Method for preparing heart organoid

Abstract

The invention discloses a method for preparing heart organoid, and relates to the technical field of organ preparation, and the method comprises the following specific steps: S1, stem cell amplification and homogenized embryoid body preparation; s2, specific induction of cardiac progenitor cells: culturing the embryoid body, driving the cells to differentiate to mesoderm, and culturing again to obtain progenitor cells; s3, cardiac field zoning and multi-lineage spontaneous differentiation: putting the progenitor cells into a culture system to induce spontaneous differentiation of the cells; s4, forming a three-dimensional self-assembly and cavity structure: transferring the differentiated tissue into a dynamic rotary culture system to form a layered organ; s5, electrophysiology maturation and function strengthening, wherein the obtained organoid is subjected to programmed electric pulse stimulation, and the highly-bionic heart organoid is obtained. Through a staged and multi-dimensional induction strategy, by simulating the time sequence and space microenvironment of human embryo heart development, stem cells are guided to form the organoid in a self-organizing manner, and the use universality of the prepared heart organoid is improved.

Description

Technical Field

[0001] This invention relates to the field of organ preparation technology, and in particular to a method for preparing heart organoids. Background Technology

[0002] Heart organoids are miniaturized tissue models obtained through in vitro three-dimensional culture technology that can simulate parts of the structure and function of the natural heart. In recent years, with the rapid development of stem cell technology and three-dimensional culture systems, heart organoids have shown broad application prospects in the fields of cardiovascular disease mechanism research, drug screening, and regenerative medicine.

[0003] Currently, common methods for preparing cardiac organoids mainly rely on directly differentiating human pluripotent stem cells into cardiomyocytes under the influence of specific inducing factors, followed by three-dimensional reconstruction using matrix gel or scaffold materials. However, induction protocols often lack precise simulation of the spatial and temporal dimensions of embryonic heart development, resulting in organoids with a single cell type, predominantly cardiomyocytes, which fails to replicate the multi-lineage composition of the natural heart, consisting of cardiomyocytes, endothelial cells, fibroblasts, and epicardial cells. Furthermore, traditional static culture systems struggle to recreate the key microenvironments present during in vivo development, such as signaling molecule concentration gradients, oxygen gradients, and biomechanical stimuli. This results in organoids lacking the regionalized characteristics of the first and second cardiac fields, failing to form chamber-like structures, exhibiting low functional maturity, and retaining electrophysiological characteristics at the early embryonic stage. These limitations restrict their application value as highly biomimetic models and affect the widespread use of cardiac organoids. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing cardiac organoids to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing heart organoids, wherein the method guides human embryonic stem cells to self-organize and form heart organoids by simulating human embryonic heart development in a phased and multi-dimensional manner, comprising the following specific steps:

[0006] S1. Stem cell expansion and preparation of homogenized embryoid bodies: Human pluripotent stem cells are expanded and cultured in an extracellular matrix-coated culture system and digested into single cells. After culture, embryoid bodies with uniform diameter are formed.

[0007] S2, Cardiac progenitor cell specialization induction: Embryomorphs are exposed to induction medium for culture to drive cell differentiation into mesoderm, and then the embryomorphs are transferred for further culture to obtain progenitor cells;

[0008] S3, Heart Field Partitioning and Spontaneous Differentiation of Multiple Lineages: Progenitor cells are placed in a culture system to simulate embryonic and lineage separation, inducing spontaneous cell differentiation;

[0009] S4. Three-dimensional self-assembly and formation of compartmentalized structures: Differentiated tissues are transferred to a dynamic rotating culture system to simulate the compartmentalization and morphogenesis of the embryonic heart, forming layered organoids;

[0010] S5. Electrophysiological maturation and functional enhancement: The obtained organoids are subjected to programmed electrical pulse stimulation to simulate the development process of the embryonic heart's electrical conduction system, driving the maturation of myocardial cells, synchronization of electrical conduction, and enhancement of contractile function, thereby obtaining highly biomimetic heart organoids.

[0011] Preferably, in step S1, human pluripotent stem cells identified as pluripotent are provided, expanded and cultured in an extracellular matrix-coated culture system, and apoptosis is inhibited by ROCK inhibitors after passage; the expanded stem cells are digested into single cells, counted, and seeded in low-absorption microplates, and statically cultured after centrifugation-assisted aggregation to form embryoid bodies of uniform diameter.

[0012] Preferably, in step S2, the induction of cardiac progenitor cell specialization includes the following steps:

[0013] S21. Early mesodermal induction: After culturing embryoid bodies in induction solution, multiple EBs were randomly selected for intermediate detection to verify the mesodermal induction effect. Through the transient activation of the Wnt pathway, the process of embryonic primitive stripe formation was simulated, driving cells to differentiate into mesoderm.

[0014] S22, Mesodermal Deposition: The embryonic bodies treated above were transferred to an induction medium containing Wnt signaling pathway inhibitors, bone morphogenetic protein 4 and fibroblast growth factor 2, and cultured for another 48 hours to simulate the mesodermal determination stage of the embryonic heart and obtain progenitor cells.

[0015] Preferably, in step S3, the obtained progenitor cell tissue is placed in a culture system with retinoic acid concentration gradient and oxygen concentration gradient constructed simultaneously, and vascular endothelial growth factor and transforming growth factor-β1 are added to the culture medium to simulate the separation process of the first / second cardiac field partition and lineage of the embryo, and to induce the cells to spontaneously differentiate into cardiomyocytes, endothelial cells, fibroblasts and epicardial cells according to their spatial location.

[0016] Preferably, in step S4, the three-dimensional self-assembly and chambering structure formation involves transferring the obtained tissue into a dynamic rotating culture system, constructing a matrix stiffness gradient and applying periodic mechanical stretching to simulate the chambering and morphogenesis process of the embryonic heart, promoting cell rearrangement and forming layered organoids with chamber-like structures.

[0017] Preferably, step S4 includes the following specific steps:

[0018] S41. Use a sterile pipette to transfer organoids into low-adsorption rotary culture flasks for dynamic rotary culture, adding 20 mL of complete culture medium to each flask.

[0019] S42. Add different concentrations of collagen I to the culture medium to create a stiffness gradient of 5 kPa-20 kPa around the organoids.

[0020] S43. Apply periodic stretching to organoids using biomechanical stretching to simulate mechanical stress during embryonic heart development;

[0021] S44. By adjusting the rotation speed, the fluid shear force on the organoid surface is maintained at 0.3 dyne / cm. 2 The culture medium was completely replaced every 2 days to maintain the gradient until 2 weeks later, when the organoid diameter reached 800-1500μm and formed a layered structure.

[0022] Preferably, in step S5, the obtained organoids are subjected to programmed electrical pulse stimulation in a culture medium containing maturation-promoting factors to simulate the development process of the embryonic heart's electrical conduction system. After simulated culture, highly biomimetic heart organoids that closely resemble natural hearts in terms of cell composition, tissue structure, contractile function, and electrophysiological characteristics are obtained.

[0023] Preferably, step S5 includes the following steps:

[0024] S51. Simulating the development and functional maturation of the embryonic cardiac electrical conduction system;

[0025] S52, Quality control of simulated postnatal cardiac function.

[0026] Preferably, step S51 includes using a mature culture medium and a customized electrostimulation culture dish with an electrode spacing of 1 cm, connecting a pulse generator to simulate in vivo cardiac pacing signals to drive the maturation of the electrophysiological system. After simulation, the culture is rotated and cultured, with the medium changed every 2 days, and T3 and dexamethasone are added to maintain the electrostimulation parameters. On day 21, immunofluorescence and optical coherence tomography are performed. From day 21 to day 30, the electrostimulation and mature culture medium are maintained, and the culture is continued until the organoids achieve stable synchronous contraction. On day 30, the electrostimulation is stopped, and the culture is rotated and cultured again, with the medium changed every 3 days to maintain the functional homeostasis of the organoids.

[0027] Preferably, step S52 includes morphological quality control, multi-lineage identification, structural quality control, and functional quality control of the organoid, used to obtain highly biomimetic heart organoids with structural stratification, multi-lineage coordination, and functional stability.

[0028] The technical effects and advantages of this invention are as follows:

[0029] (1) This invention uses a phased and multi-dimensional induction strategy to guide stem cells to self-organize into organoids by simulating the temporal and spatial microenvironment of human embryonic heart development, rather than simply artificially mixing multiple cells, thereby improving the physiological relevance of the model and increasing the versatility of the prepared heart organoids.

[0030] (2) By introducing retinoic acid gradient and vascular endothelial growth factor at a specific stage, this invention successfully induced stem cells to spontaneously differentiate into cardiomyocytes, endothelial cells, fibroblasts and epicardial cells, forming a natural spatial distribution pattern of central myocardium-peripheral fibroblasts / epidermal-scattered endothelium, and possessing a chamber-like layered structure.

[0031] (3) By introducing dynamic rotation culture, mechanical stretching and programmed electrical pulse stimulation, this invention drives the electrophysiological maturation of organoids in an in vitro environment. The obtained cardiac organoids not only have stable autonomous rhythm and drug responsiveness, but their action potential morphology and electrical conduction velocity are close to those of adult cardiomyocytes, thus solving the defect of low maturity of existing organoids. Attached Figure Description

[0032] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0033] Figure 1 This is a schematic diagram of the method steps of the present invention. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] This invention provides, for example Figure 1 The method for preparing heart organoids, as shown, guides human embryonic stem cells to self-organize into heart organoids by simulating human embryonic heart development in a phased and multi-dimensional manner. The specific steps include:

[0036] S1. Stem cell expansion and preparation of homogenized embryoid bodies: Human pluripotent stem cells are expanded and cultured in an extracellular matrix-coated culture system and digested into single cells. After culture, embryoid bodies with uniform diameter are formed.

[0037] In step S1, human pluripotent stem cells with pluripotency identification are provided and expanded in an extracellular matrix-coated culture system. After passage, apoptosis is inhibited using a ROCK inhibitor. The expanded stem cells are digested into single cells, counted, and seeded in low-absorption microplates. After centrifugation-assisted aggregation, they are statically cultured to form embryoid bodies with uniform diameter.

[0038] Human embryonic stem cells or induced pluripotent stem cells are selected to ensure that the starting cells have the potential for pluripotent differentiation, providing a prerequisite for subsequent multi-lineage differentiation of the heart. The culture dishes are pretreated before use to simulate the extracellular matrix (ECM) microenvironment in vivo, maintaining the pluripotency and adherent growth of stem cells. The culture medium is Essential8™, which provides the nutrients required for stem cell proliferation, avoids contamination, and maintains pluripotency homeostasis. The culture conditions are: 37°C, 5% CO2, and saturated humidity (95%). Fresh medium is replaced every 24 hours. The culture conditions are designed to simulate the physiological temperature and gas environment of the human body, ensuring rapid proliferation of stem cells without differentiation.

[0039] During passage, when cell confluence reaches 70%-80%, aspirate the culture medium, wash twice with PBS, add 0.5 mM EDTA, incubate at 37°C for 2 min, gently pipette to detach from the bottom of the dish, and seed into new coated culture dishes at a 1:6 ratio to avoid excessive confluence leading to spontaneous differentiation of stem cells and maintain cell homogeneity and proliferative activity. Stabilization is performed after passage; for the first 24 hours after seeding, add 10 μM of the ROCK inhibitor Y-27632 to the culture medium to inhibit apoptosis, improve passage survival rate, and ensure stem cell population homogeneity. After stabilization, perform 2-3 consecutive passage expansion cycles to obtain ≥1×10⁶ cells / years. 7 A number of homogenized pluripotent stem cells are used for subsequent embryoid body preparation to accumulate a sufficient number of functional stem cells, ensuring the efficiency and consistency of embryoid body formation.

[0040] After digesting stem cells using the above method, collect single-cell suspensions, centrifuge at 1000 rpm for 3 min, discard the supernatant to obtain uniformly dispersed single cells, avoiding uneven cell aggregation that could lead to differences in embryoid size. Then, prepare a resuspension. The embryoid formation culture medium is prepared as follows: RPMI 1640 + 20% fetal bovine serum (FBS) + 1% non-essential amino acids + 1% glutamine + 10 μM MY-27632, simulating the nutritional microenvironment of early embryonic development. Use a hemocytometer to count cells, adjusting the concentration to 4000 cells / 100 μL, and seed into low-absorption U-shaped 96-well plates, precisely adding 100 μL of suspension to each well for easy control. The number of cells in each embryoid is consistent to avoid uneven differentiation efficiency due to differences in cell number. After inoculation, the culture plate is placed in a centrifuge at 1200 rpm for 3 minutes to concentrate the cells at the center of the well bottom, promote rapid cell aggregation, and ensure the uniformity of the embryoid's spherical shape. Then, static culture is performed: the embryoids are cultured at 37℃ and 5% CO2 for 24 hours to form dense spherical embryoids. Then, the embryoids are screened by microscopic observation after 24 hours. Embryoids with a diameter of 120±20μm, smooth edges, and dense interior are selected, and broken or abnormally sized embryoids are removed to ensure the synchronicity of subsequent differentiation and reduce experimental errors.

[0041] S2, Cardiac progenitor cell specialization induction: Embryomorphs are exposed to induction medium for culture to drive cell differentiation into mesoderm, and then the embryomorphs are transferred for further culture to obtain progenitor cells;

[0042] In step S2, the induction of cardiac progenitor cell specialization includes the following steps:

[0043] S21. Early mesodermal induction: After culturing embryoid bodies in induction solution, multiple EBs were randomly selected for intermediate detection to verify the mesodermal induction effect. Through the transient activation of the Wnt pathway, the process of embryonic primitive stripe formation was simulated, driving cells to differentiate into mesoderm.

[0044] The induction solution was prepared as basal medium RPMI 1640 + B27 Supplement Minus Insulin (Gibco #A1895601) + 1% penicillin antibiotics + 8 μM CHIR99021 (Wnt / β-catenin activator). CHIR99021 activates the Wnt pathway by inhibiting GSK3β, mimicking the initiation of Wnt signaling during the gastrulation stage of embryonic cells, driving stem cells to differentiate into the mesoderm. During the medium change, on day 0 (24 hours after embryoid formation), the original medium in the 96-well plate was aspirated, and 100 μL of induction solution was added to each well, avoiding disturbance of the embryoid cells (EB) to ensure all EBs were in optimal condition. B cells were exposed to a uniform concentration of signaling molecules and cultured for 24 hours to simulate the physiological environment of mesodermal differentiation in vivo, ensuring the timeliness of signaling pathway activation. After 24 hours of induction, three EB cells were randomly selected, and the expression of mesodermal marker genes Brachyury (T) and MIXL1 was detected by qPCR. The expression level needed to be upregulated by ≥10-fold compared to the uninduced group to verify the mesodermal induction effect, ensure that cell fate progresses in the correct direction, and avoid subsequent differentiation deviations. Furthermore, through the transient activation of the Wnt pathway, the process of embryonic primitive stripe formation was simulated, enabling the cells inside the EB cells to acquire the characteristics of mesodermal precursor cells, laying the foundation for subsequent cardiac mesodermal specialization.

[0045] S22, Mesodermal Deposition: The embryonic bodies treated above were transferred to an induction medium containing Wnt signaling pathway inhibitors, bone morphogenetic protein 4 and fibroblast growth factor 2, and cultured for another 48 hours to simulate the mesodermal determination stage of the embryonic heart and obtain progenitor cells.

[0046] The induction medium is prepared in the same way as in step S21. On the first day of medium replacement (i.e., 24 hours after mesodermal induction), the entire induction medium is replaced, and 100 μL of freshly prepared induction medium is added to each well to ensure complete EB infiltration. The culture period is 48 hours of continuous culture (days 1-3). On the second day (12 hours after induction), half of the medium is replaced once to replenish fresh factors, maintain a stable concentration of signaling molecules, ensure the continuous specialization of cardiac progenitor cells, and avoid factor degradation leading to a decrease in differentiation efficiency. The key detection is performed on the third day. EB is taken for immunofluorescence staining to detect the positive rate of cardiac progenitor cell marker genes NKX2.5 and GATA4. ≥70% of cells must express these genes to verify the specialization effect of cardiac progenitor cells and ensure that most cells in EB acquire the potential for cardiac lineage differentiation. Through the signal combination of Wnt inhibition and BMP / FGF synergistic activation, the process of separation and shaping of the embryonic cardiac mesoderm from the primitive mesoderm is simulated, making the cells progenitor cells with the ability to differentiate into multiple cardiac lineages.

[0047] S3, Heart Field Partitioning and Spontaneous Differentiation of Multiple Lineages: Progenitor cells are placed in a culture system to simulate embryonic and lineage separation, inducing spontaneous cell differentiation;

[0048] In step S3, the obtained progenitor cell tissue is placed in a culture system with retinoic acid concentration gradient and oxygen concentration gradient constructed simultaneously, and vascular endothelial growth factor and transforming growth factor-β1 are added to the culture medium to simulate the separation process of the first / second cardiac field partition and lineage of the embryo, inducing cells to spontaneously differentiate into cardiomyocytes, endothelial cells, fibroblasts and epicardial cells according to their spatial location.

[0049] When performing cardiac field partitioning, the culture medium needs to be upgraded. On the third day of culture, it is replaced with insulin-containing complete B27 medium + RPMI 1640 + 1% penicillin antibody. Insulin provides the energy required for cell proliferation and differentiation, while complete B27 supplements the essential nutrients for cardiac lineage differentiation. A microfluidic chip (PDMS material, channel width 200μm) or a half-volume gradient medium replacement method is used to create a RA concentration gradient in the EB environment, for example, 0.01μM→0.1μM, increasing from the center of the EB to the periphery. RA is a key signaling molecule for cardiac field partitioning: ① Low concentration of RA (central region) induces the differentiation of first cardiac field progenitor cells into ventricular myocytes; ② High concentration of RA (peripheral region) induces the differentiation of second cardiac field progenitor cells into atrial myocytes, outflow tract cells, and epicardial cells, simulating the in vivo cardiac field spatial partitioning pattern. Co-factors are added by adding 15ng / mL VEGF-A (vascular endothelial growth factor) + 3n to the culture medium. g / mLTGF-β1; Oxygen gradient construction: The culture system was placed in a three-gas incubator, and the O2 concentration was adjusted to a gradient distribution. The culture environment of 5% CO2 and 37℃ was maintained to simulate the hypoxic microenvironment of embryonic heart development. The culture cycle was 3-7 days, and half of the medium was changed every 2 days to maintain gradient stability and drive cells to spontaneously differentiate into different lineages according to spatial location, forming a natural distribution pattern of "central myocardium - peripheral fibroblasts / epicardium - scattered endothelial cells". On the 7th day of differentiation, the diameter of EB increased to 300-500μm. Immunofluorescence detection showed that: ① the positive rate of cTnT⁺ (cardiomyocytes) in the central region was ≥60%; ② the positive rate of WT1⁺ (epicardial cells) and Vimentin⁺ (fibroblasts) in the peripheral region was ≥20%; ③ the positive rate of CD31⁺ (endothelial cells) in the scattered region was ≥10%. This achieved spontaneous differentiation of multiple lineages without the need for mixing with exogenous cells, replicating the cellular composition and spatial distribution of the natural heart.

[0050] S4. Three-dimensional self-assembly and formation of compartmentalized structures: Differentiated tissues are transferred to a dynamic rotating culture system to simulate the compartmentalization and morphogenesis of the embryonic heart, forming layered organoids;

[0051] In step S4, the three-dimensional self-assembly and chambering structure formation are achieved by transferring the obtained tissue into a dynamic rotating culture system, constructing a matrix stiffness gradient and applying periodic mechanical stretching, simulating the chambering and morphogenesis process of the embryonic heart, promoting cell rearrangement and forming layered organoids with chamber-like structures.

[0052] Step S4 includes the following specific steps:

[0053] S41. Use a sterile pipette to transfer organoids to low-adsorption rotary culture flasks for dynamic rotary culture. Add 20 mL of complete culture medium to each flask, rotate at 45 rpm, and culture at 37°C, 5% CO2, and saturated humidity. This transitions from static to dynamic rotary culture to simulate the slight movement of an embryo in amniotic fluid, promoting cell-cell interactions and self-assembly. The tip of the sterile pipette should be cut off to avoid damaging the EB.

[0054] S42. Add different concentrations of collagen I (Sigma#C3867) to the culture medium to create a stiffness gradient of 5kPa-20kPa around the organoids.

[0055] S43. Apply periodic stretching to organoids using biomechanical stretching, such as FlexcellFX-5000, with a strain of 4% and a frequency of 0.8Hz, for 8 hours a day for 7 days to simulate the mechanical stress during embryonic heart development.

[0056] S44. By adjusting the rotation speed to 30-60 rpm, the fluid shear force on the organoid surface is maintained at 0.3 dyne / cm2, simulating the shear force of blood flow in blood vessels in vivo. This promotes tight junctions of endothelial cells and accelerates the tubular maturation of vascular-like structures. The culture medium is completely replaced every 2 days to replenish fresh nutrients and factors and maintain gradient stability. After maintaining the gradient for 2 weeks, the organoid diameter reaches 800-1500 μm, forming a layered structure of dense outer layer, loose inner layer, and scattered vascular-like strands. This replicates the process of compartmentalization of the embryonic heart (cardiac tube formation → cardiac chamber expansion), achieving preliminary biomimicry in structure.

[0057] S5. Electrophysiological maturation and functional enhancement: The obtained organoids are subjected to programmed electrical pulse stimulation to simulate the development process of the embryonic heart's electrical conduction system, driving the maturation of myocardial cells, synchronization of electrical conduction, and enhancement of contractile function, thereby obtaining highly biomimetic heart organoids.

[0058] In step S5, the obtained organoids are subjected to programmed electrical pulse stimulation in a culture medium containing maturation-promoting factors to simulate the development process of the embryonic heart's electrical conduction system. After simulation culture, highly biomimetic heart organoids that closely resemble the natural heart in terms of cell composition, tissue structure, contractile function, and electrophysiological characteristics are obtained.

[0059] Step S5 includes the following steps:

[0060] S51. Simulating the development and functional maturation of the embryonic cardiac electrical conduction system; including the use of a maturation culture medium, which is prepared as complete B27 medium + RPMI 1640 + 1% penicillin antibiotics + 30nMT3 (triiodothyronine) + 0.5μM dexamethasone, and a customized electrical stimulation culture dish (with built-in platinum electrodes), with an electrode spacing of 1cm, connected to a pulse generator, to simulate in vivo cardiac pacing signals to drive the maturation of the electrophysiological system. The electrical stimulation parameters are set as a square wave pulse electric field, with a field strength of 2V / cm, a frequency of 1Hz, and a pulse width of 2ms. The stimulation mode is a cycle of 30min stimulation and 30min rest, with continuous stimulation for 12 hours per day.

[0061] After simulation, the organoids were cultured by rotation (45 rpm) every 2 days, with T3 and dexamethasone added to maintain the electrical stimulation parameters. On day 21, immunofluorescence and optical coherence tomography were performed. From day 21 to day 30, electrical stimulation and maturation medium were maintained, and the organoids were cultured until they achieved stable synchronous contraction. On day 30, electrical stimulation was stopped, and the organoids were cultured by rotation every 3 days to maintain organoid functional homeostasis.

[0062] S52. Quality control of simulated postnatal heart function, including morphological quality control, multi-lineage identification, structural quality control and functional quality control of organoids, to obtain highly biomimetic heart organoids with structural stratification, multi-lineage coordination and functional stability. Before quality control, the culture is terminated, that is, on the 30th day, electrical stimulation is stopped and rotation culture is continued, and the medium is changed every 3 days to maintain the homeostasis of organoid function.

[0063] Morphological quality control was achieved through microscopic observation. Organoids were 1-3 mm in diameter, spherically regular, with smooth edges, exhibiting spontaneous rhythmic pulsation (60-120 beats / minute), and without obvious fragmentation or liquefaction, ensuring morphological integrity and functional stability. Multi-lineage identification was verified using immunofluorescence staining: cardiomyocytes: cTnT⁺, α-MHC⁺ (positive rate ≥50%); endothelial cells: CD31⁺, VE-cadherin⁺ (positive rate ≥15%); fibroblasts: Vimentin⁺, PDGFRβ⁺ (positive rate ≥25%); epicardial cells: WT1⁺, Tbx18⁺ (positive rate ≥10%). The composition and proportions of the multi-lineage structure closely resemble those of a natural heart. Structural quality control was achieved by HE staining, which revealed clear chamber-like structures, layered wall layers, orderly arrangement of sarcomeres, and interconnected vascular-like structures, verifying the biomimicry of the structure and ensuring that the organoid replicates the histological characteristics of a natural heart. Functional quality control was achieved by recording the contraction curve using a high-speed camera, with a contraction amplitude ≥50μm and a coordinated diastolic / systolic ratio. Patch-clamp detection showed that the action potential morphology was close to that of adult cardiomyocytes, with an electrical conduction velocity ≥10cm / s. Treatment with adrenaline (1μM) increased the contraction frequency by ≥30%, while treatment with verapamil (1μM) decreased the frequency by ≥20%, ensuring that the organoid possesses the core functions of a natural heart.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing cardiac organoids, characterized in that, The method simulates human embryonic heart development in a phased and multi-dimensional manner, guiding human embryonic stem cells to self-organize into heart-like organoids, including the following specific steps: S1. Stem cell expansion and preparation of homogenized embryoid bodies: Human pluripotent stem cells are expanded and cultured in an extracellular matrix-coated culture system and digested into single cells. After culture, embryoid bodies with uniform diameter are formed. S2, Cardiac progenitor cell specialization induction: Embryomorphs are exposed to induction medium for culture to drive cell differentiation into mesoderm, and then the embryomorphs are transferred for further culture to obtain progenitor cells; S3, Heart Field Partitioning and Spontaneous Differentiation of Multiple Lineages: Progenitor cells are placed in a culture system to simulate embryonic and lineage separation, inducing spontaneous cell differentiation; S4. Three-dimensional self-assembly and formation of compartmentalized structures: Differentiated tissues are transferred to a dynamic rotating culture system to simulate the compartmentalization and morphogenesis of the embryonic heart, forming layered organoids; S5. Electrophysiological maturation and functional enhancement: The obtained organoids are subjected to programmed electrical pulse stimulation to simulate the development process of the embryonic heart's electrical conduction system, driving the maturation of myocardial cells, synchronization of electrical conduction, and enhancement of contractile function, thereby obtaining highly biomimetic heart organoids.

2. The method for preparing cardiac organoids according to claim 1, characterized in that, In step S1, human pluripotent stem cells identified as pluripotent are provided and expanded in an extracellular matrix-coated culture system. After passage, apoptosis is inhibited using a ROCK inhibitor. The expanded stem cells are digested into single cells, counted, and seeded in low-absorption microplates. After centrifugation-assisted aggregation, they are statically cultured to form embryoid bodies of uniform diameter.

3. The method for preparing cardiac organoids according to claim 1, characterized in that, In step S2, the induction of cardiac progenitor cell specialization includes the following steps: S21. Early mesodermal induction: After culturing embryoid bodies in induction solution, multiple EBs were randomly selected for intermediate detection to verify the mesodermal induction effect. Through the transient activation of the Wnt pathway, the process of embryonic primitive stripe formation was simulated, driving cells to differentiate into mesoderm. S22, Mesodermal Deposition: The embryonic bodies treated above were transferred to an induction medium containing Wnt signaling pathway inhibitors, bone morphogenetic protein 4 and fibroblast growth factor 2, and cultured for another 48 hours to simulate the mesodermal determination stage of the embryonic heart and obtain progenitor cells.

4. The method for preparing cardiac organoids according to claim 1, characterized in that, In step S3, the obtained progenitor cell tissue is placed in a culture system with retinoic acid concentration gradient and oxygen concentration gradient constructed simultaneously, and vascular endothelial growth factor and transforming growth factor-β1 are added to the culture medium to simulate the separation process of the first / second cardiac field partition and lineage of the embryo, and to induce the cells to spontaneously differentiate into cardiomyocytes, endothelial cells, fibroblasts and epicardial cells according to their spatial location.

5. The method for preparing cardiac organoids according to claim 1, characterized in that, In step S4, the three-dimensional self-assembly and chambering structure formation involves transferring the obtained tissue into a dynamic rotating culture system, constructing a matrix stiffness gradient and applying periodic mechanical stretching to simulate the chambering and morphogenesis process of the embryonic heart, promoting cell rearrangement and forming layered organoids with chamber-like structures.

6. The method for preparing cardiac organoids according to claim 1, characterized in that, Step S4 includes the following specific steps: S41. Use a sterile pipette to transfer organoids into low-adsorption rotary culture flasks for dynamic rotary culture, adding 20 mL of complete culture medium to each flask. S42. Add different concentrations of collagen I to the culture medium to create a stiffness gradient of 5 kPa-20 kPa around the organoids. S43. Apply periodic stretching to organoids using biomechanical stretching to simulate mechanical stress during embryonic heart development; S44. By adjusting the rotation speed, the fluid shear force on the organoid surface is maintained at 0.3 dyne / cm. 2 The culture medium was completely replaced every 2 days to maintain the gradient until 2 weeks later, when the organoid diameter reached 800-1500μm and formed a layered structure.

7. The method for preparing cardiac organoids according to claim 1, characterized in that, In step S5, the obtained organoids are subjected to programmed electrical pulse stimulation in a culture medium containing maturation-promoting factors to simulate the development process of the embryonic heart's electrical conduction system. After simulated culture, highly biomimetic heart organoids that closely resemble the natural heart in terms of cell composition, tissue structure, contractile function, and electrophysiological characteristics are obtained.

8. The method for preparing cardiac organoids according to claim 1, characterized in that, Step S5 includes the following steps: S51. Simulating the development and functional maturation of the embryonic cardiac electrical conduction system; S52, Quality control of simulated postnatal cardiac function.

9. The method for preparing cardiac organoids according to claim 8, characterized in that, Step S51 includes using a mature culture medium and a customized electrostimulation culture dish with an electrode spacing of 1 cm, connecting a pulse generator to simulate in vivo cardiac pacing signals to drive the maturation of the electrophysiological system. After simulation, the culture is rotated and cultured, with the medium changed every 2 days, and T3 and dexamethasone are added to maintain the electrostimulation parameters. On day 21, immunofluorescence and optical coherence tomography are performed. From day 21 to day 30, the electrostimulation and mature culture medium are maintained, and the culture is continued until the organoids achieve stable synchronous contraction. On day 30, the electrostimulation is stopped, and the culture is rotated and cultured again, with the medium changed every 3 days to maintain the functional homeostasis of the organoids.

10. The method for preparing a heart organoid according to claim 8, characterized in that, Step S52 includes morphological quality control, multi-lineage identification, structural quality control, and functional quality control of organoids, used to obtain highly biomimetic heart organoids with structural stratification, multi-lineage coordination, and functional stability.

Images