Method for preparing vascularized heart organoid containing immune cells and special kit thereof

Abstract

The invention discloses a method for preparing a vascularized heart organ containing immune cells and a special kit thereof. The kit contains a culture solution A, a culture solution B, a culture solution C, a culture solution D, a culture solution M and a culture solution H. Experiments prove that cardiac muscle cells, vascular endothelial cells and macrophages can be obtained by adopting the preparation method provided by the invention, further vascularized heart organs containing immune cells can be prepared, and the culture solution used by the preparation method not only can quickly and efficiently differentiate human pluripotent stem cells into multiple pedigree cells, but also can be used for preparing the vascularized heart organs containing immune cells. Moreover, chemical components are definite, and no animal-derived protein exists. Furthermore, by adopting the preparation method provided by the invention, the vascularized heart organoid containing the immune cells can be produced on a large scale, the quality is stable, the safety is high, and a large number of model sources are provided for tissue engineering, drug research and development, disease simulation and cell therapy. The method has great application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a method for preparing vascularized cardiac organoids containing immune cells and a special kit therefor. Background Art

[0002] Human pluripotent stem cells (including human induced pluripotent stem cells and human embryonic stem cells) can differentiate into functional cells of various human tissues and organs, which are used for studying human development, making disease models, and can replace damaged and diseased cells through cell transplantation to promote wound repair and treat diseases of the body.

[0003] Cardiomyocytes are important constituent cells of the human heart and have important application values in cardiotoxicity detection, drug development, establishing heart disease models, heart regeneration, etc. In addition to cardiomyocytes, there are also a large number of vascular endothelial cells and macrophages in the heart tissue, which also play important functions in maintaining the homeostasis of the heart and diseases. At present, it is very difficult to obtain human heart tissue, which greatly limits its application in the biomedical industry, scientific research and clinical practice. Preparing cardiac organoids using cardiomyocytes, vascular endothelial cells and macrophages differentiated from human pluripotent stem cells can replace the heart tissue obtained from donors for various R & D experiments and even for cell transplantation therapy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to prepare cardiac organoids containing immune cells (specifically macrophages) and vascular endothelial cells.

[0005] To solve the above technical problem, the present invention adopts the following technical solutions:

[0006] In the first aspect, the present invention provides a kit for preparing vascularized cardiac organoids containing immune cells using cardiomyocytes, vascular endothelial cells and macrophages.

[0007] The kit includes culture medium D, and the culture medium D may include culture medium D1 and culture medium D2.

[0008] The culture medium D1 can be any one of the following:

[0009] 1) D1-I: Composed of B27 additive with insulin added, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), human macrophage colony-stimulating factor (M-CSF), Rho-associated protein kinase (ROCK) inhibitor, and serum-free basal medium; wherein, the non-essential amino acids can be glycine, L-alanine, L-aspartic acid, L-aspartate, L-glutamic acid, L-proline and L-serine;

[0010] 2) D1-II: Composed of B27 additive with insulin added, penicillin, streptomycin, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), human macrophage colony-stimulating factor (M-CSF), Rho-associated protein kinase (ROCK) inhibitor, and serum-free basal medium;

[0011] 3) D1-III: Composed of B27 additive with insulin added, serum albumin, transferrin, vitamin C, sodium selenite, penicillin, streptomycin, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), human macrophage colony-stimulating factor (M-CSF), Rho-associated protein kinase (ROCK) inhibitor, and serum-free basal medium.

[0012] The culture medium D2 is the culture medium D1 with the ROCK inhibitor removed.

[0013] Furthermore, the culture medium D1-I can specifically be a culture medium containing B27 additive with insulin added at a volume fraction of 1% - 2%, 0.5 - 1 mM L-glutamine or its substitute, non-essential amino acids at a volume fraction of 0.5% - 1%, 50 - 100 U / mL penicillin, 50 - 100 μg / mL streptomycin, 25 - 50 ng / mL vitamin C, 5 - 20 ng / mL bFGF, 50 - 100 ng / mL VEGF, 20 - 50 ng / mL M-CSF, and 2 - 10 μM ROCK inhibitor (such as Y27632).

[0014] Even further, the 1% - 2% can be 2%, the 0.5 - 1 mM can be 1 mM, the 0.5% - 1% can be 1%, the 50 - 100 U / mL can be 100 U / mL, the 50 - 100 μg / mL can be 50 μg / mL, the 25 - 50 ng / mL can be 50 ng / mL, the 5 - 20 ng / mL can be 10 ng / mL, the 50 - 100 ng / mL can be 50 ng / mL, the 20 - 50 ng / mL can be 20 ng / mL, and the 2 - 10 μM can be 10 μM.

[0015] Furthermore, the concentration of glycine in the culture medium D1-I can be 750.0 ng / mL; the concentration of L-alanine in the culture medium D1-I can be 890 ng / mL; the concentration of L-aspartic acid in the culture medium D1-I can be 1320 ng / mL; the concentration of L-asparagine in the culture medium D1-I can be 1330 ng / mL; the concentration of L-glutamic acid in the culture medium D1-I can be 1470 ng / mL; the concentration of L-proline in the culture medium D1-I can be 1150 ng / mL; the concentration of L-serine in the culture medium D1-I can be 1050 ng / mL.

[0016] Furthermore, the culture medium D1-II can specifically be a culture medium containing B27 additive with insulin added at a volume fraction of 1%-2%, 50-100 U / mL penicillin, 50-100 μg / mL streptomycin, 5-20 ng / mL bFGF, 50-100 ng / mL VEGF, 20-50 ng / mL M-CSF, and 2-10 μM ROCK inhibitor (such as Y27632).

[0017] Furthermore, the 1%-2% can be 2%, the 50-100 U / mL can be 100 U / mL, the 50-100 μg / mL can be 50 μg / mL, the 5-20 ng / mL can be 10 ng / mL, the 50-100 ng / mL can be 50 ng / mL, the 20-50 ng / mL can be 20 ng / mL, and the 2-10 μM can be 10 μM.

[0018] Furthermore, the culture medium D1-III can specifically be a culture medium containing B27 additive with insulin added at a volume fraction of 1%-2%, 0.1-2.5 mg / mL serum albumin, 1-25 μg / mL transferrin, 0.04-1 mg / mL vitamin C, 2-50 ng / mL sodium selenite, 50-100 U / mL penicillin, 50-100 μg / mL streptomycin, 5-20 ng / mL bFGF, 50-100 ng / mL VEGF, 20-50 ng / mL M-CSF, and 2-10 μM ROCK inhibitor (such as Y27632).

[0019] Further, the 1%-2% may be 2%, the 0.1-2.5 mg / mL may be 0.5 mg / mL, the 1-25 μg / mL may be 5 μg / mL, the 0.04-1 mg / mL may be 0.2 mg / mL, the 2-50 ng / mL may be 10 ng / mL, the 50-100 U / mL may be 100 U / mL, the 50-100 μg / mL may be 50 μg / mL, the 5-20 ng / mL may be 10 ng / mL, the 50-100 ng / mL may be 50 ng / mL, the 20-50 ng / mL may be 20 ng / mL, and the 2-10 μM may be 10 μM.

[0020] Further, in the culture medium D1, the Rho-associated protein kinase (ROCK) inhibitor may specifically be Y27632.

[0021] Further, in the culture medium D1, the VEGF may specifically be VEGF-165.

[0022] Further, in the culture medium D1, the serum-free basal medium may be RPMI 1640 medium or other basal culture media commonly used for cell culture.

[0023] In a second aspect, the present invention provides a kit for preparing vascularized cardiac organoids containing immune cells.

[0024] The kit includes a culture medium A for preparing cardiomyocytes, a culture medium B for preparing vascular endothelial cells, a culture medium C for preparing macrophages, and a culture medium D for preparing vascularized cardiac organoids containing immune cells.

[0025] Among them, the culture medium A includes a culture medium A1, a culture medium A2, and a culture medium A3;

[0026] The culture medium A1 is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite, and a GSK3 inhibitor;

[0027] The culture medium A2 is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite, and an inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN);

[0028] The culture medium A3 is the culture medium A1 without the GSK3 inhibitor or the culture medium A2 without the inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN).

[0029] Furthermore, the culture medium A1 is composed of serum albumin, transferrin, vitamin C, sodium selenite, a GSK3 inhibitor, and a serum-free basal culture medium;

[0030] Furthermore, each 1 mL of the culture medium A1 may contain 0.1 - 2.5 mg of serum albumin (such as 0.1 - 0.5 mg of serum albumin, 0.5 - 2.5 mg of serum albumin, 0.1 mg of serum albumin, 0.5 mg of serum albumin, or 2.5 mg of serum albumin), 1 - 25 μg of transferrin (1 - 5 μg of transferrin, 5 - 25 μg of transferrin, 1 μg of transferrin, 5 μg of transferrin, or 25 μg of transferrin), 0.04 - 1 mg of vitamin C (0.04 - 0.2 mg of vitamin C, 0.2 - 1 mg of vitamin C, 0.04 mg of vitamin C, 0.2 mg of vitamin C, or 1 mg of vitamin C), 2 - 50 ng of sodium selenite (2 - 10 ng of sodium selenite, 10 - 50 ng of sodium selenite, 2 ng of sodium selenite, 10 ng of sodium selenite, or 50 ng of sodium selenite), and 1 - 10 nmol of a GSK3 inhibitor (1 - 5 nmol of a GSK3 inhibitor, 5 - 10 nmol of a GSK3 inhibitor, 1 nmol of a GSK3 inhibitor, 5 nmol of a GSK3 inhibitor, or 10 nmol of a GSK3 inhibitor), with the balance being a serum-free basal culture medium; the pH value of the culture medium A1 may be 7.0 - 7.6 (such as 7.0 - 7.3, 7.3 - 7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6).

[0031] In the culture medium A1, the GSK3 inhibitor may be a1) or a2) or a3) or a4): a1) CHIR-99021; a2) B216763; a3) BIO; a4) TWS119.

[0032] Furthermore, the culture medium A2 is composed of serum albumin, transferrin, vitamin C, sodium selenite, an inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN), and a serum-free basal culture medium;

[0033] Furthermore, each 1 mL of the culture medium A2 may contain 0.1 - 2.5 mg of serum albumin (such as 0.1 - 0.5 mg of serum albumin, 0.5 - 2.5 mg of serum albumin, 0.1 mg of serum albumin, 0.5 mg of serum albumin, or 2.5 mg of serum albumin), 1 - 25 μg of transferrin (1 - 5 μg of transferrin, 5 - 25 μg of transferrin, 1 μg of transferrin, 5 μg of transferrin, or 25 μg of transferrin), 0.04 - 1 mg of vitamin C (0.04 - 0.2 mg of vitamin C, 0.2 - 1 mg of vitamin C, 0.04 mg of vitamin C, 0.2 mg of vitamin C, or 1 mg of vitamin C), 2 - 50 ng of sodium selenite (2 - 10 ng of sodium selenite, 10 - 50 ng of sodium selenite, 2 ng of sodium selenite, 10 ng of sodium selenite, or 50 ng of sodium selenite), and 4 - 6 nmol of an inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN) (4 nmol of an inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN), 5 nmol of an inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN), 6 nmol of an inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN)), with the balance being a serum-free basal culture medium; the pH value of the culture medium A2 may be 7.0 - 7.6 (such as 7.0 - 7.3, 7.3 - 7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6).

[0034] In the culture medium A2, the inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN) may be IWP2.

[0035] Further, the culture medium A3 is composed of serum albumin, transferrin, vitamin C, sodium selenite, and a serum-free basal culture medium.

[0036] Furthermore, each 1 mL of the culture medium A3 may contain 0.1 - 2.5 mg of serum albumin (such as 0.1 - 0.5 mg of serum albumin, 0.5 - 2.5 mg of serum albumin, 0.1 mg of serum albumin, 0.5 mg of serum albumin, or 2.5 mg of serum albumin), 1 - 25 μg of transferrin (1 - 5 μg of transferrin, 5 - 25 μg of transferrin, 1 μg of transferrin, 5 μg of transferrin, or 25 μg of transferrin), 0.04 - 1 mg of vitamin C (0.04 - 0.2 mg of vitamin C, 0.2 - 1 mg of vitamin C, 0.04 mg of vitamin C, 0.2 mg of vitamin C, or 1 mg of vitamin C), 2 - 50 ng of sodium selenite (2 - 10 ng of sodium selenite, 10 - 50 ng of sodium selenite, 2 ng of sodium selenite, 10 ng of sodium selenite, or 50 ng of sodium selenite), with the balance being a serum-free basal culture medium.

[0037] The above-mentioned culture medium A can also be composed only of culture medium A1, culture medium A2, and culture medium A3.

[0038] Among them, the culture medium B is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF).

[0039] Furthermore, the culture medium B is composed of serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and a serum-free basal culture medium.

[0040] Even further, every 1 mL of the culture medium B may include 0.1 - 2.5 mg of serum albumin (such as 0.1 - 0.5 mg of serum albumin, 0.5 - 2.5 mg of serum albumin, 0.1 mg of serum albumin, 0.5 mg of serum albumin, or 2.5 mg of serum albumin), 1 - 25 μg of transferrin (1 - 5 μg of transferrin, 5 - 25 μg of transferrin, 1 μg of transferrin, 5 μg of transferrin, or 25 μg of transferrin), 0.04 - 1 mg of vitamin C (0.04 - 0.2 mg of vitamin C, 0.2 - 1 mg of vitamin C, 0.04 mg of vitamin C, 0.2 mg of vitamin C, or 1 mg of vitamin C), 2 - 50 ng of sodium selenite (2 - 10 ng of sodium selenite, 10 - 50 ng of sodium selenite, 2 ng of sodium selenite, 10 ng of sodium selenite, or 50 ng of sodium selenite), 10 - 250 ng of VEGF (10 - 50 ng of VEGF, 50 - 250 ng of VEGF, 10 ng of VEGF, 50 ng of VEGF, or 250 ng of VEGF), and 2 - 50 ng of bFGF (2 - 10 ng of bFGF, 10 - 50 ng of bFGF, 2 ng of bFGF, 10 ng of bFGF, or 50 ng of bFGF), with the balance being a serum-free basal culture medium. The pH value of the culture medium B can be 7.0 - 7.6 (such as 7.0 - 7.3, 7.3 - 7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6).

[0041] In the culture medium B, the VEGF can specifically be VEGF-A. The serum albumin can be recombinant human serum albumin, bovine serum albumin, or other non-animal-derived recombinant human serum albumin, such as plant-derived recombinant human serum albumin. The transferrin can be human transferrin or other non-animal-derived recombinant transferrin, such as plant-derived recombinant human transferrin.

[0042] In the culture medium B, the serum-free basal culture medium can be RPMI 1640 medium or other basal culture media commonly used in cell culture.

[0043] In the above kit, the culture medium B can specifically be culture medium B-I, culture medium B-II or culture medium B-III shown in Table 1.

[0044] Table 1

[0045]

[0046] Among them, the culture medium C includes culture medium C1, culture medium C2, culture medium C3, culture medium C4 and culture medium C5.

[0047] The culture medium C1 can be a culture medium containing B27 additive without insulin, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C and human bone morphogenetic protein 4; among them, the non-essential amino acids can be glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline and L-serine.

[0048] The culture medium C2 can be a culture medium containing a GSK3 inhibitor, and this culture medium is a liquid prepared by using the culture medium C1 as the solvent and the GSK3 inhibitor as the solute.

[0049] The culture medium C3 can be a culture medium containing B27 additive with added insulin, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor and human fibroblast growth factor; among them, the non-essential amino acids can be glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline and L-serine; the human vascular endothelial growth factor (VEGF) can specifically be VEGF-165.

[0050] The culture medium C4 can be a culture medium containing a transforming growth factor-β (TGF-β) inhibitor, and this culture medium is a liquid prepared by using the culture medium C3 as the solvent and the TGF-β inhibitor as the solute; the TGF-β inhibitor can specifically be SB431542.

[0051] The culture medium C5 can be a culture medium containing B27 additive with added insulin, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony-stimulating factor; among them, the non-essential amino acids can be glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline and L-serine.

[0052] Further, the culture medium C1 may specifically be a culture medium containing B27 additive without insulin at a volume fraction of 1%-2%, 0.5-1 mM L-glutamine or its substitute, non-essential amino acids at a volume fraction of 0.5%-1%, 50-100 U / mL penicillin, 50-100 μg / mL streptomycin, 25-50 ng / mL vitamin C, and 5-10 ng / mL human bone morphogenetic protein 4.

[0053] Further, the 1%-2% may be 2%, the 0.5-1 mM may be 1 mM, the 0.5%-1% may be 1%, the 50-100 U / mL may be 100 U / mL, the 50-100 μg / mL may be 100 μg / mL, the 25-50 ng / mL may be 50 ng / mL, and the 5-10 ng / mL may be 5 ng / mL.

[0054] Further, the concentration of glycine in culture C1 may be 750.0 ng / mL; the concentration of L-alanine in culture C1 may be 890 ng / mL; the concentration of L-aspartic acid in culture C1 may be 1320 ng / mL; the concentration of L-aspartate in culture C1 may be 1330 ng / mL; the concentration of L-glutamic acid in culture C1 may be 1470 ng / mL; the concentration of L-proline in culture C1 may be 1150 ng / mL; the concentration of L-serine in culture C1 may be 1050 ng / mL.

[0055] Further, in the culture medium C2, the content of the GSK3 inhibitor in the culture medium may be 1-2 μM or 2 μM.

[0056] Further, the culture medium C3 may specifically be a culture medium containing B27 additive with insulin added at a volume fraction of 1%-2%, 0.5-1 mM L-glutamine or its substitute, non-essential amino acids at a volume fraction of 0.5%-1%, 50-100 U / mL penicillin, 50-100 μg / mL streptomycin, 25-50 ng / mL vitamin C, 25-50 ng / mL human vascular endothelial growth factor, and 5-10 ng / mL human fibroblast growth factor.

[0057] Further, the 1%-2% may be 2%, the 0.5-1 mM may be 1 mM, the 0.5%-1% may be 1%, the 50-100 U / mL may be 100 U / mL, the 50-100 μg / mL may be 100 μg / mL, the 25-50 ng / mL may be 50 ng / mL, the 25-50 ng / mL may be 50 ng / mL, and the 5-10 ng / mL may be 10 ng / mL.

[0058] Furthermore, the concentration of glycine in culture medium C3 can be 750.0 ng / mL; the concentration of L-alanine in culture medium C3 can be 890 ng / mL; the concentration of L-aspartic acid in culture medium C3 can be 1320 ng / mL; the concentration of L-asparagine in culture medium C3 can be 1330 ng / mL; the concentration of L-glutamic acid in culture medium C3 can be 1470 ng / mL; the concentration of L-proline in culture medium C3 can be 1150 ng / mL; the concentration of L-serine in culture medium C3 can be 1050 ng / mL.

[0059] Furthermore, in culture medium C4, the content of the TGF-β inhibitor (SB431542) in the culture medium can be 5 - 10 μM or 10 μM.

[0060] Furthermore, culture medium C5 can specifically be a culture medium containing B27 additive supplemented with insulin at a volume fraction of 1% - 2%, 0.5 - 1 mM L-glutamine or its substitute, non-essential amino acids at a volume fraction of 0.5% - 1%, 50 - 100 U / mL penicillin, 50 - 100 μg / mL streptomycin, 25 - 50 ng / mL vitamin C, 10 - 20 ng / mL human interleukin-3, and 50 - 100 ng / mL human macrophage colony-stimulating factor.

[0061] Furthermore, the 1% - 2% can be 2%, the 0.5 - 1 mM can be 1 mM, the 0.5% - 1% can be 1%, the 50 - 100 U / mL can be 100 U / mL, the 50 - 100 μg / mL can be 50 ng / mL, the 25 - 50 ng / mL can be 50 ng / mL, the 10 - 20 ng / mL can be 10 ng / mL, and the 50 - 100 ng / mL can be 50 ng / mL.

[0062] Furthermore, the concentration of glycine in culture medium C5 can be 750.0 ng / mL; the concentration of L-alanine in culture medium C5 can be 890 ng / mL; the concentration of L-aspartic acid in culture medium C5 can be 1320 ng / mL; the concentration of L-asparagine in culture medium C5 can be 1330 ng / mL; the concentration of L-glutamic acid in culture medium C5 can be 1470 ng / mL; the concentration of L-proline in culture medium C5 can be 1150 ng / mL; the concentration of L-serine in culture medium C5 can be 1050 ng / mL.

[0063] The culture media C1, C2, C3, C4, and C5 can all be prepared based on RPMI 1640 medium as the basal medium.

[0064] Among them, the culture medium D is the culture medium D described in the first aspect of the present invention.

[0065] Furthermore, the kit may further include a culture medium M, and the culture medium M may include a culture medium M1 and a culture medium M2.

[0066] The culture medium M1 is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite, and bone morphogenetic protein 4 (BMP4).

[0067] The culture medium M2 is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and a glycogen synthase kinase 3 (GSK3) inhibitor.

[0068] Even further, the culture medium M1 is composed of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), and a serum-free basal medium.

[0069] Even further, each 1 mL of the culture medium M1 may include 0.1 - 2.5 mg of serum albumin (such as 0.1 - 0.5 mg of serum albumin, 0.5 - 2.5 mg of serum albumin, 0.1 mg of serum albumin, 0.5 mg of serum albumin, or 2.5 mg of serum albumin), 1 - 25 μg of transferrin (1 - 5 μg of transferrin, 5 - 25 μg of transferrin, 1 μg of transferrin, 5 μg of transferrin, or 25 μg of transferrin), 0.04 - 1 mg of vitamin C (0.04 - 0.2 mg of vitamin C, 0.2 - 1 mg of vitamin C, 0.04 mg of vitamin C, 0.2 mg of vitamin C, or 1 mg of vitamin C), 2 - 50 ng of sodium selenite (2 - 10 ng of sodium selenite, 10 - 50 ng of sodium selenite, 2 ng of sodium selenite, 10 ng of sodium selenite, or 50 ng of sodium selenite), 2 - 50 ng of BMP4 (2 - 10 ng of BMP4, 10 - 50 ng of BMP4, 2 ng of BMP4, 10 ng of BMP4, or 50 ng of BMP4), and the balance is a serum-free basal medium. The pH value of the culture medium M1 is 7.0 - 7.6 (such as 7.0 - 7.3, 7.3 - 7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6).

[0070] Further, the culture medium M2 is composed of serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 (BMP4), glycogen synthase kinase 3 (GSK3) inhibitor, and serum-free basal culture medium.

[0071] Further, each 1 mL of the culture medium M2 may contain 0.1 - 2.5 mg of serum albumin (such as 0.1 - 0.5 mg of serum albumin, 0.5 - 2.5 mg of serum albumin, 0.1 mg of serum albumin, 0.5 mg of serum albumin, or 2.5 mg of serum albumin), 1 - 25 μg of transferrin (1 - 5 μg of transferrin, 5 - 25 μg of transferrin, 1 μg of transferrin, 5 μg of transferrin, or 25 μg of transferrin), 0.04 - 1 mg of vitamin C (0.04 - 0.2 mg of vitamin C, 0.2 - 1 mg of vitamin C, 0.04 mg of vitamin C, 0.2 mg of vitamin C, or 1 mg of vitamin C), 2 - 50 ng of sodium selenite (2 - 10 ng of sodium selenite, 10 - 50 ng of sodium selenite, 2 ng of sodium selenite, 10 ng of sodium selenite, or 50 ng of sodium selenite), 2 - 50 ng of BMP4 (2 - 10 ng of BMP4, 10 - 50 ng of BMP4, 2 ng of BMP4, 10 ng of BMP4, or 50 ng of BMP4), and 1 - 10 nmol of GSK3 inhibitor (1 - 5 nmol of GSK3 inhibitor, 5 - 10 nmol of GSK3 inhibitor, 1 nmol of GSK3 inhibitor, 5 nmol of GSK3 inhibitor, or 10 nmol of GSK3 inhibitor), with the balance being serum-free basal culture medium; the pH value of the culture medium M2 may be 7.0 - 7.6 (such as 7.0 - 7.3, 7.3 - 7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6).

[0072] In the culture medium M, the serum albumin may be recombinant human serum albumin, bovine serum albumin, or other non-animal-derived recombinant human serum albumin such as plant-derived recombinant human serum albumin. The transferrin may be human transferrin or other non-animal-derived recombinant transferrin such as plant-derived recombinant human transferrin. The serum-free basal culture medium may be RPMI 1640 medium or a basal culture medium commonly used for cell culture. The GSK3 inhibitor may be a1) or a2) or a3) or a4): a1) CHIR-99021; a2) B216763; a3) BIO; a4) TWS119.

[0073] In the above kit, the culture medium M2 may specifically be the culture medium M2-I, culture medium M2-II, culture medium M2-III, culture medium M2-IV, culture medium M2-V, or culture medium M2-VI shown in Table 2.

[0074] Table 2

[0075]

[0076]

[0077] Any of the above kits may further contain culture medium H, and the culture medium H includes culture medium H1 and / or culture medium H2.

[0078] The culture medium H1 may be TeSR-E8 complete culture medium or other culture media for culturing human pluripotent stem cells;

[0079] The culture medium H2 may be TeSR-E8 complete culture medium containing 2-10 μM (such as 2-5 μM, 5-10 μM, 2 μM, 5 μM or 10 μM) ROCK inhibitor.

[0080] Specifically, any of the above TeSR-E8 complete culture media may be a product of STEM CELL Technologies, with the product catalog number 05940.

[0081] Specifically, any of the above TeSR-E8 complete culture media (500 mL) may be prepared by mixing 474 mL of TeSR-E8 basal culture medium, 25 mL of 20× supplement 1 and 1 mL of 500× supplement 2. The TeSR-E8 basal culture medium, 20× supplement 1 and 500× supplement 2 may specifically be components in the TeSR-E8 complete culture medium.

[0082] In the culture medium H2, the Rho-associated protein kinase (ROCK) inhibitor may specifically be Y27632.

[0083] In a third aspect, the present invention further provides a kit for preparing cardiomyocytes.

[0084] The kit includes the culture medium A described in the second aspect of the present invention.

[0085] In a fourth aspect, the present invention provides a method for preparing a vascularized heart organoid containing immune cells.

[0086] The method for preparing a vascularized heart organoid containing immune cells provided by the present invention includes the following steps:

[0087] (D1) Cardiomyocytes, vascular endothelial cells and macrophages differentiated from human pluripotent stem cells are mixed in a certain cell ratio and then seeded onto a 96-well ultra-low attachment plate, and culture medium D1 is added and cultured for 0.5-2 days;

[0088] (D2) Replace the culture medium in step (D1) with culture medium D2, and then maintain the medium change every other day. After culturing for at least 7 days (such as 7 - 14 days or longer), vascularized cardiac organoids containing immune cells are obtained.

[0089] In the above method, in step (D1), the cell ratios of cardiomyocytes, vascular endothelial cells, and macrophages can be 50% - 80%, 10% - 30%, and 10% - 20% respectively, and specifically can be 70%, 20%, and 10% respectively.

[0090] In the above method, in step (D1), the seeding density is 1×10 5 -3×10 5 cells / well, and specifically can be 1×10 5 cells / well.

[0091] In the above method, in step (D1), the ultra - low attachment plate can be any one of 1) the low - attachment plate with the product number 7007 produced by Corning, or 2) the low - attachment plate with the product number MS - 9096UZ produced by Sumitomo Bakelite.

[0092] In the above method, in step (D2), the culture time can be 7 - 14 days, and specifically can be 10 days.

[0093] In the above method, in step (D1), the cardiomyocytes differentiated from human pluripotent stem cells can be prepared by a method including the following steps:

[0094] (A1) Seed human pluripotent stem cells in culture medium H2 and culture for 0.5 - 1.5 days;

[0095] (A2) Replace it with culture medium H1 and culture for 2.5 - 4 days until the cell confluence reaches 90 - 95%;

[0096] (A3) Replace it with the culture medium A1 and continue to culture for 1.5 - 2.5 days;

[0097] (A4) Replace it with the culture medium A2 and continue to culture for 1.5 - 2.5 days;

[0098] (A5) Replace it with the culture medium A3 and continue to culture for at least 4 days (it can also be extended to culture for 30 days), and collect the obtained cardiomyocytes.

[0099] In the above method, the culture time in step (A1) is specifically 1 day;

[0100] In the above method, the seeding density in step (A1) can be 1.5×10 4 cells per square centimeter (cm 2 ) - 2.5×10 4 cells per square centimeter (cm2 )

[0101] In the above method, the culture time in step (A2) can specifically be 3 days;

[0102] In the above method, the culture time in steps (A3) and (A4) can specifically be 2 days;

[0103] In the above method, in step (A5), after culturing for 4 days, step (A6) can further be included: adding 4 - 6 μg / mL insulin to culture medium A3 and continuing the culture.

[0104] The insulin concentration in step (A6) can specifically be 5 μg / mL.

[0105] After step (A5), the cells can specifically be collected after 8 days of culture to prepare organoids.

[0106] In the above method, in step (D1), the vascular endothelial cells differentiated from human pluripotent stem cells can be prepared by a method including the following steps:

[0107] (B1) Inoculate human pluripotent stem cells in culture medium H2 and culture for 0.5 - 1.5 days;

[0108] (B2) Replace the cells in step (1) with culture medium M1 and culture for 0.5 - 1.5 days;

[0109] (B3) Change to culture medium M2 and culture for 1.5 - 2.5 days to obtain mesoderm cells;

[0110] (B4) Inoculate the mesoderm cells into the above - mentioned culture medium B and culture for 3 - 7 days to obtain vascular endothelial cells.

[0111] In the above method, the culture time in step (B1) can specifically be 1 day;

[0112] In the above method, the seeding density in step (B1) can be 1.5×10 4 cells / cm² (cm 2 ) - 2.5×10 4 cells / cm² (cm 2 ); the seeding density can specifically be 2×10 4 cells / cm 2 ;

[0113] In the above method, the culture time in step (B2) can specifically be 1 day;

[0114] In the above method, the culture time in step (B3) can specifically be 2 days;

[0115] In the above method, the seeding density in step (B4) can be 3×104 cells / cm 2 -1.0×10 5 cells / cm 2 .

[0116] In the above method, step (B5) may further be included in step (B4): After completing step (B4), inoculate vascular endothelial cells into the above-mentioned culture medium B and culture again.

[0117] In the above method, in step (D1), macrophages differentiated from human pluripotent stem cells can be prepared by the method including the following steps:

[0118] (C1) Inoculate human pluripotent stem cells into culture medium H2 and culture for 0.5 - 1.5 days;

[0119] (C2) Replace the cells in step (C1) with culture medium C1 and culture for 0.5 - 1.5 days;

[0120] (C3) Replace with culture medium C2 and culture for 1.5 - 2.5 days to obtain mesoderm cells;

[0121] (C4) Inoculate the mesoderm cells into the above-mentioned culture medium C3 and culture for 1.5 - 2.5 days, and then replace with culture medium C4 and culture for 3 - 5 days to obtain hematopoietic progenitor cells;

[0122] (C5) Inoculate the hematopoietic progenitor cells obtained in step (C4) into culture medium C5 and culture for 7 - 10 days to obtain macrophages.

[0123] In the above method, the culture time in step (C1) can specifically be 1 day;

[0124] In the above method, the seeding density in step (C1) can be 1.5×10 4 cells per square centimeter (cm 2 ) - 2.5×10 4 cells per square centimeter (cm 2 ); The seeding density can specifically be 2×10 4 cells / cm 2 ;

[0125] In the above method, the culture time in step (C2) can specifically be 1 day;

[0126] In the above method, the culture time in step (C3) can specifically be 2 days;

[0127] In the above method, the seeding density in step (C4) can be 0.5×10 5 cells / cm 2 - 1.0×10 5 cells / cm 2; The seeding density can specifically be 0.5×10 5 cells / cm 2 ;

[0128] In the above method, the specific culture time of the culture medium C4 in step (C4) can be 4 days;

[0129] In the above method, the seeding density in step (C5) can be 0.5×10 5 cells / cm 2 -1.0×10 5 cells / cm 2 ; The seeding density can specifically be 0.5×10 5 cells / cm 2 ;

[0130] In the above method, the specific culture time of the culture medium C5 in step (C5) can be 7 days.

[0131] In any of the above methods, the culture parameters can be: 36°C - 38°C (such as 36°C, 37°C or 38°C).

[0132] Any of the above cultures can be carried out using a CO2 incubator. The parameters set for the CO2 incubator can be: 5% CO2.

[0133] In any of the above methods, the human pluripotent stem cells can be single cells after digestion with a digestive solution. The digestive solution can specifically be Accutase. The Accutase can specifically be a product of Merk Millipore, with the catalog number SF006.

[0134] Any of the above human pluripotent stem cells can be b1) or b2) or b3) or b4) or b5) or b6):

[0135] b1) Human embryonic stem cell line;

[0136] b2) Human induced pluripotent stem cell line;

[0137] b3) Human embryonic stem cell line H1;

[0138] b4) Human embryonic stem cell line H7;

[0139] b5) Human embryonic stem cell line H9;

[0140] b6) Human induced pluripotent stem cell line CD34-iPSC.

[0141] The human embryonic stem cell line can specifically be purchased through commercial channels. The human embryonic stem cell line H1, the human embryonic stem cell line H7, and the human embryonic stem cell line H9 can all be products of the WiCell Research Institute in the United States.

[0142] Experimental results show that the preparation method provided by the present invention can obtain cardiomyocytes, vascular endothelial cells and macrophages, and thus can prepare vascularized cardiac organoids containing immune cells. Moreover, the culture medium used in this preparation method can not only enable human pluripotent stem cells to rapidly and efficiently differentiate into multiple lineages of cells, but also has a defined chemical composition and is free of animal-derived proteins. Further, the preparation method provided by the present invention can be used for large-scale production of vascularized cardiac organoids containing immune cells, with stable quality and high safety, providing a large number of model sources for tissue engineering, drug research and development, disease simulation and cell therapy. The present invention has great application value. Description of the Drawings

[0143] Figure 1 It is a clone morphology diagram of undifferentiated human pluripotent stem cells.

[0144] Figure 2 It shows the morphological changes of human pluripotent stem cells at different time points during the differentiation into cardiomyocytes.

[0145] Figure 3 It is the experimental result of immunofluorescence staining identification of cardiomyocytes.

[0146] Figure 4 It is the flow cytometry detection result of cardiomyocytes.

[0147] Figure 5 It shows the morphological changes of human pluripotent stem cells at different time points during the differentiation into vascular endothelial cells.

[0148] Figure 6 It is the experimental result of immunofluorescence staining identification of vascular endothelial cells.

[0149] Figure 7 It is the flow cytometry detection result of vascular endothelial cells.

[0150] Figure 8 It shows the morphological changes of human pluripotent stem cells at different time points during the differentiation into macrophages.

[0151] Figure 9 It is the experimental result of immunofluorescence staining identification of macrophages.

[0152] Figure 10 It is the flow cytometry detection result of macrophages.

[0153] Figure 11 It shows the morphological characteristics at different time points during the construction of vascularized cardiac organoids containing immune cells.

[0154] Figure 12 It is the immunofluorescence staining identification result of vascularized cardiac organoids containing immune cells. Detailed Embodiments

[0155] The present invention will be further described in detail below in conjunction with specific embodiments. The embodiments given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0156] In the experimental methods in the following embodiments, unless otherwise specified, they are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0157] In the following embodiments, for quantitative tests, three repeated experiments are set, and the results are averaged.

[0158] In the following embodiments, the biological materials and reagents involved are:

[0159] Human embryonic stem cell line H1 (abbreviated as H1 cells): WiCell Research Institute, USA, No.: WA01.

[0160] Induced pluripotent stem cell CD34-iPSC (abbreviated as CD34-iPSC cells): reprogrammed from human umbilical cord blood hematopoietic stem cells (CD34-positive cells) using the Sendai virus reprogramming kit (Invitrogen, catalog number: A16517). Hereinafter, the human induced pluripotent stem cell line CD34-iPSC is abbreviated as CD34-iPSC cells.

[0161] DMEM culture medium: Gibco, catalog number: 11965092.

[0162] RPMI 1640 basal medium (RPMI 1640 medium): ThermoFisher, catalog number: 11875093.

[0163] TeSR-E8 culture medium: STEMCELL, catalog number: 05990.

[0164] Non-essential amino acids (NEAA, 100×): Gibco, catalog number: 11140050

[0165] B27 supplement with insulin: Gibco, catalog number: 17504-044.

[0166] B27 minus insulin: Gibco, catalog number: A1895601.

[0167] L-Glutamine substitute (Glutamax): Gibco, Catalog No.: 35050061.

[0168] Vitamin C: Sigma Aldrich, Catalog No.: A4403.

[0169] ROCK inhibitor Y27632 (CAS146986-50-7): TargetMol, Catalog No.: T1870.

[0170] Human bone morphogenetic protein 4 (BMP4): Peprotech, Catalog No.: 120-05; the amino acid sequence of human bone morphogenetic protein 4 is SEQ ID No.1.

[0171] GSK3 inhibitor CHIR-99021: Tocris Biosciences, Catalog No.: 4423 / 10. The structural formula of CHIR-99021 is as follows:

[0172]

[0173] Wnt protein palmitoyltransferase Porcupine (PORCN) inhibitor IWP2: Tocris Bioscience, Catalog No.: 3533.

[0174] Human vascular endothelial growth factor (VEGF-165): Suzhou Novoprotein Scientific Inc., Catalog No.: C083; the amino acid sequence of vascular endothelial growth factor (VEGF-165) is SEQ ID No.2.

[0175] Human basic fibroblast growth factor (bFGF): Suzhou Novoprotein Scientific Inc., Catalog No.: C779; the amino acid sequence of human basic fibroblast growth factor (bFGF) is SEQ ID No.3.

[0176] Human interleukin-3 (IL3): Suzhou Novoprotein Scientific Inc., Catalog No.: CX90; the amino acid sequence of human interleukin-3 (IL3) is SEQ ID No.4.

[0177] Human macrophage colony-stimulating factor (M-CSF): Suzhou Novoprotein Scientific Inc., Catalog No.: C417; the amino acid sequence of human macrophage colony-stimulating factor (M-CSF) is SEQ ID No.5.

[0178] Vitamin C: Aldrich, Catalog No.: A8960.

[0179] Human transferrin: Aldrich, Catalog No.: T1147.

[0180] Sodium selenite: Sigma Aldrich, catalog number: S5261.

[0181] Recombinant human serum albumin: ORYZOGEN, catalog number: HY100C1.

[0182] DAPI: ThermoFisher, catalog number: D1306.

[0183] Cell digestive solution Accutase: Shanghai Yisheng Biotechnology Co., Ltd., catalog number: 40506ES60.

[0184] Matrigel: BD Biosciences, catalog number: 356231.

[0185] TrypLE TM Express digestive solution: ThermoFisher, catalog number: 12605010.

[0186] Type II collagenase: Worthington, catalog number: LS004194.

[0187] Mouse anti-human TNNT2 antibody: Abcam, catalog number: ab8295.

[0188] Goat anti-human TNNT2 antibody: Abcam, catalog number: ab64623.

[0189] DyLight488-labeled goat anti-mouse IgG antibody: EarthOx, catalog number: E032210.

[0190] Cy3-labeled donkey anti-goat IgG antibody: Proteintech, catalog number: SA00009-2.

[0191] FITC-labeled donkey anti-mouse IgG antibody: Proteintech, catalog number: SA00003-9.

[0192] CoraLite647-labeled donkey anti-rabbit IgG antibody: Proteintech, catalog number: SA00014-7.

[0193] FITC-labeled anti-human CD31 antibody: BD Biosciences, catalog number: 557508.

[0194] Anti-human CD31 antibody: Abcam, catalog number: ab76533.

[0195] Anti-human CD14 antibody: Abclonal, catalog number: A19011.

[0196] Anti-human CD68 antibody: Invitrogen, catalog number: 14-0688-82.

[0197] Pacific Blue-labeled CD45 antibody: Biolegend, catalog number: 982306.

[0198] APC-labeled CD14 antibody: Biolegend, catalog number: 325607.

[0199] PE-labeled CD163 antibody: Biolegend, catalog number: 326505.

[0200] Anti-human CD163-APC antibody: Biolegend, catalog number: 326509.

[0201] Cell counting chamber: Count star, catalog number: 12-0005-50.

[0202] Low-attachment plate with catalog number 7007 produced by Corning.

[0203] Dimethyl sulfoxide: MP biomedicals, catalog number: 196055.

[0204] Fetal bovine serum: Biological Industries, catalog number: 04-001-1ACS.

[0205] 4% Paraformaldehyde: PBS buffer containing 4% (4g / 100mL) paraformaldehyde.

[0206] PBST buffer: PBS buffer containing 0.1% (v / v) Tween-20.

[0207] The culture medium H1 in the following examples is TeSR-E8 complete culture medium.

[0208] E8-Y culture medium (i.e., culture medium H2) is a stem cell culture medium containing a ROCK inhibitor. Further, the stem cell culture medium can be prepared by using the ROCK inhibitor as the solute and TeSR-E8 culture medium as the solvent. The content of the ROCK inhibitor (such as Y27632) in the culture medium Ⅰ can be 5 - 10 μM or 5 μM.

[0209] The E8-Y culture medium (i.e., culture medium H2) in the following examples is specifically prepared by using Y27632 as the solute and TeSR-E8 culture medium as the solvent, and the content of Y27632 in the E8-Y culture medium (i.e., culture medium H2) is 5 μM.

[0210] Culture medium A for preparing cardiomyocytes in the kit; wherein culture medium A contains culture medium A1, culture medium A2 and culture medium A3;

[0211] Each 1 mL of the culture medium A1 can be a serum-free basal medium containing 0.1 - 2.5 mg of serum albumin, 1 - 25 μg of transferrin, 0.04 - 1 mg of vitamin C, 2 - 50 ng of sodium selenite, and 1 - 10 nmol of GSK3 inhibitor. The serum albumin can be recombinant human serum albumin, bovine serum albumin, or other recombinant human serum albumin of non-animal origin, such as recombinant human serum albumin of plant origin. The transferrin can be human transferrin or other recombinant transferrin of non-animal origin, such as recombinant human transferrin of plant origin. The serum-free basal medium can be RPMI 1640 medium or other basal media commonly used for cell culture. The GSK3 inhibitor can be a1) or a2) or a3) or a4): a1) CHIR-99021; a2) B216763; a3) BIO; a4) TWS119.

[0212] The culture medium A1 in the following examples is specifically an RPMI 1640 culture medium containing 0.5 mg / mL of recombinant human serum albumin, 5 μg / mL of human transferrin, 0.2 mg / mL of vitamin C, 10 ng / mL of sodium selenite, and 5 μM of CHIR-99021.

[0213] Each 1 mL of the culture medium A2 can be a serum-free basal medium containing 0.1 - 2.5 mg of serum albumin, 1 - 25 μg of transferrin, 0.04 - 1 mg of vitamin C, 2 - 50 ng of sodium selenite, and 4 - 6 nmol of inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN). The serum albumin can be recombinant human serum albumin, bovine serum albumin, or other recombinant human serum albumin of non-animal origin, such as recombinant human serum albumin of plant origin. The transferrin can be human transferrin or other recombinant transferrin of non-animal origin, such as recombinant human transferrin of plant origin. The serum-free basal medium can be RPMI 1640 medium or other basal media commonly used for cell culture. The inhibitor of Wnt protein palmitoyltransferase Porcupine (PORCN) can be IWP2.

[0214] The culture medium A2 in the following examples is specifically RPMI 1640 culture medium containing 0.5 mg / mL recombinant human serum albumin, 5 μg / mL human transferrin, 0.2 mg / mL vitamin C, 10 ng / mL sodium selenite, and 5 μM IWP2.

[0215] The culture medium A3 can be the culture medium A1 without the GSK3 inhibitor or the culture medium A2 without the inhibitor of the Wnt protein palmitoyltransferase Porcupine (PORCN).

[0216] The culture medium A3 in the following examples is specifically RPMI 1640 culture medium containing 0.5 mg / mL recombinant human serum albumin, 5 μg / mL human transferrin, 0.2 mg / mL vitamin C, and 10 ng / mL sodium selenite.

[0217] The culture medium B for preparing vascular endothelial cells in the kit; wherein the culture medium B includes the culture medium B1.

[0218] Each 1 mL of the culture medium B1 can be a serum-free basal culture medium containing 0.1 - 2.5 mg of serum albumin, 1 - 25 μg of transferrin, 0.04 - 1 mg of vitamin C, 2 - 50 ng of sodium selenite, 10 - 250 ng of vascular endothelial growth factor (VEGF), and 2 - 50 ng of basic fibroblast growth factor (bFGF). The serum albumin can be recombinant human serum albumin, bovine serum albumin, or other non-animal-derived recombinant human serum albumin, such as plant-derived recombinant human serum albumin. The transferrin can be human transferrin or other non-animal-derived recombinant transferrin, such as plant-derived recombinant human transferrin. The serum-free basal culture medium can be RPMI 1640 medium or other basal culture media commonly used for cell culture.

[0219] The culture medium B1 in the following examples is specifically RPMI1640 culture medium containing 0.5 mg / mL recombinant human serum albumin, 5 μg / mL human transferrin, 0.2 mg / mL vitamin C, 10 ng / mL sodium selenite, 10 ng / mL bFGF, and 50 ng / mL VEGF-165.

[0220] The culture medium C for preparing macrophages in the kit; wherein the culture medium C includes the culture medium C1, the culture medium C2, the culture medium C3, the culture medium C4, and the culture medium C5.

[0221] The culture medium C1 is a culture medium containing B27 additive without insulin at a volume fraction of 1%-2%, 0.5-1 mM L-glutamine or its substitute, non-essential amino acids at a volume fraction of 0.5%-1%, 50-100 U / mL penicillin, 50-100 μg / mL streptomycin, 25-50 ng / mL vitamin C, and 5-10 ng / mL human bone morphogenetic protein 4.

[0222] Specifically, the culture medium C1 in the following examples is RPMI 1640 culture medium containing B27 additive without insulin (B27 minus insulin) at a volume fraction of 2%, 1 mM L-glutamine substitute (Glutamax), non-essential amino acids (NEAA) at a volume fraction of 1%, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 ng / mL vitamin C, and 5 ng / mL human bone morphogenetic protein 4 (BMP4). The concentrations of each component in the non-essential amino acids in the culture medium C1 are as follows: the concentration of glycine in the culture medium C1 is 750.0 ng / mL; the concentration of L-alanine in the culture medium C1 is 890 ng / mL; the concentration of L-aspartic acid in the culture medium C1 is 1320 ng / mL; the concentration of L-asparagine in the culture medium C1 is 1330 ng / mL; the concentration of L-glutamic acid in the culture medium C1 is 1470 ng / mL; the concentration of L-proline in the culture medium C1 is 1150 ng / mL; the concentration of L-serine in the culture medium C1 is 1050 ng / mL.

[0223] The culture medium C2 is the culture medium C1 containing a GSK3 inhibitor, that is, a liquid prepared by using the culture medium C1 as a solvent and a GSK3 inhibitor as a solute. The content of the GSK3 inhibitor (such as CHIR-99021) in the culture medium III can be 1-2 μM or 2 μM.

[0224] The culture medium C2 in the following embodiments is specifically RPMI 1640 culture medium containing 2% (by volume) of B27 additive without insulin (B27 minus insulin), 1 mM L-glutamine substitute (Glutamax), 1% (by volume) of non-essential amino acids (NEAA), 100 U / mL penicillin, 100 μg / mL streptomycin, 50 ng / mL vitamin C, 5 ng / mL human bone morphogenetic protein 4 (BMP4), and 2 μM GSK3 inhibitor CHIR-99021. The concentrations of each component in the non-essential amino acids in the culture medium III are as follows: the concentration of glycine in the culture medium C2 is 750.0 ng / mL; the concentration of L-alanine in the culture medium C2 is 890 ng / mL; the concentration of L-aspartic acid in the culture medium C2 is 1320 ng / mL; the concentration of L-asparagine in the culture medium C2 is 1330 ng / mL; the concentration of L-glutamic acid in the culture medium C2 is 1470 ng / mL; the concentration of L-proline in the culture medium C2 is 1150 ng / mL; the concentration of L-serine in the culture medium C2 is 1050 ng / mL.

[0225] The culture medium C3 is a culture medium containing 1%-2% (by volume) of B27 additive with insulin added, 0.5-1 mM L-glutamine or its substitute, 0.5%-1% (by volume) of non-essential amino acids, 50-100 U / mL penicillin, 50-100 μg / mL streptomycin, 25-50 ng / mL vitamin C, 25-50 ng / mL human vascular endothelial growth factor VEGF-165, and 5-10 ng / mL human fibroblast growth factor bFGF.

[0226] The culture medium C3 in the following examples is specifically RPMI 1640 culture medium containing 2% (by volume) of B27 supplement with insulin added, 1 mM L-glutamine substitute (Glutamax), 1% (by volume) of non-essential amino acids, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 ng / mL vitamin C, 50 ng / mL human vascular endothelial growth factor VEGF-165, and 10 ng / mL human basic fibroblast growth factor (bFGF). The concentrations of each component in the non-essential amino acids in the culture medium C3 are as follows: the concentration of glycine in the culture medium C3 is 750.0 ng / mL; the concentration of L-alanine in the culture medium C3 is 890 ng / mL; the concentration of L-aspartic acid in the culture medium C3 is 1320 ng / mL; the concentration of L-asparagine in the culture medium C3 is 1330 ng / mL; the concentration of L-glutamic acid in the culture medium C3 is 1470 ng / mL; the concentration of L-proline in the culture medium C3 is 1150 ng / mL; the concentration of L-serine in the culture medium C3 is 1050 ng / mL.

[0227] The culture medium C4 is a culture medium containing a transforming growth factor-β (TGF-β) inhibitor, which is a liquid prepared by using the culture medium C3 as a solvent and the TGF-β inhibitor as a solute; the TGF-β inhibitor can specifically be SB431542. The content of the TGF-β inhibitor (SB431542) in the culture medium C4 can be 5 - 10 μM or 10 μM.

[0228] The culture medium C4 in the following examples is specifically RPMI 1640 culture medium containing 2% (by volume) of B27 supplement with insulin added, 1 mM L-glutamine substitute (Glutamax), 1% (by volume) of non-essential amino acids, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 ng / mL vitamin C, 50 ng / mL human vascular endothelial growth factor VEGF-165, 10 ng / mL human basic fibroblast growth factor (bFGF), and 10 μM SB431542. The concentrations of each component in the non-essential amino acids in the culture medium C4 are as follows: the concentration of glycine in the culture medium C4 is 750.0 ng / mL; the concentration of L-alanine in the culture medium C4 is 890 ng / mL; the concentration of L-aspartic acid in the culture medium C4 is 1320 ng / mL; the concentration of L-asparagine in the culture medium C4 is 1330 ng / mL; the concentration of L-glutamic acid in the culture medium C4 is 1470 ng / mL; the concentration of L-proline in the culture medium C4 is 1150 ng / mL; the concentration of L-serine in the culture medium C4 is 1050 ng / mL.

[0229] The culture medium C5 is a culture medium containing B27 additive added with insulin at a volume fraction of 1%-2%, 0.5-1 mM L-glutamine or its substitute, non-essential amino acids at a volume fraction of 0.5%-1%, 50-100 U / mL penicillin, 50-100 μg / mL streptomycin, 25-50 ng / mL vitamin C, 10-20 ng / mL human interleukin-3, and 50-100 ng / mL human macrophage colony-stimulating factor.

[0230] Specifically, the culture medium C5 in the following examples is RPMI 1640 culture medium containing B27 additive (B27 supplement) added with insulin at a volume fraction of 2%, 1 mM L-glutamine substitute (Glutamax), non-essential amino acids (NEAA) at a volume fraction of 1%, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 ng / mL vitamin C, 10 ng / mL human interleukin-3 (IL3), and 50 ng / mL human macrophage colony-stimulating factor (M-CSF). The concentrations of each component in the non-essential amino acids in the culture medium C5 are as follows: the concentration of glycine in the culture medium C5 is 750.0 ng / mL; the concentration of L-alanine in the culture medium C5 is 890 ng / mL; the concentration of L-aspartic acid in the culture medium C5 is 1320 ng / mL; the concentration of L-asparagine in the culture medium C5 is 1330 ng / mL; the concentration of L-glutamic acid in the culture medium C5 is 1470 ng / mL; the concentration of L-proline in the culture medium C5 is 1150 ng / mL; the concentration of L-serine in the culture medium C5 is 1050 ng / mL.

[0231] The culture medium D for preparing vascularized cardiac organoids containing immune cells in the kit, wherein the culture medium D may include culture medium D1 and culture medium D2.

[0232] The culture medium D1 can be any one of the following:

[0233] 1) D1-I: Composed of B27 additive added with insulin, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), human macrophage colony-stimulating factor (M-CSF), Rho-associated protein kinase (ROCK) inhibitor, and serum-free basal medium.

[0234] 2) D1-II: It consists of B27 additive added with insulin, penicillin, streptomycin, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), human macrophage colony-stimulating factor (M-CSF), Rho-associated protein kinase (ROCK) inhibitor, and serum-free basal medium.

[0235] 3) D1-III: It consists of B27 additive added with insulin, serum albumin, transferrin, vitamin C, sodium selenite, penicillin, streptomycin, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), human macrophage colony-stimulating factor (M-CSF), Rho-associated protein kinase (ROCK) inhibitor, and serum-free basal medium.

[0236] In the said culture medium, VEGF can specifically be VEGF-165.

[0237] In the said culture medium, the serum-free basal culture medium can be RPMI 1640 medium or other basal culture media commonly used for cell culture.

[0238] In the said culture medium D1, the said ROCK inhibitor can specifically be Y27632.

[0239] The said culture medium D2 is the culture medium D1 with the ROCK inhibitor removed.

[0240] The culture medium D1 in the following examples is specifically RPMI 1640 culture medium containing 2% (by volume) of B27 additive (B27 supplement) added with insulin, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 ng / mL human vascular endothelial growth factor VEGF-165, 10 ng / mL human fibroblast growth factor bFGF, 20 ng / mL human macrophage colony-stimulating factor M-CSF, and 10 μM ROCK inhibitor Y27632.

[0241] The culture medium D2 in the following examples is specifically RPMI 1640 culture medium containing 2% (by volume) of B27 additive (B27 supplement) added with insulin, 100 U / mL penicillin, 100 μg / mL streptomycin, 50 ng / mL human vascular endothelial growth factor VEGF-165, 10 ng / mL human fibroblast growth factor bFGF, and 20 ng / mL human macrophage colony-stimulating factor M-CSF.

[0242] The said kit contains culture medium M, and the culture medium M can contain culture medium M1 and culture medium M2.

[0243] Each 1 mL of the culture medium M1 may be a serum-free basal culture medium containing 0.1 - 2.5 mg of serum albumin (such as 0.1 - 0.5 mg of serum albumin, 0.5 - 2.5 mg of serum albumin, 0.1 mg of serum albumin, 0.5 mg of serum albumin, or 2.5 mg of serum albumin), 1 - 25 μg of transferrin (1 - 5 μg of transferrin, 5 - 25 μg of transferrin, 1 μg of transferrin, 5 μg of transferrin, or 25 μg of transferrin), 0.04 - 1 mg of vitamin C (0.04 - 0.2 mg of vitamin C, 0.2 - 1 mg of vitamin C, 0.04 mg of vitamin C, 0.2 mg of vitamin C, or 1 mg of vitamin C), 2 - 50 ng of sodium selenite (2 - 10 ng of sodium selenite, 10 - 50 ng of sodium selenite, 2 ng of sodium selenite, 10 ng of sodium selenite, or 50 ng of sodium selenite), and 2 - 50 ng of BMP4 (2 - 10 ng of BMP4, 10 - 50 ng of BMP4, 2 ng of BMP4, 10 ng of BMP4, or 50 ng of BMP4).

[0244] Each 1 mL of the culture medium M2 may be a serum-free basal culture medium containing 0.1 - 2.5 mg of serum albumin (such as 0.1 - 0.5 mg of serum albumin, 0.5 - 2.5 mg of serum albumin, 0.1 mg of serum albumin, 0.5 mg of serum albumin, or 2.5 mg of serum albumin), 1 - 25 μg of transferrin (1 - 5 μg of transferrin, 5 - 25 μg of transferrin, 1 μg of transferrin, 5 μg of transferrin, or 25 μg of transferrin), 0.04 - 1 mg of vitamin C (0.04 - 0.2 mg of vitamin C, 0.2 - 1 mg of vitamin C, 0.04 mg of vitamin C, 0.2 mg of vitamin C, or 1 mg of vitamin C), 2 - 50 ng of sodium selenite (2 - 10 ng of sodium selenite, 10 - 50 ng of sodium selenite, 2 ng of sodium selenite, 10 ng of sodium selenite, or 50 ng of sodium selenite), 2 - 50 ng of BMP4 (2 - 10 ng of BMP4, 10 - 50 ng of BMP4, 2 ng of BMP4, 10 ng of BMP4, or 50 ng of BMP4), and 1 - 10 nmol of GSK3 inhibitor (1 - 5 nmol of GSK3 inhibitor, 5 - 10 nmol of GSK3 inhibitor, 1 nmol of GSK3 inhibitor, 5 nmol of GSK3 inhibitor, or 10 nmol of GSK3 inhibitor).

[0245] In the culture medium M, the serum albumin can be recombinant human serum albumin, bovine serum albumin, or other recombinant human serum albumin of non-animal origin, such as recombinant human serum albumin of plant origin. In the culture medium M, the transferrin can be human transferrin or other recombinant transferrin of non-animal origin, such as recombinant human transferrin of plant origin. In the culture medium M, the serum-free basal medium can be RPMI 1640 medium or a basal medium commonly used for cell culture. In the culture medium M2, the GSK3 inhibitor can be a1) or a2) or a3) or a4): a1) CHIR-99021; a2) B216763; a3) BIO; a4) TWS119.

[0246] The culture medium M1 in the following examples is specifically an RPMI 1640 culture medium containing 0.5 mg / mL recombinant human serum albumin, 5 μg / mL human transferrin, 0.2 mg / mL vitamin C, 10 ng / mL sodium selenite, and 5 ng / mL BMP4.

[0247] The culture medium M2 in the following examples is specifically an RPMI 1640 culture medium containing 0.5 mg / mL recombinant human serum albumin, 5 μg / mL human transferrin, 0.2 mg / mL vitamin C, 10 ng / mL sodium selenite, 5 ng / mL BMP4, and 2 μM CHIR-99021.

[0248] Example 1: Differentiation of human pluripotent stem cells into cardiomyocytes and detection of cells during the differentiation process

[0249] I. Differentiation of human pluripotent stem cells into cardiomyocytes

[0250] (1) Take a 12-well plate, coat the plate with RPMI 1640 containing 0.5% (v / v) Matrigel, and then incubate at 37 °C for 2 hours. Inoculate human pluripotent stem cells into the coated 12-well plate (1.5×10 5 cells per well), and culture at 37 °C for 1 day using E8-Y culture medium (i.e., culture medium H2);

[0251] (2) Replace the culture medium with H1 and change the medium daily. After 2 days of culture, the morphological diagram of human pluripotent stem cells can be seen ( Figure 1 , scale bar 250 μm);

[0252] (3) Continue to culture with the culture medium H1 for 1 day until the cell confluence reaches 90 - 95% ( Figure 2 , scale bar 200 μm). At this time, replace it with the culture medium A1 and continue to culture for 2 days;

[0253] (4) After 2 days of treatment with the culture medium A1, the morphological changes of the cells can be seen at this time (Figure 2 , scale 200 microns), carefully discard the upper culture medium, and replace it with the culture medium A2 and continue culturing for 2 days;

[0254] (5) After 2 days of treatment with culture medium A2, obvious morphological changes of the cells can be observed at this time ( Figure 2 , scale 200 microns), carefully discard the upper culture medium, and replace it with the culture medium A3 and continue culturing for 8 days. At this time, cardiomyocytes with obvious pulsation can be observed ( Figure 2 , scale 200 microns);

[0255] (6) Discard the culture medium, add 500 μL of cardiomyocyte digestive solution (composed of TrypLE TM Express digestive solution and type II collagenase at a ratio of 4:1) to each 12-well plate, digest at 37 °C for 30 minutes, and then centrifuge at 300 g for 3 minutes to collect the cardiomyocytes.

[0256] II. Detection of cardiomyocytes differentiated from human pluripotent stem cells

[0257] (I). Immunofluorescence staining detection of cardiomyocytes

[0258] (1) Discard the culture medium of the cardiomyocytes differentiated in step (5) during the preparation of cardiomyocytes in Example 1 after 12 days of differentiation, add 4% paraformaldehyde and fix at room temperature for 10 minutes, then aspirate the 4% paraformaldehyde and wash 3 times with PBS buffer.

[0259] (2) Discard the PBS buffer, add 1 mL of PBS buffer containing 0.5% Triton X-100, and incubate on a shaker at room temperature for 15 minutes.

[0260] (3) Discard the supernatant, add 1 mL of PBS buffer containing 0.1% Tween 20 (0.1% PBST) to wash, discard the supernatant, and then add 1 mL of 0.1% PBST and wash on a shaker at room temperature for 5 minutes.

[0261] (4) Discard the supernatant, add 10% goat serum diluted with PBS, and incubate on a shaker at room temperature for 1 hour.

[0262] (5) Discard the goat serum, add 500 μL of primary antibody working solution, and incubate in a refrigerator at 4 °C for 16 hours. The primary antibody working solution is a 0.1% PBST solution containing mouse anti-human TNNT2 (cTnT) antibody diluted at a volume ratio of 1:200 and 1% BSA by volume.

[0263] (6) Take out the sample, discard the supernatant, add 0.1% PBST solution, and wash on a shaker at room temperature for 5 minutes, and repeat the washing 2 times.

[0264] (7) Discard the supernatant, add 500 μL of secondary antibody working solution, and incubate on a shaker at room temperature for 1 hour. The secondary antibody working solution is a 0.1% PBST solution containing goat anti-mouse IgG antibody labeled with DyLight488 fluorophore diluted at a volume ratio of 1:200 and 1% BSA by volume.

[0265] (8) Discard the secondary antibody working solution, add 1.5 mL of 0.1% PBST, wash on a shaker at room temperature for 5 minutes, and repeat the washing 2 times.

[0266] (9) Discard the supernatant, add 500 μL of DAPI working solution, and incubate on a shaker at room temperature for 5 minutes. The DAPI working solution is a 0.1% PBST solution containing DAPI stock solution diluted at a volume ratio of 1:1000.

[0267] (10) Discard the supernatant, add 1.5 mL of 0.1% PBST, wash on a shaker at room temperature for 5 minutes, and repeat the washing 2 times. Then observe the cell staining under a laser confocal microscope.

[0268] The immunofluorescence staining results of cardiomyocytes differentiated for 12 days in a 12-well plate are shown in Figure 3 . The results show that cardiomyocytes differentiated from human pluripotent stem cells express the cardiomyocyte-specific marker protein TNNT2 (cTnT).

[0269] (II) Flow cytometry of cardiomyocytes

[0270] (1) After centrifugally collecting the cardiomyocytes differentiated for 12 days obtained in step (6) during the preparation of cardiomyocytes in the example, transfer them to an EP tube, add 4% paraformaldehyde and fix at room temperature for 10 minutes, then aspirate the 4% paraformaldehyde and wash 2 times with PBS buffer.

[0271] (2) Centrifuge at 300 g for 5 minutes, discard the supernatant, add 100 μL of primary antibody working solution, and incubate on a shaker at room temperature for 1 hour. The primary antibody working solution is a washing buffer containing mouse anti-human TNNT2 (cTnT) antibody diluted at a volume ratio of 1:100. Further, the washing buffer is specifically a PBS buffer containing 1% FBS by volume and 0.1% Triton X-100 by volume.

[0272] (3) After the incubation of the primary antibody, add 1 mL of washing buffer to each tube for washing, and then centrifuge at 300 g for 5 minutes. After discarding the supernatant, wash again once.

[0273] (4) Discard the supernatant, add 100 μL of secondary antibody working solution, and incubate in the dark on a shaker at room temperature for 30 minutes. The secondary antibody working solution is washing buffer containing goat anti-mouse IgG antibody labeled with DyLight488 fluorophore diluted at a volume ratio of 1:100.

[0274] (5) After the secondary antibody incubation is completed, add 1 mL of washing buffer to each tube for washing, then centrifuge at 300 g for 5 minutes. Discard the supernatant and wash again.

[0275] (6) Discard the supernatant, add 200 μL of PBS buffer containing 1% (v / v) FBS to resuspend the cells. Then the cells can be analyzed using a flow cytometer.

[0276] The detection results of cardiomyocytes differentiated from human pluripotent stem cells are as Figure 4 shown. The results show that more than 90% of the cardiomyocytes differentiated by this method express the cardiomyocyte-specific TNNT2 (cTnT) protein marker.

[0277] Example 2: Differentiation of human pluripotent stem cells into vascular endothelial cells and detection of cells during the differentiation process

[0278] I. Differentiation of human pluripotent stem cells into vascular endothelial cells

[0279] (1) Take a 12-well plate, coat the plate with RPMI 1640 containing 0.5% (v / v) Matrigel, and then incubate at 37 °C for 2 hours. Seed human pluripotent stem cells into the coated 12-well plate at a density of 2×10 4 cells / cm 2 and culture with E8-Y medium (i.e., medium H2) for 1 day;

[0280] (2) Replace the cells in step (1) with medium M1 and culture for 1 day, then change to medium M2 and culture for 2 days. The morphological changes of the cells during this process are as Figure 5 (scale bar 200 μm) shown.

[0281] (3) Discard the medium, add 500 μL of Accutase digestion solution to each 12-well, digest at 37 °C for 1 minute, and centrifuge at 300 g for 3 minutes to obtain mesoderm cells.

[0282] (4) Before starting this step, take a 6-well plate in advance, coat the plate with RPMI 1640 containing 0.5% (v / v) Matrigel, and then incubate at 37 °C for 2 hours. Seed the mesoderm cells into the plate at a density of 3.5×10 4 cells / cm 2Inoculate into the coated 6-well plate, add the above culture medium B1 and culture for 3 days, changing the medium every day, and vascular endothelial cells on the 6th day of differentiation can be obtained. At this time, the cell morphology is as shown in Figure 5 (scale bar: 200 μm).

[0283] (5) For the cells obtained after completing step (4), inoculate them into a new uncoated 6-well plate at a density of 5×10 4 cells / cm 2 , add the above culture medium B1 and culture for 3 days, changing the medium every other day, and vascular endothelial cells on the 9th day of differentiation can be obtained. At this time, the cell morphology is as shown in Figure 5 (scale bar: 200 μm).

[0284] II. Detection of vascular endothelial cells prepared by differentiating human pluripotent stem cells

[0285] (I). Immunofluorescence staining detection of vascular endothelial cells

[0286] (1) For the vascular endothelial cells obtained after completing step (5) in the process of preparing vascular endothelial cells in Example 2, fix them with 4% paraformaldehyde at room temperature for 10 minutes, then aspirate the 4% paraformaldehyde, and wash them 3 times with PBS buffer (the purpose is to remove the residual paraformaldehyde).

[0287] (2) After completing step (1), add PBS buffer containing 5% BSA by volume and block at room temperature for 1 hour.

[0288] (3) After completing step (2), add anti-human CD31 antibody (volume ratio 1:200), incubate at room temperature for 2 hours, and then wash 3 times with 0.1% PBST buffer.

[0289] (4) After completing step (3), add secondary antibody (product of Abcam company, volume ratio 1:500), incubate at room temperature for 1 hour, and then wash 3 times with 0.1% PBST buffer.

[0290] (5) After completing step (4), add DAPI (volume ratio 1:1000), incubate on a shaker at room temperature for 5 minutes, and then wash 3 times with 0.1% PBST buffer, and observe the cell staining under a fluorescence microscope.

[0291] The results of immunofluorescence staining detection of the differentiated vascular endothelial cells are shown in Figure 6 (scale bar: 100 μm). The results show that the protein CD31 is expressed on the surface of the vascular endothelial cells cultured with culture medium B.

[0292] (II). Flow cytometry detection of vascular endothelial cells

[0293] (1) For the vascular endothelial cells at different culture days obtained after steps (4) and (5) in the preparation of vascular endothelial cells in Example 2, centrifuge at 500 g for 3 minutes, discard the supernatant, and add PBS buffer containing 1% (v / v) FBS to obtain a suspension of vascular endothelial cells.

[0294] (2) Take the cell suspension obtained in step (1) (containing 1×10 5 cells), add an antibody against human CD31 labeled with FITC, and incubate in the dark at room temperature for 20 minutes (mix well every 5 minutes during this period); then wash twice with PBS buffer containing 1% (v / v) FBS (the purpose is to remove unbound antibodies).

[0295] (3) After completing step (2), resuspend with 500 μL of PBS buffer containing 1% (v / v) FBS, and then detect using a flow cytometer.

[0296] The detection results of the differentiated vascular endothelial cells are shown in Figure 7 . The results show that after culturing with culture medium B for 3 days, more than 50% of the cell surfaces express the protein CD31 specifically expressed by endothelial cells, and after culturing for 9 days, this ratio increases to more than 90%.

[0297] Example 3: Differentiation of human pluripotent stem cells into macrophages and detection of cells during the differentiation process

[0298] I. Differentiation of human pluripotent stem cells into macrophages

[0299] (1) Take a 12-well plate, coat the plate with RPMI 1640 containing 0.5% (v / v) Matrigel, and then incubate at 37 °C for 2 hours. Inoculate human pluripotent stem cells into the 12-well plate at a density of 2×10 4 cells / cm 2 , and add culture medium H2 to culture for 1 day;

[0300] (2) Replace the culture medium of the cells in step (1) with culture medium C1 and culture for 1 day, and then change to culture medium C2 and culture for 2 days.

[0301] (3) After completing step (2), discard the supernatant, first add 500 μL of Accutase digestion solution to digest until single-cell state, and then add 3 mL of RPMI 1640 basal culture medium to terminate digestion, and centrifuge at 300 g for 3 minutes to collect cells.

[0302] (4) Before starting this step, take a 12-well plate in advance, coat the plate with RPMI 1640 containing 0.5% (v / v) Matrigel, and then incubate at 37 °C for 2 hours. Inoculate the mesoderm cells into the plate at a density of 4×10 4 cells / cm 2Inoculate into the well-coated 12-well plate. First, add culture medium C3 and culture for 2 days, then change to culture medium C4 and continue to culture for 3 days. During this process, the morphological changes of the cells are as shown in Figure 8 (scale bar: 100 μm).

[0303] (5) After completing step (4), carefully collect all the suspended cells in the culture medium, and inoculate all the obtained suspended cells at a density of 5×10 4 cells / cm 2 into a new 12-well plate, and continue to culture with culture medium C5. During this period, change half of the culture medium every day. After 7 days, macrophages can be collected. During the continuous culture process, the macrophages can show morphological changes, as shown in Figure 8 (scale bar: 100 μm).

[0304] II. Detection of macrophages prepared by differentiating human pluripotent stem cells

[0305] (I). Immunofluorescence staining detection of macrophages

[0306] (1) Fix the macrophages collected after completing step (5) in the process of preparing macrophages in Example 3 with 4% paraformaldehyde at room temperature for 10 minutes, then aspirate the 4% paraformaldehyde and wash 3 times with PBS buffer.

[0307] (2) After completing step (1), add PBS buffer containing 5% (v / v) BSA and block at room temperature for 1 hour.

[0308] (3) After completing step (2), add mouse anti-human CD68 antibody (volume ratio 1:100) and rabbit anti-human CD14 antibody (volume ratio 1:100), incubate at 4°C for 16 hours, and then wash 3 times with 0.1% PBST (PBS buffer containing 0.1% Tween-20).

[0309] (4) After completing step (3), add DAPI (volume ratio 1:1000), incubate at room temperature for 5 minutes, and then wash 3 times with 0.1% PBST buffer. Observe the cell staining under a laser confocal microscope.

[0310] The immunofluorescence results of macrophages are shown in Figure 9 (scale bar: 100 μm). The results show that the macrophages differentiated from human pluripotent stem cells express macrophage marker proteins CD14 and CD68.

[0311] II. Flow cytometry detection of macrophages

[0312] (1) For the macrophages collected after completing step (5) in the process of preparing macrophages in Example 3, resuspend the cells with PBS buffer containing 5% (v / v) FBS to obtain a cell suspension (containing 1×105 cells).

[0313] (2) Add FITC-labeled CD45 antibody, Percp-Cy5.5-labeled CD11b antibody, APC-labeled CD14 antibody, and PE-labeled CD163 antibody to the cell suspension in step (1), and incubate in the dark at room temperature for 20 minutes (mix well every 5 minutes during this period). Then wash twice with PBS buffer containing 5% (volume fraction) FBS, and centrifuge to collect the cells.

[0314] (3) After completing step 2, discard the supernatant, add 300 μL of PBS buffer containing 5% (volume fraction) FBS to resuspend the cells, and detect using a flow cytometer.

[0315] The detection results of the differentiated macrophages are as Figure 10 shown. The results show that for the macrophages differentiated using culture medium C, more than 80% of the cells co-express the typical macrophage surface markers CD45, CD11b, CD14, and CD163.

[0316] Example 4. Preparation of vascularized cardiac organoids containing immune cells and their detection

[0317] I. Preparation of vascularized cardiac organoids containing immune cells

[0318] (1) Take the cardiomyocytes, vascular endothelial cells, and macrophages differentiated from human pluripotent stem cells obtained in Example 1, Example 2, and Example 3 above, and resuspend them in culture medium D1. Subsequently, mix the three types of cells at a cell ratio of 7:2:1, and seed the mixed cells at 1×10 5 cells / well into a 96-well ultra-low attachment plate, and culture in an incubator at 37 °C and 5% CO2 for 1 day.

[0319] (2) After completing step (1), change the culture medium to culture medium D2, and subsequently change the medium every other day. After culturing for 10 days, vascularized cardiac organoids containing immune cells can be obtained. The morphological changes of the organoids during this process are as Figure 11 (scale bar 500 μm) shown.

[0320] II. Immunofluorescence staining detection of vascularized cardiac organoids containing immune cells

[0321] (1) After discarding the culture medium from the vascularized cardiac organoids containing immune cells prepared in Example 4, collect the organoids into an EP tube, fix them with 4% paraformaldehyde at room temperature for 30 minutes, then aspirate the 4% paraformaldehyde, and wash 3 times with PBS buffer.

[0322] (2) Discard the PBS buffer, add 0.5 mL of PBS buffer containing 0.5% Triton X-100, and incubate on a shaker at room temperature for 15 minutes.

[0323] (3) Discard the supernatant, add 0.5 mL of PBS buffer containing 0.1% Tween 20 (0.1% PBST) for washing, discard the supernatant, then add 1 mL of 0.1% PBST, and wash on a shaker at room temperature for 5 minutes.

[0324] (4) Discard the supernatant, add 10% goat serum diluted with PBS, and incubate on a shaker at room temperature for 1 hour.

[0325] (5) Discard the goat serum, add 500 μL of the primary antibody working solution, and incubate in a refrigerator at 4°C for 16 hours. The primary antibody working solution is a 0.1% PBST solution containing mouse anti-human CD68 antibody diluted at a volume ratio of 1:200, rabbit anti-human CD31 antibody diluted at a volume ratio of 1:200, goat anti-human TNNT2 (cTnT) antibody diluted at a volume ratio of 1:200, and 1% BSA by volume.

[0326] (6) Take out the sample, discard the supernatant, add 0.1% PBST solution, and wash on a shaker at room temperature for 5 minutes, repeating the washing 2 times.

[0327] (7) Discard the supernatant, add 500 μL of the secondary antibody working solution, and incubate on a shaker at room temperature for 1 hour. The secondary antibody working solution is a 0.1% PBST solution containing donkey anti-goat IgG antibody labeled with Cy3 fluorophore diluted at a volume ratio of 1:200, donkey anti-mouse IgG antibody labeled with FITC fluorophore diluted at a volume ratio of 1:200, donkey anti-rabbit IgG antibody labeled with CoraLite647 fluorophore diluted at a volume ratio of 1:200, and 1% BSA by volume.

[0328] (8) Discard the secondary antibody working solution, add 0.5 mL of 0.1% PBST, and wash on a shaker at room temperature for 5 minutes, repeating the washing 2 times.

[0329] (9) Discard the supernatant, add 500 μL of the DAPI working solution, and incubate on a shaker at room temperature for 5 minutes. The DAPI working solution is a 0.1% PBST solution containing DAPI stock solution diluted at a volume ratio of 1:1000.

[0330] (10) Discard the supernatant, add 1.5 mL of 0.1% PBST, and wash on a shaker at room temperature for 5 minutes, repeating the washing 2 times. Then immediately observe the cell staining under a laser confocal microscope.

[0331] The immunofluorescence staining results of the vascularized heart organoids containing immune cells are shown in Figure 12(Scale: 100 microns). The results showed that macrophages differentiated from human pluripotent stem cells expressed macrophage marker protein CD14, cardiomyocyte marker protein cTnT, and endothelial cell marker protein CD31.

[0332] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A kit for preparing vascularized cardiac organoids containing immune cells using cardiomyocytes, vascular endothelial cells and macrophages, characterized in that: The kit includes a culture solution D, and the culture solution D may include a culture solution D1 and a culture solution D2; The culture solution D1 is any one of the following: 1) D1-I: containing B27 supplement with added insulin, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C, basic fibroblast growth factor, vascular endothelial growth factor, human macrophage colony stimulating factor, Rho-associated protein kinase inhibitor and serum-free basal medium; Wherein, the non-essential amino acids are glycine, L-alanine, L-aspartic acid, L-aspartic acid, L-glutamic acid, L-proline and L-serine; 2) D1-II: contains B27 supplement with insulin, penicillin, streptomycin, basic fibroblast growth factor, vascular endothelial growth factor, human macrophage colony stimulating factor, Rho-associated protein kinase inhibitor and serum-free basal medium; 3) D1-III: contains B27 supplement with added insulin, serum albumin, transferrin, vitamin C, sodium selenite, penicillin, streptomycin, basic fibroblast growth factor, vascular endothelial growth factor, human macrophage colony stimulating factor, Rho-associated protein kinase inhibitor and serum-free basal medium; The culture solution D2 is the culture solution D1 from which the Rho-associated protein kinase inhibitor is removed.

2. The kit according to claim 1, characterized in that: The culture medium D1-I is a culture medium containing 1%-2% of B27 additive with added insulin, 0.5-1mM L-glutamine or its substitute, 0.5%-1% of non-essential amino acids, 50-100U / mL penicillin, 50-100μg / mL streptomycin, 25-50ng / mL vitamin C, 5-20ng / mL basic fibroblast growth factor, 50-100ng / mL vascular endothelial growth factor, 20-50ng / mL human macrophage colony stimulating factor, and 2-10μM Rho-associated protein kinase inhibitor; And / or, the culture medium D1-II is a culture medium containing 1%-2% by volume of B27 additive added with insulin, 50-100U / mL penicillin, 50-100μg / mL streptomycin, 5-20ng / mL basic fibroblast growth factor, 50-100ng / mL vascular endothelial growth factor, 20-50ng / mL human macrophage colony stimulating factor, and 2-10μM Rho-associated protein kinase inhibitor; And / or, the culture medium D1-III is a culture medium containing 1%-2% B27 additive with added insulin, 0.1-2.5 mg / mL serum albumin, 1-25 μg / mL transferrin, 0.04-1 mg / mL vitamin C, 2-50 ng / mL sodium selenite, 50-100 U / mL penicillin, 50-100 μg / mL streptomycin, 5-20 ng / mL bFGF, 50-100 ng / mL VEGF, 20-50 ng / mL M-CSF, and 2-10 μM Rho-associated protein kinase inhibitor.

3. A kit for preparing vascularized cardiac organoids containing immune cells, characterized in that: The kit comprises a culture solution A for preparing cardiomyocytes, a culture solution B for preparing vascular endothelial cells, a culture solution C for preparing macrophages, and a culture solution D according to claim 1 or 2; Wherein, the culture solution A includes culture solution A1, culture solution A2 and culture solution A3; The culture medium A1 is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite and GSK3 inhibitor; The culture medium A2 is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite and Wnt protein palmitoylase Porcupine inhibitor; The culture solution A3 is the culture solution obtained by removing the GSK3 inhibitor from the culture solution A1 or the culture solution obtained by removing the Wnt protein palmitoylase Porcupine inhibitor from the culture solution A2; Wherein, the culture medium B is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite, vascular endothelial growth factor, and basic fibroblast growth factor; Wherein, the culture solution C includes culture solution C1, culture solution C2, culture solution C3, culture solution C4 and culture solution C5; The culture medium C1 is a culture medium containing insulin-free B27 additive, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C and human bone morphogenetic protein 4; wherein the non-essential amino acids are glycine, L-alanine, L-aspartic acid, L-aspartic acid, L-glutamic acid, L-proline and L-serine; The culture solution C2 is a culture solution containing a GSK3 inhibitor, which is a liquid prepared by using the culture solution C1 as a solvent and the GSK3 inhibitor as a solute; The culture medium C3 is a culture medium containing B27 additives to which insulin is added, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor and human fibroblast growth factor; wherein the non-essential amino acids are glycine, L-alanine, L-aspartic acid, L-aspartic acid, L-glutamic acid, L-proline and L-serine; The culture solution C4 is a culture solution containing a transforming growth factor-β inhibitor, which is a liquid prepared by using the culture solution C3 as a solvent and the transforming growth factor-β inhibitor as a solute; The culture medium C5 is a culture medium containing B27 additives to which insulin is added, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony stimulating factor; wherein the non-essential amino acids are glycine, L-alanine, L-aspartic acid, L-aspartic acid, L-glutamic acid, L-proline and L-serine.

4. The kit according to claim 3, characterized in that: Each 1 mL of the culture medium A1 contains 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite and 1-10 nmol GSK3 inhibitor, and the remainder is a serum-free basic culture medium; the pH value of the culture medium A1 is 7.0-7.6; And / or, each 1 mL of the culture medium A2 contains 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 4-6 nmol Wnt protein palmitoylase Porcupine inhibitor, and the remainder is a serum-free basic culture medium; the pH value of the culture medium A2 is 7.0-7.6; and / or, each 1 mL of the culture medium A3 contains 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, and the remainder is a serum-free basal culture medium; And / or, each 1 mL of the culture solution B contains 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 10-250 ng vascular endothelial growth factor and 2-50 ng basic fibroblast growth factor, and the remainder is a serum-free basic culture solution; the pH value of the culture solution B is 7.0-7.

6.

5. The kit according to claim 3 or 4, characterized in that: The culture medium C1 is a culture medium containing 1%-2% of insulin-free B27 additive, 0.5-1mM L-glutamine or its substitute, 0.5%-1% of non-essential amino acids, 50-100U / mL penicillin, 50-100μg / mL streptomycin, 25-50ng / mL vitamin C and 5-10ng / mL human bone morphogenetic protein 4; and / or, the content of the GSK3 inhibitor in the culture medium C2 is 1-2 μM; And / or, the culture medium C3 is a culture medium containing 1%-2% by volume of B27 additives to which insulin is added, 0.5-1mM L-glutamine or its substitute, 0.5%-1% by volume of non-essential amino acids, 50-100U / mL penicillin, 50-100μg / mL streptomycin, 25-50ng / mL vitamin C, 25-50ng / mL human vascular endothelial growth factor and 5-10ng / mL human fibroblast growth factor; and / or, the content of the TGF-β inhibitor in the culture medium C4 is 5-10 μM; And / or, the culture medium C5 is a culture medium containing 1%-2% by volume of B27 additive added with insulin, 0.5-1mM L-glutamine or its substitute, 0.5%-1% by volume of non-essential amino acids, 50-100U / mL penicillin, 50-100μg / mL streptomycin, 25-50ng / mL vitamin C, 10-20ng / mL human interleukin-3 and 50-100ng / mL human macrophage colony stimulating factor.

6. The kit according to any one of claims 3 to 5, characterized in that: The kit further comprises a culture solution M, wherein the culture solution M comprises a culture solution M1 and a culture solution M2; The culture medium M1 is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite and bone morphogenetic protein 4; The culture medium M2 is a culture medium containing serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenetic protein 4 and GSK3 inhibitor.

7. The kit according to claim 6, characterized in that: Each 1 mL of the culture medium M1 contains 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 2-50 ng bone morphogenetic protein 4, and the remainder is a serum-free basic culture medium. The pH value of the culture medium M1 is 7.0-7.6; And / or, each 1 mL of the culture medium M2 contains 0.1-2.5 mg serum albumin, 1-25 μg transferrin, 0.04-1 mg vitamin C, 2-50 ng sodium selenite, 2-50 ng BMP4 and 1-10 nmol GSK3 inhibitor, and the remainder is a serum-free basic culture medium; the pH value of the culture medium M2 is 7.0-7.

6.

8. The kit according to any one of claims 3 to 7, characterized in that: The kit is characterized in that: the kit further comprises a culture solution H, and the culture solution H comprises a culture solution H1 and / or a culture solution H2; The culture medium H1 is TeSR-E8 complete culture medium or other culture medium for culturing human pluripotent stem cells; The culture fluid H2 is a complete TeSR-E8 culture fluid containing 2-10 μM Rho-related protein kinase inhibitor.

9. A kit for preparing cardiomyocytes, characterized in that: The kit comprises the culture solution A according to claim 3 or 4.

10. A method for preparing vascularized cardiac organoids containing immune cells, comprising the following steps: (D1) cardiomyocytes, vascular endothelial cells and macrophages differentiated from human pluripotent stem cells are mixed at a certain cell ratio and inoculated into a 96-well ultra-low adsorption plate, and the culture medium D1 described in claim 1 or 2 is added and cultured for 0.5-2 days; (D2) The culture medium D1 described in step (D1) is replaced with the culture medium D2 described in claim 1 or 2, and the culture medium is subsequently changed every other day. After culturing for at least 7 days, vascularized cardiac organoids containing immune cells are obtained.

11. The method according to claim 10, characterized in that: In the step (D1), the cell ratios of the cardiomyocytes, vascular endothelial cells and macrophages are 50%-80%, 10%-30% and 10%-20% respectively; And / or; In the step (D1), the inoculation density is 1×10 5 -3×10 5 Pieces / hole; And / or; In the step (D2), the culture time is 7-14 days.

12. The method according to claim 10 or 11, characterized in that: In the step (D1), the cardiomyocytes differentiated from human pluripotent stem cells are prepared according to a method comprising the following steps: (A1) inoculating human pluripotent stem cells in the culture medium H2 of claim 8 and culturing for 0.5-1.5 days; (A2) changing to the culture medium H1 described in claim 8 and culturing for 2.5-4 days until the cell confluence reaches 90-95%; (A3) replacing the culture medium A1 according to claim 3 or 4 and continuing culturing for 1.5-2.5 days; (A4) replacing the culture medium A2 according to claim 3 or 4 and continuing culturing for 1.5-2.5 days; (A5) Changing the culture medium to the culture medium A3 according to claim 3 or 4 and continuing the culture for at least 4 days to collect the cardiomyocytes.

13. The method according to any one of claims 10 to 12, characterized in that: In the step (D1), the vascular endothelial cells differentiated from human pluripotent stem cells can be prepared according to a method comprising the following steps: (B1) inoculating human pluripotent stem cells in the culture medium H2 of claim 8 and culturing for 0.5-1.5 days; (B2) replacing the cells in step (1) with the culture medium M1 described in claim 6 or 7 and culturing them for 0.5-1.5 days; (B3) using the culture medium M2 described in claim 6 or 7 for 1.5-2.5 days to obtain mesoderm cells; (B4) inoculating the mesoderm cells into the culture medium B according to claim 3 or 4 and culturing for 3-7 days to obtain vascular endothelial cells; And / or, in the step (D1), the macrophages differentiated from human pluripotent stem cells can be prepared according to a method comprising the following steps: (C1) inoculating human pluripotent stem cells in the culture medium H2 of claim 8 and culturing for 0.5-1.5 days; (C2) replacing the cells in step (C1) with the culture medium C1 described in any one of claims 3 to 5 and culturing them for 0.5 to 1.5 days; (C3) using the culture medium C2 according to any one of claims 3 to 5 for 1.5 to 2.5 days to obtain mesoderm cells; (C4) inoculating the mesoderm cells into the culture medium C3 described in any one of claims 3 to 5, culturing for 1.5 to 2.5 days, and then changing to the culture medium C4 described in any one of claims 3 to 5, culturing for 3 to 5 days, to obtain hematopoietic progenitor cells; (C5) The hematopoietic progenitor cells obtained in step (C4) are inoculated into the culture medium C5 described in any one of claims 3 to 5 and cultured for 7 to 10 days to obtain macrophages.

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