Single-cell microspheres prepared based on aqueous two-phase oil-free strategy and preparation method of single-cell microspheres

Abstract

The invention discloses a single-cell microsphere prepared on the basis of an aqueous two-phase oil-free strategy and a preparation method thereof, specifically, cells in an upper-layer water phase enter a lower-layer water phase through gravity induction, and hydrogel-encapsulated single cells are obtained from the cells entering the lower-layer water phase due to the adhesion effect of the cells and hydrogel and a stable interface formed by the aqueous two phases. The thickness of the gel layer can be changed by changing the concentration of the layered double aqueous phases, so that the single-cell microspheres with controllable thickness and size are obtained. The method does not involve the use of oil and a surfactant, is mild in action on cells, can realize indifference encapsulation of different types of cells, has universality, broad spectrum and ultrahigh cell encapsulation efficiency, and compared with other strategies, has the advantages of low equipment cost and high preparation efficiency, greatly improves the biocompatibility, and is suitable for industrial production. And the ultrathin hydrogel layer package which is difficult to prepare by the existing preparation strategy can be easily prepared, and can be further applied to the field of biomedicine such as cell therapy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a single-cell microsphere prepared based on a two-phase oil-free strategy and a preparation method thereof. Background Art

[0002] The main purpose of cell therapy is to replace or repair damaged cells by transplanting cells into the patient's body to promote the regeneration and repair of tissues or organs. Cells (including stem cells) are generally implanted into the damaged site by direct injection, but in many cases, direct cell transplantation is ineffective. First, due to the shear force between cells during the injection process, the mechanical pressure of the recipient tissue, and the complexity of the damaged microenvironment or blood environment, the retention rate and survival rate of cells in the lesion site are low. In addition, in some cases, directly injected cells may migrate from the injection site and differentiate into unwanted cell types, and easily cause the body's rejection reaction and be cleared by the immune system, which greatly limits the effectiveness of cell therapy. Therefore, in order to further improve the effect of treatment, the relevant research on single cell encapsulation has received widespread attention from scientific researchers in the field of regenerative medicine. Hydrogels have high water content, good biocompatibility and degradability, and their properties are very similar to those of extracellular matrix, and are widely used for cell delivery. However, the current single-cell microsphere preparation technology still faces many problems. Most researchers prepare single-cell microspheres through microfluidic devices, but microfluidic encapsulation systems are expensive and still face problems such as low cell encapsulation efficiency and difficulty in preparing ultra-thin gel layers. A sufficiently thin gel layer allows nutrients, oxygen, and water to pass freely without restricting the extension of cells, thereby promoting the realization of cell functionality. When the size of microgels increases to a certain extent, their applications will be limited, such as high-throughput analysis and affecting their pharmacokinetics after implantation. In addition, studies have shown that intravenously injected ~30μm single-cell microspheres can accumulate in the pulmonary capillary bed of mice, while ~50μm microspheres are usually physically trapped, thereby accumulating in the blood vessels of downstream tissues, leading to the risk of tissue infarction. Secondly, microfluidic strategies often cannot avoid the introduction of substances with low biocompatibility such as oils and surfactants, and the oil removal step will significantly reduce the activity of cells and the operation is cumbersome, further limiting its wide application in the biomedical field.

[0003] Another common encapsulation strategy is chemical modification, which is to modify the cell surface with exogenous substances, which is usually done by embedding corresponding groups on the cell membrane using chemical reactions. Although the current chemically engineered cells show good application prospects, they still face many challenges. For example, specific chemical reaction conditions may affect the activity of cells and the thickness controllability is poor. Secondly, exogenous substances modified on the cell surface are easily endocytosed by cells, thereby reducing cell activity and delivery efficiency. In addition, since exogenous substances occupy sites on the cell surface, this may affect the binding of cell surface receptors to specific ligands, thereby limiting the initiation and subsequent functions of related signaling pathways, and may also increase the immunogenicity of cells, leading to attack and clearance of immune cells. Furthermore, the mechanical properties of cells may be changed accordingly, resulting in reduced cell activity and changes in other physicochemical properties. These problems have also greatly limited the application of single-cell microspheres in biomedicine and other fields. Therefore, the above limitations have promoted the development of single-cell microsphere preparation technology and its transformation to small volume. Encapsulating a single cell in a nano-micron hydrogel layer can significantly increase the surface-to-volume ratio, thereby enabling better material exchange, including the transport of nutrients and oxygen, as well as the excretion of beneficial factors secreted by cells and exerting corresponding therapeutic effects. Therefore, developing a simple, universal, and highly biocompatible innovative strategy for single-cell microspheres to enhance their application in the field of biomedical engineering is a scientific problem that needs to be solved urgently. Summary of the invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0005] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0006] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a method for preparing single-cell microspheres based on a two-phase oil-free strategy.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: a method for preparing single-cell microspheres based on a two-phase oil-free strategy, comprising:

[0008] Resuspend the cells with the upper solution and slowly drip it into the container containing the lower solution to form a stable layer with the lower solution;

[0009] After the cells fall from the upper layer to the lower layer, the cell coating layer is solidified to obtain single-cell microspheres.

[0010] As a preferred solution of the method of the present invention, the upper layer solution is a protein solution or a hydrogel solution; the concentration of the upper layer solution is 0.001% to 2%.

[0011] As a preferred embodiment of the method of the present invention, the upper layer solution is one of sodium alginate solution, methacrylic anhydride gelatin solution, methacryloyl hyaluronic acid, carboxymethyl cellulose solution, matrigel solution, sericin solution, collagen solution, and fibroin solution.

[0012] As a preferred solution of the method of the present invention, the lower layer solution is a polysaccharide aqueous solution or a protein solution; the concentration of the lower layer solution is 2% to 20%.

[0013] As a preferred embodiment of the method of the present invention, the lower layer solution is one of a dextran solution, a polysucrose solution, a sucrose solution, an agarose solution, a sericin solution, a collagen solution, and a fibroin solution.

[0014] As a preferred embodiment of the method of the present invention, the method of solidifying the cell wrapping layer is dropwise addition of solution or other solidification operations.

[0015] As a preferred embodiment of the method of the present invention, the dropwise solution is a calcium chloride solution or a ferric chloride solution.

[0016] As a preferred embodiment of the method of the present invention, the other curing operation is to cure the hydrogel by ultraviolet light or temperature.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide a single-cell microsphere prepared based on a two-phase oil-free strategy.

[0018] As a preferred embodiment of the single-cell microspheres of the present invention, the encapsulation rate of the single-cell microspheres is above 98%.

[0019] Beneficial effects of the present invention:

[0020] (1) The present invention uses an aqueous solution with high biocompatibility, without the oil and surfactant used in traditional microfluidic strategies, which greatly improves the cell survival rate to more than 95%, while reducing the steps of removing oil, simplifying the experimental operation, and reducing the impact on cell activity;

[0021] (2) The single-cell microspheres prepared by the present invention can not only increase the encapsulation rate to more than 98%, but also achieve complete encapsulation;

[0022] (3) The present invention does not involve any special chemical reaction, but only relies on physical action to improve cell activity and achieve indiscriminate encapsulation, thereby solving the problems of cell surface site occupancy caused by chemical encapsulation, affecting the binding between specific ligands and receptors, and increasing immunogenicity;

[0023] (4) The present invention can control the thickness of the package by changing the concentration of the solution, and prepare ultrathin (1 μm to 5 μm) hydrogel single-cell microspheres that are difficult to obtain with traditional microfluidic strategies.

[0024] (5) The preparation method based on the two-phase oil-free strategy proposed in the present invention has excellent high throughput, biocompatibility, thickness controllability and ease of operation. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0026] Figure 1 The figure is a schematic diagram of the process of preparing single-cell microspheres based on the two-phase oil-free strategy of the present invention.

[0027] Figure 2 The stratification of different solutions.

[0028] Figure 3 Microspheres encapsulating a single HeLa cell prepared with different solution combinations.

[0029] Figure 4 Single-cell microspheres encapsulating different cells are obtained by utilizing two-phase stable stratification.

[0030] Figure 5 Single-cell microspheres with different encapsulation thicknesses were obtained by adjusting the concentrations of sodium alginate and dextran solutions.

[0031] Figure 6 Live-dead staining fluorescence images of h1299 cells under the conditions of 0.0125% Alginate and 10% Dextran. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0033] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0034] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0035] Materials and reagents used in the embodiments of the present invention: Zeiss confocal (LSM880), MSHOT microscope (MF53-N), Alginate (AR grade, Sigma), CMC V (Aladdin, JAD-C104983), Matrigel (corning, 356234), GelMA, Sericin, calcium chloride (AR grade, Damao), Dextran (Aladdin, D490149-750K), Ficoll (Sigma, F2637), Sucrose (Macklin, S818046), Gelatin (Sigma, G7041), Collagen (Macklin, C823250-100g), Silk fibroin (EFL-SF-001), Agarose (S14003-10g).

[0036] Example 1

[0037] A method for preparing single-cell microspheres based on a two-phase oil-free strategy comprises the following steps:

[0038] (1) Prepare 0.0125% Alginate solution and 10% Dextran solution

[0039] Accurately weigh 12.5 mg of Alginate solid and dissolve it in 100 ml of PBS solution to obtain 0.0125% Alginate;

[0040] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0041] In order to characterize the success of cell encapsulation, fluorescent nanoparticles were added to the Alginate solution to prepare a 0.5% fluorescent nanoparticle solution.

[0042] (2) Preparation of calcium chloride solution

[0043] Prepare a 1% calcium chloride solution by accurately weighing 1g of calcium chloride and dissolving it in 100ml of distilled water to obtain a 1% calcium chloride solution.

[0044] (3) Preparation of single cell microspheres

[0045] Pre-add 500 μL of 10% Dextran solution as the lower layer solution to a suitable container, then resuspend Hela with 500 μL of 0.0125% Alginate solution as the upper layer solution mixed with 0.5% fluorescent nanoparticles, and further slowly drip the cell suspension into the container, and stably layer it with the lower layer solution;

[0046] After the cells fall from the upper layer to the lower layer, 1% calcium chloride solution is added to solidify the cell coating layer. The coating is observed under a microscope and the size of the microspheres is recorded.

[0047] See the schematic diagram of the process for preparing single-cell microspheres based on the two-phase oil-free strategy. Figure 1 .

[0048] Example 2

[0049] A method for preparing single-cell microspheres based on a two-phase oil-free strategy comprises the following steps:

[0050] (1) Prepare 0.0125% carboxymethyl cellulose (CMC V) solution and 10% Dextran solution

[0051] Accurately weigh 12.5 mg of CMC V solid and dissolve it in 100 ml of PBS solution to obtain a 0.0125% CMC V solution;

[0052] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0053] To characterize the success of cell encapsulation, fluorescent nanoparticles were added to the CMC V solution to prepare a 0.5% fluorescent nanoparticle solution.

[0054] (2) Preparation of ferric chloride solution

[0055] Prepare a 1% ferric chloride solution by accurately weighing 1g of ferric chloride and dissolving it in 100ml of distilled water to obtain a 1% ferric chloride solution.

[0056] (3) Preparation of single cell microspheres

[0057] Pre-add 500 μL of 10% Dextran solution as the lower layer solution to a suitable container, followed by 500 μL of 0.0125% CMC V solution Hela mixed with 0.5% fluorescent nanoparticles as the upper layer solution, and further slowly drip the cell suspension into the container, and stably layer with the lower layer solution;

[0058] After the cells fall from the upper layer to the lower layer, 1% ferric chloride solution is added to solidify the cell coating layer. The coating is observed under a microscope and the size of the microspheres is recorded.

[0059] Example 3

[0060] A method for preparing single-cell microspheres based on a two-phase oil-free strategy comprises the following steps:

[0061] (1) Prepare 0.0125% Matrigel solution and 10% Dextran solution

[0062] Accurately measure 12.5 μl of Matrigel solution and dissolve it in 100 ml of PBS solution to obtain 0.0125% Matrigel solution;

[0063] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0064] To characterize the success of cell encapsulation, fluorescent nanoparticles were added to the Matrigel solution to prepare a 0.5% fluorescent nanoparticle solution.

[0065] (2) Preparation of single cell microspheres

[0066] Pre-add 500 μL of 10% Dextran solution as the lower layer solution to a suitable container, then resuspend Hela cells with 500 μL of 0.0125% Matrigel solution as the upper layer solution mixed with 0.5% fluorescent nanoparticles, and further slowly drip the cell suspension into the container and stably layer it with the lower layer solution;

[0067] After the cells fall from the upper layer to the lower layer, place them at 37°C to solidify the cell coating layer. Observe the coating under a microscope and record the size of the microspheres.

[0068] Example 4

[0069] (1) Synthesis of methacrylic anhydride gelatin (GelMA) and preparation of GelMA solution

[0070] Fish skin gelatin was dissolved in phosphate buffered saline (PBS) at 50°C at a concentration of 10%, and then methacrylic anhydride was added dropwise using a syringe pump until the concentration reached 4% (v / v); the solution was stirred on a magnetic stirrer at 50°C for 2 h to ensure homogeneity;

[0071] GelMA was diluted twice and dialyzed with deionized water at 40°C for 7 days, with the deionized water being changed every 12 hours; the solution was filtered at 40°C using a 0.22 μm sterile vacuum filtration system, and the filtered solution was divided into 25 ml batches, frozen at -80°C for 1 day, and freeze-dried in a freeze dryer for 5 days to obtain GelMA solid.

[0072] 1 g of GelMA solid was accurately weighed and dissolved in 10 ml of PBS solution to obtain a 10% GelMA solution.

[0073] In order to characterize the successful cell encapsulation, GelMA was cured by UV in this experiment, so a photoinitiator, phenyl (2,4,6-trimethylbenzoyl) lithium phosphate (LAP), was added to the GelMA solution to prepare a GelMA solution containing 0.03% LAP.

[0074] (2) Prepare 10% Dextran solution

[0075] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0076] (3) Preparation of single cell microspheres

[0077] Add 500 μL of the lower layer solution Dextran solution to a suitable container in advance, then resuspend the Hela cells with 500 μL of GelMA solution containing 0.03% LAP, and then slowly drip the cell suspension into the container and stably layer it with the lower layer solution. After the cells fall from the upper layer to the lower layer, irradiate them with an ultraviolet lamp (Sangefei X9) for 30 seconds to solidify the cell coating layer. Observe the coating under a microscope and record the size of the microspheres.

[0078] Example 5

[0079] (1) Synthesis of Sericin and Preparation of Sericin Solution

[0080] Cut the cocoons into small pieces, wash them three times with pure water, dry them and weigh 50g; measure 1L of pure water, add 2.12g of sodium carbonate (0.02M), and heat to boiling; put the cocoons into boiling water and cook for 30min, during which the cocoons are pulled apart at regular intervals; after cooling to room temperature, take out the silk, and centrifuge the remaining solution at 3500rpm, 8min, 4℃ to remove insoluble impurities;

[0081] The supernatant was placed in a dialysis bag (MW 6000-8000) and dialyzed for three days, with the water changed four times a day; the dialyzed solution was centrifuged at 3500rpm, 15min, 4°C to remove the precipitated impurities, half of the solution was placed in a dialysis bag and blown dry with an electric fan in a fume hood until the desired concentration was obtained, and the other half of the solution was frozen at -80°C and then freeze-dried in a freeze dryer for 3 days to obtain Sericin solid.

[0082] Accurately weigh 1 g of Sericin solid and dissolve it in 10 ml of PBS solution to obtain a 10% Sericin solution. To characterize the successful cell encapsulation, fluorescent nanoparticles are added to the solution to prepare a 0.5% fluorescent nanoparticle solution.

[0083] (2) Prepare 10% Dextran solution

[0084] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran.

[0085] (3) Preparation of single cell microspheres

[0086] Pre-add 500 μL of 10% Dextran solution as the lower layer solution to a suitable container, then resuspend Hela cells with 500 μL of 10% Sericin solution as the upper layer solution mixed with 0.5% fluorescent nanoparticles, and then slowly drip the cell suspension into the container and stably layer it with the lower layer solution;

[0087] After the cells fall from the upper layer to the lower layer, the light intensity is 60 mW cm -2 Irradiate with green light for 30 seconds to solidify the cell coating. Observe the coating under a microscope and record the size of the microspheres.

[0088] The stratification of 0.0125% Alginate solution, CMC V solution, Matrigel solution and 10% Dextran solution prepared in Examples 1 to 5, and the stratification of 10% GelMA solution and Sericin solution and 10% Dextran solution, respectively, are shown in FIG. Figure 2 As shown, it can be seen that both are stably stratified.

[0089] Example 6

[0090] A method for preparing single-cell microspheres based on a two-phase oil-free strategy comprises the following steps:

[0091] (1) Prepare 0.0125% methacryloyl hyaluronic acid (HAMA) solution and 10% Dextran solution

[0092] Accurately weigh 12.5 mg of HAMA solution and dissolve it in 100 ml of PBS solution to obtain a 0.0125% HAMA solution;

[0093] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0094] To characterize the success of cell encapsulation, fluorescent nanoparticles were added to the Matrigel solution to prepare a 0.5% fluorescent nanoparticle solution.

[0095] (4) Preparation of single cell microspheres

[0096] Pre-add 500 μL of 10% Dextran solution as the lower layer solution to a suitable container, then resuspend Hela cells with 500 μL of 0.0125% HAMA solution as the upper layer solution mixed with 0.5% fluorescent nanoparticles, and further slowly drip the cell suspension into the container and stably layer it with the lower layer solution;

[0097] After the cells fell from the upper layer to the lower layer, they were irradiated with UV light (Sangefei X9) for 30 seconds to solidify the cell coating layer. The coating was observed under a microscope and the size of the microspheres was recorded.

[0098] The microspheres encapsulating a single Hela prepared by combining Alginate solution, CMC V solution, Matrigel solution, GelMA solution, Sericin solution, HAMA solution and Dextran solution in Examples 1 to 6 are as follows: Figure 3 As shown, it can be seen that the cells are completely encapsulated, with a high single-cell encapsulation rate.

[0099] Example 7

[0100] The difference between this embodiment and embodiment 1 is that the encapsulated cells in step (4) are Hela, human non-small cell lung cancer cells (A549), rat cardiomyocytes (H9C2), mouse myoblasts (C2C12), human non-small cell lung cancer cells (h1299), and mesenchymal stem cells (MSC), and the remaining steps are the same as in embodiment 1.

[0101] Figure 4 The single-cell microspheres encapsulating different cells were obtained by utilizing two-phase stable stratification. It can be seen that different types of cells can be encapsulated.

[0102] Example 8

[0103] The difference between this embodiment and embodiment 1 is that the concentrations of the prepared Alginate solutions are 0.125%, 0.25%, and 0.5%, respectively, and the remaining steps are the same as those in embodiment 1.

[0104] Example 9

[0105] The difference between this embodiment and embodiment 1 is that the concentrations of the prepared Dextran solutions are 2.5%, 5%, and 7.5% respectively, and the remaining steps are the same as those in embodiment 1.

[0106] Figure 5 In Examples 8 and 9, single-cell microspheres with different coating thicknesses were obtained by adjusting the concentrations of Alginate and Dextran solutions. It can be seen that after experiments with different concentrations of permutations and combinations, the thickness of the gel layer can be controlled to be less than 5 μm.

[0107] Figure 6 This is the live-dead staining fluorescence image of h1299 cells under the conditions of 0.0125% Alginate and 10% Dextran. It can be seen that the cells have a very good survival rate, which can reach more than 95%.

[0108] Comparative Example 1

[0109] The difference between this comparative example and Example 1 is that the concentration of the prepared Alginate solution is 3%, and the remaining steps are the same as those of Example 1. The results show that the cells stay in the upper layer of the solution and cannot fall to the lower layer.

[0110] Comparative Example 2

[0111] The difference between this embodiment and embodiment 1 is that the concentration of the prepared Dextran solution is 1%, and the other steps are the same as those of embodiment 1. The results show that low concentrations of Alginate and Dextran are difficult to form a stable interface and cells cannot be encapsulated.

[0112] Comparative Example 3

[0113] The difference between this embodiment and embodiment 1 is that the Alginate solution is changed to 2% polyvinyl alcohol (PVA), and the other steps are the same as those of embodiment 1. The results show that the cells cannot be encapsulated.

[0114] Comparative Example 4

[0115] The difference between this embodiment and embodiment 4 is that the concentration of the GelMA solution is 20%, and the remaining steps are the same as those of embodiment 4. The results show that the stratification is unstable and the cells cannot be encapsulated.

[0116] Comparative Example 5

[0117] The difference between this embodiment and embodiment 1 is that the single cell microspheres are prepared by using the traditional microfluidic technology, and the specific steps are as follows:

[0118] The cells were mixed into 0.0125% Alginate solution as the dispersed phase and injected into the microfluidic chip;

[0119] Then, a fluorinated oil (fluorinated FC-40, Sigma Aldrich) (RANBioTechnologies) containing 2% w / w fluorinated surfactant was used as the continuous phase;

[0120] The hydrogel solution was sheared by oil phase to obtain single-cell microdroplets, which were then collected in 1% calcium chloride solution to form single-cell microspheres.

[0121] The results showed that the single-cell microsphere encapsulation rate obtained by microfluidic technology was 40%, and the cell survival rate was 75%, both of which were much lower than the two-phase oil-free strategy proposed in the present invention.

[0122] The present invention uses an aqueous solution with high biocompatibility and does not involve oil and surfactants used in traditional microfluidic strategies, which greatly improves the cell survival rate to more than 95%. At the same time, it reduces the steps of removing oil, simplifies experimental operations, and also reduces the impact on cell activity. The single-cell microspheres prepared by the present invention can not only increase the encapsulation rate to more than 98%, but also achieve complete encapsulation.

[0123] The present invention does not involve any special chemical reaction, but only relies on physical action to improve cell activity and achieve indiscriminate encapsulation, thereby solving the problems of cell surface site occupancy caused by chemical encapsulation, affecting the binding between specific ligands and receptors, and increasing immunogenicity. The present invention can control the thickness of the encapsulation by changing the concentration of the solution, and prepare ultrathin (1μm to 5μm) hydrogel single-cell microspheres that are difficult to obtain with traditional microfluidic strategies. The preparation method based on the two-phase oil-free strategy proposed by the present invention has excellent high throughput, biocompatibility, thickness controllability and ease of operation.

[0124] 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 preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing single-cell microspheres based on a two-phase oil-free strategy. Features: include, Resuspend the cells with the upper solution and slowly drip it into the container containing the lower solution to form a stable layer with the lower solution; After the cells fall from the upper layer to the lower layer, the cell coating layer is solidified to obtain single-cell microspheres.

2. The method according to claim 1, Features: The upper layer solution is a protein solution or a hydrogel solution; the concentration of the upper layer solution is 0.001% to 2%.

3. The method according to claim 1, Features: The upper layer solution is one of sodium alginate solution, methacrylic anhydride gelatin solution, carboxymethyl cellulose solution, matrigel solution, sericin solution, collagen solution, fibroin solution, and methacrylated hyaluronic acid.

4. The method according to claim 1, Features: The lower layer solution is a polysaccharide aqueous solution or a protein solution; the concentration of the lower layer solution is 2% to 20%.

5. The method according to claim 1, Features: The lower layer solution is one of a dextran solution, a polysucrose solution, a sucrose solution, an agarose solution, a sericin solution, a collagen solution, and a fibroin solution.

6. The method according to claim 1, Features: The method for solidifying the cell wrapping layer is to drop a solution or perform other solidification operations.

7. The method according to claim 6, Features: The dropwise addition solution is a calcium chloride solution or a ferric chloride solution.

8. The method according to claim 6, Features: The other curing operation is to cure the hydrogel by ultraviolet light or temperature.

9. Single cell microspheres prepared by the method according to any one of claims 1 to 8.

10. The single cell microsphere according to claim 9, Features: The encapsulation rate of the single cell microspheres is above 98%.

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