A gas shearing microfluidic droplet generation device for cell culture

Abstract

This invention discloses a gas shear-based microfluidic droplet preparation device for cell culture, belonging to the fields of biomedical engineering and cell culture technology. The device includes a microfluidic chip body fixed on a substrate, a sample inflow module, a gas inflow module, a droplet generation module composed of coaxially arranged sample and gas channels, and a biocompatible solidification and collection pool. This invention utilizes gas shear to avoid the toxicity of organic solvents, and the collection pool contains a mixture of culture medium and solidifying agent, achieving integrated droplet generation, solidification, and culture, thus reducing the risk of contamination. Through channel size control, inner wall cell adhesion inhibition, and multilayer coaxial structure design, it is possible to prepare cell microsphere carriers with uniform size, controllable diameter (150-400 μm), and cell viability exceeding 93%, showing broad application prospects in three-dimensional cell culture, tissue engineering, and regenerative medicine.

Description

Technical Field

[0001] This invention relates to the fields of biomedical engineering and cell culture technology, specifically to a gas shear-type microfluidic droplet preparation device for cell culture. Background Technology

[0002] Three-dimensional cell culture technology is considered an effective method to bridge the gap between traditional two-dimensional culture and the real in vivo environment. Among them, the use of microfluidic technology to prepare cell microsphere carriers and encapsulate cells in biocompatible hydrogels or polymer microspheres for three-dimensional culture has shown broad application prospects in tissue engineering, drug screening and regenerative medicine because it can simulate the in vivo extracellular matrix environment and provide a larger specific surface area for material exchange.

[0003] Droplet microfluidics is one of the effective methods for preparing highly monodisperse microdroplets. In existing technologies, immiscible liquids are typically used as the continuous and dispersed phases. A micropump drives the fluid into a microchannel, where the dispersed phase forms droplets under the shearing action of the continuous phase. However, this oil-phase shear-based droplet preparation method has several inherent drawbacks when applied to cell culture: First, the continuous phase medium (such as mineral oil or fluorinated oil) and its surfactants may have toxic effects on cells or remain on the microsphere surface, affecting subsequent cell adhesion, proliferation, and differentiation. Second, after droplet formation, complex washing and phase transfer steps are usually required before entering the culture environment, a process that is not only cumbersome but also significantly increases the risk of cell contamination. Finally, many bioactive substances (such as growth factors and proteins) are sensitive to organic solvents, and contact with the oil phase may lead to their denaturation and inactivation.

[0004] Furthermore, existing microfluidic devices often focus on droplet generation efficiency and size control in their design and material selection, while neglecting the special requirements of cell culture, such as sterile environment, biocompatibility, and in-situ immobilization culture. For example, if the chip material is not surface modified, hydrophilic materials are prone to channel wetting during the preparation of aqueous microspheres, leading to uneven droplet size; and if the device lacks sterile protection design, it will be difficult to meet the needs of long-term cell culture.

[0005] Therefore, in view of the above problems, the present invention provides a gas shear-type microfluidic droplet preparation device for cell culture, so as to solve the problems that the microdroplet preparation process in the prior art is prone to cell contamination, reduced activity, and cumbersome operation, which makes it difficult to meet the requirements of three-dimensional cell culture. Summary of the Invention

[0006] The purpose of this invention is to provide a gas shear-driven microfluidic droplet preparation device for cell culture. By using gas as the shear force source to completely avoid organic solvent contamination, and integrating a biocompatible solidification collection pool containing a mixture of cell culture medium and solidifying agent, the generated cell-containing microdroplets fall directly into the biocompatible solidification collection pool and solidify in situ into cell culture microspheres, thus achieving the integration of droplet generation, solidification, and preliminary culture. Simultaneously, by treating the inner wall of the sample channel to inhibit cell adhesion, adding a sterile environment maintenance unit, and optimizing the channel size for mass transfer, this invention can effectively maintain cell viability, ensure sterility, and prepare uniformly sized cell microsphere carriers suitable for three-dimensional culture, effectively solving the problems of cell contamination, cumbersome operation, and poor biocompatibility in existing technologies.

[0007] The objective of this invention is achieved through the following technical solution: This invention provides a gas shear-type microfluidic droplet preparation device for cell culture, comprising: a microfluidic chip body fixed on a mounting plate; a sample inflow module for carrying cell samples, disposed on the chip body, including at least one sample inlet for introducing a sample solution containing cells and a sample channel communicating therewith; a gas inflow module, disposed on the chip body, including at least one gas inlet for introducing a shear gas and a gas channel communicating therewith; a droplet generation module, including the sample channel and the gas channel coaxially arranged with a tapered outlet end, the sample channel passing through the interior of the gas channel, and its outlet end extending to the exterior of the outlet end of the gas channel to form an extension portion; and a biocompatible solidification collection pool disposed below the outlet of the droplet generation module, the pool containing a mixture of cell culture medium and solidifying agent for receiving and solidifying cell-containing microdroplets to form cell culture microspheres that can be directly used for three-dimensional culture. This invention, by using gas as the shear force source, completely avoids the toxic effects and residue problems of organic solvents on cells in traditional oil-phase shearing methods. At the same time, by mixing cell culture medium and solidifying agent in a biocompatible solidification collection tank, the cell-containing microdroplets generated can fall directly into the tank and solidify in situ into culture microspheres. This realizes an integrated process of droplet generation, solidification and initial culture, which significantly reduces the risk of cell contamination during the transfer process and provides good starting conditions for subsequent three-dimensional cell culture. The method for preparing porous polymeric spherical scaffolds for embedding cells based on the aforementioned gas shear-type microfluidic droplet preparation device is as follows: The sample channel was configured as a bilayer coaxial structure, including an inner sample channel and an outer sample channel. Cells were added to the hydrogel material to obtain an inner aqueous phase containing cells. Polymer materials and pore-forming agents were dissolved in a highly volatile organic solvent to obtain an organic phase as the shell material. The inner aqueous phase containing cells and the organic phase were injected into the inner sample channel and the outer sample channel, respectively. At the outlet, they were simultaneously cut into core-shell structured microdroplets by gas. Before the droplets fell into the receiving cell, the organic solvent evaporated due to the high gas flow rate. The polymer material rapidly precipitated, solidified, and successfully embedded the cells. In the receiving cell, the gel material further crosslinked to finally obtain a porous polymer material spherical scaffold with embedded cells.

[0008] Preferably, the hydrogel material is selected from sodium alginate and chitosan; the polymer material is selected from polylactic acid, polycaprolactone, and polylactic acid-glycolic acid copolymer; the pore-forming agent is selected from tridecane, camphene, gelatin, and sodium chloride; and the highly volatile organic solvent is selected from dichloromethane, acetonitrile, and acetone.

[0009] Preferably, the sample channel is made of rigid capillary, with an inner diameter of 50-100 μm at the tapered tip of its outlet end and an inclination angle of 5-30°. o The gas channel is made of a square glass capillary tube, with an inner diameter of 150~400μm and an inclination angle of 5~30° at its tapered tip at the outlet. o The length of the extended portion is 300-1000 μm, a size range used to control the diameter of the generated cell culture microspheres between 150-400 μm, thereby optimizing the mass transfer efficiency of oxygen and nutrients. By precisely controlling the dimensions of the sample channel and gas channel tips, this invention can flexibly adjust the particle size range of the generated microspheres. Controlling the microsphere diameter between 150-400 μm ensures that the microspheres possess sufficient mechanical strength to maintain their three-dimensional structure, while also ensuring that oxygen and nutrients can reach the interior of the microspheres through diffusion, avoiding core cell necrosis due to limited mass transfer. This is crucial for maintaining cell activity and function during long-term culture.

[0010] Preferably, the inner wall of the sample channel has a cell adhesion inhibition layer, which is formed by silanization treatment or coating with a zwitterionic polymer. This layer is used to prevent the sample solution containing cells from wetting the inner wall of the channel and to avoid non-specific cell adhesion within the channel. When preparing cell microsphere carriers using hydrophilic materials such as sodium alginate and chitosan, the sample solution easily wets the inner wall of the untreated glass channel, leading to unstable droplet formation or channel blockage. This invention, by modifying the sample channel hydrophobically or coating it with an anti-cell adhesion material, not only stabilizes the droplet formation process but, more importantly, prevents premature cell adhesion and loss within the channel, ensuring that the number of cells ultimately encapsulated within the microspheres is controllable and evenly distributed.

[0011] Preferably, the sample solution is a dispersed phase fluid containing bioactive substances, including cells, proteins, growth factors, or pharmaceutical active ingredients. The material of the sample solution is selected from one or more of polylactic acid (PLA), polylactic-co-glycolic acid copolymer (PLGA), polyglycolic acid (PGA), poly-L-lactide-caprolactone (PLCL), sodium alginate (ALG), and chitosan, with a concentration of 1wt% to 10wt%, used to regulate the mechanical strength and internal porosity of the cured microspheres. The concentration of the sample solution directly affects the physical properties of the cured microspheres: lower concentrations, such as 1wt%, yield microspheres with higher porosity and softer texture, which are beneficial for rapid cell proliferation and migration; higher concentrations, such as 10wt%, form microspheres with higher mechanical strength, suitable for culture scenarios requiring long-term structural stability. By being compatible with multiple bioactive substances and concentration ranges, this invention can meet the culture needs of different cell types and research purposes.

[0012] Preferably, the curing agent contained in the biocompatible curing collection tank is a calcium chloride solution, used to crosslink the alginate-based sample solution; the mixture of cell culture medium and curing agent also contains a surfactant, which is selected from one or more of polyvinyl alcohol (PVA), sodium dodecyl sulfonate (SDS), Tween, or Span, with a concentration of 0.5wt%~4wt%, used to maintain the spherical morphology of microdroplets and prevent aggregation. For cell culture requirements of different material systems, this invention provides a suitable curing solution composition: for alginate-based plasma crosslinking materials, calcium chloride solution can achieve rapid and gentle curing; the addition of surfactant can reduce the surface tension of microdroplets, preventing them from fusing or deforming during the process of falling into the biocompatible curing collection tank, ensuring that the final obtained cell culture microspheres have a regular spherical morphology and uniform particle size distribution.

[0013] Preferably, the device further includes a sterile environment maintenance unit, which is a transparent enclosure covering the droplet generation module and the biocompatible solidification collection pool. The enclosure maintains a positive pressure sterile state internally to maintain a sterile environment during cell culture. Cell culture requires extremely strict sterility conditions; contamination at any stage can lead to culture failure. This invention, by adding a sterile environment maintenance unit, provides a closed sterile barrier for cells throughout the entire process of droplet generation, falling into the biocompatible solidification collection pool, and initial solidification. Combined with the filtered and sterilized gas, this effectively prevents the intrusion of airborne microorganisms, meeting the hygienic requirements for long-term cell culture.

[0014] Preferably, the sample channel is a multi-layered coaxial structure, including an inner sample channel and an outer sample channel, each connected to different sample inlets, for simultaneously introducing sample solutions containing different cell types or bioactive substances to prepare cell microsphere carriers with core-shell structures or gradient distributions. To further expand the application of this invention in the construction of complex cell culture models, the multi-layered coaxial channel design allows for the one-step generation of microspheres with core-shell structures. For example, a core containing growth factors can be combined with a shell layer carrying cells, or different types of cells can be positioned in different regions of the microsphere, thereby simulating the complex microenvironment of tissues and organs in vivo, providing a powerful tool for co-culture system research and tissue engineering construction.

[0015] Preferably, the cell-containing sample solution flows in a laminar flow manner within the sample channel. At the outlet, it is subjected to the combined effects of surface tension, gravity, and the shear force of the gas ejected from the gas channel, resulting in the breakup of uniformly sized cell-containing microdroplets. These microdroplets then fall directly into the biocompatible solidification collection pool below and solidify into cell culture microspheres, achieving the integration of droplet generation, solidification, and initial culture. The working process of this invention embodies a deep integration of microfluidics and cell culture requirements: laminar flow ensures uniform force on cells within the channel, avoiding damage to cells from turbulence; gas shear, as a gentle driving force, maximizes cell viability while efficiently generating droplets; and the integrated process design eliminates intermediate transfer steps, making the transition of cells from encapsulation to the culture environment smoother and more efficient.

[0016] Preferably, the sample inlet and gas inlet are made of plastic-based dispensing needles and are fixed above the inlets of the sample channel and gas channel by bonding with biocompatible epoxy resin; the fixing plate is a glass slide, and the chip body is fixed to the fixing plate by epoxy resin. This invention utilizes readily available and inexpensive materials such as glass capillaries, fixing plates, and dispensing needles in device fabrication, and the device can be constructed through a simple assembly process, exhibiting good economic efficiency and scalability; simultaneously, all adhesive materials used have good biocompatibility, ensuring that the interface in contact with cells will not have a toxic effect on the cells.

[0017] Preferably, the gas connected to the gas channel is filtered and sterilized air, oxygen, or an inert gas, used to provide the necessary oxygen supply to the cells during the droplet formation process while providing shear force. This invention not only uses gas as a source of physical shear force but also endows it with biological functions: the introduced sterile air or oxygen can maintain normal cellular respiration and metabolism during droplet formation, avoiding hypoxic damage caused by prolonged cell residence within the device; simultaneously, the filtered and sterilized gas further enhances the sterility of the device, reflecting an overall design concept centered on cell culture.

[0018] Due to the application of the above technical solution, the present invention has the following beneficial effects compared with the prior art: 1. This invention uses gas as the shear force source, completely avoiding the toxic effects of organic solvents on cells in traditional oil-phase shearing. The cell culture medium and solidifying agent are mixed in the collection tank, so that the cell-containing microdroplets are generated and fall directly into the tank for in-situ solidification. This realizes the integration of droplet generation, solidification and culture, which significantly improves cell survival rate and reduces the risk of contamination.

[0019] 2. By precisely controlling the dimensions of the sample channel and gas channel tips, this invention allows for flexible adjustment of the cell culture microsphere diameter between 150 and 400 μm, ensuring both the mechanical strength of the microspheres and optimizing the mass transfer efficiency of oxygen and nutrients. Simultaneously, by treating the inner wall of the channel with cell adhesion inhibition, it effectively prevents sample solution wetting and non-specific cell adhesion, ensuring droplet formation stability and cell encapsulation uniformity.

[0020] 3. By adding a sterile environment maintenance unit, this invention provides a closed sterile barrier for cells throughout the entire process of droplet generation, falling into the collection pool, and initial solidification. Combined with the sterilization treatment of the introduced gas, an integrated sterile operation platform is constructed. At the same time, the introduced sterile air or oxygen can maintain the normal respiratory metabolism of cells during droplet generation and avoid hypoxia damage.

[0021] 4. This invention employs a multi-layer coaxial channel structure design, enabling the one-step generation of cell microsphere carriers with core-shell structures or gradient distributions. Different cell types or bioactive substances can be positioned in different regions of the microspheres to simulate the complex microenvironment in vivo. It is also applicable to a variety of biomedical polymer materials such as polylactic acid, poly-L-lactide-caprolactone, sodium alginate, and chitosan, with a wide range of applications.

[0022] 5. This invention uses low-cost materials such as glass capillaries, glass slides, and plastic-based dispensing needles, and the device can be constructed through a simple assembly process, which greatly reduces manufacturing costs. The adhesive materials and surface treatment layers used have good biocompatibility, ensuring the safety of the interface with cells, and have good economic efficiency and scalability. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, some of the drawings in the following description are some embodiments of the present invention. For those skilled in the art, other drawings can be made based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the gas shearing droplet microfluidic device of Embodiment 1 of the present invention; Figure 2 This is a particle size distribution diagram of poly(L-lactide-caprolactone) from Example 1 of the present invention. Figure 3 This is a scanning electron microscope image of the poly-L-lactide-caprolactone microspheres of Example 1 of the present invention; Figure 4 This is a graph showing the particle size variation trend of the poly(L-lactide-caprolactone) microspheres in Example 2 of the present invention. Figure 5 These are microscope images of polymer microspheres made of different materials from Example 3 of the present invention; Figure 6 These are microscope images of the oil-in-water microspheres from Embodiment 4 of the present invention; Figure 7 This is a scanning electron microscope image of the water-in-oil microspheres of Example 4 of the present invention; Among them, 1-sample inlet; 2-gas inlet; 3-sample channel; 4-gas channel; 5-extension section; 6-fixed plate; 7-biocompatible solidification collection pool. Detailed Implementation

[0025] To provide a clearer understanding of the technical features, objectives, and effects of this invention, specific implementation schemes are now described in detail.

[0026] The present invention will be further described below with reference to embodiments, but the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0027] Example 1 See appendix Figure 1 - Appendix Figure 3 This embodiment provides a gas shear-type microfluidic droplet preparation device for cell culture, and uses the device to prepare poly-L-lactide-caprolactone microspheres.

[0028] The device includes: a microfluidic chip body fixed on a fixing plate 6; a sample inflow module for carrying cell samples, disposed on the chip body, including a sample inlet 1 for introducing a sample solution containing cells and a sample channel 3 connected thereto; a gas inflow module, disposed on the chip body, including a gas inlet 2 for introducing shear gas and a gas channel 4 connected thereto; a droplet generation module, including the sample channel 3 and the gas channel 4 coaxially arranged with a tapered outlet end, the sample channel 3 passing through the interior of the gas channel 4, and its outlet end extending to the exterior of the outlet end of the gas channel 4 to form an extension portion 5; and a biocompatible solidification collection tank 7 disposed below the outlet of the droplet generation module, the tank containing a mixture of cell culture medium and solidifying agent for receiving and solidifying cell-containing microdroplets to form cell culture microspheres that can be directly used for three-dimensional culture. The sample channel 3 is made of rigid capillary, with an inner diameter of 60 μm at its tapered tip and a 15° inclination angle at the tapered section. The gas channel 4 is made of square glass capillary, with an inner diameter of 240 μm at its tapered tip and a 15° inclination angle at the tapered section. o The extension 5 is 500 μm long. The sample inlet 1 and the gas inlet 2 are made of plastic-based dispensing needles and are fixed above the inlets of the sample channel 3 and the gas channel 4 by bonding with biocompatible epoxy resin; the fixing plate 6 is a glass slide, and the chip body is fixed on the glass slide 6 by bonding with epoxy resin.

[0029] The method for preparing poly-L-lactide-caprolactone microspheres using the above-described apparatus includes the following steps: Poly(L-lactide-caprolactone) was dissolved in dichloromethane to prepare a 5 wt% dichloromethane solution, which served as the polymer substrate solution. Sodium dodecyl sulfate (SDS) and polyvinyl alcohol (PVA) were dissolved in deionized water to prepare a 0.5 wt% SDS and 5 wt% PVA solution, which served as the collection liquid for microemulsion droplets. The biocompatible solidification collection tank 7 contained a mixture of cell culture medium and solidifying agent, which also contained surfactants.

[0030] The dispersed phase fluid is injected into the sample inlet 1 of the gas-sheared droplet microfluidic device at a rate of 1 mL / h; nitrogen gas is injected into the gas inlet 2 of the device at a rate of 1.3 L / min, and the gas is filtered and sterilized. The height between the outlet of the sample channel 3 and the biocompatible solidification collection tank 7 is 20 cm.

[0031] The sample solution flows in laminar flow within sample channel 3. At the outlet, it is subjected to the combined effects of surface tension, gravity, and the shear force of the gas ejected from gas channel 4, resulting in the breakdown into uniformly sized microdroplets. These droplets fall directly into the biocompatible solidification collection tank 7 below and solidify into microspheres. After static solidification, the microspheres are washed five times with deionized water and freeze-dried for 24 hours to obtain poly(L-lactide-caprolactone) microspheres.

[0032] Figure 1 This is a schematic diagram of the gas shear-type microfluidic droplet preparation device in this embodiment. Figure 2 The figure shows the particle size distribution of the poly-L-lactide-caprolactone microspheres prepared in this embodiment. As can be seen from the figure, the diameter of the poly-L-lactide-caprolactone microspheres is about 108.59 μm, and the size exhibits a high degree of monodispersity. Figure 3 The image shows a scanning electron microscope (SEM) image of the poly-L-lactide-caprolactone microspheres prepared in this embodiment. As observed by the SEM, the poly-L-lactide-caprolactone microspheres are spherical and intact, with a wrinkled surface structure.

[0033] Cell viability was assessed using the prepared cell microsphere carriers: The prepared poly-L-lactide-caprolactone microspheres were sterilized overnight by immersion in 75% ethanol. The sterile microspheres were then transferred to 24-well cell culture plates and incubated in growth medium for 12 h to acclimatize to the culture environment before cell seeding. Mouse skeletal muscle cells (C2C12) were seeded at a growth medium of 2 × 10⁻⁶ cells / well. 4 Cells were seeded at a density of 1 cell / ml in growth medium and cultured for 5 days at 37°C, 95% air, and 5% CO2. Cells on the microspheres were stained using Live / Dead fluorescence staining (calcein-AM / propidium iodide double staining) and observed and counted under a fluorescence microscope. The cell viability on the microspheres was found to be 96.8%.

[0034] Example 2 See appendix Figure 4 This embodiment is based on the above embodiment 1. The similarities with embodiment 1 will not be repeated. The difference between this embodiment and embodiment 1 is that the outlet tip size of sample channel 3 and gas channel 4 is adjusted in this embodiment. Three sets of devices of different sizes are set in the preparation process. The specific parameters are shown in Table 1.

[0035] Table 1

[0036] Poly-L-lactide-caprolactone microspheres were prepared using the three sets of apparatus described above, with other preparation conditions being the same as in Example 1.

[0037] Figure 4 This figure shows the particle size variation trend of poly(L-lactide-caprolactone) microspheres prepared with three different outlet inner diameters in this embodiment. As can be seen from the figure, the diameter of the microspheres increases with the increase of the outlet inner diameters of the sample channel tip and the gas channel tip. This indicates that changing the outlet inner diameter of the device can precisely control the diameter of the polymer microspheres, further demonstrating that this device can prepare polymer microspheres with a wide size distribution, and can flexibly control the diameter of the generated cell culture microspheres between 150 and 400 μm to optimize the mass transfer efficiency of oxygen and nutrients.

[0038] Cell viability was assessed using the prepared cell microsphere carriers: The Live / Dead fluorescence staining method, identical to that used in Example 1, was employed. After 5 days of culture, cell viability on each group of microsphere carriers was measured. Results showed that the cell viability of group A microspheres (approximately 108 μm in diameter) was 96.8%, group B microspheres (approximately 152 μm in diameter) had a cell viability of 95.2%, and group C microspheres (approximately 198 μm in diameter) had a cell viability of 93.5%. These results indicate that when the microsphere diameter is within the range of 150–400 μm, the cell viability remains above 93%, demonstrating that microspheres within this size range can ensure effective mass transfer of oxygen and nutrients, maintaining high cell activity.

[0039] Example 3 See appendix Figure 5 This embodiment is based on Embodiment 1 described above. The similarities to Embodiment 1 will not be repeated. The difference between this embodiment and Embodiment 1 is that polylactic acid, sodium alginate, and chitosan are used as sample materials to prepare microspheres, and the gas flow rate has also been adjusted accordingly. When preparing sodium alginate and chitosan microspheres, the capillary tubes used in the device are hydrophobically modified. The specific steps are as follows: S1. Immerse the capillary tube in 0.1M hydrochloric acid solution and heat at 70°C for 30 minutes; S2. Remove the capillary tube, rinse it with deionized water and dry it to remove the hydrochloric acid solution; S3. Immerse the capillary obtained in step S2 in octadecyltrimethoxysilane and heat it in a vacuum oven at 65°C for 12 hours to form a hydrophobic alkyl layer. S4. Rinse and dry the capillary obtained in step S3 with deionized water to obtain a hydrophobic capillary, i.e., a cell adhesion inhibition layer is formed on the inner wall of sample channel 3. This layer is used to prevent the sample solution containing cells from wetting the inner wall of the channel and to avoid non-specific cell adhesion within the channel. The variable settings in the microsphere preparation process are shown in Table 2.

[0040] Table 2

[0041] The specific preparation methods for each group of cell microsphere carriers are as follows: Group D: Polylactic acid (PLA) was dissolved in dichloromethane to prepare a 5 wt% PLA solution as the sample solution. A 0.5 wt% SDS and 5 wt% PVA solution was mixed with DMEM cell culture medium at a 1:1 volume ratio to serve as the collection solution for biocompatible solidification collection tank 7. The sample solution was injected into sample inlet 1 at a rate of 1 mL / h; nitrogen gas was injected into gas inlet 2 at a rate of 1.0 L / min. The microdroplets fell into the collection tank and solidified to form PLA microsphere carriers.

[0042] Group E: Chitosan was dissolved in a 1% acetic acid solution to prepare a 2wt% chitosan solution. Mouse fibroblasts (L929) were mixed with the chitosan solution at a cell density of 1×10⁻⁶ cells / year. 6 The sample solution was prepared by mixing 2 wt% SDS solution with DMEM cell culture medium at a 1:1 volume ratio. This mixture served as the collection solution for the biocompatible solidification collection tank 7. The sample solution was injected into sample inlet 1 at a rate of 1 mL / h; nitrogen gas was injected into gas inlet 2 at a rate of 1.0 L / min. The cell-containing microdroplets fell into the collection tank and solidified to form chitosan cell microsphere carriers.

[0043] Group F: Sodium alginate was dissolved in deionized water to prepare a 2wt% sodium alginate solution. Mouse fibroblasts (L929) were mixed with the above sodium alginate solution at a cell density of 1×10⁻⁶ cells / year. 6 The sample solution was prepared by mixing 2 wt% calcium chloride solution with DMEM cell culture medium at a 1:1 volume ratio. This mixture served as the collection solution for the biocompatible solidification collection tank 7. The sample solution was injected into sample inlet 1 at a rate of 1 mL / h; nitrogen gas was injected into gas inlet 2 at a rate of 1.0 L / min. The cell-containing microdroplets fell into the collection tank and cross-linked and solidified to form sodium alginate cell microsphere carriers.

[0044] Group G: The preparation conditions are the same as those of Group F, except that the gas flow rate is adjusted to 0.3 L / min.

[0045] Figure 5 These are microscopic images of polymer microspheres made of different materials prepared in this embodiment. D, E, F, and G correspond to the preparation materials and conditions in Table 2, respectively. As can be seen from the images, by correspondingly changing the solvent, collecting liquid, and gas flow rate, microspheres of uniform size can be prepared from both hydrophobic polymers (polylactic acid) and hydrophilic polymers (chitosan, sodium alginate). This demonstrates that the gas shearing droplet microfluidic device described herein has wide applications, can prepare microspheres of different materials, and is suitable for various biomedical polymer materials.

[0046] Cell viability was assessed using the prepared cell microsphere carriers: The Live / Dead fluorescence staining method, identical to that used in Example 1, was employed. Cell viability was assessed in each group of microsphere carriers after 5 days of culture. Results showed that the cell viability was 95.2% in group D (polylactic acid), 94.7% in group E (chitosan), 95.8% in group F (sodium alginate, gas flow rate 1.0 L / min), and 96.2% in group G (sodium alginate, gas flow rate 0.3 L / min).

[0047] Example 4 See appendix Figure 6 - Appendix Figure 7 This embodiment is based on Embodiment 1 described above. The similarities to Embodiment 1 will not be repeated. The difference between this embodiment and Embodiment 1 is that the sample channel 3 is configured as a multi-layer coaxial structure, including an inner sample channel and an outer sample channel, each connected to different sample inlets 1, for simultaneously introducing sample solutions containing different cell types or bioactive substances to prepare cell microsphere carriers with core-shell structures or gradient distributions. Specifically, another sample channel is coaxially installed within sample channel 3. The inner sample channel has an inner diameter of 100 μm and an outer diameter of 200 μm, while the outer sample channel has an inner diameter of 750 μm. The device can prepare oil-in-water microspheres subjected to gas shear and water-in-oil microspheres subjected to gas shear.

[0048] The preparation method of oil-in-water microspheres includes the following steps: Poly(L-lactide-caprolactone) was dissolved in dichloromethane to prepare a 5 wt% dichloromethane solution of poly(L-lactide-caprolactone) as the dispersed phase solution O; sodium alginate was dissolved in deionized water to prepare a 0.5 wt% sodium alginate solution as the external aqueous phase W; calcium chloride was dissolved in deionized water to prepare a 2 wt% calcium chloride solution as the collecting liquid for the microemulsion droplets.

[0049] The dispersed phase fluid O was injected into the inner sample channel at a rate of 1 mL / h; the outer aqueous phase fluid W was injected into the outer sample channel at a rate of 10 mL / h; and nitrogen gas was injected into the gas channel 4 at a rate of 1.3 L / min. The prepared oil-in-water droplets were collected in a 2 wt% calcium chloride solution.

[0050] The preparation method of water-in-oil microspheres includes the following steps: Polyvinyl alcohol (PVA) was dissolved in deionized water to prepare a 2 wt% PVA solution as the inner aqueous phase W; poly(L-lactide-caprolactone) was dissolved in dichloromethane to prepare a 5 wt% dichloromethane solution of poly(L-lactide-caprolactone) as the oil phase solution O; sodium dodecyl sulfonate (SDS) and PVA were dissolved in deionized water to prepare a 0.5 wt% SDS and 5 wt% PVA solution as the collecting liquid for the microemulsion droplets.

[0051] The inner aqueous phase fluid W was injected into the inner sample channel at a rate of 1 mL / h; the outer oil phase fluid O was injected into the outer sample channel at a rate of 8 mL / h; and nitrogen gas was injected into the gas channel 4 at a rate of 1.3 L / min. The prepared water-in-oil droplets were collected in a solution of 0.5 wt% SDS and 5 wt% PVA.

[0052] Figure 6 These are microscopic images of the oil-in-water microspheres prepared in this embodiment. Microscopic observation shows that the oil-in-water microspheres have a clear structure and uniform size. Figure 7 This is a scanning electron microscope (SEM) image of a cross-section of the water-in-oil microspheres prepared in this embodiment. Observation using a scanning electron microscope shows that the microspheres have a clear core-shell structure.

[0053] Cell viability was assessed in the prepared cell microsphere carriers using the same Live / Dead fluorescence staining method as in Example 1. Cell viability within the microsphere carriers was measured after 5 days of culture. Results showed that the cell viability in the oil-in-water microspheres was 95.2%, and the cell viability in the water-in-oil microspheres was 95.2%.

[0054] Comparative Example 1 This comparative example is based on Example 1 above. The similarities to Example 1 will not be repeated. The difference between this comparative example and Example 1 is that this comparative example uses a traditional oil-phase shearing method to prepare the cell microsphere carrier. The specific method is as follows: Poly(L-lactide-caprolactone) was dissolved in dichloromethane to prepare a 5 wt% dichloromethane solution as the dispersed phase; mineral oil containing 2 wt% surfactant Span 80 was used as the continuous phase. The dispersed and continuous phases were injected into two inlets of a conventional T-type microfluidic chip, with a flow rate of 1 mL / h for the dispersed phase and 10 mL / h for the continuous phase. The two phases met at the junction of the microchannels, and the dispersed phase formed microdroplets under the shearing action of the continuous phase. After collecting the droplets, the oil phase was removed by centrifugation and washing, and then freeze-dried to obtain cell-loaded poly(L-lactide-caprolactone) microspheres. Cell viability on the microsphere carriers was assessed after 5 days of culture using the same Live / Dead fluorescence staining method as in Example 1.

[0055] Comparative Example 2 This comparative example is based on Example 3 above. The similarities to Example 3 will not be repeated. The difference between this comparative example and Example 3 is that this comparative example uses a traditional oil-phase shearing method to prepare sodium alginate-loaded microspheres. The specific method is as follows: Sodium alginate was dissolved in deionized water to prepare a 2 wt% sodium alginate solution. Mouse fibroblasts (L929) were mixed with the sodium alginate solution at a cell density of 1 × 10⁻⁶ cells / year. 6 A dispersion phase of sodium alginate was prepared at a flow rate of 1 mL / h, and a continuous phase of mineral oil containing 2 wt% Span 80 surfactant was prepared at a flow rate of 8 mL / h. The dispersed and continuous phases were injected into two inlets of a conventional T-type microfluidic chip, respectively. The dispersed phase flow rate was 1 mL / h, and the continuous phase flow rate was 8 mL / h. Microdroplets formed at the junction of the two phases in the microchannels. After collecting the droplets, the oil phase was removed by repeated washing, and the microspheres were then cross-linked and cured in a 2 wt% calcium chloride solution to obtain sodium alginate-loaded microspheres. Cell viability within the microsphere carriers was assessed after 5 days of culture using the same Live / Dead fluorescence staining method as in Example 1.

[0056] Comparative Example 3 This comparative example is based on Example 3 above. The similarities with Example 3 will not be repeated. The difference between this comparative example and Example 3 is that the inclination angle of the conical segment is changed to 45° to prepare sodium alginate microspheres.

[0057] The microspheres prepared in Examples 1 to 4 and Comparative Examples 1 to 3 were tested for properties such as particle size distribution, cell viability and microsphere structure. The results are shown in Table 3.

[0058] Table 3

[0059] As shown in Table 3, regarding particle size distribution, the coefficient of variation (CV%) of the cell microsphere carriers prepared in Examples 1 to 4 is all below 5%, exhibiting high monodispersity, which is far superior to the CV values ​​of Comparative Examples 1 and 2 (8.5% and 9.2%). This indicates that the gas shearing method of this invention can obtain cell microsphere carriers with more uniform size. In Example 2, as the channel tip size increases, the microsphere diameter increases accordingly, proving that this invention can precisely control the microsphere size, keeping the microsphere diameter within the range of 150-400 μm to optimize the mass transfer efficiency of oxygen and nutrients.

[0060] Regarding cell viability: After 5 days of culture, the cell microsphere carriers prepared in Examples 1-4 all maintained a cell viability of over 93%, with Example 1 reaching 96.8%, Group G of Example 3 (sodium alginate, gas flow rate 0.3 L / min) reaching 96.2%, and Group C of Example 2 (microsphere diameter approximately 198 μm) maintaining a relatively high viability of 93.5%. In contrast, the cell microsphere carriers prepared using the traditional oil-phase shearing method in Comparative Examples 1 and 2 had cell viability rates of only 78.6% and 72.4%, respectively, significantly lower than those in the examples of this invention. The cell viability of Comparative Example 3 was 85.7%, lower than Group F of Example 3. This indicates that the present invention uses gas as the shear force source, completely avoiding the toxic effects and residue problems of organic solvents on cells. Simultaneously, the integrated process design eliminates intermediate transfer steps, effectively protecting cell viability. Furthermore, the contraction gradient of the conical section of the flow channel also affects cell viability; at an inclination angle of 5-30°, it can mitigate sudden changes in shear force and fluctuations in radial pressure, effectively ensuring cell viability.

[0061] Regarding the microsphere structure: The water-in-oil and oil-in-water microspheres prepared in Example 4 all exhibited clear core-shell structures, demonstrating that the multilayer coaxial channel design can generate complex-structured cell microsphere carriers in a one-step process, providing flexible and diverse technical means for co-culture system research and tissue engineering construction. The microspheres prepared in Examples 1 to 3 were all solid homogeneous structures with regular morphology.

[0062] Regarding organic solvent residue: No organic solvent residue was detected in any of the embodiments of the present invention, while trace amounts of organic solvent residue were found in the microspheres prepared in Comparative Examples 1 to 2, requiring additional washing treatment, which increased the number of operation steps and the risk of contamination.

[0063] In summary, this invention, by employing a gas shear force source, avoids the toxic effects of organic solvents on cells. Combined with a biocompatible solidification and collection pool containing a mixture of cell culture medium and solidifying agent, it achieves an integrated process of droplet generation, solidification, and culture, significantly reducing the risk of contamination and protecting cell viability. Through precise control of channel size, the establishment of an inner wall cell adhesion inhibition layer, and the design of a multi-layered coaxial structure, this device can prepare cell microsphere carriers with uniform size (particle size variation coefficient less than 5%), controllable diameter between 150 and 400 μm, and suitable for various materials, possessing a core-shell structure. Experimental results show that the cell microsphere carriers prepared by this invention achieve a cell viability rate of over 93%, far exceeding that of traditional oil-phase shear methods, demonstrating broad application prospects in three-dimensional cell culture, tissue engineering, and regenerative medicine.

[0064] In the description of this application, it should be noted that, unless otherwise expressly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to fixed connections, detachable connections, or integral connections; they can refer to mechanical connections or electrical connections; they can refer to direct connections or indirect connections through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art will understand the specific meaning of the above terms in this application based on the specific circumstances.

[0065] The embodiments described above merely illustrate more specific and detailed implementations of the present invention, and should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A gas shear-based microfluidic droplet preparation device for cell culture, characterized in that, include: The microfluidic chip body is fixed on the fixing plate (6); A sample inflow module for carrying cell samples is disposed on the chip body and includes at least one sample inlet (1) for introducing a sample solution containing cells and a sample channel (3) connected thereto. A gas inflow module is disposed on the chip body and includes at least one gas inlet (2) for introducing shear gas and a gas channel (4) connected thereto. The droplet generation module includes a sample channel (3) and a gas channel (4) that are coaxially arranged and have a tapered outlet end. The sample channel (3) passes through the gas channel (4) and its outlet end extends to the outside of the outlet end of the gas channel (4) to form an extension portion (5). A biocompatible solidification collection tank (7) is located below the outlet of the droplet generation module. It contains a mixture of cell culture medium and solidifying agent to receive and solidify cell-containing microdroplets to form cell culture microspheres that can be directly used for three-dimensional culture. The sample channel (3) is made of rigid capillary tube, with an inner diameter of 50~100μm at the tapered tip of its outlet end and an inclination angle of 5~30°. o The gas channel (4) is made of a square glass capillary tube, with an inner diameter of 150~400μm and an inclination angle of 5~30° at its tapered tip at the outlet. o The length of the extension portion (5) is 300~1000μm, and this size range is used to control the diameter of the generated cell culture microspheres to be between 150~400μm; The biocompatible solidification collection tank (7) contains a calcium chloride solution as the solidifying agent, used to crosslink alginate-based sample solutions; the mixture of cell culture medium and solidifying agent also contains a surfactant, which is selected from one or more of polyvinyl alcohol, sodium dodecyl sulfonate, Tween or Span, with a concentration of 0.5wt%~4wt%; The cell-containing sample solution flows in a laminar flow manner in the sample channel (3). At the outlet, it is subjected to the combined effects of surface tension, gravity and the shear force of the gas ejected from the gas channel (4), and is divided into uniformly sized cell-containing microdroplets, which fall directly into the biocompatible solidification collection pool (7) below and solidify into cell culture microspheres. The method for preparing porous polymeric spherical scaffolds for embedding cells based on the aforementioned gas shear-type microfluidic droplet preparation device is as follows: The sample channel (3) is set as a double-layer coaxial structure, including an inner sample channel and an outer sample channel; cells are added to the hydrogel material to obtain an inner aqueous phase containing cells; polymer materials and pore-forming agents are dissolved in a highly volatile organic solvent to obtain an organic phase as a shell material; the inner aqueous phase containing cells and the organic phase are injected from the inner sample channel and the outer sample channel respectively, and are simultaneously cut into core-shell structured microdroplets by gas at the outlet. Before the droplets fall into the receiving pool, the organic solvent evaporates due to the high gas flow rate, the polymer material is rapidly precipitated and solidified and successfully encapsulates the cells, and the gel material is further cross-linked in the receiving pool to finally obtain a porous polymer material spherical scaffold encapsulating cells.

2. The gas shear-type microfluidic droplet preparation device for cell culture according to claim 1, characterized in that, The hydrogel material is selected from sodium alginate and chitosan; the polymer material is selected from polylactic acid, polycaprolactone, and polylactic acid-glycolic acid copolymer; the pore-forming agent is selected from tridecane, camphene, gelatin, and sodium chloride; and the highly volatile organic solvent is selected from dichloromethane, acetonitrile, and acetone.

3. The gas shear-type microfluidic droplet preparation device for cell culture according to claim 1, characterized in that, The inner wall of the sample channel (3) has a cell adhesion inhibition layer, which is formed by silanization or coating with a zwitterionic polymer.

4. The gas shear-type microfluidic droplet preparation device for cell culture according to claim 1, characterized in that, The device also includes a sterile environment maintenance unit, which is a transparent cover covering the outside of the droplet generation module and the biocompatible solidification collection pool (7). The inside of the cover is kept in a positive pressure sterile state to maintain a sterile environment during cell culture.

5. The gas shear-type microfluidic droplet preparation device for cell culture according to claim 1, characterized in that, The sample channel (3) is a multi-layer coaxial structure, including an inner sample channel and an outer sample channel, which are connected to different sample inlets (1) to simultaneously introduce sample solutions containing different cell types or bioactive substances, so as to prepare cell microsphere carriers with core-shell structure or gradient distribution.

6. The gas shear-type microfluidic droplet preparation device for cell culture according to claim 1, characterized in that, The sample solution is a dispersed phase fluid containing bioactive substances, including cells, proteins, growth factors, or pharmaceutical active ingredients; the concentration of the sample solution is 1wt% to 10wt%.

7. The gas shear-type microfluidic droplet preparation device for cell culture according to claim 1, characterized in that, The sample inlet (1) and gas inlet (2) are made of plastic-based dispensing needles and are fixed above the inlets of the sample channel (3) and gas channel (4) by bonding with biocompatible epoxy resin; the fixing plate (6) is a glass slide and the chip body is fixed on the glass slide by bonding with epoxy resin.

8. The gas shear-type microfluidic droplet preparation device for cell culture according to claim 1, characterized in that, The gas channel (4) is connected to filtered and sterilized air, oxygen, or inert gas, which is used to provide the necessary oxygen supply to the cells during the droplet generation process while providing shear force.

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