Construction method of heart organoid

Abstract

The invention relates to the technical field of organoid construction, and discloses a heart organoid construction method which comprises the following steps: preparing a culture medium I containing CD lipid, ITS-X, L-AA2P, alpha-MTG, insulin, FGF2 and a GSK-3beta inhibitor; preparing a culture medium II containing CD lipid, ITS-X, L-AA2P, alpha-MTG, insulin, FGF2 (fibroblast growth factor 2) and VEGFA (vascular endothelial growth factor A); the method comprises the following steps: digesting a human induced pluripotent stem cell mass, suspending the human induced pluripotent stem cell mass in a culture medium I, inoculating the human induced pluripotent stem cell mass into an ultra-low adsorption cell culture plate with a U-shaped hole bottom, culturing for 36-40 hours, removing the culture medium I, adding a culture medium II, and culturing until a heart organ is formed. In the method provided by the invention, two culture media are sequentially used for performing two-stage differentiation culture, namely, the heart organoid can be constructed from the human induced pluripotent stem cells dispersed after digestion, so that the construction process of the heart organoid is simplified, the construction time is shortened, and the operation difficulty is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid construction, and in particular to a method for constructing a cardiac organoid. Background Art

[0002] The heart is an important organ in the human body. Its basic function is to promote blood flow, provide sufficient blood flow to organs and tissues to supply oxygen and various nutrients, and take away the end products of metabolism so that cells can maintain normal metabolism and function. Vascular diseases, such as myocardial infarction and heart failure, have become the leading cause of death worldwide. As people age, the decline in heart function has brought a heavy burden to human health. In-depth research on the pathogenesis of cardiovascular diseases, improving the diagnosis and treatment of cardiovascular diseases, and developing new drugs have become the focus of scholars.

[0003] Two-dimensional cell models and animal models have long been used to study cardiovascular diseases. However, these traditional models have their own limitations and are not fully suitable for the study of cardiovascular diseases. For example, 2D cell models have no matrix components and organ-specific functions, and the genetic heterogeneity of original cells is gradually lost after multiple passages. Animal models are species-specific and have different structures and physiology from humans, as well as different specific organ development and pathogenesis.

[0004] Compared with the above two research models, organoids, as a new in vitro cell model, have a complex three-dimensional cell structure, which shows similar structures and functions to organs in vivo. Cardiac organoids are an effective supplement to traditional cardiovascular disease models, reflecting the biological characteristics and functions of the human heart more realistically and accurately in vitro, making them have broad application prospects and unique advantages in the fields of disease mechanism research, drug development, precision medicine and regenerative medicine. At present, some methods for constructing cardiac organoids from human induced pluripotent stem cells (iPS) have been reported, but these methods have relatively complex steps, requiring at least three culture media to be used in sequence for multi-stage differentiation culture, and the time required to construct cardiac organoids is relatively long. For example, in patent CN118345031A, three culture media need to be used at a time for three stages of differentiation culture before cardiac organoids can be constructed from human induced pluripotent stem cells. The steps are relatively complicated, and the total culture time takes about 19.5 days. Before the three-stage differentiation culture, iPS cell balls need to be formed and their cell ball morphology needs to be maintained, which further complicates the organoid construction process and is difficult to operate. Summary of the invention

[0005] In order to solve the above technical problem, that is, the existing method for constructing cardiac organoids from human induced pluripotent stem cells has complicated steps, takes a long time, and is difficult to operate, the present invention provides a method for constructing cardiac organoids. In the method of the present invention, two culture media are used in sequence for two-stage differentiation culture, and cardiac organoids can be constructed from human induced pluripotent stem cells that are discrete after digestion, which simplifies the construction process of cardiac organoids, shortens the construction time, and reduces the difficulty of operation.

[0006] The specific technical scheme of the present invention is: A method for constructing a cardiac organoid comprises the following steps: S1: prepare medium I containing CD lipid, insulin-transferrin-selenium-aminoethanol (ITS-X), L-ascorbic acid-2-phosphate (L-AA2P), 1-thioglycerol (α-MTG), insulin, fibroblast growth factor 2 (FGF2) and glycogen synthase kinase-3β (GSK-3β) inhibitor; S2: prepare medium II containing CD lipids, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2, and vascular endothelial growth factor A (VEGFA); S3: After digestion, the human induced pluripotent stem cell clusters are suspended in culture medium I and then inoculated into ultra-low attachment cell culture plates with U-shaped well bottoms. After culturing for 36 to 40 hours, culture medium I is removed and culture medium II is added to culture until cardiac organoids are formed.

[0007] In the process of constructing organoids from human induced pluripotent stem cells, various components in the culture medium will have intricate effects on the growth and differentiation of cells. The present invention uses culture medium I and culture medium II containing specific components for culture in sequence, and replaces culture medium I with culture medium II at a specific time point (i.e., when culture medium I is used for 36 to 40 hours), so that heart organoids can be successfully constructed. Using the method of the present invention, only two culture media need to be used, and cultured for about 11.5 days (the first stage differentiation culture is about 36 hours, and the second stage differentiation culture is about 10 days), and heart organoids can be constructed from human induced pluripotent stem cells, which simplifies the construction steps of heart organoids and shortens the construction time; and in the method of the present invention, heart organoids can be constructed from human induced pluripotent stem cells that are discrete after digestion, and there is no need to pre-form cell spheres and maintain the morphology of cell spheres before using culture medium I for culture, which can simplify the construction process of heart organoids to a greater extent and reduce the difficulty of operation.

[0008] Preferably, step S1 specifically comprises: adding CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and glycogen synthase kinase-3β inhibitor to DMEM / F12 culture medium, mixing, and obtaining culture medium I.

[0009] Preferably, step S2 specifically comprises: adding CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and vascular endothelial growth factor A to DMEM / F12 culture medium, mixing, and obtaining culture medium II.

[0010] Preferably, in step S1, in the culture medium I, the concentrations of CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and glycogen synthase kinase-3β inhibitor are 0.5×~2×, 0.5×~2×, 100~200 ng / mL, 350~450 μM, 1~5 μg / mL, 10~30 ng / mL and 1~10 μM, respectively.

[0011] Preferably, in step S2, in the culture medium II, the concentrations of CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and vascular endothelial growth factor A are 0.5×~2×, 0.5×~2×, 100~200 ng / mL, 350~450 μM, 1~5 μg / mL, 10~30 ng / mL and 100~200 ng / mL, respectively.

[0012] The concentration of each component in medium I and medium II will affect the growth and differentiation of cells, and thus affect the formation of cardiac organoids. The present invention found that when the concentration of 1-thioglycerol in the two culture media is not within the range of 350-450 μM, the effect of cardiac organoid construction will be poor.

[0013] Preferably, in step S3, the human induced pluripotent stem cell cluster is digested with a stem cell digestion solution.

[0014] Preferably, in step S3, the method for preparing the human induced pluripotent stem cell cluster comprises the following steps: inoculating the human induced pluripotent stem cell cluster into a culture medium, and culturing the culture medium until the cell density reaches 73-78%.

[0015] Preferably, in step S3, the human induced pluripotent stem cell cluster suspended in medium I is cultured at a concentration of 0.5×10 4 ~1.0×10 4 Pieces / cm 2The cells were seeded at a density of 100 μg / mL into an ultra-low attachment cell culture plate with a U-shaped bottom well.

[0016] Preferably, in step S3, the culture time to form cardiac organoids is 240 to 280 hours.

[0017] Preferably, in step S3, during the process of culturing until cardiac organoids are formed, culture medium II is replaced every 45 to 50 hours.

[0018] Compared with the prior art, the present invention has the following advantages: (1) In the present invention, by adopting specific culture medium components and the timing of changing the culture medium, cardiac organoids can be constructed from human induced pluripotent stem cells that are discrete after digestion by using only two culture media in sequence for two-stage differentiation culture, thereby simplifying the construction process of cardiac organoids, shortening the construction time, and reducing the difficulty of operation.

[0019] (2) In the present invention, by controlling the concentration of each component in the two culture media, the components can be better coordinated with each other, so that the cells can grow and differentiate according to ideal conditions, thereby achieving a better cardiac organoid construction effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the process of constructing the cardiac organoid in Example 1. Wherein: Figure 1 A is an image of iPS cells just seeded into a cell culture plate; Figure 1 B is a microscope image of aggregates on the second day of the first stage of differentiation culture; Figure 2 C is a microscope image of aggregates at day 10 of the second stage differentiation culture.

[0021] Figure 2 It is a state of human induced pluripotent stem cells cultured in mTeSR Plus medium.

[0022] Figure 3 is the identification result of the cardiac organoid constructed in Example 1. Figure 3 A is the HE staining identification result; Figure 3 B is the result of immunofluorescence staining.

[0023] Figure 4 This is a microscope image of the aggregates at day 10 of the second stage differentiation culture in Example 2.

[0024] Figure 5 This is a microscope image of the aggregates at day 10 of the second stage differentiation culture in Example 3.

[0025] Figure 6 This is a microscope image of the aggregates in Comparative Example 1 at the 10th day of differentiation culture.

[0026] Figure 7 This is a microscope image of the aggregates in Comparative Example 2 at the 10th day of the second stage differentiation culture.

[0027] Figure 8 This is a microscope image of the aggregates in Comparative Example 3 at the 10th day of the second stage differentiation culture.

[0028] Fig. 9 This is a microscope image of the aggregates in Comparative Example 4 at the 10th day of the second stage differentiation culture.

[0029] Fig.10 This is a microscope image of the aggregates in Comparative Example 5 at the 10th day of the second stage differentiation culture.

[0030] Fig.11 This is a microscope image of the aggregates in Comparative Example 6 at the 10th day of the second stage differentiation culture. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with the embodiments.

[0032] A method for constructing a cardiac organoid comprises the following steps: S1: prepare medium I containing CD lipids, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2, and glycogen synthase kinase-3β inhibitor; S2: prepare medium II containing CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and vascular endothelial growth factor A; S3: After digestion, the human induced pluripotent stem cell clusters are suspended in culture medium I and then inoculated into ultra-low attachment cell culture plates with U-shaped well bottoms. After culturing for 36 to 40 hours, culture medium I is removed and culture medium II is added to culture until cardiac organoids are formed.

[0033] In some specific embodiments, step S1 specifically includes: using DMEM / F12 culture medium as a solvent, preparing culture medium I containing 0.5×~2×CD lipid, 0.5×~2×insulin-transferrin-selenium-aminoethanol, 100~200 ng / mL L-ascorbic acid-2-phosphate, 350~450 μM 1-thioglycerol, 1~5 μg / mL insulin, 10~30 ng / mL fibroblast growth factor 2 and 1~10 μM glycogen synthase kinase-3β inhibitor.

[0034] In some specific embodiments, step S2 specifically includes: using DMEM / F12 culture medium as a solvent, preparing a culture medium II containing 0.5×~2×CD lipid, 0.5×~2×insulin-transferrin-selenium-aminoethanol, 100~200 ng / mL L-ascorbic acid-2-phosphate, 350~450 μM 1-thioglycerol, 1~5 μg / mL insulin, 10~30 ng / mL fibroblast growth factor 2 and 100~200 ng / mL vascular endothelial growth factor A.

[0035] In some specific embodiments, in step S3, the method for preparing human induced pluripotent stem cell clusters comprises the following steps: inoculating the human induced pluripotent stem cell clusters into a culture medium, and culturing them until the cell density reaches 73-78%.

[0036] In some specific embodiments, in step S3, the human induced pluripotent stem cell cluster is digested with a stem cell digestion solution.

[0037] In some embodiments, in step S3, the human induced pluripotent stem cell cluster suspended in medium I is cultured at a rate of 0.5×10 4 ~1.0×10 4 Pieces / cm 2 The cells were seeded at a density of 100 μg / mL into an ultra-low attachment cell culture plate with a U-shaped bottom well.

[0038] In some specific embodiments, in step S3, the time for culturing until cardiac organoids are formed is 240 to 280 hours.

[0039] In some specific embodiments, in step S3, during the process of culturing to form cardiac organoids, culture medium II is replaced every 45 to 50 hours.

[0040] The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0041] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0042] The sources of materials (brands and product numbers) used in the embodiments and comparative examples of the present invention are provided below, but the protection scope of the present invention is not limited thereto: DMEM / F12 culture medium (Cat. No.: BL305A, Biosharp, China) CD lipid (Cat. No.: 11905031, Gibco, USA) Insulin-transferrin-selenium-aminoethanol (ITS-X) (Cat. No.: C0345, Bio-Tech, China) L-Ascorbic acid-2-phosphate (L-AA2P) (Cat. No. A8960, Sigma-Aldrich, USA) 1-Methylthioglycerol (α-MTG) (Cat. No.: M6145, Sigma-Aldrich, USA) Insulin (Cat. No.: I8830, Solebao, China) Fibroblast Growth Factor 2 (FGF2) (Cat. No. 10014-HNAE, Sino Biological, China) Glycogen synthase kinase-3β (GSK-3β) inhibitor CHIR99021: (Cat. No. 53003ES10, Yisheng, China) Vascular endothelial growth factor A (VEGFA) (Cat. No. 11066-HNAH, Sino Biological, China) Human induced pluripotent stem cells (iPS) (Cat. No.: YongFu001, Rongchuang, China) Stem cell digestion solution (Cat. No.: CA1023100, Saibei, China) NSC-27 serum-free supplement (Cat. No. 60703ES10, Yisheng, China) N-Acetyl-L-cysteine ​​(NAC) (Cat. No. 50303ES05, ​​Yisheng, China) Recombinant human interleukin-5 (IL-5) (Cat. No. 90106ES60, Yisheng, China) Insulin-transferrin-selenium (ITS-G) (Cat. No. 2090250, Gibco, USA) mTeSR Plus medium: (Cat. No. 100-0276, Stemcell, Canada).

[0043] Example 1 The steps of constructing cardiac organoids in this example are as follows: Step 1: Prepare medium I CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and GSK-3β inhibitor CHIR99021 were added to DMEM / F12 medium, mixed evenly, and medium I was obtained for later use. The concentrations of the components in medium I are shown in Table 1.

[0044] Table 1 Formulation of medium I in Example 1 Element concentration DMEM / F12 medium Solvents CD lipid 2× ITS-X 2× L-AA2P 100ng / mL α-MTG 400μM insulin 5 μg / mL FGF2 30 ng / mL CHIR99021 10μM Step 2: Prepare medium II CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and VEGFA were added to DMEM / F12 medium, mixed evenly, and medium II was obtained for use. The concentrations of the components in medium II are shown in Table 2.

[0045] Table 2 Formulation of medium II in Example 1 Element concentration DMEM / F12 medium Solvents CD lipid 0.5× ITS-X 0.5× L-AA2P 100ng / mL α-MTG 400μM insulin 5 μg / mL FGF2 30 ng / mL VEGFA 200 μg / mL Step 3: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. The growth of human induced pluripotent stem cells was as follows: Figure 2 When the cell density of human induced pluripotent stem cells reaches 73%, they are digested with stem cell digestion solution, and the digestion is terminated to obtain digested iPS cells.

[0046] Step 4: First stage differentiation culture Resuspend the iPS cells digested in step 3 in medium I and then culture at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped bottom. Figure 1 A. Aggregates were observed on the second day of culture in this cell culture plate (see Figure 1 B), at 36h of culture, the first stage of differentiation culture was completed.

[0047] Step 5: Second stage differentiation culture After the first stage of differentiation culture is completed, medium I in the cell culture plate is removed and medium II is added. Culture is continued, and medium II is replaced every 2 days. From the time medium I is replaced with medium II, cardiac organoids are obtained on the 10th day of culture, and their morphology is as follows Figure 1 As shown in C, the aggregates were observed to be beating. The obtained cardiac organoids were identified by pathological staining and immunofluorescence staining. Figure 3 ( Figure 3In the figure, red fluorescence indicates endothelial cells, and blue fluorescence indicates cell nuclei. Since it is impossible to distinguish red fluorescence from blue fluorescence in the colorless figure, Figure 3 Color images were used), indicating that the cardiac organoids obtained in this example conform to the physiological structure of a normal cardiac organ.

[0048] Example 2 The steps of constructing cardiac organoids in this example are as follows: Step 1: Prepare medium I CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and GSK-3β inhibitor CHIR99021 were added to DMEM / F12 medium, mixed evenly, and medium I was obtained for later use. The concentrations of the components in medium I are shown in Table 3.

[0049] Table 3 Formulation of medium I in Example 2 Element concentration DMEM / F12 medium Solvents CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL CHIR99021 4μM Step 2: Prepare medium II CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and VEGFA were added to DMEM / F12 medium, mixed evenly, and medium II was obtained for use. The concentrations of the components in medium II are shown in Table 4.

[0050] Table 4 Formulation of Medium II in Example 2 Element concentration DMEM / F12 medium Solvents CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL VEGFA 100 μg / mL Step 3: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. When the cell density of human induced pluripotent stem cells reached 75%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0051] Step 4: First stage differentiation culture Resuspend the iPS cells digested in step 3 in medium I and then culture at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of differentiation culture was completed on the 36th hour of culture.

[0052] Step 5: Second stage differentiation culture After the first stage of differentiation culture is completed, medium I in the cell culture plate is removed and medium II is added. Culture is continued, and medium II is replaced every 2 days. From the time medium I is replaced with medium II, cardiac organoids are obtained on the 10th day of culture, and their morphology is as follows Figure 4 As shown, at this time, the aggregates were observed to be beating.

[0053] Example 3 The steps of constructing cardiac organoids in this example are as follows: Step 1: Prepare medium I CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and GSK-3β inhibitor CHIR99021 were added to DMEM / F12 medium, mixed evenly, and medium I was obtained for later use. The concentrations of the components in medium I are shown in Table 5.

[0054] Table 5 Formulation of medium I in Example 3 Element concentration DMEM / F12 medium Solvents CD lipid 0.5× ITS-X 0.5× L-AA2P 150 ng / mL α-MTG 350μM insulin 3 μg / mL FGF2 10 ng / mL CHIR99021 1μM Step 2: Prepare medium II CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and VEGFA were added to DMEM / F12 medium, mixed evenly, and medium II was obtained for use. The concentrations of the components in medium II are shown in Table 6.

[0055] Table 6 Formulation of Medium II in Example 3 Element concentration DMEM / F12 medium Solvents CD lipid 2× ITS-X 2× L-AA2P 150 ng / mL α-MTG 350μM insulin 3 μg / mL FGF2 10 ng / mL VEGFA 150 μg / mL Step 3: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. When the cell density of human induced pluripotent stem cells reached 78%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0056] Step 4: First stage differentiation culture Resuspend the iPS cells digested in step 3 in medium I and then culture at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of differentiation culture was completed on the 40th hour of culture.

[0057] Step 5: Second stage differentiation culture After the first stage of differentiation culture is completed, medium I in the cell culture plate is removed and medium II is added. Culture is continued, and medium II is replaced every 2 days. From the time medium I is replaced with medium II, cardiac organoids are obtained on the 10th day of culture, and their morphology is as follows Figure 5 As shown, at this time, the aggregates were observed to be beating.

[0058] Comparative Example 1 The difference between this comparative example and Example 2 is that: in Comparative Example 1, CHIR99021 and VEGFA are added to the same culture medium, and a one-stage differentiation culture method is adopted; the remaining materials and steps are the same as those in Example 2. Specifically, the steps for constructing cardiac organoids in this comparative example are as follows: Step 1: Prepare differentiation medium CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2, GSK-3β inhibitor CHIR99021 and VEGFA were added to DMEM / F12 medium, mixed evenly, and differentiation medium was obtained for use. The concentrations of each component in the differentiation medium are shown in Table 7.

[0059] Table 7 Formula of differentiation medium in comparative example 1 Element concentration DMEM / F12 medium Solvents CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL CHIR99021 4μM VEGFA 100 μg / mL Step 2: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. When the cell density of human induced pluripotent stem cells reached 75%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0060] Step 3: Differentiation culture Resuspend the iPS cells digested in step 3 into differentiation medium and then add them to the culture medium at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped bottom, and the differentiation medium was changed every 2 days. Aggregates were observed on the 10th day of culture, starting from when medium I was replaced with medium II. The morphology was as follows Figure 6 As shown. Figure 6 It can be seen that the aggregates formed in this comparative example are irregular in shape and uneven in size, which is not conducive to the formation of cardiac organoids.

[0061] Comparative Example 2 The difference between this comparative example and Example 2 is that in Comparative Example 2, the timing of replacing medium I with medium II is changed; the remaining materials and steps are the same as those in Example 2. Specifically, the steps of constructing cardiac organoids in this comparative example are as follows: Step 1: Prepare medium I CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and GSK-3β inhibitor CHIR99021 were added to DMEM / F12 medium, mixed evenly, and medium I was obtained for use. The concentrations of the components in medium I are shown in Table 3 above.

[0062] Step 2: Prepare medium II CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and VEGFA were added to DMEM / F12 medium, mixed evenly, and medium II was obtained for use. The concentrations of the components in medium II are shown in Table 4 above.

[0063] Step 3: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. When the cell density of human induced pluripotent stem cells reached 75%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0064] Step 4: First stage differentiation culture Resuspend the iPS cells digested in step 3 in medium I and then culture at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of differentiation culture was completed on the 96th hour of culture.

[0065] Step 5: Second stage differentiation culture After the first stage of differentiation culture, medium I in the cell culture plate was removed and medium II was added. Culture was continued, and medium II was replaced every 2 days. From the time medium I was replaced with medium II, the morphology of the aggregates was as follows on the 10th day of culture: Figure 7 As shown. Figure 7 It can be seen that most of the cells detached from the aggregates and died, which resulted in the inability to undergo subsequent differentiation and the failure of cardiac organoid construction.

[0066] Comparative Example 3 The only difference between this comparative example and Example 2 is that in Comparative Example 3, VEGFA in culture medium II is replaced with NSC-27 serum-free additive; the remaining materials and steps are the same as those in Example 2. Specifically, the steps for constructing cardiac organoids in this comparative example are as follows: Step 1: Prepare medium I CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and GSK-3β inhibitor CHIR99021 were added to DMEM / F12 medium, mixed evenly, and medium I was obtained for use. The concentrations of the components in medium I are shown in Table 3 above.

[0067] Step 2: Prepare medium II CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and NSC-27 serum-free additive were added to DMEM / F12 medium, mixed evenly, and medium II was obtained for standby use. The concentrations of the components in medium II are shown in Table 8.

[0068] Table 8 Formula of medium II in comparative example 3 Element concentration DMEM / F12 medium Solvents CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL NSC-27 Serum-Free Supplement 100 μg / mL Step 3: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. When the cell density of human induced pluripotent stem cells reached 75%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0069] Step 4: First stage differentiation culture Resuspend the iPS cells digested in step 3 in medium I and then culture at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of differentiation culture was completed on the 36th hour of culture.

[0070] Step 5: Second stage differentiation culture After the first stage of differentiation culture, medium I in the cell culture plate was removed and medium II was added. Culture was continued, and medium II was replaced every 2 days. From the time medium I was replaced with medium II, the morphology of the aggregates was as follows on the 10th day of culture: Figure 8 As shown. Figure 8 It can be seen that some cells fell off the aggregates and the construction of cardiac organoids failed.

[0071] Comparative Example 4 The only difference between this comparative example and Example 2 is that in Comparative Example 4, VEGFA in culture medium II is replaced with NAC; the remaining materials and steps are the same as those in Example 2. Specifically, the steps for constructing cardiac organoids in this comparative example are as follows: Step 1: Prepare medium I CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and GSK-3β inhibitor CHIR99021 were added to DMEM / F12 medium, mixed evenly, and medium I was obtained for use. The concentrations of the components in medium I are shown in Table 3 above.

[0072] Step 2: Prepare medium II CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and NAC were added to DMEM / F12 medium, mixed evenly, and medium II was obtained for use. The concentrations of the components in medium II are shown in Table 9.

[0073] Table 9 Formula of medium II in comparative example 4 Element concentration DMEM / F12 medium Solvents CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL NAC 100 μg / mL Step 3: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. When the cell density of human induced pluripotent stem cells reached 75%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0074] Step 4: First stage differentiation culture Resuspend the iPS cells digested in step 3 in medium I and then culture at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of differentiation culture was completed on the 36th hour of culture.

[0075] Step 5: Second stage differentiation culture After the first stage of differentiation culture, medium I in the cell culture plate was removed and medium II was added. Culture was continued, and medium II was replaced every 2 days. From the time medium I was replaced with medium II, the morphology of the aggregates was as follows on the 10th day of culture: Fig. 9 As shown. Fig. 9 It can be seen that the aggregated cells fell off, the edges were unclear, and the volume became smaller, resulting in the failure of cardiac organoid construction.

[0076] Comparative Example 5 The only difference between this comparative example and Example 2 is that in Comparative Example 5, VEGFA in culture medium II is replaced with IL-5; the remaining materials and steps are the same as those in Example 2. Specifically, the steps for constructing cardiac organoids in this comparative example are as follows: Step 1: Prepare medium I CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and GSK-3β inhibitor CHIR99021 were added to DMEM / F12 medium, mixed evenly, and medium I was obtained for use. The concentrations of the components in medium I are shown in Table 3 above.

[0077] Step 2: Prepare medium II CD lipid, ITS-X, L-AA2P, α-MTG, insulin, FGF2 and IL-5 were added to DMEM / F12 medium, mixed evenly, and medium II was obtained for standby use. The concentrations of the components in medium II are shown in Table 10.

[0078] Table 10 Formula of medium II in comparative example 5 Element concentration DMEM / F12 medium Solvents CD lipid 1× ITS-X 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL IL-5 100 μg / mL Step 3: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. When the cell density of human induced pluripotent stem cells reached 75%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0079] Step 4: First stage differentiation culture Resuspend the iPS cells digested in step 3 in medium I and then culture at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of differentiation culture was completed on the 36th hour of culture.

[0080] Step 5: Second stage differentiation culture After the first stage of differentiation culture, medium I in the cell culture plate was removed and medium II was added. Culture was continued, and medium II was replaced every 2 days. From the time medium I was replaced with medium II, the morphology of the aggregates was as follows on the 10th day of culture: Fig.10 As shown. Fig.10It can be seen that the aggregates are adhered and dead cells fall off, resulting in the failure of cardiac organoid construction.

[0081] Comparative Example 6 The difference between this comparative example and Example 2 is that in Comparative Example 6, ITS-X in Medium I and Medium II is replaced with ITS-G; the remaining materials and steps are the same as in Example 2. Specifically, the steps for constructing cardiac organoids in this comparative example are as follows: Step 1: Prepare Medium I CD lipid, ITS-G, L-AA2P, α-MTG, insulin, FGF2 and GSK-3β inhibitor CHIR99021 were added to DMEM / F12 medium, mixed evenly, and medium I was obtained for later use. The concentrations of the components in medium I are shown in Table 11.

[0082] Table 11 Formula of medium I in comparative example 6 Element concentration DMEM / F12 medium Solvents CD lipid 1× ITS-G 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL CHIR99021 4μM Step 2: Prepare medium II CD lipid, ITS-G, L-AA2P, α-MTG, insulin, FGF2 and VEGFA were added to DMEM / F12 medium, mixed evenly, and medium II was obtained for use. The concentrations of the components in medium II are shown in Table 12.

[0083] Table 12 Formulation of medium II in comparative example 6 Element concentration DMEM / F12 medium Solvents CD lipid 1× ITS-G 1× L-AA2P 200ng / mL α-MTG 450μM insulin 1 μg / mL FGF2 15 ng / mL VEGFA 100 μg / mL Step 3: Cultivating iPS cells Human induced pluripotent stem cells were inoculated into mTeSR Plus medium and placed in an incubator. The temperature in the incubator was controlled at 37°C and the CO2 concentration was 5%. The medium was changed every day. When the cell density of human induced pluripotent stem cells reached 75%, they were digested with stem cell digestion solution, and the digestion was terminated to obtain digested iPS cells.

[0084] Step 4: First stage differentiation culture Resuspend the iPS cells digested in step 3 in medium I and then culture at a cell density of 1.0×10 4 Pieces / cm 2 The cells were inoculated into a 96-well ultra-low attachment cell culture plate with a U-shaped well bottom. Aggregates were observed on the second day of culture in the cell culture plate. The first stage of differentiation culture was completed on the 36th hour of culture.

[0085] Step 5: Second stage differentiation culture After the first stage of differentiation culture, medium I in the cell culture plate was removed and medium II was added. Culture was continued, and medium II was replaced every 2 days. From the time medium I was replaced with medium II, the morphology of the aggregates was as follows on the 10th day of culture: Fig.11 As shown. Fig.11 It can be seen that black plaques appear in the aggregates and a large number of dead cells fall off, resulting in the failure of cardiac organoid construction.

[0086] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for constructing a cardiac organoid, characterized in that: The following steps are involved: S1: prepare medium I containing CD lipids, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2, and glycogen synthase kinase-3β inhibitor; S2: prepare medium II containing CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and vascular endothelial growth factor A; S3: After digestion, the human induced pluripotent stem cell clusters were suspended in culture medium I and then inoculated into ultra-low attachment cell culture plates with U-shaped well bottoms. After culturing for 36-40 h, culture medium I was removed and culture medium II was added to culture until cardiac organoids were formed.

2. The method for constructing a cardiac organoid according to claim 1, characterized in that: Step S1 specifically includes: adding CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and glycogen synthase kinase-3β inhibitor to DMEM / F12 culture medium, mixing, and obtaining culture medium I.

3. The method for constructing a cardiac organoid according to claim 1, characterized in that: Step S2 specifically includes: adding CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and vascular endothelial growth factor A to DMEM / F12 culture medium, mixing, and obtaining culture medium II.

4. The method for constructing a cardiac organoid according to claim 1 or 2, characterized in that: In step S1, in the culture medium I, the concentrations of CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and glycogen synthase kinase-3β inhibitor are 0.5×~2×, 0.5×~2×, 100~200 ng / mL, 350~450 μM, 1~5 μg / mL, 10~30 ng / mL and 1~10 μM, respectively.

5. The method for constructing a cardiac organoid according to claim 1 or 3, characterized in that: In step S2, in the culture medium II, the concentrations of CD lipid, insulin-transferrin-selenium-aminoethanol, L-ascorbic acid-2-phosphate, 1-thioglycerol, insulin, fibroblast growth factor 2 and vascular endothelial growth factor A are 0.5×~2×, 0.5×~2×, 100~200 ng / mL, 350~450 μM, 1~5 μg / mL, 10~30 ng / mL and 100~200 ng / mL, respectively.

6. The method for constructing a cardiac organoid according to claim 1, characterized in that: In step S3, the human induced pluripotent stem cell cluster is digested with a stem cell digestion solution.

7. The method for constructing a cardiac organoid according to claim 1, characterized in that: In step S3, the method for preparing the human induced pluripotent stem cell cluster comprises the following steps: inoculating the human induced pluripotent stem cell cluster into a culture medium and culturing until the cell density reaches 73-78%.

8. The method for constructing a cardiac organoid according to claim 1, characterized in that: In step S3, human induced pluripotent stem cell clusters suspended in medium I were cultured at a rate of 0.5×10 4 ~1.0×10 4 Pieces / cm 2 The cells were seeded at a density of 100 μg / mL into an ultra-low attachment cell culture plate with a U-shaped bottom well.

9. The method for constructing a cardiac organoid according to claim 1, characterized in that: In step S3, the time for culturing until cardiac organoids are formed is 240 to 280 hours.

10. The method for constructing a cardiac organoid according to claim 1, characterized in that: In step S3, during the process of culturing until cardiac organoids are formed, culture medium II is replaced every 45 to 50 hours.

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