Preparation method of self-oxygen-supply hydrogel cell scaffold as well as obtained product and application of self-oxygen-supply hydrogel cell scaffold

Abstract

The invention belongs to the technical field of biological material preparation, and particularly relates to a preparation method of a self-oxygen-supply hydrogel cell scaffold, an obtained product and application. According to the invention, stable and continuous release of oxygen and three-dimensional (3D) culture of neural stem cell spheres are realized. The method is realized by the following steps: adding compounds P-MA, P-Cys, ColMA and PEG-CaO2 into a PBS (Phosphate Buffer Solution), adding a photoinitiator, and irradiating and curing by ultraviolet light with the wavelength of 365nm to obtain the self-oxygen-supply composite hydrogel. The invention provides a novel method for applying composite hydrogel loaded with oxygen-producing nano-particles to 3D cell sphere culture and continuously releasing O2 in a slightly acidic environment to solve the problems of death of intermediate cells caused by oxygen deficit in the 3D culture process of existing cell spheres, indefinite components of matrigel, high price and the like. The problem of internal oxygen deficit in the cell ball culture process is solved, and the gel component is defined to be used for researching the effect between other components and cells. In addition, the self-oxygen-supply 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 invention belongs to the field of biomaterial preparation, and specifically relates to a preparation method of a self-oxygenating hydrogel cell scaffold loaded with CaO2 nanoparticles, and the obtained product and application. Background Art

[0002] With the development of biotechnology, researchers have found that the culture of cell spheroids can better reflect the characteristics of tissues, thus producing the culture of blood multicellular spheroids. The most remarkable thing is the successful cultivation of organoid spheroids, which can almost perfectly display the structure of micro-tissues or organs. Organoids are spherical micro-organs with three-dimensional structures composed of organ-specific cells differentiated from pluripotent stem cells or adult stem cells in vitro. They have complex structures that highly simulate real organs and can partially simulate the physiological functions of the source tissues or organs. 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 an organ or tissue in vivo. With the help of organoids, researchers can deeply observe the changes in human tissues, better understand the development process, and can 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 for fields such as regenerative medicine, and have important scientific and application value. At present, researchers have successfully constructed organoids corresponding to different organs, including the heart, liver, kidneys, intestines, and brain, for studying the functions of different organs in vitro. However, as the volume of organoids gradually increases, tissue necrosis caused by hypoxia and lack of nutrients is an urgent problem that needs to be solved. However, existing technologies have limitations in oxygen supply and improvement of the mechanical properties of scaffolds, resulting in the phenomenon of central cell apoptosis during organoid culture.

[0004] At present, Matrigel is the most commonly used colloid for organoid model cultivation. The composition of the Matrigel is unclear, the batch-to-batch difference is large, and the composition is complex. It is not easy to regulate the components to study the influence on the organoid. Therefore, the repeatability of the preparation of patterned neural tube organoids is poor, and it is difficult to adjust the Matrigel composition to improve the efficiency of organoid patterning. At the same time, the Matrigel is mainly imported domestically, and the price is expensive and the price fluctuation is large. In addition, the Matrigel is derived from mouse sarcoma, which may carry immunogens, pathogenic genes or viruses, and has a serious impact on drug screening and regenerative medicine in organoid culture. These problems greatly hinder the repeatability of neural organoid preparation and its application in the fields of developmental mechanism, drug screening, neurological disease model construction and regenerative medicine. Summary of the invention

[0005] In order to solve the problem in the prior art that the preparation of neural organoid models suffers from hypoxia during growth and relies on mouse-derived Matrigel, the present invention provides a method for preparing a 'self-oxygenating' composite hydrogel. The composition of the hydrogel prepared by the method is very clear, wherein the effective ingredients are methacryloyl polyglutamic acid P-MA, cysteine ​​functionalized polyglutamic acid P-Cys, methacryloyl type I collagen ColMA, CaO2 and photoinitiator LAP.

[0006] The present invention also provides a composite hydrogel prepared by the above-mentioned preparation method. The 'self-oxygenating' composite hydrogel can solve the problem of organoid hypoxia, and has the potential to replace Matrigel, which has unclear ingredients, is expensive, and carries pathogens or immunogens in the future. It can also be used in other 2D and 3D cell culture regenerative medicine fields.

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

[0008] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a method for preparing a self-oxygenating hydrogel cell scaffold, comprising the following steps: S1, adding methacrylate-modified γ-polyglutamic acid solid P-MA and cysteine-modified γ-polyglutamic acid solid P-Cys powder into PBS buffer, and dissolving by ultrasonication to obtain a P-MC solution; S2, adding methacrylate-modified type I collagen ColMA solid and a photoinitiator to the P-MC solution, vortexing at room temperature until completely dissolved, to obtain a mixed solution; S3, filtering the mixed solution to obtain a gel solution, then adding CaO2 nanoparticles PEG-CaO2 coated with polyethylene glycol (PEG), and obtaining a composite hydrogel solution after ultrasonic treatment; S4, curing the composite hydrogel solution to obtain the hydrogel cell scaffold P-MC-Ca.

[0009] Furthermore, in S1, the mass ratio of the P-MA to P-Cys is 1:1; and the mass volume concentrations of the P-MA and P-Cys are 2.5% to 5%, respectively.

[0010] Furthermore, in S1, the ultrasonic conditions are: 45 Hz, 25° C., for 10 minutes.

[0011] Furthermore, in S2, in the mixed solution, the concentration range of ColMA is 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%.

[0012] Furthermore, in S3, the concentration of PEG-CaO2 in the mixed solution is 200 μg / mL to 800 μg / mL.

[0013] Furthermore, in S3, filtration is performed using a 0.22 μm sterile membrane.

[0014] Furthermore, in S4, the curing treatment is performed by irradiating with an ultraviolet lamp having a wavelength of 365 nm for 20 seconds to 60 seconds.

[0015] The present invention also provides a hydrogel cell scaffold P-MC-Ca prepared by the above preparation method.

[0016] The present invention also provides the use of the hydrogel cell scaffold P-MC-Ca in cell culture The present invention also provides application of the hydrogel cell scaffold P-MC-Ca in three-dimensional culture of neuron spheres.

[0017] A further improvement of the above scheme, the methacrylylated type I collagen ColMA is derived from bovine Achilles tendon, and has a concentration range of 3 mg / mL to 10 mg / mL.

[0018] A further improvement of the above scheme, the step S1 specifically includes adding 70 mg of methacrylylated polyglutamic acid powder and 70 mg of cysteine-modified polyglutamic acid to 2 ml of phosphate buffered saline solution, stirring and dissolving to obtain a P-MC solution. Then, 10 mg of ColMA and 100 microliters of PEG-CaO2 nanoparticles with a concentration of 2 mg / mL are added to the P-MC solution, and vortexed to mix evenly to obtain a composite hydrogel solution.

[0019] The present invention uses the prepared 'self-oxygenating' composite hydrogel to culture neural stem cell spheres, specifically using the following steps: (1) Preparation of composite hydrogel: Weigh 70 mg P-MA and 70 mg P-Cys and dissolve them in 2 mL PBS buffer solution. Then, add 10 mg methacrylylated type I collagen ColMA and 2 mg photoinitiator LAP to the mixed solution in turn. Vortex at room temperature until the solid is completely dissolved to form a transparent liquid. Then, filter the composite hydrogel solution with a 0.22 μm sterile membrane to obtain a sterile gel solution. Then, add UV-sterilized (20 min) PEG-CaO2 nanoparticle solids and shake to evenly disperse them to obtain a 'self-oxygenated' composite hydrogel solution P-MC-Ca.

[0020] (2) Embedding of multipotent neural stem cell spheres: Vortex the composite hydrogel solution, transfer 300 μL of gel solutions of different concentrations to different wells of a 24-well plate, shake to completely cover the bottom of the well plate, and then irradiate with a 365nm UV lamp for 30 seconds to solidify. Then, add 300 μL of gel solutions of different concentrations to the EP tubes containing neurospheres, resuspend to obtain hydrogel cell solutions, add to the corresponding wells with gels of different concentrations, irradiate with a UV lamp for 30 seconds to solidify, and add 1 mL of culture medium for three-dimensional culture. Change the culture medium every 2 days.

[0021] (3) The prepared P-MC-Ca composite hydrogel was inoculated into the three-dimensional culture of neural stem cells, and the growth status of the neurospheres was observed on the 1st, 3rd and 6th days to preliminarily determine the optimal concentration of the gel suitable for the growth of neural stem cell spheres. Secondly, the structure of the neural tube organoids was observed on the 3rd and 6th days of neurosphere differentiation, and the expression of neural tube organoid-related markers and the structure of the organoids were detected to determine the optimal gel concentration suitable for neurosphere differentiation into neural tubes, and photos were taken to observe the budding status. After continuing to culture for one week, immunofluorescence staining was performed to analyze the types of differentiated cells.

[0022] The beneficial effects of the present invention are: (1) The present invention aims to solve the problems of internal hypoxia of existing cell spheres or organoid spheres during 3D culture and the unclear composition of Matrigel derived from mouse sarcoma. Here, a 'self-oxygenating' composite gel with clear composition is provided to replace Matrigel for 3D culture of cell spheres. The composite hydrogel prepared by the present invention has excellent biocompatibility, can solve the problem of organoid hypoxia, and has the potential to replace Matrigel with unclear composition, high price, and carrying pathogens or immunogens in the future. It can also be used in other 2D and 3D cell culture regenerative medicine fields. (2) The method provided by the present invention can not only realize the oxygen supply function, but also degrade the dynamic covalent bonds through the reductase secreted by cells to adjust the mechanical properties of the gel. This self-oxygenating hydrogel material is expected to be applied to other cell spheres or organoid spheres 3D culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a scanning electron microscope image of CaO2 nanoparticles; Figure 2 is the dynamic light scattering of CaO2 nanoparticles; Figure 3 The pictures are of gel blocks with different concentrations; Figure 4 Pictures of cell proliferation in gels of different concentrations; Figure 5Fluorescence confocal images of immunofluorescence staining of neurospheres. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] The γ-polyglutamic acid solid modified with glyceryl methacrylate used in the present invention is prepared by the following method: 1.0 g of γ-polyglutamic acid (7.75 mmol) is dissolved in 100 ml of deionized water. Subsequently, 2.0 ml of glycidyl methacrylate is slowly added to the solution and stirred to mix evenly. Then, the pH value of the reaction solution is adjusted to 4.5 by adding HCl (0.1 M) aqueous solution, and the reaction is carried out at 60°C for 10 hours. Then, the reaction solution is transferred to a dialysis bag (cutoff value 8000-14000) and thoroughly dialyzed and purified in deionized water for 3 days. Finally, a discolored solid product is obtained by freeze drying.

[0027] The cysteine-modified γ-polyglutamic acid solid used in the present invention is prepared by the following method: 1.0 g of γ-polyglutamic acid (7.75 mmol) is dissolved in 100 ml of deionized water. Subsequently, 2.0 ml of cysteine ​​is slowly added to the solution and stirred to mix evenly. Then, the pH value of the reaction solution is adjusted to 4.5 by adding HCl (0.1 M) aqueous solution, and the reaction is carried out at a temperature of 60°C for 10 hours. Then, the reaction solution is transferred to a dialysis bag (cutoff value 8000-14000) and thoroughly dialyzed and purified in deionized water for 3 days. Finally, a discolored solid product is obtained by freeze drying.

[0028] The methacrylylated type I collagen ColMA used in the present invention is prepared by the following method: 1.2 g of type I collagen (purchased from Bidex) is weighed and placed in 20 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 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 the mixture is stirred and mixed evenly. The reaction is carried out at a temperature of 55°C for 6 hours. Then, the reaction solution is transferred to a dialysis bag and thoroughly dialyzed and purified in deionized water for 3 days. Finally, a white solid product is obtained by freeze drying.

[0029] The polyethylene glycol (PEG)-coated CaO2 nanoparticles PEG-Ca used in the present invention are prepared by the following method: 0.6 g CaCl2·2H2O and 0.6 g PVP are dissolved in 30 mL anhydrous methanol under stirring. Subsequently, a mixed solution of 480 μL H2O2 and 1 mL ammonia solution is added. The mixed solution is stirred and reacted at 0°C for 1 hour. The resulting precipitate is collected by centrifugation and vacuum dried to obtain CaO2 nanoparticles. 1 mL PEG solution (10 mg mL −1 ) were added to 0.5 mL of CaO2 chloroform solution and ultrasonicated. After the chloroform was completely evaporated, the dried PEGylated PEG-Ca was dispersed in deionized water for further use.

[0030] The CaO2 nanoparticles prepared by the present invention were tested by scanning electron microscopy and dynamic light scattering (DLS): 2.5 microliters of the CaO2 nanoparticle aqueous solution was pipetted onto a conductive silicon wafer, left to stand, and allowed to dry naturally in the air before testing. Figure 1 The morphology of the nanoparticles is spherical nanoparticles.

[0031] Disperse CaO2 nanoparticles in anhydrous ethanol solution, wash with anhydrous ethanol for 3 to 5 times, and centrifuge to remove unreacted PVP and free calcium ions. Then transfer 2.5 ml of 100 μg / ml nanoparticle aqueous solution into a cuvette and perform a dynamic light scattering test quickly. Figure 2 The diameters of the nanoparticles are normally distributed, and their particle sizes are mainly concentrated in the range of 500~600 nanometers.

[0032] Example 1 This embodiment is a method for preparing a composite hydrogel loaded with CaO2 nanoparticles, comprising the following steps: (1) Weigh 70 mg of methacrylate-modified γ-polyglutamic acid solid P-MA and 70 mg of cysteine-modified γ-polyglutamic acid solid P-Cys and dissolve them in 2 mL of PBS buffer solution (pH = 7.4), and ultrasonicate at 45 Hz and 25°C for 10 minutes to obtain a P-MC solution. Then add 10 mg of methacrylylated type I collagen ColMA and 2 mg of photoinitiator LAP in turn, and vortex at room temperature until the solid is completely dissolved to form a transparent liquid. Filter the formed transparent liquid with a 0.22 μm sterile membrane to obtain a sterile gel solution; then add UV-sterilized PEG-CaO2 nanoparticles (300 μg / mL) solid, and ultrasonicate to evenly disperse it to obtain a "self-oxygenating" composite hydrogel solution, which is then stored in a sterile refrigerator at 4°C.

[0033] Example 2 (1) Weigh 50 mg of methacrylate-modified γ-polyglutamic acid solid P-MA and 50 mg of cysteine-modified γ-polyglutamic acid solid P-Cys and dissolve them in 2 mL of PBS buffer solution (pH = 7.4), and ultrasonicate at 45 Hz and 25 ° C for 10 minutes to obtain a P-MC solution. Then add 10 mg of methacrylylated type I collagen ColMA and 2 mg of photoinitiator LAP in turn, and vortex at room temperature until the solid is completely dissolved to form a transparent liquid. Filter the formed transparent liquid with a 0.22 μm sterile membrane to obtain a sterile gel solution; then add UV-sterilized PEG-CaO2 nanoparticles (300 μg / mL) solid, and ultrasonicate to evenly disperse it to obtain a "self-oxygenating" composite hydrogel solution, which is then stored in a sterile refrigerator at 4 ° C.

[0034] Example 3 (1) Weigh 60 mg of methacrylate-modified γ-polyglutamic acid solid P-MA and 60 mg of cysteine-modified γ-polyglutamic acid solid P-Cys and dissolve them in 2 mL of PBS buffer solution (pH = 7.4), and ultrasonicate at 45 Hz and 25 ° C for 10 minutes to obtain a P-MC solution. Then add 10 mg of methacrylylated type I collagen ColMA and 2 mg of photoinitiator LAP in turn, and vortex at room temperature until the solid is completely dissolved to form a transparent liquid. Filter the formed transparent liquid with a 0.22 μm sterile membrane to obtain a sterile gel solution; then add UV-sterilized PEG-CaO2 nanoparticles (300 μg / mL) solid, and ultrasonicate to evenly disperse it to obtain a "self-oxygenating" composite hydrogel solution, which is then stored in a sterile refrigerator at 4 ° C.

[0035] Example 4 (1) Weigh 80 mg of methacrylate-modified γ-polyglutamic acid solid P-MA and 80 mg of cysteine-modified γ-polyglutamic acid solid P-Cys and dissolve them in 2 mL of PBS buffer solution (pH = 7.4), and ultrasonicate at 45 Hz and 25°C for 10 minutes to obtain a P-MC solution. Then add 10 mg of methacrylylated type I collagen ColMA and 2 mg of photoinitiator LAP in turn, and vortex at room temperature until the solid is completely dissolved to form a transparent liquid. Filter the formed transparent liquid with a 0.22 μm sterile membrane to obtain a sterile gel solution; then add UV-sterilized PEG-CaO2 nanoparticles (300 μg / mL) solid, and ultrasonicate to evenly disperse it to obtain a "self-oxygenating" composite hydrogel solution, which is then stored in a sterile refrigerator at 4°C.

[0036] Example 5 The biocompatibility of the composite hydrogel loaded with CaO2 nanoparticles prepared in Example 1 was investigated, and the specific steps were as follows: (1) The self-oxygenating composite hydrogel solution P-MC-Ca is vortexed to promote uniform dispersion of the components; (2) Pipette 300 μL of gel solution into a 24-well plate to cover the bottom, and irradiate with a 365 nm UV lamp for 30 seconds to solidify. Then mix the HUVEC cells with 300 μL of gel solution and transfer them to the solidified gel wells at the bottom for 24 hours to observe cell proliferation.

[0037] The results are as follows Figure 4 As shown, the composite hydrogel loaded with PEG-CaO2 nanoparticles still has a high survival rate, indicating that the composite hydrogel has good biocompatibility.

[0038] Example 6 The 'self-oxygenating' composite hydrogel solution prepared in Example 1 was used to perform three-dimensional culture of neural stem cell spheres, and the specific steps were as follows: (1) The self-oxygenating composite hydrogel solution was vortexed and 300 μL of gel solutions of different concentrations were transferred to different wells of a 24-well plate, shaken to cover the bottom of the well plate, and then irradiated with a 365 nm UV lamp for 30 s to solidify; (2) Then add 300 μL of gel solution of different concentrations to the EP tube containing neurospheres (10-20), resuspend in the hydrogel cell solution, add to the corresponding wells with gel of different concentrations, and solidify with UV light for 30 seconds. Add 1 mL of culture medium for culture, continue to culture at 37°C, 5% CO2 concentration, change the culture medium every 2 days, and observe the growth status of neurospheres on the 1st, 3rd and 6th days to preliminarily determine the optimal concentration of gel suitable for the growth of neural stem cell balls. Secondly, observe the structure of neural tube organoids on the 3rd and 6th days of neurosphere differentiation, and detect the expression of neural tube organoid-related markers and the structure of organoids to determine the optimal gel concentration suitable for neurosphere differentiation into neural tubes, and take pictures to observe the budding state. After continuing to culture for one week, perform immunofluorescence staining to analyze the types of differentiated cells; (3) After the culture is completed, fix with 4% paraformaldehyde for 2 hours and wash three times with PBS. Then incubate with 0.1% TritonX-100 for 30 minutes, wash three times with PBS, and add blocking solution for 30 minutes. After removing the liquid, add 300μL of primary antibody: main components map2 (Rabbit) and GFAP (Mouse) solution, and incubate at 4 degrees Celsius for 24 hours. Remove the primary antibody solution, add the secondary antibody solution (594 goat anti-rabbit and 488goat anti-mouse), incubate at room temperature in the dark for 3 hours, remove the liquid, and wash three times with PBS; (4) After incubation with DAPI nuclear stain at room temperature in the dark for 10-30 min, the liquid was removed and the cells were washed three times with PBS (10 min each time). The gel blocks were transferred to a confocal microplate and fluorescent confocal photography was performed. The cells were divided into three groups: (1) 2.5% group, (2) 3.0% group, and (3) 4.0% group.

[0039] The results are as follows Figure 5 The cell spheres obtained at the three concentrations mainly differentiated into microglia and neurons.

[0040] It should be understood that the application of the present invention is not limited to the above-mentioned neural stem cell sphere culture. For this field, the components of this "self-oxygenating" gel can be improved or changed according to the above description. At the same time, this type of gel scaffold can also be used to culture other cell spheres or organoid spheres (such as vascular organoids, intestinal 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 a self-oxygenating hydrogel cell scaffold, characterized in that: The following steps are involved: S1, adding methacrylate-modified γ-polyglutamic acid solid P-MA and cysteine-modified γ-polyglutamic acid solid P-Cys powder into PBS buffer, and dissolving by ultrasonication to obtain a P-MC solution; S2, adding methacrylate-modified type I collagen ColMA solid and a photoinitiator to the P-MC solution, vortexing at room temperature until completely dissolved, to obtain a mixed solution; S3, filtering the mixed solution to obtain a gel solution, then adding CaO2 nanoparticles PEG-CaO2 coated with polyethylene glycol (PEG), and obtaining a composite hydrogel solution after ultrasonic treatment; S4, curing the composite hydrogel solution to obtain the hydrogel cell scaffold P-MC-Ca.

2. The preparation method according to claim 1, characterized in that: In S1, the mass ratio of the P-MA to P-Cys is 1:1; the mass volume concentrations of the 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 ultrasonic conditions are: 45 Hz, 25° C., for 10 minutes.

4. The preparation method according to any one of claims 1 to 3, characterized in that In S2, in the mixed solution, the concentration range of ColMA is 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%.

5. The preparation method according to claim 1 or 4, characterized in that: In S3, the concentration of PEG-CaO2 in the mixed solution is 200 μg / mL to 800 μg / mL.

6. The preparation method according to claim 1 or 5, characterized in that: In S3, filtration was performed using a 0.22 μm sterile membrane.

7. The preparation method according to any one of claims 1 to 6, characterized in that: In S4, the curing treatment is performed by irradiating an ultraviolet lamp with a wavelength of 365 nm for 20 seconds to 60 seconds.

8. A hydrogel cell scaffold P-MC-Ca prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the hydrogel cell scaffold P-MC-Ca as claimed in claim 8 in cell culture.

10. Use of the hydrogel cell scaffold P-MC-Ca as claimed in claim 8 in three-dimensional culture of neurospheres.

Images