A method for preparing cell gel microspheres based on ladder emulsification technology

Abstract

The application discloses a cell gel microsphere preparation method based on ladder emulsification technology. The microchannel surface of a microchannel array chip is subjected to hydrophobization treatment, a collagen mother liquor is used to prepare a dispersed phase containing cells, the microchannel array chip, a hydrogel container and a disposable medical syringe are assembled, the dispersed phase is injected by driving a mechanical pump, monodisperse droplet emulsion is obtained, the obtained monodisperse droplet emulsion is incubated, hydrogel droplets are induced to solidify, a pipette gun is used to pre-absorb excess oil phase, the residual oil phase and cell culture medium are further filtered through a polytetrafluoroethylene film, and pure cell gel microsphere suspension is obtained. The application simplifies the complex operation process of a traditional microfluidic device by using an integrated design microchip, can realize rapid and high-throughput preparation of cell gel microspheres, and has a wide application prospect due to the uniform particle size, stable morphology of the obtained cell gel microspheres, mature preparation process of the chip, easy acquisition of raw materials and low cost.

Description

Technical Field

[0001] This invention belongs to the field of droplet microfluidics technology, specifically relating to a rapid preparation method of cell gel microspheres based on step emulsification technology. Background Technology

[0002] Droplet microfluidics is a technology that enables precise control and manipulation of various microfluidics at the micro- and nanoscale to generate monodisperse droplets with customized sizes and diverse compositions / functions. Stepped emulsification, as an advanced droplet microfluidic technique, operates on the principle of spontaneous breakup of the dispersed phase fluid driven by a geometrically induced Laplace pressure difference. It utilizes a specific microfluidic chip with a stepped structure to achieve uniform and controllable droplet generation. Based on this technology, controllable gel microspheres can be prepared by selecting suitable hydrogel materials as the dispersed phase and crosslinking them under specific conditions. When used as cell carriers, gel microspheres with customized sizes and diverse compositions / functions can encapsulate single, multiple, or multi-cell types to simulate the specific three-dimensional microenvironments of different regions in vivo or provide cells with a relatively independent growth space different from their surrounding microenvironment. These microspheres, as novel functional biomaterials, have demonstrated significant application value and potential in biomedical fields such as three-dimensional cell culture, tissue repair, and cancer treatment.

[0003] Droplet microfluidics offers unique advantages in precisely controlling the size, composition, and function of microspheres, making it one of the most effective methods for preparing cell gel microspheres. However, achieving high-throughput production of cell gel microspheres in a rapid and portable manner remains a challenge (especially in non-specialized biomedical laboratories). Existing step emulsification devices often rely on complex peripheral equipment in practical applications, typically including multiple connecting lines for introducing the dispersed and continuous phases, as well as the use of multi-pump systems. This non-integrated and complex system architecture results in a bulky and cumbersome device. Therefore, developing a simplified platform that eliminates complex fluid control while enabling rapid and high-throughput cell gel microsphere production is crucial for promoting the widespread adoption of cell gel microspheres and their efficient application in the biomedical field. Summary of the Invention

[0004] To address the problems existing in the background technology, this invention provides a rapid preparation method for cell gel microspheres based on step emulsification technology. This method is highly integrated, simple to operate, and combines the advantages of low cost, portability, and high-throughput generation. It can rapidly prepare cell gel microspheres with uniform size and stable morphology, and has high practical application value in both laboratory research and large-scale factory manufacturing.

[0005] The technical solution adopted in this invention is: (1) Perform hydrophobic treatment on the surface of the microchannel array chip; (2) Prepare a cell-containing dispersion using collagen stock solution; (3) After assembling the designed microchannel array chip, the matching hydrogel container, and the disposable medical syringe, the dispersed phase is injected by a mechanical pump to obtain a monodisperse droplet emulsion; (4) The monodisperse droplet emulsion obtained by incubation is used to induce the solidification of hydrogel droplets; (5) After solidification, the excess oil phase is pre-absorbed by pipette, and then the residual oil phase and cell culture medium are further filtered through a polytetrafluoroethylene membrane to obtain a pure cell gel microsphere suspension.

[0006] The specific steps (1) are as follows: (11) The microchannel array chip was sequentially immersed in acetone, isopropanol and ultrapure water and sonicated. After being fully dried, it was subjected to oxygen plasma treatment to obtain a surface-activated chip. (12) The surface-activated chip was placed together with 1H, 1H, 2H, 2H-perfluorooctyltrichlorosilane placed in an open environment and incubated in a vacuum environment, and then baked on a hot plate at 120°C. (13) The chip was cleaned with isopropanol and ultrapure water in sequence and then dried at room temperature.

[0007] In step (12), the amount of 1H, 1H, 2H, 2H-perfluorooctyltrichlorosilane added is 1 mL.

[0008] Step (2) specifically involves preparing a dispersion phase by mixing collagen stock solution, 10X phosphate buffer, 1M NaOH solution, ultrapure water, fluorescent microbeads, and cell suspension in a predetermined ratio.

[0009] In step (2), at 11.61 mg / mL -1 Prepare 500 μL of collagen stock solution with 4 mg / mL -1 Taking collagen solution as an example, the pH of the solution is adjusted to be around 7.0. The volume ratio of collagen stock solution, 10X phosphate buffer, 1M NaOH solution, ultrapure water, fluorescent microbeads, and cell suspension is 172 : 50 : 4 : 124 : 50 : 100.

[0010] The collagen stock solution is specifically a high-concentration rat tail collagen type I solution, with a concentration range of 8-12 mg / mL. -1 ; The fluorescent microbeads are specifically fluorescently labeled particles with a diameter of approximately 0.87 μm, and their main purpose is to facilitate fluorescence imaging of cell gel microspheres. The cell suspension is specifically DMEM medium containing human breast cancer cell line MDA-MB-231-GFP, but the cells and culture media applicable to this invention are not limited to this.

[0011] The specific steps (3) are as follows: (31) Align the pre-reserved cylindrical through holes on the microchannel array chip with the protrusions at the bottom of the hydrogel container and press gently to complete the assembly. Then add an appropriate amount of dispersed phase into the hydrogel container. (32) Insert the injection end of a disposable medical syringe into the opening at the top of the hydrogel container, and install the syringe vertically on the mechanical pump. Then immerse the microchannel array chip in the continuous phase container, turn on the mechanical pump to drive the syringe to inject the dispersed phase in the hydrogel container into the continuous phase container to obtain a monodisperse droplet emulsion. Step (4) specifically involves: The collected monodisperse droplet emulsion was transferred to a 37°C constant temperature incubator for incubation, and the hydrogel solidification was completed after 30 minutes.

[0012] Step (5) specifically involves: Most of the oil phase was aspirated using a pipette, and the remaining emulsion was then transferred to the surface of a polytetrafluoroethylene film to further filter the residual oil phase. Finally, the cell gel microspheres were gently rinsed with cell culture medium to resuspend them, resulting in a purified cell gel microsphere suspension.

[0013] In step (2), the collagen mother liquor is a high-concentration rat tail collagen type I solution, but the hydrogel mother liquor applicable to this invention is not limited to this.

[0014] In step (3), the continuous phase is either HFE 7500 containing 2% surfactant or mineral oil.

[0015] The method employs a designed stepped emulsification device, which includes a microchannel array chip and a hydrogel container. The microchannel array chip has a through hole in the middle, and the hydrogel container has a barrel-shaped structure. The inner cavity of the barrel-shaped structure serves as the container cavity of the hydrogel container. A protrusion is provided at the bottom of the barrel-shaped structure of the hydrogel container, and an exit hole is provided on the outer surface of the protrusion. The exit hole is connected to the container cavity of the hydrogel container through a channel arranged inside the protrusion.

[0016] The microchannel array chip is a stepped emulsification chip. The overall design is based on the principle of stepped emulsification. First, a microchannel array silicon substrate with at least two stepped structures is obtained through photolithography and etching processes. Then, the microchannels are sealed at the upper end of the upper bonding glass sheet of the microchannel array silicon substrate to obtain the microchannel array chip. A cylindrical through hole for hydrogel container insertion is reserved at the bottom of the lower microchannel array silicon substrate of the microchannel array chip by laser cutting.

[0017] The injection-molded hydrogel container with a bottom protrusion structure has a bottom protrusion size that matches the size of the pre-reserved cylindrical through hole, allowing for easy fitting without the need for additional external tubing.

[0018] This invention innovatively employs an integrated design of a microchannel array chip based on the principle of stepped emulsification and a matching hydrogel container to construct a rapid cell gel microsphere preparation platform that eliminates the need for complex tubing connections, is easy to operate, and can be manufactured on a large scale. Compared with traditional microfluidic systems that rely on multiple pumps, multiple tubing, and complex peripheral equipment, this platform significantly simplifies the operation process, reduces equipment costs and the barrier to entry, while achieving rapid and high-throughput preparation of cell gel microspheres.

[0019] The method described in this invention is a rapid, high-throughput preparation method for cell gel microspheres.

[0020] This invention employs photolithography and etching processes to fabricate a microchannel array chip with a stepped structure, followed by hydrophobic treatment of the chip's microchannel surface. A cell-containing dispersion phase is prepared using collagen stock solution. The chip is then assembled with a matching hydrogel container, into which the dispersion phase is added. The hydrogel container is inserted into a disposable medical syringe, and the chip is immersed in the continuous phase. A mechanical pump drives the dispersion phase to form uniform droplets. The resulting emulsion is incubated and induced to gel, then purified using a polytetrafluoroethylene (PTFE) oleophilic-hydrophobic membrane. Finally, it is resuspended in cell culture medium to obtain a cell gel microsphere suspension.

[0021] The beneficial effects of this invention are: 1. The cell gel microsphere preparation method provided by the present invention is simple and rapid, and while avoiding complex fluid control, it also has the advantage of high throughput, and its efficiency is higher than that of ordinary flow focusing method; 2. The cell gel microspheres prepared by this invention have uniform size and stable morphology, and their size, composition and function can be customized according to specific needs. Therefore, they can be used to construct personalized three-dimensional microenvironments for the unique biochemical and mechanical properties of different types of human tissues or organs. 3. The stepped emulsification chip fabrication process used in this invention is mature and the raw materials are readily available. Processing a single 6-inch silicon wafer can yield more than 800 chips at the same time, with an average material cost of less than US$0.1.

[0022] The method of this invention simplifies the complex operation process of traditional microfluidics by using an integrated microchip, enabling rapid and high-throughput preparation of cell gel microspheres. The resulting cell gel microspheres have uniform particle size and stable morphology. Furthermore, the chip fabrication process is mature, the raw materials are readily available, and the cost is low. It has broad application prospects in three-dimensional cell culture, tumor microenvironment construction, tissue engineering, and regenerative medicine. Attached Figure Description

[0023] Figure 1 The images show physical and structural diagrams of the microchannel array chips used in Examples 1-3. The left side shows the physical image, and the right side shows the structural diagram.

[0024] Figure 2 This is a flowchart illustrating the assembly process of a microchannel array chip, a hydrogel container, and a disposable medical syringe.

[0025] Figure 3 A comparison diagram of the rapid preparation method provided by this invention and the traditional preparation method.

[0026] Figure 4 This is a schematic diagram of the process for purifying cell gel microspheres using a polytetrafluoroethylene (PTFE) film.

[0027] Figure 5 The images show the bright-field pattern and corresponding fluorescence pattern of the gel microspheres prepared in Example 1. The left side shows the bright-field pattern of the gel microspheres, and the right side shows the fluorescence pattern.

[0028] Figure 6 The images show the morphology and corresponding diameter distribution of gel microspheres prepared using different mechanical pump injection speeds in Example 1. The scale bar is 200 μm.

[0029] Figure 7 This is a bar graph showing the change in the average diameter of the gel microspheres prepared in Example 1 as a function of the mechanical pump injection speed.

[0030] Figure 8 The images show the bright-field pattern and corresponding fluorescence pattern of the cell gel microspheres prepared in Example 2. The left side shows the bright-field pattern of the cell gel microspheres, and the right side shows the fluorescence pattern.

[0031] Figure 9 The image shows the fiber structure of the composite hydrogel microenvironment with different stiffnesses constructed in Example 3 (the composite hydrogel consists of gel microspheres and a peripheral gel matrix).

[0032] Figure 10 This is a confocal fluorescence image showing the invasion behavior of tumor cells in composite hydrogel microenvironments with different stiffnesses in Example 3.

[0033] Figure 11The graphs show the changes in the average number of invading cells, the spatial distribution of invading cells, the average / maximum invasion distance with culture time and the stiffness of the surrounding matrix, and the correlation analysis between the various invasion parameters obtained from the cell gel microspheres prepared in Example 3 for simulating tumor invasion experiments. In this graph, (A) represents the change in the average number of invading cells with culture time and the stiffness of the surrounding matrix, (B) represents the change in the spatial distribution of invading cells with culture time and the stiffness of the surrounding matrix, (C) represents the physical definition of the average / maximum invasion distance, (D) represents the change in the average invasion distance with culture time and the stiffness of the surrounding matrix, (E) represents the change in the maximum invasion distance with culture time and the stiffness of the surrounding matrix, and (F) represents the correlation analysis between the various invasion parameters. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the implementation of the present invention is not limited thereto.

[0035] The embodiments of the present invention are as follows: Example 1

[0036] Patterning was defined on a 6-inch silicon wafer using traditional photolithography: AZ5140 photoresist was spin-coated onto a clean silicon wafer surface (600 rpm pre-spreading for 5 s, 4000 rpm homogenization for 30 s), pre-baked at 95°C for 90 s, and then exposed for 6.5 s using a photolithography machine (KarlSUSS MA6) with a pre-set mask. This was followed by development with 2.38% tetramethylammonium hydroxide solution for 45 s, and post-baking at 100°C for 2 min to solidify the pattern. The pattern was then transferred to the silicon substrate using inductively coupled plasma (ICP) etching. The process parameters were: C4F8 190 sccm, SF6 450 sccm, RF etching / passivation power of 50 W and 20 W respectively, ICP etching / passivation power of 2300 W and 1650 W respectively, and etching rate of 6 μm / min. -1 The wafer is cleaned sequentially with isopropanol and ultrapure water. The patterned silicon wafer and glass plate are permanently encapsulated by anodic bonding, and then laser-cut to obtain individual chips.

[0037] The microchannel array chip was sequentially immersed in acetone, isopropanol, and ultrapure water and sonicated for 10 min. After thorough drying, it was subjected to oxygen plasma treatment. The surface-activated chip was then incubated in a vacuum environment for 1 hour in an open container containing 1H,1H,2H,2H-perfluorooctyltrichlorosilane, followed by baking on a hot plate at 120°C for 5 min. The chip was then rinsed sequentially with isopropanol and ultrapure water and dried at room temperature.

[0038] By mixing 11.61 mg mL -1Prepare a dispersion with a total volume of 500 μL by mixing collagen stock solution (172 μL), 10X phosphate buffer (50 μL), 1M NaOH solution (4 μL), ultrapure water (224 μL), and fluorescent microbeads (50 μL). (Final collagen concentration: 4 mg / mL) -1 ).

[0039] Align the pre-drilled cylindrical vias on the microchannel array chip with the protrusions at the bottom of the hydrogel container, press gently to complete the assembly, and then add 70 μL of dispersed phase into the hydrogel container.

[0040] Insert the injection tip of a disposable medical syringe into the opening of the hydrogel container and fix the syringe vertically onto the mechanical pump (the flow rate is set sequentially to 0.5 mL / h). -1 1 mL h -1 2 mL h -1 3 mL h -1 4 mL h -1 5 mL h -1 6 mLh -1 7 mL h -1 Then the chip was immersed in a 24-well plate containing 500 μL of continuous phase (HFE 7500 containing 2% surfactant) to collect the product.

[0041] The collected emulsion was incubated in a 37°C constant temperature incubator for 30 min. After solidification, the lower continuous phase was discarded, and the remaining liquid was transferred to a polytetrafluoroethylene oleophilic and hydrophobic membrane to absorb the residual oil phase and surfactant. Then, it was resuspended in 2 mL of fresh culture medium to obtain a gel microsphere suspension.

[0042] The physical objects and structures of microchannel array chips fabricated on a large scale using photolithography and etching processes are shown below. Figure 1 As shown in the figure, more than 800 chips can be produced simultaneously on a 6-inch silicon wafer. The micro-device has a compact structure, with each chip measuring less than 4 mm in size.

[0043] The operation procedure for assembling the stepped emulsification unit is as follows: Figure 2 As shown in the figure, the assembly process requires no additional piping and is simple and convenient.

[0044] A comparison of the cell gel microsphere preparation method based on this step emulsification device with traditional methods, for example Figure 3 As shown, this method simplifies the operation process, greatly shortens the preparation time of cell gel microspheres, and also has the advantage of low cost.

[0045] The process for purifying cell gel microspheres using polytetrafluoroethylene (PTFE) films is as follows: Figure 4As shown, this method is gentle and mild, avoiding the structural deformation and damage to the gel microspheres caused by traditional multiple centrifugation and washing steps.

[0046] The bright-field pattern and fluorescence of the gel microspheres prepared in this embodiment are as follows: Figure 5 As shown in the figure, the purified gel microspheres have intact morphology and clear boundaries.

[0047] In this embodiment, the morphology and corresponding diameter distribution of gel microspheres prepared using different mechanical pump injection speeds are shown in the following figures. Figure 6 As shown in the figure, the microsphere products maintained excellent size uniformity with increasing flow rate, even at flow rates as high as 7 mL / h. -1 Its coefficient of variation (CV) is still below 5%.

[0048] In this embodiment, the average diameter of the gel microspheres prepared varies with the mechanical pump injection speed as follows: Figure 7 As shown in the figure, the size of the gel microspheres is positively correlated with the flow rate, and can be precisely adjusted by regulating the injection speed of the mechanical pump.

[0049] Example 2 Patterning was defined on a 6-inch silicon wafer using traditional photolithography: AZ5140 photoresist was spin-coated onto a clean silicon wafer surface (600 rpm pre-spreading for 5 s, 4000 rpm homogenization for 30 s), pre-baked at 95°C for 90 s, and then exposed for 6.5 s using a photolithography machine (KarlSUSS MA6) with a pre-set mask. This was followed by development with 2.38% tetramethylammonium hydroxide solution for 45 s, and post-baking at 100°C for 2 min to solidify the pattern. The pattern was then transferred to the silicon substrate using inductively coupled plasma (ICP) etching. The process parameters were: C4F8 190 sccm, SF6 450 sccm, RF etching / passivation power of 50 W and 20 W respectively, ICP etching / passivation power of 2300 W and 1650 W respectively, and etching rate of 6 μm / min. -1 The wafer is cleaned sequentially with isopropanol and ultrapure water. The patterned silicon wafer and glass plate are permanently encapsulated by anodic bonding, and then laser-cut to obtain individual chips.

[0050] The stepped emulsified chip was sequentially immersed in acetone, isopropanol, and ultrapure water and sonicated for 10 min. After thorough drying, it was subjected to oxygen plasma treatment. The surface-activated chip was then incubated in a vacuum environment for 1 hour in an open container containing 1H,1H,2H,2H-perfluorooctyltrichlorosilane, followed by baking on a hot plate at 120°C for 5 min. The chip was then rinsed sequentially with isopropanol and ultrapure water and dried at room temperature.

[0051] By mixing 11.61 mg mL -1Collagen stock solution (172 μL), 10X phosphate buffer (50 μL), 1M NaOH solution (4 μL), ultrapure water (124 μL), fluorescent microbeads (50 μL), and cells at a concentration of 2.5E7 mL were prepared. -1 Prepare a dispersion phase with a total volume of 500 μL using cell suspension (100 μL) (final collagen concentration: 4 mg / mL). -1 Final cell concentration: 5E6mL -1 ).

[0052] Align the pre-drilled cylindrical through-holes on the stepped emulsion chip with the protrusions at the bottom of the hydrogel container, press gently to complete the assembly, and then add 70 μL of dispersed phase into the hydrogel container.

[0053] Insert the injection tip of a disposable medical syringe into the opening of the hydrogel container and fix the syringe vertically onto the mechanical pump (flow rate set to 2 mL / h). -1 Then the chip was immersed in a 24-well plate containing 500 μL of continuous phase (HFE7500 containing 2% surfactant) to collect the product.

[0054] The collected emulsion was incubated in a 37°C constant temperature incubator for 30 min. After solidification, the lower continuous phase was discarded, and the remaining liquid was transferred to a polytetrafluoroethylene oleophilic and hydrophobic membrane to absorb the residual oil phase and surfactant. Then, the emulsion was resuspended in 2 mL of fresh culture medium to obtain a cell gel microsphere suspension.

[0055] The obtained cell gel microspheres were seeded into 4 mg mL -1 A three-dimensional tumor invasion microenvironment was constructed within a collagen hydrogel: by mixing 11.61 mg mL -1 A collagen solution with a total volume of 500 μL was prepared by mixing collagen stock solution (172 μL), 10X phosphate buffer (50 μL), 1M NaOH solution (4 μL), ultrapure water (174 μL), and cell gel microsphere suspension (100 μL).

[0056] 200 μL of prepared collagen solution was poured into the central glass area of ​​a glass-bottom culture dish and incubated in a 37°C constant temperature incubator for 30 min. After solidification, 1 mL of fresh culture medium was added.

[0057] Figure 8 The image shows the bright-field and fluorescence images of the cell gel microspheres prepared in this embodiment. As can be seen from the image, the tumor cells are uniformly encapsulated in the gel microspheres.

[0058] Example 3 Patterning was defined on a 6-inch silicon wafer using traditional photolithography: AZ5140 photoresist was spin-coated onto a clean silicon wafer surface (600 rpm pre-spreading for 5 s, 4000 rpm homogenization for 30 s), pre-baked at 95°C for 90 s, and then exposed for 6.5 s using a photolithography machine (KarlSUSS MA6) with a pre-set mask. This was followed by development with 2.38% tetramethylammonium hydroxide solution for 45 s, and post-baking at 100°C for 2 min to solidify the pattern. The pattern was then transferred to the silicon substrate using inductively coupled plasma (ICP) etching. The process parameters were: C4F8 190 sccm, SF6 450 sccm, RF etching / passivation power of 50 W and 20 W respectively, ICP etching / passivation power of 2300 W and 1650 W respectively, and etching rate of 6 μm / min. -1 The wafer is cleaned sequentially with isopropanol and ultrapure water. The patterned silicon wafer and glass plate are permanently encapsulated by anodic bonding, and then laser-cut to obtain individual chips.

[0059] The microchannel array chip was sequentially immersed in acetone, isopropanol, and ultrapure water and sonicated for 10 min. After thorough drying, it was subjected to oxygen plasma treatment. The surface-activated chip was then incubated in a vacuum environment for 1 hour in an open container containing 1H,1H,2H,2H-perfluorooctyltrichlorosilane, followed by baking on a hot plate at 120°C for 5 min. The chip was then rinsed sequentially with isopropanol and ultrapure water and dried at room temperature.

[0060] By mixing 11.61 mg mL -1 Collagen stock solution (172 μL), 10X phosphate buffer (50 μL), 1M NaOH solution (4 μL), ultrapure water (124 μL), fluorescent microbeads (50 μL), and cells at a concentration of 2.5E7 mL were prepared. -1 Prepare a dispersion phase with a total volume of 500 μL using cell suspension (100 μL) (final collagen concentration: 4 mg / mL). -1 Final cell concentration: 5E6mL -1 ).

[0061] Align the pre-drilled cylindrical vias on the microchannel array chip with the protrusions at the bottom of the hydrogel container, press gently to complete the assembly, and then add 70 μL of dispersed phase into the hydrogel container.

[0062] Insert the injection tip of a disposable medical syringe into the opening of the hydrogel container and fix the syringe vertically onto the mechanical pump (flow rate set to 2 mL / h). -1 Then the chip was immersed in a 24-well plate containing 500 μL of continuous phase (HFE7500 containing 2% surfactant) to collect the product.

[0063] The collected emulsion was incubated in a 37°C constant temperature incubator for 30 min. After solidification, the lower continuous phase was discarded, and the remaining liquid was transferred to a polytetrafluoroethylene oleophilic and hydrophobic membrane to absorb the residual oil phase and surfactant. Then, the emulsion was resuspended in 2 mL of fresh culture medium to obtain a cell gel microsphere suspension.

[0064] The obtained cell gel microspheres were seeded into collagen hydrogels of different concentrations to construct a three-dimensional tumor invasion microenvironment: by mixing 11.61 mg / mL... -1 Prepare a 500 μL collagen solution (concentrations of 2 mg / mL) using collagen stock solution (86 μL, 172 μL, 258 μL), 10X phosphate buffer (50 μL), 1M NaOH solution (2 μL, 4 μL, 6 μL), ultrapure water (262 μL, 174 μL, 86 μL), and cell gel microsphere suspension (100 μL). -1 4 mg / mL -1 6 mg mL -1 ).

[0065] 200 μL of prepared collagen solution was poured into the central glass area of ​​a glass-bottom culture dish and incubated at 37°C for 30 min. After solidification, 1 mL of fresh culture medium was added. The invasion of tumor cells was observed using an inverted microscope and a laser confocal microscope.

[0066] Figure 9 The figure shows the fiber structure of the composite hydrogel microenvironment with different stiffnesses constructed in this embodiment. The composite hydrogel is composed of gel microspheres and an outer gel matrix. As can be seen from the figure, the fiber density gradually increases with the increase of the outer matrix concentration.

[0067] Figure 10 This is a confocal fluorescence image showing the invasion behavior of tumor cells in composite hydrogel microenvironments with different stiffnesses in this embodiment. As can be seen from the image, tumor cells in different groups can gradually escape from the gel microspheres and invade the peripheral matrix, but the invasion phenomenon is more obvious in the low concentration group.

[0068] Figure 11 The graphs showing the changes in average number of invading cells, spatial distribution of invading cells, and average / maximum invasion distance with culture time and peripheral matrix stiffness obtained from the tumor invasion simulation experiment using cell gel microspheres prepared in this embodiment, as well as the correlation analysis between various invasion parameters, are presented by [the relevant source]. Figure 11 As can be seen in (A), the average number of invasive cells in different stiffness groups gradually increased with culture time, but 2 mg mL -1 The group with the highest number of invasive cells was 4 mg / mL.-1 Group 2, and 6 mg mL -1 The fewest groups. (By) Figure 11 (B) Further, it can be seen that 2 mg mL -1 In addition to having the largest number of invasive cells, this group also had these invasive cells distributed at a relatively greater distance from the microspheres. The definition of average / maximum invasion distance is as follows: Figure 11 As shown in (C), by Figure 11 (D) and Figure 11 The quantitative analysis results of (E) show that the average / maximum invasion distance of different stiffness groups gradually increased with culture time, and 2 mg mL -1 The group with the longest average / maximum invasion distance was 4 mg / mL. -1 Group 2, and 6 mg mL -1 The group is recent. Figure 11 F represents the correlation analysis of these invasion parameters. In general, as the culture time increases, tumor cells gradually invade outward from the gel microspheres. Each invasion parameter is positively correlated with the culture time. In addition, compared with the high-concentration collagen hydrogel environment, the invasion of tumor cells in the low-concentration collagen hydrogel is more intense. Each invasion parameter is negatively correlated with the matrix stiffness, indicating that the low-stiffness collagen environment significantly promotes the invasion behavior of tumor cells.

[0069] The results of the above embodiments demonstrate that the cell gel microspheres prepared by this invention possess excellent morphological stability and size uniformity, and their size, composition, and function can be customized according to requirements. Based on this, the microspheres can simulate the unique biochemical and mechanical microenvironments of different tissues and organs, providing an effective tool for constructing personalized three-dimensional culture systems. This technology has practical value for both laboratory research and large-scale factory production, and is of great significance for promoting the widespread application of cell gel microspheres in the biomedical field.

Claims

1. A method for preparing cell gel microspheres based on step emulsification technology, characterized in that: The method includes: (1) Perform hydrophobic treatment on the surface of the microchannel array chip; (2) Prepare a cell-containing dispersion using collagen stock solution; (3) After assembling the microchannel array chip and the matching hydrogel container and disposable medical syringe, the dispersed phase is injected by a mechanical pump to obtain a monodisperse droplet emulsion; (4) The monodisperse droplet emulsion obtained by incubation is used to induce the solidification of hydrogel droplets; (5) After solidification, the excess oil phase is pre-absorbed by pipette, and then the residual oil phase and cell culture medium are further filtered through a polytetrafluoroethylene membrane and resuspended to obtain a pure cell gel microsphere suspension.

2. The method for preparing cell gel microspheres based on step emulsification technology according to claim 1, characterized in that: The specific steps (1) are as follows: (11) The microchannel array chip was sequentially immersed in acetone, isopropanol and ultrapure water and sonicated. After being fully dried, it was subjected to oxygen plasma treatment to obtain a surface-activated chip. (12) The surface-activated chip was placed together with 1H, 1H, 2H, 2H-perfluorooctyltrichlorosilane placed in an open environment and incubated in a vacuum environment, and then baked on a hot plate at 120°C. (13) The chip was cleaned with isopropanol and ultrapure water in sequence and then dried at room temperature.

3. The method for preparing cell gel microspheres based on step emulsification technology according to claim 1, characterized in that: Step (2) specifically involves preparing a dispersion phase by mixing collagen stock solution, 10X phosphate buffer, 1M NaOH solution, ultrapure water, fluorescent microbeads, and cell suspension in a predetermined ratio.

4. The method for preparing cell gel microspheres based on step emulsification technology according to claim 3, characterized in that: In step (2), at 11.61 mg / mL -1 Prepare 500 μL of collagen stock solution with 4 mg / mL -1 The collagen solution was prepared by adjusting the pH to approximately 7.

0. The volume ratio of the collagen stock solution, 10X phosphate buffer, 1M NaOH solution, ultrapure water, fluorescent microbeads, and cell suspension was 172 : 50 : 4 : 124 : 50 : 100, respectively.

5. The method for preparing cell gel microspheres based on step emulsification technology according to claim 1, characterized in that: Step (3) specifically involves: (31) Align the pre-reserved through holes on the microchannel array chip with the protrusions at the bottom of the hydrogel container and press to complete the assembly. Then add the dispersed phase into the hydrogel container. (32) Insert the injection end of the syringe into the opening at the top of the hydrogel container and install the syringe vertically on the mechanical pump. Then immerse the microchannel array chip in the continuous phase, turn on the mechanical pump to drive the syringe to inject the dispersed phase in the hydrogel container into the continuous phase, and obtain a monodisperse droplet emulsion.

6. The method for preparing cell gel microspheres based on step emulsification technology according to claim 1, characterized in that: Step (4) specifically involves: The collected monodisperse droplet emulsion was transferred to a 37°C constant temperature incubator for incubation, and the hydrogel solidification was completed after 30 minutes.

7. The method for preparing cell gel microspheres based on step emulsification technology according to claim 1, characterized in that: Step (5) specifically involves: Most of the oil phase was aspirated using a pipette, and the remaining emulsion was then transferred to the surface of a polytetrafluoroethylene film for further filtration of the residual oil phase. Finally, the cell gel microspheres were rinsed and resuspended with cell culture medium to obtain a purified cell gel microsphere suspension.

8. The method for preparing cell gel microspheres based on step emulsification technology according to claim 1, characterized in that: In step (3), the continuous phase is either HFE 7500 containing 2% surfactant or mineral oil.

9. The method for preparing cell gel microspheres based on step emulsification technology according to claim 1, characterized in that: The method employs a designed stepped emulsification device, which includes a microchannel array chip and a hydrogel container. The microchannel array chip has a through hole in the middle, and the hydrogel container has a barrel-shaped structure with an inner cavity serving as a chamber. A protrusion is provided at the bottom of the hydrogel container, and an exit hole is provided on the outer surface of the protrusion. The exit hole is connected to the chamber of the hydrogel container through a channel arranged inside the protrusion.

10. The method for preparing cell gel microspheres based on step emulsification technology according to claim 8, characterized in that: The microchannel array chip is first obtained by photolithography and etching to obtain a microchannel array silicon substrate with a stepped structure. Then, a bonding glass sheet is placed on the upper layer of the microchannel array silicon substrate to seal the microchannels at the upper end to obtain the microchannel array chip. Then, a cylindrical through hole for hydrogel container insertion is reserved in the lower layer of the microchannel array silicon substrate of the microchannel array chip by laser cutting.

Images