Hydrogel for 3D culture of stem cells / organoids and preparation method thereof

Abstract

The invention discloses hydrogel for 3D culture of stem cells / organoids and a preparation method of the hydrogel, and belongs to the technical field of biological materials. The hydrogel for 3D culture of stem cells / organoids is formed by a four-arm polyethylene glycol macromolecular polymer, the polyethylene glycol macromolecular polymer has a maleimide group (PEG-4MAL) at each terminal, is coupled with an adhesive peptide containing cysteine, and is obtained by cross-linking with mercapto polyethylene glycol (PEG-SH). The hydrogel is beneficial to organoids, the stem cells are high in growth rate and short in culture period, and the obtained organoids are high in maturity, uniform in size, high in bionic capacity and high in application value. Therefore, the prepared novel hydrogel material provides a more ideal microenvironment for culture of cells and organoids, and promotes application and development of regenerative medicine and tissue engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a hydrogel for 3D culture of stem cells / organoids and a preparation method thereof. Background Art

[0002] With the rapid development of regenerative medicine and tissue engineering, three-dimensional (3D) culture technology of stem cells and organoids has become an important research field. Traditional two-dimensional culture systems are difficult to simulate the complex microenvironment in the body, so the growth characteristics and functions of cultured cells or tissues are often significantly different from the physiological state. This makes the development of effective 3D culture matrix particularly important.

[0003] Organoids are a group of cells cultured under specific conditions that can simulate the structure and function of real organs in vitro. These miniature organs not only retain the gene expression characteristics of the original tissue, but also help to study a variety of biological processes such as tissue development, disease mechanisms, and drug responses. The significance of organoids lies in their ability to provide accurate physiological models for use in disease research, drug screening, and personalized treatment. For example, tumor organoids can simulate the tumor microenvironment, help study the growth and metastasis mechanisms of tumors, and provide a more realistic in vitro model for the evaluation of anticancer drugs. In addition, organoids also have potential applications in regenerative medicine and can provide new ideas for tissue repair and regeneration.

[0004] Hydrogels have become the first choice for 3D scaffold materials due to their excellent biocompatibility, superior hydration and adjustable mechanical properties. However, Matrigel, which is currently commonly used in organoid culture, still has many shortcomings, although it can support cell growth and differentiation in some aspects.

[0005] Matrigel is derived from mouse tumor tissue. The raw materials are complex and highly variable, resulting in inconsistent batch characteristics and affecting the reliability of experimental results. Although Matrigel contains a variety of bioactive ingredients that help cell growth, its composition and function have not been fully analyzed, which limits the precise control of the microenvironment and reduces the repeatability of the experiment. In addition, the extraction and preservation process of Matrigel is complicated, the production cost is high, and the storage conditions are strict, which increases the difficulty of use and transportation. Its physical properties such as flexibility and mechanical strength are low, and it is difficult to control the degradation rate, which limits its effectiveness in certain applications. Because it is derived from animal tissue, the use of Matrigel has also caused ethical controversy, restricting its promotion in clinical research and applications.

[0006] Based on the above shortcomings, it is urgent to develop a new, customized fully synthetic hydrogel material to replace Matrigel. This new material should have good controllability and be able to simulate the in vivo microenvironment, thereby promoting the growth and functional development of cells and organoids. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, the present invention provides a hydrogel for stem cell / organoid 3D culture and a preparation method thereof.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] A method for preparing a hydrogel for 3D culture of stem cells / organoids, comprising the following steps:

[0010]

[0011] (1) A certain amount of pentaerythritol is dissolved in toluene or dichloromethane, and after the pentaerythritol is completely dissolved, polyethylene glycol and p-toluenesulfonic acid are added and heated for reaction. After the reaction is completed, the reaction solution is cooled to room temperature, and then the solvent is removed to obtain a polymer.

[0012] (2) The polymer obtained in step (1) is added to deionized water and stirred evenly, and then dialyzed in deionized water using a dialysis bag with a suitable molecular weight cutoff, and finally freeze-dried to obtain 4-arm-PEG.

[0013] (3) Dissolve 4-arm-PEG in toluene, distill under protective gas, cool to room temperature, and filter to obtain a precipitate.

[0014]

[0015] (4) dissolving the precipitate obtained by filtration in step (3) in dichloromethane, adding triethylamine to obtain a mixed solution, cooling the mixed solution, adding methanesulfonyl chloride to react at room temperature under protective gas; after the reaction is completed, adding ethanol to terminate the reaction, and then removing the solvent by rotary evaporation to obtain a precipitate, adding the precipitate to ether for slurrying, and finally filtering and collecting the precipitate and vacuum drying to obtain four-arm polyethylene glycol methyl sulfonyl ester.

[0016] (5) dissolving the 4-arm polyethylene glycol methyl sulfonyl ester obtained in step (4) in an aqueous ammonia solution containing ammonium chloride and reacting at room temperature, then extracting with dichloromethane, combining the organic phases, and performing rotary evaporation to remove the solvent to obtain a residue, adding the residue to isopropanol, and then filtering and vacuum drying to obtain the 4-arm polyethylene glycol amine.

[0017]

[0018] (6) Dissolve the four-arm polyethylene glycol amine in acetonitrile, then add maleimidopropionic acid-N-succinimide ester to react at room temperature, remove the solvent to obtain a residue, add the residue to isopropanol, filter and dry to obtain a crude four-arm polyethylene glycol maleimide.

[0019] (7) The dried crude four-arm polyethylene glycol maleimide was dialyzed with deionized water and then freeze-dried to obtain 4-arm-PEG-MAL.

[0020]

[0021] (8) PEG is dissolved in dichloromethane, and then thioglycolic acid and cyclohexylcarbodiimide are added and heated for reaction; after the reaction is completed, the solvent is removed to obtain a crude product of double-terminated thiol polyethylene glycol.

[0022] (9) The crude product double-terminated thiol polyethylene glycol is placed in water, then dialyzed, and finally freeze-dried to obtain double-terminated thiol polyethylene glycol.

[0023] (10) dissolving 4-arm-PEG-MAL in the culture medium, then adding the cysteine-containing adhesion peptide, filtering with a 0.22 μm filter to obtain a filtrate, resuspending the filtrate and the cells, and evenly mixing to obtain solution A;

[0024] (11) dissolving double-terminated thiol polyethylene glycol in the culture medium and filtering using a 0.22 μm filter to obtain solution B;

[0025] (12) Mixing the solution A obtained in step (10) and the solution B obtained in step (11), and then incubating to obtain a spontaneously vascularized hydrogel for stem cell / organoid 3D culture.

[0026] As a preferred embodiment of the present invention, in the step (1), pentaerythritol is completely dissolved in dichloromethane at a concentration of 2.5 g / L-10.0 g / L, the molar ratio of polyethylene glycol to pentaerythritol is (4-6):1; the mass ratio of p-toluenesulfonic acid to pentaerythritol is (7.53-20.08):1; the stirring speed is 600-1000 rpm; the heating reaction temperature is 80-120°C, and the time is 4-6 hours.

[0027] As a preferred embodiment of the present invention, in the step (1), the solvent is removed by rotary evaporation at a temperature ranging from 45 to 55°C.

[0028] As a preferred embodiment of the present invention, in step (2), the concentration of the polymer obtained in step (1) dissolved in water is 50-100 g / L; the molecular weight cutoff of the dialysis bag is consistent with the molecular weight of the polyethylene glycol in step (1), and the dialysis time is 3-5 days.

[0029] As a preferred embodiment of the present invention, in the step (3), the concentration of 4-arm-PEG dissolved in toluene is 50-100 g / L; the distillation temperature is 110-135° C., and the distillation time is 2-5 hours.

[0030] As a preferred embodiment of the present invention, in the step (4), the concentration of the precipitate obtained by filtration in the step (4) dissolved in dichloromethane is 100-200 g / L; the volume ratio of triethylamine to dichloromethane is (0.005-0.01):1; the molar ratio of methanesulfonyl chloride to the precipitate is (4-6):1; and the reaction time at room temperature under protective gas is 12-24 h.

[0031] As a preferred embodiment of the present invention, in the step (4), the volume ratio of ethanol to dichloromethane is (0.1-0.5):1; the temperature of rotary evaporation is 55-65°C; the volume ratio of ether to dichloromethane is (1-3):1; and drying is vacuum drying at a temperature of 25-35°C.

[0032] As a preferred embodiment of the present invention, in the step (5), the concentration of 4-arm polyethylene glycol methyl sulfonyl ester dissolved in the ammonia solution containing ammonium chloride is 50-100 g / L; the ammonium chloride in the ammonia solution containing ammonium chloride is 30-80 g / L; the reaction time at room temperature is 48-96 hours; the volume ratio of dichloromethane to ammonia solution is (2-1):1; and the solvent is removed by rotary evaporation in a water bath at 35-55°C.

[0033] As a preferred embodiment of the present invention, in the step (5), the drying is vacuum drying at a temperature of 40-60°C.

[0034] As a preferred embodiment of the present invention, in the step (6), the concentration of the four-arm polyethylene glycol amine dissolved in acetonitrile is 50-200 g / L; the molar ratio of the four-arm polyethylene glycol amine to maleimidopropionic acid-N-succinimide ester is 1:4-6; the solvent is removed by rotary evaporation at a temperature range of 45-65°C; and the mass volume ratio of the residue to isopropanol is 50-100 g / L.

[0035] As a preferred embodiment of the present invention, in the step (7), the dialysis bag for dialysis of four-arm polyethylene glycol maleimide has a molecular weight cutoff MW0: 3500; and the freeze-drying time is 3-5 days.

[0036] As a preferred embodiment of the present invention, in the step (8), the concentration of PEG dissolved in dichloromethane is 50-200 g / L; the molar ratio of thioglycolic acid to PEG is (4-6):1; the mass ratio of cyclohexylcarbodiimide to PEG is (0.01-0.1):1; the temperature of the heating reaction is 80-120°C, and the time is 8-24 hours.

[0037] As a preferred embodiment of the present invention, in the step (8), the solvent is removed by rotary evaporation at a temperature ranging from 45 to 55°C.

[0038] As a preferred embodiment of the present invention, in the step (9), the crude double-terminated thiol polyethylene glycol is dissolved in water at a mass volume ratio of 20-50 g / L; and the molecular weight cut-off MW of the dialysis bag is 1000.

[0039] As a preferred embodiment of the present invention, in the step (10), 4-arm-PEG-MAL is dissolved in the culture medium at a concentration of 100-200 g / L, and the molar ratio of the cysteine-containing adhesion peptide to 4-arm-PEG-MAL is (0.1-0.25):1; in the step (11), the double-terminated thiol polyethylene glycol is dissolved in the culture medium at a concentration of 20-50 g / L.

[0040] The adhesion peptide is an adhesion peptide containing a cysteine ​​molecule at the end.

[0041] As a preferred embodiment of the present invention, the filter is preferably 0.22 um.

[0042] As a preferred embodiment of the present invention, in step (12), the volume ratio of liquid A to liquid B is (4-1):1, the incubation temperature is 37°C, and the time is 15-30 min.

[0043] The present invention also claims protection for the hydrogel for stem cell / organoid 3D culture prepared by the hydrogel preparation method for stem cell / organoid 3D culture, wherein the hydrogel for stem cell / organoid 3D culture is formed by a four-arm polyethylene glycol macromer, wherein the polyethylene glycol macromer has a maleimide group at each end, is coupled to a cysteine-containing adhesion peptide, and is cross-linked by thiol polyethylene glycol.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention can spontaneously promote the formation of vascularization by regulating the composition and structure of the hydrogel, improving its interaction with cells and biological activity. Moreover, the cytotoxicity is low and the hydrophilicity is suitable, and there are adjustable physical properties, such as suitable stiffness and porous structure, which can meet the growth requirements of different cells and promote nutrition and gas exchange. The chemical properties of the hydrogel of the present invention are conducive to cell adhesion and proliferation, and bioactive molecules can be introduced through functional modification and simulate the extracellular matrix; it is also degradable, can be degraded in an enzyme-responsive manner, and the degradation products are harmless, which is conducive to the fast growth rate of organoids and stem cells, and the culture cycle is short. The volume of the obtained organoids breaks through the centimeter level, the maturity is high, the size is uniform, the biomimetic ability is strong, and the application value is high. The prepared new hydrogel material will provide a more ideal microenvironment for the culture of cells and organoids, and promote the application and development of regenerative medicine and tissue engineering. In addition, the storage modulus (G') and loss modulus (G") of the hydrogel prepared by the present invention are close to the mechanical properties of matrigel commonly used for culturing organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of hydrogel cross-linking for stem cell / organoid 3D culture.

[0046] Figure 2 This is a comparison chart of the storage modulus and loss modulus of the hydrogel, GelMA and Matrigel prepared in Example 1.

[0047] Figure 3 This is a diagram of the embryo-like culture of Example 2. DETAILED DESCRIPTION

[0048] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0049] Example 1

[0050] (1) Dissolve 1.0 g of pentaerythritol in 100 mL of dichloromethane. After complete dissolution, add 44.12 g of PEG1000.

[0051] (2) Weigh 7.53 g of p-toluenesulfonic acid (PTSA) as a catalyst and add it to the reaction mixture.

[0052] (3) Set the stirring speed to 1000 rpm, ensure that the mixture is evenly mixed, and then turn on the heating device. The temperature setting range is 120°C.

[0053] (4) After the reaction was continued for 6 hours and the monitoring results showed that the reaction was complete, the reaction solution was cooled to room temperature and the solvent was removed by rotary evaporation at 55°C.

[0054] (5) Dissolve 10.0 g of the polymer obtained in step (4) in 100 mL of deionized water and stir well.

[0055] (6) The precipitated polymer 4-arm-PEG was dialyzed in a dialysis bag with a molecular weight cutoff of 1000 DA against deionized water for 5 days and freeze-dried to obtain 4-arm-PEG.

[0056] (7) Take 20.0 g of the 4-arm-PEG prepared in step (6) and dissolve it in 200 mL of toluene. Under N2 protection at 135°C, perform azeotropic distillation with toluene for 5 h, then cool to room temperature and filter.

[0057] (8) After 20.0 g of the filtered 4-arm-PEG in step (7) was completely dissolved in 100 mL of dichloromethane, 1.0 mL of triethylamine was added to the reaction system by volume. The mixed solution was cooled in an ice water bath and 13.68 g of dry methanesulfonyl chloride was added dropwise, and the mixture was stirred at room temperature for 24 h in a nitrogen atmosphere.

[0058] (9) After the reaction is completed, 50 mL of anhydrous ethanol is added to terminate the reaction. The solvent is concentrated by rotary evaporation at 65° C., the precipitate is filtered out, and 300 mL of ether is added for slurrying. The precipitate is collected by filtration and dried in vacuo at 35° C. to prepare 4-arm polyethylene glycol methyl sulfonyl ester.

[0059] (10) 20 g of the 4-arm polyethylene glycol methyl sulfonyl ester obtained in step (4) was dissolved in 200 mL of an ammonia solution containing 16.0 g of ammonium chloride. The solution was stirred at room temperature for 96 hours, and then the solution was extracted 5 times with 400 mL of anhydrous dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate.

[0060] (11) The organic phase obtained in step (10) was subjected to removal of the solvent in a 55° C. water bath to obtain a residue. 20.8 g of the residue was added to 150 mL of isopropanol for slurrying, and the precipitate was collected and vacuum dried at 60° C. to prepare a 4-arm polyethylene glycol amine.

[0061] (12) 20.0 g of the four-arm polyethylene glycol amine prepared in step (11) was dissolved in 100 mL of acetonitrile, and 11.64 g of maleimide propionic acid-N-succinimide ester was added to the solution. The solution was stirred at room temperature overnight. The solvent was evaporated under vacuum at 65° C., and 24.6 g of the residue was added to 250 mL of isopropanol. The precipitate was collected and dried under vacuum to prepare a crude four-arm polyethylene glycol maleimide.

[0062] (13) The crude four-arm polyethylene glycol maleimide (4-arm-PEG-MAL) obtained in step (12) is dialyzed using a dialysis bag with a molecular weight cutoff of 3500, freeze-dried for 5 days, and stored at -20°C for later use to obtain four-arm polyethylene glycol maleimide (4-arm-PEG-MAL).

[0063] (14) Weigh 20.0 g of PEG2000 and dissolve it in 100 mL of anhydrous dichloromethane.

[0064] (15) Weigh 5.82 g of thioglycolic acid and add it to the dissolved PEG solution. Keep stirring to ensure that the thioglycolic acid is fully mixed.

[0065] (16) Weigh 2.0 g of cyclohexylcarbodiimide (DCC) and add it to the mixed solution. Heat to 120°C and react for about 8 hours.

[0066] (17) After the reaction was completed, the water bath was turned off, the round-bottom flask was removed, and the mixture was allowed to cool naturally to room temperature. With the water bath at 55 °C, dichloromethane was removed using a rotary evaporator to obtain a crude product of double-terminated thiol polyethylene glycol (SH-PEG-SH).

[0067] (18) 21.0 g of the crude product SH-PEG-SH from step (17) was dissolved in 420 mL of deionized water and filtered to remove unreacted solid matter.

[0068] (19) The filtrate was poured into a dialysis bag with a molecular weight cutoff of 1000 and dialyzed with deionized water for 5 days, with the water replaced every 12 hours.

[0069] (20) After dialysis, the product was freeze-dried and stored at −20 °C for future use to obtain SH-PEG-SH.

[0070] (21) Take 1.0 g of 4-arm-PEG-MAL obtained in step (13) and dissolve it in 5 mL of DMEM culture medium. After it is completely dissolved, add 0.028 g of an adhesion peptide containing one cysteine ​​molecule and filter using a 0.22 um filter.

[0071] (22) Take 0.1 g of SH-PEG-SH obtained in step (20) and dissolve it in 2.0 mL of DMEM culture medium. After complete dissolution, filter it with a 0.22 um filter to obtain liquid B.

[0072] (23) The solution A obtained in step (21) and the solution B obtained in step (22) were uniformly mixed at a volume ratio of 4:1, and placed in a 37° C. incubator for incubation. After incubation for 30 minutes, a stem cell / organoid 3D culture hydrogel (such as Figure 1 shown).

[0073] (24) Methacrylated hydrogel (GelMA) and Matrigel matrix gel with a mass volume ratio of 100 g / L were taken as control groups, and the storage modulus (G') and loss modulus (G") of the hydrogel in Example 1 and the control group were studied and compared using a rheometer (see Figure 2). Figure 2 As shown). The storage modulus of the methacryloyl gelatin obtained by modifying the natural biomaterial gelatin is about 860Pa; the mechanical properties of the stem cell / organoid 3D culture hydrogel prepared by the present invention are close to those of the matrigel commonly used for culturing organoids.

[0074] Example 2

[0075] (1) Dissolve 0.5 g of pentaerythritol in 100 mL of dichloromethane. After complete dissolution, add 18.25 g of PEG1000.

[0076] (2) Weigh 6.28 g of p-toluenesulfonic acid (PTSA) as a catalyst and add it to the reaction mixture.

[0077] (3) Set the stirring speed to 800 rpm, ensure that the mixture is evenly mixed, and turn on the heating device. The temperature setting range is 100 °C.

[0078] (4) After reacting for 5 hours and when the monitoring results show that the reaction is complete, the reaction solution is cooled to room temperature and the solvent is removed by rotary evaporation at 50°C.

[0079] (5) Dissolve 10.0 g of the polymer obtained in step (4) in 120 mL of deionized water and stir well.

[0080] (6) The precipitated polymer 4-arm-PEG was dialyzed in a dialysis bag with a molecular weight cutoff of 1000 DA against deionized water for 5 days and freeze-dried to obtain 4-arm-PEG.

[0081] (7) Take 20.0 g of the 4-arm-PEG prepared in step (6) and dissolve it in 300 mL of toluene. Under N2 protection at 135°C, perform azeotropic distillation with toluene for 5 h, then cool to room temperature and filter.

[0082] (8) After 20.0 g of the filtered 4-arm-PEG in step (7) was completely dissolved in 180 mL of dichloromethane, 1.0 mL of triethylamine was added to the reaction system by volume, the mixed solution was cooled in an ice-water bath, and 12.45 g of dry methanesulfonyl chloride was added dropwise, and the mixture was stirred at room temperature for 24 h in a nitrogen atmosphere.

[0083] (9) After the reaction is completed, 40 mL of anhydrous ethanol is added to terminate the reaction. The solvent is concentrated by rotary evaporation at 60° C., the precipitate is filtered out, and 200 mL of ether is added for slurrying. The precipitate is collected by filtration and dried in vacuo at 30° C. to prepare 4-arm polyethylene glycol methyl sulfonyl ester.

[0084] (10) 20.0 g of the 4-arm polyethylene glycol methyl sulfonyl ester obtained in step (4) was dissolved in 320 mL of an ammonia solution containing 20.0 g of ammonium chloride. The solution was stirred at room temperature for 72 hours, and then the solution was extracted five times with 400 mL of anhydrous dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate.

[0085] (11) The organic phase obtained in step (10) was subjected to removal of the solvent in a 55° C. water bath to obtain a residue. 18.2 g of the obtained residue was added to 120 mL of isopropanol for slurrying, and the precipitate was collected and vacuum dried at 55° C. to prepare a 4-arm polyethylene glycol amine.

[0086] (12) 12.0 g of the four-arm polyethylene glycol amine prepared in step (11) was dissolved in 100 mL of acetonitrile, and 9.7 g of maleimide propionic acid-N-succinimide ester was added to the solution. The solution was stirred at room temperature overnight. The solvent was evaporated under vacuum at 50° C., and 23.7 g of the residue was added to 150 mL of isopropanol. The precipitate was collected and dried under vacuum to prepare a crude four-arm polyethylene glycol maleimide.

[0087] (13) The crude four-arm polyethylene glycol maleimide (4-arm-PEG-MAL) obtained in step (12) is dialyzed using a dialysis bag with a molecular weight cutoff of 3500, freeze-dried and stored at -20°C for later use to obtain four-arm polyethylene glycol maleimide (4-arm-PEG-MAL).

[0088] (14) Weigh 20.0 g of PEG2000 and dissolve it in 300 mL of anhydrous dichloromethane.

[0089] (15) Weigh 4.85 g of thioglycolic acid and add it to the dissolved PEG solution. Keep stirring to ensure that the thioglycolic acid is fully mixed.

[0090] (16) Weigh 1.5 g of cyclohexylcarbodiimide (DCC) and add it to the mixed solution. Heat to 110°C and react for about 20 hours.

[0091] (17) After the reaction was completed, the water bath was turned off, the round-bottom flask was removed, and the mixture was allowed to cool naturally to room temperature. With the water bath at 30 °C, dichloromethane was removed using a rotary evaporator to obtain a crude product of double-terminated thiol polyethylene glycol (SH-PEG-SH).

[0092] (18) 21.0 g of the crude product SH-PEG-SH from step (17) was dissolved in 800 mL of deionized water and filtered to remove unreacted solid matter.

[0093] (19) The filtrate was poured into a dialysis bag with a molecular weight cutoff of 1000 and dialyzed with deionized water for 5 days, with the water replaced every 12 hours.

[0094] (20) After dialysis, the product was freeze-dried and stored at −20 °C for future use to obtain SH-PEG-SH.

[0095] (21) Take 1.0 g of 4-arm-PEG-MAL obtained in step (13) and dissolve it in 5 mL of DMEM medium. After it is completely dissolved, add 0.023 g of an adhesion peptide containing one cysteine ​​molecule and filter it using a 0.22 μm filter. Resuspend the filtrate with embryonic stem cells and mix them evenly to obtain liquid A.

[0096] (22) Take 0.1 g of SH-PEG-SH obtained in step (19) and dissolve it in 2.0 mL of DMEM medium. After complete dissolution, filter it with a 0.22 um filter to obtain liquid B.

[0097] (23) The solution A obtained in step (21) and the solution B obtained in step (22) were uniformly mixed at a volume ratio of 1:1, and placed in a 37°C incubator for incubation. After incubation for 30 minutes, a stem cell / organoid 3D culture hydrogel was obtained, and an appropriate amount of blastocyst-like culture medium was added for culture (such as Figure 3 shown).

[0098] Example 3

[0099] (1) Dissolve 0.25 g of pentaerythritol in 100 mL of dichloromethane. After complete dissolution, add 7.30 g of PEG1000.

[0100] (2) Weigh 5.02 g of p-toluenesulfonic acid (PTSA) as a catalyst and add it to the reaction mixture.

[0101] (3) Set the stirring speed to 600 rpm, turn on the heating device after ensuring uniform mixing, and the temperature setting range is 80°C.

[0102] (4) After reacting for 4 hours and when the monitoring results show that the reaction is complete, the reaction solution is cooled to room temperature and the solvent is removed by rotary evaporation at 45°C.

[0103] (5) Dissolve 10.0 g of the polymer obtained in step (4) in 200 mL of deionized water and stir well.

[0104] (6) The precipitated polymer 4-arm-PEG was dialyzed in a dialysis bag with a molecular weight cutoff of 1000DA against deionized water for 3 days and freeze-dried to obtain 4-arm-PEG.

[0105] (7) Take 20.0 g of the 4-arm-PEG prepared in step (6) and dissolve it in 400 mL of toluene. Under N2 protection at 135°C, perform azeotropic distillation with toluene for 5 h, then cool to room temperature and filter.

[0106] (8) After 20.0 g of the filtered 4-arm-PEG in step (7) was completely dissolved in 200 mL of dichloromethane, 1.0 mL of triethylamine was added to the reaction system by volume, the mixed solution was cooled in an ice-water bath and 9.12 g of dry methanesulfonyl chloride was added dropwise, and the mixture was stirred at room temperature for 12 h in a nitrogen atmosphere.

[0107] (9) After the reaction is completed, 10 mL of anhydrous ethanol is added to terminate the reaction. The solvent is concentrated by rotary evaporation at 55° C., the precipitate is filtered out, and 100 mL of ether is added for slurrying. The precipitate is collected by filtration and vacuum dried at 25° C. to prepare 4-arm polyethylene glycol methyl sulfonyl ester.

[0108] (10) 20.0 g of the 4-arm polyethylene glycol methyl sulfonyl ester obtained in step (4) was dissolved in 400 mL of an ammonia solution containing 24.0 g of ammonium chloride. The solution was stirred at room temperature for 48 hours, and then the solution was extracted 5 times with 400 mL of anhydrous dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate.

[0109] (11) The organic phase obtained in step (10) was subjected to removal of the solvent in a 35° C. water bath to obtain a residue. 19.8 g of the residue was added to 150 mL of isopropanol for slurrying, and the precipitate was collected and vacuum dried at 40° C. to prepare a 4-arm polyethylene glycol amine.

[0110] (12) 5.0 g of the four-arm polyethylene glycol amine prepared in step (11) was dissolved in 100 mL of acetonitrile, and 7.76 g of maleimide propionic acid-N-succinimide ester was added to the solution. The solution was stirred at room temperature overnight. The solvent was evaporated under vacuum at 45° C., and 22.8 g of the residue was added to 120 mL of isopropanol. The precipitate was collected and dried under vacuum to prepare a crude four-arm polyethylene glycol maleimide.

[0111] (13) The crude four-arm polyethylene glycol maleimide (4-arm-PEG-MAL) obtained in step (12) is dialyzed using a dialysis bag with a molecular weight cutoff of 3500, freeze-dried for 3 days, and stored at -20°C for later use to obtain four-arm polyethylene glycol maleimide (4-arm-PEG-MAL).

[0112] (14) Weigh 20.0 g of PEG2000 and dissolve it in 400 mL of anhydrous dichloromethane.

[0113] (15) Weigh 3.88 g of thioglycolic acid and add it to the dissolved PEG solution. Keep stirring to ensure that the thioglycolic acid is fully mixed.

[0114] (16) Weigh 0.2 g of cyclohexylcarbodiimide (DCC) and add it to the mixed solution. Heat to 80°C and react for about 24 hours.

[0115] (17) After the reaction was completed, the water bath was turned off, the round-bottom flask was removed, and the mixture was allowed to cool naturally to room temperature. With the water bath at 45 °C, dichloromethane was removed using a rotary evaporator to obtain a crude product of double-terminated thiol polyethylene glycol (SH-PEG-SH).

[0116] (18) 21.0 g of the crude product SH-PEG-SH from step (17) was dissolved in 1050 mL of deionized water and filtered to remove unreacted solid matter.

[0117] (19) The filtrate was poured into a dialysis bag with a molecular weight cutoff of 1000 and dialyzed with deionized water for 5 days, with the water replaced every 12 hours.

[0118] (20) After dialysis, the product was freeze-dried and stored at −20 °C for future use to obtain SH-PEG-SH.

[0119] (21) Take 1.0 g of 4-arm-PEG-MAL obtained in step (13) and dissolve it in 5 mL of blastocyst-like culture specific medium (wherein the factors and small compounds in the medium for dissolving the material are about 4 times that of the replacement culture medium). After complete dissolution, add 0.012 g of an adhesion peptide containing one cysteine ​​molecule and filter using a 0.22 um filter. Resuspend the filtrate with embryonic stem cells and mix evenly to obtain liquid A.

[0120] (22) Take 0.1 g of SH-PEG-SH obtained in step (20) and dissolve it in 2.0 mL of blastocyst-like culture specific medium. After complete dissolution, filter it with a 0.22 um filter to obtain liquid B.

[0121] (23) After the liquid A obtained in step (21) and the liquid B obtained in step (22) are evenly mixed at a volume ratio of 3:1, the mixture is placed in a 37°C incubator for incubation. After incubation for 30 minutes, a stem cell / organoid 3D culture hydrogel is obtained, and an appropriate amount of culture medium is added for culture.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a hydrogel for 3D culture of stem cells / organoids, characterized in that: The steps include: (1) dissolving pentaerythritol in toluene or dichloromethane, then adding polyethylene glycol and p-toluenesulfonic acid to heat for reaction, cooling the reaction solution after the reaction is completed, and then removing the solvent to obtain a polymer; (2) adding the polymer obtained in step (1) into water, stirring evenly, dialyzing, and finally freeze-drying to obtain 4-arm-PEG; (3) dissolving 4-arm-PEG in toluene, distilling under protective gas, cooling to room temperature and filtering to obtain a precipitate; (4) dissolving the precipitate obtained by filtration in step (3) in dichloromethane, adding triethylamine to obtain a mixed solution, cooling the mixed solution, adding methanesulfonyl chloride to react at room temperature under protective gas; after the reaction is completed, adding ethanol to terminate the reaction, then removing the solvent to obtain a precipitate, adding the precipitate to ether for slurrying, and finally filtering and collecting the precipitate and drying it to obtain four-arm polyethylene glycol methyl sulfonyl ester; (5) dissolving the four-arm polyethylene glycol methyl sulfonyl ester obtained in step (4) in an aqueous ammonia solution containing ammonium chloride and reacting at room temperature, then extracting with dichloromethane, combining the organic phases, removing the solvent to obtain a residue, adding the residue to isopropanol, and then filtering and drying to obtain a four-arm polyethylene glycol amine; (6) dissolving the four-arm polyethylene glycol amine in acetonitrile, then adding maleimidopropionic acid-N-succinimide ester to react at room temperature, removing the solvent to obtain a residue, adding the residue to isopropanol, filtering, and drying to obtain a crude four-arm polyethylene glycol maleimide; (7) The crude four-arm polyethylene glycol maleimide was added to water for dialysis, and then freeze-dried to obtain 4-arm-PEG-MAL; (8) dissolving PEG in dichloromethane, then adding thioglycolic acid and cyclohexylcarbodiimide and heating for reaction. After the reaction is completed, removing the solvent to obtain a crude product of double-terminated thiol polyethylene glycol; (9) placing the crude product double-terminated thiol polyethylene glycol in water, then dialyzing it, and finally freeze-drying it to obtain the double-terminated thiol polyethylene glycol; (10) dissolving 4-arm-PEG-MAL in a culture medium, then adding a cysteine-containing adhesion peptide, filtering to obtain a filtrate, resuspending the filtrate and cells, and evenly mixing to obtain a liquid A; (11) dissolving double-terminated thiol polyethylene glycol in the culture medium and filtering to obtain solution B; (12) The solution A obtained in step (10) and the solution B obtained in step (11) are mixed, and then incubated to obtain a spontaneously vascularized hydrogel for stem cell / organoid 3D culture.

2. The method for preparing a hydrogel for 3D culture of stem cells / organoids according to claim 1, characterized in that: In the step (1), pentaerythritol is completely dissolved in dichloromethane at a concentration of 2.5 g / L-10.0 g / L, the molar ratio of polyethylene glycol to pentaerythritol is (4-6):1; the mass ratio of p-toluenesulfonic acid to pentaerythritol is (7.53-20.08):1; the heating reaction temperature is 80-120° C., and the time is 4-6 hours.

3. The method for preparing a hydrogel for 3D culture of stem cells / organoids according to claim 1, characterized in that: In the step (3), the concentration of 4-arm-PEG dissolved in toluene is 50-100 g / L; the distillation temperature is 110-135° C., and the distillation time is 2-5 hours.

4. The method for preparing a hydrogel for 3D culture of stem cells / organoids according to claim 1, characterized in that: In the step (4), the concentration of the precipitate obtained by filtration in the step (3) dissolved in dichloromethane is 100-200 g / L; the volume ratio of triethylamine to dichloromethane is (0.005-0.01):1; the molar ratio of methanesulfonyl chloride to the precipitate is (4-6):1; the volume ratio of ethanol to dichloromethane is 0.1-0.5:1; and the reaction time at room temperature under protective gas is 12-24 hours.

5. The method for preparing a hydrogel for 3D culture of stem cells / organoids according to claim 1, characterized in that: In the step (5), the concentration of 4-arm polyethylene glycol methyl sulfonyl ester dissolved in the ammonia solution containing ammonium chloride is 50-100 g / L; the ammonium chloride in the ammonia solution containing ammonium chloride is 30-80 g / L; the reaction time at room temperature is 48-96 hours; and the volume ratio of dichloromethane to ammonia solution is 2-1:

1.

6. The method for preparing a hydrogel for 3D culture of stem cells / organoids according to claim 1, characterized in that: In the step (6), the concentration of 4-arm polyethylene glycol amine dissolved in acetonitrile is 50-200 g / L; the molar ratio of 4-arm polyethylene glycol amine to maleimidopropionic acid-N-succinimide ester is 1:4-6; and the mass volume ratio of the residue to isopropanol is 50-100 g / L.

7. The method for preparing a hydrogel for 3D culture of stem cells / organoids according to claim 1, characterized in that: In the step (8), the concentration of PEG dissolved in dichloromethane is 50-200 g / L; the molar ratio of thioglycolic acid to PEG is (4-6):1; the mass ratio of cyclohexylcarbodiimide to PEG is (0.01-0.1):1; the heating reaction temperature is 80-120° C. and the reaction time is 8-24 hours.

8. The method for preparing a hydrogel for 3D culture of stem cells / organoids according to claim 1, characterized in that: The 4-arm-PEG-MAL is dissolved in the culture medium at a concentration of 100-200 g / L; the molar ratio of the cysteine-containing adhesion peptide to 4-arm-PEG-MAL is (0.1-0.25):1; and the double-terminated thiol polyethylene glycol is dissolved in the culture medium at a concentration of 20-50 g / L.

9. The method for preparing a hydrogel for 3D culture of stem cells / organoids according to claim 1, characterized in that: In the step (15), the volume ratio of liquid A to liquid B is (4-1):1, the incubation temperature is 37° C., and the time is 15-30 min.

10. A hydrogel for stem cell / organoid 3D culture prepared by the method for preparing a hydrogel for stem cell / organoid 3D culture according to any one of claims 1 to 9.

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