Hydrogel for myocardial organoid culture and preparation method thereof

Abstract

The invention provides hydrogel for myocardial organoid culture and a preparation method of the hydrogel. A three-dimensional network structure of the hydrogel comprises hyaluronic acid and alginic acid; the hyaluronic acid is double-bond modified hyaluronic acid. The hydrogel has good long-term stability, can shear and remodel surrounding networks in the growth process of endothelial cells, is beneficial to growth of endothelial cells, and promotes vascularization of myocardial organs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical materials and relates to a hydrogel and a preparation method thereof, and in particular to a hydrogel for culturing myocardial organoids and a preparation method thereof. Background Art

[0002] With the continuous development of biomedical technology, the types and application scope of biomedical materials are also constantly expanding. Today, biomedical materials can be applied to various fields, such as tissue engineering, biosensors, artificial organs, medical nanotechnology, etc. Biomaterials include various materials that can be used in medical and biological applications, such as metals, ceramics, polymer materials, etc. Among them, hydrogel, as a three-dimensional scaffold material, plays an important role in the cultivation and application of organoids. It has advantages such as high biocompatibility, adjustable mechanical properties, degradability, etc. Hydrogels are widely used in the biomedical field, including but not limited to: Tissue engineering: used to construct artificial tissues and organs. Drug delivery: used to control drug release and targeted delivery. Wound dressings: used to promote wound healing and prevent infection. Soft robots: used to make flexible and deformable robot parts. These advantages of hydrogels make it have great potential and broad application prospects in the biomedical field. At present, a large number of studies have used various hydrogels as a three-dimensional cell scaffold for the cultivation of myocardial organoids for the study of the maturity and vascularization of myocardial organoids.

[0003] Disadvantages of existing hydrogels for culturing cardiac organoids:

[0004] 1) Limit the three-dimensional growth of endothelial cells. Currently, the polymer materials commonly used to construct hydrogels include chitosan, polyethylene glycol, hyaluronic acid, etc. Since polymers have more active functional groups, they can be double-bonded to form hydrogels by free radical initiation; however, many double-bonded covalently cross-linked hydrogels are difficult to support endothelial cells to dynamically respond to the hydrogel matrix and reshape the microenvironment due to the stability of covalent bonds, which limits the three-dimensional growth of endothelial cells. Therefore, it is particularly important to regulate the microstructure of the hydrogel to make it more conducive to the three-dimensional growth of endothelial cells and promote the vascularization of myocardial organoids.

[0005] 2) Instability of dynamic cross-linking bonds. Dynamically cross-linked hydrogels are more suitable for endothelial cell growth than covalently cross-linked hydrogel networks because the cross-linking points of the hydrogel network are reversible dynamic bonds such as ionic bonds, hydrogen bonds, hydrophilic and hydrophobic interactions, etc. During cell growth, the network around the cells in the hydrogel can undergo bond shearing and remodeling. However, due to the instability of dynamic bonds, it is often difficult to maintain the long-term stability of the hydrogel structure, limiting its application in promoting vascularization of myocardial organoids.

[0006] 3) Poor biocompatibility or non-degradability. The use of conductive nanofiber hydrogels or carbon nanotube hydrogels to encapsulate myocardial organoids has many disadvantages in terms of biocompatibility. It has potential cytotoxicity: carbon nanotubes may be toxic to cells, especially at high concentrations or under long-term exposure. This toxicity may cause cell apoptosis or dysfunction, thereby affecting the biocompatibility of the hydrogel; immune response: carbon nanotubes may trigger an immune response, leading to inflammation or other immune-related problems. This reaction may affect the long-term stability and function of the hydrogel in the body; toxicity of degradation products: carbon nanotubes may produce toxic byproducts when degraded in the body, which may have adverse effects on surrounding tissues. Summary of the invention

[0007] In order to solve the technical problems existing in the prior art, the present invention provides a hydrogel for the culture of myocardial organoids and a preparation method thereof. The hydrogel has good long-term stability and can shear and reshape the surrounding network during the growth of endothelial cells, which is beneficial to the growth of endothelial cells and promotes the vascularization of myocardial organoids.

[0008] In order to achieve the above technical effects, the present invention adopts the following technical solutions:

[0009] One of the purposes of the present invention is to provide a hydrogel for culturing myocardial organoids, wherein the three-dimensional network structure of the hydrogel comprises hyaluronic acid and alginate;

[0010] The hyaluronic acid is hyaluronic acid modified with double bonds, cell adhesion sites and enzyme cleavage sites.

[0011] As a preferred technical solution of the present invention, the compound used for double bond modification includes norbornene.

[0012] Preferably, the molar ratio of hyaluronic acid to norbornene is 2×10 -4 ~3×10 -4 :1.

[0013] As a preferred technical solution of the present invention, the compound used for modifying the cell adhesion site is a polypeptide having a cell adhesion site.

[0014] Preferably, the molar ratio of hyaluronic acid to the polypeptide having a cell adhesion site is 0.03-0.04:1.

[0015] As a preferred technical solution of the present invention, the compound used for modification of the cleavage site is a polypeptide having an MMP cleavage site.

[0016] Preferably, the molar ratio of hyaluronic acid to the polypeptide of the MMP cleavage site is 0.01-0.015:1.

[0017] As a preferred technical solution of the present invention, the three-dimensional network structure of the hydrogel also includes metal ions complexed with alginate.

[0018] As a preferred technical solution of the present invention, the hydrogel contains cytokines that promote the proliferation and vascularization of endothelial cells and / or promote the maturation of myocardial organoids.

[0019] As a preferred technical solution of the present invention, the hydrogel contains cells that secrete the cytokine.

[0020] The second object of the present invention is to provide a method for preparing the hydrogel for culturing myocardial organoids provided in the first object, the preparation method comprising:

[0021] Using a compound containing a double bond to react with the hyaluronic acid to obtain double-bond-modified hyaluronic acid;

[0022] The double-bond modified hyaluronic acid undergoes Michael addition reaction with a compound having a cell adhesion site and a compound having an enzyme cleavage site to obtain double-bond modified, cell adhesion site modified and enzyme cleavage site modified hyaluronic acid;

[0023] The hydrogel is obtained by mixing alginate, hyaluronic acid modified by double bond, cell adhesion site and enzyme cleavage site, an initiator and water for polymerization reaction.

[0024] As a preferred technical solution of the present invention, the alginic acid is mixed with a metal salt for reaction before being mixed for polymerization reaction.

[0025] As a preferred technical solution of the present invention, cytokines that promote endothelial cell proliferation, vascularization and / or myocardial organoid maturation are injected into the hydrogel;

[0026] Alternatively, cells secreting the cytokine are injected into the hydrogel.

[0027] Compared with the prior art, the present invention has at least the following beneficial effects:

[0028] (1) The present invention provides a hydrogel for culturing myocardial organoids and a preparation method thereof. The hydrogel has good long-term stability and can shear and reshape the surrounding network during the growth of endothelial cells, which is beneficial to the growth of endothelial cells and promotes the vascularization of myocardial organoids.

[0029] (2) The present invention provides a hydrogel for culturing myocardial organoids and a preparation method thereof, wherein the hydrogel is very beneficial for the maturation of myocardial organoids and vascularization in the host body;

[0030] (3) The present invention provides a hydrogel for culturing myocardial organoids and a preparation method thereof, wherein the hydrogel has good biocompatibility and does not cause immune response or toxicity;

[0031] (4) The present invention provides a hydrogel for culturing myocardial organoids and a preparation method thereof. The hydrogel can be gradually degraded in vivo without producing long-term residues, and is suitable for use in implants. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a schematic diagram of the three-dimensional network structure within the hydrogel for culturing myocardial organoids prepared in a specific embodiment of the present invention.

[0033] Figure 2 The swelling ratio of the hydrogel provided in Example 2 of the present invention and Comparative Examples 1 and 2 varies with time.

[0034] Figure 3 Fluorescence images of cell life and death of myocardial organoids cultured using the hydrogels provided in Example 2 of the present invention and Comparative Examples 1 and 2.

[0035] Figure 4 Fluorescence images of endothelial cell migration in myocardial organoid culture using the hydrogels provided in Example 2 of the present invention and Comparative Examples 1 and 2.

[0036] The present invention is further described in detail below. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims. DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described below through specific implementation methods.

[0038] A specific embodiment of the present invention provides a hydrogel for culturing myocardial organoids, wherein the three-dimensional network structure of the hydrogel includes hyaluronic acid and alginate;

[0039] The hyaluronic acid is hyaluronic acid modified with double bonds, cell adhesion sites and enzyme cleavage sites.

[0040] In the present invention, hyaluronic acid is modified with double bonds, and the double bonds form covalent bonds through polymerization reaction to maintain the long-term stability of the hydrogel structure. By modifying the enzyme cleavage site, dynamic cross-linking bonds are introduced, and the network around the cells can be sheared and reshaped in response to the endothelial cell growth process, which is more conducive to the growth and vascularization of endothelial cells. Hyaluronic acid and alginate are both high molecular materials of natural origin, have good biocompatibility, and will not cause immune response or toxicity; natural hydrogel materials can be gradually degraded in the body, will not produce long-term residues, and are suitable for implants.

[0041] In one embodiment of the present invention, the content of hyaluronic acid and alginate in the hydrogel is not specifically limited and can be adjusted according to the needs of myocardial organoid culture. However, the content of hyaluronic acid and alginate should meet the minimum content requirement for forming a hydrogel.

[0042] In one embodiment of the present invention, the compound used for double bond modification includes norbornene. Using norbornene as the double bond modification compound can introduce double bonds into the structure of hyaluronic acid, and the carboxyl group formed after the anhydride bond is opened can ensure the water solubility of the double bond modified hyaluronic acid and provide new hydrogen bonding sites.

[0043] In one embodiment of the present invention, the molar ratio of hyaluronic acid to norbornene is 2.0×10 -4 ~3.0×10 -4 :1, such as 2×10 -4 :1,2.1×10 -4 :1,2.2×10 -4 :1,2.3×10 -4 :1,2.4×10 -4 :1,2.5×10 -4 :1,2.6×10 -4 :1,2.7×10 -4 :1,2.8×10 -4 :1,2.9×10 -4 :1 or 3.0×10 -4 :1, etc., but are not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0044] In one embodiment of the present invention, the compound used for cell adhesion site modification is a polypeptide having a cell adhesion site. The present invention does not specifically limit the amino acid sequence of the polypeptide, and any polypeptide containing a cell adhesion site is suitable for cell adhesion site modification, such as CIKVAV polypeptide.

[0045] In a specific embodiment of the present invention, the molar ratio of hyaluronic acid to the polypeptide having a cell adhesion site is 0.03-0.04:1, such as 0.03:1, 0.31:1, 0.032:1, 0.033:1, 0.034:1, 0.035:1, 0.036:1, 0.037:1, 0.038:1, 0.039:1 or 0.04:1, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0046] In one embodiment of the present invention, the compound used for modification of the cleavage site is a polypeptide having an MMP cleavage site. The present invention does not specifically limit the amino acid sequence of the polypeptide, and polypeptides containing an MMP cleavage site are suitable for modification of cell adhesion sites, such as GCRDGPQGIWGQDRCG polypeptide.

[0047] In a specific embodiment of the present invention, the molar ratio of hyaluronic acid to the polypeptide of the MMP cleavage site is 0.01 to 0.015:1, such as 0.01:1, 0.011:1, 0.012:1, 0.013:1, 0.014:1 or 0.015:1, etc., but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable. In a specific embodiment of the present invention, the three-dimensional network structure of the hydrogel also includes metal ions complexed with alginate. Among them, the present invention does not specifically limit the type of metal ions, but the metal ions used should not have biological toxicity, nor should they affect the culture of myocardial trachea, and the remaining metal ions that can be complexed with functional groups in alginate are applicable to this application, such as calcium ions.

[0048] In one embodiment of the present invention, the hydrogel contains cytokines that promote the proliferation and vascularization of endothelial cells and / or promote the maturation of myocardial organoids. For example, the cytokines may be VEGF and TGF-β.

[0049] In one embodiment of the present invention, the hydrogel contains cells that secrete cytokines that promote endothelial cell proliferation, vascularization and / or myocardial organoid maturation, for example, M2 macrophages.

[0050] A specific embodiment of the present invention provides a method for preparing a hydrogel for culturing myocardial organoids, the preparation method comprising:

[0051] Using a compound containing a double bond to react with the hyaluronic acid to obtain double-bond-modified hyaluronic acid;

[0052] The double-bond modified hyaluronic acid undergoes Michael addition reaction with a compound having a cell adhesion site and a compound having an enzyme cleavage site to obtain double-bond modified, cell adhesion site modified and enzyme cleavage site modified hyaluronic acid;

[0053] The hydrogel is obtained by mixing alginate, hyaluronic acid modified by double bond, cell adhesion site and enzyme cleavage site, an initiator and water for polymerization reaction.

[0054] In one specific embodiment of the present invention, the anhydride group in norbornene reacts with the hydroxymethyl group in hyaluronic acid, and the reaction equation is shown below.

[0055]

[0056] In a specific embodiment of the present invention, the cell adhesion site modification and enzyme cleavage site modification of hyaluronic acid are carried out by Michael addition reaction between the thiol group in the cysteine ​​segment in the polypeptide having the cell adhesion site and the polypeptide having the MMP enzyme cleavage site and the carbon-carbon double bond in the double-bond modified hyaluronic acid. Among them, the reaction conditions of the Michael addition reaction are conventional reaction conditions in the field of organic synthesis, which can be adaptively adjusted according to the different reaction raw materials, and are not specifically limited here. As an example, the Michael addition reaction in the present invention can be shown as follows.

[0057]

[0058] In one embodiment of the present invention, the alginic acid is mixed with a metal salt for reaction before being mixed for polymerization, and the functional groups in the alginic acid react with the metal salt (such as calcium salt) to form cross-linked bonds.

[0059] In a specific embodiment of the present invention, the initiator can be a photoinitiator, and the photoinitiator can be LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) or 1173 (2-hydroxy-2-methyl-1-phenyl-1-propanone) or PBZ (4-phenylbenzophenone) and other structurally modified benzophenone initiators.

[0060] In one specific embodiment of the present invention, cytokines that promote endothelial cell proliferation, vascularization and / or promote myocardial organoid maturation are injected into the hydrogel; or, cells that secrete the cytokines are injected into the hydrogel.

[0061] In the present invention, cytokines such as VEGF and TGF-β are directly injected into the hydrogel, or cells that can secrete cytokines such as VEGF and TGF-β, such as M2 macrophages, are injected to promote the proliferation and vascularization of endothelial cells and the maturation of myocardial organoids.

[0062] To better illustrate the present invention and facilitate understanding of the technical solution of the present invention, typical but non-limiting embodiments of the present invention are as follows:

[0063] Example 1

[0064] Double bond modification of hyaluronic acid:

[0065] 3.333×10 -6 0.01218 mol hyaluronic acid was reacted with 0.01218 mol norbornene according to the conditions shown in the following formula. After the reaction, the mixture was washed three times and dialyzed with a 14 kDa dialysis bag for 72 h. After purification and freeze-drying, the double-bonded modified hyaluronic acid was obtained with a yield of 44.64%.

[0066] A new peak at 6-6.5 ppm can be observed in its NMR test spectrum, indicating the presence of carbon-carbon double bonds in norbornene.

[0067]

[0068] Example 2

[0069] Preparation of hydrogel:

[0070] 1.78 mL of water, 9.254 × 10 -5 mol alginate, 1.424×10 -6 mol CIKVAV, 4.692×10 -6 molMMPscp and 2×10 -7 mol calcium chloride was mixed to react, and then 6.798×10 -7 mol photoinitiator, 4.2656×10 -8 The hyaluronic acid prepared by mol is mixed and polymerized, and a hydrogel is obtained after the polymerization reaction. The schematic diagram of the three-dimensional network inside the hydrogel is shown in Figure 1 As shown, M2 macrophages were seeded into the hydrogel.

[0071] Comparative Example 1

[0072] In this comparative example, except that Alg-Ca 2+ Except for the system, other conditions are the same as those in Example 2.

[0073] Comparative Example 2

[0074] In this comparative example, except that M2 macrophages were not inoculated, other conditions were the same as those in Example 3.

[0075] The stability of the hydrogels prepared in Example 2 and Comparative Examples 1 and 2 was tested by immersing the prepared hydrogels in deionized water, measuring the radius and height of the three columnar hydrogels after swelling with water absorption at different times, and thus calculating the swelling ratio. The test results showed that the three hydrogels reached a stable state after 15 hours, as shown in FIG. Figure 2 (HMC hydrogel is the hydrogel of comparative example 1, HMCA is the hydrogel of comparative example 2, and HMCA-M2 is the hydrogel of example 2.)

[0076] The hydrogels prepared in Example 2 and Comparative Examples 1 and 2 were used to culture myocardial organoids. The myocardial organoids were inoculated into Example 2 and Comparative Examples 1 and 2. After three days of culture under the hydrogel scaffold system, the myocardial organoids were stained with Calcein-AM / PI dye for live-dead staining. After 30 minutes, laser confocal imaging was used. Culture Result 1 (as shown in FIG. Figure 3) shows that Comparative Example 1, Comparative Example 2 and Example 2 have almost no effect on the survival of myocardial organoids. From the fluorescence image, Example 2 is more conducive to the culture of myocardial organoids, and the cultured myocardial organoids have better activity.

[0077] The hydrogels prepared in Example 2 and Comparative Examples 1 and 2 were used to culture myocardial organoids. The myocardial organoids were inoculated into Example 2 and Comparative Examples 1 and 2. After the inoculation was completed, 1w of fluorescent endothelial cell suspension was dripped onto the surface of the three hydrogels respectively. After 5 minutes, the culture medium was added and the cells were cultured in an incubator for three days. Then, fluorescence images were taken using a laser confocal microscope. Culture Result 2 (as shown in FIG. Figure 4 ) shows that Example 2 can better promote the migration of endothelial cells compared with Comparative Examples 1 and 2, reflecting the advantages of the hydrogel in Example 2.

[0078] The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned detailed structural features, that is, it does not mean that the present invention must rely on the above-mentioned detailed structural features to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the components selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0079] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0080] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0081] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A hydrogel for culturing myocardial organoids, characterized in that: The three-dimensional network structure of the hydrogel includes hyaluronic acid and alginate; The hyaluronic acid is double-bond modified, cell adhesion site modified and enzyme cleavage site modified hyaluronic acid.

2. The hydrogel according to claim 1, characterized in that The compound used for the double bond modification includes norbornene; The molar ratio of the hyaluronic acid to the norbornene is 2×10 -4 ~3×10 -4 :

1.

3. The hydrogel according to claim 1, characterized in that The compound used for modifying the cell adhesion site is a polypeptide having a cell adhesion site; The molar ratio of the hyaluronic acid to the polypeptide having the cell adhesion site is 0.03-0.04:

1.

4. The hydrogel according to claim 1, characterized in that The compound used for modification of the cleavage site is a polypeptide having an MMP cleavage site; The molar ratio of the hyaluronic acid to the polypeptide having the MMP cleavage site is 0.01-0.015:

1.

5. The hydrogel according to claim 1, characterized in that The three-dimensional network structure of the hydrogel also includes metal ions complexed with alginate.

6. The hydrogel according to claim 1, characterized in that The hydrogel contains cytokines that promote the proliferation and vascularization of endothelial cells and / or promote the maturation of myocardial organoids.

7. The hydrogel according to claim 6, characterized in that The hydrogel contains cells that secrete the cytokine.

8. A method for preparing a hydrogel for culturing cardiac organoids according to any one of claims 1 to 9, characterized in that: The preparation method comprises: Using a compound containing a double bond to react with the hyaluronic acid to obtain double-bond-modified hyaluronic acid; The double-bond modified hyaluronic acid is subjected to Michael addition reaction with a compound having a cell adhesion site and a compound having an enzyme cleavage site to obtain the double-bond modified, cell adhesion site modified and enzyme cleavage site modified hyaluronic acid; The alginate, the hyaluronic acid modified with double bonds, cell adhesion sites and enzyme cleavage sites, an initiator and water are mixed to carry out polymerization reaction to obtain the hydrogel.

9. The preparation method according to claim 8, characterized in that: The alginic acid is mixed with a metal salt for reaction before being mixed for polymerization reaction.

10. The preparation method according to claim 8, characterized in that: injecting cytokines that promote endothelial cell proliferation, vascularization and / or myocardial organoid maturation into the hydrogel; Alternatively, cells secreting the cytokine are injected into the hydrogel.

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