Organ chip based on microsphere cell culture and preparation method and application thereof

Abstract

The invention provides an organ chip based on microsphere cell culture and a preparation method and application thereof, and belongs to the technical field of cell in-vitro culture. By constructing an AHAMA + GelMA hydrogel microsphere system and optimizing the chip structure design, the problems of cell shedding, disordered microsphere flowing, cross contamination among different chambers and the like in the existing organ chip technology are successfully solved. Experimental results show that the AHAMA + GelMA microspheres have good cell compatibility, good initial adhesion rate and good anchoring effect, the chip design can effectively fix the microspheres and prevent the microspheres from moving among different cavities, and the stability and repeatability of experiments are remarkably improved. The innovative improvement provides a new thought and method for the development of the organ chip technology, and has important scientific significance and wide application prospects.

Description

Technical Field

[0001] This invention belongs to the field of in vitro cell culture technology, specifically relating to an organ-on-a-chip based on microsphere cell culture, its preparation method, and its application. Background Technology

[0002] Three-dimensional cell culture can more realistically simulate the in vivo cell growth environment, providing a more accurate model for cell behavior research and drug screening. Therefore, three-dimensional cell culture has enormous application potential in the biomedical field. Placing cells in a three-dimensional culture scaffold (such as hydrogel) provides a three-dimensional growth space, allowing them to grow under conditions closer to those in vivo. However, constructing a three-dimensional culture scaffold that highly mimics the extracellular matrix remains an ongoing challenge.

[0003] Microfluidics is a biomanufacturing technology with microscale resolution, capable of manufacturing specific micro-sized structures and creating biomimetic microenvironments for biological tissues. Microfluidics primarily utilizes cell microencapsulation and organ-on-a-chip technology to construct cellular microenvironments, providing reliable tools for biomedical research. Cell microencapsulation uses droplet microfluidics to encapsulate single or multiple cells in micrometer-sized capsules, forming a protective microenvironment. Organ-on-a-chip technology refers to biomimetic physiological organ systems built on microfluidic chips. Culturing various cell types on the chip allows for the simulation of cellular composition and structure within organs, enabling the study of cell-cell interactions and their impact on physiological and pathological processes. Microfluidic channels simulate blood flow and other bodily fluid flows, providing a dynamic microenvironment that allows cells to better mimic in vivo functions.

[0004] In the field of organ-on-a-chip (OoC), accurately simulating the in vivo microenvironment is one of the key bottlenecks that current cell culture technology urgently needs to overcome. Traditional two-dimensional cell culture methods, such as Matrigel or coating techniques, can only provide a planar cell attachment interface and cannot reproduce the complex three-dimensional structure of cells in vivo and the dynamic interactions between cells. Therefore, it is difficult to accurately simulate the physiological functions of cells in vivo, which seriously limits in-depth research on complex biological processes such as tissue development, disease mechanisms, and drug responses.

[0005] While three-dimensional hydrogel culture technology can provide cells with a three-dimensional growth environment that more closely resembles that in vivo, existing hydrogel systems still face many unresolved challenges when applied to organ-on-a-chip systems. On the one hand, the large size of hydrogels leads to uneven cell distribution within them, with significant differences between central and peripheral cells in nutrient acquisition, metabolic waste removal, and signal transduction. This cellular heterogeneity severely affects the accuracy and reliability of experimental results. On the other hand, the exchange of substances between cells inside the hydrogel and the external culture medium is limited. Nutrients and biological signals are difficult to efficiently transfer into the hydrogel, and cellular metabolic waste is difficult to remove in a timely manner, further affecting cell growth and functional performance.

[0006] The physical properties of hydrogels also present numerous challenges to their application in organ-on-a-chip systems. Hydrogels have a high water content and a liquid-like texture, making them prone to diffusion into other channels or chambers during single-channel injection, leading to uneven cell distribution and uncontrollable experimental results. While increasing the degree of cross-linking to improve stability and make them more solid can reduce deformation and diffusion under hydrodynamic conditions to some extent, it significantly increases the difficulty of injection, making precise injection and positioning via microchannels or microneedles challenging. This delicate balance between liquid and solid states is difficult to maintain precisely, further limiting the application scope of hydrogels in organ-on-a-chip systems.

[0007] In contrast, hydrogel microspheres possess excellent biocompatibility and a high specific surface area, enabling them to load various drugs, active agents, and nanomaterials, thus demonstrating great potential in applications such as drug sustained release and cell culture. Furthermore, the microscale size of hydrogel microspheres facilitates injection, effectively promoting the exchange of substances between cells and the external environment, providing a three-dimensional culture environment that mimics the in vivo cell growth microenvironment. However, in actual cell culture processes, after cells are seeded on hydrogel microspheres and cultured on a chip for a period of time, they easily detach from the microspheres and spread across the chip surface. This phenomenon not only causes cells to detach from their original three-dimensional microsphere environment, losing the intercellular interactions and tissue structures formed on the microspheres, but may also lead to non-specific cell attachment and growth on the chip surface, further interfering with the accuracy and reproducibility of experimental results.

[0008] Furthermore, existing chip manufacturing technologies have significant limitations in achieving high-precision structures. Traditional soft lithography struggles to create sufficiently tall chip chambers, while multilayer lithography, although theoretically capable of achieving even taller structures, suffers from complex processes, high costs, and difficulty in guaranteeing high-precision manufacturing results. While current 3D printing technology has addressed the height issue to some extent, it still faces challenges in achieving precise micropillar spacing between chambers. Micropillar spacing is difficult to make very small, failing to completely prevent microsphere movement between chambers. Especially under dynamic cultivation conditions, microspheres can easily deform and squeeze through the spacing, leading to cross-contamination between chambers.

[0009] On the other hand, existing chip chamber designs cannot effectively fix microspheres, which will flow randomly in the chamber under the action of hydrodynamics. This not only affects the accuracy and repeatability of experiments, but also limits the application potential of organ-on-a-chip in simulating complex physiological and pathological processes.

[0010] In summary, existing technologies have several shortcomings in integrating hydrogel microsphere systems with organ-on-a-chip platforms that urgently need to be addressed. Currently, no mature technology or product can perfectly combine hydrogel microsphere systems with organ-on-a-chip to form a complete platform suitable for complex biomedical research. These problems not only limit the application scope of hydrogel microsphere systems in the organ-on-a-chip field but also hinder their widespread application in important research areas such as disease model construction, drug screening, and tissue engineering, thus restricting the overall development and application prospects of organ-on-a-chip technology.

[0011] Therefore, designing a microsphere-based organ-on-a-chip to achieve precise simulation of the three-dimensional microenvironment of cells and efficient research on multi-cell and multi-tissue interactions, and solving problems such as cell shedding, disordered flow of microspheres, and cross-contamination between different chambers in existing organ-on-a-chip technologies, has become an urgent technical challenge. Summary of the Invention

[0012] This invention aims to solve the aforementioned technical problems by providing an organ-on-a-chip based on microsphere cell culture, its preparation method, and its applications. The technical objective of this invention is to address the issues of easy cell detachment, disordered microsphere flow, and cross-contamination between different chambers in existing organ-on-a-chip technologies, as well as the low bonding degree between existing organ-on-a-chips and microspheres, which hinders cell culture and proliferation. This invention provides a microsphere-based organ-on-a-chip to achieve precise simulation of the three-dimensional microenvironment of cells and efficient research on multi-cell and multi-tissue interactions, thereby enabling efficient cell growth.

[0013] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows: This invention first provides a method for preparing organ-on-a-chip based on microsphere cell culture, comprising the following steps: (1) Aldehyde-modified hyaluronic acid was prepared by oxidation reaction using hyaluronic acid and sodium periodate as raw materials, and then esterified with methacrylic anhydride to obtain aldehyde-modified methacrylic hyaluronic acid. (2) Aldehyde-modified methacrylated hyaluronic acid and methacrylamide gelatin were mixed at a weight ratio of 1:2. The resulting mixed solution and photoinitiator were used as the continuous phase and emulsified in the dispersed phase. Microdroplets were prepared by microfluidic device and then photocrosslinked and cured to obtain hydrogel microspheres. (3) A mold is prepared by 3D printing, and then the mixture of PDMS base liquid and curing agent is poured into the mold. After curing reaction, the mold is demolded to obtain a PDMS chip mold. (4) Multiple independent chambers are set on the PDMS chip mold, and a partition is set between the multiple independent chambers. The chambers are 100-200 micrometers higher than the partitions. Multiple micropores are set in each independent chamber. The diameter of the micropores is 0.46-0.50 mm. Then, the PDMS mold and the substrate are treated with plasma. After the treatment is completed, the PDMS mold and the substrate are tightly bonded and solidified to prepare an organ chip for cell culture.

[0014] This invention constructs an aldehyde-modified methacrylated hyaluronic acid (AHAMA) + methacrylamide gelatin (GelMA) hydrogel microsphere system, and combines it with a unique chip structure design to achieve precise simulation of the three-dimensional microenvironment of cells and efficient research on multi-cell and multi-tissue interactions. It achieves good cell culture results and excellent cell growth, and effectively solves the key bottleneck problems existing in the current organ-on-a-chip technology.

[0015] In existing technologies, traditional cell culture methods struggle to replicate the complex three-dimensional structures and dynamic interactions of cells in vivo. While three-dimensional hydrogel culture technology offers certain advantages, it still suffers from numerous shortcomings. For example, cells are prone to detachment from hydrogel microspheres, resulting in the loss of their three-dimensional microsphere environment; the disordered flow of microspheres within the chip chambers affects the accuracy and reproducibility of experimental results; and current chip chamber designs fail to effectively prevent the movement of microspheres between different chambers, leading to cross-contamination and other problems. These issues severely limit the application potential of organ-on-a-chip systems in simulating complex physiological and pathological processes.

[0016] To address the aforementioned issues, this invention constructs AHAMA+GelMA hydrogel microspheres. The RGD peptides on the surface of these microspheres provide excellent initial cell attachment, while the aldehyde groups on the surface of the microspheres can dynamically covalently bind with the amino groups on the cell surface. This enables cells to be firmly anchored on the microspheres, effectively preventing cell detachment and ensuring stable cell growth in the three-dimensional microsphere environment while maintaining cell-cell interactions and tissue structure.

[0017] In terms of chip structure design, this invention introduces multiple appropriately sized micro-well structures, each capable of accommodating a microsphere. This design not only achieves uniform dispersion of the microspheres but also physically restricts their disordered flow within the chambers, significantly improving experimental stability and repeatability. Furthermore, this invention innovatively employs an integral partition structure between the chambers, with a 100-200 micrometer gap between the partition and the top of the chip chamber. This design allows liquid to pass freely, but due to their large size, the microspheres cannot float and pass horizontally through this gap, effectively preventing their movement between different chambers and avoiding cross-contamination.

[0018] The chip of this invention is also designed with multiple independent chambers, which can be combined with a dynamic perfusion system. This system can simulate the interactions between multiple cells and tissues, providing a more accurate and reliable platform for studying complex physiological and pathological processes. Through dynamic cyclic perfusion, this invention can monitor the physiological and pathological changes of cells in a dynamic environment in real time, providing an experimental environment that more closely resembles in vivo physiological conditions for research such as disease model construction, drug screening, and tissue engineering.

[0019] In summary, this invention, through the construction of an AHAMA+GelMA hydrogel microsphere system, optimization of the chip chamber structure, and the introduction of a dynamic circulation perfusion system, not only solves the problems of cell shedding, disordered microsphere flow, and cross-contamination existing in current technologies, but also significantly improves the accuracy and reproducibility of experiments and expands the application scope of organ-on-a-chip in biomedical research. These innovative improvements provide new ideas and methods for the development of organ-on-a-chip technology, and have significant scientific value and broad application prospects.

[0020] Furthermore, the mass-to-volume ratio of hyaluronic acid to sodium periodate in step (1) is 1 g: 5 mL, and the oxidation reaction is carried out at room temperature with stirring in the dark for 2 h.

[0021] Furthermore, the mass-to-volume ratio of the aldehyde-modified hyaluronic acid to methacrylic anhydride is 1 g: 1 mL, and the esterification reaction is carried out under ice bath stirring for 12–24 h.

[0022] Furthermore, the degree of amino substitution of the methacrylamide gelatin described in step (2) is 90 ± 5%.

[0023] Furthermore, the photoinitiator mentioned in step (2) is LAP, Irgacure 2959, Irgacure 754, Irgacure 819 or Darocur 1173.

[0024] Furthermore, the weight ratio of the mixed solution of aldehyde-modified methacrylated hyaluronic acid and methacrylated gelatin to the photoinitiator is 10:1.

[0025] Furthermore, the dispersed phase in step (2) is a mixture of 95 wt% paraffin oil and 5 wt% Span 80.

[0026] Furthermore, the curing agent in step (3) includes a thermosetting agent, a platinum catalyst curing agent, or a silane crosslinking agent; the thermosetting agent is Sylgard 184 or Dow Corning 184; the platinum catalyst curing agent is a Karstedt catalyst or a Speier catalyst; and the silane crosslinking agent is 3-methacryloyloxypropyltrimethoxysilane or vinyltrimethoxysilane.

[0027] Furthermore, the weight ratio of the PDMS base liquid to the curing agent is 10:1, and the curing reaction is carried out at 60°C for 12 hours.

[0028] Furthermore, the parameters for plasma treatment in step (4) are: oxygen flow rate of 10-20 sccm, vacuum degree of 100-200 mTorr, power of 50-100 W, and treatment time of 1-3 minutes.

[0029] Furthermore, in step (4), the chamber is 150 micrometers higher than the partition. Specifically, the height of the chamber can be set to 1.5 mm, the height of the partition to 1.35 mm, the micropore depth to 0.24 mm, and the micropore diameter to 0.48 mm.

[0030] Furthermore, the substrate in step (4) is a glass sheet or a PDMS sheet, and the curing bonding condition is to cure at room temperature for 24 hours.

[0031] The second objective of this invention is to provide an organ-on-a-chip prepared by the method described above, which is used for microsphere cell culture.

[0032] A third objective of this invention is to provide the application of the organ-on-a-chip as described above in in vitro cell culture.

[0033] The beneficial effects of this invention are as follows: This invention successfully solves the problems of cell detachment, disordered microsphere flow, and cross-contamination between different chambers in existing organ-on-a-chip technologies by constructing an AHAMA+GelMA hydrogel microsphere system and optimizing the chip structure design. Experimental results show that the AHAMA+GelMA microspheres have good cell compatibility, excellent initial adhesion rate, and good anchoring effect. The chip design effectively fixes the microspheres and prevents their movement between different chambers, significantly improving the stability and reproducibility of the experiments. These innovative improvements provide new ideas and methods for the development of organ-on-a-chip technology, and have significant scientific value and broad application prospects. Attached Figure Description

[0034] Figure 1 This is AHAMA's proton nuclear magnetic resonance image.

[0035] Figure 2 Optical microscope image of AHAMA+GelMA microspheres.

[0036] Figure 3 For chip design structure; A) Chip planar design diagram; B) Chip local design parameters; C) Chip 3D rendering diagram; D) Chamber height diagram; E) Separator height diagram; F) Microvia depth diagram.

[0037] Figure 4 A) 3D printed mold; B) Lower chip after PDMS molding; C) Partial view of the lower chip; D) Upper chip; E) Chip after bonding; F) Partial view of the sealed chip.

[0038] Figure 5 For microporous structures and microsphere loading; A) Micropores of 3D printed molds under light microscopy; B) Micropores of the lower chip; C) Light microscopic images of micropores with different diameters under initial microsphere loading and dynamic culture conditions; D) Comparative analysis of microsphere loading rate and retention rate of micropores with different diameters.

[0039] Figure 6 Live / dead staining of cells on microspheres within the chip; A) Live / dead staining of GelMA microspheres with cell load; B) Live / dead staining of AHAMA microspheres with cell load; C) Live / dead staining of AHAMA+GelMA microspheres with cell load; D) Initial attachment rate and anchoring rate of composite microspheres with different ratios.

[0040] Figure 7 The diagram illustrates the dynamic perfusion process on the chip; A) The culture medium is slowly perfused using a micro-pump; B) Perfusion channels are established on the chip. Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention. Example

[0042] I. Experimental Methods 1. Preparation and characterization of AHAMA 1 gram of hyaluronic acid was dissolved in 100 mL of deionized water, followed by the addition of 5 mL of sodium periodate solution. The mixture was stirred at room temperature in the dark for 2 hours to achieve aldehyde methylation of the hyaluronic acid. After the reaction was complete, 1 mL of ethylene glycol was added to deactivate the unreacted iodate. The reaction mixture was purified by membrane dialysis to remove unreacted small molecule impurities, finally yielding aldehyde-modified hyaluronic acid. Next, 1 gram of aldehyde-modified hyaluronic acid was dissolved in 100 mL of deionized water, and 1 mL of methacrylic anhydride was added under ice bath conditions. The mixture was stirred overnight to achieve esterification. After the reaction was complete, the precipitate was removed by centrifugation, and the mixture was purified again by membrane dialysis to obtain the final aldehyde-modified methacrylic hyaluronic acid (AHAMA). AHAMA was lyophilized and stored, and analyzed using hydrogen nuclear magnetic resonance (HNMR). 1 The structure of the product was identified by H-NMR, confirming that its chemical structure was consistent with the expected structure.

[0043] 2. Preparation of AHAMA+GelMA microspheres The GelMA used was a finished product with an amino substitution degree of 90±5%. The AHAMA+GelMA premix was mixed with a photoinitiator at a ratio of 10:1 (by weight) to form a continuous phase. 95% by weight of paraffin oil and 5% by weight of Span 80 were mixed as the dispersed phase. The continuous phase was emulsified within the dispersed phase, forming pre-gel droplets at the channel junctions of the microfluidic device. The resulting droplets were exposed to ultraviolet light to undergo a cross-linking reaction, forming hydrogel microspheres. After cross-linking, the hydrogel microspheres were sequentially washed with acetone (3 times) and deionized water (3 times) to remove residual organic matter and impurities from the surface. Finally, the washed hydrogel microspheres were lyophilized to obtain dried AHAMA+GelMA microspheres for long-term storage and subsequent use.

[0044] 3. Chip mold printing Chip mold printing was performed using a Formlabs Stereolithography 3D printer and BioMed Clear Resin material. First, the mold model was precisely designed using professional 3D modeling software (SolidWorks), ensuring accurate dimensions and a reasonable structure, and then exported as an STL file. Next, the printer status was checked to ensure proper operation, and the BioMed Clear Resin material was installed. The STL file was imported into PreForm software, the model's position and angle were adjusted, printing parameters were optimized (e.g., layer thickness set to 100 micrometers), and the support structure was generated and optimized. The printer was started, and after calibration, the printing job began. After printing, the mold was carefully removed and cleaned with isopropyl alcohol for 20 minutes to remove any residual uncured resin. The mold was then placed in a Form Cure device and cured at 60°C for 60 minutes to enhance its mechanical properties and stability. After curing, the support structure was removed, and the mold surface was sanded and polished to ensure a smooth and flat surface, meeting the requirements for subsequent molding operations.

[0045] 4. Chip casting Accurately weigh the PDMS base solution and curing agent in a 10:1 ratio, pour them into a clean container, and stir thoroughly until the mixture is completely transparent and free of particles. Clean the surface of the printed mold with isopropanol to remove any residual dust or impurities. Then, slowly pour the well-stirred PDMS mixture into the mold, ensuring it completely covers all parts of the mold. Place the mold in a vacuum drying oven and evacuate for 30 minutes to remove air bubbles from the PDMS mixture and prevent the formation of pores during molding. Afterward, place the mold in a 60°C oven for curing for 12 hours. Once cured, check that the PDMS is completely cured. Carefully peel the PDMS from the edge of the mold to demold it, and clean the surface of the PDMS mold to remove any remaining PDMS fragments or other impurities, ensuring the integrity and surface quality of the chip.

[0046] 5. Chip bonding When drilling holes in a PDMS mold, first use a marker to accurately mark the hole positions on the mold surface. Then, select a drilling tool of appropriate size (such as a micro drill bit) and fix it on the worktable. Apply pressure slowly and evenly to drill, ensuring the drilling tool is perpendicular to the mold surface. Perform multiple gentle operations until the hole is penetrated, avoiding unnecessary damage to the mold. For plasma bonding, place the PDMS mold and the substrate to be bonded (such as a glass slide or other PDMS sheet) into a plasma treatment machine. Set the oxygen flow rate to 10-20 sccm, the vacuum level to 100-200 mTorr, the power to 50-100 W, and the treatment time to 1-3 minutes. After treatment, quickly align and tightly adhere the PDMS mold to the substrate, applying appropriate pressure to ensure no air bubbles and good contact. Cure at room temperature for 24 hours to form a strong bond between the PDMS and the substrate. After curing, check the bonding effect to ensure there is no separation or leakage, providing a reliable structural basis for subsequent cell culture and experimental operations.

[0047] 6. Chip cultivation After completing the punching and bonding operations of the PDMS mold, specific cells were seeded onto the surface of the hydrogel microspheres according to the experimental protocol. The seeded microspheres were placed in a suitable cell culture environment, maintaining a constant temperature (typically 37°C) and carbon dioxide concentration (5% CO2), and cultured for 24 hours to ensure sufficient cell attachment and good growth on the hydrogel microspheres. During culture, the cell growth status was observed regularly to ensure that cell viability and distribution met experimental requirements. After cell culture, the prepared microfluidic chip was autoclaved. After sterilization, the chip was removed and placed in a sterile operating table to allow it to cool naturally to room temperature, ensuring the chip remained sterile during subsequent operations. Subsequently, the cultured cell microspheres were slowly added into the chip chamber through the inlet above the chamber using aseptic techniques. The operation must be gentle and precise to avoid mechanical damage to the cell microspheres. After adding the cell microspheres, quickly use a solid silicone rubber sealing cap that has been autoclaved and tightly insert it into the opening at the top of the chip chamber to ensure a good seal and prevent external contamination from entering the chip. This provides a stable sterile environment for subsequent cell culture and experimental operations within the chip. After completing the inoculation of cell microspheres and the sterilization and sealing of the chip, select either static or dynamic culture mode according to experimental requirements. If static culture is selected, place the chip in a cell culture incubator and maintain an environment of 37°C and 5% CO2 to ensure continuous cell growth and metabolism on the microspheres. If dynamic culture is selected, connect the chip to a circulating pump system. By precisely controlling the flow rate and volume, the culture medium circulates within the chip chamber, providing a continuous supply of nutrients and removing metabolic waste from the cell microspheres, simulating the dynamic physiological environment in vivo.

[0048] II. Experimental Results 1. Preparation of AHAMA The structure of aldehyde-modified methacrylated hyaluronic acid (AHAMA) was identified by proton nuclear magnetic resonance (¹H-NMR). The structure was determined from the proton nuclear magnetic resonance image (¹H-NMR). Figure 1 As can be seen from the results, aldehyde groups (chemical shifts 4.9 ppm and 5.0 ppm) and methacrylic acid groups (chemical shifts 5.7 ppm and 6.1 ppm) were successfully grafted onto the hyaluronic acid backbone. This result indicates that the synthesis of AHAMA successfully achieved the expected chemical modification.

[0049] 2. Preparation of AHAMA+GelMA microspheres AHAMA+GelMA hydrogel microspheres prepared using microfluidic technology were observed using an optical microscope. Figure 2 The data shows that the microspheres are uniform in size, have intact morphology, and have a particle size of approximately 350 micrometers.

[0050] 3. Chip Design From the plan view of the chip mold ( Figure 3 As can be seen from (A), the chip consists of four independent chambers, each with multiple micropores inside. Figure 3 (B). The chamber is designed to be 1.5 mm deep. Figure 3 (D), the partition height is 1.35 mm ( Figure 3 The design incorporates a 150-micron height difference between the partitions and the top of the chambers. This allows for free liquid flow while restricting microsphere movement, effectively preventing cross-contamination between the microspheres in different chambers. Furthermore, the micropore depth is 0.24 mm (…). Figure 3 The medium (F) can effectively fix the microspheres and limit their disordered flow within the chamber, significantly improving the stability and repeatability of the experiment.

[0051] 4. Chip fabrication Molds made using Formlabs Stereolithography 3D printer and BioMed Clear Resin material ( Figure 4 (A) has high-precision structural features. After molding with PDMS, the edges and corners are cut off to ensure a smooth interface. Figure 4 (B) Drill holes at the corresponding openings on the PDMS film. Figure 4 After plasma treatment, PDMS can achieve good bonding with the substrate. Figure 4 (E). The chamber opening can be sealed with a silicone rubber sealing cap. Figure 4 (F) to ensure the chip's sealing and sterility during cell culture.

[0052] 5. Loading of microspheres Micropores prepared by 3D printing mold casting Figure 5 Both A and B are uniform in size and morphology. After optimizing the micropore diameter, it was found that micropores with a diameter of 0.48 mm could achieve a microsphere loading and retention rate of up to 90%. In the initial static culture state, each micropore can precisely accommodate one microsphere, while in the dynamic culture process, this micropore design can effectively restrict the position of microspheres within the chip, ensuring the uniform distribution and fixation of microspheres within the chip. This design provides a stable and uniform three-dimensional microenvironment for cell culture. Figure 5 (C, D), thus laying a solid foundation for subsequent cell culture and research.

[0053] 6. Chip cultivation We observed cell growth on microspheres within the chip using live / dead staining experiments. The results showed that the GelMA hydrogel microspheres provided excellent initial cell attachment; however, with prolonged culture time, cells gradually migrated from the microsphere surface to the chip surface. Figure 6 (A). In contrast, AHAMA hydrogel microspheres exhibit a significant cell anchoring effect, effectively preventing cells from detaching from the microsphere surface, thereby maintaining cell growth and intercellular interactions within the three-dimensional microsphere environment. However, due to the lack of RGD peptides in AHAMA microspheres, their initial cell attachment efficiency is relatively low ( Figure 6 (B). Further research revealed that by adjusting the ratio of AHAMA to GelMA, when the ratio was 2:1, both the initial cell attachment rate and anchoring rate reached approximately 85%. Figure 6 (C, D) This indicates that the prepared AHAMA+GelMA composite hydrogel microspheres perfectly integrate the advantages of both, achieving an optimal balance in terms of initial cell attachment and anchoring effects. This property is crucial for maintaining cell growth and intercellular interactions in the three-dimensional microsphere environment, significantly improving the accuracy and reliability of experimental results.

[0054] 7. Introduce a dynamic irrigation system Figure 7 This diagram illustrates the integration of the chip with a dynamic perfusion system, demonstrating the slow perfusion of culture medium using a circulating pump. Figure 7 (A) and establishing injection channels on the chip ( Figure 7(B) This invention can effectively simulate the interactions between multiple cells and tissues, providing a more accurate and reliable platform for studying complex physiological and pathological processes. Through dynamic circulatory perfusion, this invention can monitor the physiological and pathological changes of cells in a dynamic environment in real time, providing an experimental environment that more closely resembles in vivo physiological conditions for research such as disease model construction, drug screening, and tissue engineering.

[0055] III. Conclusion This invention successfully solves the problems of cell detachment, disordered microsphere flow, and cross-contamination between different chambers in existing organ-on-a-chip technologies by constructing an AHAMA+GelMA hydrogel microsphere system and optimizing the chip structure design. Experimental results show that the AHAMA+GelMA microspheres have good cell compatibility, excellent initial adhesion rate, and good anchoring effect. The chip design effectively fixes the microspheres and prevents their movement between different chambers, significantly improving the stability and reproducibility of the experiments. These innovative improvements provide new ideas and methods for the development of organ-on-a-chip technology, and have significant scientific value and broad application prospects.

Claims

1. A method for preparing organ-on-a-chip based on microsphere cell culture, characterized in that, Includes the following steps: (1) Aldehyde-modified hyaluronic acid was prepared by oxidation reaction using hyaluronic acid and sodium periodate as raw materials, and then esterified with methacrylic anhydride to obtain aldehyde-modified methacrylic hyaluronic acid. (2) Aldehyde-modified methacrylated hyaluronic acid and methacrylamide gelatin were mixed at a weight ratio of 1:

2. The resulting mixed solution and photoinitiator were used as the continuous phase and emulsified in the dispersed phase. Microdroplets were prepared by microfluidic device and then photocrosslinked and cured to obtain hydrogel microspheres. (3) A mold is prepared by 3D printing, and then the mixture of PDMS base liquid and curing agent is poured into the mold. After curing reaction, the mold is demolded to obtain a PDMS chip mold. (4) Multiple independent chambers are set on the PDMS chip mold, and a partition is set between the multiple independent chambers. The chambers are 100-200 micrometers higher than the partitions. Multiple micropores are set in each independent chamber. The diameter of the micropores is 0.46-0.50 mm. Then, the PDMS mold and the substrate are treated with plasma. After the treatment is completed, the PDMS mold and the substrate are tightly bonded and solidified to prepare an organ chip for cell culture.

2. The method according to claim 1, characterized in that, The mass-to-volume ratio of hyaluronic acid to sodium periodate in step (1) is 1 g: 5 mL, and the oxidation reaction is carried out at room temperature with stirring in the dark for 2 h.

3. The method according to claim 1, characterized in that, The mass-to-volume ratio of aldehyde-modified hyaluronic acid to methacrylic anhydride in step (1) is 1 g: 1 mL, and the esterification reaction is carried out under ice bath with stirring for 12 to 24 h.

4. The method according to claim 1, characterized in that, The degree of amino substitution of the methacrylamide gelatin in step (2) is 90±5%; preferably, the photoinitiator is LAP, Irgacure 2959, Irgacure 754, Irgacure 819 or Darocur 1173; more preferably, the weight ratio of the mixed solution of the aldehyde-modified methacrylamide hyaluronic acid and methacrylamide gelatin to the photoinitiator is 10:

1.

5. The method according to claim 1, characterized in that, The dispersed phase in step (2) is a mixture of 95 wt% paraffin oil and 5 wt% Span 80.

6. The method according to claim 1, characterized in that, The curing agent in step (3) includes a thermosetting agent, a platinum catalyst curing agent, or a silane crosslinking agent; the thermosetting agent is Sylgard 184 or Dow Corning 184; the platinum catalyst curing agent is a Karstedt catalyst or a Speier catalyst; the silane crosslinking agent is 3-methacryloyloxypropyltrimethoxysilane or vinyltrimethoxysilane; preferably, the weight ratio of the PDMS base liquid to the curing agent is 10:1; more preferably, the curing reaction is performed at 60°C for 12 hours.

7. The method according to claim 1, characterized in that, The parameters for plasma treatment in step (4) are: oxygen flow rate of 10-20 sccm, vacuum degree of 100-200 mTorr, power of 50-100 W, and treatment time of 1-3 minutes.

8. The method according to claim 1, characterized in that, The substrate in step (4) is a glass sheet or a PDMS sheet, and the curing and bonding conditions are curing at room temperature for 24 hours; preferably, the chamber is 150 micrometers higher than the partition; more preferably, the height of the chamber is 1.5 mm, the height of the partition is 1.35 mm, the micropore depth is 0.24 mm, and the micropore diameter is 0.48 mm.

9. An organ-on-a-chip prepared by the method according to any one of claims 1-8.

10. The use of the organ-on-a-chip according to claim 9 in in vitro cell culture.

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