Medium and method for constructing and culturing vascular organoid and application of medium and method

Abstract

The invention relates to the technical field of organs, and discloses a culture medium and a method for constructing and culturing vascular organs and application of the culture medium and the method. By utilizing the culture medium set provided by the invention, the vascular organoid with complete functions and structures can be constructed through iPSC induction, and matrigel does not need to be adopted in the construction and culture process, so that the construction operation of the organoid is greatly simplified, and the cost is saved. The blood vessel organoid constructed by the invention has a basic structure of a blood vessel, has corresponding response to medicine components which are known in the field and have influence on the blood vessel, and can be used as a blood vessel in-vitro model.

Description

Technical Field

[0001] The present invention relates to the field of organ technology, and in particular to a culture medium and method for constructing and culturing vascular organoids and applications thereof. Background Art

[0002] Blood vessel formation is one of the earliest events during embryonic organogenesis. Angioblasts, originating from the mesoderm, first differentiate into endothelial cells. Adjacent endothelial cells connect to form a primary vascular plexus, a process known as angiogenesis. This primary vascular plexus then expands through angiogenesis to form the embryonic vascular network, which further specializes into arteries and veins. However, the pattern of arterial formation during mammalian growth and development is diverse, and the specific arteriovenous specialization process during early embryonic development is still not fully understood.

[0003] All organs require a vascular system for adequate nutrient and oxygen supply, and a significant number of diseases are associated with vascular dysfunction. Therefore, establishing a comprehensive in vitro model of the vascular system is crucial for the development of biomedicine. Wimmer et al. reported the generation of self-organizing three-dimensional human vascular organoids derived from pluripotent stem cells. This method used a type I collagen / Matrigel mixture to induce glomerular sprouting, which acted as a buffer and barrier between the organoids and the plastic material of the well plate. Schmidt et al. disclosed a culture method that does not use Matrigel, using conical agarose coatings in 96-well plates to aggregate pluripotent stem cells and subsequently culture the organoids. However, this method used a single, high-dose of vascular endothelial growth factor (VEGF) for a prolonged period during the vascular induction phase and did not continue the induction of the vascular organoids. This protocol produced fewer, loosely packed mural cells, surrounded by a large number of mesenchymal-like cells, and failed to accurately simulate the morphology and function of blood vessels in vitro.

[0004] Lack of vascularization has been a major obstacle to generating functional and viable biomimetic tissue constructs suitable for clinical and research applications. Researchers have explored vascularization in various organoids, such as vascularized brain organoids, liver, blood-brain barrier, and kidney organoids, but issues such as poor reproducibility, large batch variability, and low throughput remain to be addressed. Summary of the Invention

[0005] The present invention aims to overcome the existing problems of vascular organoid construction, such as the cumbersome process, low throughput, complex culture medium components, high culture costs, and operational difficulties. The present invention provides a culture medium and method for constructing and culturing vascular organoids, as well as their applications. The culture medium provided by the present invention includes a culture medium kit composed of multiple culture media with relatively simple components. During the vascular organoid construction process, the appropriate culture medium is selected based on the construction progress. This convenient and low-cost method can efficiently construct vascular organoids with multiple cell types unique to vascular tissue, forming distinct reticular and tubular structures.

[0006] To achieve the above objectives, the present invention provides, in a first aspect, a culture medium kit for constructing and culturing vascular organoids, the culture medium kit comprising the following culture medium:

[0007] Culture medium A: comprising basal culture medium A and additive I, wherein the basal culture medium A is mTeSR1 culture medium, and the additive I comprises Y-27632;

[0008] Culture medium B: comprising basal culture medium B and additive II, wherein the basal culture medium B is DMEM / F12 culture medium, and the additive II comprises at least one of mTeSR1, non-essential amino acids, β-mercaptoethanol, glutamine, serum, and serum substitute;

[0009] Medium C: comprising medium B and additive III, wherein the additive III comprises CHIR-99021;

[0010] Culture medium D: comprising culture medium B and additive IV, wherein the additive IV comprises BMP-4;

[0011] Culture medium E: comprising culture medium B and additive V, wherein the additive V comprises VEGF-A;

[0012] Culture medium F: comprising culture medium B and additive VI, wherein the additive VI comprises FGF-2;

[0013] Medium G: comprising medium B and additive VII, wherein the additive VII comprises at least one of VEGF-A, FGF-2, and SB43152;

[0014] Culture medium H: comprises culture medium B and additive VIII, wherein the additive VIII comprises at least one of serum, serum replacement, VEGF-A and FGF-2.

[0015] A second aspect of the present invention provides a method for constructing vascular organoids, comprising culturing stem cells in a culture medium to induce them to form vascular organoids, wherein the culture medium is selected from the culture medium described in the first aspect.

[0016] The third aspect of the present invention provides a vascular organoid constructed according to the method described in the second aspect.

[0017] The fourth aspect of the present invention provides the use of the vascular organoid described in the third aspect in drug screening and / or disease modeling.

[0018] The fifth aspect of the present invention provides the culture medium described in the first aspect, and / or the method described in the second aspect of the present invention for use in at least one of improving the success rate of vascular organoid construction, prolonging the culture time of vascular organoids, reducing the cost of vascular organoid construction, increasing the flux of vascular organoid construction and / or not using matrix gel.

[0019] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0020] (1) The culture medium provided by the present invention has simple components and can be selected according to the construction process, which can effectively reduce the amount of additives in the culture medium and reduce the cost of vascular organoid construction.

[0021] (2) The vascular organoids obtained by the method provided by the present invention are composed of multiple cells, and after successful construction, a reticular structure can be clearly seen, and the shape is close to the real vascular morphology. They are suitable for use as in vitro models for research or testing such as screening of vascular drugs, disease modeling, and toxicological testing.

[0022] (3) The method provided by the present invention does not require the use of matrix gel and can be completed by directly placing the culture in the culture medium, thereby better protecting the culture and enabling it to be maintained in culture for a longer period of time. In addition, since the use of matrix gel significantly increases the cost of organoid culture, the method of the present invention can reduce the culture cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a schematic diagram of the process for constructing vascular organoids in Example 1;

[0024] Figure 2 is a morphological diagram of the vascular organoid constructed in Example 1 at different culture stages;

[0025] Figure 3 These are the HE, MASSON, and VG staining results of paraffin sections of the vascular organoids constructed in Example 1;

[0026] Figure 4 This is a diagram showing the results of multiple immunofluorescence staining of paraffin sections of vascular organoids constructed in Example 1;

[0027] Figure 5 This is the overall immunofluorescence staining result of the vascular organoid constructed in Example 1;

[0028] Figure 6 This is a graph showing the CD31 immunofluorescence staining results of the vascular organoid constructed in Example 1;

[0029] Figure 7 1 is the immunofluorescence staining result of CD31, Calponin, PDGF and SMA of the vascular organoid constructed in Example 1;

[0030] Figure 8 This is a graph showing the immunofluorescence staining results of F4 / 80 and CD4 of the vascular organoid constructed in Example 1;

[0031] Figure 9 This is a diagram showing the results of immunofluorescence staining of tissue clearing of the vascular organoid constructed in Example 1;

[0032] Figure 10 is a transmission electron micrograph of the vascular organoid constructed in Example 1;

[0033] Figure 11 This is a graph showing the results of CD31 immunofluorescence staining of vascular organoids after long-term culture in Example 3;

[0034] Figure 12 This is a graph showing the vitality trend of vascular organoids treated with norepinephrine in Test Example 1;

[0035] Figure 13 This is a trend chart showing the restoration of vascular organoid vitality using Tanshinone IIA in Test Example 1. DETAILED DESCRIPTION

[0036] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0037] The inventors of the present invention cleverly discovered in their research that during the construction of vascular organoids, by using culture media containing different small molecule additives in accordance with the differentiation conditions of the organoids, the construction process of vascular organoids can be effectively simplified, and culture conditions and equipment can be optimized, thereby reducing costs and improving the convenience of vascular organoid construction, providing a basis for the widespread application of organoids in disease modeling and drug development.

[0038] A first aspect of the present invention provides a culture medium kit for constructing and culturing vascular organoids, the culture medium kit comprising the following culture medium:

[0039] Culture medium A: comprising basal culture medium A and additive I, wherein the basal culture medium A is mTeSR1 culture medium, and the additive I comprises Y-27632;

[0040] Culture medium B: comprising basal culture medium B and additive II, wherein the basal culture medium B is DMEM / F12 culture medium, and the additive II comprises at least one of mTeSR1, non-essential amino acids, β-mercaptoethanol, glutamine, serum, and serum substitute;

[0041] Medium C: comprising medium B and additive III, wherein the additive III comprises CHIR-99021;

[0042] Culture medium D: comprising culture medium B and additive IV, wherein the additive IV comprises BMP4;

[0043] Culture medium E: comprising culture medium B and additive V, wherein the additive V comprises VEGF-A;

[0044] Culture medium F: comprising culture medium B and additive VI, wherein the additive VI comprises FGF-2;

[0045] Medium G: comprising medium B and additive VII, wherein the additive VII comprises at least one of VEGF-A, FGF-2, and SB43152;

[0046] Culture medium H: comprises culture medium B and additive VIII, wherein the additive VIII comprises at least one of serum, serum replacement, VEGF-A and FGF-2.

[0047] In the present invention, mTeSR1 medium is a commercially available medium with a defined formula. Those skilled in the art can purchase it commercially or prepare it themselves according to its published formula.

[0048] Y-27632 is a RHO / ROCK pathway inhibitor with a CAS number of 146986-50-7 and a structural formula as shown in the following formula (1).

[0049]

[0050] In the present invention, DMEM / F12 medium is a commercially available medium with a defined formula. Those skilled in the art can purchase it commercially or prepare it themselves according to its disclosed formula.

[0051] Non-essential amino acids (NEAAs) are commercially available, defined complex amino acid preparations that primarily include seven non-essential amino acids required for cell culture. The non-essential amino acids used in this invention can be purchased directly from a commercial source or prepared using a custom formulation.

[0052] β-Mercaptoethanol can provide a source of sulfur groups, has a CAS number of 60-24-2, and has a structural formula as shown in the following formula (2).

[0053]

[0054] Serum substitutes are cell / tissue in vitro culture additives with clear ingredients used in the organoid culture process that can replace FBS. The purpose of using serum substitutes in the present invention is to replace fetal bovine serum (FBS), and any compound or composition that can achieve this goal can be used in the present invention. The present invention does not particularly limit the source of the serum substitute used. It can be directly obtained commercially or prepared by itself according to its formula, as long as it has the aforementioned efficacy. For example, commercially available KnockOut serum substitute reagent (also known as Knockout SR, abbreviated as KOSR) can be used, which is a cell culture additive whose ingredients clearly do not contain fetal bovine serum (FBS).

[0055] Glutamine is a non-essential amino acid involved in metabolic processes and provides a carbon source for oxidation reactions in certain cells. The CAS number is 56-85-9, and the structural formula is shown in the following formula (3). Generally, when performing organoid culture, glutamine can be used directly, or its substitutes (such as L-alanyl-L-glutamine dipeptide, etc.) can be used to provide glutamine. In the present invention, commercially available glutamine or its substitutes can be used, or related products prepared by oneself can be used. For example, commercially available GlutaMAX additive (the main component of which is L-alanyl-L-glutamine dipeptide) can be used to provide glutamine for the culture medium.

[0056]

[0057] CHIR-99021 is a GSK-3α / β inhibitor with a CAS number of 252917-06-9 and a structural formula as shown in the following formula (4).

[0058]

[0059] BMP-4 (bone morphogenetic protein 4) is a pleiotropic ligand protein belonging to the TGF-β family. It participates in vascular system circulation and can activate receptors on vascular cells.

[0060] VEGF-A is a member of the vascular endothelial growth factor (VEGF) family, produced by alternative splicing or proteolytic cleavage, with distinct receptor-binding and matrix-binding properties.

[0061] FGF is the abbreviation of fibroblast growth factor, which is a general term for a family of proteins that can bind to fibroblast growth factor receptors, including multiple different proteins such as FGF1 to FGF21.

[0062] SB43152 is a TGF-β receptor kinase inhibitor with a CAS number of 301836-41-9 and a structural formula as shown in the following formula (5).

[0063]

[0064] Serum is a rich source of proteins and growth factors that support cell growth. Fetal bovine serum (FBS) can be used in the present invention.

[0065] According to the present invention, preferably, in culture medium A, the amount of additive I is such that the final concentration of Y-27632 in culture medium A is not less than 5 μM, preferably 5-50 μM.

[0066] For example, in culture medium A, the final concentration of Y-27632 can be 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 18 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, or 50 μM, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0067] According to the present invention, preferably, in medium B, the amount of additive II is such that the concentration of non-essential amino acids in medium B is not less than 1% by weight, preferably 1-3% by weight; the final concentration of mTeSR1 is not less than 10% by weight, preferably 10-20% by weight; the final concentration of β-mercaptoethanol is not less than 1×10 -6 Volume%, preferably 1×10 -6 -1×10 -4 volume %; the final concentration of serum and / or serum substitute is not less than 10% by weight, preferably 10-20% by weight; the final concentration of glutamine is not less than 1 mM, preferably 1-5 mM.

[0068] For example, in culture medium B, the final concentration of non-essential amino acids can be 1 weight%, 1.2 weight%, 1.4 weight%, 1.6 weight%, 1.8 weight%, 2 weight%, 2.2 weight%, 2.4 weight%, 2.6 weight%, 2.8 weight%, or 3 weight%, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0069] For example, in culture medium B, the final concentration of mTeSR1 can be 10 weight%, 11 weight%, 12 weight%, 13 weight%, 14 weight%, 15 weight%, 16 weight%, 17 weight%, 18 weight%, 19 weight%, or 20 weight%, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0070] For example, in medium B, the final concentration of β-mercaptoethanol can be 1×10 -6 Volume%, 2×10 -6 Volume%, 3×10 -6 Volume%, 4×10 -6 Volume%, 5×10 -6 Volume%, 6×10 -6 Volume%, 7×10 -6 Volume%, 8×10 -6 Volume%, 9×10 -6 Volume%, 1×10 -5 Volume%, 2×10 -5 Volume%, 3×10 -5 Volume%, 4×10 -5 Volume%, 5×10 -5 Volume%, 6×10 -5 Volume%, 7×10 -5 Volume%, 8×10 -5 Volume%, 9×10 -5 Volume%, 1×10 -4 The content may be in a range of any two of the above values, or any intermediate value in the range.

[0071] For example, the final concentration of serum and / or serum replacement in culture medium B can be 10% by weight, 11% by weight, 12% by weight, 13% by weight, 14% by weight, 15% by weight, 16% by weight, 17% by weight, 18% by weight, 19% by weight, or 20% by weight, or can be a range consisting of any two of the above values, or any intermediate value in the range.

[0072] For example, in culture medium B, the final concentration of glutamine can be 1 mM, 1.2 mM, 1.4 mM, 1.6 mM, 1.8 mM, 2 mM, 2.2 mM, 2.4 mM, 2.6 mM, 2.8 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, or 5 mM, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0073] According to the present invention, preferably, in the culture medium C, the amount of additive III used is such that the final concentration of CHIR-99021 in the culture medium C is not less than 5 μM, preferably 5-20 μM.

[0074] For example, in culture medium C, the final concentration of CHIR-99021 can be 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 11 μM, 12 μM, 13 μM, 14 μM, 15 μM, 18 μM, or 20 μM, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0075] According to the present invention, preferably, the amount of additive IV in culture medium D is such that the final concentration of BMP-4 in culture medium D is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL.

[0076] For example, the final concentration of BMP-4 in culture medium D can be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or can be a range consisting of any two of the above values, or any intermediate value in the range.

[0077] According to the present invention, preferably, the amount of additive V in culture medium E is such that the final concentration of VEGF-A in culture medium E is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL.

[0078] For example, in culture medium E, the final concentration of VEGF-A can be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0079] According to the present invention, preferably, in culture medium F, the amount of additive VI is such that the final concentration of FGF-2 in culture medium VI is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL.

[0080] For example, in culture medium F, the final concentration of FGF-2 can be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0081] According to the present invention, preferably, in culture medium G, the amount of additive VII is such that the final concentration of VEGF-A in culture medium VII is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; the final concentration of FGF-2 is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; and the final concentration of SB43152 is not less than 5 μM, preferably 5 μM-50 μM.

[0082] For example, in culture medium G, the final concentration of FGF-2 can be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0083] For example, in culture medium E, the final concentration of VEGF-A can be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0084] For example, in culture medium G, the final concentration of SB43152 can be 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 40 μM, or 50 μM, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0085] According to the present invention, preferably, in culture medium H, the amount of additive VIII is such that the final concentration of VEGF-A in culture medium VIII is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; the final concentration of FGF-2 is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; and the final concentration of serum and / or serum substitute is not less than 5% by weight, preferably 5-15% by weight.

[0086] For example, the final concentration of FGF-2 in culture medium H can be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0087] For example, the final concentration of VEGF-A in culture medium H can be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, or 100 ng / mL, or can be a range consisting of any two of the above values, or any intermediate value in the range.

[0088] For example, the final concentration of serum and / or serum replacement in culture medium H can be 10% by weight, 11% by weight, 12% by weight, 13% by weight, 14% by weight, 15% by weight, 16% by weight, 17% by weight, 18% by weight, 19% by weight, or 20% by weight, or can be a range consisting of any two of the above values, or any intermediate value in the range.

[0089] According to the present invention, preferably, each culture medium in the culture medium set further contains antibiotics.

[0090] More preferably, the antibiotic is selected from penicillin and / or streptomycin. Preferably, the antibiotics in each culture medium may be the same or different.

[0091] In the present invention, a variety of different antibiotics that can be used for in vitro culture of cells, organoids, etc. (such as penicillin, streptomycin, etc.) can be used in combination, and commercially available compound antibiotic products can also be used.

[0092] In the present invention, there is no particular limitation on the dosage of antibiotics, as long as it ensures that the organoids are not susceptible to bacterial infection during construction and culture. Those skilled in the art will be able to determine the dosage based on the actual antibiotic selected, or refer to the instructions of commercially available antibiotic products.

[0093] A second aspect of the present invention provides a method for constructing vascular organoids, comprising culturing pluripotent stem cells in a culture medium to induce them to form vascular organoids, wherein the culture medium is selected from the culture medium described in the first aspect.

[0094] According to the present invention, preferably, the stem cells are pluripotent stem cells, preferably induced pluripotent stem cells.

[0095] In the present invention, pluripotent stem cells can be any pluripotent stem cell known in the art for use in organoid construction, such as commercially available pluripotent stem cells or those prepared in-house using existing techniques. The present invention also does not particularly limit the type of pluripotent stem cells; for example, induced pluripotent stem cells (iPSCs) can be used.

[0096] According to the present invention, preferably, the method comprises the following steps:

[0097] (1) Embryoid culture: Pluripotent stem cells are inoculated into medium A for the first culture to obtain embryoid bodies;

[0098] (2) Optional pre-differentiation preparation: inoculating the embryoid bodies obtained in step (1) into culture medium B for an optional second culture to obtain differentiated embryoid bodies;

[0099] (3) Vascular organoid differentiation, including:

[0100] (3-1) Vascular organoid differentiation I: The embryoid bodies obtained in step (1) or the differentiated embryoid bodies obtained in step (2) are inoculated into culture medium C for a third culture to obtain a first-stage culture;

[0101] (3-2) Vascular organoid differentiation II: The first-stage culture obtained in step (3-1) was inoculated into medium D for a fourth culture to obtain a second-stage culture;

[0102] (3-3) Vascular organoid differentiation III: The second-stage culture obtained in step (3-2) was inoculated into medium E for fifth culture to obtain a third-stage culture;

[0103] (3-4) Vascular organoid differentiation IV: The third stage culture obtained in step (3-3) is inoculated into medium F for the sixth culture to obtain a fourth stage culture;

[0104] (3-5) Vascular organoid differentiation V: The fourth stage culture obtained in step (3-4) is inoculated into medium G for seventh culture to obtain a fifth stage culture;

[0105] (3-6) Vascular organoid differentiation VI: The fifth stage culture obtained in step (3-5) is inoculated into medium H for eighth culture to obtain vascular organoids;

[0106] Preferably, the method further comprises:

[0107] (4) Maintenance culture of vascular organoids: The vascular organoids obtained in steps (3-6) are cultured long-term using culture medium H.

[0108] In the present invention, vascular organoids prepared using the above-described method can be cultured and maintained long-term, maintaining their corresponding functions. After extensive research, the inventors discovered that the inclusion of serum in Medium H is more conducive to prolonging the culture time of vascular organoids, while the use of serum and serum replacement in Medium B provides similar results. Based on this, preferably, in the culture medium set of the present invention, Medium B can contain serum and / or serum replacement, while Medium H contains serum.

[0109] According to the present invention, preferably, in step (1), the culturing time of the first culturing is 24-100 h.

[0110] Further preferably, during the first culture process, culture medium A is replaced every 20-30 hours.

[0111] According to the present invention, preferably, in step (2), the second culture time is 20-30 hours.

[0112] According to the present invention, preferably, in step (3-1), the third culture time is 20-30 hours.

[0113] According to the present invention, preferably, in step (3-2), the fourth culture time is 40-50 hours.

[0114] According to the present invention, preferably, in step (3-3), the fifth culture time is 40-50 hours.

[0115] According to the present invention, preferably, in step (3-4), the sixth culture time is 40-50 hours.

[0116] According to the present invention, preferably, in step (3-5), the seventh culture time is 40-50 hours.

[0117] According to the present invention, preferably, in step (3-6), the eighth culture time is 40-50 hours.

[0118] According to the present invention, preferably, in step (4), the long-term culture time is 1-3 months.

[0119] More preferably, during long-term culture, the culture medium H is replaced every 20-30 hours.

[0120] According to the present invention, preferably, the method further comprises removing the culture medium and washing the culture before each step. Since each step uses a culture medium containing different additives, the purpose of the washing process is mainly to remove the culture medium remaining on the cells.

[0121] According to the present invention, preferably, the method does not include the process of embedding the culture obtained in each step using matrigel. When organoids are constructed in this area, it is usually necessary to embed the culture using matrigel to provide nutrients and to support the maintenance of the culture morphology. However, the presence of matrigel makes the operation of organoid construction more complicated, and the matrigel needs to be replaced regularly during the culture process. Temperature changes and mechanical forces during the replacement process can damage the culture and affect the response of the culture to drugs, causing certain experimental errors. In addition, the use of matrigel also significantly increases the cost of organoid culture. After extensive research, the inventors of the present invention have found that when vascular organoids are constructed and cultured using the method and culture medium provided by the present invention, there is no need to use matrigel, and the culture can be directly placed in the culture medium to complete, so that the present invention can better protect the culture and maintain it for a longer time. The method and culture medium of the present invention can reduce the cost of culture and promote the response of the culture to drugs.

[0122] The third aspect of the present invention provides a vascular organoid constructed according to the method described in the second aspect.

[0123] The fourth aspect of the present invention provides the use of the vascular organoid described in the third aspect in drug screening and / or disease modeling.

[0124] In the present invention, the vascular organoids comprise at least endothelial cells, fibroblasts, pericytes, and smooth muscle cells, and their shape resembles that of a real blood vessel, presenting an elongated tubular structure. These cells and structures can be detected and confirmed using conventional techniques in the art. For example, immunofluorescence assays can be used to detect various cell markers in the obtained vascular organoids, and transmission electron microscopy can be used to observe the microstructure of the vascular organoids to confirm their presence.

[0125] The vascular organoids obtained using the methods provided herein are composed of multiple cell types, possess the basic structural and functional properties of blood vessels, and respond appropriately to pharmaceutical ingredients known in the art to affect vascular function. Therefore, the vascular organoids provided by the present invention can be used as in vitro models for screening vascular drugs and / or disease modeling.

[0126] The fifth aspect of the present invention provides the culture medium described in the first aspect, and / or the method described in the second aspect of the present invention for use in at least one of improving the success rate of vascular organoid construction, prolonging the culture time of vascular organoids, reducing the cost of vascular organoid construction, increasing the flux of vascular organoid construction and / or not using matrix gel.

[0127] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0128] In the following examples, iPSCs (induced pluripotent stem cells) were purchased from Beijing Saibei Biotechnology Co., Ltd.; serum replacement was provided by KOSR reagent, which was purchased from Gibco with a catalog number of 10828028; non-essential amino acids were provided by NEAA reagent, which was purchased from Gibco with a catalog number of 11140050; β-mercaptoethanol was purchased from Gibco with a catalog number of 31350010; glutamine was provided by GlutaMAX reagent, which was purchased from Gibco with a catalog number of 25030149; CHIR-99021 Purchased from MCE Company, catalog number HY10182; Y-27632 was purchased from MCE Company, catalog number HY-10071; SB43152 was purchased from MCE Company, catalog number HY-10431; FGF-2 was purchased from MCE Company, catalog number HY-P7004; VEGF-A was purchased from MCE Company, catalog number HY-P7429; BMP-4 was purchased from MCE Company, catalog number HY-P7007; serum (FBS) was purchased from Gibco, catalog number A5670701.

[0129] Other reagents or materials not otherwise specified were commercially available from regular chemical or biological reagent / material suppliers, and all reagents were of analytical grade.

[0130] Preparation Example 1

[0131] Additives were weighed or measured according to the final concentrations listed in Table 1 and added to the basal medium to prepare a medium set consisting of medium A, B, C, D, E, F, G, and H. Each medium contained 1 wt% penicillin-streptomycin solution (Gibco, 15140122).

[0132] Table 1

[0133]

[0134]

[0135] Example 1

[0136] This example is used to illustrate the effect of the method provided by the present invention on the construction of vascular organoids.

[0137] (1) Construction of vascular organoids

[0138] The culture medium formula used is shown in Table 1. Figure 1 The culture process of vascular organoids is as follows:

[0139] (1) Embryoid culture: The pre-cultured iPSCs were washed three times with preheated DPBS, digested and separated into single cell suspensions, and prepared into a cell suspension of 10,000 cells / 150 μL using culture medium A. The cell suspension was then added to a 96-well ultra-low attachment plate (U-shaped bottom) (200 μL per well). At this time, the cell morphology in the culture plate was as follows: Figure 2 As shown in DAY0 in the figure.

[0140] After inoculation, the culture plate was centrifuged at 300g for 5 min at 4°C and then placed in a 37°C incubator (5% CO2) for 48 h. Cell morphology changes were observed every 24 h. Figure 2 As shown in Day 2, spherical cell aggregates were formed after 24 hours of culture. After 48 hours, the culture medium was removed from the culture plate and 200 μL / well of culture medium A was added. Culture was continued for another 24 hours to obtain embryoid bodies.

[0141] (2) Preparation before differentiation: The embryoid bodies obtained in step (1) were inoculated into medium A for a second culture to prepare for differentiation. This stage lasted for 24 hours.

[0142] (3) Vascular organoid differentiation

[0143] (3-1) The inducer obtained in step (1) or (2) was inoculated into medium C for the third culture, and then placed in a 37°C incubator (5% CO2) for 24 h to obtain the first stage culture (morphology reference Figure 2 DAY4 in the

[0144] (3-2) Vascular organoid differentiation II: The first stage culture obtained in step (3-1) was inoculated into medium D for the fourth culture and cultured in a 37°C incubator (5% CO2) for 48 h to obtain the second stage culture (morphology reference Figure 2 DAY6 in the video);

[0145] (3-3) Vascular organoid differentiation III: The second stage culture obtained in step (3-2) was inoculated into medium E for the fifth culture and cultured in a 37°C incubator (5% CO2) for 48 h to obtain the third stage culture (morphology reference Figure 2 DAY8 in the video);

[0146] (3-4) Vascular organoid differentiation IV: The third stage culture obtained in step (3-3) was inoculated into medium F for the sixth culture and cultured in a 37°C incubator (5% CO2) for 48 h to obtain the fourth stage culture (morphology reference Figure 2 DAY10 in the video);

[0147] (3-5) Vascular organoid differentiation V: The fourth stage culture obtained in step (3-4) was inoculated into medium G for the seventh culture and placed in a 37°C incubator (5% CO2) for 48 hours to obtain the fifth stage culture (morphology reference Figure 2 DAY12 in the figure), at this time, the shape change of the spheroid can be gradually observed under bright field;

[0148] (3-6) Vascular organoid differentiation VI: The fifth stage culture obtained in step (3-5) was inoculated into medium H for the eighth culture and placed in a 37°C incubator (5% CO2) for 48 hours to obtain vascular organoids. At this time, the spheroids can become a network-like 3D structure (morphology reference Figure 2 DAY14 in the experiment), which preliminarily indicated that the construction of vascular organoids was completed;

[0149] (4) Maintenance culture of vascular organoids: The vascular organoids obtained in steps (3-6) were cultured for a long time using culture medium H (morphology reference Figure 2 DAY20 in the video).

[0150] (II) Structural and functional testing of vascular organoids

[0151] (1) Paraffin sections of vascular organoids were stained with HE, MASSON, and VG. Figure 3 As for the staining results, in the HE staining sample, the outline of the vascular organoids is clear, the cells are densely distributed, and the cell nuclei are obviously blue-purple, which indicates that the tissue structure remains intact and the cell nucleus and cytoplasm can be clearly distinguished; the Masson staining sample shows the collagen fibers in the connective tissue in the vascular organoids; the VG staining sample shows the distribution of a small amount of collagen fibers in the vascular organoids.

[0152] (2) Multiple immunofluorescence staining of paraffin sections was used to detect other cell-specific markers in the vascular organoids obtained by the above method. Figure 4As shown in the figure, blue is the DAPI staining result, representing the position of the cell nucleus, CD31 (vascular endothelial cells), SMA (one of the smooth muscle cell markers) and VEGF (vascular endothelial growth factor) staining results are green, Calponin (one of the smooth muscle cell markers) and PDGF (platelet-derived growth factor) staining results are red; the rightmost (Merge) is a fusion image of multiple staining results. Vascular organoids contain not only vascular endothelial cells (marked by CD31), but also smooth muscle cells and matrix-related components (marked by Calponin and SMA). In addition, the expression of PDGF and VEGF shows their potential activity in angiogenesis and tissue repair.

[0153] (3) Whole-body immunofluorescence staining was used to detect cell-specific markers in the vascular organoids obtained by the above method. Figure 5 As shown in the figure, the blue color is the DAPI staining result, which represents the location of the cell nucleus, the green color is the VE-Cadherin (vascular endothelial cadherin) staining result, and the red color is the α-SMA (a smooth muscle cell marker) staining result. It can be seen that there are clear vascular endothelial cells (marked by VE-Cadherin) and smooth muscle cells (marked by α-SMA) in the vascular organoids, and they are distributed in different regions of the vascular organoids.

[0154] (4) Continue to use immunofluorescence to detect cell-specific markers in the vascular organoids obtained by the above method. Figure 6 The CD31 immunofluorescence staining results of vascular organoids are shown, in which the red signal of CD31 (vascular endothelial cells) is relatively clear and concentrated, showing a morphological structure similar to a vascular network, and the lumen can be clearly seen. Figure 7 In the figure, red represents endothelial cells, blue represents cell nuclei, and calponin (a smooth muscle cell marker) is labeled with green fluorescence and is mainly distributed in the peripheral area of ​​the organoid, representing the distribution of smooth muscle cells. PDGF (a pericyte marker) is represented by green fluorescence, indicating the presence of pericytes in the vascular organoids. α-SMA (a smooth muscle cell marker) is green fluorescence, further confirming the presence of smooth muscle cells in the organoids. The presence of multiple blood vessel and smooth muscle-related markers in vascular organoids demonstrates the formation of vascular-like structures and their biological activity, with good angiogenesis and repair potential, which makes vascular organoids have potential application value in the fields of regenerative medicine and tissue engineering.

[0155] (5) Immunofluorescence staining of F4 / 80 and CD4 was used to detect other cell-specific markers in the vascular organoids obtained by the above method. Figure 8As shown, the F4 / 80 marker is used to identify macrophages, and the CD4 marker is used to identify helper T cells. Both macrophages (F4 / 80 positive) and T cells (CD4 positive) are distributed within the vascular organoids, suggesting that these immune cells may participate in the organoid's immune regulation or repair processes. DAPI nuclear labeling shows a relatively uniform distribution of cells within the organoids, with immune cells clustering in specific areas of the vascular organoids, suggesting that their immune function may be more active in these areas.

[0156] (6) The overall structure of blood vessels in vascular organoids was observed using tissue clearing technology and immunofluorescence technology. Figure 9 As shown, CD31-positive signals are distributed along the vessel wall, demonstrating the continuity and integrity of the vessel-like structure. The α-SMA signal clearly shows the presence of smooth muscle cells surrounding the vessel wall, indicating that the presence of these smooth muscle cells supports the contractile function and physical stability of the vessel-like structure. Calponin signals are located at the periphery of the vessel-like structure, indicating that these cells may provide mechanical support and contractile function to vascular organoids, ensuring the integrity and functionality of the vascular system. The signal distribution of VEGF and PDGF reveals the key role of these factors in promoting endothelial cell proliferation and angiogenesis.

[0157] (7) Transmission electron microscopy was used to observe the microstructure of vascular organoids. Figure 10 As shown, the ultrastructure of vascular organoids includes an intact endothelial cell layer, a smooth muscle cell layer, and a basement membrane. The vascular organoids obtained by this method possess similar organizational structure and functional properties to in vivo microvessels. The tight junctions of the endothelial cells, the contractile ability of the smooth muscle cells, and the presence of the basement membrane all demonstrate that the organoids possess favorable biomimetic characteristics.

[0158] (III) Success rate of vascular organoid culture

[0159] The development of vascular organoids was observed using bright-field microscopy, and it was found that within 15 days of culture, the average construction success rate of vascular organoids on each 96-well ultra-low adsorption plate exceeded 90% (construction success rate = number of vascular organoids successfully constructed on a single 96-well ultra-low adsorption plate / number of wells inoculated with iPSCs on a single 96-well ultra-low adsorption plate × 100%).

[0160] Example 2

[0161] according to Figure 11As shown, this example describes a method for detecting the morphological structure of vascular organoids and the expression of the specific marker CD31 (endothelial cell marker) after long-term culture (40 days) to verify that the method of the present invention can achieve long-term stable culture of vascular organoids. On the 40th day of culture, the vascular organoids were able to maintain a complete three-dimensional structure, with regular cell arrangement and clear vascular morphology. Over time, the vascular network became more complex, with an increased number of branches and a stable network structure.

[0162] This example demonstrates that the method of the present invention can achieve long-term stable culture of vascular organoids while maintaining the normal morphology and functionality of the organoids through the expression detection of the endothelial cell marker CD31 and the analysis of their shape characteristics after long-term culture. Figure 11 The results shown in this paper indicate that the present invention is suitable for the development and application of long-term biological tissue models, including the fields of drug screening and regenerative medicine.

[0163] Example 3

[0164] Vascular organoids were constructed according to the method of Example 1, except that the pre-differentiation preparation of step (2) was omitted and the vascular organoids were directly cultured for differentiation. Ultimately, vascular organoids with a structure similar to that of Example 1 were obtained. The overall culture time was shortened by 2 days, and the culture success rate remained unchanged, without affecting the subsequent differentiation process.

[0165] Test Example 1

[0166] This test example is used to illustrate the response of the vascular organoids provided by the present invention to drugs.

[0167] This experiment uses Assay (Promega, catalog number: G7572) was used to detect the ATP levels in different drug-treated groups.

[0168] First, the vascular organoids obtained in Example 1 were treated with norepinephrine, and then the ATP levels of the treated organoids were detected. The results showed that when the final concentration was in the range of 100-150 μM, the activity of the vascular organoids was significantly reduced after treatment with norepinephrine, as shown in Figure 2. Figure 11 After 24 hours of intervention with 150 μM norepinephrine in vascular organoids, the vascular organoids were treated with tanshinone ⅡA, and the ATP levels of the organoids were tested again. The results are shown in FIG. Figure 12 It can be seen that the comparison Figure 11 and Figure 12 The results show that Tanshinone IIA has the effect of restoring the vitality of organoids.

[0169] It can be seen that the vascular organoids provided by the present invention have a good response to drugs and can be used as an in vitro model for drug screening.

[0170] The same experiment was conducted using vascular organoids cultured for 40 days, and the results were similar to those of Figure 11-12 Similar results were obtained in the previous study, indicating that the vascular organoids of the present invention can still maintain a good response to drugs after long-term culture.

[0171] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A culture medium kit for constructing and culturing vascular organoids, characterized in that: The culture medium set includes the following culture medium: Culture medium A: comprising basal culture medium A and additive I, wherein the basal culture medium A is mTeSR1 culture medium, and the additive I comprises Y-27632; Culture medium B: comprising basal culture medium B and additive II, wherein the basal culture medium B is DMEM / F12 culture medium, and the additive II comprises at least one of mTeSR1, non-essential amino acids, β-mercaptoethanol, glutamine, serum, and serum substitute; Medium C: comprising medium B and additive III, wherein the additive III comprises CHIR-99021; Culture medium D: comprising culture medium B and additive IV, wherein the additive IV comprises BMP-4; Culture medium E: comprising culture medium B and additive V, wherein the additive V comprises VEGF-A; Culture medium F: comprising culture medium B and additive VI, wherein the additive VI comprises FGF-2; Medium G: comprising medium B and additive VII, wherein the additive VII comprises at least one of VEGF-A, FGF-2, and SB43152; Culture medium H: comprises culture medium B and additive VIII, wherein the additive VIII comprises at least one of serum, serum replacement, VEGF-A and FGF-2.

2. The culture medium set according to claim 1, wherein: In medium A, the amount of additive I is such that the final concentration of Y-27632 in medium A is not less than 5 μM, preferably 5-50 μM; and / or, in medium B, the amount of additive II is such that the concentration of non-essential amino acids in medium B is not less than 1% by weight, preferably 1-3% by weight; the final concentration of mTeSR1 is not less than 10% by weight, preferably 10-20% by weight; the final concentration of β-mercaptoethanol is not less than 1×10 -6 Volume%, preferably 1×10 -6 -1×10 -4 volume %; the final concentration of serum and / or serum substitute is not less than 10% by weight, preferably 10-20% by weight; the final concentration of glutamine is not less than 1 mM, preferably 1-5 mM; and / or, in medium C, the amount of additive III is such that the final concentration of CHIR-99021 in medium C is not less than 5 μM, preferably 5-20 μM; and / or, in the culture medium D, the amount of additive IV is such that the final concentration of BMP-4 in the culture medium D is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; and / or, in the culture medium E, the amount of additive V is such that the final concentration of VEGF-A in the culture medium E is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; and / or, in culture medium F, the amount of additive VI is such that the final concentration of FGF-2 in culture medium VI is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; and / or, in medium G, the amount of additive VII is such that the final concentration of VEGF-A in medium VII is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; the final concentration of FGF-2 is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; and the final concentration of SB43152 is not less than 5 μM, preferably 5 μM-50 μM; And / or, in culture medium H, the amount of additive VIII is such that the final concentration of VEGF-A in culture medium VIII is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; the final concentration of FGF-2 is not less than 10 ng / mL, preferably 10 ng / mL-100 ng / mL; and the final concentration of serum and / or serum substitute is not less than 5 weight%, preferably 5-15 weight%.

3. The culture medium set according to claim 1 or 2, wherein: Each culture medium in the culture medium set also contains antibiotics; Preferably, the antibiotic is selected from penicillin and / or streptomycin.

4. A method for constructing vascular organoids, characterized in that: The method comprises culturing stem cells in a culture medium to induce them to form vascular organoids, wherein the culture medium is selected from the culture medium according to any one of claims 1 to 3.

5. The method according to claim 4, wherein The stem cells are pluripotent stem cells, preferably induced pluripotent stem cells; Preferably, the method comprises the following steps: (1) Embryoid culture: Pluripotent stem cells are inoculated into medium A for the first culture to obtain embryoid bodies; (2) Optional pre-differentiation preparation: inoculating the embryoid bodies obtained in step (1) into culture medium B for an optional second culture to obtain differentiated embryoid bodies; (3) Vascular organoid differentiation, including: (3-1) Vascular organoid differentiation I: The embryoid bodies obtained in step (1) or the differentiated embryoid bodies obtained in step (2) are inoculated into culture medium C for a third culture to obtain a first-stage culture; (3-2) Vascular organoid differentiation II: The first-stage culture obtained in step (3-1) was inoculated into medium D for a fourth culture to obtain a second-stage culture; (3-3) Vascular organoid differentiation III: The second-stage culture obtained in step (3-2) was inoculated into medium E for fifth culture to obtain a third-stage culture; (3-4) Vascular organoid differentiation IV: The third stage culture obtained in step (3-3) is inoculated into medium F for the sixth culture to obtain a fourth stage culture; (3-5) Vascular organoid differentiation V: The fourth stage culture obtained in step (3-4) is inoculated into medium G for seventh culture to obtain a fifth stage culture; (3-6) Vascular organoid differentiation VI: The fifth stage culture obtained in step (3-5) is inoculated into medium H for eighth culture to obtain vascular organoids; Preferably, the method further comprises: (4) Maintenance culture of vascular organoids: The vascular organoids obtained in steps (3-6) are cultured long-term using culture medium H.

6. The method according to claim 5, wherein: In step (1), the first culture is performed for 24-100 hours; Preferably, during the first culture process, culture medium A is replaced every 20-30 hours; And / or, in step (2), the second culture time is 20-30h; And / or, in step (3-1), the third culture time is 20-30h; And / or, in step (3-2), the fourth culture time is 40-50h; And / or, in step (3-3), the fifth culture time is 40-50h; And / or, in step (3-4), the sixth culture time is 40-50h; And / or, in step (3-5), the seventh culture time is 40-50h; And / or, in step (3-6), the eighth culture time is 40-50h; And / or, in step (4), the long-term culture time is 1-3 months; Preferably, during long-term culture, the culture medium H is replaced every 20-30 hours.

7. The method according to claim 5 or 6, wherein: The method further comprises removing the culture medium and washing the culture before each step; And / or, the method does not include embedding the culture obtained in each step with matrix gel.

8. A vascular organoid constructed and obtained according to the method of any one of claims 4 to 7.

9. Use of the vascular organoid according to claim 8 in drug screening and / or disease modeling.

10. Use of the culture medium of any one of claims 1-3, and / or the method of any one of claims 5-7, in at least one of improving the success rate of vascular organoid construction, prolonging the culture time of vascular organoids, reducing the cost of vascular organoid construction, increasing the throughput of vascular organoid construction, and / or eliminating the use of matrix gel.

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