Preparation method of cell-loaded alginate composite hydrogel microspheres with excellent sphericity degree

Abstract

The invention discloses a preparation method of cell-loaded alginate composite hydrogel microspheres with excellent sphericity, and belongs to the technical field of biology. The preparation method comprises the following steps: firstly, generating water-in-oil liquid drops from a uniformly mixed alginate composite material solution containing cells through a microfluidic device, and enabling the water-in-oil liquid drops to fall onto a hydrophobic hydrogel substrate containing a cross-linking agent; the cross-linking agent in the hydrophobic hydrogel substrate containing the cross-linking agent diffuses into the water phase of the water-in-oil liquid drops through the contact surface of the cross-linking agent and the water-in-oil liquid drops, in-situ solidification of the liquid drops is achieved, and then the monodisperse alginate composite material hydrogel microspheres encapsulating the cells are obtained. The method supports in-situ crosslinking of multi-component alginate material micro-droplets, and the cured hydrogel microspheres are high in sphericity degree and good in monodispersity; in the solidification process of the micro-droplets, organic or inorganic acid is prevented from being used as a pH regulator, and the high activity of cells can be kept. The hydrogel microspheres can be applied to the fields of tumor drug screening, biomedical research and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular is a method for preparing cell-carrying alginate composite material hydrogel microspheres with excellent sphericity. Background Art

[0002] Hydrogel microspheres are micro-nano-scale spheres made of cross-linked polymer materials. They have a controllable network structure and can provide the required mechanical environment and sites for adhesion, migration and degradation for the cells encapsulated therein. Microspheres have a high specific surface area, which is conducive to the transmission of nutrients and can achieve large-scale amplification using a small amount of clinical samples in a short period of time. Therefore, hydrogel microspheres have shown great advantages in three-dimensional cell modeling. Alginate is a common material for preparing cell microsphere scaffolds. It can react and gel rapidly with metal ions in solutions under physiological conditions, and thus has great advantages.

[0003] There are many methods for preparing hydrogel microspheres using alginate, but they can be divided into two categories, namely external gelation and internal gelation.

[0004] In the existing preparation process of alginate-based microspheres, the most commonly used method is to use divalent metal salt ions to induce the solidification of sodium alginate microdroplets in an aqueous solution, and the electrostatic droplet process is a typical preparation method. This process disperses the sodium alginate solution into fine droplets through electric field force, and these droplets then fall into the metal salt solution, where the droplets solidify and settle. This method can prepare hydrogel microspheres with regular morphology and uniform particle size under cell-friendly conditions. However, when other solutions containing polymers such as proteins and polysaccharides are added to the sodium alginate solution, it will form a great steric hindrance to the cross-linking of sodium alginate and metal ions, greatly prolonging the solidification time of the sodium alginate microdroplets, so during the preparation process, the microdroplets are prone to fusion or floating on the surface of the metal salt solution, destroying the original morphology of the droplets, so this method is not suitable for preparing hydrogel microspheres of sodium alginate composite materials.

[0005] In contrast, the internal gelation method is to pre-incorporate insoluble or insoluble metal salts into the sodium alginate solution. Generally, it is necessary to induce the metal salt to release ions under the action of acid to cross-link the sodium alginate molecules. For example, the emulsification method and the microfluidic method use the oil phase to break up the sodium alginate solution to form an oil-in-water emulsion. The acetic acid contained in the oil phase can diffuse into the aqueous phase droplets and promote the release of metal ions. Although this method can prepare hydrogel microspheres of sodium alginate composite materials with regular morphology, acid treatment has a very large damage to cell viability. In addition, due to the limited amount of acid, the sodium alginate in the microdroplets cannot be fully cross-linked, and the prepared hydrogel microspheres are usually of poor strength. In order to avoid the effect of acid on cell activity, some researchers have added metal ion solution pipelines to the microfluidic pipelines, trying to achieve cross-linking by fusing metal ion droplets with alginate droplets, but the results cannot be accurately controlled, and irregular microspheres such as gourd-shaped and dumbbell-shaped are often formed, which affects the subsequent application of the microspheres.

[0006] Therefore, the prior art still lacks a method for preparing alginate-based composite hydrogel microspheres with good sphericity and monodispersity, which can maintain good cell activity and achieve in situ cell loading. Summary of the invention

[0007] To solve the above problems, the purpose of the present invention is to provide a method for preparing cell-carrying alginate composite hydrogel microspheres with excellent sphericity. The present invention uses an alginate composite solution as an aqueous phase, adopts a microfluidic pipeline to prepare alginate composite oil-in-water droplets with good sphericity, and uses a hydrophobic hydrogel substrate containing metal ions as a receiving carrier of the microdroplets. The alginate composite oil-in-water droplets are cross-linked and solidified on the hydrophobic surface, which avoids the disadvantage of the introduction of acid in the existing internal gelation process that affects cell activity, and eliminates the problem of irregular morphology of the existing external gelation.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] The invention provides a method for preparing cell-carrying alginate composite material hydrogel microspheres, comprising the following steps: a uniformly mixed alginate composite material solution containing cells is used to generate oil-in-water droplets through a microfluidic device, and the droplets are dropped onto a hydrophobic hydrogel substrate containing a crosslinking agent, and the crosslinking agent in the hydrophobic hydrogel substrate containing the crosslinking agent diffuses into the water phase of the oil-in-water droplets through a contact surface with the oil-in-water droplets, thereby realizing in-situ solidification of the droplets, and then obtaining monodisperse alginate composite material hydrogel microspheres encapsulating the cells.

[0010] Based on the above technical solution, further, the monodisperse alginate composite hydrogel microspheres encapsulating cells are cultured in an organoid culture medium to obtain organoid hydrogel microspheres.

[0011] Based on the above technical solution, further, the hydrophobic hydrogel substrate containing a crosslinking agent is prepared by preparing a crosslinking agent and a hydrophobic high molecular polymer into a uniform solution, adding the solution into a well plate, and curing the solution to obtain the hydrophobic hydrogel substrate.

[0012] Based on the above technical solution, further, the hydrophobic high molecular polymer is a hydrophobic protein or a protein or polysaccharide polymer modified with hydrophobic groups, including but not limited to gelatin, agar, starch, bovine serum albumin, chitosan, xanthan gum, silk fibroin, cyclodextrin and pectin, and the hydrophobic group modification includes but is not limited to the introduction of methyl, ethyl, propane, octane, methylpropylene, acetyl, octenyl succinate groups at the hydroxyl and / or carboxyl ends of the polymer.

[0013] Based on the above technical solution, further, the crosslinking agent in the hydrophobic hydrogel substrate is a divalent cation or a trivalent cation, and the divalent cation includes Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ , Sr 2+ 、Zn 2+ and Ba 2+ , trivalent cations include Fe 3+ and Ga 3 + .

[0014] Based on the above technical solution, further, the concentration of the cross-linking agent in the hydrophobic hydrogel substrate is 2-100 mmol / L.

[0015] Based on the above technical solution, further, the alginate composite material solution containing cells is prepared by uniformly mixing alginate, composite material and cells in a culture medium, wherein the concentration of alginate in the solution is 0.1-50 mg / mL, and the cell density is 0.1-9×10 6 / mL, and the mass ratio of alginate to the composite material is 1:1 to 100:1.

[0016] Based on the above technical solution, further, the cells include thyroid cells, breast cells, pancreatic cells, liver cells, thymus cells, lung cells, kidney cells, stomach cells, intestinal cells and tumor cells of the above organs.

[0017] Based on the above technical solution, further, the alginate includes sodium alginate and potassium alginate.

[0018] Based on the above technical solution, further, the composite material includes but is not limited to physically mixed protein polymer materials or polysaccharide polymer materials, and the polymer materials include but are not limited to collagen, matrix glue, gelatin, silk fibroin, chitosan, dextran, heparin and hyaluronic acid.

[0019] Based on the above technical solution, further, the alginate is connected with a chemical modification group, and the chemically modified alginate includes but is not limited to groups modified with biologically active functions or covalent cross-linking sites on the carboxyl and / or hydroxyl groups of alginate, including RGD peptide, QPQGLAK polypeptide, galactose, PEG, cysteine, (4-(1,2,4,5-tetrazine-3-yl)phenyl)methylamine, norbornenemethylamine, dopamine, thiol and groups containing terminal olefin structures.

[0020] Based on the above technical solution, further, the chemically modified alginate includes methacrylylated alginate and methacrylylated alginate connected with RGD peptide.

[0021] Based on the above technical scheme, further, when the chemically modified alginate is methacrylated alginate, a photoinitiator is added to the cell-containing alginate composite material solution, and the microdroplets are irradiated with an ultraviolet light source during the in situ curing of the microdroplets to cross-link the groups containing the terminal olefin structure, thereby accelerating the in situ curing of the microdroplets.

[0022] Based on the above technical solution, further, the microfluidic device mainly includes "T"-shaped, "Y"-shaped, "cross"-shaped, "U"-shaped, and arrow-shaped microfluidic pipelines that can realize oil-in-water droplets.

[0023] Based on the above technical solution, further, the oil phase in the water-in-oil droplets includes but is not limited to volatile liquid oils, including n-hexane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, electronic fluorinated liquid and fluorocarbon oil.

[0024] Based on the above technical solution, further, the particle size of the monodisperse alginate composite hydrogel microspheres encapsulating cells is 30-1000 μm, and the number of cells in each microsphere is 1-10000.

[0025] The present invention has the following beneficial effects:

[0026] (1) During the droplet generation process, no acid is added to the oil phase as a pH regulator, and the oil phase does not contain a surfactant, so there is no need for washing or centrifugation. Therefore, the cells encapsulated in the hydrogel microspheres can maintain a high activity.

[0027] (2) In the present invention, a hydrophobic hydrogel substrate is used as a carrier for receiving microdroplets. The surface of the substrate is hydrophobic without the need for hydrophobic treatment. This hydrophobicity can maintain the original spherical shape of the droplets, and the volume of each droplet is uniform. Therefore, the prepared hydrogel microspheres have high sphericity, uniform particle size, and good monodispersity.

[0028] (3) The cell-laden microdroplets can stay on the substrate for a long enough time without changing the spherical morphology, which provides sufficient cross-linking time for the alginate in the microdroplets and realizes the in situ solidification of the cell-laden alginate composite microspheres. During long-term culture, cells are not easy to leak from the microspheres.

[0029] (4) The present invention realizes a one-step operation of cell-laden alginate composite material microdroplets from generation, distribution, in situ solidification to cultivation, thereby improving the efficiency of in vitro modeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention, the drawings related to the embodiments are briefly introduced below.

[0031] Figure 1 Schematic diagram of the droplet generation, gelation and distribution process in an embodiment of the present invention.

[0032] Figure 2 The morphology of the liquid at different stages in Example 5, wherein A is the morphology of the microdroplets dropped into the well plate on the surface of the gelatin hydrogel, B is the morphology of the microspheres on the surface of the gelatin hydrogel after the n-hexane evaporates, and C is the morphology of the microspheres after the culture medium is added.

[0033] Figure 3 The lung cancer organoids (A) and the staining image of the cell viability (B) preliminarily formed in the microspheres under a light microscope in Example 7, wherein the bright spots are living cells.

[0034] Figure 4 is the IC of osimertinib and gemcitabine on the two-component calcium alginate microspheres in Example 3 50 Value result. DETAILED DESCRIPTION

[0035] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative labor all fall within the protection scope of the present invention.

[0036] Example 1

[0037] This embodiment provides a method for preparing matrix gel / calcium alginate two-component hydrogel microspheres, such as Figure 1 As shown, the following steps are included:

[0038] (1) A solution of 5 mmol / L calcium chloride and 60 mg / mL gelatin (BBI LIFE SCIENCES, Shanghai) was prepared with sterile water, filtered through a 0.22 μm filter and placed in a 60°C water bath. Then, 6 μL of the solution was added to each well of a 384-well plate and cooled on ice for 30 min.

[0039] (2) Matrigel (CORNING, USA) was thawed on ice, and then primary lung cancer cell suspension was added to the Matrigel;

[0040] (3) Prepare a solution with a sodium alginate concentration of 20 mg / mL using DMEM medium, and then mix it with the solution in step (2). After mixing, the final concentration of sodium alginate is 10 mg / mL. The cell amount is 10 5 / mL, the volume of matrix gel accounts for 50% of the total volume of the solution.

[0041] (4) The solution in step (3) was transferred to a 1 mL syringe, and 20 mL of electronic fluoride liquid (boiling point of about 75° C.) was transferred to a 50 mL syringe, and both were installed in a microinjection pump, followed by the installation of a microfluidic device and capillary line sterilized by high temperature and high pressure, and the water phase flow rate was adjusted to 0.6 μL / min and the oil phase flow rate was adjusted to 100 μL / min;

[0042] (5) After the pipeline has been running stably for 10 min, an automatic stepper is used to drip a drop of water-in-oil solution into each well of the 384-well plate in step (1). The dripping process lasts for about 10 min.

[0043] (6) After the addition is completed, the gelation is continued for 5 min, and then the plate is turned upside down to discharge the oil phase (the gel sticks to the surface of the gelatin and will not fall off), and then the excess oil phase is evaporated;

[0044] (7) Using the stepper again, 20 μL of lung cancer organoid culture medium was added to each well of the 384-well plate, and the well plate was transferred to a cell culture incubator and cultured for 10 days to obtain a primary lung cancer organoid microsphere array (eg, Figure 3 shown).

[0045] Example 2

[0046] This embodiment provides a method for preparing two-component calcium alginate microspheres with different hardness, comprising the following steps:

[0047] (1) Prepare a solution with a calcium chloride concentration of 2 mmol / L and a gelatin concentration of 60 mg / mL with sterile water, filter through a 0.22 μm filter and place in a water bath at 60°C. Then add 6 μL of the solution to each well of a 384-well plate and cool on ice for 30 min.

[0048] (2) Dissolve methacrylated gelatin (GelMA, reference: Hu N, Cai Z, Jiang X, Wang C, Tang T, Xu T, Chen H, Li X, Du X, Cui W. Hypoxia-pretreated ADSC-derived exosome-embedded hydrogels promote angiogenesis and accelerate diabetic wound healing. Acta Biomater. 2023 Feb; 157: 175-186.) in water to prepare a solution with a concentration of 50 mg / mL, and add photoinitiator Irgacure 2959 to the solution at a concentration of 5 mg / mL. After dissolution, filter through a 0.22 μm filter membrane;

[0049] (3) A solution with a sodium alginate concentration of 30 mg / mL was prepared using DMEM medium, diluted with DMEM medium, mixed with the solution in step (2) respectively, and added to the cell suspension respectively to prepare three cell suspensions with different sodium alginate concentrations; the final concentrations of sodium alginate after mixing were 5 mg / mL (Group A, compression modulus of about 10 kPa), 10 mg / mL (Group B, compression modulus of about 30 kPa) and 15 mg / mL (Group C, compression modulus of about 52 kPa), and the cell weight was 10 5 / mL, and the concentration of GelMA was 50mg / mL;

[0050] (4) Transfer the solution of group A in step (3) into a 1 mL syringe, and then transfer 20 mL of n-hexane into a 50 mL syringe, and install both of them on a microinjection pump, and then install a microfluidic device and capillary line sterilized by high temperature and high pressure, and adjust the water phase flow rate to 0.6 μL / min and the oil phase flow rate to 150 μL / min;

[0051] (5) After the pipeline has been running stably for 10 min, an automatic stepper is used to drip a drop of water-in-oil solution into each well of the 384-well plate in step (1). The dripping process lasts for about 10 min.

[0052] (6) After the addition is completed, the mixture is allowed to stand for gelation for 5 min, and then irradiated with a 365 nm ultraviolet lamp for 10 min. The plate is then turned upside down to discharge the oil phase, and the excess oil phase is then evaporated;

[0053] (7) Add the cell suspensions of group B and group C to two other 384-well plates in the same manner.

[0054] (8) Using the stepper again, 20 μL of organoid culture medium was added to each well of the 384-well plate, and the well plate was transferred to a cell culture incubator and cultured for 7 days to obtain a primary breast cancer organoid microsphere array.

[0055] The difference between Example 2 and Example 1 is that in Example 1, the hardness of the hydrogel microspheres is at the same level, while in Example 2, by changing the concentration of sodium alginate, different groups of hydrogel microspheres have different hardnesses.

[0056] Example 3

[0057] In this example, the constructed two-component calcium alginate microsphere array is used for drug screening, comprising the following steps:

[0058] (1) Organoid culture medium containing osimertinib or gemcitabine was prepared, and five drug concentration gradients were set, namely 0, 0.01, 0.1, 1, and 10 mM.

[0059] (2) Take the lung cancer organoid microsphere array of step (7) in Example 1, add the medium containing the drug in step (1), and set 3 parallel samples for each drug concentration. After continuing to culture for 7 days, use CellTiter-Glo and a microplate reader to measure the activity of the organoids after different drug treatments. The specific method is to take the microsphere culture well plate treated with drugs, add 50 μL of CellTiter-Glo detection solution to each well according to the instructions of the reagent manufacturer, and then react at 37°C in the dark for 30 minutes, take 80 μL of the reaction solution to a light-proof black well plate, use a bioluminescence instrument (BERTHOLD, Germany) to detect the fluorescence value of the reaction solution, import the fluorescence value into GraphPad software, and calculate the IC of different drugs. 50 Value, the result is Figure 4 shown.

[0060] Example 4

[0061] In this example, the constructed two-component calcium alginate microsphere array is used for drug screening, comprising the following steps:

[0062] (1) Prepare temozolomide solution using organoid culture medium. The concentration of temozolomide is 4 mmol / L.

[0063] (2) The breast cancer organoid microsphere array of step (8) in Example 2 was added with the medium containing the drug in step (1), and the culture was continued for 7 days. The activity of the organoids after drug treatment under different hardness environments was measured using CellTiter-Glo and an ELISA reader.

[0064] Example 5

[0065] This embodiment provides a method for rapidly constructing a three-component calcium alginate microsphere array for cell adhesion and growth on the surface of the microsphere, comprising the following steps:

[0066] (1) Prepare a solution with a calcium chloride concentration of 5 mmol / L and a gelatin concentration of 60 mg / mL using sterile water, filter the membrane and place it in a water bath at 60°C. Then add 6 μL of the above solution to each well of a 384-well plate and cool it on ice for 30 min.

[0067] (2) Thaw Matrigel on ice;

[0068] (3) preparing a solution with a sodium alginate concentration of 20 mg / mL and a hyaluronic acid concentration of 2 mg / mL using DMEM medium, and then mixing with the solution in step (2), wherein the final concentrations of sodium alginate and hyaluronic acid after mixing are 10 mg / mL and 1 mg / mL, respectively, and the volume of the matrix gel accounts for 50% of the total volume of the solution;

[0069] (4) The solution in step (3) was transferred to a 1 mL syringe, and then 20 mL of electronic fluoride liquid (boiling point is about 75° C.) was transferred to a 50 mL syringe, and both were installed in a microinjection pump, followed by the installation of a microfluidic device and capillary line sterilized by high temperature and high pressure, and the water phase flow rate was adjusted to 0.6 μL / min and the oil phase flow rate was adjusted to 100 μL / min.

[0070] (5) After the pipeline has been running stably for 10 minutes, an automatic stepper is used to drip water-in-oil droplets into each well of the 384-well plate in step (1). The dripping process lasts for about 10 minutes. The state of the microdroplets on the gelatin surface is as follows: Figure 2 As shown in A.

[0071] (6) After the addition is completed, the gelation is continued for 5 minutes, and then the plate is turned upside down to discharge the oil phase, and then the excess oil phase is evaporated. The state of the microspheres on the gelatin surface is as follows Figure 2 As shown in B.

[0072] (7) Using the stepper again, 20 μL of culture medium was added to each well of the 384-well plate. The empty microsphere model without encapsulated cells was as follows. Figure 2 As shown in C.

[0073] Example 6

[0074] This embodiment provides a method for preparing photo-crosslinked calcium alginate composite hydrogel microspheres, comprising the following steps:

[0075] (1) Methacrylylated sodium alginate (AlgMA, reference: Yin T, Zhang X, Shao S, Xiang T, Zhou S. Covalently crosslinked sodium alginate / poly(sodium p-styrenesulfonate)cryogels for selective removal of methylene blue. Carbohydr Polym. 2023 Feb 1; 301(Pt B): 120356.) and methacrylylated gelatin (GelMA, reference: Cai C, Zhang X, Li Y, Liu X, Wang S, Lu M, Yan X, Deng L, Liu S, Wang F, Fan C. Self-Healing Hydrogel Embodied with Macrophage-Regulation and Responsive-Gene-Silencing Properties for Synergistic Prevention of Peritendinous Adhesion. Adv Mater. 2022 Feb; 34(5): e2106564.) and photoinitiator Irgacure 2959 were dissolved in organoid culture medium and filtered through a 0.22 μm filter. The concentration of AlgMA was 20 mg / mL, the concentration of GelMA was 2 mg / mL, and the concentration of photoinitiator was 5 mg / mL;

[0076] (2) Take 100uL of the precursor solution of the hydrogel microspheres in step (1), and add 50uL of primary lung cancer cell suspension (total cell volume is 10 5 After mixing, the solution was drawn into a 1 mL syringe and then installed in a microinjection pump;

[0077] (3) placing the gelatin and calcium chloride mixed solution in step (1) of Example 1 into each well of a 384-well plate and cooling on ice for 30 min;

[0078] (4) referring to steps (4)-(5) of Example 1, preparing and dripping water-in-oil droplets into the orifice plate;

[0079] (6) After the microdroplets were gelled for 10 minutes, the microspheres were irradiated with a 365 nm ultraviolet light source for 5 minutes, and then the culture medium was added to the well plate according to the steps of Example 1.

[0080] (7) The well plate of step (6) was placed in an incubator and cultured for 10 days. Lung cancer organoids were formed in the microspheres.

[0081] Example 7

[0082] This embodiment provides a method for preparing matrix gel / calcium alginate two-component hydrogel microspheres, such as Figure 1 As shown, the following steps are included:

[0083] (1) Prepare a solution with a calcium chloride concentration of 5 mmol / L and octenylsuccinic anhydride modified agar (reference: Chen Fuquan, Chen Huijing, Hong Qinglin, et al. Improvement of the embedding effect of sodium alginate microcapsules by octenylsuccinic anhydride modified agar [J]. Modern Food Science and Technology, 2021, 37(05): 130-138.) with sterile water. After high temperature and high pressure sterilization, add 6 μL of the above solution to each well of a 384-well plate while it is still hot and place it on ice for 30 min;

[0084] (2) Thawing the matrix gel on ice, and then adding the primary lung cancer cell suspension into the matrix gel;

[0085] (3) Prepare a solution with a sodium alginate concentration of 20 mg / mL using DMEM medium, and then mix it with the solution in step (2). After mixing, the final concentration of sodium alginate is 10 mg / mL. The cell amount is 10 5 / mL, the volume of matrix gel accounts for 50% of the total volume of the solution;

[0086] (4) The solution in step (3) was transferred to a 1 mL syringe, and then 20 mL of electronic fluoride liquid (boiling point is about 75° C.) was transferred to a 50 mL syringe, and both were installed in a microinjection pump, followed by the installation of a microfluidic device and capillary line sterilized by high temperature and high pressure, and the water phase flow rate was adjusted to 0.6 μL / min and the oil phase flow rate was adjusted to 100 μL / min.

[0087] (5) After the pipeline has been running stably for 10 min, an automatic stepper is used to drip a drop of water-in-oil solution into each well of the 384-well plate in step (1). The dripping process lasts for about 10 min.

[0088] (6) After the addition is completed, the gelation is continued for 5 min, and then the orifice plate is turned upside down to discharge the oil phase, and then the excess oil phase is evaporated;

[0089] (7) Using the stepper again, 20 μL of lung cancer organoid culture medium was added to each well of the 384-well plate, and the well plate was transferred to a cell culture incubator and cultured for 10 days to obtain a primary lung cancer organoid microsphere array. Figure 3 As shown in A, lung cancer organoids were formed in the microspheres under an upright microscope, and after staining, the cells had a high survival rate ( Figure 3 B).

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing cell-carrying alginate composite hydrogel microspheres, It is characterized in that The method comprises the following steps: a uniformly mixed alginate composite material solution containing cells is passed through a microfluidic device to generate oil-in-water droplets, which are dropped onto a hydrophobic hydrogel substrate containing a cross-linking agent; the cross-linking agent in the hydrophobic hydrogel substrate containing the cross-linking agent diffuses into the aqueous phase of the oil-in-water droplets through the contact surface with the oil-in-water droplets, thereby achieving in-situ solidification of the droplets, and further obtaining monodisperse alginate composite material hydrogel microspheres encapsulating cells.

2. The preparation method according to claim 1, It is characterized in that The monodisperse alginate composite hydrogel microspheres encapsulating cells are cultured in an organoid culture medium to obtain organoid hydrogel microspheres.

3. The preparation method according to claim 1, It is characterized in that The hydrophobic hydrogel substrate containing a crosslinking agent is prepared by preparing a crosslinking agent and a hydrophobic high molecular polymer into a uniform solution, adding the solution into a well plate, and then solidifying the solution to obtain the hydrogel substrate; The hydrophobic high molecular polymer is a hydrophobic protein or a protein or polysaccharide polymer modified with hydrophobic groups, including but not limited to gelatin, agar, starch, bovine serum albumin, chitosan, xanthan gum, silk fibroin, cyclodextrin and pectin. The hydrophobic group modification includes but is not limited to the introduction of methyl, ethyl, propane, octane, methylpropylene, acetyl, octenyl succinate groups at the hydroxyl and / or carboxyl ends of the polymer.

4. The preparation method according to claim 1, It is characterized in that The crosslinking agent in the hydrophobic hydrogel substrate is a divalent cation or a trivalent cation, and the divalent cation includes Mg 2+ , Ca 2+ , Cu 2+ , Fe 2+ , Sr 2+ 、Zn 2+ and Ba 2+ , trivalent cations include Fe 3+ and Ga 3+ ; The concentration of the cross-linking agent in the hydrophobic hydrogel substrate is 2-100 mmol / L.

5. The preparation method according to claim 1, It is characterized in that The alginate composite material solution containing cells is prepared by mixing alginate, composite material and cells in a culture medium, wherein the concentration of alginate in the solution is 0.1-50 mg / mL and the cell density is 0.1-9×10 6 / mL, and the mass ratio of alginate to composite material is 1:1 to 100:1; The composite material includes but is not limited to physically mixed protein polymer materials or polysaccharide polymer materials, and the polymer materials include but are not limited to collagen, matrix glue, gelatin, silk protein, chitosan, dextran, heparin and hyaluronic acid.

6. The preparation method according to claim 1, It is characterized in that The cells include thyroid cells, breast cells, pancreatic cells, liver cells, thymus cells, lung cells, kidney cells, stomach cells, intestinal cells and tumor cells of the above organs.

7. The preparation method according to claim 1, It is characterized in that The alginate is connected with a chemical modification group, and the chemically modified alginate includes but is not limited to a group modified with a biologically active function or a covalent cross-linking site on the carboxyl and / or hydroxyl groups of the alginate, including RGD peptide, QPQGLAK polypeptide, galactose, PEG, cysteine, (4-(1,2,4,5-tetrazine-3-yl)phenyl)methylamine, norbornenemethylamine, dopamine, thiol and a group containing a terminal olefin structure.

8. The preparation method according to claim 1, It is characterized in that The microfluidic device mainly includes "T"-shaped, "Y"-shaped, "cross"-shaped, "U"-shaped, and arrow-shaped microfluidic pipelines that can realize oil-in-water droplets.

9. The preparation method according to claim 1, It is characterized in that The oil phase in the water-in-oil droplets includes, but is not limited to, volatile liquid oils, including n-hexane, octane, nonane, decane, undecane, dodecane, tridecane, tetradecane, electronic fluorinated liquid and fluorocarbon oil.

10. The preparation method according to claim 1, It is characterized in that The particle size of the monodisperse alginate composite material hydrogel microspheres encapsulating cells is 30-1000 μm, and the number of cells in each microsphere is 1-10000.

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