Cardiovascular organoid differentiation culture method

Abstract

The invention relates to a differentiation culture method for cardiovascular organs, which comprises the following steps: using stem cells as starting cells, respectively carrying out seven-day cardiac cell induced differentiation culture and seven-day blood vessel induced differentiation culture, mixing, carrying out fusion culture, culturing for four days, carrying out dynamic culture from eleventh day to twentieth day, and harvesting the cardiovascular organs. Deepened vascularization can be seen in the central region of the organ. Compared with the prior art, the heart-like organ and the blood vessel-like organ are combined, various heart cell types and blood vessel cell types are included, and the implementation time is shorter. Through verification, the cardiovascular organoid model with improved maturity is obtained, the central area of the obtained cardiovascular organoid has a vascularization effect of penetration growth, and multiple culture and construction prove that the cardiovascular organoid model has stability and repeatability.

Description

Technical Field

[0001] This invention relates to a tissue culture method, and more particularly to an in vitro induced differentiation culture method for organoids. Background Technology

[0002] Over the past two decades, the number of new compounds has increased by 1,000 annually, with over 90% lacking toxicity data. Currently, most toxicity data for chemicals is obtained from laboratory animals, but animal testing presents several significant challenges: 1. It requires substantial financial resources; 2. The experimental cycle is long; 3. It requires a large number of laboratory animals; 4. Due to numerous in vivo influencing factors, it is difficult to conduct metabolic and mechanistic studies. For animal protection reasons, the "3R" principle—Reduction, Refinement, and Replacement—is strongly advocated. From both a scientific and economic perspective, the toxicological substitution method is of paramount importance for the hazard assessment and management of exogenous chemicals.

[0003] Organoids are tissue analogs with a specific spatial structure formed through three-dimensional in vitro culture of adult stem cells or pluripotent stem cells. They can simulate real organs in structure and function, maximally mimicking the structure and function of in vivo tissues. Compared to traditional two-dimensional cell lines, organoids have the following advantages: 1. Cell diversity; 2. Three-dimensional structure and cell interaction; 3. Physiological function relevance; 4. Disease model construction; 5. Immune microenvironment simulation, etc. From the perspective of toxicological alternative methods, establishing a safety evaluation system and methods that accurately reflect human biological characteristics is of great significance for safeguarding human health. In fact, in 2022, the United States passed the FDA Modernization Act 2.0; in 2020, organoid research projects were included in the EU's Horizon 2020 strategic plan; and in November 2021, the Center for Drug Evaluation of my country's National Medical Products Administration included organoids in its guiding principles for the first time. As one of the emerging in vitro alternative testing methods, organoids have received strong policy support from countries around the world.

[0004] The cardiovascular system, also known as the circulatory system, is a crucial system in the human body responsible for transporting blood, oxygen, nutrients, and metabolic waste. Its main components include the heart, blood vessels, and the lymphatic system. From 1990 to 2019, the prevalence and mortality rates of cardiovascular disease gradually increased, ranking first in global mortality. Cardiovascular disease is also a leading cause of disability, placing a severe burden on the global healthcare economy. Existing in vitro cardiac organoids can be categorized based on different culture methods into scaffold-based and scaffold-free cell spherical aggregates. Based on growth mode, they can be classified as self-assembly growth and cell line co-culture growth; based on maturity, they can be classified as early embryonic cardiac organoids and more mature cardiac organoids. However, in the field of mature cardiac organoids, no cardiac organoid model comparable in maturity to human heart tissue has yet been developed.

[0005] Cardiac organoid models also have some common drawbacks: (1) the number of efficient and stable 3D heart constructs is limited; (2) the three-dimensional heart constructs reported in the literature currently lack tissue microenvironment, especially the immune system and vascular network; (3) they lack the ability to dynamically sense and respond to mechanical stretching; and (4) the maturity of cardiac organoids is still far lower than that of human heart tissue. Cardiac organoids have been used for toxicity testing and drug development research, but the above-mentioned defects mean that cardiac organoid models are still some distance from clinical application. Literature review shows that there are communication mechanisms among endothelial cells, pericytes, fibroblasts and cardiomyocytes that promote the maturation of cardiac organoids. Crosstalk between cardiac cells is crucial to the function of the heart. Vascular cells enhance the maturity and contractility of cardiac cells and have unique utility in disease modeling. Co-culturing cardiac and vascular cells can increase the maturity of organoid models, making them closer to the phenotype of real human organs. Summary of the Invention

[0006] One object of the present invention is to provide a method for differentiating and culturing cardiovascular organoids, so as to shorten the time required to obtain various constituent cells of the heart and various constituent cells of the vascular network.

[0007] Another objective of this invention is to provide a method for differentiating and culturing cardiovascular organoids, thereby obtaining cardiac organoids with deep central vascularization in a relatively short time.

[0008] A method for differentiating and culturing cardiovascular organoids uses stem cells (such as human embryonic stem cells) as starting cells. The cells are first cultured for seven days to induce differentiation of cardiac cells and seven days to induce differentiation of blood vessels, then mixed and fused. After culturing for another four days, dynamic culture is carried out from the eleventh day (e.g., placed on a shaker). On the twentieth day, cardiovascular organoids are harvested, and deep vascularization can be seen in the central region of the organ.

[0009] The cells were cultured in culture medium #2 in a 37°C, 5% CO2 incubator in a cell container that was oriented towards differentiation of heart organoids.

[0010] Cells were cultured in culture medium #1 in a 37°C, 5% CO2 incubator in a container oriented towards vascular organoid differentiation.

[0011] The addition of culture medium #1 or culture medium #2 is recorded as day 1 of differentiation culture.

[0012] On day 2 of differentiation, cells oriented towards cardiac organoid differentiation were cultured using culture medium #3, and cells oriented towards vascular organoid differentiation were cultured using culture medium #5.

[0013] On day 3 of differentiation, cells oriented towards heart organoid differentiation were cultured again using culture medium #3 containing IWP2.

[0014] On day 5 of differentiation, cells oriented towards cardiac organoid differentiation were cultured in culture medium #3, and cells oriented towards vascular organoid differentiation were cultured in culture medium #6.

[0015] Early differentiation was completed on day 7 of differentiation, resulting in precardiac organoids and prevascular organoids.

[0016] Seed anterior cardiac organoids at a density of 3-6 cell spheres / 100μL in low-adhesion containers (e.g., 96-well plates), and anterior vascular organoids at a density of 1-2 cell spheres / 100μL in low-adhesion containers. Co-culture the anterior cardiac organoids with the anterior cardiac organoids using culture medium containing 50 v / v% culture medium #4 and 50 v / v% culture medium #7. Continue culturing the cells in a 37°C, 5% CO2 incubator.

[0017] On day eleven (day four of co-culture), the fused pre-cardiovascular organoids were transferred to dynamic culture (e.g., a magnetic stirrer) using culture medium #4. Culture continued until day twentieth, at which point the cardiovascular organoids were harvested. The frequency of medium replacement was determined based on the number of organoids and the color of the culture medium.

[0018] Before inducing differentiation culture, stem cells (or those that have been passaged) are divided into cardiac organoids and vascular organoids in a ratio of 6:1.

[0019] The method of this invention uses culture medium #2, which comprises: 98 v / v % RPMI-1640 medium + 2 v / v % B-27. TM -Insulin supplement +1ng / mL Activin A +1.25ng / mL BMP4 +3μM CHIR99021.

[0020] The method of this invention uses culture medium #3, which comprises: 98 v / v RPMI-1640 medium + 2 v / v β-27. TM -Insulin supplement, and IWP2 added as needed, to a concentration of 2μM.

[0021] The method of this invention uses culture medium #4, which comprises: 90 v / v% RPMI-1640 medium + 5 v / v% KSR + 2 v / v% B-27. TM Supplements.

[0022] The method of this invention uses culture medium #5, which comprises: 48 v / v% DMEM / F12 medium + 48% neurobasal medium + 2 v / v% B-27. TM Supplement + 1v / v% N-2 Supplement + 1v / v% GlutaMAX TM +12mMCHIR99021+30ng / mL BMP-4.

[0023] The method of this invention uses culture medium #6, which comprises: 48 v / v% DMEM / F12 medium + 48 v / v% neurobasal medium + 2 v / v% B-27. TM Supplement + 1% N-2 supplement + 1v / v% GlutaMAX TM +100ng / mLVEGF-A+2μM forskolin.

[0024] The method of the present invention uses the following components of culture medium #7: 98 v / v% x-vivo15 medium + 2 v / v% FBS.

[0025] To obtain more hES cells, they need to be passaged. Cell separation medium A is used for passage, consisting of 5 mg / mL IV collagenase dissolved in RPMI-1640 medium. hES cells are then cultured using culture medium #1, consisting of mTeSR. TM 1 Complete culture medium + 10 μM Y27632.

[0026] The method of this invention obtains cardiovascular organoids in approximately twenty days. Compared with existing technologies, it combines cardiac and vascular organoids, incorporating multiple cardiac and vascular cell types, and requires less time. Verification has shown that it yields cardiovascular organoid models with improved maturity. The central region of the obtained cardiovascular organoids exhibits vascularization effects through infiltration growth, and multiple culture and construction studies demonstrate its stability and reproducibility.

[0027] The method of this invention uses a single cytokine and requires no extracellular matrix, significantly reducing the cost of culture and differentiation. This facilitates the widespread use of the model. It is suitable for large-scale organoid production, and a single differentiation can meet the testing needs of various subsequent experiments.

[0028] Toxicity testing of compounds / drugs demonstrates that the cardiovascular organoids obtained by this invention are suitable for testing compounds / drugs. Compared to single-heart and single-vessel organoids, cardiovascular organoids offer the advantage of quantifiable concentration testing for toxicity.

[0029] The technical effects of the technical solution provided by this invention are as follows:

[0030] 1. Improve the maturity and function of organoids

[0031] By co-culturing with vascular organoids, cardiomyocytes can obtain a physiological environment closer to that in vivo, thereby improving their contractile function and electrophysiological properties; promoting vascular maturation: co-culturing endothelial cells and smooth muscle cells can better simulate the structure and function of blood vessels, promoting vascular maturation and stability.

[0032] 2. Increase the structural complexity of organoids

[0033] Multiple cell types coexist: Cardiovascular organoids include multiple cell types such as cardiomyocytes, smooth muscle cells, fibroblasts, epithelial cells, progenitor cells, endothelial cells, and pericytes, which can more comprehensively simulate the complexity of cardiovascular tissues; Promote intercellular interactions: Interactions between different cell types can promote cell differentiation and functional maturation. For example, signal transduction between cardiomyocytes and endothelial cells can enhance the contractile function of cardiomyocytes.

[0034] 3. Enhance research and application value

[0035] Drug screening and toxicity testing: Cardiovascular organoids can be used for drug screening and toxicity testing, enabling more accurate prediction of the effects and safety of drugs in the human cardiovascular system.

[0036] 4. Improve culture efficiency and reproducibility

[0037] A phased culture strategy, culturing pre-cardiac organoids and pre-vascular organoids separately before co-culturing them, allows for better control of the culture process and improves efficiency and reproducibility. Standardized culture methods provide a standardized operating procedure for the culture of cardiovascular organoids, facilitating replication and comparison between different laboratories.

[0038] 5. Promotes cell differentiation and functional maturation

[0039] Enhanced intercellular signaling: Co-culture enables better signal transduction and interaction between different cell types, promoting cell differentiation and functional maturation. Improved cell metabolism and function: The co-culture environment provides metabolic conditions closer to those in vivo, promoting cell metabolism and functional maturation.

[0040] 6. Reduce research costs and time

[0041] Reduce the use of laboratory animals: Cardiovascular organoids can serve as in vitro models, reducing reliance on laboratory animals, lowering research costs, and alleviating ethical concerns. Attached Figure Description

[0042] Figure 1 This is a schematic diagram illustrating the method for culturing cardiovascular organoids according to the present invention;

[0043] Figure 2 Characterization diagram of cardiovascular organoid biomarkers;

[0044] Figure 3 A schematic diagram of the cardiovascular network in cardiovascular organoids;

[0045] Figure 4 Statistical graph of biomarker expression in cardiovascular organoids;

[0046] Figure 5 A statistical chart showing the lactate dehydrogenase content in the supernatant of cardiac and cardiovascular organoids after exposure to the reference compound;

[0047] Figure 6 A statistical graph showing the staining results of live and dead cells in cardiac and cardiovascular organoids after exposure to the reference compound. Detailed Implementation

[0048] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0049] The main experimental materials used in the embodiments of this invention are from the following sources:

[0050] The human embryonic stem cell (hES) line H9 (WA09) was purchased from the Weill Institute in the United States.

[0051] The culture medium was RPMI-1640 (Roswell Park Memorial Institute 1640 Medium), mTeSR TM 1( Complete Kit), KSR (KnockOut) TM Serum Replacement, DMEM / F12 medium, neurobasal medium, and x-vivo15 are common reagents that can be purchased directly from companies as finished products (e.g., ThermoFisher, Stem Cell, etc.).

[0052] B-27 TM The supplement (serum-free) was purchased from Gibco, catalog number: 17504044.

[0053] N-2 supplements were purchased from Gibco, product number: 17502048.

[0054] The recombinant human cytokine BMP-4 and recombinant human Activin A used for hES culture and induced differentiation were purchased from ThermoFisher, Inc., USA.

[0055] The compounds CHIR99021 and IWP2 used for hES culture and induced differentiation were purchased from Beyotime International Co., Ltd. in China.

[0056] The overall steps of culturing cardiovascular organoids using the method of this invention are as follows: Figure 1 As shown, the cell culture medium and its composition used in the following embodiments of the present invention are as follows:

[0057] 1) Cell separation solution A, with the following components:

[0058] 5 mg / mL IV collagenase was dissolved in RPMI-1640 medium.

[0059] 2) Culture medium #1

[0060] mTeSR TM 1. Complete culture medium for routine culture of hES.

[0061] 3) Culture medium #2

[0062] The components used for the first stage (seven days) of induction differentiation of hES centripetal organoids are as follows:

[0063] 98% RPMI-1640 medium + 2% B27-insulin supplement + 1 ng / mL Activin A + 1.25 ng / mL BMP4 + 3 μM CHIR99021.

[0064] 4) Culture medium #3

[0065] The components used for the second stage (seven days) of induction differentiation of hES centripetal organoids are as follows:

[0066] 98% RPMI-1640 medium + 2% B27-insulin supplement, and IWP2 added as needed to a concentration of 2 μM.

[0067] 5) Culture medium #4

[0068] The components used for the third-stage (seven-day) induction of hES into cardiovascular organoids are as follows:

[0069] 90% RPMI-1640 medium + 5% KSR + 2% B27 supplement.

[0070] 6) Culture medium #5

[0071] The following components were used for the first stage (seven days) of hES-induced differentiation into vascular organoids:

[0072] 48% DMEM / F12 medium + 48% neurobasal medium + 2% B-27 TM Supplement + 1% N-2 Supplement + 1% GlutaMAX TM +12mM CHIR99021 +30ng / mL BMP-4

[0073] 7) Culture medium #6

[0074] The following components were used for the second stage (seven days) of hES-induced differentiation into vascular organoids:

[0075] 48% DMEM / F12 medium + 48% neurobasal medium + 2% B-27 TM Supplement + 1% N-2 supplement + 1% Glutamax + 100 ng / mL VEGF-A + 2 μM forskolin.

[0076] 8) Culture medium #7

[0077] The components used for the third-stage (seven-day) induction of hES into cardiovascular organoids are as follows:

[0078] 98% x-vivo15 medium + 2% FBS.

[0079] Example 1: Culture of human embryonic stem cells

[0080] hES cells will be provided in a state of normal growth after resuscitation. Once the cell density reaches more than 70%, the cells will be seeded in clumps into Matrigel-coated (1:15 dilution) culture dishes.

[0081] hES passage: Cells were passaged using cell separation solution A.

[0082] On days 4-5 after hES inoculation, the number of stem cells reaches a high level of proliferation, and large-diameter, high-density clones (about 70%) can be observed in the culture dish. At this time, passage is performed.

[0083] Aspirate the hES culture medium intended for passage and wash twice with D-PBS.

[0084] Add 1 ml of cell separation solution A to the culture dish and incubate in a 37°C, 5% CO2 incubator. After 8-10 minutes, remove the dish and observe that the clonal structure is obviously loose and the outer edge of the clonal curls. Add 1 ml of culture solution #1 to stop digestion. Transfer the obtained cell clumps to a centrifuge tube and centrifuge at 300 rpm for 3 minutes at room temperature.

[0085] Remove the supernatant and passage the cells to pre-coated Matrigel culture dishes at a ratio of 1:4 to 1:8, depending on the cell quantity, and culture with an appropriate amount of hES medium.

[0086] Example 2: Human embryonic stem cells were induced to differentiate into procardiac organoids and provascular organoids.

[0087] When the cells passaged in cell separation medium A reached 70% confluence, the cells were divided into cardiac organoids and vascular organoids at a ratio of 6:1. Cardiac organoid cells were cultured in 37°C, 5% CO2 incubators using 5 ml of culture medium #2 per 6 cm dish.

[0088] The vascular organoid differentiated cells were cultured in a 37°C, 5% CO2 incubator using 5 ml of culture medium #1 per 6 cm dish.

[0089] This is recorded as day 1 of differentiation.

[0090] On day 2 of differentiation, the heart organoid differentiated cells were cultured again using 5 ml of culture medium #3 per 6 cm dish. IWP2 was not added to culture medium #3 at this time. Then, the heart organoid differentiated cells were cultured again using 5 ml of culture medium #5 per 6 cm dish.

[0091] On day 3 of differentiation, the heart organoid differentiated cells were cultured again using 5 ml of culture medium #3 per 6 cm dish. At this time, IWP2 was added to culture medium #3.

[0092] On day 5 of differentiation, the heart organoid differentiated cells were cultured again using 5 ml of culture medium #3 per 6 cm dish. IWP2 was not added to culture medium #3 at this stage. Then, the heart organoid differentiated cells were cultured again using 5 ml of culture medium #6 per 6 cm dish.

[0093] The pre-differentiation was completed on the 7th day of differentiation, and pre-cardiac organoids and pre-vascular organoids were obtained, which prepared for the subsequent construction of cardiovascular organoids.

[0094] Example 3: Induction of precardiac organoids and prevascular organoids into cardiovascular organoids

[0095] On day 7 of differentiation, anterior cardiac organoids were seeded at a density of 3-6 cell spheroids / 100 μL in low-adhesion 96-well plates (purchased from NEST Biotechnology, China). Anterior vascular organoids were seeded at a density of 1-2 cell spheroids / 100 μL in the same low-adhesion 96-well plate, with each well containing 200 μL of culture medium containing 50% culture medium #4 and 50% culture medium #6. The cells were cultured further at 37°C in a 5% CO2 incubator.

[0096] On day 11 of differentiation, the fused pre-cardiovascular organoids were transferred to a magnetic stirrer for dynamic culture. 80 mL of culture medium #4 was added to each culture flask. The frequency of culture medium replacement was determined based on the number of organoids and the color of the culture medium. Culture was completed on day 20.

[0097] Example 4: Characterization and Identification of Differentiated Cardiovascular Organoids

[0098] The cells used in this embodiment are derived from Example 3.

[0099] (1) After cell culture is completed, add 4% paraformaldehyde, place at room temperature for 20 minutes, wash twice with PBS, and prepare frozen sections.

[0100] (2) Cardiovascular organoids were collected, and RNA was extracted to perform qPCR to determine the expression levels of various markers, including platelet-derived growth factor receptor β (PDGFRB), vascular endothelial growth factor receptor (VEGFR), platelet-endothelial cell adhesion molecule 1 (PECAM1), myocyte enhancer factor 2C (MEF2C), cardiac troponin T2 (TNNT2), NK2 homeobox transcription factor 5 (NKX2.5), connexin 43 (CX43), natriuretic peptide precursor A (NPPA), matrix metalloproteinase 9 (MMP9), cardiac and neural crest derivative expression transcription factor 1 (HAND1), insulin gene enhancer-binding protein 1 (ISL1), bone morphogenetic protein 4 (BMP4), and T-box transcription factor 5 (TBX5). The expression levels of these markers in simple COs and VOs models were compared as controls. Figure 4 As shown.

[0101] Compared to COs and VOs alone, CVOs exhibited altered expression across multiple markers. PECAM1, a cell adhesion molecule primarily located on the surface of endothelial cells, was typically upregulated during these cells' maturation and functional enhancement. PDGFRβ was primarily expressed in vascular smooth muscle cells (SMCs) and pericytes. PDGFRβ is generally associated with the proliferation, migration, and maturation of these cell types. The PDGFRβ signaling pathway is a crucial regulator of angiogenesis and remodeling. VEGFR, the major receptor for vascular endothelial growth factor (VEGF), often shows decreased expression levels as endothelial cells mature and differentiate. This downregulation of VEGFR may signify the transition of endothelial cells from a proliferative to a mature state. The observed changes in the expression patterns of these three markers collectively suggest that the CVOs model enhances vascular maturation compared to the VOs model.

[0102] In terms of cardiac-related indicators, MEF2C is a key transcription factor primarily expressed in cardiomyocytes. Its upregulation is generally closely associated with cardiomyocyte maturation and enhanced function. Elevated MYH6 expression levels indicate enhanced cardiomyocyte contractility, enabling more efficient pumping function of the heart. Upregulation of GATA4 is typically associated with cardiomyocyte differentiation and maturation, and promotes cardiomyocyte proliferation and survival. Decreased TNNT may indicate a transition from a proliferative to a mature state in cardiomyocytes, with reduced dependence on Ca2+ regulation. Therefore, the upregulation of MEF2C, MYH6, and GATA4, and the downregulation of TNNT reflect the transition of cardiomyocytes from a proliferative and differentiation phase to a maturation and functional optimization phase. These changes collectively reflect enhanced CVO vascular networks and improved myocardial maturity.

[0103] (3) After preparation, add antibodies (cardiomyocytes (TNNT)) used to identify different cells. + Sacro-α-actinin + ), smooth muscle cells (α-SMA) + ), fibroblasts (COL1A1) + ), epithelial cells (KRT18) + ), progenitor cells (NKX2.5) + ) and endothelial cells representing vascular organoids (CD31) + ) and pericytes (PDGFR-β) + After incubating at 4°C for 18 hours, wash three times with PBS for 5 minutes each time. Then incubate with fluorescent secondary antibody at 37°C for 2 hours, followed by washing three times with PBS for 5 minutes each time. Finally, stain with DAPI solution, wash five times with PBS, and observe under a fluorescence microscope. Figure 2 and Figure 3 As shown, the expression of these markers indicates that the generated cardiovascular organoids possess at least seven cell types: cardiomyocytes, smooth muscle cells, fibroblasts, epithelial cells, progenitor cells, and endothelial cells and pericytes, which represent vascular organoids. Pericytes are not found in the single COs model, and cardiomyocytes are not found in the single VOs model. This demonstrates the successful construction of cardiovascular organoids.

[0104] Example 5: Verification of the toxicity of the cardiovascular reference compound to cardiovascular organoids derived from hES.

[0105] The reference compounds for cardiovascular toxicity are shown in Table 1 below. The main determining factors for the reference content include: 1. Commonly used drugs in clinical practice; 2. Drugs with certain potential vascular toxicity in addition to the target organ; 3. Compounds with three or more publications in existing human case reports, mammalian experiments, and in vitro cell experiments demonstrating corresponding cardiovascular toxicity are selected as positive compounds. Compounds with no case reports or cardiovascular toxicity-related reports in existing literature are selected as negative compounds; 4. Four data gradients are selected for experiments based on clinical dosage and in vitro / in vivo data.

[0106] Table 1. Cardiovascular organoid toxicity testing mode, compound toxicity, and dosage selection.

[0107]

[0108] During the culture process, differentiating cells were exposed to the toxicity test culture media at the concentrations shown in Table 1 for 72 hours. After exposure, the supernatant was collected for lactate dehydrogenase (LDH) content determination and live / dead cell assay. The results are as follows: Figure 5 and Figure 6 As shown.

[0109] Amiodarone is one of the most commonly used drugs for treating arrhythmias. However, higher concentrations of amiodarone can also cause cardiovascular toxicity. Amiodarone is a biphasic compound. In the amiodarone MH and H groups, a significant increase in cell death in both COs and CVOs was observed. In contrast, COs exhibited higher levels of LDH release and earlier cell death. The overall trend of COs was almost linear, with no "S"-shaped change observed. However, in the CVOs group, cell death was significantly increased between the MH and H groups, showing an "S"-shaped change similar to IC50, which better reflects the safe dosage range required for clinical use. This highlights the better predictive ability of the CVOs model compared to the drug's IC50 value.

[0110] Cisplatin, a platinum-based compound, is a cell cycle-nonspecific antitumor drug with broad-spectrum antitumor activity and is a typical cardiotoxic compound. Results showed that LDH levels in the supernatant of all CVO groups were significantly increased, approximately two-fold higher than in the amiodarone group. Live and dead cell staining revealed a cell death rate of approximately 25% in the MH group and nearly 40% in the H group. However, LDH release from COs was lower than that from amiodarone. Compared to the COs model, the CVOs model exhibited greater toxicity in both LDH release and cell death. We attribute this to cisplatin-induced vascular endothelial dysfunction, characterized by endothelial cell damage, increased oxidative stress, and activated inflammatory responses. This endothelial dysfunction leads to vasoconstriction and reduced blood flow, thereby exacerbating myocardial ischemia. The insufficient myocardial blood supply caused by endothelial dysfunction further impairs vascular function, producing a synergistic destructive effect on the heart and blood vessels. Therefore, it exhibits stronger toxicity to CVOs. This indicates that CVOs can not only reflect cardiotoxicity but also test vascular toxicity, a toxicity endpoint, thus broadening the applicability of the model. In the negative markers dexamethasone and vitamin C, CVOs showed a higher proportion of live cells than COs, demonstrating good activity of CVOs.

[0111] Based on the above results, we can conclude that cardiovascular organoids have advantages over single-heart and single-vessel organoids for toxicity testing.

Claims

1. A method for differentiating and culturing cardiovascular organoids, characterized in that, The stem cells used as the starting cells were first cultured for seven days to induce differentiation of cardiac cells and seven days to induce differentiation of blood vessels, then mixed and cultured for fusion. After another four days of culture, dynamic culture was carried out from the eleventh day until the twentieth day, when cardiovascular organoids were harvested. Deep vascularization could be seen in the central region of the organ.

2. The method according to claim 1, characterized in that... Before inducing differentiation culture, stem cells were divided into cardiac organoids and vascular organoids at a ratio of 6:

1.

3. The method according to claim 1, characterized in that... Culture medium #2 was used in cell containers that differentiate into heart-like organoids. The components of culture medium #2 include: 98 v / v RPMI-1640 medium, 2 v / v β-27. TM -Insulin supplement, 1 ng / mL Activin A, 1.25 ng / mL BMP4 and 3 μM CHIR99021.

4. The method according to claim 1, characterized in that... Culture medium #1 was used in cell containers that differentiate into vascular organoids. The components of culture medium #1 include mTeSR TM 1. Complete culture medium containing 10 μM Y27632.

5. The method according to claim 1, characterized in that... On the second day of differentiation, cells oriented towards cardiac organoid differentiation were cultured in culture medium #3, and cells oriented towards vascular organoid differentiation were cultured in culture medium #5. Culture medium #3 consists of: 98 v / v RPMI-1640 medium and 2 v / v B-27. TM -insulin.

6. The method according to claim 1, characterized in that... On day 3 of differentiation, cells oriented towards heart organoid differentiation were cultured again using culture medium #3 containing IWP2. Culture medium #3 consists of: 98 v / v RPMI-1640 medium and 2 v / v B-27. TM -insulin.

7. The method according to claim 1, characterized in that... On day 5 of differentiation, cells oriented towards cardiac organoid differentiation were cultured in culture medium #3, and cells oriented towards vascular organoid differentiation were cultured in culture medium #6. Culture medium #3 consists of: 98 v / v RPMI-1640 medium and 2 v / v B-27. TM -insulin; The components of culture medium #5 include: 48 v / v% DMEM / F12 medium, 48 v / v% neurobasal medium, and 2 v / v% B-27. TM ,1v / v%N-2,1v / v%GlutaMAX TM 12mM CHIR99021 and 30ng / mL BMP-4.

8. The method according to claim 1, characterized in that... On day 7 of differentiation, anterior cardiac organoids were seeded in low-adhesion containers at a density of 3-6 cell spheres / 100μL, and anterior vascular organoids were seeded in low-adhesion containers at a density of 1-2 cell spheres / 100μL. They were then co-cultured with anterior cardiac organoids using culture media containing 50 v / v% culture medium #4 and 50 v / v% culture medium #7. The components of culture medium #4 include: 90 v / v RPMI-1640 medium, 5 v / v % KSR and 2 v / v % B-27. TM ; The components of culture medium #7 include: 98 v / v% x-vivo15 medium + 2 v / v% FBS.

9. The method according to claim 1, characterized in that... On day eleven, the fused pre-cardiovascular organoids were transferred to dynamic culture using culture medium #4 and cultured until day twentieth. The components of culture medium #4 include: 90 v / v RPMI-1640 medium, 5 v / v % KSR and 2 v / v % B-27. TM .

10. The method according to claim 1, characterized in that... Stem cells were passaged using cell separation solution A, which included 5 mg / mL IV collagenase dissolved in RPMI-1640 medium.

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