A composition for rapidly high-throughput culturing of atrial organoids and a culturing method and application thereof

Abstract

This application discloses a composition for rapid, high-throughput culture of atrial organoids, its culture method, and its applications, belonging to the field of cell biology technology. Specifically, it discloses a culture medium for atrial organoids, a culture medium assembly for atrial organoids, and a method for preparing atrial organoids. This method replaces IWP-4 in the culture medium with IWP-2 during Day-2 of culture, enabling the culture of large quantities of atrial and cardiac muscle organoids within 9-11 days without the need for coating the culture plates with Matrigel. This method combines the advantages of low cost and high efficiency, shortens the time required, simplifies experimental procedures, and greatly improves its applicability.

Description

Technical Field

[0001] This invention patent belongs to the field of cell biology technology, specifically relating to a composition for rapid high-throughput culture of atrial organoids, its culture method, and its application. Background Technology

[0002] Heart organoids are three-dimensional cardiac micro-tissues formed by in vitro induced differentiation of human pluripotent stem cells. They can mimic the structure, electrophysiological function, and cell interactions of the human heart. In recent years, they have developed rapidly due to breakthroughs in stem cell technology and bioengineering.

[0003] Traditional cardiac organoid culture procedures are complex (requiring initial two-dimensional culture followed by digestion into single cells for three-dimensional culture or culture in individual low-absorption U-shaped plates; for example, CN202510139407 requires matrix gel embedding and six culture medium additives; CN202411574358 requires a low-absorption 96-well U-shaped plate and eight culture medium additives; CN202410237953 requires 16 culture medium additives), time-consuming (approximately 15 days, as in CN202410237953), and currently available reagent kits are expensive (e.g., the Acro cardiac organoid kit is expensive and can only differentiate 26 organoids, resulting in low cost-effectiveness), making it unsuitable for large-scale promotion. This method eliminates the need for matrix gel embedding or low-absorption U-shaped plate formation, requires fewer reagents, and is simpler to operate, making it easier to implement and promote. Summary of the Invention

[0004] In view of the above-mentioned shortcomings of at least one of the prior art, the purpose of this application is to provide a composition for rapid high-throughput culture of atrial organoids, a culture method thereof, and its application. This application replaces IWP-4 in the culture medium during Day-2 with IWP-2, enabling the culture of large quantities of atrial myocardial organoids to be cultured within 9-11 days without the need for coating the cultured stem cells with Matrigel during Day-2 culture, thus combining the advantages of low cost and high efficiency.

[0005] In a first aspect, this application provides a first culture medium, said culture medium comprising IWP2.

[0006] Secondly, this application provides a culture medium group, which includes the first culture medium described above, and also includes a second culture medium, the second culture medium including mTeSR1 culture medium and Y27632; in a specific embodiment, the content of Y27632 is 5-15 μM, and in a specific embodiment, the content of Y27632 is 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, or 15 μM.

[0007] Thirdly, this application provides a dry powder culture medium prepared from the first culture medium described above or any of the culture medium groups described above.

[0008] Fourthly, this application provides a method for preparing a heart organoid, the method comprising culturing stem cells using the first culture medium described above or any of the culture medium groups described above.

[0009] Fifthly, this application provides the use of IWP-2, the first culture medium described above, or any of the culture medium groups described above in the preparation of cardiac organoids.

[0010] The beneficial effects of this application are as follows:

[0011] 1. The preparation method of this application can achieve the culture of a large number of atrial myocardial organoids in 9-11 days, that is, about 20+ organoids can be harvested per well of a 6-well plate, which has the advantages of low cost and high efficiency.

[0012] 2. Compared with the traditional method of differentiating cardiac organoids (i.e., seeding stem cells, maintaining two-dimensional culture and differentiating until day 15 when digesting into single cells, and seeding into 96-well low-adsorption U-shaped plates, which requires another 6-8 days to form a three-dimensional structure), the preparation method of this application shortens the time and simplifies the experimental operation, greatly improving its accessibility.

[0013] 3. Cells prepared using the above method were verified by immunofluorescence staining to have a high expression rate of atrial cardiomyocytes (MYL7) and specific cardiac tissue structures, including an endothelial cell (CD31) layer and a cardiomyocyte (cTnT) layer. Attached Figure Description

[0014] Figure 1 This is an organoid diagram of the atrium.

[0015] Figure 2 Immunofluorescence images of atrial marker (MYL7) and cell nucleus (DAPI).

[0016] Figure 3 Immunofluorescence images of cardiomyocyte marker (cTnT) and endothelial cell marker (CD31).

[0017] Figure 4 A diagram of stem cell colonies that meet the passage standard (thickening in the center of the colony).

[0018] Figure 5 To generate a colony map of spontaneously differentiated stem cells to be eliminated, marked with a marker.

[0019] Figure 6 This is a diagram of a pseudoembryosome.

[0020] Figure 7This is a record of the culture process of cardiomyocytes and organoids. Figure 7 In the image, A represents two-dimensional cardiomyocytes cultured on the coated surface. Figure 7 In the image, B represents a three-dimensional cardiac organoid formed on an uncoated surface.

[0021] Figure 8 A diagram documenting the three-dimensional culture process of cardiac organoids.

[0022] Figure 9 This is an image of a mature heart organoid.

[0023] Figure 10 This is a record of the state of cells that failed to differentiate on Day 2 in Comparative Example 1. Detailed Implementation

[0024] The specific embodiments of this application will be further described in detail below with reference to the accompanying drawings. These embodiments are only for illustrating this application and are not intended to limit the scope of the invention.

[0025] In the description of this invention, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0026] In existing technologies, traditional cardiac organoid culture procedures are complex (requiring two-dimensional culture to mature, digestion into single cells, and then three-dimensional culture or culture in a single low-absorption U-shaped plate, such as CN202510139407 which requires matrix gel coating and 6 kinds of culture medium additives; CN202411574358 which requires low-absorption 96-well U-shaped plates and 8 kinds of culture medium additives; CN202410237953 which requires 16 kinds of culture medium additives), time-consuming (requiring about 15 days, such as CN202410237953), and the existing finished kits are expensive (such as the Acro cardiac organoid kit, which is expensive and can only differentiate 26 organoids, resulting in low cost-effectiveness), making it unsuitable for large-scale promotion.

[0027] The inventors accidentally discovered in experiments that, in the early stages of stem cell culture (Day-2), it was not necessary to use matrigel to coat the culture plate for culturing stem cells, and the stem cells could still be cultured into spherical embryoids.

[0028] This application provides a method for preparing an embryoid body, the method comprising:

[0029] Step (1): Expose the stem cells to the second culture medium as described in claim 3 for culture;

[0030] Step (2): After completing step (1), expose the culture to the third culture medium of claim 3 for culture to obtain spherical embryoids.

[0031] The culture environment in step (1) is 37°C, 5% CO2, and the culture time is 12-36h, preferably 24h;

[0032] In a specific embodiment, the culture environment in step (2) is 37°C, 5% CO2, and the culture time is 12-36h, preferably 24h.

[0033] This process significantly reduces culture costs because it eliminates the need for matrigel, and the time required to form embryoids is identical to that of conventional techniques.

[0034] The spherical embryoids were further cultured in a fourth culture medium to obtain cardiac progenitor cells or cardiac progenitor cell populations. The culture environment was 37°C, 5% CO2, and the culture time was 36-60 hours; in a specific embodiment, the culture time was 48 hours.

[0035] Studies have found that when the above-cultured cardiac progenitor cell population is further cultured in conventional culture medium (1640, B27 without insulin, 5uM IWP4 medium), differentiation stops. Through extensive screening and optimization of various substances in the culture medium (1640, B27 without insulin, IWP4 medium), the inventors discovered that if IWP4 in the culture medium is replaced with IWP2, the cardiac progenitor cell population that has stopped differentiating can continue to grow and differentiate.

[0036] This application provides culture media for cardiac cells, tissues, and / or organoids, including IWP2.

[0037] In a specific embodiment, the culture medium further includes B27; in a specific embodiment, the B27 does not contain insulin, and preferably, the B27 content is 1x concentration;

[0038] In a specific embodiment, the culture medium further includes a basal culture medium, specifically a 1640 medium, and the concentration of the basal culture medium is 1:1. In a specific embodiment, the basal culture medium contains glutamine and HEPES. In a specific embodiment, the basal culture medium does not contain pyruvate. In a specific embodiment, the 1640 medium is purchased from Gibco lot #22400-089. In a specific embodiment, the glutamine is L-glutamine.

[0039] The aforementioned culture medium can culture cardiac progenitor cells into cardiac progenitor cells. IWP2, as a Wnt signaling pathway inhibitor, efficiently induces cardiac progenitor cell formation. It can induce uncoated cardiac progenitor cells to develop into cardiac progenitor cells, and these cells are able to develop into cardiac progenitor cells. Other types of Wnt signaling pathway inhibitors (such as IWP4) cannot induce uncoated cardiac progenitor cells to develop into cardiac progenitor cells. Therefore, the selection of IWP2 has achieved an unexpected technical effect.

[0040] It should be understood that the 1640 medium component in the culture medium can be replaced with DMEM / F-12 basal medium, DMEM / F-12, RPMI 1640DMEM basal medium, mTeSR1 medium, etc., according to the culture requirements, or one or more components of 1640 medium, DMEM / F-12 basal medium, DMEM / F-12, RPMI 1640DMEM basal medium, mTeSR1 medium can be replaced, as long as the expected culture effect can be achieved, it is within the scope of this application.

[0041] It should be understood that the mTeSR1 medium component in the culture medium 2 can be replaced with DMEM / F-12 basal medium, DMEM / F-12, RPMI 1640DMEM basal medium, 1640 medium, etc., according to the culture requirements, or one or more components of mTeSR1 medium, DMEM / F-12 basal medium, DMEM / F-12, RPMI 1640DMEM basal medium, 1640 medium can be replaced, as long as the expected culture effect can be achieved, it is within the scope of this application.

[0042] In a specific embodiment, the IWP2 content is 3-7 μM, and in a specific embodiment, the IWP2 content is 3 μM, 4 μM, 5 μM, 6 μM, or 7 μM.

[0043] Based on this, the use of the above-mentioned cardiac cell, tissue and / or organoid culture media can overcome the problem of Day-2 cardiac progenitor cell differentiation arrest caused by the lack of use of matrigel to coat the culture plates of cultured stem cells.

[0044] Furthermore, this application provides a culture medium group, which includes a first culture medium, the first culture medium being the culture medium according to claim 1, and a second culture medium, the second culture medium including a basal culture medium and / or Y27632. In a specific embodiment, the basal culture medium is mTeSR1 culture medium. In a specific embodiment, the content of Y27632 is 5-15 μM; in a specific embodiment, the content of mTeSR1 culture medium is 1x concentration, and in a specific embodiment, the content of Y27632 is 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, or 15 μM.

[0045] In a specific embodiment, the culture medium group further includes a third culture medium, which includes a basal culture medium. In a specific embodiment, the basal culture medium is mTeSR1 culture medium.

[0046] In a specific embodiment, the culture medium group further includes a fourth culture medium, which includes B27; in a specific embodiment, the B27 does not contain insulin, and in a specific embodiment, the B27 content is 1x concentration;

[0047] In a specific embodiment, the culture medium group further includes a fourth culture medium, which comprises basal culture medium, B27, CHIR99021, and / or matrigel; in a specific embodiment, the B27 does not contain insulin, and the B27 content is 1x concentration; in a specific embodiment, the CHIR99021 content is 5-15 μM; in a specific embodiment, the basal culture medium is 1640 culture medium, and the basal culture medium content is 1x concentration; in a specific embodiment, the basal culture medium contains glutamine and HEPES; in a specific embodiment, the basal culture medium does not contain pyruvate; in a specific embodiment, the 1640 culture medium is purchased from Gibco lot #22400-089; in a specific embodiment, the matrigel content is 0.5-1.5% based on the total volume of the fourth culture medium. In a specific embodiment, the matrigel content is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, and 1.5%. In a specific embodiment, the volume percentage of the matrigel is 1%. In a specific embodiment, the matrigel is purchased from Corning lot #354277; in a specific embodiment, the content of CHIR99021 is 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, and 15 μM.

[0048] In a specific embodiment, the culture medium group further includes a fifth culture medium, which includes a basal culture medium and / or B27; in a specific embodiment, the B27 does not contain insulin; in a specific embodiment, the B27 content is 1x concentration; in a specific embodiment, the basal culture medium is 1640 culture medium; in a specific embodiment, the basal culture medium content is 1x concentration; in a specific embodiment, the basal culture medium contains glutamine or HEPES; in a specific embodiment, the basal culture medium does not contain pyruvate; in a specific embodiment, the 1640 culture medium is purchased from Gibco lot #22400-089.

[0049] In a specific embodiment, the culture medium group further includes a sixth culture medium, which includes a basal culture medium and / or B27; in a specific embodiment, the B27 content is 1x concentration; in a specific embodiment, the basal culture medium is 1640 culture medium; in a specific embodiment, the basal culture medium content is 1x concentration; in a specific embodiment, the basal culture medium contains L-glutamine or HEPES; in a specific embodiment, the basal culture medium does not contain pyruvate; in a specific embodiment, the 1640 culture medium is purchased from Gibco lot #22400-089.

[0050] In a specific embodiment, the composition and content of the 1640 culture medium are shown in Table 1.

[0051] Using the above-mentioned culture medium can provide the material basis for culturing stem cells into heart organoids, and it eliminates the need to use Matrigel to coat the culture plates of stem cells in the early stage of stem cell culture (Day-2), which greatly saves costs and improves efficiency.

[0052] This application provides dry powder culture media prepared from the above-described culture media or any of the culture media groups described above.

[0053] In a specific embodiment, the moisture content of the dry powder culture medium can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, ..., 50%.

[0054] This application provides a method for preparing cardiac cells, tissues, and / or organoids, the method comprising the step of culturing stem cells with the above-described culture medium and / or the above-described culture medium group.

[0055] It should be understood that the stem cells include cell line DYR0100, globular embryoids, cardiac progenitor cells, and cardiac progenitor cells. The aforementioned globular embryoids develop under conditions without matrigel. The aforementioned cardiac progenitor cells and cardiac progenitor cells develop under conditions without matrigel during the globular embryoid stage.

[0056] In a specific embodiment, the method includes:

[0057] (1) Expose stem cells to the second culture medium described above for culture;

[0058] (2) After completing step (1), the culture is exposed to the third culture medium mentioned above for culture to obtain globular embryoids;

[0059] (3) Expose the spherical embryoids to the fourth culture medium described above for culture;

[0060] (4) After completing step (3), expose the culture to the first culture medium described above for further culture;

[0061] (5) After completing step (4), expose the culture to the fifth culture medium described above for further culture;

[0062] (6) After completing step (5), expose the culture to the sixth culture medium mentioned above for further culture.

[0063] In a specific embodiment, the culture environment in step (1) is 37°C, 5% CO2, and the culture time is 24h;

[0064] In a specific embodiment, the culture environment in step (2) is 37°C, 5% CO2, and the culture time is 24h;

[0065] In a specific embodiment, the culture environment in step (3) is 37°C, 5% CO2, and the culture time is 48h;

[0066] In a specific embodiment, the culture environment in step (4) is 37°C, 5% CO2, and the culture time is 48h;

[0067] In a specific embodiment, the culture environment in step (5) is 37°C, 5% CO2, and the culture time is 48h;

[0068] In a specific embodiment, the culture environment in step (6) is 37°C, 5% CO2, and the culture time is 48h.

[0069] In a specific embodiment, the cardiac cells, tissues and / or organoids or cultures develop atrial myocardial organoids on days 9-11 of culture.

[0070] The above-described preparation method can achieve the culture of a large number of atrial cardiomyocyte organoids within 9-11 days, that is, approximately 20+ organoids can be harvested per well in a 6-well plate, combining the advantages of low cost and high efficiency. Compared with the traditional cardiac organoid differentiation method (i.e., seeding stem cells, maintaining two-dimensional culture until day 15 when digested into single cells, and then seeding into 96-well low-absorption U-shaped plates for another 6-8 days to form a three-dimensional structure), the preparation method of this application shortens the time and simplifies the experimental operation, greatly improving its applicability. Cells prepared using the above method have been verified by immunofluorescence staining to have a high expression rate of atrial cardiomyocytes (MYL7) and specific cardiac tissue structures, including an endothelial cell (CD31) layer and a cardiomyocyte (cTnT) layer.

[0071] This application provides the use of IWP-2, the first culture medium described above, or any of the culture medium groups described above in the preparation of cardiac organoids, cardiac tissues, or cardiac cells.

[0072] It should be understood that the first culture medium is suitable for culturing cardiac progenitor cells or cell populations that have not undergone the process of development from globular embryoids, which develop under matrigel-free conditions. The stem cells include the cell line DYR0100, globular embryoids, cardiac progenitor cells, and cardiac progenitor cells. The aforementioned globular embryoids develop under matrigel-free conditions. The aforementioned cardiac progenitor cells and cardiac progenitor cells develop under matrigel-free conditions during the globular embryoid stage.

[0073] In a specific embodiment, the cardiac organoid has MYL7 cardiomyocyte markers, cTnT cardiomyocyte markers, and / or CD31 endothelial cell markers.

[0074] In a specific embodiment, the use includes culturing stem cells.

[0075] In a specific embodiment, the stem cell is the stem cell line DYR0100.

[0076] In a specific embodiment, the stem cells are differentiated stem cell line DYR0100.

[0077] In a specific embodiment, the differentiation conditions include culturing at 37°C and 5% CO2 until the center of the colony thickens.

[0078] It should be understood that step (1) is to cultivate from Day-2 to Day-1, step (2) is to cultivate from Day-1 to Day-0, step (3) is to cultivate from Day-0 to Day-2, step (4) is to cultivate from Day-2 to Day-4, step (5) is to cultivate from Day-4 to Day-6, and step (6) is to cultivate from Day-8 to Day-10 and Day-10+.

[0079] It should be understood that step (2) can promote cell aggregation and initial differentiation orientation of the embryoid body by regulating cell adhesion and signaling pathways. Step (3) can cause the embryoid body to differentiate into the mesoderm under the influence of specific signaling molecules, developing into a cardiac progenitor cell population. Step (4) can culture the cardiac progenitor cell population into cardiac progenitor cells.

[0080] The term "stem cell" in this document refers to totipotent or pluripotent cells, that is, cells capable of differentiating into many final differentiated cell types. Totipotent stem cells typically have the ability to develop into any cell type. Totipotent stem cells can be embryonic or non-embryonic. Pluripotent stem cells are typically cells capable of differentiating into several different final differentiated cell types. Unipotent stem cells can only produce one cell type but possess self-renewal properties, which distinguishes them from non-stem cells. These stem cells can originate from various tissue or organ systems, including the heart, blood, nerves, muscles, skin, intestines, bones, kidneys, liver, pancreas, thymus, etc. In a specific embodiment, the stem cell is the stem cell line DYR0100, which is derived from the Institute of Stem Cell Research, Chinese Academy of Sciences. The stem cell line DYR0100 is disclosed in the following literature: "Liu T, Li Q, Wang S, et al. Transplantation of ovarian granulosa like cells derived from human induced pluripotent stem cells for the treatment of murinepremature ovarian failure[J]. Molecular medicine reports, 2016, 13(6): 5053-5058." In this literature, it is named "DYR0100 iPSCs".

[0081] On the other hand, this application provides the use of a culture medium containing the IWP-2 additive in culturing cardiac organoids, cardiac tissues, or cardiac cells.

[0082] In a specific embodiment, the culture medium comprises: a basal culture medium, and B-27 additive and IWP-2 added thereto; preferably, the final concentration of B-27 is 0.5-1.5×; preferably, the IWP2 content is 3-7 μM; preferably, the basal culture medium is 1640 medium; more preferably, the 1640 medium is 0.5-1.5×; preferably, the culture medium further comprises: glutamine and / or HEPES; preferably, the culture medium does not contain pyruvate;

[0083] In a specific embodiment, the cardiac organoid has MYL7 cardiomyocyte markers, cTnT cardiomyocyte markers, and / or CD31 endothelial cell markers.

[0084] On the other hand, this application provides a culture medium group suitable for culturing cardiac organoids, cardiac tissues or cardiac cells, the culture medium group comprising: a first culture medium, the first culture medium being the culture medium containing IWP-2 additive as described in claim 1 or 2, and the culture medium group further comprising: at least one of a second culture medium, a third culture medium, a fourth culture medium, a fifth culture medium or a sixth culture medium.

[0085] In a specific embodiment, the second culture medium comprises: a basal culture medium, and Y27632 dihydrochloride added thereto. In a specific embodiment, the final concentration of the Y27632 dihydrochloride is 5-15 μM. In a specific embodiment, the basal culture medium is mTeSR1 medium. In a specific embodiment, the final concentration of the mTeSR1 medium is 0.5-1.5 × 10⁻⁶.

[0086] In a specific embodiment, the third culture medium comprises: a basal culture medium. In a specific embodiment, the basal culture medium is mTeSR1 medium. In a specific embodiment, the final concentration of the mTeSR1 medium is 0.5-1.5×;

[0087] In a specific embodiment, the fourth culture medium comprises: a basal culture medium, and B-27 additive, IWP-2, CHIR99021, and matrigel added thereto. In a specific embodiment, the B27 additive does not contain insulin. In a specific embodiment, the final concentration of B27 is 0.5-1.5×. In a specific embodiment, the final concentration of CHIR99021 is 5-15 μM. In a specific embodiment, the basal culture medium is 1640 medium. In a specific embodiment, the final concentration of the 1640 medium is 0.5-1.5×. In a specific embodiment, the fourth culture medium further comprises glutamine and HEPES. In a specific embodiment, the fourth culture medium does not contain pyruvate. In a specific embodiment, the volume percentage of matrigel, based on the total volume of the fourth culture medium, is 0.5-1.5%.

[0088] In a specific embodiment, the fifth culture medium comprises: a basal culture medium and a B-27 additive added thereto. In a specific embodiment, the B27 does not contain insulin. In a specific embodiment, the final concentration of the B27 is 0.5-1.5×. In a specific embodiment, the basal culture medium is 1640 medium. In a specific embodiment, the final concentration of the 1640 medium is 0.5-1.5×. In a specific embodiment, the fourth culture medium further contains glutamine and / or HEPES. In a specific embodiment, the fifth culture medium does not contain pyruvate.

[0089] In a specific embodiment, the sixth culture medium comprises: a basal culture medium and a B-27 additive added thereto. In a specific embodiment, the final concentration of the B27 is 0.5-1.5×. In a specific embodiment, the basal culture medium is 1640 medium. In a specific embodiment, the final concentration of the 1640 medium is 0.5-1.5×. In a specific embodiment, the sixth culture medium further contains glutamine and / or HEPES. In a specific embodiment, the sixth culture medium does not contain pyruvate.

[0090] On the other hand, this application provides a dry powder culture medium group or a concentrated liquid group prepared from the above-mentioned culture medium group.

[0091] On the other hand, this application provides the use of the above-mentioned culture medium group, or the above-mentioned dry powder culture medium group or concentrated liquid group, in culturing cardiac organoids, cardiac tissues or cardiac cells.

[0092] On the other hand, this application provides a method for preparing an embryoid body, the method comprising:

[0093] Step (1): Expose the stem cells to the second culture medium in the culture medium group of claim 4 for culture;

[0094] Step (2): After completing step (1), the culture is exposed to the third culture medium in the culture medium group of claim 4 for culture to obtain globular embryoids.

[0095] On the other hand, this application provides a method for preparing cardiac cells, tissues and / or organoids, the method comprising: culturing stem cells or their cultures using a culture medium containing IWP-2 additive as described in any of the above uses, the above-described culture medium group, or the above-described dry powder culture medium group or concentrate group.

[0096] In a specific embodiment, the method includes: the stem cells are selected from pluripotent stem cells; preferably, the stem cells are selected from human induced pluripotent stem cells;

[0097] In a specific embodiment, the culture is selected from globular embryoids, cardiac progenitor cells or cell populations, and cardiac progenitor cells or cell populations. In a specific embodiment, the globular embryoids are developed in an environment without matrigel.

[0098] In a specific embodiment, the method for preparing the cardiac progenitor cells or cardiac progenitor cell population includes:

[0099] Step (1): Expose the stem cells to the second culture medium described above for culture;

[0100] Step (2): After completing step (1), expose the culture to the third culture medium mentioned above for culture to obtain globular embryoids;

[0101] Step (3): The spherical embryoids are exposed to the fourth culture medium described above for culture to obtain a heart culture. In a specific embodiment, the heart culture is selected from precursor cells or cardiac precursor cell populations;

[0102] Step (4): After completing step (3), the culture is exposed to the culture medium containing IWP-2 additive as described above or the first culture medium described above for culturing to obtain a heart culture. In a specific embodiment, the heart culture is selected from cardiac progenitor cells;

[0103] In a specific embodiment, the method further includes step (5): after completing step (4), the culture is exposed to the fifth culture medium described above for culture to obtain a heart culture;

[0104] In a specific embodiment, the method further includes step (6): after completing step (5), the culture is exposed to the sixth culture medium described above for culturing to obtain a heart culture. In a specific embodiment, the heart culture is selected from three-dimensional heart organoids.

[0105] In a specific embodiment, the glutamine and HEPES are added to the basal culture medium or packaged separately.

[0106] In a specific embodiment, the culture time of step (1) is 24 hours, the culture temperature is 37°C, and the culture conditions are 5% CO2.

[0107] In a specific embodiment, the culture time of step (2) is 24 hours, the culture temperature is 37°C, and the culture conditions are 5% CO2.

[0108] In a specific embodiment, the culture time of step (3) is 48h, the culture temperature is 37℃, and the culture conditions are 5%CO2.

[0109] In a specific embodiment, the culture time of step (4) is 48h, the culture temperature is 37℃, and the culture conditions are 5% CO2.

[0110] In a specific embodiment, the culture time of step (5) is 24 hours, the culture temperature is 37°C, and the culture conditions are 5% CO2.

[0111] In a specific embodiment, the culture time of step (6) is 48 hours, the culture temperature is 37°C, and the culture conditions are 5% CO2.

[0112] In this application, "1x concentration" or "1×" is synonymous, referring to a concentration or content that is 1 times the actual working concentration. Similarly, in this application, "50x concentration" or "50×" is synonymous, referring to a concentration or content that is 50 times the actual working concentration.

[0113] B27 was purchased from Gibco lot #17504044.

[0114] B27 without insulin was purchased from Gibco lot #A1895601.

[0115] 1640 culture medium was purchased from Gibco lot #22400-089.

[0116] The CAS number for IWP-2 is 686770-61-6.

[0117] The CAS number for CHIR99021 (or CHIR-99021) is 252917-06-9.

[0118] Matrigel was purchased from Corning lot #354277.

[0119] The CAS number for L-glutamine is 56-85-9.

[0120] mTeSR1 medium: purchased from Stemcell lot #85850.

[0121] HEPES, also known as 4-hydroxyethylpiperazine ethanesulfonic acid, has the CAS number 7365-45-9.

[0122] The CAS number for pyruvate is 127-17-3.

[0123] Y27632, also known as Y-27632 dihydrochloride, has the CAS number 146986-50-7.

[0124] The 1640 medium (also known as RPMI 1640 medium or Gibco RPMI 1640 medium) was purchased from Gibco lot #22400-089. It contains L-glutamine and HEPES, but does not contain pyruvate.

[0125] Table 1. Composition of Gibco RPMI 1640 medium at 1x concentration

[0126]

[0127]

[0128]

[0129] Preparation of 1640+B27 without insulin+CHIR99021+1% matrigel (corning lot #354277) medium: Add B27 without insulin (50×) to 49ml of 1640 medium to a final concentration of 1×, add CHIR99021 to a final concentration of 10uM, and add matrigel to a final concentration of 1ml / 100ml to obtain 1640+B27 without insulin+CHIR99021+1% matrigel medium (also known as 1640, B27 without insulin, 10uM CHIR99021 medium or fourth medium).

[0130] Preparation of 1640, B27 without insulin, IWP4 medium: Add B27 without insulin (50×) to 49 ml of 1640 medium to make a final concentration of 1×, and add IWP-4 to make a final concentration of 5 uM to obtain 1640, B27 without insulin, IWP4 medium.

[0131] 1640, B27 without insulin, IWP2 medium: Add B27 without insulin (50×) to 49 ml of 1640 medium to a final concentration of 1×, and add IWP-2 to a final concentration of 5 uM to obtain 1640, B27 without insulin, IWP2 medium (also known as the first medium or medium containing IWP-2 additive).

[0132] 1640, B27 without insulin medium: Add B27 without insulin (50×) to 49 ml of 1640 medium to make a final concentration of 1×, to obtain 1640, B27 without insulin medium (also known as the fifth medium).

[0133] 1640 and B27 medium: Add B27 (50×) to 49 ml of 1640 medium to make a final concentration of 1×, and obtain 1640 and B27 medium (also known as the sixth medium).

[0134] Stem cell culture medium + 10uM Y27632 (second culture medium): Add 50ul of 10mM Y-27632 to 50ml of mTeSR1 culture medium to make the final concentration 10uM.

[0135] Stem cell culture medium (excluding Y-27632): mTeSR1 medium (also known as the third medium).

[0136] The DYR0100 cell line was cultured and expanded at 37℃ and 5% CO2 until the colony center thickened and a dense center appeared (results are shown in Figure 1). Figure 4 As shown), the differentiated areas were removed under the microscope (the differentiated areas were less than 5%, and the morphology of the differentiated areas was as shown). Figure 5 The remaining undifferentiated areas were used to propagate cells from cell line DYR0100 for subsequent culture experiments. It is worth noting that the above propagation process is only for increasing cell number; there is no functional difference between cell line DYR0100 and its propagated cells. Using unpropagated cell line DYR0100 in the following experiments will also achieve the same effect.

[0137] Example 1

[0138] The experiment was divided into two groups: a positive control group and an optimized group. Both groups were cultured in the same batch of experimental equipment.

[0139] Experimental group:

[0140] Day-2: Dilute Matrigel (corning lot #354277) with DMEM / F12 medium on ice to prepare the coating solution (find the dilution factor on the official website according to the lot number). Add 2 ml of coating solution to each well of a 6-well plate and coat the wells at 37°C for 1 h. Wash the DYR0100 cell line twice with pre-warmed (37°C) DPBS. Add 1 ml of GCDR (ready-to-use type, stemcell lot #100-0485) to each well and incubate at 37°C for 6 min. Collect single cells, centrifuge, discard the supernatant, resuspend in stem cell medium + 10 uM Y27632, count, and seed at a concentration of 300,000 cells per square centimeter into 24-well plates (pre-coated with Matrigel), marking this as day-2. Incubate at 37°C and 5% CO2 for 24 h until Day-1.

[0141] Day 1: The culture medium was changed to stem cell culture medium (excluding Y-27632). It was observed that stem cells spontaneously formed spherical embryoid bodies in uncoated well plates (specifically as follows...). Figure 3 As shown), it was placed at 37℃ and 5% CO2 and incubated for 24 hours until Day 0.

[0142] Day 0: Change the culture medium to 1640, B27 without insulin, 10uM CHIR99021 medium and incubate at 37℃ and 5% CO2 for 48h until Day 2.

[0143] Day 2: Observe the culture progress, results are as follows: Figure 7 As shown in A, Figure 7 Figure A shows two-dimensional cardiomyocytes cultured on the coated surface. The culture medium was then changed to 1640, B27 without insulin, IWP4 medium and cultured at 37°C with 5% CO2 for 48 hours until Day 4.

[0144] Day 4: Change the culture medium to 1640, B27 without insulin medium and incubate at 37℃ and 5% CO2 for 48 hours until Day 6.

[0145] Day 6: The culture medium was changed to 1640 and B27. At this time, pulsation was observed in both coated wells (planar growth) and uncoated wells (three-dimensional growth). Therefore, it was found that three-dimensional cardiac organoids could be formed even on uncoated surfaces. Subsequent verification showed that the expression of cardiac troponin and endothelial cell markers was observed. The culture was carried out at 37°C and 5% CO2 for 48 hours until Day 8.

[0146] Day 8+: Replace with fresh 1640+B27 medium every 2 days, and culture can continue for more than 60 days.

[0147] The pulsation begins between day 7 and day 9.

[0148] Optimization Group:

[0149] Day-2: Wash DYR0100 cell line twice with preheated (37℃) DPBS. Add 1 ml of GCDR (ready-to-use type, stemcell lot #100-0485) to each well. Incubate at 37℃ for 6 min, collect single cells, centrifuge, discard the supernatant, and resuspend in stem cell culture medium + 10 uM Y27632 (also known as the second medium). After counting, seed at a concentration of 300,000 cells per square centimeter into 24-well plates (without Matrigel coating), and mark as day-2. Incubate at 37℃ and 5% CO2 for 24 h until Day-1.

[0150] Day 1: The culture medium was changed to mTeSR1 medium (without Y-27632) (also known as the third medium). It was observed that stem cells spontaneously formed spherical embryoid bodies in the uncoated well plates (specifically as follows...). Figure 6 As shown), the embryoids were cultured at 37°C and 5% CO2 for 24 hours until Day 0. During this period, the embryoids contained a variety of cell types, laying the foundation for subsequent differentiation. This stage mainly promoted cell aggregation and preliminary differentiation orientation by regulating cell adhesion and signaling pathways.

[0151] Day 0: The culture medium was changed to 1640, B27 without insulin, 10uM CHIR99021 medium (also known as the fourth medium) and cultured at 37℃ and 5% CO2 for 48h. During this period, the embryoids began to differentiate into the mesoderm under the influence of specific signaling molecules and developed into the cardiac progenitor cell population.

[0152] Day 2: Observe the culture progress, results are as follows: Figure 7 As shown, Figure 7 Figure B shows a three-dimensional cardiac organoid formed on an uncoated surface. The culture medium was then changed to 1640, B27 without insulin, IWP2 medium (also known as the first medium or medium containing IWP-2 additive) and cultured at 37°C with 5% CO2 for 48 hours until Day 4. During this period, the culture developed into cardiac progenitor cells under the influence of specific signaling molecules.

[0153] Day 4: Change the culture medium to 1640, B27 without insulin medium (also known as the fifth medium) and incubate at 37°C and 5% CO2 for 48 hours until Day 6.

[0154] Day 6: The culture medium was changed to 1640 and B27 medium (also known as the sixth medium). At this time, pulsation was observed in both coated wells (planar growth) and uncoated wells (three-dimensional growth). Therefore, it was found that three-dimensional cardiac organoids could be formed even on uncoated surfaces. Subsequent verification showed that the expression of cardiac troponin and endothelial cell markers was observed. The culture was carried out at 37°C and 5% CO2 for 48 hours until Day 8.

[0155] Day 8+: Observe the culture progress, results as follows Figure 8 As shown, the culture medium is replaced with a fresh 1640+B27 medium every 2 days, and the culture can be carried out for more than 60 days.

[0156] Days 7-9: Pulsation begins between days 7 and 9. During this period, pacemaker cardiomyocytes begin to appear. As culture continues, the cardiomyocytes mature, and the pulsation area and frequency gradually stabilize, forming partial spatial tissue distribution (such as stratification of endothelial cells and cardiomyocytes). Results are as follows... Figure 9 As shown.

[0157] Days 9-11: Observe the culture progress, results are as follows: Figure 1 As shown, the culture method in this embodiment can achieve the culture of a large number of atrial myocardial organoids in a relatively short time (9-11 days) (approximately 20+ organoids can be harvested per well in a 6-well plate). Figure 1 (With a 4x objective lens in one field of view), it combines the advantages of low cost and high efficiency. Compared with the traditional method of differentiating cardiac organoids (inoculating stem cells, maintaining two-dimensional culture until day 15 when digesting them into single cells, and then inoculating them into 96-well low-adsorption U-shaped plates, which require another 6-8 days to form a three-dimensional structure), it shortens the time and simplifies the experimental operation, greatly improving its accessibility.

[0158] Will Figure 1 The obtained atrial organoids were verified by immunofluorescence staining, and the results were as follows: Figure 2 ( Figure 2 In the image, red indicates MYL7 and blue indicates DAPI. Figure 3 ( Figure 3 As shown in the figure (green represents cTnT and red represents CD31), it has a high expression rate of atrial cardiomyocytes (MYL7) and a specific cardiac tissue structure, with an endothelial cell (CD31) layer and a cardiomyocyte (cTnT) layer.

[0159] Figure 2 The immunofluorescence staining verification steps are as follows:

[0160] 1. Transfer the organoids to be stained to a 1-inch confocal dish, discard the culture medium, and wash three times with DPBS for 5 minutes each time. Discard the DPBS.

[0161] 2. Add 1 ml of 4% paraformaldehyde and let it stand at room temperature for 1 hour to fix. Discard the paraformaldehyde.

[0162] 3. Add 1 ml of DPBS and wash three times at 100 rpm for 5 min each time on a shaker. Discard the DPBS.

[0163] 4. Add 1 ml of 3% Triton X-100, allow it to pass through for 15 minutes, then discard the liquid.

[0164] 5. Add 1 ml of DPBS and wash 3 times at 100 rpm on a shaker for 5 min each time.

[0165] 6. Discard the liquid after 1 hour of sealing on a shaker at 100 rpm with 3% BSA at room temperature.

[0166] 7. Prepare 1 ml of 1:200 primary antibody (MYL7, proteintech lot #17283-1-AP) solution with 3% BSA, add it to a confocal dish and incubate overnight at 4°C.

[0167] 8. Discard the primary antibody solution, prepare 0.1% Tween 20 PBST, add 1 ml, and wash three times on a shaker at 100 rpm for 5 min each time. Discard the PBST.

[0168] 9. Prepare 1 ml of a 1:500 secondary antibody (goat anti rabbit 594, proteintech lot #RGAM004) solution, add it all to a confocal dish, and incubate at room temperature in the dark for 1 hour. Discard the secondary antibody solution.

[0169] 10. Add 1 ml of PBST and wash three times at 100 rpm for 5 minutes each time. Discard the liquid.

[0170] 11. Add 200 μL of ready-to-use DAPI (proteintech lot #PR30021), incubate at room temperature for 5 minutes, and discard the liquid.

[0171] 12. Add 1 ml of PBST and wash three times at 100 rpm for 5 min each time.

[0172] 13. Observe under a microscope.

[0173] Figure 3 The immunofluorescence staining verification steps are as follows:

[0174] 1. Transfer the organoids to be stained to a 1-inch confocal dish, discard the culture medium, and wash three times with DPBS for 5 minutes each time. Discard the DPBS.

[0175] 2. Add 1 ml of 4% paraformaldehyde and let it stand at room temperature for 1 hour to fix. Discard the paraformaldehyde.

[0176] 3. Add 1 ml of DPBS and wash three times at 100 rpm for 5 min each time on a shaker. Discard the DPBS.

[0177] 4. Add 1 ml of 3% Triton X-100, allow it to pass through for 15 minutes, then discard the liquid.

[0178] 5. Add 1 ml of DPBS and wash 3 times at 100 rpm on a shaker for 5 min each time.

[0179] 6. Discard the liquid after 1 hour of sealing on a shaker at 100 rpm with 3% BSA at room temperature.

[0180] 7. Prepare 1 ml of 1:200 primary antibody (cTnT, proteintech lot #68300-1-Ig; CD31, proteintech lot #11265-1-AP) solution with 3% BSA, add it to a confocal dish and incubate overnight at 4°C.

[0181] 8. Discard the primary antibody solution, prepare 0.1% Tween 20 PBST, add 1 ml, and wash three times on a shaker at 100 rpm for 5 min each time. Discard the PBST.

[0182] 9. Prepare 1 ml of a 1:500 secondary antibody solution (goat anti rabbit 594, proteintech lot #RGAM004; goat anti mouse 488, proteintech lot #RGAR002), add it all to a confocal dish, and incubate at room temperature in the dark for 1 hour. Discard the secondary antibody solution.

[0183] 10. Add 1 ml of PBST and wash three times at 100 rpm for 5 minutes each time. Discard the liquid.

[0184] 11. Add 200 μL of ready-to-use DAPI (proteintech lot #PR30021), incubate at room temperature for 5 minutes, and discard the liquid.

[0185] 12. Add 1 ml of PBST and wash three times at 100 rpm for 5 min each time.

[0186] 13. Observe under a microscope.

[0187] Comparative Example 1

[0188] Day-2: Dilute Matrigel (corning lot #354277) with 24 ml of DMEM / F12 medium on ice to prepare the coating solution at the dilution factor (find the dilution factor on the official website according to the lot number). Add 2 ml of coating solution to each well of a six-well plate and coat the plate at 37°C for 1 hour.

[0189] The stem cell line DYR0100 was washed twice with preheated (37°C) DPBS. 1 ml of GCDR (ready-to-use, stemcell lot #100-0485) was added to each well. After incubation at 37°C for 6 min, single cells were collected, centrifuged, and the supernatant was discarded. The cells were resuspended in stem cell culture medium + 10 μM Y27632. After counting, the cells were seeded at a concentration of 300,000 cells per square centimeter into 24-well plates (pre-coated with Matrigel), designated as day-2. The plates were incubated at 37°C and 5% CO2 for 24 h until day-1.

[0190] Day-1: Change the culture medium to mTeSR1 (without Y-27632) and incubate at 37℃ and 5% CO2 for 24 hours until Day 0.

[0191] Day 0: Change the culture medium to 1640, B27 without insulin, 10uM CHIR99021 medium. Incubate at 37℃ and 5% CO2 for 48 hours until Day 2.

[0192] Day 2: The culture medium was changed to 1640, B27 without insulin, and IWP4 medium. Before adding the IWP4-containing medium, the differentiation status was as expected. After adding the IWP4-containing medium, cell differentiation stopped, so this round of experiments was terminated. Details are as follows: Figure 10 As shown.

[0193] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and substitutions can be made without departing from the technical principles of the present invention, and these improvements and substitutions should also be considered within the scope of protection of the present invention.

Claims

1. Use of culture media containing IWP-2 additive in culturing cardiac organoids, cardiac tissues or cardiac cells.

2. The use as described in claim 1, characterized in that, The culture medium comprises: a basal culture medium, and B-27 additive and IWP-2 added thereto; preferably, the final concentration of B-27 is 0.5-1.5×; preferably, the IWP2 content is 3-7 μM; preferably, the basal culture medium is 1640 medium; more preferably, the 1640 medium is 0.5-1.5×; preferably, the culture medium further comprises: glutamine and / or HEPES; preferably, the culture medium does not contain pyruvate; And / or, the cardiac organoids have MYL7 cardiomyocyte markers, cTnT cardiomyocyte markers and / or CD31 endothelial cell markers.

3. A culture medium assembly, characterized in that, The culture medium group is suitable for culturing cardiac organoids, cardiac tissues or cardiac cells, and the culture medium group comprises: a first culture medium, which is the culture medium containing IWP-2 additive as described in claim 1 or 2, and the culture medium group further comprises: at least one of a second culture medium, a third culture medium, a fourth culture medium, a fifth culture medium or a sixth culture medium.

4. The culture medium assembly as described in claim 3, characterized in that, The second culture medium comprises: a basal culture medium, and Y27632 dihydrochloride added thereto; preferably, the final concentration of the Y27632 dihydrochloride is 5-15 μM; preferably, the basal culture medium is mTeSR1 medium; more preferably, the final concentration of the mTeSR1 medium is 0.5-1.5 × 10⁻⁶. And / or, the third culture medium comprises: a basal culture medium, preferably mTeSR1 medium, more preferably, the final concentration of the mTeSR1 medium is 0.5-1.5×; And / or, the fourth culture medium comprises: a basal culture medium, and B-27 additive, IWP-2, CHIR99021, and matrigel added thereto; preferably, the B27 additive does not contain insulin; preferably, the final concentration of the B27 is 0.5-1.5×; preferably, the final concentration of the CHIR99021 is 5-15 μM; preferably, the basal culture medium is 1640 medium; more preferably, the final concentration of the 1640 medium is 0.5-1.5×; preferably, the fourth culture medium further comprises glutamine and HEPES; preferably, the fourth culture medium does not contain pyruvate; preferably, the volume percentage of matrigel, based on the total volume of the fourth culture medium, is 0.5-1.5%; And / or, the fifth culture medium comprises: a basal culture medium, and a B-27 additive added thereto; preferably, the B27 does not contain insulin; preferably, the final concentration of the B27 is 0.5-1.5×; preferably, the basal culture medium is 1640 medium; more preferably, the final concentration of the 1640 medium is 0.5-1.5×; preferably, the fourth culture medium also contains glutamine and / or HEPES; preferably, the fifth culture medium does not contain pyruvate; And / or, the sixth culture medium comprises: a basal culture medium, and a B-27 additive added thereto; preferably, the final concentration of the B27 is 0.5-1.5×; preferably, the basal culture medium is 1640 culture medium; more preferably, the final concentration of the 1640 culture medium is 0.5-1.5×; preferably, the sixth culture medium further contains glutamine and / or HEPES; preferably, the sixth culture medium does not contain pyruvate.

5. A dry powder culture medium or a concentrated solution prepared from the culture medium group described in claim 3 or 4.

6. Use of the culture medium group of claim 3 or 4, or the dry powder culture medium group or concentrate group of claim 5, in culturing cardiac organoids, cardiac tissues or cardiac cells.

7. A method for preparing an embryoid, characterized in that, The method includes: Step (1): Expose the stem cells to the second culture medium in the culture medium group of claim 4 for culture; Step (2): After completing step (1), the culture is exposed to the third culture medium in the culture medium group of claim 4 for culture to obtain globular embryoids.

8. A method for preparing cardiac cells, tissues, and / or organoids, characterized in that, The method includes culturing stem cells or their cultures using a culture medium containing IWP-2 additive as described in any of the uses of claims 1-2, a culture medium group as described in claim 3 or 4, or a dry powder culture medium group or concentrate group as described in claim 5.

9. The method as described in claim 8, characterized in that, The method includes: the stem cells are selected from pluripotent stem cells; preferably, the stem cells are selected from human induced pluripotent stem cells; And / or, the culture is selected from globular embryoids, cardiac progenitor cells or cell populations, cardiac progenitor cells or cell populations; preferably, the globular embryoids are developed in an environment without matrigel.

10. The method as described in claim 8, characterized in that, The method for preparing the cardiac progenitor cells or cardiac progenitor cell population includes: Step (1): Expose the stem cells to the second culture medium as described in claim 3 or 4 for culture; Step (2): After completing step (1), expose the culture to the third culture medium as described in claim 3 or 4 for culture to obtain globular embryoids; Step (3): Expose the spherical embryoid body to the fourth culture medium described in claim 3 or 4 for culture to obtain a heart culture; preferably, the heart culture is selected from precursor cells or heart precursor cell populations; Step (4): After completing step (3), the culture is exposed to the culture medium containing IWP-2 additive as described in claim 1 or 2 or the first culture medium as described in claim 3 or 4 to obtain a heart culture; preferably, the heart culture is selected from cardiac progenitor cells; Optionally, the method further includes step (5): after completing step (4), the culture is exposed to the fifth culture medium as described in claim 3 or 4 for culture to obtain a heart culture; Optionally, the method further includes step (6): after completing step (5), the culture is exposed to the sixth culture medium as described in claim 3 or 4 for culture to obtain a heart culture; preferably, the heart culture is selected from three-dimensional heart organoids.

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