Heart function preparation as well as preparation method and application thereof
By fusing endothelial progenitor cells with cardiac extracellular matrix composite cell spheres and cardiomyocyte-like cell membranes, a cardiac function preparation was formed, which solved the problem of insufficient number of surviving cells in stem cell transplantation and achieved a significant improvement in myocardial repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-26
- Publication Date
- 2026-03-13
AI Technical Summary
Existing stem cell transplantation methods suffer from insufficient number of surviving cells and low survival rates, which limit myocardial repair function and fail to effectively reverse myocardial cell death and cardiac function decline.
Endothelial progenitor cells were cultured with cardiac extracellular matrix to form composite cell spheres, which were then fused with myocardial-like cell membrane sheets. After being cut into particles, the spheres were mixed with poloxamer P407 solution to form a cardiac function preparation, which was then injected intraperitoneally to cover the myocardial infarction area.
It significantly improved the retention and survival rate of stem cells in the infarct area, enhanced paracrine function, reduced surgical trauma, and improved the effect of myocardial repair.
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Figure CN121648162A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of myocardial repair stem cell preparation technology, specifically relating to a cardiac function preparation, its preparation method, and its application. Background Technology
[0002] Cardiovascular disease is the leading cause of death and disability, with myocardial infarction being the most common and critical heart disease. This type of disease not only consumes enormous medical resources but also places an increasingly heavy burden on society. Current clinical treatments for myocardial infarction mainly include percutaneous coronary intervention, thrombolysis, and bypass grafting. While these methods can alleviate symptoms by restoring blood flow to the ischemic area, they cannot regenerate the infarcted myocardium, nor can they reverse the decline in cardiac function and ventricular remodeling caused by massive myocardial cell death, making end-stage heart failure difficult to avoid. Drug therapy can only slow disease progression and cannot reverse myocardial necrosis and cardiac function deterioration; while heart transplantation offers a glimmer of hope for end-stage patients, the severe shortage of donors prevents it from being used as a routine treatment.
[0003] Against this backdrop, cell transplantation, as a core implementation method of stem cell repair technology in the field of tissue engineering therapy, has brought new possibilities for myocardial repair. This technology precisely delivers stem cells to the site of myocardial injury through specific delivery methods, mediating myocardial repair. Numerous studies have confirmed that stem cell-based cell transplantation can exert a cardioprotective effect through the core mechanisms of stem cell repair: on the one hand, transplanted stem cells can differentiate into cardiomyocyte-like cells, directly replacing necrotic cardiomyocytes in the infarcted area; on the other hand, they can release bioactive molecules through paracrine effects, synergistically improving tissue elasticity in the infarcted area, stimulating angiogenesis, and limiting the thinning of the infarcted area, thereby blocking the progression of left ventricular enlargement and the occurrence of progressive heart failure. However, stem cell transplantation-mediated cardiac repair still faces severe challenges, with the extremely low survival rate of transplanted cells being the core bottleneck—studies show that approximately 90% of stem cells successfully injected into the heart almost completely disappear within about 48 hours after transplantation. This problem directly restricts both core pathways of stem cell repair: insufficient number of surviving cells makes it difficult to meet the total number of cells differentiated towards myocardium for tissue repair needs, while the paracrine function of surviving cells is also significantly limited due to massive cell apoptosis, ultimately severely restricting the clinical therapeutic effect of stem cell transplantation.
[0004] Hydrogels, as a class of polymeric materials with a three-dimensional network structure, possess advantages such as mimicking the extracellular matrix, excellent biocompatibility, and porous structure. They not only provide mechanical support to infarcted areas but also serve as carriers for targeted drug and cell delivery, showing great promise in myocardial repair research. However, existing formulations still have shortcomings in enhancing the myocardial phenotypic differentiation of stem cells, improving paracrine function, and further increasing the long-term retention and survival of cells in infarcted areas; an efficient and stable solution has not yet been formed. Therefore, developing a stem cell formulation that can effectively enhance the myocardial phenotype of stem cells and improve cell survival rate to strengthen paracrine function is of great significance for promoting the clinical application of stem cell myocardial repair therapy. Summary of the Invention
[0005] In order to overcome the shortcomings of the prior art, the present invention aims to provide a cardiac function preparation, its preparation method and application, to solve the problem that the number of surviving cells is insufficient in stem cell transplantation to repair myocardium, resulting in limited repair function.
[0006] To achieve the above objectives, the present invention employs the following technical solution: The first aspect of this invention discloses a method for preparing a cardiac function preparation, comprising the following steps: 1) Endothelial progenitor cells were mixed with cardiac extracellular matrix and cultured to obtain composite cell spheres; 2) Mix the myocardial cell membrane sheet with the composite cell spheres obtained in step 1) and culture them. After the composite cell spheres are evenly distributed on the cell membrane sheet and show a tendency to fuse, cut them into pieces to obtain cell membrane sheet-cell sphere composite particles. 3) Mix the cell membrane sheet-cell sphere composite particles obtained in step 2) with poloxamer P407 solution to obtain a cardiac function preparation.
[0007] Preferably, in step 1), the ratio of endothelial progenitor cells to cardiac extracellular matrix is (0.5~2)×10⁻⁶. 6 Cells: 1 mg.
[0008] Preferably, in step 1), the extracellular matrix of the heart cells is porcine extracellular matrix of the heart cells.
[0009] More preferably, the method for preparing the porcine heart extracellular matrix is as follows: fresh adult porcine hearts are washed, frozen, and then thawed, followed by sequential reverse perfusion with 2×PBS, a mixed solution of 0.02% trypsin / 0.05% EDTA / 0.05% sodium azide, and 3%... Triton X The extracellular matrix of porcine heart cells was obtained by sterilizing a mixture of 100 / 0.05% EDTA / 0.05% sodium azide and 4% deoxycholic acid solution with a mixture of 0.1% peracetic acid / 4% ethanol solution, washing with PBS and ultrapure water in sequence, lyophilizing the sections, pulverizing and sieving, retaining 0.2~0.5 mm particles.
[0010] Preferably, in step 1), the cardiac extracellular matrix is pre-cultured in α-MEM medium containing a penicillin-streptomycin mixed solution for 22-26 h to obtain matrix microcarriers; endothelial progenitor cell suspension is inoculated into the matrix microcarriers and cultured for 3-5 days to obtain composite cell spheres.
[0011] More preferably, the concentration of the endothelial progenitor cell suspension is (1~5)×10⁻⁶. 6 Endothelial progenitor cell suspension per mL.
[0012] Preferably, in step 2), the cardiomyocytes are induced from mesenchymal stem cells.
[0013] Preferably, in step 2), the method for preparing the cardiomyocyte-like cell sheet is as follows: a concentration of 1×10⁻⁶ cells is used. 5 A suspension of cardiomyocytes per mL was seeded into a petri dish. After 2-4 days of confluence, the culture medium was replaced with complete medium containing 50 μg / mL vitamin C. The medium was changed every other day, and the culture was continued until a membrane was formed to obtain a cardiomyocyte membrane.
[0014] Preferably, in step 2), during mixed culture, 0.5 to 5 mg of composite cell spheres are added to each square centimeter of myocardial-like cell membrane.
[0015] Preferably, in step 2), the diameter of the cell membrane sheet-cell sphere composite particle is 0.3~1 mm.
[0016] Preferably, in the cardiac function preparation, the mass concentration of cell membrane sheet-cell sphere composite particles is 2%~20%, and the mass concentration of poloxamer P407 is 16%~18%.
[0017] Preferably, the cardiac function preparation also includes a nutrient solution, wherein the cell membrane sheet-cell sphere composite particles, poloxamer P407 solution and nutrient solution are mixed to obtain the cardiac function preparation; The nutrient solution contains one or more of the following components: collagen, hyaluronic acid, fibronectin, laminin, and elastin.
[0018] More preferably, the nutrient solution has a mass concentration of 2% to 15%.
[0019] In a second aspect, the present invention discloses a cardiac function preparation obtained by the above preparation method.
[0020] A third aspect of the present invention discloses the use of the above-mentioned cardiac function preparation in the preparation of a drug for treating cardiovascular diseases.
[0021] Preferably, the cardiovascular disease is myocardial infarction.
[0022] Preferably, the cardiac function preparation is injected into the pericardial infarction tissue on one side under ultrasound guidance.
[0023] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing a cardiac function preparation. 1) It adopts a composition primarily composed of cardiomyocytes, supplemented by endothelial progenitor cells and cardiac extracellular matrix components, which better reflects the physiological characteristics of myocardial tissue under natural conditions: abundant cells, little matrix, and cell composition dominated by cardiomyocytes and supplemented by endothelial cells; 2) To enable the xenogeneic cardiac extracellular matrix to be rapidly absorbed and transformed by the body, endothelial progenitor cells are pre-attached to the matrix for culture. With a carrier, the survival time of endothelial progenitor cells after entering the human body is significantly increased, effectively promoting angiogenesis in cardiac tissue and facilitating the repair of infarcted tissue; 3) It employs a method of fusing composite cell spheres with cardiomyocyte-like cell membranes and then cutting them into smaller pieces. This method allows for the pre-induction of stem cells into cardiomyocyte-like cells in vitro, saving the time required for stem cells to differentiate into cardiomyocyte-like cells in vivo and then repair infarcted myocardial tissue. Furthermore, the cell membranes exhibit significantly stronger adhesion to tissues than the composite cell spheres. As the P407 gel injected into the infarct site degrades in vivo, it adheres better to the outer side of the infarcted area; and the cut cell membranes can be used for pericardial injection via 16... The G-injector, without requiring open-chest surgery, directly covers the infarcted area of the myocardium under ultrasound guidance in a minimally invasive manner. Compared to existing methods such as intravenous stem cell infusion and coronary stem cell injection, it significantly improves the cell survival rate at the infarct site. This not only reduces surgical damage, but the transplanted cells can also be well covered by the thermosensitive gel in the infarct area, allowing them to act directly on the infarcted myocardial tissue. Because the tissue at the myocardial infarction site is inherently thinner than in other locations, currently used myocardial injection methods cause significant trauma to the heart itself. The intraperitoneal injection method used in conjunction with this approach to cover the infarcted area significantly reduces the clinical risks of stem cell cardiac repair methods. Attached Figure Description
[0024] Figure 1 This is a morphological image of umbilical cord mesenchymal stem cells. Figure 2 Morphological image of cardiomyocytes induced by umbilical cord mesenchymal stem cells; Figure 3 This is a graph showing the results of PCR detection of myocardial-specific biomarkers. Figure 4The images show the morphology of cell membrane sheets formed by stem cell-induced cardiomyocytes; where A is a macroscopic view of the cell membrane sheet and B is a microscopic view. Figure 5 Morphology of extracellular matrix powder from porcine heart cells; Figure 6 Morphological diagram of endothelial progenitor cells derived from the human umbilical vein; Figure 7 Morphological image of a composite cell sphere formed by the combination of endothelial progenitor cells and porcine cardiac extracellular matrix powder; Figure 8 Microscopic observation of the fusion of cell membrane sheets and composite cell spheres; Figure 9 Diagram showing the formation of 0.3–1 mm cell membrane sheet-cell sphere composite particles through cutting. Figure 10 A photograph of the prepared cardiac function preparation; Figure 11 The image shows a comparison of cell membrane and cell spheroid structures with the cell viability of corresponding single-cell suspensions cultured in serum-free conditions; where A represents cardiomyocytes and B represents endothelial progenitor cells. Detailed Implementation
[0025] To enable those skilled in the art to understand the features and effects of the present invention, the following descriptions and definitions are only general descriptions of the terms and expressions mentioned in the specification and claims. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in the event of any conflict, the definitions in this specification shall prevail.
[0026] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.
[0027] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values (including integers and fractions) within those ranges.
[0028] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”
[0029] In this article, endothelial progenitor cells (EPCs) are the precursor cells of vascular endothelial cells, whose core function is vascular repair and regeneration in the adult stage.
[0030] In this article, the myocardial extracellular matrix (ECM) is the collective term for the non-cellular components surrounding parenchymal cells such as cardiomyocytes and endothelial cells in cardiac tissue, and is a key "supporting network" for maintaining the structure and function of the heart.
[0031] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.
[0032] This invention provides a method for preparing a cardiac function preparation, comprising the following steps: 1. Preparation of cardiomyocyte-like cell membranes 1) Induction of cardiomyocytes: P3-P5 generation human umbilical cord mesenchymal stem cells were seeded in petri dishes and induction culture was started after 24 h. The culture was carried out at 37℃ and 5% CO2. The induction medium was changed every 2-3 days and the induction was continued for 7-15 days. Cell morphology changes were observed and the expression of myocardial markers was detected to ensure successful induction. The induction medium contained 2% fetal bovine serum, 50 ng / mL fibroblast growth factor 2 (FGF-2), 5 ng / mL insulin-like growth factor 1 (IGF-1), 25 ng / mL bone morphogenetic protein 2 (BMP-2), and 0.1 μmol / L angiotensin II (AngII).
[0033] 2) Preparation of cardiomyocyte-like cell sheets: Take the induced cardiomyocyte-like cells and prepare a solution with a concentration of 1×10⁻⁶. 5 Cell suspensions of 1 cell / mL were seeded into Petri dishes; after 2-4 days of confluence, the culture medium was replaced with complete medium containing 50 μg / mL vitamin C. The medium was changed every other day, and the culture was continued until a membrane was formed to obtain myocardial-like cell membranes.
[0034] 2. Preparation of stem cell-cardiac extracellular matrix composite cell spheres (referred to as composite cell spheres) 1) Place cardiac extracellular matrix powder in a complete culture medium containing 1× antibiotics and pre-culture for 22-26 h to obtain matrix microcarriers.
[0035] 2) Endothelial progenitor cells derived from the human umbilical vein were prepared to a concentration of (1~5)×10⁻⁶. 6Endothelial progenitor cell suspension, at a rate of (0.5~2) × 10⁻⁶ cells / mL. 6 The ratio of 1 endothelial progenitor cell to 1 mg cardiac extracellular matrix powder was used to inoculate the endothelial progenitor cell suspension into the matrix microcarrier obtained in step 1), and cultured for 3-5 days to obtain composite cell spheres.
[0036] 3. Preparation of cell membrane sheet-cell sphere composite particles (hereinafter referred to as composite particles). The composite cell spheres prepared in step 2 were suspended in α-MEM medium containing 2% serum and then mixed with the cardiomyocyte-like cell sheets obtained in step 1. 0.5–5 mg of composite cell spheres were added to each square centimeter of cardiomyocyte-like cell sheet. When the composite cell spheres were evenly distributed on the cell sheet and showed a tendency to fuse with it, they were cut into pieces of 0.3–1 mm in size to obtain cell sheet-cell sphere composite particles. These particles were washed 3–5 times with physiological saline and set aside.
[0037] 4. Preparation of temperature-sensitive gel formulation 1) Prepare a 30% (m / m) poloxamer P407 solution using physiological saline; 2) Under aseptic conditions, the cell membrane sheet-cell sphere composite particles prepared in step 3 were suspended in physiological saline and then mixed with poloxamer P407 solution to obtain a cardiac function preparation. The composite particles have a mass concentration of 2% to 20%, and the poloxamer P407 concentration is 16% to 18% (m / m). The cardiac function preparation is a low-viscosity fluid at low temperature (0 to 10°C), and its viscosity gradually increases with increasing temperature. At body temperature (36 to 37°C), a phase change occurs, and the viscosity increases rapidly, changing from a liquid to a semi-solid gel.
[0038] Preferably, in step 2), a nutrient solution may be added. This nutrient solution is physiological saline containing one or more of the following components: collagen, hyaluronic acid, fibronectin, laminin, and elastin, with a concentration of 20%–30% (m / m). Under aseptic conditions, the cell sheet-cell sphere composite particles prepared in step 3 are suspended in physiological saline, mixed with poloxamer P407 solution, and then the nutrient solution is added and mixed thoroughly to obtain a cardiac function preparation. In this cardiac function preparation, the mass concentration of the composite particles is 2%–20%, the concentration of poloxamer P407 is 16%–18% (m / m), and the final concentration of the nutrient solution is 2%–15% (m / m).
[0039] 5. Intrapericardial injection covering the infarcted area. After identifying the infarcted area, the cardiac function preparation prepared in step 4 and pre-cooled is drawn into the pericardium using a syringe and introduced under ultrasound guidance. The gel, which is liquid at low temperature, is pushed out on one side of the infarcted area. Upon contact with the heart, it forms a non-flowing semi-solid gel at body temperature, covering the infarcted area and promoting repair.
[0040] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading this description, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.
[0041] The human umbilical cord mesenchymal stem cells and endothelial progenitor cells used in this article are both commercially available and can also be isolated using conventional methods in the art. For example, in the following embodiments, the method for isolating human umbilical cord mesenchymal stem cells is as follows: Umbilical cord tissue from full-term cesarean section newborns is collected, placed in umbilical cord preservation solution, and processed within 24 hours. The umbilical cord is rapidly cleaned and disinfected with 75% alcohol (v / v), transferred to a laminar flow hood for aseptic processing, rinsed three times with saline containing penicillin and antibiotics, the umbilical vein and umbilical artery are removed, Wharton's jelly is torn off with toothed forceps and cut into pieces to approximately 2 mm. 3Small pieces of stem cells were evenly distributed in 10 cm culture dishes, and 3 mL of stem cell culture medium was added. The dishes were then incubated at 37°C with 5% CO2. Stem cell culture medium was added again after 24 hours. After 3 days, another 5 mL of stem cell culture medium was added, and the dishes were gently agitated to cover the tissue pieces. The dishes were then incubated at 37°C with 5% CO2. The medium was changed every 3.5 days thereafter. When the confluence of cells around most of the tissue pieces reached 80%, the culture medium was aspirated, and the tissue pieces were transferred to new culture dishes. The current culture dish was washed once with PBS, and 3 mL of trypsin digestion solution was added until the cells shrank and rounded. Stem cell culture medium was then added to stop the digestion. The cells were collected and centrifuged to obtain a cell pellet. After identification as qualified mesenchymal stem cells according to the "General Requirements for Stem Cells," the cells were passaged, expanded, cultured, and cryopreserved to obtain human umbilical cord mesenchymal stem cells. For use, umbilical cord mesenchymal stem cells were resuscitated in culture flasks and cultured in stem cell culture medium (α-MEM medium containing 10% serum and 1× antibiotics) at 37°C in a cell culture incubator containing 5% CO2. The cardiac extracellular matrix powder used in this article is derived from sources including, but not limited to, porcine cardiac extracellular matrix powder, and can also be derived from the cardiac extracellular matrix powder of livestock such as sheep and cattle. It can be obtained commercially or prepared using the methods described in the following examples. Conventional instruments and equipment in the art were used in the following examples. Experimental methods in the following examples, unless otherwise specified, were generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercial products with specifications in the art.
[0042] I. Preparation of Cardiac Function Pharmacology Example 1 1. Preparation of cardiomyocyte-like cell membranes 1) Induction of cardiomyocytes like Figure 1 As shown, P3-P5 generation human umbilical cord mesenchymal stem cells were prepared to a concentration of 5×10⁻⁶. 4Cell suspensions were prepared at 1 / mL and seeded into Petri dishes. The cells were cultured at 37°C and 5% CO2 for 24 h. Once the cell density reached approximately 70%, the induction medium was replaced and cultured further. The induction medium was α-MEM containing 2% fetal bovine serum, 50 ng / mL FGF-2, 5 ng / mL IGF-1, 25 ng / mL BMP-2, and 0.1 μmol / L AngII. The induction medium was changed every 2 days. Cells cultured in α-MEM served as a blank control group. Induction continued for 7 days, and cell morphology changes were observed to obtain cardiomyocyte-like cells. After 7 days of induction, RT-qPCR was used to detect markers of human umbilical cord mesenchymal stem cell differentiation into the myocardium stage: cTnT, Cx43, GATA4, and NKx2.5. Cell morphology results are shown below. Figure 2 As shown in the figure, after induction culture, the cell morphology gradually becomes spindle-shaped and rod-shaped, approaching the morphology of cardiomyocytes. RT-qPCR detection results are as follows... Figure 3 As shown, cTnT, Cx43, GATA4 and NKx2.5 were expressed in the induced group, indicating that the human umbilical cord mesenchymal stem cells induced by the drug continuously differentiated into cardiomyocytes.
[0043] 2) Preparation of cardiomyocyte-like cell sheets: Take the cardiomyocyte-like cells obtained in step 1) and prepare a solution with a concentration of 1×10⁻⁶. 5 Cardiac cardiomyocyte suspensions of 100 cells / mL were seeded into Petri dishes containing induction medium; after 2-4 days of confluence, the medium was replaced with α-MEM containing 50 μg / mL vitamin C, and the medium was changed every other day. Culture was continued for 7 days until a monolayer cell sheet of approximately 15 μm thickness was formed, thus obtaining the cardiomyocyte membrane. Figure 4 ).
[0044] 2. Preparation of endothelial progenitor cell-cardiac cell extracellular matrix composite cell spheres (referred to as composite cell spheres) 1) Preparation of porcine heart extracellular matrix powder. The steps are as follows: After removing an adult porcine heart, immediately rinse it in physiological saline at 4°C to remove blood and surface fat; freeze at -80°C for 24 h, then thaw in ultrapure water at room temperature; insert a catheter into the root of the porcine aorta and perfuse retrogradely for 20 min at a flow rate of 1 L / min, then replace with 2×PBS and circulate perfusion at 1 L / min for 15 min; preheat a mixed solution of 0.02% trypsin / 0.05% EDTA / 0.05% sodium azide to 37°C, then circulate perfusion at 1 L / min for 2 h; subsequently, use 3%... Triton X A mixture of 100% / 0.05% EDTA / 0.05% sodium azide and 4% deoxycholic acid solution was circulated and perfused for 2 h at room temperature at a flow rate of 1.3 L / min. After each perfusion, the heart was perfused with ultrapure water for about 5 min, followed by perfusion with 2×PBS for 15 min to aid cell lysis and remove cell debris and chemical residues. Disinfection was completed by perfusion with a mixture of 0.1% peracetic acid / 4% ethanol at a flow rate of 1.7 L / min for 1 h. Subsequently, the intact matrix was perfused twice with PBS, followed by three perfusions with ultrapure water, each at a flow rate of 1.7 L / min for 15 min, to neutralize and remove acidic substances from the matrix. The matrix was cut into thin slices of about 2 mm, laid flat on non-stick aluminum foil, freeze-dried, and then cut into fine powder with a paper cutter. The powder was sieved to retain particles of 0.2-0.5 mm. Figure 5 ), to obtain porcine heart extracellular matrix powder, for later use; 2) After sterilizing the porcine heart cell extracellular matrix powder obtained in step 1), the microcarrier was placed in α-MEM medium containing 1× penicillin-streptomycin mixed solution and pre-cultured at 37℃ and 5% CO2 for 24 h to obtain the matrix microcarrier. 3) Obtain endothelial progenitor cells derived from the human umbilical vein ( Figure 6 ), add complete culture medium to prepare a solution with a concentration of 1×10 6 An endothelial progenitor cell suspension of 1 / mL was inoculated into a matrix microcarrier obtained in step 2), wherein the matrix microcarrier was cardiac extracellular matrix powder with a dry weight of 1 mg; then the matrix microcarrier inoculated with endothelial progenitor cells was placed in a carbon dioxide cell culture incubator and cultured for another 3 days to obtain composite cell spheres ( Figure 7 ).
[0045] 3. Preparation of cell membrane sheet-cell sphere composite particles The composite cell spheres obtained in step 2 were suspended in α-MEM medium containing 2% fetal bovine serum. 2 mg of composite cell spheres were added to each square centimeter of myocardial cell membrane and placed in the culture container of the myocardial cell membrane from step 1. The cells were cultured normally for 24 h until the composite cell spheres were evenly distributed on the cell membrane and showed a tendency to fuse. Figure 8 The whole is cut into pieces of 0.3~1 mm in size to obtain cell membrane sheet-cell sphere composite particles. Figure 9 Rinse three times with saline solution and set aside.
[0046] 4. Preparation of temperature-sensitive gel formulation 1) Mix 30 g of poloxamer P407 with 70 mL of physiological saline to obtain a 30% (m / m) poloxamer P407 solution. Place the solution in a refrigerator at 4°C overnight to ensure the homogeneity of the solution.
[0047] 2) Under aseptic conditions, take 2.67 mL of the 30% (m / m) poloxamer P407 solution obtained in step 1) and dilute it with 1.5 mL of physiological saline. Filter to remove bacteria. Then add 0.5 g of the cell membrane sheet-cell sphere composite particles prepared in step 3, and then add physiological saline to make up to 5 mL. Mix well to obtain the cardiac function preparation. In this cardiac function preparation, the concentration of cell membrane-cell sphere composite particles is 10% (m / m), and the concentration of P407 is 16% (m / m).
[0048] The cardiac function preparation prepared by this method is a low-viscosity fluid at low temperatures. At 29.7℃, a phase transition occurs, with a rapid increase in viscosity, changing from a liquid to a solid. A cell membrane sheet-cell sphere composite particle structure can be observed within the gel. Figure 10 Its temperature sensitivity was verified by a 37℃ water bath, and the gel solidification time was 1 min 34 s.
[0049] 5. Intrapericardial injection covering the infarcted area. After identifying the infarcted area, a 16G syringe is used to draw up the cardiac function preparation prepared in step 4 and pre-cooled to 0-4°C. Under ultrasound guidance, the preparation is introduced into the pericardium. The gel preparation, which is liquid at low temperature, is pushed out on one side of the infarcted area. At body temperature, it forms a non-flowing semi-solid gel state, covering the infarcted area and promoting repair.
[0050] Example 2 1. Preparation of cardiomyocyte-like cell membranes The preparation method is the same as in Example 1.
[0051] 2. Preparation of endothelial progenitor cell-cardiac cell extracellular matrix composite cell spheres The preparation method is the same as in Example 1.
[0052] 3. Preparation of cell membrane sheet-cell sphere composite particles The preparation method is the same as in Example 1.
[0053] 4. Preparation of temperature-sensitive gel formulation 1) Mix 30 g of poloxamer P407 with 70 mL of physiological saline to obtain a 30% (m / m) poloxamer P407 solution. Place the solution in a refrigerator at 4°C overnight to ensure the homogeneity of the solution.
[0054] 2) Mix 5 g of hyaluronic acid, 1 g of type I collagen with 94 mL of physiological saline to obtain a nutrient solution containing 5% hyaluronic acid (m / m) and 1% type I collagen (m / m).
[0055] 3) Take 6.4 mL of the poloxamer P407 solution obtained in step 1) and 4 mL of the nutrient solution obtained in step 2), mix them evenly, filter to sterilize, and obtain a mixed solution for later use.
[0056] 4) Under aseptic conditions, add 0.5 g of the cell membrane sheet-cell sphere composite particles obtained in step 3 to 4.34 mL of the mixed solution obtained in step 3), add physiological saline to make up to 5 mL, mix well, and obtain the cardiac function preparation; In this cardiac function preparation, the concentration of cell membrane-cell sphere composite particles is 10% (m / m), the concentration of P407 is 16% (m / m), and the concentration of nutrient solution is 2% (m / m).
[0057] II. Efficacy Testing 1. Verification of enhanced cell activity The MTT assay was used to compare the cell viability and survival time of cardiomyocyte suspensions and cardiomyocyte membranes, and endothelial progenitor cell suspensions and endothelial progenitor cell spheres with the same cell number under serum-free culture conditions.
[0058] 1) Experimental Grouping Control group 1: Cardiac-like cell suspension; Control group 2: Endothelial progenitor cell suspension + cardiac extracellular matrix; Experimental group 1: Cardiac-like cell suspension; Experimental group 2: Cardiac-like cell membrane sheet; Experimental group 3: Endothelial progenitor cell suspension + cardiac extracellular matrix; Experimental group 4: Endothelial progenitor cell complex spheroids; Among them, experimental group 1 and experimental group 2 had the same cell count, experimental group 3 and experimental group 4 had the same cell count and the same extracellular matrix content of cardiac cells, the only difference being whether or not they had cellular structure; the experimental groups were used to simulate the situation of nutritional deficiency after cell transplantation into the body.
[0059] 2) Experimental Procedure Samples from each group were seeded into 6-well plates. The control group was cultured in serum-containing medium, while the experimental group was cultured in serum-free medium. The plates were incubated at 37°C and 5% CO2. Three wells were taken from each well at 1, 2, and 3 days of incubation to measure the OD value. The cell viability of control group 1 and control group 2 was used as the baseline.
[0060] 3) Experimental Results The comparison revealed that the cell viability of cardiomyocyte-like cell sheets (experimental group 2) and endothelial progenitor cell composite spheres (experimental group 4) after 1 day of serum-free culture was basically consistent with that of the same volume of single-cell suspension (experimental groups 1 and 3) after 1 day of serum-free culture. However, the cell viability in the serum-free environment was maintained for a much longer time than that in the single-cell suspension group (experimental groups 1 and 3). After 3 days, the cell viability was still maintained at more than 70%, while the cell viability of the single-cell suspension group had decreased to below 10% after 3 days. Figure 11 This result indicates that after single cells are constructed into composite cell spheres and cardiomyocyte-like cell sheets, the cells are more tolerant of nutrient deficiency, meaning they survive for longer periods and secrete more factors.
[0061] 2. Validation of the therapeutic effect on myocardial infarction 1) Establishment of a mouse model of myocardial infarction Thirty-five male C57 mice aged 8-10 weeks and weighing 25±5 g were anesthetized with isoflurane and then entered the thoracic cavity through the left 3 / 4 intercostal space. The left anterior descending coronary artery was ligated to establish a myocardial infarction model. The 24-hour survival rate was 88.6% (31 / 35). TTC staining confirmed the occurrence of infarction 24 hours after the operation. The average size of the infarct area (weight of infarcted myocardium / weight of the whole ventricle) was (20±3.7)%, indicating that the myocardial infarction model was successfully established.
[0062] 2) Experimental Grouping The myocardial infarction mice obtained in step 1) were randomly divided into four groups: Group A: Control group, 40 μL of physiological saline was injected into the pericardium of the myocardial infarction area of mice under ultrasound guidance; Group B: Material group, 40 μL of porcine cardiac extracellular matrix powder gel was injected into the pericardium of the myocardial infarction area of mice under ultrasound guidance; the porcine cardiac extracellular matrix powder gel was formed by mixing 16% poloxamer P407 solution, 10% porcine cardiac extracellular matrix powder and physiological saline by weight percentage. Group C: Single-cell suspension + material group. Under ultrasound guidance, 40 μL of a mixture of single-cell suspension, P407, and cardiac extracellular matrix was injected into the infarcted area of the pericardium in mice. The mixture of single-cell suspension, P407, and cardiac extracellular matrix consisted of: 40 μL of the cardiac function preparation prepared in Example 1, with an equal amount of cardiomyocytes and endothelial progenitor cells (4.2 cells each). 10 5 5.8 10 5 The solution was prepared by mixing 16% poloxamer P407 solution, 10% porcine cardiac extracellular matrix powder, and physiological saline. Group D: Cellular composite particles + P407 group, 40 μL of the cardiac function preparation obtained in Example 1 was injected into the infarcted area of the pericardium of mice under ultrasound guidance.
[0063] 3) Cardiac function testing Six weeks after each group of mice was injected with the corresponding preparation, the wall thickening rate of the left ventricular infarction area was detected by MRI. Images were acquired by high-frequency ultrasound, and the heart wall motion score index (WMSI) was calculated based on the 10-segment model. The left ventricular ejection fraction was measured by echocardiography.
[0064] Table 1 Comparison of cardiac function improvement results
[0065] The results of cardiac function improvement are shown in Table 1. In the cell composite particles + P407 group (group D), the wall thickening rate of the left ventricular infarction area increased from (-2.8±0.6)% to (6.9±0.4)%, the wall motion score index decreased from 2.2±0.3 to 1.8±0.2, and the left ventricular ejection fraction increased from (35.7±1.8)% to (46.2±2.3)%, which were significantly higher than those in the material group (group B) and the single cell suspension + material group (group C). The control group (group A) showed no significant improvement.
[0066] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.
Claims
1. A method for preparing a cardiac function preparation, characterized in that, Includes the following steps: 1) Endothelial progenitor cells were mixed with cardiac extracellular matrix and cultured to obtain composite cell spheres; 2) Mix the myocardial cell membrane sheet with the composite cell spheres obtained in step 1) and culture them. After the composite cell spheres are evenly distributed on the cell membrane sheet and show a tendency to fuse, cut them into pieces to obtain cell membrane sheet-cell sphere composite particles. 3) Mix the cell membrane sheet-cell sphere composite particles obtained in step 2) with poloxamer P407 solution to obtain a cardiac function preparation.
2. The method for preparing a cardiac function preparation according to claim 1, characterized in that, In step 1), the ratio of endothelial progenitor cells to cardiac extracellular matrix is (0.5~2)×10⁻⁶. 6 Cells: 1 mg.
3. The method for preparing a cardiac function preparation according to claim 1, characterized in that, In step 2), the method for preparing the cardiomyocyte-like cell sheet is as follows: a concentration of 1×10⁻⁶ cells is used. 5 Cardiac myocardial cell suspensions of 1 cell / mL were seeded into Petri dishes. After 2-4 days of confluence, the culture medium was replaced with complete medium containing 50 μg / mL vitamin C. The medium was changed every other day and cultured until a membrane was formed to obtain cardiac myocardial cell membranes.
4. The method for preparing a cardiac function preparation according to claim 1, characterized in that, In step 2), during mixed culture, 0.5–5 mg of composite cell spheres are added to each square centimeter of cardiomyocyte-like cell sheet.
5. A method for preparing a cardiac function preparation according to claim 1, characterized in that, In step 2), the diameter of the cell membrane sheet-cell sphere composite particle is 0.3~1 mm.
6. The method for preparing a cardiac function preparation according to claim 1, characterized in that, In cardiac function preparations, the mass concentration of cell membrane sheet-cell sphere composite particles is 2%~20%, and the mass concentration of poloxamer P407 is 16%~18%.
7. A method for preparing a cardiac function preparation according to any one of claims 1 to 6, characterized in that, The cardiac function preparation also includes a nutrient solution. The cardiac function preparation is obtained by mixing cell membrane sheet-cell sphere composite particles, poloxamer P407 solution and nutrient solution. The nutrient solution contains one or more of the following components: collagen, hyaluronic acid, fibronectin, laminin, and elastin.
8. The cardiac function preparation obtained by the preparation method according to any one of claims 1 to 7.
9. The use of the cardiac function preparation according to claim 8 in the preparation of a drug for treating cardiovascular diseases.
10. The application according to claim 9, characterized in that, The cardiovascular disease mentioned is myocardial infarction.