Single-cell microsphere prepared on basis of double-aqueous-phase oil-free strategy and preparation method therefor

Abstract

Provided are a single-cell microsphere prepared on the basis of a double-aqueous-phase oil-free strategy and a preparation method therefor. Specifically, by means of gravity induction, cells in an upper-layer aqueous phase enter a lower-layer aqueous phase; due to an adhesion effect of the cells and a hydrogel and a stable interface formed by the double aqueous phases, the cells entering the lower-layer aqueous phase become single cells encapsulated by the hydrogel; and the thickness of a gel layer can be changed by changing the concentration of the layered double aqueous phases, such that the single-cell microsphere with the thickness and size controllable is obtained. The method does not involve the use of oil and a surfactant, has mild effects on the cells, can realize undifferentiated encapsulation of different types of cells, and has universality and broad applicability, and an ultrahigh cell encapsulation rate; and compared with other strategies, the method features low device cost and high preparation efficiency, while significantly improving biocompatibility, and can be used for easily preparing an ultrathin hydrogel coating difficult to prepare according to an existing preparation strategy, which can be further applied to the field of biomedicine such as cell treatment.

Description

Single-cell microspheres prepared based on a two-phase oil-free strategy and preparation method thereof 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, thereby promoting the regeneration and repair of tissues or organs. Cells (including stem cells) are generally implanted into the damaged area 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 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, which can easily cause rejection reactions in the body and lead to clearance by the immune system, which greatly limits the effectiveness of cell therapy. Therefore, in order to further improve the effectiveness of treatment, related research on single-cell encapsulation has attracted widespread attention from 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, making them widely used for cell delivery. However, the current single-cell microsphere preparation technology still faces many problems. Most researchers prepare single-cell microspheres using 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 cell expansion, thereby promoting the realization of cell functionality. When the size of the microgel increases to a certain extent, its application is limited, such as high-throughput analysis and affecting its pharmacokinetics after implantation. In addition, studies have shown that intravenously injected single-cell microspheres of ~30μm can accumulate in the lung capillary bed of mice, while microspheres of ~50μm are usually physically trapped and accumulate 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 significantly reduces cell activity and is cumbersome, further limiting its widespread application in the biomedical field.

[0003] Another common encapsulation strategy is chemical modification, which involves modifying the cell surface with exogenous substances. This is typically done by using chemical reactions to embed corresponding groups into the cell membrane. Although current chemically engineered cells show promising application prospects, they still face many challenges. For example, specific chemical reaction conditions may affect cell activity and the thickness is poorly controllable. Secondly, exogenous substances modified on the cell surface are easily internalized by the cells, thereby reducing cell activity and delivery efficiency. In addition, because exogenous substances occupy sites on the cell surface, they may affect the binding of cell surface receptors to specific ligands, thereby limiting the initiation of related signaling pathways and subsequent functions. They may also increase the immunogenicity of the cells, leading to attack and clearance by immune cells. Furthermore, the mechanical properties of the cells may be altered, resulting in reduced cell activity and changes in other physicochemical properties. These and other issues have greatly limited the application of single-cell microspheres in biomedicine and other fields. Therefore, the above limitations have driven the development of single-cell microsphere preparation technology and the transformation to smaller volumes. Encapsulating single cells within a nano- to micro-scale hydrogel layer significantly increases the surface-to-volume ratio, enabling better material exchange, including the transport of nutrients and oxygen, as well as facilitating the excretion of beneficial factors secreted by cells and their corresponding therapeutic effects. Therefore, developing a simple, versatile, and highly biocompatible innovative strategy for single-cell microspheres to enhance their application in biomedical engineering is a scientific issue that needs to be addressed urgently.

[0004] Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] 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.

[0008] 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:

[0009] 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;

[0010] After the cells fall from the upper layer to the lower layer, the cell coating layer is solidified to obtain single-cell microspheres.

[0011] As a preferred embodiment of the method of the present invention, the upper layer solution is a protein solution or a hydrogel solution; and the concentration of the upper layer solution is 0.001% to 2%.

[0012] 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, methacrylated hyaluronic acid, carboxymethyl cellulose solution, matrigel solution, sericin solution, collagen solution, and fibroin solution.

[0013] As a preferred embodiment of the method of the present invention, the lower layer solution is a polysaccharide aqueous solution or a protein solution; and the concentration of the lower layer solution is 2% to 20%.

[0014] As a preferred embodiment of the method of the present invention, the lower layer solution is one of dextran solution, polysucrose solution, sucrose solution, agarose solution, sericin solution, collagen solution, and fibroin solution.

[0015] As a preferred embodiment of the method of the present invention, the method for solidifying the cell wrapping layer is dropwise addition of solution or other solidification operations.

[0016] As a preferred embodiment of the method of the present invention, the dropwise added solution is a calcium chloride solution or a ferric chloride solution.

[0017] 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.

[0018] Another object of the present invention is to overcome the deficiencies in the prior art and provide a single-cell microsphere prepared based on an oil-free two-phase aqueous strategy.

[0019] As a preferred embodiment of the single-cell microspheres of the present invention, the encapsulation efficiency of the single-cell microspheres is above 98%.

[0020] Beneficial effects of the present invention:

[0021] (1) The present invention uses a highly biocompatible aqueous solution, without the oil and surfactant used in traditional microfluidic strategies, which greatly improves the cell survival rate to over 95%. It also reduces the oil removal step, simplifies the experimental operation, and reduces the impact on cell activity.

[0022] (2) The single-cell microspheres prepared by the present invention can not only increase the encapsulation efficiency to more than 98%, but also achieve complete encapsulation;

[0023] (3) The present invention does not involve any special chemical reactions, but relies solely on physical effects to enhance cell activity and achieve indiscriminate encapsulation, thereby resolving issues such as cell surface site occupancy caused by chemical encapsulation, affecting the binding between specific ligands and receptors, and increasing immunogenicity.

[0024] (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.

[0025] (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

[0026] 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. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0027] FIG1 is a schematic diagram of the process for preparing single-cell microspheres based on the oil-free two-phase aqueous strategy of the present invention.

[0028] Figure 2 shows the stratification of different solutions.

[0029] FIG3 shows microspheres encapsulating a single HeLa cell prepared using different solution combinations.

[0030] FIG4 shows single-cell microspheres encapsulating different cells obtained by two-phase stable stratification.

[0031] FIG5 shows single-cell microspheres with different coating thicknesses obtained by adjusting the concentrations of sodium alginate and dextran solutions.

[0032] FIG6 is a fluorescence image of live-dead staining of h1299 cells under the conditions of 0.0125% Alginate and 10% Dextran. DETAILED DESCRIPTION

[0033] 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.

[0034] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. 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.

[0035] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0036] 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).

[0037] Example 1

[0038] A method for preparing single-cell microspheres based on an aqueous two-phase oil-free strategy comprises the following steps:

[0039] (1) Prepare 0.0125% Alginate solution and 10% Dextran solution

[0040] Accurately weigh 12.5 mg of Alginate solid and dissolve it in 100 ml of PBS solution to obtain 0.0125% Alginate;

[0041] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0042] To demonstrate the success of cell encapsulation, fluorescent nanoparticles were added to the Alginate solution to prepare a 0.5% fluorescent nanoparticle solution.

[0043] (2) Preparation of calcium chloride solution

[0044] Prepare a 1% calcium chloride solution by accurately weighing 1g of calcium chloride and dissolving it in 100ml of distilled water.

[0045] (3) Preparation of single-cell microspheres

[0046] Pre-add 500 μL of 10% Dextran solution as the lower layer to a suitable container. Then, resuspend Hela cells in 500 μL of 0.0125% Alginate solution as the upper layer mixed with 0.5% fluorescent nanoparticles. Slowly drip the cell suspension into the container and stably separate it from the lower layer.

[0047] After the cells have fallen from the upper layer to the lower layer, 1% calcium chloride solution is added to solidify the cell coating. The coating is observed under a microscope and the size of the microspheres is recorded.

[0048] The schematic diagram of the process for preparing single-cell microspheres based on the two-phase oil-free strategy is shown in Figure 1.

[0049] Example 2

[0050] A method for preparing single-cell microspheres based on an aqueous two-phase oil-free strategy comprises the following steps:

[0051] (1) Prepare 0.0125% carboxymethyl cellulose (CMC V) solution and 10% Dextran solution

[0052] 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;

[0053] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0054] To characterize the success of cell encapsulation, fluorescent nanoparticles were added to the CMC V solution to prepare a 0.5% fluorescent nanoparticle solution.

[0055] (2) Preparation of ferric chloride solution

[0056] 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.

[0057] (3) Preparation of single-cell microspheres

[0058] 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 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;

[0059] After the cells have fallen from the upper layer to the lower layer, 1% ferric chloride solution is added dropwise to solidify the cell coating. The coating is observed under a microscope and the size of the microspheres is recorded.

[0060] Example 3

[0061] A method for preparing single-cell microspheres based on an aqueous two-phase oil-free strategy comprises the following steps:

[0062] (1) Prepare 0.0125% Matrigel solution and 10% Dextran solution

[0063] Accurately measure 12.5 μl of Matrigel solution and dissolve it in 100 ml of PBS solution to obtain a 0.0125% Matrigel solution;

[0064] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0065] To characterize the success of cell encapsulation, fluorescent nanoparticles were added to the Matrigel solution to prepare a 0.5% fluorescent nanoparticle solution.

[0066] (2) Preparation of single-cell microspheres

[0067] Pre-add 500 μL of 10% Dextran solution as the lower layer to a suitable container. Then, resuspend Hela cells in 500 μL of 0.0125% Matrigel solution as the upper layer mixed with 0.5% fluorescent nanoparticles. Slowly drip the cell suspension into the container and stably layer it with the lower layer.

[0068] After the cells have fallen from the upper layer to the lower layer, they are placed at 37°C to allow the cell coating to solidify. The coating is observed under a microscope and the size of the microspheres is recorded.

[0069] Example 4

[0070] (1) Synthesis of methacrylic anhydride gelatin (GelMA) and preparation of GelMA solution

[0071] Fish skin gelatin was dissolved in phosphate-buffered saline (PBS) at 10% at 50°C, and 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.

[0072] GelMA was diluted twice and dialyzed with deionized water at 40°C for 7 days, with the deionized water changed every 12 hours. The solution was filtered at 40°C using a 0.22 μm sterile vacuum filtration system. 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.

[0073] Accurately weigh 1 g of GelMA solid and dissolve it in 10 ml of PBS solution to obtain a 10% GelMA solution.

[0074] To demonstrate the success of cell encapsulation, GelMA was cured by UV in this experiment. Therefore, a photoinitiator, lithium phenyl (2,4,6-trimethylbenzoyl) phosphate (LAP), was added to the GelMA solution to prepare a GelMA solution containing 0.03% LAP.

[0075] (2) Prepare 10% Dextran solution

[0076] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0077] (3) Preparation of single-cell microspheres

[0078] Pre-add 500 μL of the lower layer solution, Dextran, to a suitable container. Then, resuspend HeLa cells in 500 μL of GelMA solution containing 0.03% LAP. Slowly drip the cell suspension into the container, allowing it to form a stable layer with the lower layer solution. After the cells fall from the upper layer to the lower layer, irradiate with a UV lamp (Sanger X9) for 30 seconds to solidify the cell coating. Observe the coating under a microscope and record the size of the microspheres.

[0079] Example 5

[0080] (1) Synthesis of Sericin and Preparation of Sericin Solution

[0081] Cut the cocoons into small pieces, wash them three times with pure water, dry them, and weigh 50g. Add 2.12g of sodium carbonate (0.02M) to 1L of pure water and heat to boiling. Place the cocoons in the boiling water and cook for 30 minutes, splitting the cocoons at regular intervals. After cooling to room temperature, remove the silk and centrifuge the remaining solution at 3500 rpm for 8 minutes at 4°C to remove insoluble impurities.

[0082] 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 3500 rpm for 15 minutes at 4°C to remove precipitated impurities. Half of the solution was placed in the dialysis bag and blown dry with an electric fan in a fume hood until the desired concentration was achieved. 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.

[0083] 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.

[0084] (2) Prepare 10% Dextran solution

[0085] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran.

[0086] (3) Preparation of single-cell microspheres

[0087] Pre-add 500 μL of 10% Dextran solution (lower layer) to a suitable container. Then, resuspend Hela cells with 500 μL of 10% Sericin solution (upper layer) mixed with 0.5% fluorescent nanoparticles. Slowly drip the cell suspension into the container and stably separate it from the lower layer.

[0088] After the cells fall from the upper layer to the lower layer, the light intensity is 60mW 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.

[0089] The stratification of 0.0125% Alginate solution, CMC V solution, and Matrigel solution prepared in Examples 1 to 5 with 10% Dextran solution, and the stratification of 10% GelMA solution and Sericin solution with 10% Dextran solution, respectively, are shown in FIG2 . It can be seen that all of them are stably stratified.

[0090] Example 6

[0091] A method for preparing single-cell microspheres based on an aqueous two-phase oil-free strategy comprises the following steps:

[0092] (1) Prepare 0.0125% methacryloyl hyaluronic acid (HAMA) solution and 10% Dextran solution

[0093] Accurately weigh 12.5 mg of HAMA solution and dissolve it in 100 ml of PBS solution to obtain a 0.0125% HAMA solution;

[0094] Accurately weigh 10 g of Dextran solid and dissolve it in 100 ml of PBS solution to obtain 10% Dextran;

[0095] To characterize the success of cell encapsulation, fluorescent nanoparticles were added to the Matrigel solution to prepare a 0.5% fluorescent nanoparticle solution.

[0096] (4) Preparation of single-cell microspheres

[0097] Pre-add 500 μL of 10% Dextran solution as the lower layer to a suitable container. Then, resuspend Hela cells with 500 μL of 0.0125% HAMA solution as the upper layer mixed with 0.5% fluorescent nanoparticles. Slowly drip the cell suspension into the container and stably separate it from the lower layer.

[0098] After the cells fall from the upper layer to the lower layer, they are irradiated with a UV lamp (Sangerfei X9) for 30 seconds to solidify the cell coating. The coating is observed under a microscope and the size of the microspheres is recorded.

[0099] The microspheres encapsulating a single HeLa cell prepared by combining Alginate solution, CMC V solution, Matrigel solution, GelMA solution, Sericin solution, HAMA solution with Dextran solution in Examples 1 to 6 are shown in FIG3 . It can be seen that the cell encapsulation is complete and has a high single cell encapsulation efficiency.

[0100] Example 7

[0101] The difference between this embodiment and Example 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 Example 1.

[0102] Figure 4 shows single-cell microspheres encapsulating different cells obtained by two-phase stable stratification. It can be seen that different types of cells can be encapsulated.

[0103] Example 8

[0104] 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. The remaining steps are the same as those in embodiment 1.

[0105] Example 9

[0106] 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. The remaining steps are the same as those in embodiment 1.

[0107] FIG5 shows single-cell microspheres with different coating thicknesses obtained by adjusting the concentrations of Alginate and Dextran solutions in Examples 8 and 9. It can be seen that after experiments with different concentrations, the thickness of the gel layer can be controlled to be below 5 μm.

[0108] FIG6 is a fluorescence image of live-dead staining 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%.

[0109] Comparative Example 1

[0110] 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 Example 1. The results show that the cells remain in the upper layer of the solution and cannot fall to the lower layer.

[0111] Comparative Example 2

[0112] The difference between this example and Example 1 is that the concentration of the prepared dextran solution is 1%, and the remaining steps are the same as Example 1. The results show that low concentrations of Alginate and Dextran are difficult to form a stable interface and cells cannot be encapsulated.

[0113] Comparative Example 3

[0114] The difference between this example and Example 1 is that the Alginate solution is changed to 2% polyvinyl alcohol (PVA), and the remaining steps are the same as Example 1. The results show that the cells cannot be encapsulated.

[0115] Comparative Example 4

[0116] 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 in embodiment 4. The results show that the stratification is unstable and the cells cannot be encapsulated.

[0117] Comparative Example 5

[0118] The difference between this embodiment and embodiment 1 is that the single-cell microspheres are prepared using traditional microfluidic technology. The specific steps are as follows:

[0119] The cells were mixed with 0.0125% Alginate solution as the dispersed phase and injected into the microfluidic chip;

[0120] Then, fluorinated oil (fluorinated FC-40, Sigma Aldrich) (RAN BioTechnologies) containing 2% w / w fluorinated surfactant was used as the continuous phase;

[0121] The hydrogel solution was sheared using an oil phase to obtain single-cell microdroplets, which were then collected in a 1% calcium chloride solution to form single-cell microspheres.

[0122] The results showed that the single-cell microsphere encapsulation efficiency obtained by microfluidic technology was 40%, and the cell survival rate was 75%, both of which were far lower than the two-phase oil-free strategy proposed in the present invention.

[0123] The present invention uses an aqueous solution with high biocompatibility and does not involve the 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.

[0124] The present invention does not involve any special chemical reactions, relying solely on physical interactions to enhance cell activity and achieve indiscriminate encapsulation, thereby addressing issues such as chemical encapsulation-induced cell surface site occupancy, the impact on binding between specific ligands and receptors, and increased immunogenicity. By varying the solution concentration, the present invention can control the encapsulation thickness, enabling the preparation of ultrathin (1μm to 5μm) hydrogel single-cell microspheres that are difficult to achieve using traditional microfluidic strategies. The proposed preparation method, based on an oil-free aqueous two-phase strategy, offers excellent high throughput, biocompatibility, thickness controllability, and ease of operation.

[0125] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit 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 solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: 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, characterized in that: The dropwise addition solution is a calcium chloride solution or a ferric chloride solution.

8. The method according to claim 6, characterized in that: 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, characterized in that: The encapsulation rate of the single cell microspheres is above 98%.

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