Large-size high-activity cell spheres and a preparation method thereof

Abstract

The application belongs to the technical field of biological medicine engineering, and particularly relates to a large-size high-activity cell ball and a preparation method thereof. The method first utilizes an ATPS system and osmotic pressure to pre-agglomerate cells into microsphere modules, and then utilizes osmotic pressure reduction to induce inter-module interface bio-orthogonal reaction, so that millimeter-level tissues are rapidly constructed within 2 hours. The cell ball (200-1000 µm) prepared by the method has an internal live cell proportion of greater than 90%, and effectively overcomes the diffusion limit bottleneck in the preparation of traditional large balls. qPCR verification shows that the AML12 assembly has an expression of key functional genes such as albumin, CYP1A2 and TDO2 increased by dozens of times compared with a 2D group, proving that the preparation process has a significant advantage in maintaining the biological activity and function of large-size tissues.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a large-sized, highly active cell sphere and its preparation method. Background Technology

[0002] In regenerative medicine and in vitro pathological model research, three-dimensional (3D) cell spheroids serve as the core unit for simulating in vivo tissue structure and function, possessing irreplaceable value. Compared to traditional two-dimensional (2D) monolayer adherent culture, 3D cell spheroids can provide more realistic cell-cell communication, cell-matrix interactions, and nutrient and signal gradients similar to those in vivo. However, as tissue engineering advances towards large-scale, organ-level operations, the preparation of cell spheroids with diameters exceeding 200 µm while maintaining high bioactivity has become a key bottleneck restricting the development of this field.

[0003] Existing 3D cell culture techniques, such as the hanging drop method, low-attachment surface method, and roller bottle method, mainly rely on spontaneous aggregation mediated by adhesion molecules (such as cadherins) on the cell surface. These methods perform well in preparing small cell spheroids (diameter <200µm), but face the following serious limitations when constructing large-sized tissues: First, there's the issue of the "necrotic core" caused by the metabolic diffusion limit. According to biophysical modeling studies, the effective diffusion depth of oxygen, nutrients, and metabolic waste in dense cell layers is typically limited to 150-200 µm. In traditional methods of preparing large spheres, the cell aggregation process is slow (usually requiring 3-7 days). During this period, cells in the central region of the sphere, due to prolonged hypoxia and nutrient deprivation, undergo irreversible necrosis or apoptosis, forming the so-called "necrotic core." This not only weakens the bioactivity of the implant but may also trigger an inflammatory response, affecting transplantation outcomes.

[0004] Secondly, there is a contradiction between aggregation efficiency and structural stability. Traditional spontaneous aggregation processes are time-consuming, and the resulting tissue structures are loose with low mechanical strength, making them unable to withstand physical compression from the implantation site (such as the spleen or liver parenchyma). Although adding scaffold materials can enhance strength, the introduction of scaffolds often dilutes cell density, interferes with direct cell-to-cell contact, and may trigger foreign body reactions.

[0005] Furthermore, there is the complexity of cross-scale construction. Current bioprinting or scaffold technologies still face challenges in achieving precise control of tissues at the millimeter level, including high costs, complex equipment, and small throughput. Bottom-up assembly using micro-modules is considered an effective approach to achieving large-scale tissue construction, but an ideal chemically driven solution is still lacking for achieving rapid, gentle, and robust connections at the module interfaces.

[0006] Click chemistry, particularly copper-free catalyzed tensiylene-azide cycloaddition reactions (SPAAC), offers novel approaches to cell assembly due to its extremely high reaction selectivity, rapid kinetics under physiological conditions, and good biocompatibility. Introducing click chemical tags onto cell membranes can achieve directional, covalent connections between cells. However, how to combine click chemistry with macroscopic physical driving forces (such as osmotic pressure) to achieve the transformation from microsphere modules to large-scale compact tissues in an extremely short time (<2 hours) has not yet been systematically reported in patents. Summary of the Invention

[0007] The core objective of this invention is to provide a method for preparing large-sized, highly active cell spheres based on the concept of "rapid assembly of microsphere modules." This method, through a multi-level assembly strategy, decomposes the cell sphere construction process into two stages: "micro-module prefabrication" and "macro-compaction coupling," effectively bypassing the hypoxic damage caused by long-term, slow aggregation.

[0008] The technical solution and core mechanism of this invention are as follows: First, a high-density encapsulation of micromodules is achieved using an aqueous two-phase system (ATPS). ATPS is formed by mixing two thermodynamically incompatible polymers (such as dextran and polyethylene oxide (PEO)) in water. When the concentration exceeds a critical point, the system spontaneously separates into phases. Because cells have a better affinity for the dextran phase, a high-cell-density droplet microenvironment can be generated by suspending cells in the dextran phase (dispersed phase) and pumping them into the PEO phase (continuous phase).

[0009] Second, the invention utilizes the osmotic compression generated by a high concentration of polymer. In this invention, by adjusting the concentration of PEO in the outer phase, an extremely high osmotic pressure difference is generated, driving the rapid expulsion of water from the inner phase droplets. This physical pressure forces the cells within the droplets to complete a dense arrangement within hours, forming microsphere modules.

[0010] Third, bioorthogonal membrane anchoring click chemistry is introduced to achieve covalent coupling between modules. Chol-PEG-DBCO and Chol-PEG-N3 molecules are inserted into the cell membrane using the hydrophobic interaction of cholesterol. When the microsphere modules with complementary groups come into close contact under osmotic pressure, a spontaneous SPAAC reaction occurs at the interface, generating stable 1,2,3-triazole covalent bonds, thereby locking the microsphere modules into macroscopic large-scale spheres.

[0011] To achieve the above objectives, this application adopts the following technical solution: A first aspect of the present invention provides a method for preparing three-dimensional cell aggregates, comprising: 1) Primary microsphere modules were prepared by placing cells in an aqueous two-phase system; 2) The primary microsphere module is divided into two parts, each modified with complementary bioorthogonal reactive groups; 3) Mix the two primary microsphere modules, which are respectively modified with complementary bioorthogonal reactive groups, and place them in an aqueous two-phase system to form a secondary droplet containing multiple primary microsphere modules; 4) The secondary droplets are physically compressed so that the two primary microsphere modules come into contact with each other and undergo a biological orthogonal reaction to obtain the three-dimensional cell aggregate.

[0012] In some embodiments of the present invention, in step 1), the concentration of the cells in the aqueous two-phase system is 1 × 10⁻⁶. 7 ~1×10 9 per mL.

[0013] In some embodiments of the present invention, in step 1), the aqueous two-phase system includes a first aqueous polymer and a second aqueous polymer.

[0014] In some embodiments of the present invention, the first aqueous polymer is dextran.

[0015] In some embodiments of the present invention, the second aqueous polymer is polyethylene oxide.

[0016] In some embodiments of the present invention, the dextran has a mass fraction of 10wt%-15wt% and a molecular weight of 400-600 kDa.

[0017] In some embodiments of the present invention, the polyethylene oxide has a mass fraction of 4wt%-7wt% and a molecular weight of 100-300 kDa.

[0018] In step 1), the primary microsphere module is prepared using an aqueous two-phase system (ATPS). ATPS is formed by mixing two thermodynamically incompatible polymers (dextran and polyethylene oxide (PEO)) in water. When the concentration exceeds a critical point, the system spontaneously separates into phases. Since cells have a better affinity for the dextran phase, a high-cell-density droplet microenvironment can be generated by suspending cells in the dextran phase (dispersed phase) and pumping them into the PEO phase (continuous phase).

[0019] In some embodiments of the present invention, in step 2), the complementary bioorthogonal reactive groups include a group of azides and tensilides, tetrazides and tensilides.

[0020] In some embodiments of the present invention, the complementary bioorthogonal reactive groups include azides and tensilylene.

[0021] In some embodiments of the present invention, the complementary bioorthogonal reactive groups include cholesterol-polyethylene glycol-dibenzocyclooctylene (Chol-PEG-DBCO) and cholesterol-polyethylene glycol-azide (Chol-PEG-N3).

[0022] In some embodiments of the present invention, the specific steps of the modification include: Incubate with primary microsphere modules using Chol-PEG-DBCO or Chol-PEG-N3.

[0023] In some embodiments of the present invention, the concentration of Chol-PEG-DBCO or Chol-PEG-N3 is 5-20 μM.

[0024] In some embodiments of the present invention, the incubation temperature is 35~40°C.

[0025] In some embodiments of the present invention, the incubation time is 5 to 30 minutes.

[0026] In step 2), the reactive groups are precisely positioned on the cell membrane surface by anchoring lipid molecules through a membrane, ensuring that the cross-linking reaction occurs only at the cell's contact interface without interfering with the physiological and biochemical processes inside the cell. The use of copper-free bioorthogonal click chemistry avoids the toxicity of copper catalysts to cells while ensuring high selectivity and rapid kinetics under physiological conditions. This allows the rate of covalent bond formation to match the rate of physical compression densification, achieving simultaneous locking of structure and function.

[0027] In some embodiments of the present invention, in step 3), the ratio of the number of the two complementary microsphere units is 1:(0.5~2).

[0028] In step 3), the primary microsphere module has already inserted DBCO and N3 molecules into the cell membrane through the hydrophobic interaction of cholesterol. When the microsphere modules with complementary groups come into close contact under osmotic pressure, a SPAAC reaction spontaneously occurs at the interface, generating stable 1,2,3-triazole covalent bonds, thereby locking the primary microsphere module into a larger macroscopic secondary microsphere unit.

[0029] In some embodiments of the present invention, in step 4), the physical compression includes osmotic pressure compression.

[0030] In some embodiments of the present invention, the osmotic pressure compression specifically refers to: This invention utilizes the osmotic compression generated by a high concentration of polymer. By adjusting the concentration of the second aqueous polymer, PEO, an extremely high osmotic pressure difference is generated, driving the rapid expulsion of water from the inner phase droplet. This physical pressure forces the cells within the droplet to complete a dense arrangement within several hours, forming a three-dimensional cell aggregate.

[0031] In some embodiments of the present invention, the concentration of the second aqueous polymer PEO is increased to 10-15 wt%.

[0032] In a second aspect, the present invention provides a three-dimensional cell aggregate prepared by the preparation method described in the first aspect of the present invention.

[0033] In some embodiments of the present invention, the diameter of the three-dimensional cell aggregate is 200-1000 µm.

[0034] A third aspect of the present invention provides a method for regulating the size of three-dimensional cell aggregates, comprising preparing the aggregates using the preparation method described in the first aspect of the present invention, wherein the size of the three-dimensional cell aggregates is regulated by adjusting the volume fraction of the primary microsphere module in the aqueous two-phase system described in step 3).

[0035] In some embodiments of the present invention, φ is used sph This represents the volume fraction of the primary microsphere module in the aqueous two-phase system.

[0036] In some embodiments of the present invention, when φ sph When φ is 0.10, the equivalent diameter of the obtained three-dimensional cell aggregates is mainly 200–400 µm; when φ sph When φ is 0.20, the equivalent diameter of the obtained three-dimensional cell aggregates is mainly 400–600 µm; when φ sph When the value is 0.30, the equivalent diameter of the obtained three-dimensional cell aggregates is mainly 600-900 µm.

[0037] Beneficial effects: This invention constructs a novel paradigm for the assembly of three-dimensional cell aggregates across scales and driven by a dual approach: the introduction of physical compression force driven by osmotic pressure difference and the chemical locking force of membrane-anchored bioorthogonal reactions. First, utilizing the osmotic pressure difference generated by the polymer medium as a macroscopic physical engine, it can forcibly drive the dehydration and densification of cell units or primary modules within hours, compressing the traditional spontaneous aggregation process, which typically takes several days, into an extremely short time window. This fundamentally cuts off the path of necrosis of cells in the central region due to prolonged hypoxia. Second, the covalent bond network formed at the cell interface by membrane-anchored bioorthogonal groups provides permanent microscopic support for the rapidly densified structure, solving the problem of easy rebound and loosening of the structure after the removal of simple physical compression, and endowing large-sized aggregates with excellent mechanical stability. Finally, this rapid assembly strategy preserves the initial viability and metabolic potential of cells to the greatest extent, resulting in a cell survival rate of over 90% and no necrotic core in the prepared three-dimensional cell aggregates with diameters ranging from 200 μm to 1000 μm. It also shows significantly better physiological relevance than traditional culture systems in key indicators such as liver function gene expression, providing reliable technical support for large-scale tissue engineering construction and high-fidelity in vitro drug screening models. Attached Figure Description

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 This is a schematic diagram of the modular preparation process of the large-sized cell aggregates of the present invention, including the preparation of primary microsphere modules, surface modification of bioorthogonal reactive groups, and secondary assembly of primary microsphere modules.

[0039] Figure 2 The NMR spectra of Chol-PEG-DBCO and its precursor Chol-PEG-NH2 are shown in the form of proton NMR spectra.

[0040] Figure 3 Fourier transform infrared spectra of Chol-PEG-N3 and its precursor materials.

[0041] Figure 4 The image shows the microstructure of a primary microsphere module prepared using a two-phase aqueous system.

[0042] Figure 5 The images show fluorescence imaging of the primary microsphere modules after modification with Chol-PEG-DBCO or Chol-PEG-N3.

[0043] Figure 6 Bright-field and fluorescence imaging images of two types of primary microsphere modules modified with Chol-PEG-DBCO and Chol-PEG-N3, respectively, after being mixed and formed into a secondary assembly.

[0044] Figure 7 The images show the microstructures of secondary assemblies formed under different volume fractions of primary microsphere modules, where the volume fractions of primary microsphere modules are 0.10, 0.20, and 0.30, respectively.

[0045] Figure 8 Histological images of the secondary assembly after paraffin embedding, sectioning, and hematoxylin-eosin staining.

[0046] Figure 9 This figure shows the results of detecting the relative expression levels of hepatocyte function-related genes in the two-dimensional culture group and the three-dimensional assembly group. Detailed Implementation

[0047] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0048] In tissue engineering, the effective diffusion distance of oxygen L It can be estimated using the following simplified formula:

[0049] in D The diffusion coefficient of oxygen in tissues (approximately 2 × 10⁻⁶) -5 cm 2 / s), C 0 represents the dissolved oxygen concentration in the medium. Q This refers to the cellular oxygen consumption rate. Under normal conditions, the effective diffusion distance for high-density hepatocyte or tumor cell clusters is approximately 150-200 µm. Traditional preparation methods, such as the hanging drop method, require several days of spontaneous settling and aggregation of cells to form spheres larger than 500 µm. During these days, the initially formed outer dense layer rapidly hinders oxygen entry, leading to necrosis of the incompletely assembled internal cells. The "modular" strategy of this invention involves: first preparing microsphere modules with a diameter of approximately 100 µm, a size far below the metabolic limit, ensuring initial activity within the module; then utilizing the extremely fast kinetics of bioorthogonal chemistry (second-order reaction rate constants are typically 0.1-1.0 M). -1 s -1Within two hours, these highly active modules are "locked" into a tight, large sphere. Due to the extremely rapid assembly speed, the cells have already established a certain degree of stress resistance before being "buried" inside the large sphere, and the micro-interstitial spaces within the assembly still allow for some fluid exchange in the early stages until the cells are finally rearranged.

[0050] The specific experimental process of this invention is shown in the following embodiments.

[0051] Example 1: Synthesis and Identification of Bioorthogonal Membrane Anchoring Molecules The key chemical reagents required for this invention, Chol-PEG-DBCO and Chol-PEG-N3, are synthesized through the following steps: 1. Preparation of Chol-PEG-DBCO Weigh out 100 mg of cholesterol-polyethylene glycol-amino (Chol-PEG) 3400 -NH2 (approximately 0.0294 mmol) was dissolved in 5 mL of anhydrous DMF, and DBCO-COOH, EDC, and NHS were added sequentially, with Chol-PEG present. 3400 The molar ratio of -NH2, DBCO-COOH, EDC, and NHS was 1:1.2:1.5:1.5. The reaction was carried out at room temperature in the dark for 12 hours. After the reaction was completed, pre-cooled diethyl ether was added to precipitate the product. The precipitation was repeated three times, and the product was dialyzed in deionized water for 48 hours to remove unreacted small molecules. Then, it was freeze-dried under vacuum to obtain a white powder of Chol-PEG-DBCO.

[0052] 2. Evaluation Result: Passed 1 H NMR characterization ( Figure 2 A significant characteristic aromatic proton peak of DBCO was observed at δ 7.2–7.7 ppm, and its integral ratio with the cholesterol skeletal signal was as expected.

[0053] First, cholesterol-polyethylene glycol-amino (Chol-PEG) 3400 Bromoacetylation was performed using bromoacetic acid (-NH2). 12.3 mg bromoacetic acid (0.0882 mmol), 33.8 mg EDC (0.176 mmol), and 30.5 mg NHS (0.265 mmol) were added to 2 mL of dichloromethane, with a molar ratio of bromoacetic acid, EDC, and NHS of 1:2:3. Activation was performed at room temperature for 30 minutes. Subsequently, 100 mg of Chol-PEG was added. 3400 -NH2 (0.0294 mmol), enabling Chol-PEG 3400 The molar ratio of -NH2 to bromoacetic acid was 1:3, and the reaction was carried out at room temperature for 24 hours. After the reaction was completed, pre-cooled diethyl ether was added to precipitate the product. The precipitation was repeated 3 times to obtain the crude Chol-PEG-Br product.

[0054] The obtained crude Chol-PEG-Br product was dissolved in 2 mL of DMF, and 45.9 mg of sodium azide (0.706 mmol) was added. The molar ratio of sodium azide to bromoacetic acid was 8:1. The reaction was carried out overnight at 60 °C. After the reaction was completed, undissolved sodium azide was removed by centrifugation or filtration. The supernatant was dialyzed against deionized water for 48 hours and then lyophilized to obtain Chol-PEG-N3.

[0055] 4. Identification Results: FTIR spectroscopy showed that the obtained Chol-PEG-N3 was within the range of 2100–2130 cm⁻¹. - The characteristic absorption peak of the azide group appears near ¹, while no corresponding characteristic peak is observed in the precursor Chol-PEG-NH2 at this position, indicating that the azide group was successfully introduced. Figure 3 ).

[0056] Example 2: Preparation of high-density microsphere modules based on ATPS To obtain high-quality assembled modules, the uniformity of the microsphere modules is crucial.

[0057] 1. Preparation of ATPS An equilibrium aqueous system containing 12 wt% dextran (500 kDa) and 5.5 wt% polyethylene oxide (PEO, 200 kDa) was prepared. This system utilizes the steric hindrance effect between dextran and PEO to form a stable two-phase system under physiological pH conditions.

[0058] 2. Cell inoculation AML12 mouse hepatocytes were collected and resuspended in 12 wt% Dextran phase solution to achieve a final cell concentration of 1 × 10⁻⁶ cells in the resulting cell-Dextran suspension. 7 Cells / mL. Subsequently, this cell-Dextran suspension was used as the dispersion phase for the preparation of primary droplets and primary microsphere modules.

[0059] 3. Microsphere formation Using a 1 mL precision syringe, a cell-containing Dextran solution was pumped into a 5.5 wt% PEO continuous phase at a flow rate of 0.5 mL / min to generate droplets.

[0060] 4. Osmotic compression The generated droplets were collected and placed in a 14 wt% PEO solution. Due to the extremely high osmotic pressure of 14 wt% PEO, water was rapidly drawn out of the droplets, causing the cells to collapse and aggregate tightly within 8 hours, ultimately forming compact microspheres with a diameter of 100 ± 10 µm. Figure 4 Due to the protective effect of ATPS, this process causes minimal damage to the cell membrane.

[0061] Example 3: Cross-scale assembly of large-scale cell spheres 1. Surface marking The microsphere modules from Example 2 were collected and incubated for 10 minutes in physiological buffer (such as PBS) containing 10 µM Chol-PEG-DBCO or Chol-PEG-N3. Cholesterol-anchored molecules spontaneously inserted into the cell membrane on the microsphere surface within 10 minutes. Confocal microscopy confirmed that the microsphere surface exhibited a strong circumferential fluorescence distribution. Figure 5 ).

[0062] 2. Mixing and Secondary Encapsulation: Two types of primary microsphere modules modified with Chol-PEG-DBCO and Chol-PEG-N3 were mixed in a 1:1 ratio and suspended in a 12 wt% Dextran dispersion. The microsphere module volume fraction φ was defined as the ratio of the total volume of the microsphere module to the total volume of the microsphere-Dextran suspension. sph In this embodiment, the volume fraction of the microsphere modules is 0.10–0.30, preferably 0.20. A volume fraction of 0.20 indicates that the total volume of the primary microsphere modules accounts for 20% of the total volume of the microsphere-Dextran suspension. Subsequently, the microsphere-Dextran suspension is pumped into a 5.5 wt% PEO continuous phase to form secondary droplets containing multiple primary microsphere modules.

[0063] 3. Covalent-driven compaction: A 14wt% PEO solution is added to the external continuous phase. During this stage, strong osmotic compressive forces the microsphere modules within the secondary droplets to squeeze against each other, rapidly eliminating gaps. Once the DBCO-containing microspheres come into physical contact with the azide-containing microspheres, the SPAAC reaction occurs immediately, establishing an irreversible covalent bond at the interface.

[0064] 4. Formation sequence: After 2 hours, a large, compact ball with a complete shape and smooth surface was formed. Figure 6 This speed is nearly 40 times faster than the traditional self-aggregation method.

[0065] Example 4 Performance evaluation and bioactivity of large-scale cell spheroids This embodiment focuses on the impact of modular assembly technology on the physical scale and bioactivity of large-sized cell spheroids: 1. Precise control of size distribution (see...) Figure 7 ): With an equivalent diameter of 100±10 µm for the primary microsphere module, a 1:1 ratio of the two types of labeled microspheres, and consistent secondary droplet generation parameters and osmotic compression conditions, the size of the final assembly can be adjusted by regulating the volume fraction of the primary microsphere module in the Dextran dispersion phase. The volume fraction of the primary microsphere module refers to the ratio of the total volume of the primary microsphere module to the total volume of the microsphere module and the Dextran dispersion phase. When the volume fraction of the primary microsphere module is 0.10, 0.20, and 0.30, the ratio of the total volume of the primary microsphere module to the volume of the Dextran dispersion phase is 1:9, 1:4, and 3:7, respectively. For example, when the total volume of the microsphere-Dextran suspension is 1 mL, the total volume of the primary microsphere module is 0.10 mL, 0.20 mL, and 0.30 mL, respectively, and the corresponding volumes of the Dextran dispersion phase are 0.90 mL, 0.80 mL, and 0.70 mL, respectively. When the volume fraction of the primary microsphere module is 0.10, the equivalent diameter of the resulting assembly is mainly 200–400 µm; when the volume fraction is 0.20, the equivalent diameter is mainly 400–600 µm; and when the volume fraction is 0.30, the equivalent diameter is mainly 600–900 µm. This indicates that, while keeping other assembly conditions constant, increasing the volume fraction of the primary microsphere module can increase the size of the final assembly.

[0066] 2. Histological sections (HE staining): Histological sections of cell spheres of different sizes formed by secondary assembly were prepared and stained with HE. HE staining results showed that the internal tissue was evenly distributed, and good material integration had been achieved between the microsphere modules. No obvious nuclear condensation or tissue voids were found in the central region of the spheres, indicating that the central cells successfully maintained metabolic activity and were not damaged by hypoxia during the rapid assembly process of 2 hours. Figure 8 ).

[0067] 3. Gene Expression Analysis (qPCR): To assess the functional maturity of cells after 3D assembly, key liver function-related genes were detected. Compared with the traditional 2D culture group, the significant upregulation of albumin and transthyretin demonstrated that the assembly had a very strong protein synthesis and metabolism level; the 40-fold upregulation of cytochrome P4501A2 indicated that this 3D model had higher physiological relevance in drug metabolism evaluation; the expression level of tryptophan 2,3-dioxygenase (TDO2), a liver-specific amino acid metabolism enzyme, was increased by approximately 72-fold; in addition, the relative expression levels of Gpt and Got were increased, indicating that the three-dimensional assembly environment has a promoting effect on the amino acid metabolism-related phenotype of AML12 cells.

[0068] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for preparing three-dimensional cell aggregates, comprising the following steps: 1) Primary microsphere modules were prepared by placing cells in an aqueous two-phase system; 2) The primary microsphere module is divided into two parts, each modified with complementary bioorthogonal reactive groups; 3) Mix the two primary microsphere modules, which are respectively modified with complementary bioorthogonal reactive groups, and place them in an aqueous two-phase system to form a secondary droplet containing multiple primary microsphere modules; 4) The secondary droplets are physically compressed so that the two primary microsphere modules come into contact with each other and undergo a biological orthogonal reaction to obtain the three-dimensional cell aggregate.

2. The preparation method according to claim 1, characterized in that: In step 1), the concentration of the cells in the aqueous two-phase system is 1×10⁻⁶. 7 ~1×10 9 per mL.

3. The preparation method according to claim 1, characterized in that: In step 1), the aqueous two-phase system includes a first aqueous polymer and a second aqueous polymer; The first aqueous polymer is dextran; The second aqueous polymer is polyethylene oxide.

4. The preparation method according to claim 3, characterized in that: The dextran has a mass fraction of 10wt%-15wt% and a molecular weight of 400-600 kDa. The polyethylene oxide has a mass fraction of 4wt%-7wt% and a molecular weight of 100-300 kDa.

5. The preparation method according to claim 1, characterized in that: In step 2), the complementary bioorthogonal reactive groups include a group of azides and tensilides, tetrazides and tensilides.

6. The preparation method according to claim 1, characterized in that: In step 3), the ratio of the number of the two complementary primary microsphere modules is 1:(0.5~2).

7. The preparation method according to claim 1, characterized in that: In step 4), the physical compression includes osmotic compression.

8. A three-dimensional cell aggregate prepared by the preparation method according to any one of claims 1 to 7.

9. The three-dimensional cell aggregate according to claim 8, characterized in that: The diameter of the three-dimensional cell aggregates is 200-1000 µm.

10. A method for controlling the size of three-dimensional cell aggregates, comprising preparing them using the preparation method according to any one of claims 1 to 7, wherein, By adjusting the volume fraction of the primary microsphere module described in step 3) in the aqueous two-phase system, the size of the three-dimensional cell aggregate can be controlled.

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