Preparation method of self-oxygen-supplying water gel cell scaffold, and product and application thereof

Abstract

The present application belongs to the technical field of biomaterial preparation, and particularly relates to a preparation method of a self-oxygen-supplying hydrogel cell scaffold, the obtained product and application. The present application realizes stable and continuous release of oxygen and three-dimensional (3D) culture of neural stem cell spheres. The method is realized through the following steps: compounds P-MA, P-Cys, ColMA and PEG-CaO2 are added to a PBS solution, a photoinitiator is added, and a'self-oxygen-supplying' composite hydrogel is obtained by irradiation with ultraviolet light of a wavelength of 365 nm. The present application provides a composite hydrogel loaded with oxygen-producing nanoparticles for 3D cell sphere culture and a new method for continuous release of O2 in a slightly acidic environment, aiming at the problems of intermediate cell death caused by hypoxia in the process of 3D culture of existing cell spheres, unclear matrigel composition, high price and the like. The present application solves the problem of internal hypoxia in the process of cell sphere culture, and clearly defines the gel composition for studying the interaction between other components and cells. In addition, the self-oxygen-supplying hydrogel material prepared by the technology is expected to be applied to 3D culture of other cell spheres or organoid spheres.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomaterial preparation, and particularly relates to a preparation method of a self-oxygen-supplying hydrogel cell scaffold loaded with CaO2 nanoparticles and the obtained product and application. BACKGROUND

[0002] With the development of biotechnology, researchers found that the culture of cell spheroids can better reflect the characteristics of tissues, and thus the culture of blood cell spheroids was developed. The most remarkable is the successful cultivation of organoid spheroids, which can almost perfectly exhibit the structure of micro-tissues or organs. An organoid is a spheroid micro-organ with a three-dimensional structure composed of organ-specific cells differentiated from pluripotent stem cells or adult stem cells in an in vitro environment, which has a complex structure highly similar to a real organ and can partially simulate the physiological function of the source tissue or organ. Moreover, stem cells can self-organize into a 3D structure similar to an in vivo organ or tissue through cell sorting and spatially restricted lineage differentiation in a 3D environment, similar to in vivo. With the help of organoids, researchers can observe the changes in human tissues in depth, better understand the development process, and be used for drug efficacy screening and pathological research.

[0003] In addition, organoids can provide an ideal physiological model between animal models and human clinical experiments in the field of regenerative medicine, and have important scientific and application values. At present, researchers have successfully constructed organoids corresponding to different organs, including heart, liver, kidney, intestine and brain, etc., for in vitro research on the functions of different organs. However, when the volume of organoids gradually increases, tissue necrosis caused by hypoxia and lack of nutrients is a problem that needs to be solved. However, the existing technology has limitations in oxygen supply and improvement of scaffold mechanical properties, resulting in the phenomenon of central cell apoptosis during organoid culture.

[0004] At present, Matrigel is the most commonly used gel for organoid model culture. The composition of Matrigel is not clear, has large batch differences, and is complex, and it is not easy to regulate the composition to study the influence on organoids. Therefore, the reproducibility of the preparation of patterned neural tube organoids is poor, and it is difficult to adjust the composition of Matrigel to improve the efficiency of organoid patterning. At the same time, Matrigel is mainly imported in China, which is expensive and has large price fluctuations. In addition, Matrigel is derived from mouse sarcoma and may carry immunogens, pathogenic genes or viruses, which can have a serious impact on the culture of organoids for drug screening and regenerative medicine. These problems greatly hinder the reproducibility of neural organoid preparation and its application in the fields of developmental mechanisms, drug screening, construction of neural disease models and regenerative medicine. SUMMARY

[0005] In order to solve the problem of oxygen deficiency in the growth process of the preparation of the neural organoid model in the prior art, and the problem of relying on the Matrigel matrix glue of the mouse source, the application provides a preparation method of a'self-oxygen supply' composite hydrogel, and the components of the hydrogel prepared by the method are very clear, and the effective components are P-MA, P-Cys, ColMA, CaO2 and LAP.

[0006] The application further provides a composite hydrogel prepared by the preparation method, and the'self-oxygen supply' composite hydrogel can solve the problem of oxygen deficiency of the organoid, and has the potential to replace the matrix glue Matirgel in the future, which has unclear components, is expensive, carries pathogens or immunogens, and can also be used in the field of regenerative medicine of other 2D and 3D cell culture.

[0007] The application further provides an application of the composite hydrogel in cell culture.

[0008] The application further provides an application of the composite hydrogel in cell culture.

[0009] The application provides a preparation method of a self-oxygen supply hydrogel cell scaffold, which comprises the following steps:

[0010] S1, glyceryl methacrylate modified gamma-polyglutamic acid solid P-MA and cysteine modified gamma-polyglutamic acid solid P-Cys powder are added into a PBS buffer solution, and are ultrasonically dissolved to obtain a P-MC solution;

[0011] S2, the methacrylate modified type I collagen ColMA solid and a photoinitiator are added into the P-MC solution, and are shaken and vortexed at room temperature until completely dissolved to obtain a mixed solution;

[0012] S3, the mixed solution is filtered to obtain a gel solution, and then polyethylene glycol (PEG) coated CaO2 nanoparticles PEG-CaO2 are added, and after ultrasonic treatment, a composite hydrogel solution is obtained;

[0013] S4, the composite hydrogel solution is subjected to solidification treatment to obtain a hydrogel cell scaffold P-MC-Ca.

[0014] Further, in S1, the mass ratio of P-MA to P-Cys is 1:1, and the mass / volume concentrations of P-MA and P-Cys are 2.5% to 5%, respectively.

[0015] Further, in S1, the ultrasonic treatment is performed at 45 Hz and 25 DEG C for 10 minutes.

[0016] Further, in S2, the concentration of ColMA in the mixed solution is 5mg / mL-20mg / mL; the photoinitiator is LAP or I2959; the mass fraction of the photoinitiator in the mixed solution is 0.1%.

[0017] Further, in S3, the concentration of PEG-CaO2 in the mixed solution is 200μg / mL-800μg / mL.

[0018] Further, in S3, the filtration is performed by using a 0.22μm sterile membrane.

[0019] Further, in S4, the solidification treatment is performed by using a 365nm UV lamp for 20s-60s.

[0020] The application further provides a hydrogel cell scaffold P-MC-Ca prepared by the above preparation method.

[0021] The application further provides application of the above hydrogel cell scaffold P-MC-Ca in cell culture.

[0022] The application further provides application of the above hydrogel cell scaffold P-MC-Ca in three-dimensional culture of neural cell spheres.

[0023] In further improvement of the above scheme, the methacrylated I-type collagen ColMA is derived from bovine Achilles tendon, and the concentration is 3mg / mL-10mg / mL.

[0024] In further improvement of the above scheme, S1 is specifically adding 70mg of methacrylated polyglutamic acid powder and 70mg of cysteine-modified polyglutamic acid into 2ml of phosphate buffered saline solution, stirring and dissolving to obtain a P-MC solution. Then, 10mg of ColMA and 100μl of PEG-CaO2 nanoparticles with a concentration of 2mg / mL are added into the P-MC solution, and vortexed to mix uniformly to obtain a composite hydrogel solution.

[0025] The application uses the prepared'self-oxygen supply' composite hydrogel to culture neural stem cell spheres, and the following steps are specifically used:

[0026] (1) Preparation of composite hydrogel: 70 mg of P-MA and 70 mg of P-Cys were dissolved in 2 mL of PBS buffer solution, then 10 mg of methacrylated type I collagen ColMA and 2 mg of photoinitiator LAP were added to the above mixed solution in turn, and the solution was shaken and vortexed at room temperature until the solid was completely dissolved to form a transparent liquid. Then, the composite hydrogel solution was filtered through a 0.22 μm sterile membrane to obtain a sterile gel solution. Then, the UV-sterilized (20 min) PEG-CaO2 nanoparticle solid was added and shaken to uniformly disperse to obtain a'self-oxygenated' composite hydrogel solution P-MC-Ca.

[0027] (2) Multi-potent neural stem cell sphere embedding: The composite hydrogel solution was shaken and vortexed, and 300 μL of different concentrations of gel solution was transferred to different wells of a 24-well plate, respectively, and shaken to completely cover the bottom of the plate, then irradiated with a 365 nm ultraviolet lamp for 30 s to solidify. Then, 300 μL of different concentrations of gel solution was added to the EP tube containing the neural spheres, respectively, to resuspend the hydrogel cell solution, and added to the corresponding gel wells with different concentrations of gel, and irradiated with a ultraviolet lamp for 30 s to solidify, and 1 mL of culture medium was added for three-dimensional culture, and the culture medium was changed every 2 days.

[0028] (3) The prepared P-MC-Ca composite hydrogel was connected to the three-dimensional culture of neural stem cells, and the growth state of the neural spheres was observed on the 1st, 3rd and 6th day, and the optimal concentration of the gel suitable for the growth of the neural sphere was preliminarily determined. Secondly, the structure of the neural tube organoid was observed on the 3rd and 6th day of neural sphere differentiation, respectively, and the expression of neural tube organoid related markers and the structure of the organoid were detected to determine the optimal gel concentration suitable for the differentiation of neural spheres to grow neural tubes, and the outgrowth state was observed by taking pictures. After one week of continuous culture, the differentiated cell types were analyzed by immunofluorescence staining.

[0029] The beneficial effects of the present application are:

[0030] (1) The present application aims to solve the problems of internal hypoxia of existing cell spheres or organoid spheres in 3D culture and the unclear composition of Matrigel derived from mouse sarcoma, and provides a'self-oxygenated' composite gel with clear composition for replacing Matrigel for 3D culture of cell spheres. The composite hydrogel prepared by the present application has excellent biocompatibility, can solve the problem of hypoxia of organoids, and has the potential to replace Matrigel with unclear composition, high price, and carrying pathogens or immunogens in the future, and can also be used in other 2D and 3D cell culture regenerative medicine fields;

[0031] (2) The method provided by the present invention can not only realize the oxygen supply function, but also use the reductase secreted by the cell to degrade the dynamic covalent bond to regulate the mechanical properties of the gel. This self-oxygenated hydrogel material is expected to be applied to the 3D culture of other cell spheres or organoid spheres. Attached Figure Description

[0032] Figure 1 Scanning electron microscope image of CaO2 nanoparticles;

[0033] Figure 2 Dynamic light scattering of CaO2 nanoparticles;

[0034] Figure 3 Images of gel blocks at different concentrations;

[0035] Figure 4 Images of cell proliferation in gels of different concentrations;

[0036] Figure 5 Confocal fluorescence image of neurosphere immunofluorescence staining. Detailed Implementation

[0037] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] The γ-polyglutamic acid solid modified with glyceryl methacrylate used in this invention was prepared by the following method: 1.0 g of γ-polyglutamic acid (7.75 mmol) was dissolved in 100 mL of deionized water. Then, 2.0 mL of glycidyl methacrylate was slowly added to the solution, and the mixture was stirred until homogeneous. The pH of the reaction solution was then adjusted to 4.5 by adding 0.1 M HCl aqueous solution, and the reaction was carried out at 60 °C for 10 hours. The reaction solution was then transferred to a dialysis bag (with a cutoff value of 8000-14000) and thoroughly purified by dialysis with deionized water for 3 days. Finally, the discolored solid product was obtained by freeze-drying.

[0040] The cysteine-modified γ-polyglutamic acid solid used in this invention was prepared by the following method: 1.0 g of γ-polyglutamic acid (7.75 mmol) was dissolved in 100 mL of deionized water. Then, 2.0 mL of cysteine ​​was slowly added to the solution and stirred until homogeneous. The pH of the reaction solution was then adjusted to 4.5 by adding 0.1 M HCl aqueous solution, and the reaction was carried out at 60°C for 10 hours. The reaction solution was then transferred to a dialysis bag (retention value 8000-14000) and thoroughly purified by dialysis in deionized water for 3 days. Finally, the discolored solid product was obtained by freeze-drying.

[0041] The methacrylated type I collagen ColMA used in the present application is prepared by the following method: 1.2 g of type I collagen (purchased from Becton Dickinson) is weighed and dissolved in 20 mL of 1.5 mmol / L HCl aqueous solution by magnetic stirring. Then, the pH value of the reaction solution is adjusted to 7.0 by adding sodium hydroxide (2.0 M) 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 stirred to mix uniformly. The reaction is carried out at 55°C for 6 hours. Then, the reaction solution is transferred to a dialysis bag and purified by dialysis in deionized water for 3 days. Finally, the white solid product is obtained by freeze-drying.

[0042] The polyethylene glycol (PEG) coated CaO2 nanoparticles PEG-Ca used in the present application are prepared by the following method: 0.6 g of CaCl2·2H2O and 0.6 g of PVP are dissolved in 30 mL of anhydrous methanol under stirring. Then, a mixed solution of 480 μL of H2O2 and 1 mL of ammonia water solution is added. The mixed solution is stirred at 0°C for 1 hour. The obtained precipitate is collected by centrifugation, and vacuum dried to obtain CaO2 nanoparticles. 1 mL of PEG solution (10 mg / mL) is added to 0.5 mL of CaO2 chloroform solution, respectively, and ultrasonically treated. After the chloroform is completely evaporated, the dried PEGylated PEG-Ca is redispersed in deionized water for further use. −1 ) is added to 0.5 mL of CaO2 chloroform solution, respectively, and ultrasonically treated. After the chloroform is completely evaporated, the dried PEGylated PEG-Ca is redispersed in deionized water for further use.

[0043] The CaO2 nanoparticles prepared in the present application are tested by scanning electron microscopy and dynamic light scattering (DLS) experiments: 2.5 μL of CaO2 nanoparticle aqueous solution is dropped on a conductive silicon wafer with a pipette, left to stand, and naturally dried in air, and then tested. The morphology of the obtained nanoparticles is spherical nanoparticles. Figure 1

[0044] The CaO2 nanoparticles are dispersed in anhydrous ethanol solution, washed with anhydrous ethanol for 3-5 times, and centrifuged to remove unreacted PVP and free calcium ions. Then, 2.5 mL of nanoparticle aqueous solution with a concentration of 100 μg / mL is taken to a cuvette, and dynamic light scattering test is quickly performed. The obtained nanoparticles have a normal distribution of diameters, and the particle size is mainly concentrated in 500-600 nm. Figure 2

[0045] Example 1

[0046] The present example is a preparation method of composite hydrogel loaded with CaO2 nanoparticles, which comprises the following steps: ​​

[0047] (1) 70 mg of glyceryl methacrylate-modified γ-polyglutamic acid solid P-MA and 70 mg of cysteine-modified γ-polyglutamic acid solid P-Cys were weighed and dissolved in 2 mL of PBS buffer solution (pH = 7.4) under ultrasonic waves at 45 MHz and 25°C for 10 minutes to obtain a P-MC solution. Then, 10 mg of methacrylated type I collagen ColMA and 2 mg of a photoinitiator LAP were sequentially added, and the mixture was shaken and vortexed at room temperature until the solids were completely dissolved to form a transparent liquid. The formed transparent liquid was filtered using a 0.22 μm sterile membrane to obtain a sterile gel solution, and then ultraviolet-sterilized PEG-CaO2 nanoparticles (300 μg / mL) solid was added and ultrasonically shaken to uniformly disperse the PEG-CaO2 nanoparticles to obtain a'self-oxygen supply' composite hydrogel solution, which was stored in a sterile refrigerator at 4°C.

[0048] Example 2

[0049] (1) 70 mg of glyceryl methacrylate-modified γ-polyglutamic acid solid P-MA and 70 mg of cysteine-modified γ-polyglutamic acid solid P-Cys were weighed and dissolved in 2 mL of PBS buffer solution (pH = 7.4) under ultrasonic waves at 45 MHz and 25°C for 10 minutes to obtain a P-MC solution. Then, 10 mg of methacrylated type I collagen ColMA and 2 mg of a photoinitiator LAP were sequentially added, and the mixture was shaken and vortexed at room temperature until the solids were completely dissolved to form a transparent liquid. The formed transparent liquid was filtered using a 0.22 μm sterile membrane to obtain a sterile gel solution, and then ultraviolet-sterilized PEG-CaO2 nanoparticles (300 μg / mL) solid was added and ultrasonically shaken to uniformly disperse the PEG-CaO2 nanoparticles to obtain a'self-oxygen supply' composite hydrogel solution, which was stored in a sterile refrigerator at 4°C.

[0050] Example 3

[0051] (1) 70 mg of glyceryl methacrylate-modified γ-polyglutamic acid solid P-MA and 70 mg of cysteine-modified γ-polyglutamic acid solid P-Cys were weighed and dissolved in 2 mL of PBS buffer solution (pH = 7.4) under ultrasonic waves at 45 MHz and 25°C for 10 minutes to obtain a P-MC solution. Then, 10 mg of methacrylated type I collagen ColMA and 2 mg of a photoinitiator LAP were sequentially added, and the mixture was shaken and vortexed at room temperature until the solids were completely dissolved to form a transparent liquid. The formed transparent liquid was filtered using a 0.22 μm sterile membrane to obtain a sterile gel solution, and then ultraviolet-sterilized PEG-CaO2 nanoparticles (300 μg / mL) solid was added and ultrasonically shaken to uniformly disperse the PEG-CaO2 nanoparticles to obtain a'self-oxygen supply' composite hydrogel solution, which was stored in a sterile refrigerator at 4°C.

[0052] Example 4

[0053] (1) 80 mg of glyceryl methacrylate modified γ-polyglutamic acid solid P-MA and 80 mg of cysteine modified γ-polyglutamic acid solid P-Cys were weighed and dissolved in 2 mL of PBS buffer solution (pH = 7.4) under ultrasonic for 10 minutes at 45 MHz and 25°C to obtain a P-MC solution. Then 10 mg of methacrylated type I collagen ColMA and 2 mg of a photoinitiator LAP were sequentially added, and the mixture was shaken and vortexed at room temperature until the solids were completely dissolved to form a transparent liquid. The formed transparent liquid was filtered with a 0.22 μm sterile membrane to obtain a sterile gel solution; then ultraviolet sterilized PEG-CaO2 nanoparticle (300 μg / mL) solids were added and ultrasonically shaken to uniformly disperse to obtain a'self-oxygenated' composite hydrogel solution, which was stored in a sterile refrigerator at 4°C.

[0054] Example 5

[0055] The biocompatibility of the composite hydrogel loaded with CaO2 nanoparticles prepared in Example 1 was investigated, and the specific steps were as follows:

[0056] (1) The'self-oxygenated' composite hydrogel solution P-MC-Ca was shaken and vortexed to uniformly disperse the components;

[0057] (2) 300 μL of the gel solution was transferred to a 24-well plate to cover the bottom, and was irradiated with a 365 nm ultraviolet lamp for 30 s to solidify. Then HUVEC cells were mixed with 300 μL of the gel solution and moved to the gel well with the solidified bottom, and were cultured for 24 h to observe cell proliferation.

[0058] The results are shown in Figure 4 , and the composite hydrogel loaded with PEG-CaO2 nanoparticles still has a relatively high survival rate, indicating that the composite hydrogel has good biocompatibility.

[0059] Example 6

[0060] The'self-oxygenated' composite hydrogel solution prepared in Example 1 was used for three-dimensional culture of neural stem cell spheres, and the specific steps were as follows:

[0061] (1) The'self-oxygenated' composite hydrogel solution was shaken and vortexed, and 300 μL of gel solution of different concentrations was transferred to different 24-well plate wells, which were shaken to cover the bottom of the plate, and then were irradiated with a 365 nm ultraviolet lamp for 30 s to solidify;

[0062] (2) Then, 300 μL of different concentrations of gel solution were added to the EP tube containing neural spheres (10-20) to resuspend the hydrogel cell solution, and were added to the corresponding gel hole with different concentrations, and were irradiated with an ultraviolet lamp for 30 s to solidify. 1 mL of medium was added for culture, and the culture was continued at 37°C, 5% CO2 concentration, and the medium was changed every 2 days, and the growth state of the neural spheres was observed on the 1st day, the 3rd day and the 6th day, and the optimal concentration of the gel suitable for the growth of the neural stem cell spheres was preliminarily determined. Secondly, the structure of the neural tube organoids was observed on the 3rd day and the 6th day of the neural sphere differentiation, and the expression of the neural tube organoid related markers and the structure of the organoids were detected to determine the optimal gel concentration suitable for the differentiation of the neural spheres to grow neural tubes, and the outgrowth state was observed by taking pictures. After one week of continuous culture, immunofluorescence staining was performed to analyze the types of differentiated cells;

[0063] (3) After the culture was completed, 4% paraformaldehyde was used for fixation for 2 h, and then PBS was washed three times. Then 0.1% Triton X-100 was used for incubation for 30 min, and then PBS was washed three times, and blocking solution was added for blocking for 30 min. After removing the liquid, 300 μL of primary antibody solution: main components map2 (rabbit) and GFAP (mouse) was added, and incubated at 4°C for 24 h. The primary antibody solution was removed, and secondary antibody solution (594 goat anti-rabbit and 488 goat anti-mouse) was added, and incubated at room temperature for 3 h in the dark, and then the liquid was removed, and PBS was washed three times;

[0064] (4) Subsequently, DAPI nuclear staining agent was used for incubation at room temperature in the dark for 10-30 min, and then the liquid was removed and PBS was washed three times (10 min each time). The gel block was transferred to a confocal dish for fluorescence confocal photography. There were mainly three groups: (1) 2.5% group, (2) 3.0% group, and (3) 4.0% group.

[0065] The results are as follows Figure 5 The cell spheres mainly differentiated into microglial cells and neurons at the three concentrations.

[0066] It should be understood that the application of the present application is not limited to the above-mentioned neural stem cell sphere culture, and those skilled in the art can improve or change the composition of the'self-oxygen supply' gel according to the above description, and at the same time, such gel scaffolds can also be used for the culture of other cell spheres or organoid spheres (such as vascular organoids, intestinal organoids, etc.), and all these improvements and changes should belong to the protection scope of the claims of the present application.

Claims

1. A method for preparing a self-oxygenated hydrogel cell scaffold, characterized in that, Includes the following steps: S1, add γ-polyglutamic acid solid P-MA modified with glyceryl methacrylate and γ-polyglutamic acid solid P-Cys powder modified with cysteine ​​to PBS buffer, and dissolve by sonication to obtain P-MC solution. S2, Add methacrylic anhydride-modified type I collagen ColMA solid and photoinitiator to P-MC solution, shake and vortex at room temperature until completely dissolved to obtain a mixed solution; S3. Filter the mixed solution to obtain a gel solution, then add polyethylene glycol-encapsulated CaO2 nanoparticles (PEG-CaO2), and sonicate to obtain a composite hydrogel solution. S4, the composite hydrogel solution is solidified to obtain the hydrogel cell scaffold P-MC-Ca; In S2, the concentration of ColMA in the mixed solution ranges from 5 mg / mL to 20 mg / mL; the photoinitiator is LAP or I2959; and the mass-volume fraction of the photoinitiator in the mixed solution is 0.1%. In S3, the concentration of PEG-CaO2 in the mixed solution is 200 μg / mL to 800 μg / mL.

2. The preparation method according to claim 1, characterized in that, In S1, the mass ratio of P-MA to P-Cys is 1:1; the mass-volume concentrations of P-MA and P-Cys are 2.5% to 5%, respectively.

3. The preparation method according to claim 1 or 2, characterized in that, In S1, the ultrasound conditions are: 45 Hz, 25°C for 10 minutes.

4. The preparation method according to claim 1, characterized in that, In S3, filtration is performed using a 0.22μm sterile membrane.

5. The preparation method according to claim 1, characterized in that, In S4, the curing process involves irradiating the product with a 365nm ultraviolet lamp for 20s to 60s.

6. A hydrogel cell scaffold P-MC-Ca prepared using the preparation method according to any one of claims 1-5.

7. The application of the hydrogel cell scaffold P-MC-Ca as described in claim 6 in cell culture.

8. The application of the hydrogel cell scaffold P-MC-Ca as described in claim 7 in the three-dimensional culture of neural cell spheres.

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