Culture medium for preparing 3D heart organoid and preparation method of 3D heart organoid

Abstract

The invention belongs to the technical field of biology, 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 have a synergistic effect, the CM-1 culture medium enables 3D cell spheres to form primordial germ layer structures in the culture medium, the CM-2 culture medium promotes myocardial precursor cells to differentiate into functional myocardial cells, and the CM-3 culture medium helps to form contraction structures of mature myocardial cells and supports functional maturation; the CM-4 culture medium improves the cell activity in the induced differentiation process and the structural stability and functional maturity of 3D heart organs. By utilizing the culture medium provided by the invention, the 3D heart organoid can be prepared in a 3D dynamic environment, a 2D culture step is omitted, the operation complexity is reduced, the biomimetic property is improved, and the culture period of the 3D heart organoid is shortened.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a culture medium for preparing 3D cardiac organoids and a method for preparing the 3D cardiac organoids. Background Art

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

[0003] In the research process of cardiac cell culture, the traditional two-dimensional (2D) cell culture model is an important research method. This model is relatively simple to operate, low-cost and easy to carry out, and has long provided basic experimental support for basic research on cardiac cells. However, the two-dimensional planar structure cannot fully present the true three-dimensional spatial configuration of the heart, and it is difficult to accurately simulate the complex interactions of cells in a three-dimensional environment, such as signal transmission between cells, maintenance of cell polarity, and three-dimensional dynamic interactions between cells and the extracellular matrix. These shortcomings limit the application of traditional two-dimensional culture in simulating the true physiological and pathological state of the heart.

[0004] Compared to traditional two-dimensional culture, three-dimensional culture offers a significantly improved cell growth environment, better simulating the three-dimensional spatial distribution of cells in vivo and some intercellular interactions, and has made some progress in cardiac cell research. However, some current three-dimensional culture methods still have limitations, such as long culture cycles and limited ability to simulate complex tissue features such as cardiac chamber structure and cell types, making it difficult to fully and accurately reproduce the physiological state of the heart. Summary of the Invention

[0005] The purpose of the present invention is to prepare 3D cardiac organoids in a 3D dynamic environment, skipping the 2D culture step, reducing operational complexity and improving biomimetic properties, thereby shortening the 3D cardiac organoid culture cycle.

[0006] The present invention provides a culture medium for preparing 3D cardiac organoids, comprising a basal culture medium, a CM-1 culture medium, a CM-2 culture medium, a CM-3 culture medium, and a CM-4 culture medium;

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

[0008] The CM-1 culture medium comprises 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 comprises 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 comprises 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 comprises 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 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 balance of the basal 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 of 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 culture 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 balance of the basic culture medium.

[0017] Preferably, the volume ratio of IMDM medium to F-12 medium in the basal 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 use of the culture medium described in the above technical solution in preparing 3D cardiac organoids.

[0020] The present invention also provides a method for preparing a 3D cardiac organoid, comprising the following steps:

[0021] After the hPSCs cell suspension was seeded into a low-adsorption 96-well plate, the plate was centrifuged and pre-cultured for 24 hours to obtain a pre-treated plate.

[0022] The culture medium in the pre-treated well plate is replaced with the CM-1 culture medium in the culture medium described in the above technical solution, and the first culture is carried out for 36 to 40 hours to obtain a first treated well plate;

[0023] The culture medium in the first treatment well plate was replaced with the CM-2 culture medium in the culture medium described in the above technical solution, and the culture was carried out for a second time for 4 days to obtain a second treatment well plate;

[0024] The culture medium in the second treatment well plate was replaced with the CM-3 culture medium in the culture medium described in the above technical solution, and the culture was carried out for a third time for 2 days to obtain a third treatment well plate;

[0025] The culture medium in the third treatment well plate is replaced with the CM-4 culture medium described in the above technical solution, and the culture medium is cultured for at least 2 days to obtain 3D cardiac organoids.

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

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

[0028] Preferably, the method for preparing the hPSCs cell suspension comprises: inoculating hPSCs cells in a culture plate coated with vitronectin, culturing with a pre-culture medium, and when the hPSCs confluence reaches 70%, digesting and resuspending the cells to obtain the hPSCs cell suspension;

[0029] The preculture medium includes 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 seeding density is 5000 to 10000 cells / well.

[0031] Preferably, the first culture is carried out at a rotation speed of 50-100 rpm, a temperature of 37° C., and a CO 2 concentration of 5%;

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

[0033] The third culture has a rotation speed of >40 rpm and <100 rpm, a temperature of 37° C., and a CO 2 concentration of 5%;

[0034] The fourth culture was performed at a rotation speed of 50-100 rpm, a temperature of 37° C., and a CO 2 concentration of 5%.

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

[0036] During the third culture process, fresh CM-3 culture medium was replaced every day;

[0037] During the fourth culture, 50% of the fresh CM-4 medium was replaced every day; the replacement of 50% of the fresh differentiation medium was to discard 50% V / V of the old medium and replace it with fresh CM-4 medium.

[0038] Beneficial effects:

[0039] The present invention provides a culture medium for preparing 3D cardiac organoids, comprising a basal culture medium, a differentiation culture medium, a CM-1 culture medium, a CM-2 culture medium and a CM-3 culture medium; the basal culture medium comprises an IMDM culture medium and an F-12 culture medium; the differentiation culture medium comprises the basal culture medium, bovine serum albumin, β-mercaptoethanol and an insulin-transferrin-selenium complex; the CM-1 culture medium comprises the differentiation culture medium, activin A, bone morphogenetic protein, a GSK-3β small molecule inhibitor, a PI3K inhibitor and an insulin-transferrin-selenium complex; the CM-2 culture medium comprises the differentiation culture medium, bone morphogenetic protein, a fibroblast growth factor, a Tankyrase inhibitor, retinoic acid and an insulin-transferrin-selenium complex; and the CM-3 culture medium comprises the differentiation culture medium, a bone morphogenetic protein, a fibroblast growth factor and an insulin-transferrin-selenium complex. The components of the CM-1 culture medium described in the present 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; 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 maturity of 3D cardiac organoids. Using the culture medium provided by the present invention, it is possible to prepare 3D cardiac organoids in a 3D dynamic environment, skipping the 2D culture step, reducing operational complexity and improving biomimetic properties, and shortening the 3D cardiac organoid culture cycle. The results of the example show that 3D cardiac organoids obtained by culturing with the culture medium of the present invention for 10.5 days contain cardiomyocytes and have a chamber structure similar to that of the heart, with an average diameter of 996 microns, an average autonomous rhythm of 51 beats / minute, and a coefficient of variation of organoid diameter of <15%. They can more fully simulate natural cardiac cell components and have a short culture cycle. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0041] Figure 1 The effect of different concentrations of Vetronectin on the early recovery efficiency of stem cells in the presence or absence of BSA;

[0042] Figure 2 This is a flow chart of Example 2 3D culture of cardiac organoids;

[0043] Figure 3 This is the flow chart of 2D to 3D culture of cardiac organoids in Comparative Example 1;

[0044] Figure 4 Statistical graph of cardiac organoid diameters obtained in Example 2 and Comparative Example 1;

[0045] Figure 5 Statistical graphs of the autonomous rhythms of cardiac organoids obtained in Example 2 and Comparative Example 1;

[0046] Figure 6 These are the immunofluorescence staining results of the cardiac organoid cardiomyocyte marker cTnt obtained in Example 2 and Comparative Example 1; wherein, the scale bar is 100 μm. DETAILED DESCRIPTION

[0047] The present invention provides a culture medium for preparing 3D cardiac organoids, comprising a basal culture medium, a CM-1 culture medium, a CM-2 culture medium, a CM-3 culture medium, and a CM-4 culture medium;

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

[0049] The CM-1 culture medium comprises 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 comprises 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 comprises 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 comprises 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 a basal culture medium, which has the effect of promoting rapid cell proliferation at the embryoid body / suspension stage and initiating germ layer induction.

[0055] As an embodiment, the CM-1 culture medium includes 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 of the basal culture medium.

[0056] The bovine serum albumin in the CM-1 culture medium of the present invention has the effects of improving cell adhesion and proliferation, reducing oxidative damage, and forming a reversible bond with growth factors to prevent their degradation or adsorption on the plastic wall. β-mercaptoethanol has the effect of reducing oxidative damage.

[0057] In one embodiment, the concentration of activin A in the CM-1 medium of the present invention is 30 to 80 ng / ml; in another embodiment, the concentration of activin A in the CM-1 medium of the present invention is 40 to 60 ng / ml; and in another embodiment, the concentration of activin A in the CM-1 medium of the present invention is 50 ng / ml. Activin A in the CM-1 medium of the present invention maintains stem cell pluripotency and specifies 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 to 20 ng / ml; in another embodiment, the concentration of bone morphogenetic protein in the CM-1 culture medium of the present invention is 6 to 15 ng / ml; in another embodiment, the concentration of bone morphogenetic protein in the CM-1 culture medium of the present invention is 8 to 10 ng / ml. The bone morphogenetic protein in the CM-1 culture medium of the present invention has the effect of promoting the chamberization of cardiac organoids.

[0059] In one embodiment, the GSK-3β small molecule inhibitor described herein includes CHIR99021. The GSK-3β small molecule inhibitor in the CM-1 culture medium described herein plays a role in determining cardiac chamber compartmentalization and ensuring maximum differentiation efficiency. Furthermore, the concentration of the GSK-3β small molecule inhibitor is crucial for cardiac chamber compartmentalization and ensuring maximum differentiation efficiency. Improper concentration adjustment can affect cell differentiation.

[0060] In one embodiment, the concentration of the PI3K inhibitor in the CM-1 medium of the present invention is 2.5 to 10 μM; in another embodiment, the concentration of the PI3K inhibitor in the CM-1 medium of the present invention is 5 to 8 μM; in another embodiment, the concentration of the PI3K inhibitor in the CM-1 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 medium of the present invention has the effect of promoting the differentiation of cardiomyocyte progenitor cells.

[0061] In one embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-1 medium of the present invention is 27.5 to 110 μg / ml; in another embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-1 medium of the present invention is 30 to 100 μg / ml; in another embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-1 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 to 2000):0.67. The insulin-transferrin-selenium complex in the CM-1 medium of the present invention has the effects of promoting metabolism, reducing free radicals, and alleviating oxidative damage.

[0062] The 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 act synergistically to enable the 3D cell spheres to form a primitive germ layer structure in the culture medium.

[0063] As an embodiment, the CM-2 culture medium of the present invention includes 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 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 to 15 ng / ml; in another embodiment, the concentration of bone morphogenetic protein in the CM-2 culture medium of the present invention is 8 to 12 ng / ml; in 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 has the effect of promoting the chamberization of cardiac organoids.

[0065] In one embodiment, the concentration of the fibroblast growth factor in the CM-2 culture medium of the present invention is 6 to 15 ng / ml; in another embodiment, the concentration of the fibroblast growth factor in the CM-2 culture medium of the present invention is 8 to 10 ng / ml. The fibroblast growth factor in the CM-2 culture medium of the present invention supports the expansion of mesoderm 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 another embodiment, the Tankyrase inhibitor of the present invention comprises XAV-939. The Tankyrase inhibitor in the CM-2 culture medium of the present invention promotes Axin protein stabilization, inhibits Wnt / β-catenin signaling pathway activity, promotes the refined induction of cardiomyocyte lineages, and prevents the overexpansion of non-cardiomyocyte lineages.

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

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

[0069] The bone morphogenetic protein, fibroblast growth factor, Tankyrase inhibitor, retinoic acid and insulin-transferrin-selenium complex in the CM-2 culture medium of the present invention act synergistically to promote the differentiation of myocardial progenitor cells into functional myocardial cells.

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

[0071] In one embodiment, the concentration of bone morphogenetic protein in the CM-3 culture medium of the present invention is 6 to 15 ng / ml; in another embodiment, the concentration of bone morphogenetic protein in the CM-3 culture medium of the present invention is 8 to 12 ng / ml; in 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 has the effect of promoting the chamberization of cardiac organoids.

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

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

[0074] The bone morphogenetic protein, fibroblast growth factor and insulin-transferrin-selenium complex in the CM-3 culture medium of the present invention act synergistically to help form the contractile structure of mature myocardial cells and support functional maturation.

[0075] As an 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] As an embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-4 medium of the present invention is 30 to 100 μg / ml. As another embodiment, the concentration of the insulin-transferrin-selenium complex in the CM-4 medium of the present invention is 35 to 40 μg / ml. As an embodiment, the mass ratio of insulin, transferrin, and sodium selenite in the insulin-transferrin-selenium complex of the present invention is 1000:(550 to 2000):0.67. The insulin-transferrin-selenium complex in the CM-4 medium of the present invention has the effects of promoting metabolism, reducing free radicals, and reducing oxidative damage.

[0077] The CM-4 culture medium of the present invention comprises bovine serum albumin, β-mercaptoethanol and insulin-transferrin-selenium complex, which act synergistically to improve cell viability, structural stability and functional maturity of organoids during the induction of differentiation.

[0078] The present invention also provides the use of the culture medium described in the above technical solution in preparing 3D cardiac organoids.

[0079] The present invention also provides a method for preparing a 3D cardiac organoid, comprising the following steps:

[0080] After the hPSCs cell suspension was seeded into a low-adsorption 96-well plate, the plate was centrifuged and pre-cultured for 24 hours to obtain a pre-treated plate.

[0081] The culture medium in the pre-treated well plate is replaced with the CM-1 culture medium in the culture medium described in the above technical solution, and the first culture is carried out for 36 to 40 hours to obtain a first treated well plate;

[0082] The culture medium in the first treatment well plate was replaced with the CM-2 culture medium in the culture medium described in the above technical solution, and the culture was carried out for a second time for 4 days to obtain a second treatment well plate;

[0083] The culture medium in the second treatment well plate was replaced with the CM-3 culture medium in the culture medium described in the above technical solution, and the culture was carried out for a third time for 2 days to obtain a third treatment well plate;

[0084] The culture medium in the third treatment well plate is replaced with the CM-4 culture medium described in the above technical solution, and the culture medium is cultured for at least 2 days to obtain 3D cardiac organoids.

[0085] In one embodiment, the hPSC cell suspension of the present invention comprises hPSC cells, an 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 to 20 μM; in another embodiment, the concentration of the ROCK inhibitor in the hPSC cell suspension of the present invention is 6 to 15 μM; in another embodiment, the concentration of the ROCK inhibitor in the hPSC cell suspension of the present invention is 8 to 12 μM; in 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 may be Y-27632.

[0086] As one embodiment, the method for preparing the hPSCs cell suspension of the present invention includes: inoculating hPSCs cells in a culture plate coated with Vitronectin, culturing with a pre-culture medium, and when the hPSCs reach a confluency of 70%, digesting and resuspending 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. As one 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 of the hPSCs cell culture medium. As one embodiment, the hPSCs cell culture medium of the present invention can be E8 culture medium. The present invention uses Vitronectin coating to simulate the mechanical and biochemical signals of natural heart ECM and promote the self-organization of chamber structure; using hPSCs cells containing bovine serum albumin, fibroblast growth factor and TGFβ1 to culture hPSCs cells can increase hPSCs cell activity, ensure hPSCs cell health and pluripotency, while shortening hPSCs passaging time, inhibiting hPSCs premature differentiation, and thus reducing variability.

[0087] The present invention seeded a hPSC cell suspension into a low-adhesion 96-well plate, centrifuged, and pre-incubated for 24 hours to obtain a pretreated plate. In one embodiment, the seeding density is 5,000 to 10,000 cells / well; in another embodiment, the seeding density is 6,000 to 8,000 cells / well. This seeding density is limited to trigger integrin-adhesion molecule signaling, promoting stable cell survival and synchronized proliferation.

[0088] In one embodiment, the centrifugation speed of the present invention is 300×g; in another embodiment, the centrifugation time of the present invention is 3 minutes. In the present invention, the hPSC cell suspension is seeded into a low-adhesion 96-well plate and then centrifuged to promote uniform aggregation of hPSC cells to form cell clusters.

[0089] In one embodiment, the rotation speed of the pre-culture of the present invention is 300 rpm. In one embodiment, the temperature of the pre-culture of the present invention is 37° C. In one embodiment, the CO 2 concentration of the pre-culture of the present invention is 5%.

[0090] After obtaining the pretreatment well plate, the present invention replaces the culture medium in the pretreatment well plate with the CM-1 culture medium in the culture medium described in the above technical solution, and performs a first culture for 36 to 40 hours to obtain a first treatment well plate.

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

[0092] In the present invention, the culture medium in the pre-treated well plate is replaced with the CM-1 culture medium in the 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 treatment well plate, the present invention replaces the culture medium in the first treatment well plate with the CM-2 culture medium in the culture medium described in the above technical solution, and performs a second culture for 4 days to obtain a second treatment well plate.

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

[0095] In the present invention, the culture medium in the first treatment well plate is replaced with the CM-2 culture medium in the 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 treatment well plate, the present invention replaces the culture medium in the second treatment well plate with the CM-3 culture medium in the culture medium described in the above technical solution, and cultures the well plate for a third time for 2 days to obtain the third treatment well plate.

[0097] In one embodiment, the rotation speed of the third culture of the present invention is greater than 40 rpm and less than 100 rpm. In another embodiment, the rotation speed of the third culture of the present invention is. In one embodiment, the temperature of the third culture of the present invention is 37°C. In one embodiment, the CO2 concentration of the third culture of the present invention is 5%. In one embodiment, fresh CM-3 medium is replaced daily during the third culture of the present invention. In one embodiment, fresh CM-3 medium is replaced once daily during the third culture of the present invention.

[0098] In the present invention, the culture medium in the second treatment well plate is replaced with the CM-3 culture medium in the 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 treatment well plate, the present invention replaces the culture medium in the third treatment well plate with the CM-4 culture medium in the culture medium described in the above technical solution, and cultured for at least 2 days to obtain 3D heart organoids.

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

[0101] To further illustrate the present invention, a culture medium for preparing 3D cardiac organoids and a method for preparing 3D cardiac organoids provided by the present invention are described in detail below with reference to the accompanying drawings and examples. However, these 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 the early recovery efficiency of stem cells in the presence or absence of BSA

[0104] 1.ECM biomimetic coating

[0105] The wells of a Corning tissue culture treated plate (Corning #3516) were pre-coated with vitronectin (Vitronectin, VTN, Gibco #A14700, 5-20 μg / ml) for 2 to 4 hours to obtain a coated plate.

[0106] 2. hPSCs were seeded onto the coated plates prepared in step 1 at a density of 160,000 to 175,000 cells / well. The plates were cultured in the experimental medium and serum-free medium at 37°C, 5% CO2, and saturated humidity for 3 days. The cell growth status was observed daily. Figure 1 As shown. Figure 1 It can be seen that vitronectin coating can promote the adhesion of hPSCs cells, and BSA can significantly promote the proliferation of hPSCs cells. Considering the economic efficiency, 10 μg / ml vitronectin and BSA are preferred to work synergistically 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 example are as follows:

[0108] Experimental culture medium: E8 medium was used as the basal medium, supplemented with 0.5% fetal bovine serum albumin (BSA) (m / v), 2 ng / ml fibroblast growth factor (FGF2), and 1 ng / ml TGFβ1, mixed thoroughly, and then sterilized by filtration.

[0109] Serum-free medium: Use E8 medium as the basal medium, add 2 ng / ml fibroblast growth factor (FGF2) and 1 ng / ml TGFβ1, mix thoroughly, and filter sterilize.

[0110] Example 2

[0111] 3D culture of cardiac organoids

[0112] 1. Culture medium configuration:

[0113] CM-1 medium: IMDM medium and F-12 medium were mixed at a volume ratio of 1:1. The following components were added after mixing: 0.5% BSA (m / v), 1‰ β-mercaptoethanol (v / v), 50 ng / ml Activin A (Gibco #120-14-10UG), 10 ng / ml BMP4 (Gibco #PHC9534), 3 μM CHIR99021 (H9 cell line, abcam #Ab120890), 2 μM LY294002 (Selleck #S1105), and 30 μg / ml Insulin-Transferrin-Selenium. The mass ratio of insulin, transferrin, and sodium selenite in Insulin-Transferrin-Selenium was 1000:550:0.67.

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

[0115] CM-3 medium: IMDM medium and F-12 medium were mixed in a volume ratio of 1:1. The following components were added after mixing: 0.5% BSA (m / v), 1‰ β-mercaptoethanol (v / v), 10ng / ml BMP4, 8ng / ml FGF2 and 40μg / ml Insulin-Transferrin-Selenium.

[0116] CM-4 medium: IMDM medium and F-12 medium were mixed at a volume ratio of 1:1. After mixing, the following components were added: 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 treated plates (Corning #3516) for 2 to 4 hours to obtain coated plates.

[0118] 3. hPSCs Subculture

[0119] hPSCs were seeded at a density of 160,000–175,000 cells / well onto the coated plates prepared in step 1 and cultured in assay medium at 37°C, 5% CO₂, and saturated humidity. When the hPSCs reached 70% confluency, they were digested with stem cell mild digestion enzymes for 3–5 minutes. After centrifugation, the cells were resuspended in assay medium (based on E8 medium supplemented with 0.5% fetal bovine serum albumin (BSA) (volume / volume), 2 ng / ml fibroblast growth factor (FGF2), and 1 ng / ml TGFβ1) containing 5 μM ROCK inhibitor (Y-27632) to create a cell suspension. This cell suspension can be seeded onto pre-coated plates for cell passage. After 24 hours of culture, the assay medium should be replaced with ROCK inhibitor-free assay medium. Alternatively, the cell suspension can be used directly for subsequent 3D cardiac organoid culture. The culture conditions throughout the subculture process were 37°C, 5% CO₂, and saturated humidity. Note: Mycoplasma contamination should be regularly tested throughout the process of hPSC subculture to ensure the stability of the cells.

[0120] 4. Follow Figure 2 3D cell spheroid culture

[0121] 4.1.3D Spheroid Formation

[0122] The resulting cell suspension was seeded into a low-adhesion 96-well plate (Corning #7007) at 5,000 to 10,000 cells / well and centrifuged at 300 × g for 3 minutes to promote uniform aggregation and form cell clusters. After culturing for 24 hours, differentiation culture was performed according to the following steps to obtain differentiated cardiomyocyte clusters:

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

[0124] (2) The culture medium was replaced with CM-2 medium and cultured continuously for 4 days in a shaking incubator (60 rpm) at 37°C and 5% CO2, with the medium changed every day to promote the differentiation of myocardial progenitor cells into mature myocardial cells.

[0125] (3) The medium was replaced with CM-3 medium and cultured continuously for 2 days in a shaking incubator (60 rpm) at 37°C and 5% CO2, with the medium changed every day to promote cell polarity.

[0126] 4.2 3D cardiac organoid self-organization and maturation

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

[0128] Comparative Example 1

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

[0130] 1. Prepare the culture medium as in Example 2, perform ECM biomimetic coating and hPSCs subculture to obtain a cell suspension;

[0131] 2. Follow Figure 3 2D to 3D culture

[0132] 2.12D cardiomyocyte differentiation

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

[0134] (1) Replace with CM-1 medium and culture at 37°C, 5% CO2 for 36 hours;

[0135] (2) The culture medium was replaced with CM-2 medium and cultured continuously at 37°C and 5% CO2 for 4 days, with the medium changed every day.

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

[0137] (4) The culture medium was replaced with CM-4 medium and cultured continuously at 37°C and 5% CO2 for 21 days, with the medium changed every day. This step is time-consuming and aims to gradually remove undifferentiated stem cells and maintain mature differentiated cardiomyocytes.

[0138] 2.2 2D cardiomyocytes assembled into 3D cell clusters

[0139] (1) Mature differentiated cardiomyocytes were digested with mild enzymes and seeded into AggreWell800 plates (STEMCELL #34850) at a density of 1000 cells / well. The plates were centrifuged at 200 × g for 3 minutes to promote uniform aggregation and cultured in CM-4 medium for 24–48 hours to form uniform cell spheres.

[0140] (2) The cell spheres obtained in step (1) were transferred to ultra-low attachment 6-well plates (Corning #3471) and cultured in CM-4 medium on a shaker (60 rpm) at 37°C and 5% CO2 for at least 2 days, with 50% of the medium replaced daily to obtain mature cardiomyocyte clusters (cardiac organoids primarily containing cardiomyocytes).

[0141] Test Example 1

[0142] Functional characterization of cardiac organoids

[0143] 1. Organoid Beating Recording

[0144] After 10 days of culture in CM-4 medium in Example 2 and 10 days of culture in ultra-low adsorption 6-well plates in step 2.2 of Comparative Example 1, 3D organoids were taken and their autonomous beating function was observed, and the diameter and average autonomous rhythm were calculated. Figure 4 and Figure 5 shown.

[0145] according to Figure 4 and Figure 5 It can be seen that the 3D heart organoids obtained by the culture method of Comparative Example 1 beat autonomously, with an average diameter of 1029 microns and an average autonomous rhythm of 38 beats per minute ( Figure 4 and Figure 5 The 3D heart organoids obtained by the culture method of Example 2 beat autonomously, with an average diameter of 996 microns and an average autonomous rhythm of 51 beats per minute ( Figure 4 and Figure 5 Compared with Comparative Example 1, the number of beats of the 3D cardiac organoids obtained in Example 2 was significantly increased.

[0146] 2. Immunofluorescence Staining

[0147] After 10 days of culture in CM-4 medium in Example 2 and 10 days of culture in ultra-low adsorption 6-well plates in step 2.2 of Comparative Example 1, 3D organoids were taken to make slices, and cardiomyocytes were labeled with cTnT antibodies for fluorescent staining to confirm the stratification and overall structure of the cardiac organoids. Figure 6 shown.

[0148] according to Figure 6 As can be seen, the 3D cardiac organoids obtained using the culture method of Example 2 showed similar fluorescence staining results to those obtained using the culture method of Comparative Example 1. Both contain cardiomyocytes, and the organoids possess a chamber structure similar to that of the heart. The 3D cardiac organoids obtained using the culture method of Example 2 contain other cell types in addition to cardiomyocytes, more fully simulating the cell components of a natural heart.

[0149] Example 3

[0150] Cardiac organoid 3D culture was prepared as in Example 2, except that the concentration of CHIR99021 in the CM-1 culture medium used was different. The concentration of CHIR99021 in the CM-1 culture medium used in this example was 5 μM.

[0151] The diameter of the 3D cardiac organoids obtained by the culture method of this example increased and began to beat at d12.

[0152] Comparative Example 2

[0153] Cardiac organoid 3D culture was prepared in the same manner as in Example 2, except that the concentration of CHIR99021 in the CM-1 culture medium used was different. The concentration of CHIR99021 in the CM-1 culture medium used in this comparative example was 1 μM.

[0154] The 3D heart organoids obtained by the culture method of this comparative example did not beat.

[0155] Comparative Example 3

[0156] Cardiac organoid 3D culture was prepared in the same manner as in Example 2, except that the concentration of CHIR99021 in the CM-1 culture medium used was different. The concentration of CHIR99021 in the CM-1 culture medium used in this comparative example was 2 μM.

[0157] The 3D heart organoids obtained by the culture method of this comparative example did not beat.

[0158] Based on the above content, it can be seen that the technical solution provided by the present invention skips the 2D culture step, and the process from stem cell expansion to organoid maturation is completed in a 3D dynamic environment, successfully culturing 3D cardiac organoids and shortening the 3D cardiac organoid culture cycle.

[0159] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A culture medium for preparing 3D cardiac organoids, characterized in that Including basal medium, CM-1 medium, CM-2 medium, CM-3 medium and CM-4 medium; The basal culture medium includes IMDM culture medium and F-12 culture medium; The CM-1 culture medium comprises 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; The CM-2 culture medium comprises the basal culture medium, bovine serum albumin, β-mercaptoethanol, bone morphogenetic protein, fibroblast growth factor, Tankyrase inhibitor, retinoic acid and insulin-transferrin-selenium complex; The CM-3 culture medium comprises the basal culture medium, bovine serum albumin, β-mercaptoethanol, bone morphogenetic protein, fibroblast growth factor and insulin-transferrin-selenium complex; The CM-4 culture medium comprises the basal culture medium, bovine serum albumin, β-mercaptoethanol and insulin-transferrin-selenium complex; The insulin-transferrin-selenium complex comprises insulin, transferrin and sodium selenite.

2. The culture medium according to claim 1, characterized in that 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 balance of the 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 balance of the 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 balance of the basal culture medium; The CM-4 culture 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 balance of the basic culture medium.

3. The culture medium according to claim 1 or 2, characterized in that The volume ratio of IMDM medium to F-12 medium in the basic 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.

4. Use of the culture medium according to any one of claims 1 to 3 in preparing 3D cardiac organoids.

5. A method for preparing a 3D cardiac organoid, characterized in that: The following steps are involved: After the hPSCs cell suspension was seeded into a low-adsorption 96-well plate, the plate was centrifuged and pre-cultured for 24 hours to obtain a pre-treated plate. replacing the culture medium in the pre-treated well plate with the CM-1 medium in any one of claims 1 to 3, and culturing for 36 to 40 hours to obtain a first treated well plate; replacing the culture medium in the first treatment well plate with the CM-2 culture medium among the culture mediums according to any one of claims 1 to 3, and culturing for a second time for 4 days to obtain a second treatment well plate; replacing the culture medium in the second treatment well plate with the CM-3 culture medium among the culture mediums according to any one of claims 1 to 3, and culturing for a third time for 2 days to obtain a third treatment well plate; The culture medium in the third treatment well plate is replaced with the CM-4 culture medium according to any one of claims 1 to 3, and the culture medium is cultured for at least 2 days to obtain a 3D cardiac organoid.

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

7. The preparation method according to claim 5 or 6, characterized in that: The preparation method of the hPSCs cell suspension comprises: inoculating hPSCs cells in a culture plate coated with vitronectin, culturing with a pre-culture medium, and when the hPSCs confluence reaches 70%, digesting and resuspending to obtain the hPSCs cell suspension; The preculture medium includes 0.5% m / V bovine serum albumin, 15-60 ng / ml fibroblast growth factor and 0.9-3.6 ng / ml TGFβ1.

8. The preparation method according to claim 5, characterized in that The inoculation density is 5000 to 10000 cells / well.

9. The preparation method according to claim 5, characterized in that The first culture was performed at a rotation speed of 50-100 rpm, a temperature of 37° C., and a CO 2 concentration of 5%; The second culture was performed at a rotation speed of 50-100 rpm, a temperature of 37°C, and a CO2 concentration of 5%; The third culture has a rotation speed of >40 rpm and <100 rpm, a temperature of 37° C., and a CO 2 concentration of 5%; The fourth culture was performed at a rotation speed of 50-100 rpm, a temperature of 37° C., and a CO 2 concentration of 5%.

10. The preparation method according to claim 5 or 9, characterized in that: During the second culture process, fresh CM-2 culture medium was replaced every day; During the third culture process, fresh CM-3 culture medium was replaced every day; During the fourth culture, 50% of the fresh CM-4 medium was replaced every day; the replacement of 50% of the fresh differentiation medium was to discard 50% V / V of the old medium and replace it with fresh CM-4 medium.

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