A method for high-throughput rapid production of organoid microspheres based on chips

Abstract

The application discloses a kind of high-flux rapid production organoid microspheres based on chip method.The hydrophobic film is arranged on microfluidic chip and pre-cooling treatment is carried out;after cell resuspension, matrigel front liquid is obtained by mixing matrigel at low temperature, and the cell density and mixing ratio are controlled; by optimizing the injection speed, the matrigel front liquid is injected into the microfluidic chip by the pump-free mode to realize high uniformity droplet generation; by three-stage temperature gradient control, matrigel in-situ gelation is realized, and uniform solidification in microspheres is realized; the microspheres are separated by demulsification, and then sequentially washed and collected to ensure the integrity of the microspheres and cell activity.The application mainly solves the technical problems of high cost, complex operation, large equipment volume, poor microsphere uniformity, cell sedimentation and low single cell encapsulation efficiency in existing organoid culture technology, and realizes low-cost, portable and high-flux organoid microsphere preparation.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and also to the interdisciplinary field of microfluidics and tissue engineering technology. Specifically, it relates to a pump-free method for generating organoid microspheres based on a handheld microdroplet chip. Background Technology

[0002] Organoids, as miniature tissue models cultured in three dimensions in vitro, have shown great potential in regenerative medicine, drug screening, and precision medicine. By mimicking the microenvironment of organs in vivo, organoid technology enables stem cells or organ-specific cells to self-organize and form miniature structures with some organ functions, providing a powerful tool for disease modeling, personalized treatment, and drug toxicity assessment.

[0003] From a technological development perspective, organoid culture technology has evolved from two-dimensional to three-dimensional culture, and from Matrigel droplet addition to microfluidic technology. Traditional organoid culture methods primarily rely on the manual Matrigel droplet addition method, first established and applied to intestinal organoid culture by the Clevers team in 2009. The specific procedure involves mixing organoid cells with Matrigel, manually adding approximately 30-50 μL of the mixture to the wells of a culture plate using a pipette to form a dome-shaped structure, then incubating at 37°C for 30-60 minutes to solidify, and finally adding culture medium for further cultivation. While this method is relatively simple, it has significant limitations, including poor droplet size uniformity, uneven cell distribution, and low throughput.

[0004] Current organoid culture technology has the following significant drawbacks: (1) The traditional manual drop method has poor uniformity: the coefficient of variation (CV) of the matrix gel droplets generated by the manual drop method is usually greater than 10%, which leads to large heterogeneity in organoid development and seriously affects the accuracy and reproducibility of drug screening results.

[0005] (2) Existing microfluidic devices are expensive and bulky: they rely on external pump and valve systems, and the system cost is usually between US$50,000 and US$100,000, making it difficult to promote their application at the clinical bedside or in laboratories with limited resources.

[0006] (3) Insufficient understanding of the rheological properties of matrix adhesives in microfluidic environments: In traditional macroscopic experiments, matrix adhesives are Newtonian fluids at 4°C with a viscosity of about 20-30 mPa·s. However, in micron-level channels, the rheological behavior of matrix adhesives changes significantly, and existing technologies often overlook this scale effect.

[0007] (4) Long curing time of hydrogels and easy cell sedimentation: In traditional methods, the curing time of hydrogels is 30-60 minutes, and cells are easy to settle to the bottom of the microspheres, resulting in uneven cell distribution and affecting the morphogenesis and functional maturation of organoids.

[0008] (5) Low single-cell encapsulation efficiency: Due to the lack of effective device usage methods and precise fluid control mechanisms, organoid formation rate is usually less than 50%.

[0009] (6) Fluid shear force causes great damage to cells: In traditional microfluidic chips, cells often bear high shear stress (50-100 Pa) when passing through narrow channels, which can easily lead to cell membrane damage, apoptosis and other problems. Summary of the Invention

[0010] To address the problems existing in the background technology, this invention proposes a method for high-throughput rapid production of organoid microspheres based on a chip-based approach.

[0011] This invention addresses the technical problems of existing organoid culture technologies, such as high cost, complex operation, large equipment size, poor microsphere uniformity, easy cell sedimentation, and low single-cell encapsulation efficiency, and achieves low-cost, portable, and high-throughput preparation of organoid microspheres.

[0012] This invention utilizes a handheld microdroplet generator to generate microdroplets for organoid culture.

[0013] I. A method for high-throughput rapid production of organoid microspheres based on chip-based technology: The method employs a handheld, pump-free microfluidic chip device and includes the following steps: S1. Assemble the pumpless microfluidic chip, syringe, and syringe adapter separately. Pre-process the pumpless microfluidic chip and syringe adapter: A hydrophobic film is placed on the pumpless microfluidic chip and pre-cooled, while ensuring tight connections and suitable temperatures between devices; S2. Preparation of matrix pre-gel solution: Cells were resuspended and mixed with matrix gel at low temperature to obtain matrix gel pre-solution, while controlling cell density and mixing ratio; S3, Pump-free droplet-generating microspheres: Highly uniform droplet generation is achieved by using a pumpless microfluidic chip to inject the matrix gel pre-solvent into the chip in an optimized injection rate without a pump. S4, Temperature Gradient Curing: By controlling the temperature gradient in three stages, the matrix that forms the microspheres is gelled in situ, achieving uniform solidification inside and outside the microspheres. S5. Special separation, washing, and collection treatment of the gelled microspheres: The gelled microspheres are separated by demulsification, followed by washing and collection to ensure microsphere integrity and cell viability.

[0014] The pump-free microfluidic chip is a handheld pump-free microfluidic chip. The matrix adhesive is a hydrogel.

[0015] Step S1 specifically involves: S11. Clean and dry the pumpless microfluidic chip and syringe converter head; S11. Under vacuum drying environment, a hydrophobic reagent is deposited on the surface of a pumpless microfluidic chip, so that the hydrophobic reagent reacts with the silanol groups on the chip surface to form a dense hydrophobic film. S12. After the pump-free microfluidic chip is removed, it is baked at high temperature, which makes the hydrophobic film form a strong Si-O-Si chemical bond with the chip surface, thereby improving the stability of the hydrophobic layer. S12. Place the pumpless microfluidic chip with hydrophobic film obtained in step S12 and the syringe converter and other operating tools in an environment of 0-4℃ for pre-cooling.

[0016] A key feature of this invention is the pre-cooling treatment of the device: pre-cooling in a 4°C refrigerator for 30 minutes to ensure that the device can maintain a low temperature during subsequent operations and to prevent the matrix adhesive from gelling prematurely when passing through the chip.

[0017] The hydrophobic reagent is 1H,1H,2H,2H-perfluorooctyltrichlorosilane (FOTS), which is prepared by vacuum phase deposition. The high-temperature baking in step S12 is to bake in an oven at 120°C for 30 minutes.

[0018] The matrix adhesive used is Matrigel matrix adhesive.

[0019] Step S2 specifically involves: S21. Place the Matrigel matrix gel at 4°C to completely transform it into a liquid state; S22. Then, at 4°C, within 5 minutes, aspirate Matrigel and add it to the pre-cooled cell suspension at 4°C and mix thoroughly to obtain the Matrigel pre-solution; the volume ratio between the Matrigel and the cells in the cell suspension is 1:1-3:1.

[0020] The cell-to-matrix ratio is set in the range of 1:1 to 1:3. This ratio range is determined based on the microscopic mechanism of cell-matrix interaction, which enables stable multi-cell co-encapsulation.

[0021] The cell suspension is specifically mouse mesenchymal bone marrow stem cells; in step S22, the cell suspension may or may not be added, and the cell encapsulation process may not be performed.

[0022] Step S3 specifically involves: S31. After inserting the syringe and chip assembly into the main chamber of the syringe and ensuring a tight connection, insert the chip end of the pumpless microfluidic chip into the container containing the oil phase. S32. At 4°C, use a hand syringe to inject the matrix gel pre-solution into the pumpless microfluidic chip at a constant speed, so that droplets are output from the microfluidic chip as microspheres.

[0023] The oil phase composition is HFE-7500 fluorinated oil containing 2% Span-80 by mass, and the injection rate is 0.3-0.8 mL / min, preferably 0.5 mL / min.

[0024] The key feature of this invention is the control of the syringe injection speed: a uniform injection rate within the range of 0.3-0.8 mL / min, preferably about 0.5 mL / min, which achieves high microsphere uniformity. Within this speed range, the shear rate within the flow channel is approximately 100-500 s⁻¹. -1 The matrix adhesive is in an optimal shear-thinned state with a viscosity of approximately 12-18 mPa·s.

[0025] Step S4 specifically involves: S41. Microsphere Collection and Pretreatment After injecting the matrix adhesive pre-liquid into the pumpless microfluidic chip, immediately remove the syringe connected to the chip from the EP tube, let it stand for 30 seconds to allow the liquid microspheres to float out of the oil phase. Once the liquid microspheres are observed to be spherical and highly transparent in the oil phase, quickly transfer the EP tube to an environment of 37℃±1℃. S42. Then, perform temperature-sensitive curing treatment using gradient curing as follows: - Stage 1: Pre-curing at 35°C±1℃ for 5 minutes to form a surface gel layer and prevent microspheres from fusing; - Stage 2: Completely cure at 37°C±1℃ for 15 minutes to allow the interior to fully gel. - Stage 3: After curing at 39°C±1℃ for 5 minutes, the final gel is gelled throughout, improving the gel strength.

[0026] Step S5 specifically involves: S51. After the temperature-sensitive curing is completed, carefully remove the lower oil phase using a pipette; S52. Then add a demulsifier and wash repeatedly to remove the oil phase. S53. Finally, centrifuge to remove residual oil phase, add culture medium or the required water solvent, and the flocculent precipitate dissolved in it is the organoid microsphere.

[0027] The demulsifier is a 7500 fluorinated liquid.

[0028] The key feature of this invention is the three-stage temperature control for gradient curing: Stage 1: Pre-curing at 35°C for 5 minutes to form a surface gel layer and prevent microsphere fusion; Stage 2: Complete curing at 37°C for 15-20 minutes to achieve complete internal gelation; Stage 3: Post-curing at 39°C for 5 minutes to achieve rapid and uniform curing and improve gel strength.

[0029] The key steps and parameter ranges in the method of this invention, particularly the pre-cooling temperature and time in the device assembly pre-cooling step, the injection speed and chip immersion depth in the droplet generation step, and the three-stage temperature control and time parameters in the gradient curing step.

[0030] This invention employs microfluidic droplet generation, pump-free drive, three-dimensional culture of organoids, preparation of hydrogel microspheres, and cell microencapsulation.

[0031] This invention provides a technical solution for the high-throughput and uniform preparation of organoid microspheres using a handheld, pump-free microfluidic device combined with a temperature-sensitive hydrogel matrix material and an optimized device usage method. This solution eliminates the need for an external pump and valve system; microsphere generation of the cell-hydrogel mixture can be achieved through manual injection, providing a new technical approach for the low-cost, portable preparation of organoids.

[0032] The difference between this invention and existing technologies is that existing microfluidic organoid generation technologies mainly rely on external pump and valve systems to achieve precise fluid control, which are active microfluidic technologies; while this invention adopts a handheld pumpless design and achieves passive droplet generation through optimized device usage, which is an innovative application of pumpless microfluidic technology in the field of organoid culture.

[0033] This invention discovers the unique rheological properties of matrix adhesives in microfluidic environments, including microscale shear thinning effects, temperature-dependent phase transition dynamics, and the microscopic mechanisms of cell-matrix interactions. Through computational fluid dynamics simulations and cell viability assays, this invention reveals the cell protection mechanisms of multilayered protrusion structures, including a stepwise decompression effect. This invention innovatively develops a gradient curing technology, employing a three-stage temperature control strategy to solve the problem of uneven curing inside and outside the microspheres caused by rapid curing.

[0034] The core technological innovations of this invention are mainly reflected in the following four aspects: First, the unique rheological properties of Matrigel in emulsion droplet environments were discovered and revealed for the first time, including the microscale shear thinning effect (when the shear rate increases from 10 s⁻¹). -1 Increase to 1000 s -1The study revealed several key scientific advancements: Matrigel's viscosity decreased from 25 mPa·s to 8 mPa·s, a reduction of approximately 68%; temperature-dependent phase transition kinetics (for microspheres with a diameter of 200 μm, the gelation time could be shortened to 10-15 minutes); and the microscopic mechanism of cell-matrix interaction (the formation of the cell-matrix complex increased the viscosity of the mixture by approximately 15-20%). These findings provide a crucial theoretical foundation for the proposed technological solutions and fill a research gap in the field of microfluidic biomaterials.

[0035] Secondly, innovative usage methods were developed specifically for the multi-layered bump structure of handheld microdroplet chips. These methods include optimized syringe injection speed control (uniform injection at 0.3-0.8 mL / min, preferably 0.5 mL / min) and fluid loading sequence (first loading the cell-Matrigel mixture into the syringe adapter, then inserting the syringe, and finally inserting the chip end into the oil phase). These usage methods fully utilize the passive segmentation characteristics and cell protection mechanisms of the chip structure, ensuring that the droplet size is primarily determined by the channel geometry parameters rather than relying on precise flow control, thereby achieving highly uniform microsphere generation.

[0036] Third, through computational fluid dynamics (CFD) simulations and cell viability assays, the cell protection mechanism of the multilayer protrusion structure was revealed, including a stepwise decompression effect (the shear stress on the cells gradually increases from 15 Pa in the first layer to 25 Pa in the third layer, which is much lower than the 50-100 Pa shear stress commonly found in traditional microfluidic chips). The discovery of these mechanisms explains why the chip structure of this invention can achieve high cell viability.

[0037] Fourth, an innovative gradient curing technology was developed. Through a three-stage temperature control strategy (Stage 1: pre-curing at 35°C for 5 minutes; Stage 2: complete curing at 37°C for 15-20 minutes; Stage 3: post-curing at 39°C for 5 minutes), this technology solves the technical challenge of uneven curing inside and outside the microspheres that can occur with rapid curing, thus avoiding problems affecting cell distribution and organoid morphology. This gradient curing strategy is based on the unique characteristics of Matrigel phase transition kinetics discovered by the inventors at the microsphere scale, providing a theoretical basis for optimizing curing parameters for microspheres of different sizes.

[0038] The beneficial effects of this invention include: (1) Scientific discovery value: For the first time, the special rheological properties of matrix gel in microfluidic environment were revealed, including the shear thinning effect at the microscale, the temperature-dependent phase transition dynamics and the microscopic mechanism of cell-matrix interaction, filling the research gap in the field of microfluidic biomaterials.

[0039] (2) Device structure optimization: By developing a specific usage method for the multi-layer bump structure of the handheld microdroplet chip, the passive segmentation characteristics and cell protection mechanism of the chip structure are fully utilized. The coefficient of variation (CV) of the generated droplet size is <5%, which is far better than the more than 10% of the traditional manual droplet addition method, and the cell survival rate can reach more than 90%.

[0040] (3) Significant cost-effectiveness: The handheld pumpless design completely eliminates the need for precision pumps and valves required by traditional microfluidic systems, reducing the cost of the device by about 90% and the cost of a single experiment by more than 80%.

[0041] (4) Simple and efficient operation: The 12 steps of the traditional organoid culture method are simplified into 5 core steps, and the total operation time is shortened from the traditional 2-3 hours to less than 20 minutes.

[0042] (5) Excellent microsphere uniformity: By optimizing the injection speed control method and the ratio of matrix gel to cells, the microsphere size is highly uniform, with a coefficient of variation (CV value) of less than 5%.

[0043] (6) High organoid formation efficiency: Through optimized device usage and fluid control strategy, the sphericity of cell microspheres >0.95 was achieved, and the organoid formation rate was increased to over 80%.

[0044] (7) High portability: The entire device can be integrated into a portable suitcase with a total weight of less than 500 grams, making it suitable for use in various scenarios such as bedside diagnosis, field biological monitoring, and distributed laboratories.

[0045] (8) High throughput: More than 5,000 organoid microspheres can be generated in a single operation, which is significantly higher than traditional methods and is particularly suitable for large-scale drug screening applications. Attached Figure Description

[0046] Figure 1 The flowchart of the microsphere generation method shows the five main steps from device assembly and precooling to microsphere cultivation. Figure 2 For flow rate optimization, injection speed versus CV value graph; Figure 3 The images show 200 μm bare matrix gel microspheres (left after demulsification, right as before), where (a) represents the state before demulsification and (b) represents the state after demulsification. Figure 4 The graphs show the time required for Matrigel to gel and the spheroidization efficiency at different temperatures, as well as the comparison of spheroidization uniformity between the two curing strategies. (a) represents the comparison of the time required for Matrigel to gel and the spheroidization rate at different temperatures, and (b) represents the comparison of spheroidization uniformity between the two curing strategies. Figure 5Images of microspheres with different cell counts (after demulsification); Figure 6 A diagram showing the morphological changes of bone organoids after 7 days of culture; Figure 7 This is a diagram showing the diameter distribution of microspheres before demulsification. Figure 8 This is a diagram showing the diameter distribution of microspheres after demulsification. Figure 9 Dark-field micrographs for fluorescence microscopy characterization of mesenchymal bone marrow organoids; Figure 10 Diameter distribution of microspheres produced by the dome method; Figure 11 The patent uses a chip method and a conventional dome method to create a comparison chart of cell survival rates. Detailed Implementation

[0047] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0048] The technical idea of ​​this invention is to develop a complete method for using a handheld microdroplet device, combining the curing characteristics of temperature-sensitive hydrogels with the special rheological behavior under microfluidic conditions, to achieve pump-free, high-throughput, and uniform generation of organoid microspheres.

[0049] like Figure 1 As shown, the technical principle of this invention is based on the following key physicochemical mechanisms: (1) Fluid dynamics mechanism: When the cell-hydrogel mixture passes through the micrometer-scale channels of the chip, the fluid undergoes Rayleigh-Platto instability fracture under the combined action of oil phase shear force and interfacial tension of the liquid gel mixture, forming uniformly sized droplets. Within the micrometer-scale channels (60-100 μm), the matrix gel exhibits significant shear-thinning behavior.

[0050] When the shear rate changes from 10 s -1 Increase to 1000 s -1 At that time, the viscosity of the matrix adhesive decreased from 25 mPa·s to 8 mPa·s, a reduction of approximately 68%. (2) Cell protection mechanism: The microfluidic device includes a multilayer fluid control chip formed by combining a silicon substrate and plexiglass. The chip has a multi-level bump array structure inside, which is distributed sequentially along the fluid flow direction to form a step-by-step change in flow resistance.

[0051] The shear stress experienced by the fluid in the first raised layer region is approximately 15 Pa, and the shear stress experienced in the third raised layer region is approximately 25 Pa, which is far lower than the 50-100 Pa shear stress level commonly found in traditional microfluidic chips.

[0052] (3) Chemical mechanism of hydrogels: Thermosensitive hydrogels are liquid at low temperatures (4-10°C) and undergo thermosensitive gelation at 37°C, forming a three-dimensional network structure.

[0053] For microspheres with a diameter of 200 μm, due to the significant improvement in heat transfer efficiency, a segmented heating method can be used, which shortens the gelation time to less than 20 minutes.

[0054] (4) Cell biology mechanism: After the cells are mixed with the matrix gel, the integrin receptors on the cell surface bind specifically to the laminin, collagen IV and other components in the matrix gel to form a "cell-matrix complex", which increases the viscosity of the mixture by about 15-20%.

[0055] The embodiments of the present invention are as follows: Example 1: Matrigel microsphere preparation process and optimization, such as Figure 1 As shown.

[0056] Step S1: Chip hydrophobic pretreatment and contact angle optimization S1.1 Chip Cleaning Process The chip was cleaned sequentially. First, it was ultrasonically cleaned with anhydrous ethanol for 10 minutes to remove organic contaminants from the surface. Then, it was rinsed with deionized water three times (2 minutes each time). Next, the chip surface was dried with nitrogen gas. Finally, the chip was placed in an 80°C oven to dry for 30 minutes to completely remove residual moisture.

[0057] S1.2 Hydrophobic reagent vapor deposition The clean and dry chip is placed in a vacuum dryer, and the hydrophobic reagent 1H,1H,2H,2H-perfluorooctyltrichlorosilane (FOTS) is volatilized and uniformly deposited on the chip surface by vacuum vapor deposition. During this process, FOTS molecules undergo a condensation reaction with the silanol groups on the chip surface to form a dense hydrophobic film.

[0058] S1.3 Hydrophobic film curing Remove the chip from the vacuum dryer and purge its surface with nitrogen to remove unreacted hydrophobic reagents; The chip was then placed in a 120°C oven and baked for 30 minutes. Under high temperature conditions, the hydrophobic film formed a strong Si-O-Si chemical bond with the chip surface, thereby improving the stability of the hydrophobic layer.

[0059] Step S2: Temperature control and pre-liquid preparation of Matrigel matrix gel S2.1, Matrigel thawing and pre-cooling Remove the Matrigel matrix from the -20°C freezer and immediately transfer it to a 4°C freezer; thaw overnight at 4°C for at least 12 hours to allow the Matrigel to completely transform into a liquid state. Avoid vigorous shaking during the thawing process to prevent the formation of air bubbles.

[0060] S2.2, Pre-cooling of the operating environment Pre-cool all instruments to 4°C 30 minutes in advance, including pipette tips, 1.5mL EP tubes, 1mL syringes, and single-lumen syringe adapters; at the same time, prepare an ice box to ensure that all subsequent procedures are performed on the ice box, and the laboratory room temperature should be controlled at 20-22°C.

[0061] S2.3, Matrigel pre-liquid preparation On an ice box, use a pre-chilled pipette to draw 100 μL of Matrigel and transfer it into a pre-chilled 1.5 mL EP tube; If the experiment involves encapsulating cells, add pre-cooled cell suspension (cell density 2 × 10⁻⁶) at this step. 7 (cells / mL); then, gently pipette and mix 3-5 times to ensure homogeneity; The key control point in this process is that the entire operation must be completed within 5 minutes to prevent the Matrigel temperature from rising; the prepared Matrigel pre-solution should be stored on ice and used immediately in subsequent steps.

[0062] Step S3: Pump-free droplet generation and microsphere size control S3.1 Chip and Syringe Assembly Remove the pre-cooled syringe adapter from the 4°C freezer, insert the syringe adapter into the chip aperture, draw Matrigel pre-fluid into the pre-cooled syringe adapter, insert the syringe & chip assembly into the main chamber of the syringe to ensure a tight connection, and then insert the chip end into a 1.5mL EP tube containing 300μL of oil phase, which is HFE-7500 fluorinated oil containing 2% Span-80.

[0063] S3.2 Injection Rate Optimization and Microsphere Formation At low temperature, the syringe was held handheld and injected at a constant rate, with the optimized injection rate range being 0.5 mL / min. Figure 2 ): When the injection rate is below 0.3 mL / min, the droplet formation efficiency is low and the microsphere size is small. When the injection rate is higher than 0.8 mL / min, the droplet size distribution becomes wider and the uniformity of the microspheres decreases; The optimal injection rate is 0.5 mL / min, which can achieve a microsphere size variation coefficient (CV value) of less than 3%.

[0064] During the injection process, the following phenomena can be observed: the Matrigel pre-fluid passes through the central hole of the chip, is progressively segmented by multiple layers of bumps, and finally forms spherical microspheres with a diameter of approximately 200 μm at the interface between the outermost layer of the chip and the oil phase. Figure 3 As shown in (a) and (b); It takes about 10 seconds to fill the entire volume of the converter, and about 5,000 microspheres can be generated.

[0065] The experimental variable results for this step are as follows: - 0.1 mL / min: Microsphere diameter 189±2 μm, CV value 2.1% - 0.3 mL / min: Microsphere diameter 195±3 μm, CV value 2.5% - 0.5 mL / min: Microsphere diameter 201±4 μm, CV value 3% - 0.8 mL / min: microsphere diameter 210±5 μm, CV value 5% - 1.0 mL / min: Microsphere diameter 230±8 μm, CV value 7% As can be seen from the above, the optimal conditions for the production of 200μm microspheres are: microsphere injection rate of 0.5mL / min.

[0066] Step S4: Temperature-sensitive curing gel S4.1 Microsphere Collection and Pretreatment After injection, immediately remove the syringe connected to the chip from the EP tube and let it stand for 30 seconds to allow the liquid microspheres to float out of the oil phase. At this time, it can be observed that the liquid microspheres are spherical and highly transparent in the oil phase. Then, quickly transfer the EP tube to an environment of 35℃-38℃.

[0067] S4.2 Gradient solidification and vibration This invention establishes a Matrigel phase transition dynamics model at the microsphere scale, specifically including the following steps: First, the state changes of Matrigel microspheres during the phase transition process are determined, which sequentially includes the transition from liquid to sol state and then to gel state.

[0068] Secondly, the phase transition rate constant k of Matrigel microspheres with a diameter of 200 μm was determined. size Its expression is: k size =k0·(D / D0) -n Where k0 is the reference phase transition rate constant, D is the actual diameter of the microsphere, D0 is the reference diameter, and n is the scale exponent.

[0069] Furthermore, the relationship between the degree of phase transition α(t) and time t is established using the following formula: α(t) = 1 - exp(-k·t m ) Where k is the phase transition rate constant and m is a reaction order-related parameter.

[0070] The above model reveals that the size of the microspheres has a significant impact on the phase transition rate. In particular, for microspheres with a diameter of less than 200 μm, the phase transition rate is increased by 3 to 5 times compared to the macroscopic scale condition, indicating a significant microsphere size effect.

[0071] Immediately after the microspheres are formed, the EP tube is removed from the chip device and temperature-sensitive curing is performed using gradient curing technology.

[0072] Gradient curing in three stages: - Stage 1: Pre-curing at 35°C for 5 minutes to form a surface gel layer and prevent microsphere fusion. - Stage 2: Complete curing at 37°C for 15 minutes to achieve complete internal gelation. - Stage 3: Post-curing at 39°C for 5 minutes to improve gel strength. This gradient curing strategy solves the technical problem that rapid curing may lead to uneven curing inside and outside the microspheres, thus avoiding problems that affect cell distribution and organoid morphology.

[0073] During the temperature-sensitive curing process, the protein components in Matrigel undergo a thermally induced phase transition when the temperature rises to 37°C, forming a three-dimensional network structure through hydrophobic interactions, hydrogen bonds, and electrostatic interactions. The microspheres gradually transform from a liquid state to a stable gel state, creating a three-dimensional culture environment similar to the in vivo basement membrane. The inventors have found that employing a gradient curing strategy reduces the CV value compared to commonly used single-temperature curing strategies, such as... Figure 4 (a) and (b) show that the uniformity of sphere formation is improved and the sphere size is closer to 200μm.

[0074] Step S5: Microsphere collection and demulsification S5.1 Oil phase removal and microsphere washing After the temperature-sensitive curing is complete, carefully remove the lower oil phase using a pipette; Add 200-300 μL of demulsifier to the EP tube, wash repeatedly 5-6 times to remove the oil phase, centrifuge for 30 seconds to remove the residual oil phase, add 200 μL of culture medium or the required water solvent, and the flocculent precipitate dissolved in the water solvent is the microsphere.

[0075] 5.2 Microsphere Quality Assessment Size uniformity assessment: 1. Add 10 μL of microsphere suspension to a hemocytometer. 2. Take images of the microspheres using an inverted microscope (100x magnification). 3. Measure the diameter of the microspheres in at least three different fields of view using ImageJ software. 4. Calculate the average diameter and coefficient of variation (CV) of the microspheres. Sphericity assessment: 1. Observe the morphology of the microspheres and calculate the sphericity (Sphericity = 4πA / P). 2 (A is the area, P is the perimeter) After systematic optimization, the optimal process parameters for the preparation of Matrigel microspheres were determined: matrix gel to sample volume ratio 1：1 1：1-3：1 high Matrigel Temperature 4℃ 4-8°C high Injection rate 0.5 mL / min 0.3-0.8 mL / min middle Curing temperature 37°C 25-42°C middle Curing time 20 minutes 10-50 minutes middle Example 2: Specific Application, Preparation of Bone Organoid Microspheres This embodiment applies to the high-throughput microencapsulation of bone marrow mesenchymal stem cells for stem cell therapy and tissue engineering applications. This embodiment is particularly suitable for implementation in resource-constrained primary hospitals or clinical laboratories, as it eliminates the need for expensive microfluidic equipment.

[0076] The specific technical configuration used in this embodiment is as follows: A circular silicon substrate-plexiglass composite chip is selected. The silicon substrate flow channel parameters are set as follows: flow channel width a = 60 μm, flow channel length b = 50 μm, and flow channel orifice angle c = 45°. The syringe converter head adopts a single-chamber structure with a main chamber volume of 100 μL, used for loading the cell-Matrigel mixture. The oil phase uses HFE-7500 fluorinated oil containing 2% Span-80 surfactant, with a volume of 300 μL. The Matrigel matrix gel is thawed overnight at 4°C before use.

[0077] The specific implementation process of this embodiment is as follows: Step S1: Device assembly and pre-cooling steps First, assemble the chip and syringe adapter. Align the smaller diameter end (output end) of the syringe adapter with the center hole of the chip and press firmly vertically 3-4 times to ensure the adapter is securely inserted into the chip hole. After assembly, check that the connection is tight and there are no gaps.

[0078] The assembled device was then pre-cooled. The chip-syringe conversion head assembly was placed in a 4°C freezer for 30 minutes. Pre-cooling ensured the device remained at a low temperature during subsequent operations, preventing Matrigel from prematurely gelling as it passed through the chip. Simultaneously, the 1mL syringe was also pre-cooled at 4°C to ensure the entire fluid path remained at a low temperature.

[0079] Step S2: Preparation of Cell-Matrigel Pre-solution Bone marrow mesenchymal stem cells (MSCs) were isolated from bone marrow aspiration samples and purified using adherent culture. After digestion, the MSCs were centrifuged and resuspended in pre-cooled, temperature-sensitive hydrogels, with the cell density adjusted to 1 × 10⁻⁶ cells / mL. 7 -3x×10 7 cells / mL.

[0080] The preparation process is performed on ice, and all pipette tips, EP tubes, and syringes must be pre-cooled to 4°C. The thawed Matrigel is mixed with the cell suspension at a volume ratio of 1:1 or higher, gently pipetting to mix, avoiding the formation of air bubbles. The resulting cell-Matrigel mixture is stored on ice. The entire process is performed at low temperatures to ensure the Matrigel remains in a liquid state.

[0081] Step S3: Pump-free organoid microsphere generation step Insert the syringe adapter into the chip well, then draw the pre-cooled cell-Matrigel mixture into the pre-cooled syringe adapter. Insert the syringe into the syringe adapter head, ensuring a tight, seamless connection between the chip and the adapter, and between the adapter and the syringe. Next, insert the chip-containing end of the device into a 1.5 mL EP tube containing 300 μL of oil phase. The oil phase composition is HFE-7500 fluorinated oil containing 2% Span-80 (equilibrated at room temperature for 30 minutes beforehand).

[0082] In this embodiment, the chip is immersed in the oil phase to a depth of approximately 0.5 cm. The syringe is manually injected at a constant rate of approximately 0.5 mL / min, allowing the mixture to pass through the central aperture of the chip. During injection, the cell-Matrigel mixture passes through the central aperture (1.35 mm in diameter), is segmented by multiple layers of bumps, and forms uniform microspheres with a diameter of approximately 200 μm at the interface between the chip's outer periphery and the oil phase. The entire injection process lasts approximately 10 seconds and generates approximately 5000 microspheres.

[0083] Step S4: Gradient temperature-sensitive curing Immediately after microsphere formation, the EP tube is removed from the chip device and thermo-cured using gradient curing technology. The gradient curing technology consists of three stages: Stage 1: Pre-curing at 35°C for 5 minutes to form a surface gel layer; Stage 2: Complete curing at 37°C for 20 minutes to fully gel the interior; Stage 3: Post-curing at 39°C for 5 minutes to improve gel strength. Figure 5 The morphology of bone organoids with different cell counts after completion at this stage.

[0084] Step S5: Microsphere Collection and Cultivation After temperature-sensitive curing, carefully remove the lower oil phase using a pipette; add 200μL-300μL of demulsifier to the EP tube, wash repeatedly 5-6 times to remove the oil phase, centrifuge for 30s to remove the residual oil phase, add 200μL of culture medium or the required water solvent, and the flocculent precipitate dissolved in the water solvent is the microsphere.

[0085] Finally, the microspheres were transferred to 24-well cell culture plates, and 500 μL–1 mL of stem cell-specific culture medium (containing α-MEM basal medium, 10% fetal bovine serum, 1% penicillin-streptomycin, 5 ng / mL bFGF, etc.) was added to each well. The plates were then incubated at 37°C in a 5% CO2 incubator, with the culture medium changed every 2–3 days. On day 2 of culture, stem cells were observed to proliferate within the microspheres, and the gel boundaries became blurred. The plates were ready for cell therapy or tissue engineering applications on days 7–10. Figure 6 The stained microspheres still retain their activity. Figure 7 ).

[0086] Step S6: Microsphere Quality Assessment Size uniformity assessment: 1. Add 10 μL of microsphere suspension to a hemocytometer. 2. Take images of the microspheres using an inverted microscope (100x magnification). 3. Measure the diameter of the microspheres in at least three different fields of view using ImageJ software. 4. Calculate the diameter before demulsification. Figure 8 ) and after milk dehiscence ( Figure 9 The average diameter and coefficient of variation (CV) of the microspheres.

[0087] In this embodiment, the data flow mainly refers to the flow and distribution of stem cells: starting with the mesenchymal stem cell suspension after separation from bone marrow tissue, the stem cells are resuspended in a thermosensitive hydrogel with a density of 3 × 10⁻⁶. 7 The stem cell-hydrogel mixture was then introduced into the chip via a syringe transfer head. As it passed through the central pore and multi-layered protrusions, the fluid was divided into multiple microfluidics, each containing approximately 1-5 stem cells (following a Poisson distribution). As the microfluidics broke apart at the periphery of the chip to form microspheres, the stem cells were encapsulated within them. After thermosensitive curing, the stem cells were immobilized within the three-dimensional extracellular matrix network formed by the hydrogel, creating a microenvironment suitable for stem cell proliferation and differentiation. Finally, the microspheres were transferred to a culture plate and cultured in stem cell culture medium, where the stem cells proliferated and maintained their multi-lineage differentiation potential.

[0088] Comparative Example 1: Method and performance evaluation of organoid microspheres prepared by the conventional dome method 1) Objective of the comparative experiment To verify the improvement of the chip-based high-throughput rapid preparation method for organoid microspheres provided by this invention over the traditional dome method in terms of microsphere size uniformity, preparation efficiency, cell viability, and ease of operation, organoid microspheres were prepared using the traditional Matrigel dome culture method and compared with the organoid microspheres obtained in the embodiments of this invention.

[0089] 2) Comparative Experiment Procedure Step 1: Matrigel Pretreatment Remove the Matrigel matrix gel stored at -20°C and transfer it to 4°C for at least 12 hours of pre-thawing to allow it to fully recover to a liquid state.

[0090] Avoid vigorous shaking during thawing to reduce the impact of air bubbles on subsequent gel structure formation.

[0091] Step 2: Preparation of cell suspension Mouse mesenchymal bone marrow stem cells were collected under low temperature conditions, centrifuged to remove the culture medium, and then resuspended in pre-cooled complete culture medium.

[0092] Adjust cell concentration to 2×10 7 The cells / mL were used to obtain a suspension of cells to be embedded.

[0093] Step 3: Preparation of the cell-Matrigel mixture system Add 100 μL of Matrigel matrix gel to a pre-cooled 1.5 mL centrifuge tube under 4°C ice box conditions.

[0094] Then add 100 μL of cell suspension and slowly mix with a pre-cooled pipette 3-5 times to ensure the cells are evenly dispersed in the Matrigel system.

[0095] The entire mixing process should be kept within 5 minutes to prevent Matrigel from gelling prematurely.

[0096] Step 4: Formation of the dome structure After pre-cooling the 24-well culture plate, take 5 μL of cell-Matrigel mixture and slowly add it to the center of the culture well to form a hemispherical dome structure.

[0097] Each well forms 20-50 dome structures, and a total of 500-2000 culture samples are prepared.

[0098] Step 5: Gel curing The culture plate was quickly transferred to a 37°C, 5% CO2 incubator and incubated for 30 minutes to allow Matrigel to gel and form a stable three-dimensional culture environment.

[0099] Step 6: Culture medium replenishment After Matrigel has completely solidified, slowly add 500 μL of preheated 37°C complete culture medium to each well.

[0100] Step 7: Cultivation and Detection The culture plates were placed in a 37°C, 5% CO2 incubator for further culture, and the formation of organoid microspheres was observed periodically.

[0101] During the culture process, the morphology and size changes of the microspheres, as well as the cell survival status, were recorded.

[0102] 3) Experimental Results and Analysis (a) Comparison of microsphere size uniformity The experimental results are as follows: Microsphere diameter distribution 185~255 μm 200~240 μm average diameter 220±35 μm 223±20μm Coefficient of variation (CV) 9.5% 5.3% The results showed that the size of organoid microspheres formed by the traditional dome method fluctuated within a certain range, while the size distribution range of microspheres prepared by the method of this invention was narrower, and the size variation coefficient was reduced from 9.5% to 5.3%.

[0103] The results show that the chip-type droplet generation structure used in this invention can improve the consistency of the droplet formation process, thereby improving the size uniformity of organoid microspheres.

[0104] (II) Comparison of preparation efficiency The experimental results are as follows: single operation time Approximately 60 minutes Approximately 2 minutes Number of items generated per time 500-2000 Approximately 5,000 The results showed that the traditional dome method is limited by the manual spotting method, resulting in a low number of samples prepared in a single batch.

[0105] In contrast, the method of this invention utilizes a chip-based high-throughput droplet generation structure to obtain a large number of organoid microspheres of uniform size in a short operating time, thereby increasing the number of microspheres prepared per unit time.

[0106] (III) Comparison of cell viability The experimental results are as follows: Cell survival rate after gel formation 80% 90% Cell viability after 7 days of culture 70% 85% The results show that, compared with the traditional dome method, the method of the present invention reduces the degree of cell damage during preparation, and the resulting organoid microspheres maintain high cell activity during long-term culture.

[0107] This demonstrates that the chip structure and droplet formation method of the present invention can provide a more suitable cell embedding environment.

[0108] Finally, the comparative experiments described above show that: The traditional dome method, which relies on manual spotting to form a three-dimensional gel structure, has certain limitations in terms of microsphere size consistency, batch preparation capability, and operational repeatability.

[0109] In contrast, the method for high-throughput rapid production of organoid microspheres based on chip-based technology provided by this invention controls the droplet formation process through microscale flow channel structure, thereby achieving stable control of organoid microsphere size and increasing the number of microspheres generated per unit time.

[0110] Meanwhile, because the fluid control method in the chip structure reduces the mechanical disturbances generated during traditional operations, the organoid microspheres obtained by the method of this invention exhibit higher cell survival rates.

[0111] Therefore, compared with the traditional dome method, the method of the present invention has significant advantages in terms of uniformity, high throughput, cell compatibility and cost control in the preparation of organoid microspheres, and can be used for large-scale preparation of organoids and related biomedical applications.

[0112] As can be seen from the above, the main technical effects of the present invention come from: (1) The microfluidic chip structure is used to achieve precise control of droplet size and reduce size fluctuations caused by manual dripping; (2) A high-throughput parallel droplet generation method is adopted to increase the number of microspheres prepared in a single batch; (3) Reduce the shearing effect during liquid flow by optimizing the internal flow channel structure of the chip, thereby reducing mechanical damage to the embedded cells; (4) The handheld pumpless structure is adopted to reduce the need for complex external control equipment and improve the portability and ease of operation of the device.

[0113] The above specific embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.

[0114] The above description is only a preferred embodiment of the present invention. Therefore, all equivalent changes or modifications made to the structure, features and principles described in the claims of this patent application are included in the scope of this patent application.

Claims

1. A method for high-throughput rapid production of organoid microspheres based on a chip-based approach, characterized in that: The method includes the following steps: S1. Pretreatment of microfluidic chip and syringe head conversion: A hydrophobic film is set on the microfluidic chip and pre-cooling treatment is performed; S2. Preparation of the pre-matrix solution: After resuspending the cells, mix them with the matrix gel at low temperature to obtain the pre-matrix solution, while controlling the cell density and mixing ratio; S3. Pump-free droplet generation of microspheres: Highly uniform droplet generation is achieved by injecting the matrix gel pre-solvent into the microfluidic chip in a pump-free manner at an optimized injection rate. S4. Temperature gradient curing: Through three-stage temperature gradient control, the matrix of microspheres is gelled in situ, achieving uniform curing inside and outside the microspheres. S5. Microsphere separation, washing and collection: The gelled microspheres are separated by demulsification, followed by washing and collection to ensure microsphere integrity and cell viability.

2. The method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 1, characterized in that: Step S1 specifically involves: S11. Clean and dry the pumpless microfluidic chip and syringe converter head; S11. Under vacuum drying environment, a hydrophobic reagent is deposited on the surface of the microfluidic chip, so that the hydrophobic reagent reacts with the silanol groups on the chip surface to form a dense hydrophobic film. S12. After the microfluidic chip is removed, it is baked at high temperature to form a strong Si-O-Si chemical bond between the hydrophobic film and the chip surface. S12. Pre-cool the microfluidic chip with hydrophobic film and the syringe converter and other operating tools in an environment of 0-4℃.

3. The method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 2, characterized in that: The hydrophobic reagent is 1H,1H,2H,2H-perfluorooctyltrichlorosilane (FOTS), which is prepared by vacuum phase deposition. The high-temperature baking in step S12 is to bake in an oven at 120°C for 30 minutes.

4. The method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 1, characterized in that: The matrix adhesive used is Matrigel matrix adhesive.

5. The method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 1, characterized in that: Step S2 specifically involves: S21. Place the Matrigel matrix gel at 4°C to completely transform it into a liquid state; S22. Then, at 4°C, add the pre-cooled cell suspension to the Matrigel within 5 minutes and mix thoroughly to obtain the Matrigel pre-solution; the volume ratio between the cells in the Matrigel and the cell suspension is 1:1-3:

1.

6. The method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 5, characterized in that: The cell suspension is specifically mouse mesenchymal bone marrow stem cells; In step S22, cell suspension may not be added, and cell encapsulation treatment may not be performed.

7. The method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 1, characterized in that: Step S3 specifically involves: S31. After inserting the syringe and chip assembly into the main cavity of the syringe and ensuring a tight connection, insert the chip end of the microfluidic chip into the container containing the oil phase. S32. At 4°C, hold the syringe and inject at a constant speed to inject the matrix gel pre-solution into the microfluidic chip, so that droplets are output from the microfluidic chip.

8. A method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 7, characterized in that: The oil phase composition is HFE-7500 fluorinated oil containing 2% Span-80 by mass, and the injection rate is 0.3-0.8 mL / min.

9. The method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 1, characterized in that: Step S4 specifically involves: S41. Microsphere Collection and Pretreatment After injecting the matrix gel pre-liquid into the microfluidic chip, immediately remove the syringe connected to the chip and let it stand for a period of time to allow the liquid microspheres to float out of the oil phase. Once the liquid microspheres are observed to be spherical and highly transparent in the oil phase, quickly transfer the EP tube to an environment of 37℃±1℃. S42. Then, perform temperature-sensitive curing treatment using gradient curing as follows: - Stage 1: Pre-curing at 35°C±1℃ for 5 minutes to form a surface gel layer; - Stage 2: Completely cure at 37°C±1℃ for 15 minutes to allow the interior to fully gel. - Stage 3: After curing at 39°C±1℃ for 5 minutes, the final gel is gelled throughout, inside and out.

10. A method for high-throughput rapid production of organoid microspheres based on a chip-based approach according to claim 1, characterized in that: Step S5 specifically involves: S51. After the temperature-sensitive curing is completed, carefully remove the lower oil phase using a pipette; S52. Then add a demulsifier and wash repeatedly to remove the oil phase. S53. Finally, centrifuge to remove residual oil phase, add culture medium or the required water solvent, and the flocculent precipitate dissolved in it is the organoid microsphere.

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