Preparation method and application of ECM protein-HA-Gel composite hydrogel for three-dimensional culture of vascular organs

Abstract

The invention belongs to the technical field of cross culture of biological materials and organoids, and particularly relates to an ECM protein for three-dimensional culture of vascular organoids. HA-; the invention relates to a preparation method and application of Gel composite hydrogel. In order to solve the problems that in the 3D culture process of existing cell spheres or organoid spheres, matrigel derived from mouse sarcoma seriously depends on, components are undefined, the price is high, and immunogenicity exists, the composite gel with the definite components is provided and used for replacing Matrigel to conduct 3D culture of vascular organoids. The composite hydrogel prepared by the invention has the characteristics of short gelation time, good biocompatibility, adjustable gel components and the like under the synergistic effect of all the raw materials, can solve the problem of matrigel used for 3D culture of organoids, can replace the potential of matrigel in the future, reduces the price of 3D culture cell scaffold materials, and has broad application prospects. Meanwhile, the method can also be used in the regenerative medicine field of other two-dimensional or three-dimensional cell and organoid culture.

Description

Technical Field

[0001] The present invention belongs to the cross-technical field of biomaterials and organoid culture, and specifically relates to a preparation method and application of an ECM protein-HA-Gel composite hydrogel for three-dimensional culture of vascular organoids. Background Art

[0002] Organoids are a new model that can highly simulate the process of organogenesis in the human body. They are spherical micro-organs with three-dimensional structures composed of organ-specific cells produced by directed induction differentiation of pluripotent stem cells or adult stem cells. Moreover, stem cells can perform cell sorting and spatially restricted lineage differentiation in a 3D environment in a manner similar to that in vivo, thereby self-organizing into a 3D structure similar to organs or tissues in the body. Therefore, organoids can provide an ideal physiological model between animal models and human clinical experiments for the fields of developmental biology, drug development, biological toxicity evaluation, and regenerative medicine, and have important scientific and application value. At present, many methods have successfully constructed organoids corresponding to different organs, including the brain, heart, liver, kidney, intestine, and bile duct, so as to realize the study of the functions of different human organs in vitro.

[0003] In recent years, organoids have become the preferred model carrier for new drug development and mechanism exploration. With the development of regenerative medicine technology and the gradual deepening of organoid-related research, organoids in the vascularized microenvironment are limited by factors such as lack of oxygen and nutrients in the later stage of culture, resulting in cell apoptosis or even tissue necrosis, making it difficult to maintain structure and function for a long time, which has become a difficult technical problem to solve in the culture of vascular organoids.

[0004] At present, Matrigel is the most commonly used colloid for organoid model culture. The composition of the matrix gel is unclear, the batch-to-batch differences are large, and the composition is complex. The composition cannot be regulated to study the effects on the differentiation and growth of organoids. The large batch-to-batch differences lead to poor reproducibility in the preparation of patterned vascular organoids, lack of rigor for subsequent comparative experiments, and heterogeneity of organoids in size, shape and cellular composition. At the same time, the commonly used matrix gel is mainly imported in China, and the high price and large price fluctuations have seriously hindered the development of basic scientific research. In addition, Matrigel is derived from mouse tumors and may carry immunogens, pathogenic genes or viruses. Since most of the components are undefined, batch-to-batch changes and local changes in mechanical properties in matrix gel preparations will introduce differences into organoid cultures, leading to variability and poor reproducibility of organoids, which have serious effects on the construction of disease models, drug screening and regenerative medicine in different types of organoid cultures; in addition, the addition of Matrigel to the culture system increases the difficulty of organoid delivery, genetic manipulation, whole genome genetic screening and high-throughput drug screening. These problems greatly hinder the reproducibility of vascular organoid preparation and its application in developmental mechanisms, drug screening, vascular disease model construction, and regenerative medicine. Summary of the invention

[0005] In order to solve the problems that the preparation of vascular organoid models in the prior art is too dependent on mouse-derived matrix gel Matrigel, expensive and poor reproducibility, the present invention provides a method for preparing an ECM protein-HA-Gel composite hydrogel for three-dimensional culture of vascular organoids. The composition of the composite hydrogel is very clear, and it has the potential to replace the matrix gel Matirgel with unclear composition, high price, and carrying pathogens and immunogens, and can be applied to the field of regenerative medicine for 2D and 3D cell or organoid culture.

[0006] The present invention further provides the use of the above-mentioned ECM protein-HA-Gel composite hydrogel in vascular organoid culture.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a method for preparing an ECM protein-HA-Gel composite hydrogel for three-dimensional vascular organoid culture, comprising the following steps: (1) Adding hyaluronic acid HAMA modified with glycidyl methacrylate and methacrylylated gelatin GelMA into a mixed culture medium and dissolving them by ultrasonication to obtain a HA-Gel gel solution; (2) Adding glycidyl methacrylate-modified protein and photoinitiator to the HA-Gel solution, vortexing at room temperature until completely dissolved, to obtain a mixed solution; or Adding a protein modified with glycidyl methacrylate to a HA-Gel gel solution, heating and incubating to obtain a composite hydrogel solution, and then adding a photoinitiator to the composite hydrogel solution to obtain a mixed solution; (3) The mixed solution is heated, filtered, and then photocured to obtain a hydrogel cell scaffold ECM protein-HA-Gel composite hydrogel.

[0008] Preferably, in step (1), the mass ratio of HAMA to GelMA is 1-2:2-3; the mass volume concentrations of HAMA and GelMA in the mixed culture medium are 1.0% to 10%, respectively. Preferably, in step (1), the molecular weight of the HAMA is greater than 2000 KDa.

[0009] Preferably, in step (1), the mixed culture medium solution is prepared by StemPro-34SFM complete culture medium (50 mL + 1.3 mL additive + 0.5 mL Glutamax + 0.5 mL P / S) + 15% FBS + 100 ng / mL VEGF165 + 100 ng / mL FGF2.

[0010] Preferably, the additives in the mixed culture medium are purchased from Thermo Fisher Scientific (Cat. No. 10639011).

[0011] Preferably, in step (2), the concentration of the protein modified with glycidyl methacrylate in the mixed solution is in the range of 2 mg / mL to 20 mg / mL; the protein is type I collagen, elastin, fibronectin or laminin; preferably, the protein is type I collagen.

[0012] Preferably, in step (2), the mass fraction of the photoinitiator in the mixed solution is 0.1% to 0.5%; the photoinitiator is LAP or I2959.

[0013] Preferably, in step (3), the water bath heating conditions are: heating at 50-60°C for 10-20 min; and filtering is performed using a 0.22 μm sterile membrane.

[0014] Preferably, in step (3), the curing treatment is performed by irradiating with an ultraviolet lamp having a wavelength of 405 or 365 nm for 20 seconds to 50 seconds.

[0015] The present invention also provides an application of the ECM protein-HA-Gel composite hydrogel obtained by the above preparation method in the three-dimensional culture of vascular organoids.

[0016] In some embodiments, multiple groups of the hydrogel cell scaffolds are prepared through steps (1)-(3), and the protein concentration in each group of the hydrogel cell scaffolds is different. Then, the multiple groups of the hydrogel scaffolds are screened to select the hydrogel cell scaffolds with the most suitable protein concentration.

[0017] In some embodiments, the glycidyl methacrylate-modified type I collagen ColMA is derived from bovine Achilles tendon, has an average MW of 300,000, and a concentration range of 2 mg / mL to 10 mg / mL.

[0018] Most preferably, 0.1% by weight of LAP photoinitiator is slowly added to the HA-Gel solution prepared in step (1), and the solution is incubated and shaken in a 60° C. water bath to dissolve and mix evenly.

[0019] Most preferably, the step (1) is specifically as follows: 40 mg of hyaluronic acid solid HAMA modified with glycidyl methacrylate and 60 mg of methacrylylated gelatin GelMA are added to 2 mL of culture medium solution, stirred to dissolve, and HA-Gel hydrogel solution is obtained. Then, 20 mg of type I collagen ColMA modified with glycidyl methacrylate is added to the HA-Gel solution, and vortexed to mix evenly, to obtain a composite hydrogel HA-Gel-ColMA solution.

[0020] The method of using the prepared composite hydrogel for culturing vascular organoids in the present invention specifically adopts the following steps: (1) Embedding of vascular organoids: Vortex the composite hydrogel HA-Gel-ColMA solution, transfer 300 μL of the gel solution of different components to different wells of 24-well plates, shake to completely cover the bottom of the well plate, and then solidify with a 365nm UV lamp for 30 seconds. Then, add 250 μL of gel solutions of different concentrations to the EP tubes containing vascular organoids, resuspend to obtain hydrogel solutions containing vascular organoids, add to the corresponding well plates with different concentrations, solidify with UV light for 30 seconds, and add 1 mL of vascular organoid culture medium for three-dimensional culture. Change the culture medium every 2 days.

[0021] (2) The prepared HA-Gel-ColMA composite hydrogel was inoculated into vascular organoids for 3D culture, and the differentiation and sprouting growth of vascular organoids were observed on the 1st, 3rd and 6th days to determine the optimal concentration of gel suitable for the growth of vascular organoids. Secondly, the morphology, structure and volume changes of vascular organoids were observed on the 1st, 3rd and 6th days of vascular organoid differentiation, and the expression of vascular organoid-related markers and the structure of organoids were detected to determine the optimal gel concentration suitable for vascular organoid differentiation into microvessels, and photos were taken to observe the sprouting state. After 6 days of continuous culture, immunofluorescence staining was performed to analyze the types of differentiated cells and their distribution.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention aims to solve the problems of unclear composition, high price, and immunogenicity of existing cell spheres or organoid spheres that rely heavily on Matrigel derived from mouse sarcoma during 3D culture. Here, a composite gel with clear composition is provided to replace Matrigel for 3D culture of organoids. The composite hydrogel prepared by the present invention has the characteristics of fast gelation time, good biocompatibility, and adjustable gel components under the synergistic effect of various raw materials. It can solve the problems of Matrigel used in 3D culture of organoids, and has the potential to replace Matrigel in the future, reducing the price of 3D culture cell scaffold materials. At the same time, it can also be used in other 2D and 3D cell and organoid culture regenerative medicine fields; (2) Compared with the traditional Matrigel which relies on imported brands, the effective ingredients of the composite hydrogel prepared by the present invention do not need to be imported, but are all domestically produced, and the price of raw materials is low, saving about 80% compared with the price of Matrigel, and the cost of use is lower, which is very suitable for a large number of scientific research such as organoid culture, drug screening, 3D printing, etc.; (3) The HA-Gel-ColMA composite hydrogel prepared by the present invention has a porous structure similar to that of the cytoplasmic matrix (ECM), and can effectively exchange nutrients and waste.

[0023] (4) The collagen in the HA-Gel-ColMA composite hydrogel prepared by the present invention can provide abundant attachment sites for organoid cells, thereby being more conducive to the differentiation and growth of organoids.

[0024] (5) The mature vascular organoids obtained by culturing the HA-Gel-ColMA composite hydrogel prepared in the present invention have a 3D structure and molecular characteristics that can achieve a culture effect similar to that of Matrigel, and have the potential to replace the more expensive Matrigel for basic and clinical research.

[0025] (6) The HA-Gel-ColMA composite hydrogel prepared by the present invention can change the cross-linking degree of the composite gel by adjusting the mass fraction of GelMA and HAMA and adjusting the UV irradiation cross-linking time in the range of 15s to 60s, thereby changing the mechanical properties of the HA-Gel-ColMA composite hydrogel to obtain the optimal conditions for the growth and differentiation of organoids.

[0026] (7) The preparation and culture methods of the artificial matrix gel provided by the present invention can improve the efficiency of vascular organoid differentiation into tubes, thereby expanding the application of human vascular organoids in the fields of developmental mechanism, drug screening, model construction and treatment of myocardial infarction and cerebral infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the nuclear magnetic hydrogen spectrum of methacryloylated type I collagen ColMA; Figure 2 is a scanning electron micrograph of HA-Gel-ColMA; Figure 3 Here are pictures of gel blocks with and without collagen added; Figure 4 Figure 2 shows the development process of vascular organoids in HA-Gel-ColMA at different times. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] The GelMA used in the present invention has a porous structure similar to the extracellular matrix (ECM), which can not only provide a three-dimensional (3D) space for cell proliferation and differentiation, but also can be used as a carrier of various biologically active substances such as cytokines and drugs. The raw material gelatin of the GelMA used in the present invention can be either type A or type B. The synthesis of methacryloyl gelatin GelMA used in the present invention is mainly based on the literature reported Materials 2016, 9 , 797, which is mainly obtained by the reaction of methacrylic anhydride and gelatin.

[0031] The hyaluronic acid HAMA solid modified with glycidyl methacrylate used in the present invention is prepared by the following method: 1.0 g hyaluronic acid (7.75 mmol) is dissolved in 100 ml of deionized water. Subsequently, 2.0 ml of glycidyl methacrylate (purchased from McLean) is slowly added to the solution and stirred to mix evenly. Then, the pH value of the reaction solution is adjusted to 4.7 by adding HCl (0.1 M) aqueous solution, and the reaction is carried out at a temperature of 60°C for 10 hours. Finally, the reaction solution is transferred to a dialysis bag with a cutoff value of 8000-14000 and dialyzed and purified in deionized water for 3 days. Finally, a white solid product is obtained by freeze drying.

[0032] The methacrylylated type I collagen ColMA used in the present invention is prepared by the following method: 1.0 g of type I collagen (purchased from Yuanye Company) is weighed and placed in 100 ml of 1.5 mmol / L HCl aqueous solution, and magnetic stirring is used to completely dissolve it. Then, the pH value of the reaction solution is adjusted to 7.0 by dropping 2.0 M sodium hydroxide aqueous solution, and then 0.36 g of glycidyl methacrylate is slowly added to the solution, and 0.8 g of lithium bromide is added, and the mixture is vigorously stirred to completely dissolve it. React at 60°C for 6 hours. Then, the reaction solution is transferred to a dialysis bag (cutoff value 8000-14000) and dialyzed and purified in deionized water for 3 days. Finally, a white solid product is obtained by freeze drying. It was characterized by nuclear magnetic hydrogen spectrum, and by comparison with collagen, it was found that two obvious peaks belonging to double bond hydrogen protons appeared at 5.5~6.5ppm, indicating that ColMA was successfully synthesized. The results are shown in Figure 1 .

[0033] Example 1 (1) Add 60 mg of methacryloyl gelatin GelMA solid powder and 40 mg of methacryloyl hyaluronic acid HAMA into 2 mL of mixed culture medium solution, and incubate in a 55°C water bath for 10 min to mix evenly to obtain a mixed hydrogel HA-Gel solution; (2) adding 20 mg of methacryloyl-type I collagen to the obtained HA-Gel solution, mixing and incubating in a 60°C water bath for 20 min to obtain a composite hydrogel HA-Gel-ColMA solution, wherein the concentration of the collagen solution in the prepared composite hydrogel solution is 10 mg / mL; (3) Add 2 mg of photoinitiator LAP to the prepared composite hydrogel HA-Gel-ColMA solution, heat it in a water bath and filter it with a 0.22 μm microporous filter membrane while it is hot to obtain a composite hydrogel solution that can be photocured and cross-linked. After photocuring to form a gel, it is characterized by scanning electron microscopy (SEM), and the results show a porous structure. The results are shown in Figure 2 .

[0034] Example 2 (1) Add 60 mg of methacryloyl gelatin GelMA solid powder and 40 mg of methacryloyl hyaluronic acid HAMA into 2 mL of mixed culture medium solution, and incubate in a 55°C water bath for 10 min to mix evenly to obtain a mixed hydrogel HA-Gel solution; (2) adding 10 mg of methacryloyl-type I collagen to the obtained HA-Gel solution, mixing and incubating in a 60°C water bath for 20 min to obtain a composite hydrogel HA-Gel-ColMA solution, wherein the concentration of the collagen solution in the prepared composite hydrogel solution is 10 mg / mL; (3) Add 2 mg of the photoinitiator LAP to the composite hydrogel HA-Gel-ColMA solution prepared by the above HA-Gel-ColMA, heat it in a water bath, and filter it with a 0.22 μm microporous filter membrane while it is hot to obtain a composite hydrogel solution that can be photocured and cross-linked.

[0035] Example 3 The hydrogel prepared in Example 1-2 was used for encapsulation of vascular organoids for 3D culture, and the specific steps were as follows: (1) Vortex the filtered composite hydrogel solution, transfer 300 μL of the gel solution into different wells of a 24-well plate, shake to cover the bottom of the well plate, and then irradiate with a 365 nm UV lamp for 30 seconds to solidify; (2) Then add 300 μL of gel solution to the EP tube containing vascular organoids, resuspend in the hydrogel cell solution, add to the corresponding wells with different concentrations of gel, and solidify under 365nm UV light for 30 seconds. Add 1mL of mixed culture medium for culture, continue to culture at 37°C, 5% CO2 concentration, change the culture medium every 2 days, and observe the vascular organoids sprouting to form a vascular network on the 1st to 3rd day, and preliminarily determine the optimal concentration of gel suitable for the growth of vascular organoids. Secondly, observe the structure of neural tube organoids on the 3rd and 6th days of vascular organoid differentiation, and detect the expression of neural tube organoid-related markers and the structure of the organoids to determine the optimal gel concentration suitable for vascular organoid differentiation to grow microvessels, and take pictures to observe the sprouting state. After continuing to culture for one week, immunofluorescence staining was performed to analyze the types of differentiated cells. Figure 3 These are pictures of the gel after light curing with and without collagen added; Figure 4 This is a diagram of the development process of vascular organoids at different times after encapsulation.

[0036] It should be understood that the above describes the basic principles, main features and advantages of the present application. The application of the present invention is not limited to the culture of the above-mentioned vascular organoids. For the art, the components of the photo-crosslinked composite hydrogel can be modified with different groups or the content of the components according to the above description. At the same time, this type of gel scaffold can also be used to culture other cells or organoids (such as neural organoids, intestinal organoids, tumor organoids, etc.). All these improved and transformed applications should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for preparing an ECM protein-HA-Gel composite hydrogel for three-dimensional culture of vascular organoids, characterized in that: The following steps are involved: (1) Adding glycidyl methacrylate-modified hyaluronic acid HAMA and methacrylylated gelatin GelMA into a mixed culture medium and dissolving them by ultrasonication to obtain a HA-Gel gel solution; (2) Adding glycidyl methacrylate-modified protein and photoinitiator to the HA-Gel solution, vortexing at room temperature until completely dissolved, to obtain a mixed solution; Alternatively, a protein modified with glycidyl methacrylate is added to the HA-Gel gel solution, and after heating and incubation, a composite hydrogel solution is obtained, and then a photoinitiator is added to the composite hydrogel solution to obtain a mixed solution; (3) The mixed solution is heated, filtered, and then photocured to obtain a hydrogel cell scaffold ECM protein-HA-Gel composite hydrogel.

2. The preparation method according to claim 1, characterized in that: In step (1), the mass ratio of HAMA to GelMA is 1-2:2-3; the mass volume concentrations of HAMA and GelMA in the mixed culture medium are 1.0% to 10%, respectively.

3. The preparation method according to claim 1 or 2, characterized in that: In step (1), the molecular weight of the HAMA is greater than 2000 KDa.

4. The preparation method according to any one of claims 1 to 3, characterized in that In step (1), the mixed culture medium solution is prepared by StemPro-34SFM complete culture medium (50mL + 1.3mL additive + 0.5mL Glutamax + 0.5mL P / S) + 15% FBS + 100ng / mL VEGF165 + 100ng / mL FGF2.

5. The preparation method according to claim 1, characterized in that: In step (2), the concentration of the protein modified with glycidyl methacrylate in the mixed solution is in the range of 2 mg / mL to 20 mg / mL; the protein is type I collagen, elastin, fibronectin or laminin; preferably, the protein is type I collagen.

6. The preparation method according to claim 1 or 5, characterized in that In step (2), the mass fraction of the photoinitiator in the mixed solution is 0.1% to 0.5%; the photoinitiator is LAP or I2959.

7. The preparation method according to any one of claims 1 to 6, characterized in that: In step (3), the water bath heating conditions are: heating at 50-60°C for 10-20 min; and filtering is performed using a 0.22 μm sterile membrane.

8. The preparation method according to claim 1 or 7, characterized in that: In step (3), the curing treatment is performed by irradiating with an ultraviolet lamp having a wavelength of 405 or 365 nm for 20 seconds to 50 seconds.

9. An application of the ECM protein-HA-Gel composite hydrogel obtained by the preparation method described in any one of claims 1 to 7 in the three-dimensional culture of vascular organoids.

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