A culture medium for preparing a 3D cardiac organoid and a method for preparing a 3D cardiac organoid

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a culture medium for preparing a 3D heart organoid and a preparation method of the 3D heart organoid. The components in the culture medium provided by the application have a synergistic effect, the CM-1 culture medium enables 3D cell balls to form a primitive germ layer structure in the culture medium, the CM-2 culture medium promotes differentiation of myocardial precursor cells into functional myocardial cells, the CM-3 culture medium helps to form a contraction structure of mature myocardial cells and supports functional maturation, and the CM-4 culture medium improves cell viability in the process of induction and differentiation, as well as structural stability and functional maturation of the 3D heart organoid. By using the culture medium provided by the application, the 3D heart organoid can be prepared in a 3D dynamic environment, a 2D culture step is skipped, operation complexity is reduced, bionics is improved, and a 3D heart organoid culture period is shortened.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a culture medium for preparing 3D heart organoids and a method for preparing 3D heart organoids. Background Technology

[0002] Cardioids, as three-dimensional (3D) self-organizing models constructed from human pluripotent stem cells (hPSCs), have shown great potential in the field of cardiovascular disease research. They can simulate key stages of human heart development, complex structural features, and important physiological functions, providing a valuable tool for in-depth exploration of heart-related mechanisms.

[0003] In the research of cardiac cell culture, traditional two-dimensional (2D) cell culture models are an important research tool. These models are relatively simple to operate, low in cost, and easy to implement, and have long provided fundamental experimental support for basic research on cardiac cells. However, the two-dimensional planar structure cannot fully represent the true three-dimensional spatial configuration of the heart, nor can it accurately simulate the complex interactions between cells in a three-dimensional environment, such as intercellular signal transduction, maintenance of cell polarity, and the three-dimensional dynamic interaction between cells and the extracellular matrix. These limitations restrict the application of traditional two-dimensional culture in simulating the true physiological and pathological states of the heart.

[0004] Compared to traditional two-dimensional culture, three-dimensional culture improves the cell growth environment to some extent, better simulating the three-dimensional spatial distribution of cells in vivo and some cell-cell interactions, and has made some progress in cardiac cell research. However, some three-dimensional culture methods still have limitations, such as long culture cycles, limited ability to simulate complex tissue features such as cardiac chamber structure and cell types, and difficulty in comprehensively and accurately reproducing the physiological state of the heart. Summary of the Invention

[0005] The purpose of this invention is to demonstrate how to prepare 3D heart organoids in a 3D dynamic environment, skipping the 2D culture step, reducing operational complexity and improving biomimicry, and shortening the culture cycle of 3D heart organoids.

[0006] This invention provides a culture medium for preparing 3D heart organoids, including a basal culture medium, CM-1 culture medium, CM-2 culture medium, CM-3 culture medium and CM-4 culture medium;

[0007] The basal culture media include IMDM medium and F-12 medium;

[0008] The CM-1 culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, activin A, bone morphogenetic protein, GSK-3β small molecule inhibitor, PI3K inhibitor, and insulin-transferrin-selenium complex.

[0009] The CM-2 culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, bone morphogenetic protein, fibroblast growth factor, Tankyrase inhibitor, retinoic acid, and insulin-transferrin-selenium complex.

[0010] The CM-3 culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, bone morphogenetic protein, fibroblast growth factor, and insulin-transferrin-selenium complex.

[0011] The CM-4 culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, and insulin-transferrin-selenium complex;

[0012] The insulin-transferrin-selenium complex comprises insulin, transferrin, and sodium selenite.

[0013] Preferably, the CM-1 culture medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 25–100 ng / ml activin A, 5–20 ng / ml bone morphogenetic protein, 1–5 μM GSK-3β small molecule inhibitor, 2.5–10 μM MPI3K inhibitor, 27.5–110 μg / ml insulin-transferrin-selenium complex, and the balance being the basal culture medium;

[0014] The CM-2 culture medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 5–20 ng / ml bone morphogenetic protein, 4–16 ng / ml fibroblast growth factor, 2.5–10 μM Tankyrase inhibitor, 0.25–1 μM retinoic acid, 27.5–110 μg / ml insulin-transferrin-selenium complex, and the balance being the basal culture medium.

[0015] The CM-3 culture medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 5–20 ng / ml bone morphogenetic protein, 4–16 ng / ml fibroblast growth factor, 27.5–110 μg / ml insulin-transferrin-selenium complex, and the balance of the basal culture medium.

[0016] The CM-4 medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 27.5–110 μg / ml insulin-transferrin-selenium complex, and the remainder being the basal medium.

[0017] Preferably, the volume ratio of IMDM medium to F-12 medium in the basal culture medium is 1:(1-3);

[0018] The mass ratio of insulin, transferrin, and sodium selenite in the insulin-transferrin-selenium complex is 1000:(550-2000):0.67.

[0019] The present invention also provides the application of the culture medium described in the above technical solution in the preparation of 3D heart organoids.

[0020] This invention also provides a method for preparing 3D heart organoids, comprising the following steps:

[0021] After seeding hPSCs cell suspension into low-adsorption 96-well plates, centrifuging and pre-culturing for 24 hours, pre-treated well plates were obtained.

[0022] The culture medium in the pretreated well plate was replaced with CM-1 culture medium as described in the above technical solution, and the first culture was carried out for 36 to 40 hours to obtain the first treated well plate.

[0023] The culture medium in the first treatment plate was replaced with CM-2 culture medium as described in the above technical solution, and cultured for 4 days to obtain the second treatment plate.

[0024] Replace the culture medium in the second treatment plate with the CM-3 culture medium described in the above technical solution, and culture for 2 days to obtain the third treatment plate.

[0025] The culture medium in the third processing plate is replaced with CM-4 culture medium as described in the above technical solution, and the fourth culture is carried out for at least 2 days to obtain 3D heart organoids.

[0026] Preferably, the hPSCs cell suspension comprises hPSCs cells, hPSCs cell culture medium, and ROCK inhibitor;

[0027] The concentration of ROCK inhibitor in the hPSCs cell suspension was 5–20 μM.

[0028] Preferably, the method for preparing the hPSCs cell suspension includes: seeding hPSCs cells in a culture plate coated with telogen effluvium, culturing them in a pre-culture medium, and when the hPSCs confluence reaches 70%, digesting and resuspending them to obtain the hPSCs cell suspension.

[0029] The pre-culture medium comprises 0.5% m / V bovine serum albumin, 15–60 ng / ml fibroblast growth factor, and 0.9–3.6 ng / ml TGFβ1.

[0030] Preferably, the inoculation density is 5,000 to 10,000 cells / well.

[0031] Preferably, the rotation speed of the first culture is 50-100 rpm, the temperature is 37°C, and the CO2 concentration is 5%.

[0032] The second culture was carried out at a rotation speed of 50-100 rpm, a temperature of 37°C, and a CO2 concentration of 5%.

[0033] The third culture was conducted at a rotation speed >40 rpm and <100 rpm, a temperature of 37°C, and a CO2 concentration of 5%.

[0034] The fourth culture was carried out at a rotation speed of 50-100 rpm, a temperature of 37°C, and a CO2 concentration of 5%.

[0035] Preferably, the CM-2 culture medium is replaced daily during the second culture process;

[0036] During the third culture process, the CM-3 medium should be replaced with fresh medium daily.

[0037] During the fourth culture process, 50% of the CM-4 medium is replaced daily; replacing 50% of the differentiation medium with fresh CM-4 medium means discarding 50% of the old V / V medium and replacing it with fresh CM-4 medium.

[0038] Beneficial effects:

[0039] This invention provides a culture medium for preparing 3D cardiac organoids, comprising a basal culture medium, a differentiation culture medium, CM-1 culture medium, CM-2 culture medium, and CM-3 culture medium; the basal culture medium includes IMDM culture medium and F-12 culture medium; the differentiation culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, and insulin-transferrin-selenium complex; the CM-1 culture medium includes the differentiation culture medium, activin A, bone morphogenetic protein, GSK-3β small molecule inhibitor, PI3K inhibitor, and insulin-transferrin-selenium complex; the CM-2 culture medium includes the differentiation culture medium, bone morphogenetic protein, fibroblast growth factor, Tankyrase inhibitor, retinoic acid, and insulin-transferrin-selenium complex; and the CM-3 culture medium includes the differentiation culture medium, bone morphogenetic protein, fibroblast growth factor, and insulin-transferrin-selenium complex. The components of the CM-1 culture medium described in this invention work synergistically to enable 3D cell spheres to form primitive germ layer structures in the culture medium; the components of the CM-2 culture medium work synergistically to promote the differentiation of cardiac progenitor cells into functional cardiomyocytes; the components of the CM-3 culture medium work synergistically to help form the contractile structure of mature cardiomyocytes and support functional maturation; and the components of the CM-4 culture medium work synergistically to improve cell viability during the induced differentiation process, as well as the structural stability and functional maturation of 3D cardiac organoids. Using the culture medium provided by this invention, 3D cardiac organoids can be prepared in a dynamic 3D environment, skipping the 2D culture step, reducing operational complexity, improving biomimicry, and shortening the 3D cardiac organoid culture cycle. The results of the examples show that 3D cardiac organoids obtained by culturing in the culture medium of this invention for 10.5 days contain cardiomyocytes and have a chamber structure similar to the heart, with an average diameter of 996 micrometers, an average autonomous rhythm of 51 beats / minute, and an organoid diameter variation coefficient of <15%, which can more fully simulate the components of natural heart cells and has a short culture cycle. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0041] Figure 1 The effect of different concentrations of vetronectin on early stem cell resuscitation efficiency in the presence and absence of BSA;

[0042] Figure 2 This is a flowchart of the 3D culture process for cardiac organoids in Example 2;

[0043] Figure 3 This is a flowchart of the 2D to 3D culture process for cardiac organoids in Comparative Example 1.

[0044] Figure 4 The following are statistical graphs of the diameter of cardiac organoids obtained in Example 2 and Comparative Example 1;

[0045] Figure 5 The above are statistical graphs of the spontaneous rhythms of cardiac organoids obtained in Example 2 and Comparative Example 1.

[0046] Figure 6 The results of immunofluorescence staining of cTnt, a marker of cardiac organoid cardiomyocytes, obtained in Example 2 and Comparative Example 1 are shown; the scale bar is 100 μm. Detailed Implementation

[0047] This invention provides a culture medium for preparing 3D heart organoids, including a basal culture medium, CM-1 culture medium, CM-2 culture medium, CM-3 culture medium and CM-4 culture medium;

[0048] The basal culture media include IMDM medium and F-12 medium;

[0049] The CM-1 culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, activin A, bone morphogenetic protein, GSK-3β small molecule inhibitor, PI3K inhibitor, and insulin-transferrin-selenium complex.

[0050] The CM-2 culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, bone morphogenetic protein, fibroblast growth factor, Tankyrase inhibitor, retinoic acid, and insulin-transferrin-selenium complex.

[0051] The CM-3 culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, bone morphogenetic protein, fibroblast growth factor, and insulin-transferrin-selenium complex.

[0052] The CM-4 culture medium includes the basal culture medium, bovine serum albumin, β-mercaptoethanol, and insulin-transferrin-selenium complex;

[0053] The insulin-transferrin-selenium complex comprises insulin, transferrin, and sodium selenite.

[0054] In one embodiment, the volume ratio of IMDM medium to F-12 medium in the basal culture medium of the present invention is 1:(1-3); in another embodiment, the volume ratio of IMDM medium to F-12 medium in the basal culture medium of the present invention is 1:2. The present invention uses a mixture of IMDM medium and F-12 medium as the basal culture medium, which promotes rapid cell proliferation in the embryo / suspension stage and initiates germ layer induction.

[0055] In one embodiment, the CM-1 culture medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 25–100 ng / ml activin A, 5–20 ng / ml bone morphogenetic protein, 1–5 μM MSK-3β small molecule inhibitor, 2.5–10 μM PI3K inhibitor, 27.5–110 μg / ml insulin-transferrin-selenium complex, and the balance of the basal culture medium.

[0056] The bovine serum albumin in the CM-1 culture medium described in this invention improves cell adhesion and proliferation, reduces oxidative damage, and forms a reversible binding with growth factors, preventing their degradation or adsorption onto the plastic wall. β-mercaptoethanol also helps reduce oxidative damage.

[0057] In one embodiment, the concentration of activator A in the CM-1 culture medium of the present invention is 30–80 ng / ml; in another embodiment, the concentration of activator A in the CM-1 culture medium of the present invention is 40–60 ng / ml; in yet another embodiment, the concentration of activator A in the CM-1 culture medium of the present invention is 50 ng / ml. Activator A in the CM-1 culture medium of the present invention plays a role in maintaining stem cell pluripotency and determining germ layer fate.

[0058] In one embodiment, the concentration of bone morphogenetic protein in the CM-1 culture medium of the present invention is 5–20 ng / ml; in another embodiment, the concentration of bone morphogenetic protein in the CM-1 culture medium of the present invention is 6–15 ng / ml; in yet another embodiment, the concentration of bone morphogenetic protein in the CM-1 culture medium of the present invention is 8–10 ng / ml. The bone morphogenetic protein in the CM-1 culture medium of the present invention promotes the compartmentalization of cardiac organoids.

[0059] As one embodiment, the GSK-3β small molecule inhibitor of the present invention includes CHIR99021. The GSK-3β small molecule inhibitor in the CM-1 culture medium of the present invention plays a crucial role in determining the compartmentalization of the cardiac chambers and ensuring maximum differentiation efficiency; furthermore, the concentration of the GSK-3β small molecule inhibitor is critical for the compartmentalization of the cardiac chambers and ensuring maximum differentiation efficiency, and improper concentration adjustment will affect cell differentiation.

[0060] In one embodiment, the concentration of the PI3K inhibitor in the CM-1 culture medium of the present invention is 2.5–10 μM; in another embodiment, the concentration of the PI3K inhibitor in the CM-1 culture medium of the present invention is 5–8 μM; in yet another embodiment, the concentration of the PI3K inhibitor in the CM-1 culture medium of the present invention is 6 μM. In one embodiment, the PI3K inhibitor of the present invention includes LY294002. The PI3K inhibitor in the CM-1 culture medium of the present invention has the effect of promoting myocardial progenitor differentiation.

[0061] In one embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-1 culture medium of the present invention is 27.5–110 μg / ml; in another embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-1 culture medium of the present invention is 30–100 μg / ml; in yet another embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-1 culture medium of the present invention is 35 μg / ml. In one embodiment, the mass ratio of insulin, transferrin, and sodium selenite in the insulin-transferrin-selenium complex of the present invention is 1000:(550–2000):0.67. The insulin-transferrin-selenium complex in the CM-1 culture medium of the present invention promotes metabolism, reduces free radicals, and reduces oxidative damage.

[0062] The synergistic effect of activin A, bone morphogenetic protein, GSK-3β small molecule inhibitor, PI3K inhibitor and insulin-transferrin-selenium complex in the CM-1 culture medium of the present invention enables 3D cell spheres to form primitive germ layer structures in the culture medium.

[0063] In one embodiment, the CM-2 culture medium of the present invention comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 5–20 ng / ml bone morphogenetic protein, 4–16 ng / ml fibroblast growth factor, 2.5–10 μM Tankyrase inhibitor, 0.25–1 μM retinoic acid, 27.5–110 μg / ml insulin-transferrin-selenium complex, and the balance of the basal culture medium.

[0064] In one embodiment, the concentration of bone morphogenetic protein in the CM-2 culture medium of the present invention is 6–15 ng / ml; in another embodiment, the concentration of bone morphogenetic protein in the CM-2 culture medium of the present invention is 8–12 ng / ml; in yet another embodiment, the concentration of bone morphogenetic protein in the CM-2 culture medium of the present invention is 10 ng / ml. The bone morphogenetic protein in the CM-2 culture medium of the present invention promotes the compartmentalization of cardiac organoids.

[0065] In one embodiment, the concentration of fibroblast growth factor in the CM-2 culture medium of the present invention is 6–15 ng / ml; in another embodiment, the concentration of fibroblast growth factor in the CM-2 culture medium of the present invention is 8–10 ng / ml. The fibroblast growth factor in the CM-2 culture medium of the present invention supports mesoderm expansion and promotes differentiation into cardiomyocyte progenitor cells.

[0066] In one embodiment, the concentration of the Tankyrase inhibitor in the CM-2 culture medium of the present invention is 5-8 ng / ml; in another embodiment, the concentration of the Tankyrase inhibitor in the CM-2 culture medium of the present invention is 6 ng / ml. In one embodiment, the Tankyrase inhibitor of the present invention includes XAV-939. The Tankyrase inhibitor in the CM-2 culture medium of the present invention promotes the stabilization of Axin protein, inhibits the activity of the Wnt / β-catenin signaling pathway, promotes the fine induction of myocardial cell lineages, and avoids the excessive expansion of non-myocardial cell lineages.

[0067] In one embodiment, the concentration of retinoic acid in the CM-2 culture medium of the present invention is 0.4–0.8 μM; in another embodiment, the concentration of retinoic acid in the CM-2 culture medium of the present invention is 0.5–0.6 μM. The retinoic acid in the CM-2 culture medium of the present invention promotes the tissue formation of organoid morphology and the formation of luminal structures.

[0068] In one embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-2 culture medium of the present invention is 30–100 μg / ml; in another embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-2 culture medium of the present invention is 35 μg / ml. In one embodiment, the mass ratio of insulin, transferrin, and sodium selenite in the insulin-transferrin-selenium complex of the present invention is 1000:(550–2000):0.67. The insulin-transferrin-selenium complex in the CM-2 culture medium of the present invention promotes metabolism, reduces free radicals, and reduces oxidative damage.

[0069] The CM-2 culture medium described in this invention contains bone morphogenetic proteins, fibroblast growth factor, Tankyrase inhibitors, retinoic acid, and insulin-transferrin-selenium complex, which work synergistically to promote the differentiation of myocardial progenitor cells into functional cardiomyocytes.

[0070] In one embodiment, the CM-3 culture medium of the present invention comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 5–20 ng / ml bone morphogenetic protein, 4–16 ng / ml fibroblast growth factor, 27.5–110 μg / ml insulin-transferrin-selenium complex, and the balance of the basal culture medium.

[0071] In one embodiment, the concentration of bone morphogenetic protein in the CM-3 culture medium of the present invention is 6–15 ng / ml; in another embodiment, the concentration of bone morphogenetic protein in the CM-3 culture medium of the present invention is 8–12 ng / ml; in yet another embodiment, the concentration of bone morphogenetic protein in the CM-3 culture medium of the present invention is 10 ng / ml. The bone morphogenetic protein in the CM-3 culture medium of the present invention promotes the compartmentalization of cardiac organoids.

[0072] In one embodiment, the concentration of fibroblast growth factor in the CM-3 culture medium of the present invention is 5–15 ng / ml; in another embodiment, the concentration of fibroblast growth factor in the CM-3 culture medium of the present invention is 8–12 ng / ml; in yet another embodiment, the concentration of fibroblast growth factor in the CM-3 culture medium of the present invention is 10 ng / ml. The fibroblast growth factor in the CM-3 culture medium of the present invention supports mesoderm expansion and promotes differentiation into cardiomyocyte progenitor cells.

[0073] In one embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-3 culture medium of the present invention is 30–100 μg / ml; in another embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-3 culture medium of the present invention is 35–40 μg / ml. In one embodiment, the mass ratio of insulin, transferrin, and sodium selenite in the insulin-transferrin-selenium complex of the present invention is 1000:(550–2000):0.67. The insulin-transferrin-selenium complex in the CM-3 culture medium of the present invention promotes metabolism, reduces free radicals, and reduces oxidative damage.

[0074] The CM-3 culture medium described in this invention contains bone morphogenetic proteins, fibroblast growth factors, and insulin-transferrin-selenium complex, which work synergistically to help form the contractile structure of mature cardiomyocytes and support functional maturation.

[0075] In one embodiment, the CM-4 culture medium of the present invention comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 27.5–110 μg / ml insulin-transferrin-selenium complex, and the balance of the basal culture medium.

[0076] In one embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-4 culture medium of the present invention is 30–100 μg / ml. In another embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-4 culture medium of the present invention is 35–40 μg / ml. In one embodiment, the mass ratio of insulin, transferrin, and sodium selenite in the insulin-transferrin-selenium complex of the present invention is 1000:(550–2000):0.67. The insulin-transferrin-selenium complex in the CM-4 culture medium of the present invention promotes metabolism, reduces free radicals, and reduces oxidative damage.

[0077] The CM-4 culture medium described in this invention, with its synergistic effect of bovine serum albumin, β-mercaptoethanol, and insulin-transferrin-selenium complex, can enhance cell viability, organoid structural stability, and functional maturation during induced differentiation.

[0078] The present invention also provides the application of the culture medium described in the above technical solution in the preparation of 3D heart organoids.

[0079] This invention also provides a method for preparing 3D heart organoids, comprising the following steps:

[0080] After seeding hPSCs cell suspension into low-adsorption 96-well plates, centrifuging and pre-culturing for 24 hours, pre-treated well plates were obtained.

[0081] The culture medium in the pretreated well plate was replaced with CM-1 culture medium as described in the above technical solution, and the first culture was carried out for 36 to 40 hours to obtain the first treated well plate.

[0082] The culture medium in the first treatment plate was replaced with CM-2 culture medium as described in the above technical solution, and cultured for 4 days to obtain the second treatment plate.

[0083] Replace the culture medium in the second treatment plate with the CM-3 culture medium described in the above technical solution, and culture for 2 days to obtain the third treatment plate.

[0084] The culture medium in the third processing plate is replaced with CM-4 culture medium as described in the above technical solution, and the fourth culture is carried out for at least 2 days to obtain 3D heart organoids.

[0085] In one embodiment, the hPSC cell suspension of the present invention comprises hPSC cells, hPSC cell culture medium, and a ROCK inhibitor. In one embodiment, the concentration of the ROCK inhibitor in the hPSC cell suspension of the present invention is 5–20 μM; in another embodiment, the concentration of the ROCK inhibitor in the hPSC cell suspension of the present invention is 6–15 μM; in yet another embodiment, the concentration of the ROCK inhibitor in the hPSC cell suspension of the present invention is 8–12 μM; and in yet another embodiment, the concentration of the ROCK inhibitor in the hPSC cell suspension of the present invention is 10 μM. The ROCK inhibitor of the present invention can be Y-27632.

[0086] In one embodiment, the method for preparing the hPSCs cell suspension of the present invention includes: seeding hPSCs cells in a culture plate coated with Vitronectin, culturing them in a pre-culture medium, and when the hPSC confluence reaches 70%, digesting and resuspending them to obtain the hPSCs cell suspension; the pre-culture medium includes 0.5% m / V bovine serum albumin, 15-60 ng / ml fibroblast growth factor, and 0.9-3.6 ng / ml TGFβ1. In another embodiment, the pre-culture medium of the present invention includes 0.5% m / V bovine serum albumin, 30 ng / ml fibroblast growth factor, 1.8 ng / ml TGFβ1, and the remainder being hPSCs cell culture medium. In yet another embodiment, the hPSCs cell culture medium of the present invention can be E8 medium. This invention uses Vitronectin coating to mimic the mechanical and biochemical signals of the natural heart ECM, promoting the self-organization of the chamber structure; by culturing hPSCs containing bovine serum albumin, fibroblast growth factor and TGFβ1, it is possible to increase the activity of hPSCs, ensure the health and pluripotency of hPSCs, while shortening the passage time of hPSCs and inhibiting early differentiation of hPSCs, thereby reducing variability.

[0087] In this invention, hPSC cell suspension is seeded into low-adsorption 96-well plates, centrifuged, and pre-cultured for 24 hours to obtain pre-treated plates. In one embodiment, the seeding density is 5000–10000 cells / well; in another embodiment, the seeding density is 6000–8000 cells / well. This invention limits the seeding density, which triggers integrin-adhesion molecule signaling, promoting stable cell survival and synchronous proliferation.

[0088] In one embodiment, the centrifugation speed is 300 × g; in another embodiment, the centrifugation time is 3 minutes. Centrifugation of hPSC cell suspension seeded into a low-adsorption 96-well plate promotes uniform aggregation of hPSC cells to form cell clusters.

[0089] In one embodiment, the rotation speed of the pre-culture in this invention is 300 rpm. In another embodiment, the temperature of the pre-culture in this invention is 37°C. In yet another embodiment, the CO2 concentration of the pre-culture in this invention is 5%.

[0090] After obtaining the pretreated well plate, the present invention replaces the culture medium in the pretreated well plate with the CM-1 culture medium in the above-mentioned technical solution, and incubates for 36 to 40 hours to obtain the first treated well plate.

[0091] In one embodiment, the first culture time of the present invention is 38 hours. In another embodiment, the rotation speed of the first culture is 50-100 rpm; in yet another embodiment, the rotation speed is 60-80 rpm. In one embodiment, the temperature of the first culture is 37°C. In one embodiment, the CO2 concentration of the first culture is 5%.

[0092] The present invention replaces the culture medium in the pretreated plate with the CM-1 culture medium described in the above technical solution. The first culture has the effect of promoting cell proliferation and initiating mesoderm induction.

[0093] After obtaining the first processed plate, the present invention replaces the culture medium in the first processed plate with the CM-2 culture medium described in the above technical solution, and then incubates for 4 days to obtain the second processed plate.

[0094] In one embodiment, the rotation speed of the second culture medium is 50-100 rpm; in another embodiment, the rotation speed of the second culture medium is 60-80 rpm. In one embodiment, the temperature of the second culture medium is 37°C. In one embodiment, the CO2 concentration of the second culture medium is 5%. In one embodiment, the CM-2 medium is replaced daily during the second culture process; in another embodiment, the CM-2 medium is replaced once daily during the second culture process. The daily replacement of the CM-2 medium in this invention means completely removing the old CM-2 medium and replacing it with fresh CM-2 medium daily.

[0095] The present invention replaces the culture medium in the first treatment plate with the CM-2 culture medium described in the above technical solution, and the second culture has the effect of promoting the differentiation of myocardial progenitor cells into mature myocardial cells.

[0096] After obtaining the second processed plate, the present invention replaces the culture medium in the second processed plate with the CM-3 culture medium described in the above technical solution, and then incubates for 2 days to obtain the third processed plate.

[0097] In one embodiment, the rotation speed of the third culture in this invention is >40 rpm and <100 rpm; in another embodiment, the rotation speed of the third culture in this invention is [not specified]. In one embodiment, the temperature of the third culture in this invention is 37°C. In one embodiment, the CO2 concentration of the third culture in this invention is 5%. In one embodiment, the CM-3 medium is replaced daily during the third culture process. In another embodiment, the CM-3 medium is replaced once daily during the third culture process.

[0098] The present invention replaces the culture medium in the second treatment plate with the CM-3 culture medium described in the above technical solution, and the third culture has the effect of promoting cell polarity arrangement.

[0099] After obtaining the third processing plate, the present invention replaces the culture medium in the third processing plate with the CM-4 culture medium described in the above technical solution, and culturees for at least 2 days to obtain 3D heart organoids.

[0100] In one embodiment, the rotation speed of the fourth culture medium is 50-100 rpm; in another embodiment, the rotation speed is 60-80 rpm. In one embodiment, the temperature of the fourth culture medium is 37°C. In one embodiment, the CO2 concentration of the fourth culture medium is 5%. In one embodiment, 50% of the CM-4 medium is replaced daily during the fourth culture process; in another embodiment, 50% of the CM-4 medium is replaced once daily during the fourth culture process. Replacing the 50% fresh differentiation medium with fresh CM-4 medium means discarding the 50% v / v old medium and replacing it with fresh CM-4 medium.

[0101] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, provides a culture medium for preparing 3D heart organoids and a method for preparing 3D heart organoids, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0102] Example 1

[0103] Effects of different concentrations of vetronectin on early stem cell resuscitation efficiency with and without BSA

[0104] 1. ECM bionic swaddle

[0105] Pre-coat the wells of Corning tissue culture plates (Corning#3516) with vitronectin (VTN, Gibco#A14700, 5-20 μg / ml) for 2-4 hours to obtain coated plates.

[0106] 2. hPSCs were seeded at a density of 160,000–175,000 cells / well on the coated plates from step 1. They were cultured for 3 days at 37°C, 5% CO2, and saturated humidity using both experimental and serum-free medium. Cell growth was observed daily. Results are as follows: Figure 1 As shown. According to Figure 1 It can be seen that polinecin coating can promote the adhesion of hPSCs cells, and BSA can significantly promote the proliferation of hPSCs cells. Considering the economy, the synergistic effect of 10 μg / ml polinecin and BSA is preferred to promote the adhesion and proliferation of stem cells.

[0107] The compositions of the experimental culture medium and serum-free culture medium used in this embodiment are as follows:

[0108] Experimental culture medium: E8 medium was used as the basal medium, with the addition of 0.5% fetal bovine serum albumin (BSA) (m / V), 2 ng / ml fibroblast growth factor (FGF2) and 1 ng / ml TGFβ1. After thorough mixing, the mixture was filtered to remove bacteria.

[0109] Serum-free culture medium: E8 medium was used as the base medium, with 2 ng / ml fibroblast growth factor (FGF2) and 1 ng / ml TGFβ1 added. After thorough mixing, the medium was filtered to remove bacteria.

[0110] Example 2

[0111] 3D culture of heart organoids

[0112] 1. Preparation of culture medium:

[0113] CM-1 medium: Mix IMDM medium and F-12 medium at a 1:1 volume ratio, and then add the following components: 0.5% BSA (m / V), 1‰ β-mercaptoethanol (V / V), 50 ng / ml Activin A (Gibco#120-14-10UG), 10 ng / ml bone morphogenetic protein (BMP4, Gibco#PHC9534), 3 μM CHIR99021 (H9 cell line, abcam#Ab120890), 2 μM LY294002 (Selleck#S1105), and 30 μg / ml insulin-transferrin-selenium complex; wherein the mass ratio of insulin, transferrin, and sodium selenite in insulin-transferrin-selenium is 1000:550:0.67.

[0114] CM-2 medium: Mix IMDM medium and F-12 medium at a volume ratio of 1:1, and then add the following components: 0.5% BSA (m / V), 1‰ β-mercaptoethanol (V / V), 10 ng / ml BMP4, 8 ng / ml fibroblast growth factor (FGF2, TargetMol#TMPY-00749), 5 μM XAV-939 (Selleck#S1180), 0.5 μM retinoic acid (Sigma#R2625), and 27.5 μg / ml Insulin-Transferrin-Selenium;

[0115] CM-3 medium: Mix IMDM medium and F-12 medium at a volume ratio of 1:1, and then add the following components: 0.5% BSA (m / V), 1‰ β-mercaptoethanol (V / V), 10 ng / ml BMP4, 8 ng / ml FGF2 and 40 μg / ml Insulin-Transferrin-Selenium.

[0116] CM-4 medium: Mix IMDM medium and F-12 medium at a volume ratio of 1:1, and then add the following components: 0.5% BSA (m / V), 1‰ β-mercaptoethanol (V / V) and 40 μg / ml Insulin-Transferrin-Selenium.

[0117] 2. ECM biomimetic coating: Vitronectin (VTN, Gibco#A14700, 5-20 μg / ml) was pre-coated in the wells of Corning tissue culture plate (Corning#3516) for 2-4 hours to obtain the coated plate.

[0118] 3. Passage culture of hPSCs

[0119] hPSCs were seeded at a density of 160,000–175,000 cells / well on the coated plates from step 1 and cultured in experimental medium at 37°C, 5% CO2, and saturated humidity. When the hPSC confluence reached 70%, the cells were digested with a mild stem cell digestive enzyme for 3–5 minutes, centrifuged, and resuspended in experimental medium containing 5 μM ROCK inhibitor (Y-27632) (based on E8 medium supplemented with 0.5% fetal bovine serum albumin (BSA) (m / V), 2 ng / ml fibroblast growth factor (FGF2), and 1 ng / ml TGFβ1) to obtain a cell suspension. This cell suspension can be seeded into pre-coated culture plates for cell passage. When used for cell passage, the medium should be replaced with ROCK inhibitor-free experimental medium after 24 hours of culture. It can also be used directly for subsequent direct 3D culture of cardiac organoids. The culture conditions throughout the passage process were: 37°C, 5% CO2, and saturated humidity. Note: Mycoplasma contamination should be checked regularly throughout the hPSC passage culture process to ensure cell stability.

[0120] 4. According to Figure 2 The process of culturing 3D cell spheres.

[0121] 4.1.3D cell spheroid formation

[0122] The obtained cell suspension was seeded at 5000–10000 cells / well in a low-adsorption 96-well plate (Corning #7007), centrifuged at 300×g for 3 minutes to promote uniform aggregation and form cell clusters. After culturing for 24 hours, differentiation culture was carried out according to the following steps to obtain differentiated cardiomyocyte clusters:

[0123] (1) Replace with CM-1 medium and incubate for 36 hours on a shaker (60 rpm) at 37°C and 5% CO2.

[0124] (2) Replace with CM-2 medium and culture continuously for 4 days in a shaker (60 rpm) at 37°C and 5% CO2, changing the medium daily to promote the differentiation of myocardial progenitor cells into mature myocardial cells.

[0125] (3) Replace with CM-3 medium and culture continuously for 2 days in a shaker (60 rpm) at 37°C and 5% CO2, changing the medium daily to promote cell polarity.

[0126] 4.23D Heart Organoid Self-Organization and Maturation

[0127] Differentiated cardiomyocyte clusters were transferred to ultra-low adsorption 96-well plates (Corning#7007), and the culture medium was changed to CM-4 medium. They were cultured continuously for at least 2 days on a shaker (60 rpm) at 37°C and 5% CO2, with 50% fresh medium replaced daily, for long-term culture of mature organoids.

[0128] Comparative Example 1

[0129] 2D to 3D culture of heart organoids

[0130] 1. Prepare the culture medium according to the method in Example 2, perform ECM biomimetic coating and hPSC passage culture to obtain cell suspension;

[0131] 2. According to Figure 3 The process involves 2D to 3D cultivation.

[0132] 2.12D Directed Differentiation of Cardiomyocytes

[0133] The obtained cell suspension was seeded at 200,000 cells / well in a six-well plate (Corning #3516) pre-coated with 10 μg / ml vitrin. After culturing in E8 medium containing 5 μM ROCK inhibitor (Y-27632) for 24 hours, cardiomyocytes were induced according to the following steps to obtain differentiated cardiomyocytes:

[0134] (1) Replace with CM-1 medium and incubate statically at 37℃ and 5% CO2 for 36 hours;

[0135] (2) Replace with CM-2 medium and culture continuously at 37℃ and 5% CO2 for 4 days, changing the medium every day.

[0136] (3) Replace with CM-3 medium and culture continuously at 37℃ and 5% CO2 for 2 days, changing the medium every day.

[0137] (4) Replace with CM-4 medium and culture continuously at 37℃ and 5% CO2 for 21 days, changing the medium every day. This step takes a long time, and the purpose is to gradually remove undifferentiated stem cells and maintain mature differentiated cardiomyocytes.

[0138] 2.22D cardiomyocytes assemble into 3D cell clusters

[0139] (1) After digesting mature differentiated cardiomyocytes with mild digestive enzymes, seed them at a density of 1000 cells / well into AggreWell 800 plates (STEMCELL#34850), centrifuge at 200×g for 3 minutes to promote uniform aggregation, and culture in CM-4 medium for 24-48 hours to form uniform cell spheres.

[0140] (2) Transfer the cell spheres obtained in step (1) to a 6-well plate with ultra-low adsorption (Corning#3471), and culture them in CM-4 medium at 37°C and 5% CO2 for at least 2 days on a shaker (60 rpm) with 50% fresh medium replaced daily to obtain mature cardiomyocyte clusters (cardiac organoids mainly containing cardiomyocytes).

[0141] Test Example 1

[0142] Functional identification of cardiac organoids

[0143] 1. Organoid beating records

[0144] In Example 2, after culturing in CM-4 medium for 10 days, and in Comparative Example 1, after culturing in ultra-low adsorption 6-well plates in step 2.2 for 10 days, 3D organoids were harvested to observe their spontaneous beating function, and the diameter and average spontaneous rhythm were statistically analyzed. The results are as follows: Figure 4 and Figure 5 As shown.

[0145] according to Figure 4 and Figure 5 It can be seen that the 3D heart organoids obtained by the culture method in Comparative Example 1 exhibited spontaneous beating with an average diameter of 1029 micrometers and an average spontaneous rhythm of 38 beats per minute. Figure 4 and Figure 5 (Comparative example); 3D heart organoids obtained by the culture method in Example 2 exhibited spontaneous beating, with an average diameter of 996 micrometers and an average spontaneous rhythm of 51 beats per minute. Figure 4 and Figure 5 In the example (in the middle example), compared with Comparative Example 1, the number of beats of the 3D heart organoid obtained in Example 2 was significantly increased.

[0146] 2. Immunofluorescence staining

[0147] In Example 2, after culturing in CM-4 medium for 10 days, and in Comparative Example 1, after culturing in ultra-low adsorption 6-well plates in step 2.2 for 10 days, 3D organoids were harvested and sectioned. Cardiomyocytes were labeled with cTnT antibody and stained with fluorescence to confirm the layering and overall structure of the cardiac organoids. Results are as follows: Figure 6 As shown.

[0148] according to Figure 6 As can be seen, the 3D heart organoids obtained by the culture method in Example 2 show similar fluorescence staining results to those obtained by the culture method in Comparative Example 1. Both contain cardiomyocytes, and the heart organoids already possess a chamber structure similar to that of the heart. In addition to cardiomyocytes, the 3D heart organoids obtained by the culture method in Example 2 also contain other cell types, thus more fully mimicking the components of natural heart cells.

[0149] Example 3

[0150] The heart organoids were prepared and cultured in 3D according to the method of Example 2, the only difference being the concentration of CHIR99021 in the CM-1 medium used. In this example, the concentration of CHIR99021 in the CM-1 medium used was 5 μM.

[0151] The 3D heart organoids obtained by the culture method in this embodiment have increased diameter and achieve beating at d12.

[0152] Comparative Example 2

[0153] The heart organoids were prepared and cultured in 3D according to the method of Example 2. The only difference was the concentration of CHIR99021 in the CM-1 medium used. The concentration of CHIR99021 in the CM-1 medium used in this comparative example was 1 μM.

[0154] The 3D heart organoids obtained using this comparative culture method did not beat.

[0155] Comparative Example 3

[0156] The heart organoids were prepared and cultured in 3D according to the method of Example 2. The only difference was the concentration of CHIR99021 in the CM-1 medium used. The concentration of CHIR99021 in the CM-1 medium used in this comparative example was 2 μM.

[0157] The 3D heart organoids obtained using this comparative culture method did not beat.

[0158] As can be seen from the above, the technical solution provided by the present invention skips the 2D culture step, and completes the process from stem cell expansion to organoid maturation in a 3D dynamic environment, successfully culturing 3D heart organoids and shortening the 3D heart organoid culture cycle.

[0159] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a 3D heart organoid, characterized in that, Includes the following steps: After seeding hPSCs cell suspension into low-adsorption 96-well plates, centrifuging and pre-culturing for 24 hours, pre-treated well plates were obtained. The culture medium in the pretreated well plate was replaced with CM-1 culture medium, and the first culture was carried out for 36-40 hours to obtain the first treated well plate. The culture medium in the first treatment plate was replaced with CM-2 culture medium, and the second culture was carried out for 4 days to obtain the second treatment plate. Replace the culture medium in the second treatment plate with CM-3 culture medium, and culture for 2 days to obtain the third treatment plate. Replace the culture medium in the third processing plate with CM-4 medium, and culture for at least 2 days to obtain 3D heart organoids. The CM-1 culture medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 25~100 ng / ml activin A, 5~20 ng / ml bone morphogenetic protein, 1~5 µM GSK-3β small molecule inhibitor, 2.5~10 µM PI3K inhibitor, 27.5~110 µg / ml insulin-transferrin-selenium complex, and the remainder being basal culture medium; The CM-2 culture medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 5-20 ng / ml bone morphogenetic protein, 4-16 ng / ml fibroblast growth factor, 2.5-10 µM Tankyrase inhibitor, 0.25-1 µM retinoic acid, 27.5-110 µg / ml insulin-transferrin-selenium complex, and the remainder being basal culture medium; The CM-3 culture medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 5~20 ng / ml bone morphogenetic protein, 4~16 ng / ml fibroblast growth factor, 27.5~110 µg / ml insulin-transferrin-selenium complex, and the remainder being basal culture medium; The CM-4 medium comprises 0.5% m / V bovine serum albumin, 1‰ V / V β-mercaptoethanol, 27.5~110 µg / ml insulin-transferrin-selenium complex, and the remainder being basal medium; The basal culture media include IMDM medium and F-12 medium; The insulin-transferrin-selenium complex includes insulin, transferrin, and sodium selenite; The first culture was carried out at a rotation speed of 50-100 rpm, a temperature of 37°C, and a CO2 concentration of 5%. The second culture was conducted at a rotation speed of 50-100 rpm, a temperature of 37°C, and a CO2 concentration of 5%. The third culture was conducted at a rotation speed >40 rpm and <100 rpm, a temperature of 37°C, and a CO2 concentration of 5%. The fourth culture was carried out at a rotation speed of 50-100 rpm, a temperature of 37°C, and a CO2 concentration of 5%.

2. The preparation method according to claim 1, characterized in that, The volume ratio of IMDM medium to F-12 medium in the basal culture medium is 1:(1~3). The mass ratio of insulin, transferrin, and sodium selenite in the insulin-transferrin-selenium complex is 1000:(550~2000):0.

67.

3. The preparation method according to claim 1, characterized in that, The hPSCs cell suspension includes hPSCs cells, hPSCs cell culture medium, and ROCK inhibitor; The concentration of ROCK inhibitor in the hPSCs cell suspension was 5-20 μM.

4. The preparation method according to claim 1 or 3, characterized in that, The method for preparing the hPSCs cell suspension includes: seeding hPSCs cells in a culture plate coated with viscosin, culturing them in a pre-culture medium, and when the hPSCs confluence reaches 70%, digesting and resuspending them to obtain the hPSCs cell suspension. The pre-culture medium comprises 0.5% m / V bovine serum albumin, 15-60 ng / ml fibroblast growth factor, and 0.9-3.6 ng / ml TGFβ1.

5. The preparation method according to claim 1, characterized in that, The seeding density is 5000~10000 cells / well.

6. The preparation method according to claim 1, characterized in that, During the second culture process, the CM-2 medium should be replaced with fresh medium daily; During the third culture process, the CM-3 medium should be replaced with fresh medium daily. During the fourth culture process, 50% of the CM-4 medium is replaced daily; replacing 50% of the differentiation medium with fresh medium means discarding 50% of the old V / V medium and replacing it with fresh CM-4 medium.

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