Physiological mixed gel precursor solution, gel material, preparation method of gel material and application of gel material in organoids

Abstract

The invention relates to the technical field of organoid culture, in particular to a physiological mixed gel precursor solution for organoid culture, which comprises a culture medium, spleen hydrogel, 0.8-1.2 mg / mL of type I collagen, 0.1-0.5 mg / mL of type IV collagen, 2.5-7.5 mg / mL of fibrinogen and 1-5 [mu] g / mL of aprotinin. The culture medium is a special liquid culture medium for the budding and growth of a vascular organ embryoid body; the spleen hydrogel is hydrogel taking animal spleen extracellular matrix as a raw material, and the concentration of the spleen hydrogel in the precursor solution is 2.5-12.5 mg / mL. According to the present invention, the physiological mixed gel precursor solution is gelatinized to obtain the matrix gel material, and the matrix gel material is used to wrap and culture the vascular organ embryoid body ball, such that the safety risk caused by the tumor-derived material can be reduced, the preparation cost can be reduced, and the technical support can be provided for the commercial preparation of the vascular organ.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a physiological mixed gel precursor solution, a gel material, and a preparation method and application thereof. Background Art

[0002] Blood vessels are the basis of animal life. The construction of a vascular network with branching structure and perfusion function in vitro can be widely used in scientific research and clinical applications. Literature reports that embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can self-organize in vitro to form three-dimensional vascular organoids with in vitro capillary network structure and function. Existing methods for culturing vascular organoids in vitro require biological matrix materials as the support of the three-dimensional structure. Usually, a three-dimensional mixed gel material composed of Matrigel and collagen in different proportions is used, but Matrigel is a biological material derived from animal tumors and is not suitable for clinical use due to safety risks. In addition, there are also some technical reports that three-dimensional mixed gel materials prepared with fibrin and collagen in different proportions can be used, but the three-dimensional support structure of this mixed gel is not stable enough, resulting in insufficient encapsulation of vascular organoids, affecting the budding growth of vascular organoids.

[0003] In the prior art, patent application CN111065731B discloses a mixed hydrogel material mainly composed of type I and type IV collagen, and CN116836911A discloses a mixed hydrogel material mainly composed of type I collagen and gelatin. Both of these technical solutions use high-concentration collagen materials, which will bring high costs for the commercial preparation of vascular organoids. CN117143359A discloses a synthetic hydrogel, which can achieve the growth of vascular organoids while reducing the preparation cost, but the material is not a physiological material and has unsafety, limited applicability and instability. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a physiological mixed gel precursor solution and a preparation method and application thereof, which solve the technical problems existing in the prior art such as unsafe, high cost and insufficient stability of three-dimensional matrix materials used for vascular organoid culture.

[0006] (II) Technical solution

[0007] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0008] In the first aspect, the present invention provides a physiological mixed gel precursor solution, which is composed of the following ingredients: culture medium, spleen hydrogel, type I collagen 0.8-1.2 mg / mL, type IV collagen 0.1-0.5 mg / mL, fibrinogen 2.5-7.5 mg / mL, aprotinin 1-5 μg / mL; wherein the concentration of spleen hydrogel is 2.5-12.5 mg / mL; the culture medium is a special liquid culture medium for the budding growth of vascular organoid embryoids; the spleen hydrogel is a hydrogel made of animal spleen extracellular matrix. The preparation method of the spleen hydrogel stock solution can be found in the prior patent CN107224617B, and existing commercial products can also be purchased.

[0009] According to a preferred embodiment of the present invention, the preparation method of the spleen hydrogel stock solution is as follows:

[0010] S1. Preparation of biological matrix materials: spleen tissue of animals of the same or different species is processed to prepare biological matrix materials; the processing method is to remove the fat tissue in the spleen tissue and cut it into 1-3 mm 3 The tissue pieces were soaked in physiological saline, rinsed, and then decellularized to obtain decellularized spleen extracellular matrix;

[0011] S2, digestion: wash the spleen extracellular matrix obtained in step S1 with deionized water, shake and rinse, change the water every 40-60 minutes, for a total of 15-25 times, remove the residual decellularization reagent, vacuum freeze-dry, weigh, add 0.01-0.2M hydrochloric acid solution and grind into a homogenate, add pepsin, and digest with constant temperature stirring to obtain a uniform and viscous spleen extracellular matrix solution;

[0012] S3. Incubate to form gel: In an ice bath, adjust the pH of the spleen extracellular matrix solution to neutral with 1M sodium hydroxide solution, then add PBS solution, incubate at 37°C to form gel, and store at 4°C.

[0013] According to a preferred embodiment of the present invention, the special liquid culture medium for the budding and growth of vascular organoid embryoids is the STEMdiffTM Blood Vessel Organoid Kit. The kit contains a variety of basal culture media and added factors. In the actual culturing process of vascular organoids, different basal culture media and factors can be selected for culturing according to different culturing stages. The vascular organoid maturation medium in the STEMdiffTM Blood Vessel Organoid Kit is used in the culturing stage involved in the specific embodiment of the present invention.

[0014] According to a preferred embodiment of the present invention, the spleen hydrogel is a hydrogel made from at least one of pig spleen and bovine spleen extracellular matrix.

[0015] In a second aspect, the present invention provides a method for preparing a physiological mixed gel precursor solution, comprising the following steps:

[0016] (1) diluting the spleen hydrogel stock solution with a special liquid culture medium for budding growth of vascular organoid embryoids to obtain a first dispersion; mixing a fibrinogen solution, a type I collagen solution, a type IV collagen solution, and a special liquid culture medium for budding growth of vascular organoid embryoids to obtain a second dispersion; mixing aprotinin solution with a special liquid culture medium for budding growth of vascular organoid embryoids to obtain a third dispersion;

[0017] (2) The first dispersion liquid, the second dispersion liquid, and the third dispersion liquid are mixed to obtain the physiological mixed gel precursor solution.

[0018] According to a preferred embodiment of the present invention, in step (1), the aprotinin solution is obtained by dissolving aprotinin lyophilized powder in sterile pure water; the fibrinogen solution is obtained by dissolving at least one of bovine fibrinogen and human fibrinogen in physiological saline at 30-38°C and filtering with a 0.22 μm filter membrane; the type I collagen solution is obtained by dissolving rat tail collagen in a low concentration acetic acid solution; and the type IV collagen solution is obtained by dissolving human collagen in a low concentration acetic acid solution and then diluting with a sterile 1×PBS solution.

[0019] According to a preferred embodiment of the present invention, in step (1), the fibrinogen solution, type I collagen solution and type IV collagen solution are commercial products purchased separately, and the protein concentration in the solution is about 4-10 mg / mL.

[0020] According to a preferred embodiment of the present invention, in step (1), the special liquid culture medium for budding growth of vascular organoid embryoids and the spleen hydrogel stock solution are first refrigerated in a 4-8°C refrigerator for 12-24 hours, and then the special liquid culture medium for budding growth of vascular organoid embryoids and the spleen hydrogel stock solution are mixed, gently stirred, mixed evenly, and centrifuged to remove bubbles to obtain a first dispersion.

[0021] According to a preferred embodiment of the present invention, in step (1), the special liquid culture medium for budding and growing vascular organoid embryoids is fully mixed with the type I collagen solution and the type IV collagen solution on ice, and then the fibrinogen solution is added and slowly shaken to mix evenly to obtain a second dispersion.

[0022] According to a preferred embodiment of the present invention, in step (1), the special liquid culture medium for budding and growth of vascular organoid embryoids and the aprotinin solution are reheated in an environment of 20-25° C. for 5-10 minutes and then fully mixed to obtain a third dispersion.

[0023] According to a preferred embodiment of the present invention, in step (2), the first dispersion and the second dispersion are respectively placed at 8-12° C. for 5-10 minutes, and then the two are fully mixed, and then the third dispersion is added and continued to mix to obtain the physiological mixed gel precursor solution.

[0024] In a third aspect, the present invention also provides a method for culturing vascular organoids, comprising: a first step: adding a thrombin solution to the physiological mixed gel precursor solution, wherein the amount of thrombin added is 10-30 U / mL, and after uniform mixing, a physiological mixed gel prepolymer solution is obtained; a special liquid culture medium for budding growth of vascular organoids is mixed with type I collagen to form a gel mixed culture solution, wherein the concentration of type I collagen is 0.1-0.3 mg / mL;

[0025] The second step: disperse and wrap the vascular organoid embryoid body spheres in a physiological mixed gel prepolymerization solution for gel solidification treatment, the solidification treatment temperature is 25-37°C, the solidification treatment time is 1-2h, and then the solidified gel material wrapped with the vascular organoid embryoid body spheres is immersed in the gel mixed culture solution and cultured for 24h, and then the special liquid culture medium for the budding growth of vascular organoids is replaced, and static culture is carried out for 5-15 days to obtain vascular organoid spheres with branched vascular lumen-like structures; wherein the dispersion density of the vascular organoid embryoid body spheres in the solidified gel material is 60-100 / mL.

[0026] According to a preferred embodiment of the present invention, in the second step, the vascular organoid embryoid spheres and the cardiomyocytes, hepatocytes or glomerular cells are dispersed and encapsulated in a physiological mixed gel prepolymer solution for solidification.

[0027] According to a preferred embodiment of the present invention, the operation process of the second step is as follows:

[0028] Step 1: Take a portion of the physiological mixed gel prepolymer solution and spread it evenly in a culture well plate, a culture dish or an organ culture chip, and then perform gel solidification treatment to form a culture medium bottom layer. The solidification treatment temperature is 25-37° C., and the solidification treatment time is 1-2 hours. After solidification, the obtained culture medium bottom layer has a thickness of 2-3 mm;

[0029] Step 2: adding the vascular organoid embryoid body spheres to another portion of the physiological mixed gel prepolymer solution, shaking to evenly disperse the vascular organoid embryoid body spheres in the physiological mixed gel prepolymer solution, spreading the obtained mixture evenly on the bottom layer of the culture medium, and then performing gel curing treatment to form a wrapping layer, the curing treatment temperature is 25-37° C., the curing treatment time is 1-2 h, and the thickness of the cured wrapping layer is 2-3 mm;

[0030] Step 3: adding the gel mixed culture solution to the solidified wrapping layer, and the coverage depth of the gel mixed culture solution on the wrapping layer reaches 1-2 mm; after culturing for 24 hours, removing the liquid on the upper layer of the wrapping layer and replacing it with a special liquid culture medium for the sprouting growth of vascular organoids, and then replacing it with fresh special liquid culture medium for the sprouting growth of vascular organoids every day until the culture is 5-15 days;

[0031] Step 4: After the culture is completed, the vascular organoids are peeled off from the physiological matrix gel material to obtain vascular organoids with branched vascular lumen-like structures.

[0032] In a fourth aspect, the present invention provides a method for preparing a physiological matrix gel material, comprising: adding a thrombin solution to the above-mentioned physiological mixed gel precursor solution, the thrombin addition amount being 10-30 U / mL, and mixing evenly to obtain a physiological mixed gel prepolymer solution; and then curing the solution at a temperature of 25-37°C for 1-2 hours to obtain a physiological matrix gel material.

[0033] In a fifth aspect, the present invention relates to the use of the above-mentioned physiological mixed gel precursor solution and the physiological matrix gel material prepared therefrom in constructing vascularized engineered myocardial tissue, vascularized artificial liver lobule tissue or vascularized artificial glomerular tissue.

[0034] Specifically, vascular organoids or vascular organoid embryoid spheres with branched vascular lumen-like structures can be encapsulated with cardiomyocytes, hepatocytes or glomerular cells in the physiological matrix gel material for co-culture, thereby constructing vascularized engineered myocardial tissue, vascularized artificial liver lobule tissue or vascularized artificial glomerular tissue.

[0035] (III) Beneficial effects

[0036] The present invention adopts for the first time a physiological mixed gel precursor solution composed of spleen hydrogel made from pig or bovine spleen, bovine fibrinogen and human fibrinogen, type I collagen, type IV collagen, etc., for preparing a physiological matrix gel material for vascular organoid culture, replacing the biological materials and synthetic hydrogels derived from animal tumors in the prior art. Compared with the biological materials derived from animal tumors, the physiological matrix gel material of the present invention is safer and more suitable for clinical use; compared with synthetic hydrogels, the gel material of the present invention is a physiological material and is more suitable for cell culture; compared with the use of high-concentration collagen materials (mainly type I collagen), the cost of this type of gel material can be reduced.

[0037] The matrix gel material prepared by the present invention can reduce the safety risks brought by tumor-derived materials, lower the preparation cost, and broaden the application scope of the gel material without affecting the growth and structural maturation of vascular organoids, thereby providing technical support for the commercial preparation of vascular organoids.

[0038] The vascular organoid embryoid spheres are wrapped with the physiological matrix gel material of the present invention, and are immersed in a gel mixed culture medium containing a special liquid culture medium for the sprouting growth of vascular organoids and type I collagen for static culture. The vascular organoid embryoid spheres can grow buds after only one day of culture, and the bud density is higher than that of the gel material without spleen hydrogel and type IV collagen. After the culture is completed, a vascular lumen-like structure with a branched structure can be obtained, the length of which reaches 1 mm, and the overall size of the vascular organoid can reach several millimeters.

[0039] In summary, the present invention achieves the technical objectives of reducing the cost of preparing vascular organoids and improving the safety of using vascular organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The figure is a schematic diagram of the preparation process of the physiological mixed gel precursor solution of the present invention.

[0041] Figure 2 Schematic diagram of the method for forming matrix gel material and culturing vascular organoids using a physiological mixed gel precursor solution.

[0042] Figure 3 These are pictures of vascular organoids obtained by culturing vascular organoid embryoid spheres using the matrix condensation material obtained in Example 1 and Comparative Example 1 for 1 day, 3 days, and 5 days, and then peeling them from the condensation material after 5 days of culturing and culturing for 3 days.

[0043] Figure 4 This is an immunofluorescence identification diagram of vascular organoids obtained by culturing vascular organoid embryoid spheres for 5 days using the matrix condensation materials obtained in Example 1 and Comparative Example 1 (co-staining CD31 / DAPI). DETAILED DESCRIPTION

[0044] In order to better understand the above technical solution, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a clearer and more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0045] The physiological mixed gel precursor solution provided by the present invention is composed of: culture medium, spleen hydrogel, type I collagen 0.8-1.2 mg / mL, type IV collagen 0.1-0.5 mg / mL, fibrinogen 2.5-7.5 mg / mL, and aprotinin 1-5 μg / mL; wherein the concentration of the spleen hydrogel is 2.5-12.5 mg / mL; the culture medium is a special liquid culture medium for the sprouting growth of vascular organoid embryoids; and the spleen hydrogel is a hydrogel using animal spleen extracellular matrix as raw material.

[0046] In a preferred embodiment of the present invention, the special liquid culture medium for budding and growing embryoid bodies of vascular organoids is the STEMdiff™ Blood Vessel Organoid Kit. In other embodiments, other brands of vascular organoid culture medium can also be used.

[0047] There are many kinds of basal culture media and added factors in the STEMdiffTM Blood Vessel Organoid Kit. Different basal culture media and added factors are selected for culture according to different culture stages. The culture stage involved in the specific embodiment of the present invention uses the vascular organoid maturation medium in the STEMdiffTM Blood Vessel Organoid Kit.

[0048] The spleen hydrogel is a hydrogel made of at least one of pig spleen and bovine spleen extracellular matrix. The preparation method of the spleen hydrogel stock solution can be found in the prior patent CN107224617B, and existing commercial products can also be purchased.

[0049] Referring to the prior patent CN107224617B, the preparation method of spleen hydrogel stock solution is as follows:

[0050] S1. Preparation of biological matrix materials: spleen tissue of animals of the same or different species is processed to prepare biological matrix materials; the processing method is to remove the fat tissue in the spleen tissue and cut it into 1-3 mm 3The tissue pieces were soaked in physiological saline, rinsed, and then decellularized to obtain decellularized spleen extracellular matrix;

[0051] S2, digestion: wash the spleen extracellular matrix obtained in step S1 with deionized water, shake and rinse, change the water every 40-60 minutes, for a total of 15-25 times, remove the residual decellularization reagent, vacuum freeze-dry, weigh, add 0.01-0.2M hydrochloric acid solution and grind into a homogenate, add pepsin, and digest with constant temperature stirring to obtain a uniform and viscous spleen extracellular matrix solution;

[0052] S3. Incubate to form gel: In an ice bath, adjust the pH of the spleen extracellular matrix solution to neutral with 1M sodium hydroxide solution, then add PBS solution, incubate at 37°C to form gel, and store at 4°C.

[0053] like Figure 1 , which is a schematic diagram of the preparation process of the physiological mixed gel precursor solution of the present invention, including separately preparing a first dispersion liquid, a second dispersion liquid and a third dispersion liquid, and then mixing them.

[0054] Specifically, the preparation process is as follows:

[0055] (1) First, the special liquid culture medium for budding growth of vascular organoid embryoids and the spleen hydrogel stock solution are refrigerated in a refrigerator at 4-8°C for 12-24 hours, and then the special liquid culture medium for budding growth of vascular organoid embryoids and the spleen hydrogel stock solution are mixed, gently stirred, mixed evenly, and centrifuged to remove bubbles to obtain a first dispersion.

[0056] (2) The special liquid culture medium for budding and growth of vascular organoid embryoids is fully mixed with the type I collagen solution and the type IV collagen solution on ice, and then the fibrinogen solution is added and slowly shaken to mix evenly to obtain a second dispersion.

[0057] The fibrinogen solution is obtained by dissolving at least one of bovine fibrinogen and human fibrinogen in physiological saline at 30-38°C and filtering with a 0.22μm filter membrane. The type I collagen solution is obtained by dissolving rat tail collagen in a low-concentration acetic acid solution; the type IV collagen solution is obtained by dissolving human collagen in a low-concentration acetic acid solution and then diluting with a sterile 1×PBS solution.

[0058] In addition, the fibrinogen solution, type I collagen solution and type IV collagen solution are purchased from commodities, respectively, and the protein concentration in the solution is about 4-10 mg / mL.

[0059] (3) The special liquid culture medium for budding and growth of vascular organoid embryoids and the aprotinin solution are rewarmed in an environment of 20-25° C. for 5-10 minutes, and then fully mixed to obtain a third dispersion. The aprotinin solution is obtained by dissolving aprotinin lyophilized powder in sterile pure water.

[0060] (4) The first dispersion and the second dispersion are placed at 8-12° C. for 5-10 minutes respectively, and then the two are fully mixed, and then the third dispersion is added and the mixing is continued to obtain the physiological mixed gel precursor solution.

[0061] Combination Figure 1-Figure 2 As shown, it is a schematic diagram of the method of culturing vascular organoids using the above-mentioned physiological mixed gel precursor solution of the present invention. According to the figure, the steps are as follows:

[0062] (1) Add thrombin solution to the physiological mixed gel precursor solution, the amount of thrombin added is 10-30 U / mL, and mix well to obtain a physiological mixed gel prepolymer solution. Divide the physiological mixed gel prepolymer solution into two parts for standby use.

[0063] (2) Mixing a special liquid culture medium for vascular organoid sprouting growth with type I collagen to form a gel mixed culture medium, wherein the concentration of type I collagen is 0.1-0.3 mg / mL.

[0064] (3) Take a portion of the physiological mixed gel prepolymer solution and spread it evenly in a culture well plate, a culture dish or an organ culture chip, and then perform gel solidification treatment to form a culture medium bottom layer. The solidification treatment temperature is 25-37°C and the solidification treatment time is 1-2 hours. After solidification, the thickness of the obtained culture medium bottom layer is 2-3 mm.

[0065] Among them, thrombin can react with fibrinogen in the physiological mixed gel precursor solution, catalyzing the conversion of fibrinogen into insoluble fibrin monomers, which then aggregate into a fibrin network, causing the physiological mixed gel precursor solution to be prepolymerized to obtain a physiological mixed gel prepolymer solution. The spleen hydrogel is easily gelated at a temperature of 25-37°C to obtain a physiological matrix gel material.

[0066] (4) Taking another portion of the physiological mixed gel prepolymer solution to encapsulate the vascular organoid embryoid body spheres or other cells to be cultured, specifically, the vascular organoid embryoid body spheres (together with cardiomyocytes / hepatocytes, glomerular cells, etc.) are dispersed and encapsulated in the physiological mixed gel prepolymer solution, and gently shaken to evenly disperse the vascular organoid embryoid body spheres in the physiological mixed gel prepolymer solution, and the obtained mixture is evenly spread on the bottom layer of the culture medium, and then gel-curing treatment is performed to form a coating layer. The same gel curing conditions as in the above (3) are used for the treatment, the curing treatment temperature is 25-37°C, the curing treatment time is 1-2h, and the thickness of the cured coating layer is 2-3mm.

[0067] During the encapsulation process, the dispersion density of vascular organoid embryoid body spheres in the solidified gel material was 60-100 / mL.

[0068] Among them, the type of encapsulated cells or organoid embryoids depends on the biological material that needs to be constructed. The vascular organoid culture method of the present invention can be used to construct but is not limited to vascularized engineered myocardial tissue, vascularized artificial liver lobule tissue and vascularized artificial glomerular tissue.

[0069] (5) Adding the gel mixed culture medium prepared in step (2) to the solidified encapsulation layer, the coverage depth of the gel mixed culture medium on the encapsulation layer reaches 1-2 mm; after culturing for 24 hours, removing the liquid on the upper layer of the encapsulation layer and replacing it with a special liquid culture medium for the sprouting growth of vascular organoids, and then replacing it with fresh special liquid culture medium for the sprouting growth of vascular organoids once a day until the culture is 5-15 days.

[0070] (6) After the culture is completed, the vascular organoids are peeled off from the physiological matrix gel material to obtain vascular organoid spheres with branched vascular lumen-like structures.

[0071] Of course, the physiological matrix gel material prepared by the present invention can also be used for the culture of other biological cells or tissues. The preparation method of the physiological matrix gel material is as follows: adding thrombin solution to the physiological mixed gel precursor solution composed of the above-mentioned special liquid culture medium for the budding growth of vascular organoid embryoids, spleen hydrogel, type I collagen, type IV collagen, fibrinogen, aprotinin, etc., and then adding thrombin 10-30U / mL, mixing evenly, to obtain a physiological mixed gel prepolymer solution; then curing treatment at 25-37°C for 1-2h, the physiological matrix gel material can be obtained. When in use, vascular organoids or vascular organoid embryoids with branched vascular lumen-like structures can be wrapped with cardiomyocytes, hepatocytes or glomerular cells in the physiological matrix gel material for co-culture, thereby constructing vascularized engineered myocardial tissue, vascularized artificial liver lobule tissue or vascularized artificial glomerular tissue.

[0072] The following is a description of the present invention in combination with Example 1 and Comparative Example 1.

[0073] Example 1

[0074] The composition of the physiological mixed gel precursor solution of this embodiment is: a special liquid culture medium for the budding growth of vascular organoid embryoids, 5 mg / mL of spleen hydrogel, 1 mg / mL of type I collagen, 0.3 mg / mL of type IV collagen, 4.5 mg / mL of fibrinogen, and 3 μg / mL of aprotinin. The special liquid culture medium is the vascular organoid maturation culture medium in the STEMdiffTM Blood Vessel Organoid Kit (item #100-0651). The spleen hydrogel is a hydrogel prepared from bovine spleen. For the preparation method of the hydrogel stock solution, please refer to the prior patent CN107224617B. Type I collagen is rat tail-derived type I collagen. Type IV collagen is human collagen. Fibrinogen is bovine fibrinogen.

[0075] The preparation method of the above physiological mixed gel precursor solution is:

[0076] (1) First, the special liquid culture medium for budding growth of vascular organoid embryoids and the spleen hydrogel stock solution are refrigerated in a 4°C refrigerator for 24 hours, and then the special liquid culture medium for budding growth of vascular organoid embryoids and the spleen hydrogel stock solution are mixed, gently stirred, mixed evenly, and centrifuged to remove bubbles to obtain a first dispersion.

[0077] (2) The special liquid culture medium for budding and growth of vascular organoid embryoids is fully mixed with the type I collagen solution and the type IV collagen solution on ice, and then the fibrinogen solution is added and slowly shaken to mix evenly to obtain a second dispersion.

[0078] The fibrinogen solution is obtained by dissolving at least one of bovine fibrinogen and human fibrinogen in physiological saline at 35°C and filtering with a 0.22 μm filter membrane. The type I collagen solution is obtained by dissolving rat tail collagen in a low-concentration acetic acid solution; the type IV collagen solution is obtained by dissolving human collagen in a low-concentration acetic acid solution and then diluting with a sterile 1×PBS solution.

[0079] (3) The special liquid culture medium for budding and growth of vascular organoid embryoids and the aprotinin solution were rewarmed in a 20°C environment for 10 minutes, and then fully mixed to obtain a third dispersion. The aprotinin solution was obtained by dissolving aprotinin lyophilized powder in sterile pure water.

[0080] (4) The first dispersion and the second dispersion are placed at 10° C. for 10 minutes respectively, and then the two are fully mixed, and then the third dispersion is added and the mixing is continued to obtain the physiological mixed gel precursor solution.

[0081] Comparative Example 1

[0082] The composition of the physiological mixed gel precursor solution provided in this comparative example is based on Example 1 except that the spleen hydrogel and type IV collagen are removed, and the other compositions and proportions are the same as those in Example 1.

[0083] Application Examples

[0084] The physiological mixed gel precursor solutions provided in Example 1 and Comparative Example 1 are respectively used for pre-gelation to obtain gel prepolymer solutions, and the gel prepolymer solutions obtained in Example 1 and Comparative Example 1 are used to wrap vascular organoid embryoid spheres and then gelated for culturing vascular organoids. The vascular organoid culturing method is as follows:

[0085] (1) Add thrombin solution to the rationally mixed gel precursor solution, the amount of thrombin added is 20 U / mL, and after mixing evenly, a gel prepolymer solution is obtained. The gel prepolymer solution is divided into two parts for standby use. The special liquid culture medium for vascular organoid sprouting growth is mixed with type I collagen to form a gel mixed culture medium, wherein the concentration of type I collagen is 0.2 mg / mL.

[0086] (2) Take a portion of the gel prepolymerization solution, spread it evenly in a culture dish, and treat it for coagulation at 30° C. for 2 h. After solidification, the thickness of the bottom layer of the culture medium obtained is 3 mm.

[0087] (3) Taking another portion of the gel prepolymer solution to encapsulate the vascular organoid embryoid body spheres or other cells to be cultured, specifically, encapsulating the vascular organoid embryoid body spheres in the gel prepolymer solution at a dispersion density of 80 spheres / mL, gently shaking to evenly disperse the vascular organoid embryoid body spheres in the gel prepolymer solution, and evenly spreading the obtained mixture on the culture medium bottom layer formed in step (2), and coagulating it at 30°C for 2 hours. The thickness of the encapsulation layer after solidification is 3 mm.

[0088] (4) adding the gel mixed culture medium prepared in step (1) to the solidified encapsulation layer, and the coverage depth of the gel mixed culture medium on the encapsulation layer reaches 2 mm; after culturing for 24 hours (the first day of culture), removing the liquid on the upper layer of the encapsulation layer and replacing it with a special liquid culture medium for the sprouting growth of vascular organoids, and then replacing it with fresh special liquid culture medium for the sprouting growth of vascular organoids once a day for 2-5 days.

[0089] The morphological changes of budding growth of vascular organoid embryoids were recorded during the culture period from 1 day to 5 days. Figure 3 .like Figure 3 As shown, when the gel-wrapped vascular organoid embryoid body spheres were cultured for 1 day, more sprouts could be observed in the matrix coagulation material formed based on the physiological mixed gel precursor solution of Example 1, specifically, a number of burr-like sprouts extended from the surface of the vascular organoid embryoid body spheres, while only a small number of sprouts were found on the surface of the vascular organoid embryoid body spheres in the matrix coagulation material formed based on the physiological mixed gel precursor solution of Comparative Example 1. This indicates that encapsulating the vascular organoid embryoid body spheres in the matrix coagulation material formed in Example 1 is conducive to the production of higher density sprouts.

[0090] After culturing the gel-wrapped vascular organoid embryoid spheres for 5 days, obvious branched vascular lumen-like structures have been formed around the vascular organoid embryoid spheres in the matrix coagulation material formed based on the physiological mixed gel precursor solution of Example 1, while no obvious branched vascular lumen-like structures are observed on the surface of the vascular organoid embryoid spheres in the matrix coagulation material formed based on the physiological mixed gel precursor solution of Comparative Example 1. This indicates that wrapping the vascular organoid embryoid spheres in the matrix coagulation material formed in Example 1 can culture vascular organoids with complete structures more quickly.

[0091] After 5 days of culture, the vascular organoid embryoid spheres were separated from the matrix gel material and cultured for another 3 days. Figure 3 It can be observed that the vascular organoids peeled off from the matrix coagulation material formed in Example 1 have grown to a diameter of more than 2 mm, while the diameter of the vascular organoids peeled off from the matrix coagulation material formed in Comparative Example 1 is just over 1 mm. It can be seen that after the vascular organoid embryoid body spheres are wrapped in the matrix coagulation material formed in Example 1 and cultured for a period of time, even if the vascular organoid embryoid body spheres are peeled off from the matrix coagulation material, they will have a faster growth rate in the subsequent culture process.

[0092] Combination Figure 4 As shown, after culturing the gel-wrapped vascular organoid embryoid spheres for 5 days, a rich vascular lumen-like network structure has been formed around the vascular organoids cultured based on the matrix condensation material formed by the physiological mixed gel precursor solution of Example 1, while the vascular organoids cultured based on the matrix condensation material formed by the gel precursor solution of Comparative Example 1 have an incomplete structure and no vascular lumen-like network structure is observed.

[0093] In summary, by encapsulating the vascular organoid embryoid spheres in the matrix condensation material formed in Example 1 and culturing them, higher density buds and faster sprouting growth rates can be obtained, and ultimately vascular organoids with better morphological integrity and support can be obtained.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A physiological mixed gel precursor solution, characterized in that: The invention is composed of the following ingredients: culture medium, spleen hydrogel, type I collagen 0.8-1.2 mg / mL, type IV collagen 0.1-0.5 mg / mL, fibrinogen 2.5-7.5 mg / mL, and aprotinin 1-5 μg / mL; wherein the concentration of spleen hydrogel is 2.5-12.5 mg / mL; the culture medium is a special liquid culture medium for the budding growth of vascular organoid embryoids; and the spleen hydrogel is a hydrogel made from animal spleen extracellular matrix.

2. The physiological mixed gel precursor solution according to claim 1, characterized in that: The preparation method of the spleen hydrogel stock solution is as follows: S1. Preparation of biological matrix materials: spleen tissue of animals of the same or different species is processed to prepare biological matrix materials; the processing method is to remove the fat tissue in the spleen tissue and cut it into 1-3 mm 3 The tissue pieces were soaked in physiological saline, rinsed, and then decellularized to obtain decellularized spleen extracellular matrix; S2, digestion: wash the spleen extracellular matrix obtained in step S1 with deionized water, shake and rinse, change the water every 40-60 minutes, for a total of 15-25 times, remove the residual decellularization reagent, vacuum freeze-dry, weigh, add 0.01-0.2M hydrochloric acid solution and grind into a homogenate, add pepsin, and digest with constant temperature stirring to obtain a uniform and viscous spleen extracellular matrix solution; S3. Incubate to form gel: In an ice bath, adjust the pH of the spleen extracellular matrix solution to neutral with 1M sodium hydroxide solution, then add PBS solution, incubate at 37°C to form gel, and store at 4°C.

3. The physiological mixed gel precursor solution according to claim 1, characterized in that: The special liquid culture medium for the sprouting and growth of vascular organoid embryoids is the STEMdiffTM Blood Vessel Organoid Kit.

4. The physiological mixed gel precursor solution according to claim 1, characterized in that: The spleen hydrogel is a hydrogel using at least one of the extracellular matrix of pig spleen and bovine spleen as raw material.

5. A method for preparing the physiological mixed gel precursor solution according to any one of claims 1 to 4, characterized in that: The steps include: (1) diluting the spleen hydrogel stock solution with a special liquid culture medium for budding growth of vascular organoid embryoids to obtain a first dispersion; mixing a fibrinogen solution, a type I collagen solution, a type IV collagen solution, and a special liquid culture medium for budding growth of vascular organoid embryoids to obtain a second dispersion; mixing aprotinin solution with a special liquid culture medium for budding growth of vascular organoid embryoids to obtain a third dispersion; (2) The first dispersion liquid, the second dispersion liquid, and the third dispersion liquid are mixed to obtain the physiological mixed gel precursor solution.

6. A method for culturing vascular organoids, characterized in that: include: The first step is to add a thrombin solution to the physiological mixed gel precursor solution according to any one of claims 1 to 4, wherein the amount of thrombin added is 10-30 U / mL, and after mixing evenly, a physiological mixed gel prepolymer solution is obtained; a special liquid culture medium for budding growth of vascular organoids is mixed with type I collagen to form a gel mixed culture medium, wherein the concentration of type I collagen is 0.1-0.3 mg / mL; The second step: disperse and wrap the vascular organoid embryoid body spheres in a physiological mixed gel prepolymerization solution for gel solidification treatment, the solidification treatment temperature is 25-37°C, the solidification treatment time is 1-2h, and then the solidified gel material wrapped with the vascular organoid embryoid body spheres is immersed in the gel mixed culture solution and cultured for 24h, and then the special liquid culture medium for the budding growth of vascular organoids is replaced, and static culture is carried out for 5-15 days to obtain vascular organoid spheres with branched vascular lumen-like structures; wherein the dispersion density of the vascular organoid embryoid body spheres in the solidified gel material is 60-100 / mL.

7. The culture method according to claim 6, characterized in that: In the second step, the vascular organoid embryoid spheres are dispersed and encapsulated with cardiomyocytes, stem cells or glomerular cells in a physiological mixed gel prepolymer solution for solidification.

8. The culture method according to claim 6, characterized in that: The second step is as follows: Step 1: Take a portion of the physiological mixed gel prepolymer solution and spread it evenly in a culture well plate, a culture dish or an organ culture chip, and then perform gel solidification treatment to form a culture medium bottom layer. The solidification treatment temperature is 25-37° C., and the solidification treatment time is 1-2 hours. After solidification, the obtained culture medium bottom layer has a thickness of 2-3 mm; Step 2: adding the vascular organoid embryoid body spheres to another portion of the physiological mixed gel prepolymer solution, shaking to evenly disperse the vascular organoid embryoid body spheres in the physiological mixed gel prepolymer solution, spreading the obtained mixture evenly on the bottom layer of the culture medium, and then performing gel curing treatment to form a wrapping layer, the curing treatment temperature is 25-37° C., the curing treatment time is 1-2 h, and the thickness of the cured wrapping layer is 2-3 mm; Step 3: adding the gel mixed culture solution to the solidified wrapping layer, and the coverage depth of the gel mixed culture solution on the wrapping layer reaches 1-2 mm; after culturing for 24 hours, removing the liquid on the upper layer of the wrapping layer and replacing it with a special liquid culture medium for the sprouting growth of vascular organoids, and then replacing it with fresh special liquid culture medium for the sprouting growth of vascular organoids every day until the culture is 5-15 days; Step 4: After the culture is completed, the vascular organoids are peeled off from the physiological matrix gel material to obtain vascular organoids with branched vascular lumen-like structures.

9. A method for preparing a physiological matrix gel material, characterized in that: include: Add thrombin solution to the physiological mixed gel precursor solution according to any one of claims 1 to 5, the amount of thrombin added is 10-30 U / mL, mix well to obtain a physiological mixed gel prepolymer solution; then solidify it at 25-37° C. for 1-2 hours to obtain a physiological matrix gel material.

10. Use of the physiological mixed gel precursor solution according to any one of claims 1 to 5 or the physiological matrix gel material prepared by the preparation method according to claim 9 in constructing vascularized engineered myocardial tissue, vascularized artificial liver lobule tissue or vascularized artificial glomerular tissue.

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