Method for preparing cell-containing hydrogel microspheres and preparation device

Abstract

The application discloses a preparation method and a preparation device of cell-containing hydrogel microspheres, and belongs to the field of gel material preparation, and comprises the following steps: S1, constructing a double-layer liquid phase reaction system; S2, assembling a printing tube and a nozzle; S3, preparing hydrogel microspheres; S4, cross-linking the hydrogel microspheres after sedimentation; and S5, collecting the hydrogel microspheres. When the cell-containing hydrogel microspheres are prepared, the mixing system of cells and hydrogels is only processed under mild conditions; after forming, the structure is stabilized through ion cross-linking, so that the damage of heat, light and mechanical stress to the cells can be reduced, and the device required by the method has a simple structure, low production cost and good economy. The cell-containing hydrogel microspheres prepared by the method can be applied to multiple fields such as tissue engineering, organoid construction, cell therapy carriers, drug evaluation three-dimensional cell models and biological printing basic units, and have both scientific research value and industrial application potential.

Description

Technical Field

[0001] This invention belongs to the field of gel material preparation, and particularly relates to a method and apparatus for preparing cell-containing hydrogel microspheres. Background Technology

[0002] In recent years, the fields of biology, chemistry, medicine, cosmetics, food, and functional materials have all been developing various technologies based on micro-sized hydrogel particles. These hydrogel microspheres can be applied to multiple fields such as drug carriers, cell culture platforms, functional materials, and encapsulation systems, among which particle size uniformity and morphological stability are the core key indicators.

[0003] Furthermore, the demand for three-dimensional cell culture carriers has increased significantly in the fields of tissue engineering, regenerative medicine, organoid preparation, and bioprinting. Cell-loaded hydrogel microspheres, in particular, can induce intercellular and cell-matrix interactions, and compared to traditional two-dimensional culture systems, they can better simulate the in vivo microenvironment. Therefore, the demand for technologies to mass-produce cell-loaded microspheres with uniform particle size continues to grow.

[0004] The currently known mainstream methods for preparing hydrogel microspheres can be categorized as follows: The first method is microfluidic chip fabrication. This method can generate droplets within microchannels to prepare particles with relatively uniform particle size, but the equipment structure is complex and maintenance is difficult; the microfluidic chip fabrication process is cumbersome, the purification process after using the oil phase is complicated, and the equipment processing and operating costs are high. At the same time, the high complexity of the equipment makes it difficult to adapt to conventional bioprinting systems; for viscoelastic hydrogels containing cells, the flow rate is difficult to control stably, making long-term mass production impossible.

[0005] The second method is inkjet / ultrasonic jetting. This method can generate tiny droplets and has a fast jetting speed, but it is difficult to adapt to high-viscosity hydrogels and has poor process stability. Cell-loaded viscoelastic hydrogels are limited by viscosity and cannot be jetted stably, easily leading to nozzle clogging. Furthermore, this method makes it difficult for droplets to directly contact the curing liquid, and droplets formed in the air are prone to particle deformation upon contact with the curing liquid surface.

[0006] The third method is the oil-phase emulsification method / simple droplet method. This method can be achieved with only simple equipment, but the particle size distribution is wide and the size controllability is poor; the shape of the droplets is unstable after formation, the formation and cross-linking processes are not separated, and the particles will deform and agglomerate due to pressure and interface differences when the droplets come into contact with the cross-linking solution, making it difficult to ensure uniform particle size.

[0007] Existing hydrogel microsphere preparation technologies have the following drawbacks: First, there is a structural defect where molding and cross-linking occur simultaneously. In most technologies, the hydrogel solution solidifies immediately upon contact with the cross-linking liquid, and the droplets deform and collapse before forming a stable spherical shape.

[0008] Second, it is difficult to guarantee uniform particle size. The droplet formation process of simple dropping and emulsification methods is easily affected by external stirring and viscosity fluctuations, resulting in a wide particle size distribution range.

[0009] Third, the process controllability is poor. Existing technologies make it difficult to precisely control process parameters such as pressure, time, and position, resulting in poor reproducibility of repeated production under the same conditions.

[0010] Fourth, the range of applicable materials is limited. Some technologies are only suitable for specific photocurable materials or hydrogels within a specific viscosity range, and cannot be extended to a variety of material systems.

[0011] Fifth, the process is complex and the cost is relatively high. The processing and maintenance costs of microfluidic equipment are high; the post-processing of emulsification is cumbersome.

[0012] In summary, existing technologies have significant room for improvement in terms of morphological stability, particle size uniformity, production reproducibility, process simplicity, and material versatility. Summary of the Invention

[0013] To address the aforementioned issues, this invention provides a method and apparatus for preparing cell-containing hydrogel microspheres. Employing a novel microfluidic technology, even using the most basic and inexpensive sodium alginate-gelatin hydrogel, calcium chloride crosslinking curing solution, and traditional pneumatic extrusion preparation methods, micron-sized standard hydrogel microspheres can be efficiently prepared.

[0014] The objective of this invention is achieved through the following technical solution: An apparatus for preparing cell-containing hydrogel microspheres, comprising a layered reaction vessel, a bio-3D printing device, and a hydrogel microsphere collection device; The layered reaction vessel includes an upper opening, a bottom drain outlet, and a switch control structure located at the drain outlet, serving as a reaction vessel for the hydrogel microspheres. The bio-3D printing equipment includes a printing tube, a printing nozzle, and a 3D printer, and is used for 3D printing hydrogel microspheres. The hydrogel microsphere collecting device is used to collect the prepared hydrogel microspheres.

[0015] A method for preparing cell-containing hydrogel microspheres, comprising the following steps using the aforementioned preparation apparatus: Step S1: Construct a two-layer liquid-phase reaction system; Calcium chloride solution and non-toxic bio-oil are added to a layered reaction vessel. After standing and separating, an upper oil phase and a lower water phase are formed. The interface between the oil phase and the upper air is defined as the first interface, and the interface between the oil phase and the lower calcium chloride solution is defined as the second interface. Step S2: Assemble the printing tube and printhead; The pre-prepared and uniformly mixed hydrogel solution containing cells is injected into the printing tube. A printing nozzle with a suitable diameter is selected and connected to the printing tube. The printing tube is then assembled onto the 3D printer. The machine tool is controlled to lower the printing tube nozzle and immerse it in the oil phase to a preset depth, ensuring that the nozzle does not contact the calcium chloride solution. Step S3: Prepare hydrogel microspheres; An intermittent pneumatic extrusion method is adopted, which controls a micro air pump to introduce airflow into the printing tube at a preset outlet air pressure value, release time, and number of release air pressures, so that a preset micro amount of hydrogel is extruded at the nozzle. Before the volume of the hydrogel reaches a critical value, the nozzle port is kept connected in the oil phase. When the volume of the hydrogel in the oil phase reaches the preset standard, the 3D printer is controlled to vertically lift the nozzle upward through the first interface into the air. The hydrogel is retained in the oil phase due to the cross-sectional resistance of the first interface. Only when the nozzle continues to rise and enters the air, the hydrogel forms standard hydrogel microspheres in the oil phase by relying on surface tension. Step S4: Cross-linking of hydrogel microspheres after sedimentation; The hydrogel microspheres settle naturally under gravity, cross the second interface, and enter the lower calcium chloride solution layer. The density difference between the hydrogel microspheres and the calcium chloride solution, along with the cross-linking reaction, controls the settling resistance and cross-linking rate of the microspheres as they cross the second interface. Once the hydrogel microspheres have completely crossed the second interface, the settling rate returns to normal, and the cross-linking reaction continues. When the microspheres reach the bottom of the container, they possess sufficient structural strength and will not deform. Step S5: Collection of hydrogel microspheres; After the hydrogel microspheres settle to the bottom of the stratified reaction vessel, the switch control structure of the stratified reaction vessel is opened to discharge and collect the hydrogel microspheres.

[0016] Preferably, the collected hydrogel microspheres also need to be washed and then placed in a cell culture medium for culture.

[0017] Preferably, in step S1, the non-toxic bio-oil is 100cst dimethyl silicone oil or edible oil.

[0018] Preferably, in step S2, the hydrogel solution is sodium alginate-gelatin hydrogel; the printing nozzle is a 27-32G needle; the outlet air pressure is 0.2-1.5 MPa; and the release time is set to 0.1-1 s.

[0019] Preferably, in step S3, the interfacial resistance of the first interface is used to implement the separation of the hydrogel from the nozzle. In step S4, the hydrogel microspheres begin to cross-link immediately when they cross the second interface, and the settling speed is slowed down due to the cross-linking effect. After they have completely crossed the interface, the normal settling speed is restored. An interfacial lubricant can also be added to the lower aqueous phase to reduce the interfacial tension at the second interface, promote the falling of microspheres, and reduce the deformation of microspheres.

[0020] Preferably, when the hydrogel solution contains a photocurable material, the device is further equipped with a light irradiation unit for irradiating the settled hydrogel microspheres to cure them.

[0021] Preferably, the drain port of the layered reaction vessel is used to discharge the cross-linked hydrogel microspheres, and the upper oil phase is prevented from being discharged by a switch control structure.

[0022] Preferably, the stratified reaction vessel is equipped with a temperature control unit on the outside to regulate the internal temperature of the vessel.

[0023] Preferably, the stratified reaction vessel is further equipped with a vibration generating unit on the outside, which applies vibration to the inside of the vessel when the hydrogel microspheres settle, accelerating the passage of the microspheres through the interface.

[0024] Through the above design scheme, the present invention can bring the following beneficial effects: The method and apparatus for preparing hydrogel microspheres of this invention separate the droplet forming process from the cross-linking process, which can minimize the deformation of the hydrogel microspheres. Particle size can be precisely controlled, stably producing microspheres with uniform particle size. It is adaptable to various types of hydrogel materials, with a wide range of applications. The same process flow can be repeated, with excellent reproducibility in batch production. Combined with an automatic nozzle position control module, it can achieve fully automated, continuous, and quantitative preparation of large quantities of hydrogel microspheres. The microspheres can be discharged separately from the upper oil phase, eliminating oil phase residue issues and simplifying post-processing. When preparing cell-loaded hydrogel microspheres, the cell-mixed hydrogel is processed only under mild conditions; after forming, stabilization is achieved through ionic cross-linking, reducing damage to cells from heat, light, and mechanical stress; and with precise temperature control, the cells can be maintained in a uniformly dispersed state. It eliminates the need for expensive photocurable hydrogels and complex microfluidic chips, requiring only low-cost biocompatible raw materials and conventional dispensing equipment. The equipment structure is simple, the production cost is low, and it is economical.

[0025] The cell-carrying hydrogel microspheres prepared by this invention can be applied to multiple fields such as tissue engineering, organoid construction, cell therapy carriers, three-dimensional cell models for drug evaluation, and basic units for bioprinting, and have both high scientific research value and industrial application potential. Attached Figure Description

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments: Figure 1 Flowchart for assembling the printing tube and nozzle of the present invention and adding a low-viscosity hydrogel containing cells; Figure 2 This is a schematic diagram illustrating the complete preparation process of the first hydrogel microsphere of the present invention; Figure 3 This is a schematic diagram illustrating the process of repeatedly mass-producing hydrogel microspheres of approximately the same diameter according to the present invention; Figure 4 This is a schematic diagram illustrating the process of removing all hydrogel microspheres in an oil-free environment according to the present invention; Figure 5 Images of the hydrogel microspheres printed according to this invention; Figure 6 The images show living cells inside microspheres under a confocal fluorescence microscope, as described in this invention.

[0027] In the diagram, 110 is the printhead, 10 is the oil phase, 20 is the aqueous phase, 200 is the layered reaction vessel, 202 is the top opening, 204 is the bottom drain, 210 is the switch control structure, and 300 is the illumination unit. Detailed Implementation

[0028] Exemplary embodiments of the present disclosure will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

[0029] It should be noted that, unless otherwise stated, the technical or scientific terms used in this invention should have the ordinary meaning as understood by one of ordinary skill in the art.

[0030] This invention provides a method and apparatus for preparing cell-containing hydrogel microspheres. The invention is further illustrated below with reference to the accompanying drawings and through Example 1.

[0031] 1. Weigh 0.1g of pharmaceutical-grade sodium alginate and 0.9g of gelatin, take 10mL of 1640 complete culture medium, mix thoroughly, and heat in a water bath for 2 days to prepare sodium alginate-gelatin hydrogel.

[0032] 2. Extract 1 mL of 10⁶-level mouse MIN6 cell suspension. Thoroughly mix the cell suspension with a mature hydrogel at 37°C to prepare a sodium alginate-gelatin hydrogel containing MIN6 cells. Note that the hydrogel temperature must be strictly controlled at approximately 37°C during cell implantation. Excessive temperature may damage the cells, while insufficient temperature increases the hydrogel viscosity, leading to uneven cell distribution within the hydrogel.

[0033] 3. For example Figure 1 As shown, the printing tube is assembled and connected to the selected printing nozzle 110. In this embodiment, a 27G specification printing nozzle 110 (outer diameter 0.41 mm, inner diameter approximately 0.21 mm, length 13 mm) is used. Under the premise that other experimental conditions remain unchanged, the smaller the inner diameter of the printing nozzle 110, the smaller the diameter of the prepared hydrogel microspheres. The prepared cell-containing hydrogel solution is injected into the printing tube, and then it is installed into the bio-3D printer. The temperature of the hydrogel is maintained at a constant 37°C using a heating module.

[0034] 4. For example Figure 2 As shown, the nozzle of the bio-3D printer is slowly immersed into the oil phase 10 at 37°C (edible oil is used in this embodiment) through the upper opening 202 of the layered reaction container 200 (e.g., a separatory funnel), with the nozzle tip approximately 5 mm from the first interface. Subsequently, the outlet pressure of the micro-pump is set to 1.5 MPa, the air pressure release time is set to 0.1 s, and the number of releases is 1. Under these parameters, a small amount of hydrogel can be observed being extruded from the nozzle, but no automatic detachment occurs. Theoretically, the greater the depth to which the printing nozzle 110 is immersed in the oil phase, the greater the resistance at the nozzle, and the corresponding decrease in the amount of water extruded. When the printing tube is raised vertically, as the nozzle passes through the first interface, the hydrogel is intercepted in the oil phase 10, forming a standard microsphere under the action of surface tension. Subsequently, the hydrogel microsphere settles naturally under gravity.

[0035] 5. For example Figure 3 As shown, the hydrogel microspheres decelerate due to increased resistance when passing through the second interface, but still maintain their downward settling trend. After the previous microsphere has sunk to a certain depth, the printing tube is lowered again, so that the nozzle tip returns to a position 5 mm from the first interface. Maintaining the above process parameters, the printing process is repeated. By repeating this cycle, the required number of hydrogel microspheres can be prepared as needed.

[0036] 6. For example Figure 4As shown, 30 hydrogel microspheres were continuously prepared according to the above-described cyclic printing process, with a total printing time not exceeding 3 minutes. After the last hydrogel microsphere settled to the bottom of the aqueous phase 20, it was allowed to stand for 10 minutes. During this period, the cross-linking reaction continued, allowing the mechanical strength and degradation rate of the microsphere scaffold to gradually reach the target requirements. The container switch control structure 210 was then turned on, allowing the hydrogel microspheres to flow out with the calcium chloride solution from the bottom drain port 204 and be collected. Immediately afterwards, the switch control structure 210 was turned off to minimize the loss of calcium chloride solution.

[0037] 7. The collected hydrogel microspheres were rapidly washed with distilled water to remove residual calcium chloride solution, which is toxic to cells. Microscopic measurements showed that the diameter of the hydrogel microspheres ranged from 520 to 570 μm, with an error not exceeding 50 μm. The washed microspheres were then transferred to cell culture medium and cultured at 37°C with 5% CO2.

[0038] The above describes the complete preparation process of hydrogel microspheres. In actual operation, thanks to the precision and efficiency of a bio-3D printer, this process can be rapidly repeated. Since a large number of hydrogel microspheres do not interfere with each other in the liquid environment, precise quantitative acquisition of the number of microspheres can be achieved. After all the microspheres have settled to the bottom of the container, the surface of the microspheres has completed the oil washing process. Opening the valve at the bottom of the separatory funnel allows the microspheres to flow out of the container almost instantly along with the calcium chloride solution. The valve is then closed to minimize the amount of calcium chloride solution carried out, while strictly preventing leakage of the upper oil phase to ensure that no oil residue remains on the surface of the microspheres. Example 2

[0039] A 27G needle was selected as the print head 110, and a 5mL EP tube was used as the printing container. The air pressure was set between 0.2-0.37Mpa, and the release time was set between 0.1-1 seconds.

[0040] In this embodiment, the lower aqueous phase is 3 mL of calcium chloride crosslinking solution containing Tween-80 (1.5-2 mL of Tween-80 added to 100 mL of 0.02 g / mL calcium chloride solution); the upper layer is 2 mL of 100 cSt dimethyl silicone oil. The printing container is equipped with a temperature control unit to preheat the entire liquid phase to approximately 34°C. The printing needle is located approximately 10 mm below the oil surface.

[0041] The printing material was a gelatin-sodium alginate composite hydrogel (0.9 g gelatin + 0.1 g sodium alginate dissolved in 10 mL deionized water), and the collected microspheres had a diameter distribution of about 200-800 micrometers. Example 3

[0042] When the hydrogel solution contains a photocurable material, a light-irradiation unit 300 can be added outside the reaction vessel to photocur the hydrogel microspheres during the sedimentation process. Example 4

[0043] The stratified reaction vessel may also be equipped with a vibration generating unit to apply vibration to the inside of the vessel during the sedimentation of hydrogel microspheres, thereby accelerating the microspheres through the interface.

[0044] Example 5 In this embodiment, an interfacial lubricant is added to the lower aqueous phase. This interfacial lubricant reduces the interfacial tension at the second interface, which can both accelerate the falling speed of the microspheres and reduce the deformation generated during the falling process.

Claims

1. An apparatus for preparing cell-containing hydrogel microspheres, characterized in that: The preparation apparatus includes a layered reaction vessel, a bio-3D printing device, and a hydrogel microsphere collection device; The layered reaction vessel includes an upper opening, a bottom drain outlet, and a switch control structure located at the drain outlet, serving as a reaction vessel for the hydrogel microspheres. The bio-3D printing equipment includes a printing tube, a printing nozzle, and a 3D printer, and is used for 3D printing hydrogel microspheres. The hydrogel microsphere collecting device is used to collect the prepared hydrogel microspheres.

2. A method for preparing cell-containing hydrogel microspheres, characterized in that, The cell-containing hydrogel microspheres are prepared using the preparation apparatus as described in claim 1, wherein the preparation method includes the following steps: Step S1: Construct a two-layer liquid-phase reaction system; Calcium chloride solution and non-toxic bio-oil are added to a layered reaction vessel. After standing and separating, an upper oil phase and a lower water phase are formed. The interface between the oil phase and the upper air is defined as the first interface, and the interface between the oil phase and the lower calcium chloride solution is defined as the second interface. Step S2: Assemble the printing tube and printhead; The pre-prepared and uniformly mixed hydrogel solution containing cells is injected into the printing tube. A printing nozzle with a suitable diameter is selected and connected to the printing tube. The printing tube is then assembled onto the 3D printer. The machine tool is controlled to lower the printing tube nozzle and immerse it in the oil phase to a preset depth, ensuring that the nozzle does not contact the calcium chloride solution. Step S3: Prepare hydrogel microspheres; An intermittent pneumatic extrusion method is adopted, which controls a micro air pump to introduce airflow into the printing tube at a preset outlet air pressure value, release time, and number of release air pressures, so that a preset micro amount of hydrogel is extruded at the nozzle. Before the volume of the hydrogel reaches a critical value, the nozzle port is kept connected in the oil phase. When the volume of the hydrogel in the oil phase reaches the preset standard, the 3D printer is controlled to vertically lift the nozzle upward through the first interface into the air. The hydrogel is retained in the oil phase due to the cross-sectional resistance of the first interface. Only when the nozzle continues to rise and enters the air, the hydrogel forms standard hydrogel microspheres in the oil phase by relying on surface tension. Step S4: Cross-linking of hydrogel microspheres after sedimentation; The hydrogel microspheres settle naturally under gravity, cross the second interface, and enter the lower calcium chloride solution layer. The density difference between the hydrogel microspheres and the calcium chloride solution, along with the cross-linking reaction, controls the settling resistance and cross-linking rate of the microspheres as they cross the second interface. Once the hydrogel microspheres have completely crossed the second interface, the settling rate returns to normal, and the cross-linking reaction continues. When the microspheres reach the bottom of the container, they possess sufficient structural strength and will not deform. Step S5: Collection of hydrogel microspheres; After the hydrogel microspheres settle to the bottom of the stratified reaction vessel, the switch control structure of the stratified reaction vessel is opened to discharge and collect the hydrogel microspheres.

3. The method for preparing cell-containing hydrogel microspheres according to claim 2, characterized in that: The collected hydrogel microspheres also need to be cleaned and then placed in cell culture medium for further cultivation.

4. The method for preparing cell-containing hydrogel microspheres as described in claim 2, characterized in that: In step S1, the non-toxic bio-oil is 100cst dimethyl silicone oil or edible oil.

5. The method for preparing cell-containing hydrogel microspheres according to claim 2, characterized in that: In step S2, the hydrogel solution is sodium alginate-gelatin hydrogel; the printing nozzle is a 27-32G needle; the outlet air pressure is 0.2-1.5 MPa, and the release time is set to 0.1-1 s.

6. The method for preparing cell-containing hydrogel microspheres as described in claim 2, characterized in that: In step S3, the interfacial resistance of the first interface is used to implement the separation of the hydrogel from the nozzle. In step S4, the hydrogel microspheres begin to cross-link immediately when they cross the second interface, and the settling speed is slowed down due to the cross-linking effect. After they have completely crossed the interface, the normal settling speed is restored. An interfacial lubricant can also be added to the lower aqueous phase to reduce the interfacial tension at the second interface, promote the falling of microspheres, and reduce the deformation of microspheres.

7. The method for preparing cell-containing hydrogel microspheres according to claim 2, characterized in that: When the hydrogel solution contains a photocurable material, the device is also equipped with a light irradiation unit for irradiating the settled hydrogel microspheres to cure them.

8. The apparatus for preparing cell-containing hydrogel microspheres according to claim 2, characterized in that: The drain port of the layered reaction vessel is used to discharge the cross-linked hydrogel microspheres, and the upper oil phase is prevented from being discharged by a switch control structure.

9. The method for preparing cell-containing hydrogel microspheres according to claim 2, characterized in that: The stratified reaction vessel is equipped with a temperature control unit on the outside to regulate the internal temperature of the vessel.

10. The method for preparing cell-containing hydrogel microspheres according to claim 2, characterized in that: The stratified reaction vessel is also equipped with a vibration generating unit on the outside, which applies vibration to the inside of the vessel when the hydrogel microspheres settle, accelerating the microspheres to pass through the interface.

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