Method for high-throughput preparation of heart organoid

Abstract

The invention discloses a method for high-throughput preparation of heart organoid. Relates to the technical field of biology. Comprising the following steps: culturing iPSC; performing iPSC digestion, inoculation and culture before induction; performing induction; and continuously culturing. The method can be used for simply preparing a large number of heart organs. The preparation method is simple and convenient, has low requirements on materials and equipment, and needs few reagents. And a large number of organoid can be obtained in the same batch. The organoid is composed of various cell types such as myocardial cells, fibroblasts, vascular endothelial cells and the like. The contraction of the cardiac organoids is induced in response to a drug.

Description

Technical Field

[0001] This invention relates to the field of biological technology, and more specifically to a method for high-throughput preparation of cardiac organoids. Background Technology

[0002] Heart organoids, as an emerging biotechnology tool, have demonstrated significant advantages in scientific research and medicine. First, heart organoids can highly simulate the three-dimensional structure and some functions of the real heart, such as the contractile properties of cardiomyocytes and calcium signaling characteristics, providing an ideal platform for studying cardiac physiological and pathological mechanisms. Second, heart organoids contain various cardiac cell types, such as cardiomyocytes, endothelial cells, and fibroblasts. This cellular diversity makes them closer to real heart tissue and can more comprehensively reflect the complex functions of the heart. Furthermore, heart organoids generated using patient-derived induced pluripotent stem cells (iPSCs) can be used to study personalized disease mechanisms, providing an important tool for precision medicine. In drug development, heart organoids can be used for high-throughput drug screening, assessing the effects of drugs on cardiac function, and more accurately predicting drug toxicity to the heart, thereby reducing risks in clinical trials. Compared to traditional animal models, heart organoids better reflect the biological characteristics of the human heart, providing a more reliable research platform for scientific research and drug development.

[0003] Despite the numerous advantages of heart organoids, their practical application still faces several challenges. The induction process requires costly growth factors, which are highly dependent on their quality. A common induction method involves first forming embryoids and then inducing them into heart organoids, which carries the risk of organoid loss during the induction process.

[0004] Therefore, whether a high-throughput method for preparing cardiac organoids can be provided is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a high-throughput method for preparing cardiac organoids. To reduce the production cost of cardiac organoids and improve operational convenience, the organoid induction method developed in this invention eliminates the need for additional growth factors in the basal culture medium throughout the induction process, thus reducing costs. During induction, the cells remain in a two-dimensional adherent state, reducing the risk of cell loss during medium changes. The constructed cardiac organoids can respond to the effects of drugs on heart rate and can be used for drug toxicity testing.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for high-throughput preparation of cardiac organoids includes the following steps:

[0008] (1) Cultivate iPSCs;

[0009] (2) iPSC digestion, inoculation and pre-induction culture;

[0010] (3) Induction:

[0011] 31) Replace the culture medium with RPMI 1640 and add insulin-free B27 and CHIR99021, then culture.

[0012] 32) Subsequently, the culture medium was changed to RPMI 1640 with the addition of insulin-free B27 and IWR1.

[0013] 33) Change the culture medium to RPMI 1640 and add insulin-free B27 for culture;

[0014] (4) Continued culture: On the 8th day of induction, the culture medium was changed to RPMI 1640 and insulin B27 was added for culture; cell clumps were obtained and transferred to a horizontal shaker for culture to obtain heart organoids.

[0015] Preferred: Step (1) includes:

[0016] 1) Prepare the culture medium: dissolve mTeSR TM Plus 5X Additives; mTeSR TM Plus 5X additive added to mTeSR TM Add the antibiotics to the Plus basal medium and mix thoroughly to obtain mTeSR. TM Plus complete culture medium;

[0017] 2) Coated culture dish: Dissolve Vitronectin XF TM Using CellAdhere TM Dilute with dilution buffer; add to non-tissue culture plates; discard coating solution before use. CellAdhere TM Wash with dilution buffer to prepare for iPSC inoculation;

[0018] 3) Cell digestion and passage: Discard the culture medium in the culture dish, add Gentle Cell Dissociation Reagent, and let it stand until the cells detach from the bottom of the dish. Discard the Gentle Cell Dissociation Reagent and add mTeSR. TM Plus complete medium, aspirate and pipette the medium; seed cells into new culture wells at the appropriate density; passage; add ROCK inhibitor; change medium daily (mTeSR). TM Plus complete culture medium until cell confluence reaches 80-95% for the preparation of heart organoids.

[0019] Preferred: Step 1) Bispecific antibodies: penicillin, streptomycin; Step 2) Vitronectin XF TM and CellAdhere TM The dilution buffer ratio is 40 μl: 960 μl; the inoculation ratio for subculturing in step 3) is 1:5 to 1:20.

[0020] Furthermore, in step 3), the inoculation ratio for passage is 1:5.

[0021] Preferred: Step (2) includes:

[0022] 21) Discard the culture medium, add Accutase until the cells detach from the bottom of the dish, discard the Accutase, and add mTeSR. TM Plus, complete culture medium is pipetted to single cells, and the cells are counted and plated.

[0023] 22) Inoculation with Vitronectin XF TM In coated 96-well plates, the inoculation density is 3000–6000 cells per well; using mTeSR TM After adding Y-27632 to the Plus culture medium, the medium was changed; the medium was then replaced with mTeSR. TM Plus complete culture medium, change the medium daily until day 4.

[0024] Preferred conditions: Step 21) Conditions for cells to detach from the bottom of the dish: 37°C for 3-5 minutes; Step 22) Seeding density: 3000 cells per well; Culture time: 24 hours.

[0025] Preferred step (3) specifically includes:

[0026] 31) Replace the culture medium with 200 μl RPMI 1640 and add 1 / 50 volume of insulin-free B27 and 6-8 μM CHIR99021, and incubate for 48 hours;

[0027] 32) Subsequently, the culture medium was changed to 200 μl RPMI 1640, and 1 / 50 of the volume of insulin-free B27 and 3-7 μM IWR1 were added and cultured for 48 hours;

[0028] 33) Replace the culture medium with 200 μl RPMI 1640 and add 1 / 50 of the volume of insulin-free B27 and culture for 72 hours.

[0029] Preferred: Step (4) Shaking culture: culture at 50-100 rpm for 2 days.

[0030] The present invention also provides cardiac organoids prepared by any of the above methods.

[0031] This invention also provides the application of any of the above methods or the above-described cardiac organoids in pharmaceutical and basic research.

[0032] Preferred: For drug toxicity testing.

[0033] As can be seen from the above technical solution, compared with the prior art, this invention discloses a method for high-throughput preparation of cardiac organoids. The technical effect achieved is that this invention can easily prepare a large number of cardiac organoids. The preparation method is simple, with low requirements for materials and equipment, and requires few reagents. A large number of organoids can be obtained in the same batch. The organoids are composed of various cell types such as cardiomyocytes, fibroblasts, and vascular endothelial cells. The contraction frequency of cardiac organoids is responsive to drug induction. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0035] Figure 1 The attached figures are representative diagrams of the construction process and induction process of the cardiac organoids provided by the present invention, wherein A: construction process of the cardiac organoids; B: representative diagram of the induction process.

[0036] Figure 2 The attached figure is a statistical graph of the contraction frequency of the heart organoid provided by the present invention.

[0037] Figure 3 The attached figure shows the analysis of cardiac organoid-related mRNA expression levels provided by this invention. A: Analysis of mRNA expression levels of cardiomyocyte markers in two-dimensional induced cardiomyocytes and iPSCs; B: Analysis of mRNA expression levels of pluripotent stem cell markers in two-dimensional induced cardiomyocytes and iPSCs; C: Analysis of mRNA expression levels of fibroblast and vascular endothelial cell markers in two-dimensional induced cardiomyocytes and iPSCs; Unpaired t-test, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

[0038] Figure 4The attached figure shows the response of cardiac organoids provided by this invention to drugs that alter cardiac contraction frequency. A: Effect of 1 nM and 100 nM on the contraction frequency of cardiac organoids before induction with isoproterenol; B: Effect of 10 μM and 100 μM on the contraction frequency of cardiac organoids before induction with Ivabradine; C: Effect of 1 μM and 10 μM on the contraction frequency of cardiac organoids before induction with Amiodarone; D: Statistical results, unpaired t-test, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, nsp>0.05.

[0039] Figure 5 The attached figure shows the response of cardiac organoids prepared by the conventional method provided by this invention to drugs that alter cardiac contraction frequency. A: Statistical results of the effects of 1 nM and 100 nM isoproterenol on the contraction frequency of cardiac organoids before induction; B: Statistical results of the effects of 10 μM and 100 μM isoproterenol on the contraction frequency of cardiac organoids before induction; C: Statistical results of the effects of 1 μM and 10 μM isoproterenol on the contraction frequency of cardiac organoids before induction; Unpaired t-test, ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05, nsp>0.05. Detailed Implementation

[0040] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0041] This invention discloses a method for high-throughput preparation of cardiac organoids.

[0042] Example 1

[0043] A method for high-throughput preparation of cardiac organoids (process flow is shown in...) Figure 1 (A) includes the following steps:

[0044] (1) Cultivate iPSCs (amplify them to the required number):

[0045] 1) Prepare the culture medium: Dissolve mTeSR at room temperature (15-25℃). TMPlus 5X Additives (STEMCELL Catalog #100-0276); Add 10 mL of mTeSR TM Plus 5X additive was added to 40 mL of mTeSR TM Add the antibiotics (penicillin and streptomycin) to the Plus basal medium (STEMCELL Catalog #100-0276), mix thoroughly, and this is mTeSR. TM Plus complete culture medium; sterilize by filtration with a 0.22-micron low-protein filter; allow to warm to room temperature for half an hour before each use;

[0046] 2) Coated culture dishes: Dissolve Vitronectin XF at room temperature TM (STEMCELL), using CellAdhere TM Dilute with (STEMCELL Catalog #07183) dilution buffer; ratio: 40 μl: 960 μl; add 500 μl to each well of a 12-well non-tissue culture plate; gently shake the plate back and forth to ensure the coating solution evenly covers the surface; allow to incubate at room temperature (15-25℃) for 1 hour before use; discard the coating solution before use and dilute with 500 μl CellAdhere buffer. TM Wash once with dilution buffer and prepare to inoculate iPSC cells (Chinese Academy of Sciences Cell Bank, catalog number: SCSP-1301);

[0047] 3) Cell digestion and passage: Discard the culture medium in the culture dish, add 500 μl of room temperature GCDR (STEMCELL Gentle Cell Dissociation Reagent Catalog #100-0485), and incubate at room temperature for 5-10 minutes (8 minutes in this example) until the cells detach from the bottom of the dish. Discard the GCDR and add mTeSR. TM 1 mL of Plus complete culture medium was used to detach cells by aspiration; cells were then seeded into new culture wells at the appropriate density; the seeding ratio was 1:5 to 1:20 for passage (1:5 in this example); 10 μM of Y-27632 (ROCK inhibitor) was added; 1 mL of mTeSR medium was changed daily. TM Plus complete culture medium, until the cell confluence reaches 80-95% (90% in this example), can be used to prepare for the preparation of heart organoids;

[0048] (2) iPSC digestion, inoculation, and pre-induction culture:

[0049] 21) Discard the culture medium and add 500 μl of Accutase (Gibco). TMIncubate at 37°C for 3-5 minutes (3 minutes in this example) until cells detach from the bottom of the dish. Discard Accutase and add 1 mL mTeSR. TM Plus, complete culture medium is pipetted to single cells, and the cells are counted and plated.

[0050] 22) Inoculation with Vitronectin XF TM In the coated 96-well plate, the seeding density is 3000–6000 cells per well (3000 cells per well in this example); 100 μm TeSR TM After adding 10 μL MY-27632 to the Plus culture medium and culturing for 24 hours, the medium was changed; the medium was then replaced with 100 μL lmTeSR. TM Plus complete culture medium, change the medium daily until day 4;

[0051] (3) Induction:

[0052] 31) Replace the culture medium with 200 μl RPMI 1640 (Gibco, c11875500bt) and add 1 / 50 volume of B27 (insulin-free, Gibco, A1895601) and 6-8 μM CHIR99021 (8 μM in this example), and incubate for 48 hours;

[0053] 32) The culture medium was then changed to 200 μl RPMI 1640, with 1 / 50 of the volume of B27 (without insulin) and 3-7 μM IWR1 (5 μM in this example; IWR1 source: Mce, HY-12238) added and cultured for 48 hours;

[0054] 33) Replace the culture medium with 200 μl RPMI 1640 and add 1 / 50 of the volume of B27 (without insulin) and culture for 72 hours;

[0055] (4) Continued culture: On day 8 of induction, replace the culture medium with 200 μl RPMI 1640 and add 1 / 50 volume of B27 (containing insulin, Gibco, 17504044) for culture (change the medium every 2-3 days); to obtain beating cell clumps, transfer them to a horizontal shaker and culture at 50-100 rpm for 2 days to obtain suspended and beating heart organoids. Figure 1 (B)

[0056] The statistical results of heart organoid beating frequency are as follows: Figure 2 ;

[0057] To identify the cell composition of the obtained cardiac organoids, cardiac organoids were collected on day 20 of induction and their mRNA expression levels were routinely analyzed (RNA was extracted from the organoids using the Trizol method and reverse transcribed into cDNA; mRNA expression levels were detected using quantitative real-time PCR).

[0058] Results: Compared with iPSCs, the expression of the cardiac organoid stem cell markers SSEA and SOX2 in this invention was significantly reduced. Figure 3 (B) The expression of cardiomyocyte markers TNNT2, MYL2, MYH6, and MYH7 was significantly increased, and the MYH7 / MYH6 ratio was significantly higher than that in two-dimensional cells, indicating that the organoids were more mature. Figure 3 (A). Fibroblast marker VIM was significantly increased, and vascular endothelial cell marker CD31 was significantly increased. Figure 3 The presence of C indicates that the constructed heart organoids contain cardiomyocytes, fibroblasts, and vascular endothelial cells.

[0059] To verify whether the constructed cardiac organoids could respond to drug induction and alter their contraction frequency, isoproterenol (which induces an increase in contraction frequency), ivabradine (which induces a decrease in contraction frequency), and amiodarone (which induce a decrease in contraction frequency) were applied to the cardiac organoids at different concentrations.

[0060] result:

[0061] First, the response of cardiac organoids to Isoproterenol was verified. Before induction, the contraction of the cardiac organoids was recorded. Then, 1 nM Isoproterenol was added, and after 20 minutes, the contraction frequency of the cardiac organoids significantly increased. Next, Isoproterenol was added to a final concentration of 100 nM; after 20 minutes, the contraction frequency of the cardiac organoids increased slightly, but the difference was not statistically significant. Figure 4 (A, D)

[0062] To verify the response of cardiac organoids to Ivabradine, the contraction of the cardiac organoids was recorded before induction. Subsequently, 10 μM Ivabradine was added; after 20 minutes, the contraction frequency of the cardiac organoids decreased slightly, but the difference was not significant. Next, Ivabradine was added to a final concentration of 100 μM; after 20 minutes, the contraction frequency of the cardiac organoids decreased significantly, and all heartbeats stopped. Figure 4 (B, D)

[0063] The response of cardiac organoids to amiodarone was verified. Contraction of the cardiac organoids was recorded before induction. Then, 1 μM amiodarone was added; after 20 minutes, the contraction frequency of the cardiac organoids significantly decreased, and some stopped beating. Next, amiodarone was added to a final concentration of 10 μM; after 20 minutes, all cardiac organoids stopped beating. Figure 4 (C, D)

[0064] This invention demonstrates that the organoids prepared by this invention can be used for screening or validation of drugs that induce changes in heart rate.

[0065] Comparative Experiment 1

[0066] Comparison with existing commonly used preparation methods

[0067] Common preparation method: Embryomorphs are prepared using U-shaped low-adhesion 96-well plates. Then, B27-insulin is added to RPMI 1640 as the basal medium, and mesoderm is induced with Bmp4, Activin A, and CHIR99021. Cardiac mesoderm is induced with a Wnt signaling pathway inhibitor, and cardiomyocytes are further cultured on RPMI 1640 with added B27-insulin to form cardiomyocytes.

[0068] The heart organoids prepared by this invention are more sensitive to drugs that induce changes in cardiac contraction frequency than heart organoids prepared by commonly used methods. Figure 4 and Figure 5 (Tables 1-3). Isoproterenol increases heart rate; the cardiac organoids prepared in this invention show an increasing heart rate at a concentration of 1 nM, while commonly used methods show abnormal responses at the same concentration. Ivabradine decreases heart rate; the cardiac organoids prepared in this invention show a decreasing heart rate at a concentration of 100 μM, while commonly used methods show no response at the same concentration. Amiodarone decreases heart rate; the cardiac organoids prepared in this invention show a decreasing heart rate at a concentration of 10 μM, while commonly used methods show no response at the same concentration.

[0069] Table 1

[0070] Isoproterenol concentration 0 1nM 100nM Common methods for preparing cardiac organoid responses / Significantly reduced Indifference The present invention provides a response for preparing cardiac organoids. / Significantly increased Significantly increased

[0071] Table 2

[0072] Ivabradine concentration 0 10μM 100μM Common methods for preparing cardiac organoid responses / Indifference Indifference The present invention provides a response for preparing cardiac organoids. / Indifference Significantly reduced

[0073] Table 3

[0074] Amiodarone concentration 0 1μM 10μM Common methods for preparing cardiac organoid responses / Indifference Indifference The present invention provides a response for preparing cardiac organoids. / Significantly reduced Significantly reduced

[0075] Comparative Experiment 2

[0076] Table 4 compares the effects of changing the type and amount of culture medium raw materials.

[0077] This indicates that changing the concentration of CHIR99021 resulted in different rates of heart organoid beating, with a concentration of 8 μM being the optimal.

[0078] Table 4

[0079] CHIR99021 concentration 6μM 7μM 8μM Initial cell count 3000 100％(n＝16) 100％(n＝14) 100％(n＝12) Initial cell count 6000 0％(n＝6) 0％(n＝8) 20％(n＝6)

[0080] Furthermore, based on an initial cell count of 3000, changing the IWR1 concentration caused all heart-like organoids to beat.

[0081] IWR 13μM, fluctuation ratio 100% (n=11)

[0082] IWR 15μM, fluctuation ratio 100% (n=21)

[0083] IWR 17μM, fluctuation ratio 100% (n=10)

[0084] This indicates that changing the IWR1 concentration did not change the effect.

[0085] Comparative Experiment 3

[0086] Comparison of effects after changing culture steps and parameters

[0087] Changing the initial cell count resulted in different proportions of jumping organoids. With an initial cell count of 3000 cells per well, the organoid jumping rate was 100% (all 42 organoids jumped). With an initial cell count of 6000 cells per well, the organoid jumping rate was 25% (5 out of 20 organoids jumped).

[0088] This indicates that an initial cell count of 3000 cells resulted in better induction.

[0089] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0090] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for high-throughput preparation of cardiac organoids, characterized in that, Includes the following steps: (1) Cultivate iPSCs; (2) iPSC digestion, inoculation and pre-induction culture; (3) Induction: 31) Replace the culture medium with RPMI 1640 and add insulin-free B27 and CHIR99021, then culture. 32) Subsequently, the culture medium was changed to RPMI 1640 with the addition of insulin-free B27 and IWR1. 33) Change the culture medium to RPMI 1640 and add insulin-free B27 for culture; (4) Continued culture: On the 8th day of induction, the culture medium was changed to RPMI 1640 and insulin B27 was added for culture; cell clumps were obtained and transferred to a horizontal shaker for culture to obtain heart organoids.

2. The method as described in claim 1, characterized in that, Step (1) includes: 1) Prepare the culture medium: dissolve mTeSR TM Plus 5X Additives; mTeSR TM Plus 5X additive added to mTeSR TM Add the antibiotics to the Plus basal medium and mix thoroughly to obtain mTeSR. TM Plus complete culture medium; 2) Coated culture dish: Dissolve Vitronectin XF TM Using CellAdhere TM Dilute with dilution buffer; add to non-tissue culture plates; discard coating solution before use. CellAdhere TM Wash with dilution buffer to prepare for iPSC inoculation; 3) Cell digestion and passage: Discard the culture medium in the culture dish, add Gentle Cell Dissociation Reagent, and let it stand until the cells detach from the bottom of the dish. Discard the Gentle Cell Dissociation Reagent and add mTeSR. TM Plus complete medium, aspirate and pipette the medium; seed cells into new culture wells at the appropriate density; passage; add ROCK inhibitor; change medium daily (mTeSR). TM Plus complete culture medium until cell confluence reaches 80-95% for the preparation of heart organoids.

3. The method as described in claim 2, characterized in that: Step 1) Double antibiotics: penicillin and streptomycin; Step 2) Vitronectin XF TM and CellAdhere TM The dilution buffer ratio is 40 μl: 960 μl; the inoculation ratio for subculturing in step 3) is 1:5 to 1:

20.

4. The method as described in claim 3, characterized in that: Step (2) includes: 21) Discard the culture medium, add Accutase until the cells detach from the bottom of the dish, discard the Accutase, and add mTeSR. TM Plus, complete culture medium is pipetted to single cells, and the cells are counted and plated. 22) Inoculation with Vitronectin XF TM In coated 96-well plates, the inoculation density is 3000–6000 cells per well; using mTeSR TM After adding Y-27632 to the Plus culture medium, the medium was changed; the medium was then replaced with mTeSR. TM Plus complete culture medium, change the medium daily until day 4.

5. The method as described in claim 4, characterized in that: Step 21) Conditions for cells to detach from the bottom of the dish: 37℃ for 3-5 minutes; Step 22) Seeding density: 3000 cells per well; Culture time: 24 hours.

6. The method as described in claim 5, characterized in that: Step (3) specifically involves: 31) Replace the culture medium with 200 μl RPMI 1640 and add 1 / 50 of insulin-free B27 and 6-8 μM CHIR99021, and incubate for 48 hours; 32) Subsequently, the culture medium was changed to 200 μl RPMI 1640, and 1 / 50 of the volume of insulin-free B27 and 3-7 μM IWR1 were added and cultured for 48 hours; 33) Replace the culture medium with 200 μl RPMI 1640 and add 1 / 50 of the volume of insulin-free B27 and culture for 72 hours.

7. The method as described in claim 6, characterized in that: Step (4) describes the shaker culture: culture at 50-100 rpm for 2 days.

8. The heart organoid prepared by any one of claims 1 to 7.

9. The application of any method of claims 1 to 7 or the cardiac organoid of claim 8 in pharmaceutical and basic research.

10. The application as described in claim 9, characterized in that, Used for drug toxicity testing.

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