[0032] The mixed digestion solution contains active enzymes such as collagenase IV and trypsin. 37°C is the physiological temperature of the human body, the optimal temperature for enzyme activity. A rotation speed of 150-200 rpm ensures sufficient contact between the digestion solution and the tissue block, while avoiding mechanical damage to cells caused by excessive rotation speed. Due to the complex composition of tissues, too short a digestion time will lead to incomplete tissue breakdown and reduced cell yield, while too long a digestion time will cause excessive enzymatic digestion, resulting in cell membrane damage and decreased cell viability. Microscopic observation of the tissue digestion after digestion ensures that the tissue block is fully digested and controls the quality of mesenchymal stem cell extraction.

[0033] In one embodiment, in step S3, the immunomagnetic bead sorting is based on mesenchymal stem cell surface markers, which include at least three of CD29, CD44, CD90, and CD105.

[0034] Specifically, CD29, CD44, CD90, and CD105 are all internationally recognized specific surface markers for human mesenchymal stem cells (MSCs). CD29, also known as integrin β1, participates in cell adhesion and matrix interactions, and is stably expressed in MSCs but not in hematopoietic stem cells or immune cells. CD44 is a hyaluronic acid receptor involved in the regulation of MSC proliferation and migration. CD90, also known as Thy-1, is a glycoprotein molecule highly expressed in undifferentiated MSCs, gradually downregulated with cell differentiation, and can serve as a marker of the undifferentiated state of MSCs. CD105 is an endothelial glycoprotein involved in the regulation of the TGF-β signaling pathway and is closely related to the immunomodulatory function of MSCs.

[0035] In addition to mesenchymal stem cells, placental tissue contains various other cell types. Screening with a single biomarker may result in cross-reactivity; for example, CD44 is also expressed at low levels in some hematopoietic cells. Therefore, using a combination of at least three biomarkers for immunomagnetic bead sorting helps improve the cell purity of mesenchymal stem cells, remove contaminating hematopoietic cells, fibroblasts, and other impurities, and avoid their interference with subsequent induced differentiation.

[0036] In one embodiment, the specific operation of immunomagnetic bead sorting in step S3 includes: washing the pretreated cells 2-3 times with PBS buffer; sorting the cells at 5×10⁻⁶ beads per cell. 6 -1×10 7 Cells are mixed with 100-200 μL of magnetic bead suspension coated with any one of the antibodies of CD29, CD44, CD90, and CD105, and incubated at 4°C for 15-30 minutes. Positive cells are then screened using a magnetic bead sorting column. The positive cells obtained are then further sorted using magnetic beads coated with another surface marker antibody of CD29, CD44, CD90, and CD105 to obtain positive cells. This stepwise sorting process is repeated until positive cells with the target surface marker combination are finally obtained.

[0037] Specifically, washing with PBS buffer 2-3 times can effectively remove human serum albumin components and residual factors from the pretreated culture medium, preventing them from interfering with the specific binding of antibodies to cell surface markers and affecting antibody binding efficiency. (Following a 5×10⁻⁶ ppm ratio...) 6 -1×10 7 Cells are mixed with 100-200 μL of magnetic bead suspension containing surface marker antibodies. This ratio ensures sufficient contact between the cells and the sorting magnetic beads and avoids non-specific binding caused by excessive antibody.

[0038] Magnetic bead sorting columns are essential consumables in immunomagnetic bead sorting technology. They are filled with magnetic particles, and when placed in a strong magnetic field, a high-intensity, gradient magnetic field is created within the column. When the mixture of labeled magnetic beads and cells flows through the column, the target cells labeled with the beads are attracted to the magnetic particles within the column—these are positive cells. Unlabeled cells, unaffected by the magnetic field, flow directly through the column—these are negative cells. After separation, the column is removed from the magnetic field, and the adsorbed positive cells are eluted, yielding highly purified target cells. Immunomagnetic bead sorting cannot label and identify multiple cells; therefore, it requires a step-by-step sorting process.

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

[0040] Specifically, the differentiation of pancreatic β-like cells is a gradual maturation process. Therefore, adding different differentiation-inducing substances at different stages is a scientific regulation of the differentiation process of mesenchymal stem cells based on the biological laws of cell differentiation. Specifically, stage 1 induces endoderm differentiation, which is the origin layer of pancreatic islet cells; stage 2 induces the formation of pancreatic islet precursor cells; and stage 3 induces the maturation of pancreatic β-like cells, enabling them to secrete insulin.

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

[0042] Specifically, cell cryopreservation requires careful attention to avoid mechanical damage to cell membranes and organelles caused by ice crystal formation. Rapid cooling can cause sharp ice crystals to form inside and outside the cell, piercing the cell membrane and leading to cell death. Conversely, slow cooling can cause excessive dehydration, disrupting osmotic pressure balance. Therefore, a suitable cooling rate is necessary to allow intracellular water to seep out in an orderly manner and form a vitrified state outside the cell, thereby maximizing the preservation of cell viability.

[0043] Setting the temperature to 4°C as the initial cooling stage helps cells gradually adapt to the low-temperature environment, reducing the stress response caused by a sudden temperature drop. A 30-minute pre-cooling time is sufficient to reduce the cell metabolic rate, preventing cellular shock caused by direct exposure to low temperatures. A slow cooling rate of 1°C / min helps prevent intracellular ice crystal formation because extracellular water freezes first, and the resulting osmotic pressure gradient causes intracellular water to slowly seep out. Maintaining a temperature of -20°C for 1 hour allows for sufficient and stable extracellular ice crystal formation, while simultaneously allowing the remaining intracellular water to further balance through osmosis. Once the temperature drops below -20°C, the extracellular fluid is completely solidified; at this point, the cooling rate can be increased to 5°C / min, promoting the formation of amorphous glassy states from the remaining intracellular water. Maintaining a temperature of -80°C for at least 12 hours ensures complete temperature equilibrium between the inside and outside of the cell, while also verifying the initial stability of the cells at ultra-low temperatures, avoiding the temperature shock caused by direct transfer to liquid nitrogen. The ultra-low temperature environment of liquid nitrogen completely halts cell metabolism, preventing cell aging, apoptosis, or functional decline, significantly increasing cell preservation time.

[0044] This application also provides a mesenchymal stem cell preparation, obtained by the preparation method of the mesenchymal stem cell preparation provided in this application, comprising placental mesenchymal stem cells, pancreatic β-like cells induced to differentiate from placental mesenchymal stem cells, and a cell protectant. The cell protectant comprises 5%-8% trehalose, 2%-3% human serum albumin, 0.1-0.3 mmol / L vitamin E, 0.5%-1% hydroxyethyl starch with a molecular weight of 200 kDa, and the balance being physiological saline.

[0045] Specifically, the mesenchymal stem cell preparation of this application contains pancreatic β-like cells derived from placental mesenchymal stem cells and with high insulin secretion; and improves cell activity under long-term storage conditions by improving the cell protectant formulation.

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

[0047] Specifically, type 1 diabetes is mainly caused by autoimmune abnormalities leading to the destruction of pancreatic β cells and an absolute lack of insulin. The pancreatic β-like cells in the mesenchymal stem cell preparation of this application can directly replenish insulin-secreting cells, while the immunomodulatory function of placental mesenchymal stem cells can reduce autoimmune attacks and delay further damage to pancreatic β cells. Type 2 diabetes is characterized by insulin resistance and pancreatic β cell dysfunction. The mesenchymal stem cell preparation of this application can promote insulin secretion by pancreatic β-like cells and improve insulin sensitivity in tissues such as the liver and muscles through cytokines secreted by placental mesenchymal stem cells, thereby reducing insulin resistance.

[0048] Diabetes-related complications arise from long-term hyperglycemia damaging multiple tissues and organs throughout the body. Among these, diabetic retinopathy is caused by microvascular damage and ischemia / hypoxia in the retina due to hyperglycemia. The mesenchymal stem cell preparation described in this application can promote vascular repair through the paracrine effects of placental mesenchymal stem cells, while simultaneously inhibiting abnormal neovascularization in the retina. Diabetic nephropathy is caused by long-term hyperglycemia leading to glomerular mesangial proliferation and decreased renal function. Placental mesenchymal stem cells can reduce the levels of inflammatory factors such as TNF-α and IL-6, inhibiting renal inflammatory responses and reducing mesangial matrix deposition.

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

[0050] Specifically, intravenous injection allows pancreatic β-like cells and mesenchymal stem cells in the preparation to be directly delivered to target organs throughout the body via the bloodstream. Compared to local injection, intravenous injection is simpler to perform, less invasive, has higher patient compliance, and can act on multiple lesion sites simultaneously. Intravenous injection is a routine clinical method of drug administration, requires no complex surgical equipment, and can be performed even in primary care hospitals.

[0051] The present invention will be further illustrated below through specific embodiments: Example 1 The preparation method of the mesenchymal stem cell preparation in this embodiment includes the following steps: S1. Collection and isolation of mesenchymal stem cells: Placenta from healthy full-term mothers at 37 weeks of gestation was selected. Under aseptic conditions, the chorionic villus was removed, the amnion and blood vessels were removed, and the placenta was rinsed 5 times with 0.9% saline until no blood was visible. The placenta was then cut into pieces of approximately 2 mm. 3 Tissue fragments were collected; the tissue fragments were placed in centrifuge tubes and digested with a mixed digestion solution containing 0.15% collagenase IV, 0.25% trypsin and 2 mmol / L EDTA. The digestion conditions were 37°C and 150 rpm for 45 minutes on a shaker. The digestion was then observed under a microscope. The digestion was terminated by adding DMEM / F12 high glucose medium containing 10% human platelet lysate. After filtration through a 200-mesh sieve, the mixture was centrifuged at 1100 rpm for 9 minutes, the supernatant was discarded, and the precipitate was resuspended in PBS (phosphate buffer) to obtain the initial mesenchymal stem cell extract.

[0052] S2. Pretreatment of mesenchymal stem cells: The initial extract of mesenchymal stem cells was processed at 5×10⁻⁶... 4 Cells / cm² were seeded in low-adsorption culture dishes. The culture medium contained pretreatment factors consisting of 5 ng / mL fibroblast growth factor, 5 ng / mL insulin-like growth factor, and 3 mmol / L β-hydroxybutyric acid. The culture dishes were placed in an incubator and cultured under a low-oxygen environment of 37°C, 3% O2, and 5% CO2.

[0053] S3. Purification and Expansion of Mesenchymal Stem Cells: Mesenchymal stem cells with a purity ≥98% were obtained through immunomagnetic bead sorting; the purified mesenchymal stem cells were then expanded and cultured, passaged for 3 generations. Immunomagnetic bead sorting is based on the surface markers of mesenchymal stem cells, namely CD29, CD44, CD90, and CD105. The specific operation is as follows: Wash the pretreated cells three times with PBS buffer; at a dose of 1×10⁻⁶ cells / day. 7 Cells were added to 200 μL of magnetic bead suspension coated with CD29 antibody and mixed. After incubation at 4°C for 30 minutes, positive cells were screened using a MACS sorting column. The selected CD29 cells were then... +Positive cells were then further sorted using magnetic beads coated with CD44 antibody to obtain positive cells; the selected CD29 cells were then further sorted. + CD44 + Positive cells were then further sorted using magnetic beads coated with CD90 antibody to obtain positive cells; the selected CD29 cells were then further sorted. + CD44 + CD90 + Positive cells were then sorted stepwise using magnetic beads coated with CD105 antibody to finally obtain CD29. + CD44 + CD90 + CD105 + Positive cells.

[0054] S4. Induction of mesenchymal stem cells into pancreatic β-like cells: Expanded mesenchymal stem cells were seeded in induction differentiation medium and cultured for 21 days; the specific steps for induction differentiation were as follows: Phase 1: Culture for 1-7 days in DMEM / F12 high-glucose medium containing 15 ng / mL nerve growth factor and 10 ng / mL hepatocyte growth factor; Phase 2: Culture for 8-14 days, adding 50 nmol / L FLI-06 and 10 mmol / L nicotinamide to the Phase 1 culture medium; Phase 3: Culture for 15-21 days, adding 1 μmol / L retinoic acid and 3 μg / mL insulin to the Phase 2 culture medium.

[0055] S5. Formulation preparation: Undifferentiated placental mesenchymal stem cells and induced differentiated pancreatic β-like cells were mixed with a cell protectant at a volume ratio of 1:2:2 to prepare the formulation, and the formulation was cryopreserved using a programmed cooling method. The specific cooling program was as follows: 4℃ for 30 minutes, cooling to -20℃ at a rate of 1℃ / min and holding for 1 hour, then cooling to -80℃ at a rate of 5℃ / min and holding for at least 12 hours, and finally transferring to liquid nitrogen for long-term storage.

[0056] The cell protectant consists of 5% trehalose, 2% human serum albumin, 0.1 mmol / L vitamin E, 1% hydroxyethyl starch with a molecular weight of 200 kDa, and the remainder physiological saline.

[0057] The mesenchymal stem cell preparation of this embodiment is obtained using the preparation method of the mesenchymal stem cell preparation of this embodiment.

[0058] The mesenchymal stem cell preparation in this embodiment is applied to type 1 diabetes and is administered via intravenous injection.

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

[0060] Example 3 Based on Example 1, the difference is that the induction of mesenchymal stem cells into pancreatic β-like cells in step S4 is not divided into stages, that is: the expanded mesenchymal stem cells are seeded into the induction differentiation medium and cultured for 14-21 days; the induction differentiation medium is based on high glucose medium, with the addition of 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.

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

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

[0063] Based on Example 1, the difference is that in step S5, the cell protectant consists of 10% DMSO (dimethyl sulfoxide) and 20% fetal bovine serum.

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

[0065] This experiment used a human insulin (ultra-sensitive) enzyme-linked immunosorbent assay kit, purchased from Beyotime Biotechnology, catalog number PI608. The specific steps are as follows, referring to the kit instructions: (1) Group settings: For each example and comparative example, a low sugar group and a high sugar group were set up, with 3 replicates for each group; a blank control group was also set up with 3 replicates; and 3 background correction wells, i.e. blank wells, were set up, with only TMB solution and stop solution added to the wells.

[0066] (2) Take the pancreatic β-like cell culture dishes obtained from the induced differentiation of each example and comparative example, remove the induction differentiation medium, add 1×PBS to wash 3 times, and discard the PBS. Operate according to the following settings: Low glucose group: Add 10 mL of DMEM / F12 medium containing 2.8 mM glucose; High glucose group: Add 10 mL of DMEM / F12 medium containing 16.7 mM glucose; Blank control group: Take a new culture dish and add 10 mL of DMEM / F12 medium.

[0067] (3) Then, incubate the culture dishes of each group in a cell culture incubator for 2 hours. Collect the supernatant of each group, centrifuge the supernatant at 500g for 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, let it equilibrate at room temperature (25-28℃) for 20 minutes. Dilute the washing buffer (20X) with double-distilled water or deionized water to 1X. Add the standard diluent to one bottle of standard according to the volume indicated on the standard label and incubate at room temperature for 15 minutes. Take 5 clean 1.5 ml centrifuge tubes, add 250 μL of standard diluent to each tube in advance, and then perform serial dilution of the standard to obtain six standard concentrations of 200, 100, 50, 25, 12.5 and 6.25 mIU / L. Finally, add the diluted standard to the wells of the pre-coated plate in sequence. The standard diluent is added directly as the 0 mIU / L concentration, for a total of seven standard concentrations.

[0069] (5) Sample addition: Take out the pre-coated strips required for the experiment and place them in the 96-well frame; add the test sample or different concentration standards to the corresponding wells at 25 μL / well; immediately add horseradish peroxidase-labeled human insulin antibody at 100 μL / well; seal the reaction wells with a sealing film (clear) and incubate at room temperature for 120 minutes; wash the plate 5 times, and pat dry on thick absorbent paper on the last wash; add chromogenic reagent TMB solution at 100 μL / well, seal the reaction wells with a sealing film (white), and incubate at room temperature in the dark for 10 minutes. (6) Detection: Add stop solution at 50 μL / well, mix well and immediately measure the A450 absorbance using an ELISA reader.

[0070] (7) Data processing: The absorbance value of each standard or test sample should be reduced by the absorbance value of the background correction well. Use the test data of the standard to create a standard curve, and then calculate the insulin secretion of the test sample based on the standard curve.

[0071] (8) Results Analysis: Figure 1 This indicates the insulin secretion levels of pancreatic β-like cells under low / high glucose conditions using different mesenchymal stem cell preparations. For example... Figure 1 As shown, under high-glucose environment stimulation, the insulin secretion of pancreatic β-like cells in Examples 1-3 was significantly higher than that in Comparative Examples 1-3, indicating that the mesenchymal stem cell preparation of this application has the potential to treat diabetes; the insulin secretion of pancreatic β-like cells in Example 1 was significantly higher than that in Examples 2-3, indicating that placenta derived from healthy full-term mothers at 37-40 weeks of gestation or staged induction differentiation steps can further improve the insulin secretion function of pancreatic β-like cells.

[0072] Experiment Example 2 The efficacy of mesenchymal stem cell preparations in treating diabetes was verified using a type 1 diabetic rat model.

[0073] In this experiment, 6-8 week old SPF-grade male SD rats, weighing 200-250g, were used. Streptozotocin (STZ, dissolved in 0.1mol / L citrate buffer) was used to establish the rat model. The specific steps are as follows: (1) Modeling: The rats were fasted for 12 hours before administration, but water was allowed. The rats were administered the drug via intraperitoneal injection while fasting. The model group, each example, and the comparative group were injected with STZ solution at a dose of 60 mg / kg based on the rats' body weight; the control group was injected with an equal volume of 0.1 mol / L citrate buffer. After the injection, the rats were returned to their cages and fed normally, with sufficient food, water, and space for activity.

[0074] (2) Blood was collected from the tail vein 72 hours later (fasting for 6 hours before blood collection). Fasting blood glucose was measured with a blood glucose meter. If the blood glucose was ≥16.7mmol / L for two consecutive times, the diabetes model was considered successful and could be included in the experiment.

[0075] (3) Group setup: A control group, a model group, and control and comparative groups were set up. Rats that successfully developed the model were randomly assigned to each group, with 10 rats in each group. The control and model groups were injected with 0.2 mL of 0.9% saline via the tail vein; the control and comparative groups were injected with a mesenchymal stem cell preparation with a cell concentration of 5 × 10⁻⁶ cells / mL. 6 The dosage is 0.2 mL, cells / mL. Before administration, allow the mesenchymal stem cell preparation to reach room temperature and mix gently to avoid cell aggregation.

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

[0077] (5) Results Analysis: Figure 2 This indicates the blood glucose change trends in type 1 diabetic rats 1-8 weeks after administration of different mesenchymal stem cell preparations. For example... Figure 2As shown, the blood glucose level in the control group remained within the normal range (5.1-5.3 mmol / L), while the blood glucose level in the model group continued to rise over time, verifying the stability of the diabetes model. The decrease in blood glucose level at each time point in Examples 1-3 was significantly better than that in their comparative groups 1-3, and after 8 weeks of treatment, it was close to that of the control group, demonstrating the excellent efficacy of this application in treating type 1 diabetes. In the examples, the therapeutic effect of Example 1 was better than that of Examples 2-3, corresponding to the results of Experiment 1, indicating that placenta derived from healthy full-term mothers with a gestational age of 37-40 weeks or that the induction differentiation process can further improve the therapeutic effect of mesenchymal stem cell preparations on type 1 diabetes.

[0078] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing a mesenchymal stem cell preparation, characterized in that, Includes the following steps: S1. Collection and isolation of mesenchymal stem cells: Placental tissue was selected, refined, and then digested with a mixed digestive solution containing collagenase IV and trypsin. The digestion was then terminated by adding a high-glucose culture medium containing human platelet lysate. After filtration and centrifugation, the initial extract of mesenchymal stem cells was obtained. The placental tissue was selected from the placenta of a healthy full-term mother at 37 weeks of gestation. S2. Pretreatment of mesenchymal stem cells: The primary extract of mesenchymal stem cells is inoculated into a culture medium containing pretreatment factors and cultured under hypoxic conditions; the pretreatment factors consist 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 ≥98% were obtained by immunomagnetic bead sorting; the purified mesenchymal stem cells were expanded and cultured, passaged for 3-5 generations; wherein, the immunomagnetic bead sorting was based on surface markers of mesenchymal stem cells, namely CD29, CD44, CD90, and CD105; S4. Induction Differentiation of Mesenchymal Stem Cells into Pancreatic β-like Cells: The expanded mesenchymal stem cells were seeded in an induction differentiation medium and cultured for 14-21 days; the induction differentiation medium was based on a high-glucose medium supplemented with 15-20 ng / mL nerve growth factor, 10-15 ng / mL hepatocyte growth factor, and 50 The induction of differentiation involved the following steps: -100 nmol / L FLI-06, 10-15 mmol / L nicotinamide, 1-3 μmol / L retinoic acid, and 3-5 μg / mL insulin; Stage 1: Culture for 1-7 days in DMEM / F12 high-glucose medium containing 15 ng / mL nerve growth factor and 10 ng / mL hepatocyte growth factor; Stage 2: Culture for 8-14 days, adding 50 nmol / L FLI-06 and 10 mmol / L nicotinamide to the Stage 1 medium; Stage 3: Culture for 15-21 days, adding 1 μmol / L retinoic acid and 3 μg / mL insulin to the Stage 2 medium. S5. Formulation preparation: Undifferentiated placental mesenchymal stem cells and induced differentiated pancreatic β-like cells are mixed with a cell protectant at a volume ratio of 1:2:2 to 1:2:3 to prepare a formulation, which is then frozen and stored. The cell protectant consists of 5%-8% trehalose, 2%-3% human serum albumin, 0.1-0.3 mmol / L vitamin E, 0.5%-1% hydroxyethyl starch with a molecular weight of 200 kDa, and the remainder being physiological saline.

2. The method for preparing the mesenchymal stem cell preparation as described in claim 1, characterized in that, In step S1, the mixed digestion solution also contains 2 mmol / L ethylenediaminetetraacetic acid. The digestion conditions are 37°C, shaker at 150-200 rpm for 30-45 minutes, and then the tissue digestion is observed under a microscope.

3. The method for preparing the mesenchymal stem cell preparation as described in claim 1, characterized in that, The specific procedures for immunomagnetic bead sorting in step S3 include: washing the pretreated cells 2-3 times with PBS buffer; sorting the cells at 5 × 10⁻⁶ beads per cell. 6 -1×10 7 Cells are mixed with 100-200 μL of magnetic bead suspension coated with any one of the antibodies of CD29, CD44, CD90, or CD105, and incubated at 4°C for 15-30 minutes. Positive cells are then sorted using a magnetic bead sorting column. The positive cells obtained from the screening are then further sorted using magnetic beads coated with another surface marker antibody of CD29, CD44, CD90, or CD105 to obtain positive cells in a stepwise manner. This stepwise sorting is repeated until positive cells with the target surface marker combination are finally obtained.

4. The method for preparing the mesenchymal stem cell preparation as described in claim 1, characterized in that, In step S5, the cryopreservation adopts a programmed cooling method. The specific cooling program is as follows: maintain 4°C for 30 minutes, cool down to -20°C at a rate of 1°C / min and maintain for 1 hour, then cool down to -80°C at a rate of 5°C / min and maintain for at least 12 hours, and finally transfer to liquid nitrogen for long-term preservation.

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