A cell spheroid culture organ chip and a preparation method thereof

Abstract

The application relates to the chip design and manufacturing technical field, and provides a cell ball culture organ chip and a preparation method thereof, the chip comprises a first chip structure and a second chip structure; the first chip structure is arranged above the second chip structure; the first chip structure is provided with a medicine inlet, a concentration dispersion structure and a first flow channel array; the medicine inlet is connected with one end of the concentration dispersion structure, and the other end of the concentration dispersion structure is communicated with the first flow channel array; the second chip structure is provided with a second flow channel array and a cell culture cavity array; the cell culture cavity array is distributed in a second flow channel of the second flow channel array; the position of the first flow channel array in the first chip structure corresponds to and overlaps with the position of the second flow channel array in the second chip structure. Based on the embodiment of the application, the concentration dispersion structure is arranged between the medicine inlet and the first flow channel array, a uniform concentration gradient can be formed, the preparation of medicines with different concentrations can be omitted in the medicine screening process, and high-throughput screening can be realized.

Description

Technical Field

[0001] This invention relates to the field of chip design and fabrication technology, and in particular to a cell sphere culture organ-on-a-chip and its preparation method. Background Technology

[0002] Since the first report of organ-on-a-chip technology in 2004, over a decade of research has yielded tremendous success, simulating and demonstrating a wide range of human biological behaviors in unprecedented ways. It holds significant promise for applications in new drug development, toxicity testing, stem cell research, and understanding disease mechanisms. However, planar cultured cells differ from truly in vivo physiological characteristics. Until 2009, the first 3D cell culture system—organoids—emerged. These are highly similar to in vivo source tissues or organs, capable of replicating the complex spatial morphology of differentiated tissues and exhibiting cell-to-cell and cell-to-matrix interactions. Ideally, organoids possess similar physiological responses to in vivo differentiated tissues. Organoids can enable effective detection of drug efficacy and toxicity. Furthermore, they can be directly generated from human cells, avoiding unreliable test results due to differences between animal and human cells and reducing the need for animal use in clinical trials. However, existing organoids have many shortcomings in drug screening, such as the difficulty in achieving high-throughput screening. Summary of the Invention

[0003] This application provides a cell sphere culture organ-on-a-chip and its preparation method, which can form a uniform concentration gradient, eliminate the need for preparing drugs of different concentrations during drug screening, and achieve high-throughput screening.

[0004] This application provides a cell sphere culture organ chip, including: a first chip structure and a second chip structure;

[0005] The first chip structure is positioned above the second chip structure;

[0006] The first chip structure includes a drug inlet, a concentration dispersion structure, and a first flow channel array.

[0007] The drug inlet is connected to one end of the concentration dispersion structure, and the other end of the concentration dispersion structure is connected to the first flow channel array.

[0008] The second chip structure includes a second flow channel array and a cell culture chamber array;

[0009] The cell culture chamber array is distributed within the second channel of the second flow channel array;

[0010] The position of the first channel array on the first chip structure corresponds to the position of the second channel array on the second chip structure.

[0011] Furthermore, the first chip structure is provided with multiple first sample inlet / outlet groups;

[0012] Each of the multiple first inlet / outlet groups includes a first inlet / outlet and a second inlet / outlet;

[0013] For any one of the first channels in the first channel array, the first sample inlet / outlet is set at one end of any one of the first channels;

[0014] The second inlet / outlet is located at the other end of any one of the first flow channels;

[0015] The first and second sample inlets / outlets are disposed on the first chip structure.

[0016] Furthermore, the second chip structure is provided with multiple second sample inlet / outlet groups;

[0017] Each of the multiple second inlet / outlet groups includes a third inlet / outlet and a fourth inlet / outlet;

[0018] The first inlet / outlet in the first inlet / outlet group corresponds one-to-one with the third inlet / outlet in the second inlet / outlet group, and the second inlet / outlet in the first inlet / outlet group corresponds one-to-one with the fourth inlet / outlet in the second inlet / outlet group.

[0019] The position of the third sample inlet / outlet on the second chip structure corresponds to the position of the first sample inlet / outlet on the first chip structure, and the position of the fourth sample inlet / outlet on the second chip structure corresponds to the position of the second sample inlet / outlet on the second chip structure.

[0020] For any one of the second channels in the second channel array, the third inlet / outlet is connected to one end of any one of the second channels. The third inlet / outlet is used to inject or export the cell suspension to be cultured into the second channel.

[0021] The fourth inlet / outlet is connected to the other end of the second flow channel. The fourth inlet / outlet is used to inject or discharge culture medium into the second flow channel.

[0022] Furthermore, the first channel in the first channel array corresponds one-to-one with the second channel in the second channel array;

[0023] The first channel spacing in the first channel array is set on the first chip structure;

[0024] The second channel spacing in the second channel array is set on the second chip structure;

[0025] The multiple first channels in the first channel array are isolated from each other, and the corresponding first channels are connected to the second channels;

[0026] The positions of the first flow channel on the first chip structure and the positions of the second flow channel on the second chip structure are corresponding and coincide.

[0027] Furthermore, the concentration dispersion structure includes multiple concentration dispersion layers;

[0028] Each of the multiple concentration dispersion layers corresponds one-to-one with the first flow channel in the first flow channel array.

[0029] The concentration dispersion layer, which corresponds to a single concentration, is connected to the first flow channel.

[0030] The concentration of the drug flowing through each of the multiple concentration dispersion layers is different.

[0031] Furthermore, the diameter of the first inlet / outlet is smaller than the diameter of the second inlet / outlet;

[0032] The diameter of the first inlet / outlet is within the range [1.5mm, 2.5mm];

[0033] The diameter of the second inlet / outlet is within the range of [3.5mm, 4.5mm].

[0034] Furthermore, the cell culture chambers in the cell culture chamber array are U-shaped grooves with their openings facing upwards;

[0035] The diameter of the cell culture chamber is within the range of [550μm, 650μm];

[0036] The depth of the cell culture chamber is within the range of [550 μm, 650 μm].

[0037] Accordingly, embodiments of this application provide a method for preparing a cell sphere culture organ-on-a-chip, comprising:

[0038] Obtain the chip to be processed and the mold; the mold has a channel array region to be prepared and a groove to be injected in each channel to be prepared in the channel array to be prepared.

[0039] Photolithography is performed on the chip to be processed to fabricate a drug inlet, a concentration dispersion structure and a first flow channel array on the chip to be processed, thereby obtaining a first chip structure;

[0040] Polydimethylsiloxane is cured in a mold to obtain a second chip structure; the second chip structure includes a second flow channel array and a cell culture chamber array, with the cell culture chamber array distributed within the second flow channel of the second flow channel array.

[0041] The first and second chip structures were bonded together to obtain a cell sphere culture organ chip.

[0042] Further, polydimethylsiloxane is cured in the mold to obtain a second chip structure, including:

[0043] Polydimethylsiloxane is injected into the mold and left to stand until the polydimethylsiloxane solidifies to obtain the chip structure to be prepared.

[0044] Polydimethylsiloxane is injected into the groove to be injected in the chip structure to be prepared. Excess polydimethylsiloxane is removed. Surface tension is used to make the polydimethylsiloxane adhere to the sidewalls and bottom of the groove to be injected, forming a U-shaped groove, thus obtaining the second chip structure.

[0045] Furthermore, the chip to be processed is subjected to photolithography to fabricate a drug inlet, a concentration dispersion structure, and a first flow channel array on the chip to be processed, and after obtaining the first chip structure, the process further includes:

[0046] Drop tridecylfluorooctyltriethoxyalkylene onto a glass slide;

[0047] The glass slide and the first chip structure are placed in a vacuum for vacuum treatment, so that tridecylfluorooctyltriethoxyalkylene adheres to the surface of the first chip structure.

[0048] The embodiments of this application have the following beneficial effects:

[0049] This application provides a data processing method, apparatus, electronic device, and storage medium, including a first chip structure and a second chip structure. The first chip structure is disposed above the second chip structure. The first chip structure has a drug inlet, a concentration dispersion structure, and a first flow channel array. The drug inlet is connected to one end of the concentration dispersion structure, and the other end of the concentration dispersion structure is connected to the first flow channel array. The second chip structure has a second flow channel array and a cell culture chamber array. The cell culture chamber array is distributed within the second flow channel of the second flow channel array. The positions of the first flow channel array and the second flow channel array on the first chip structure correspond and coincide. Based on this application embodiment, by setting a concentration dispersion structure between the drug inlet and the first flow channel array, a uniform concentration gradient can be formed, eliminating the need for preparing drugs of different concentrations during drug screening and enabling high-throughput screening. Attached Figure Description

[0050] To more clearly illustrate the technical solutions and advantages in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 This is a schematic diagram of the structure of a cell sphere culture organ-on-a-chip provided in an embodiment of this application;

[0052] Figure 2This is an effect verification diagram of a concentration dispersion structure provided in an embodiment of this application;

[0053] Figure 3 This is a linear relationship diagram of a concentration dispersion structure provided in an embodiment of this application;

[0054] Figure 4 This is a schematic diagram of injecting a suspension of cells to be cultured, provided in an embodiment of this application;

[0055] Figure 5 This is a schematic flowchart of a method for preparing a cell sphere culture organ chip according to an embodiment of this application;

[0056] Figure 6 This is a schematic diagram illustrating the fabrication of a first chip structure provided in an embodiment of this application;

[0057] Figure 7 This is a schematic diagram illustrating the fabrication of a second chip structure provided in an embodiment of this application;

[0058] Figure 8 This is a cross-sectional schematic diagram of a groove to be injected before processing, provided in an embodiment of this application;

[0059] Figure 9 This is a schematic cross-sectional view of a groove to be injected, provided in an embodiment of this application;

[0060] Figure 10 This is a bright-field image of cell culture after 7 days, provided in an embodiment of this application;

[0061] Figure 11 This is a distribution diagram of cell spheroid diameter provided in an embodiment of this application. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings. Obviously, the described embodiments are merely one embodiment of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0063] The term "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of this application. In the description of the embodiments of this application, it should be understood that the terms "first," "second," and "third," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," and "third," etc., may explicitly or implicitly include one or more of that feature. Furthermore, the terms "first," "second," and "third," etc., are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of this application described herein can be implemented in orders other than those illustrated or described herein. In addition, the terms "comprising," "having," and "being," and any variations thereof, are intended to cover non-exclusive inclusion.

[0064] The development of cell biology, molecular biology, and other disciplines has provided new methods for drug screening, and numerous molecular and cellular-level drug screening models have emerged and been applied in drug research and screening practices. The main advantages of molecular and cellular-level drug screening models lie in achieving high-throughput sample screening and enabling multiple screenings of a single drug. Although traditional two-dimensional cell culture has been used in biomedical research for many years and has significant value, it cannot study tissue specificity, differentiation function, or accurately predict certain functions of in vivo tissues or drug biopsies. However, the development of organ-on-a-chip and organoid technologies enables the use of multi-cell co-culture and 3D cell culture to simulate the in vivo microenvironment, making the cell growth environment closer to the physiological environment and cellular functional characteristics, thereby making the drug screening results more accurate and reliable.

[0065] Organ-on-a-chip, a microfluidic chip platform, is based on in vitro cell culture to construct small organ models. Under appropriate growth factors and mechanical conditions, cells differentiate and self-organize into organ-specific cell types and tissues, reproducing organ structure and function. This allows for research in human biology and pathology. An organ-on-a-chip is a microfluidic device for in vitro tissue culture under relevant physiological and microenvironment engineering conditions. This device is called a "chip" because it integrates controllable channels, various pumps, valves, circuits, and sensors based on the principle of microfluidic chip technology. It is called an "organ" because it involves stimulating and simulating organ function through the microenvironment. For example, simulating fluid shear forces in blood vessels to culture vascular cells, or simulating circulatory physical load stretching to culture cardiomyocytes. In organ-on-a-chip models, as the type and size of organoids increase, their cores move further away from the surface when in contact with fresh culture medium. This makes the simple diffusion process insufficient to provide sufficient oxygen and nutrients to the growing cells, and also limits the timely removal of metabolic waste by the fluid. This results in only cells in contact with fresh culture medium growing well. With the continuous advancement of organ-on-a-chip technology, temperature and pH levels can be optimized, and the delivery of nutrients, oxygen, and waste removal can be carried out in a controlled environment. Compared to animal models, organ-on-a-chip models can be scaled up, enabling high-throughput testing at a lower cost while reducing ethical concerns. In summary, in organ-on-a-chip design, the structure of the channels, the shear forces from the fluid, the types of cells, relevant cell growth factors, and cell-cell and cell-tissue interactions all affect the biomimicry of the model. Therefore, in the construction of organ-on-a-chip systems, key mechanical conditions and environmental factors should be determined and controlled using methods from engineering mechanics and cell biology to obtain normal biological responses from the model, and to provide cells and tissues with more realistic expressions and more accurate data closer to the human body than traditional in vitro models.

[0066] The following describes a specific embodiment of a cell spheroid culture organ-on-a-chip based on this application. Figure 1 This is a schematic diagram of a cell spheroid culture organ-on-a-chip provided in an embodiment of this application. This specification provides the structural composition as shown in the embodiments or accompanying drawings, but based on conventional or non-inventive labor, more or fewer mechanisms, modules, or structures may be included. The structural composition listed in the embodiments is merely one of many possible compositions and does not represent the only possible composition. In actual implementation, the structural composition shown in the embodiments or accompanying drawings can be followed.

[0067] Specifically, such as Figure 1As shown, the cell spheroid culture organ-on-a-chip may include a first chip structure 100 and a second chip structure 200. The first chip structure 100 may be disposed above the second chip structure 200. The first chip structure 100 may be provided with a drug inlet 110, a concentration dispersion structure 120, and a first flow channel array 130. The drug inlet 110 may be connected to one end of the concentration dispersion structure 120, and the other end of the concentration dispersion structure 120 may be connected to the first flow channel array 130. The second chip structure 200 may be provided with a second flow channel array 210 and a cell culture chamber array 220, the cell culture chamber array 220 being distributed within the second flow channel of the second flow channel array 210. The position of the first flow channel array 130 on the first chip structure 100 may correspond to the position of the second flow channel array 210 on the second chip structure 200.

[0068] In some possible implementations, the first chip structure 100 has an upper surface and a lower surface, and the second chip structure 200 has an upper surface and a lower surface. The upper surface of the first chip structure 100 may be provided with a drug inlet 110, a concentration dispersion structure 120, and a first flow channel array 130. The upper surface of the second chip structure 200 may be provided with a second flow channel array 210 and a cell culture chamber array 220. The lower surface of the first chip structure 100 may be bonded to the upper surface of the second chip structure 200. Optionally, the first chip structure 100 and the second chip structure 200 may be bonded using plasma to obtain a cell sphere culture organ chip.

[0069] In practical applications, since cell spheroid culture organ-on-a-chip utilizes three-dimensional cell culture technology, the materials used in these chips need to be low in toxicity, biocompatibility, and permeability. Furthermore, the materials must be easy to process, possess a certain degree of elasticity, and be easy to seal. Optionally, polydimethylsiloxane (PDMS) can be selected, as it possesses characteristics such as low toxicity, good permeability, good optical properties, strong biocompatibility, low cost, and ease of processing. To meet experimental requirements, six first channels can be arranged on the first chip structure 100, forming a first channel array 130, and six second channels can be arranged on the second chip structure 200, forming a second channel array 210. Each second channel can contain 96 cell culture chambers. Alternatively, each second channel can have three rows of cell culture chambers, with 29 cell culture chambers per row.

[0070] In some possible implementations, the diameter of the inlet 110 can be 2 mm, the length of the first channel in the first channel array 130 can be 2 cm, and the width can be 3 mm. The length of the second channel in the second channel array 210 can be 2 cm, and the width can be 3 mm. That is, the dimensions of the channels in the first chip structure 100 can be the same as the dimensions of the channels in the second chip structure 200.

[0071] In this embodiment, the first chip structure 100 may be provided with a plurality of first sample inlet / outlet groups 140. Each of the plurality of first sample inlet / outlet groups 140 may include a first sample inlet / outlet 141 and a second sample inlet / outlet 142. For any one of the first channels in the first channel array 130, the first sample inlet / outlet 141 may be disposed at one end of the first channel, and the second sample inlet / outlet 142 may be disposed at the other end of the first channel. Furthermore, both the first sample inlet / outlet 141 and the second sample inlet / outlet 142 may be disposed throughout the first chip structure 100.

[0072] In some possible implementations, the diameter of the first inlet / outlet 141 may be smaller than the diameter of the second inlet / outlet 142. The diameter of the first inlet / outlet 141 may be within the range of [1.5mm, 2.5mm], and the diameter of the second inlet / outlet 142 may be within the range of [3.5mm, 4.5mm]. Optionally, the diameter of the first inlet / outlet 141 may be 2mm, and the diameter of the second inlet / outlet 142 may be 4mm.

[0073] In this embodiment, the second chip structure 200 may be provided with a plurality of second sample inlet / outlet groups 230. Each of the plurality of second sample inlet / outlet groups 230 may include a third sample inlet / outlet 231 and a fourth sample inlet / outlet 232. The first sample inlet / outlet 141 in the first sample inlet / outlet group 140 may correspond one-to-one with the third sample inlet / outlet 231 in the second sample inlet / outlet group 230, and the second sample inlet / outlet 142 in the first sample inlet / outlet group 140 may correspond one-to-one with the fourth sample inlet / outlet 232 in the second sample inlet / outlet group 230. The position of the one-to-one corresponding third sample inlet / outlet 231 on the second chip structure 200 may coincide with the position of the first sample inlet / outlet 141 on the first chip structure 100, and the position of the one-to-one corresponding fourth sample inlet / outlet 232 on the second chip structure 200 may coincide with the position of the second sample inlet / outlet 142 on the first chip structure 100. For any one of the second channels in the second channel array 210, the third inlet / outlet 231 can be connected to one end of that second channel. The third inlet / outlet 231 can be used to inject the cultured cell suspension into the second channel, and can also be used to remove the cultured cell suspension from the second channel. The fourth inlet / outlet 232 can be connected to the other end of that second channel. The fourth inlet / outlet 232 can be used to inject culture medium into the second channel, and can also be used to remove culture medium from the second channel.

[0074] In some possible implementations, the diameter of the third inlet / outlet 231 may be smaller than the diameter of the fourth inlet / outlet 232. The diameter of the third inlet / outlet 231 may be within the range of [1.5mm, 2.5mm], and the diameter of the fourth inlet / outlet 232 may be within the range of [3.5mm, 4.5mm]. Optionally, the diameter of the third inlet / outlet 231 may be 2mm, and the diameter of the fourth inlet / outlet 232 may be 4mm.

[0075] In some possible implementations, the cell culture chambers in the cell culture chamber array 220 can be U-shaped grooves with their openings facing upwards. The diameter of the cell culture chamber can be within the range of [550 μm, 650 μm], and the depth of the cell culture chamber can be within the range of [550 μm, 650 μm]. Optionally, the opening diameter of the cell culture chamber can be 600 μm, and the depth can be 600 μm.

[0076] In this embodiment, the first channels in the first channel array 130 can correspond one-to-one with the second channels in the second channel array 310. The first channels in the first channel array 130 can be spaced apart on the first chip structure 100, and the second channels in the second channel array 310 can be spaced apart on the second chip structure 200. The multiple first channels in the first channel array 130 are isolated from each other, and the multiple second channels in the second channel array 310 are also isolated from each other. The one-to-one corresponding first and second channels can be connected, and the positions of the one-to-one corresponding first channels on the first chip structure can correspond to and coincide with the positions of the second channels on the second chip structure. Optionally, the six channels on the first chip structure can be relatively independent and spaced apart, and the six channels on the second chip structure can be relatively independent and spaced apart.

[0077] In this embodiment, the concentration dispersion structure 120 may include multiple concentration dispersion layers. Each concentration dispersion layer corresponds one-to-one with a first flow channel in the first flow channel array 130. The corresponding concentration dispersion layers are connected to the first flow channels, and the concentration of the drug flowing through each concentration dispersion layer is different. Optionally, the concentration dispersion structure 120 may be distributed in a stepped manner, and the height of the concentration dispersion structure 120 may be 100 μm.

[0078] Figure 2 This is an effect verification diagram of a concentration dispersion structure provided in an embodiment of this application. Figure 3 This is a linear relationship diagram of a concentration dispersion structure provided in an embodiment of this application. A syringe pump connected to one of the inlets 110 can inject a PBS buffer solution, and a syringe pump connected to the other inlet 110 can inject a 0.1% FITC isothiocyanate solution. The lengths of the connecting tubes for the two inlets can be kept equal, and the volumes of liquid in the syringe pumps can be equal. When the syringe pump is turned on, a flow rate of 30 μL / min can be achieved. -1 Two solutions were injected into the concentration dispersion structure 120 at a certain rate. After the solution filled the entire concentration dispersion layer of the concentration dispersion structure 120, the concentration dispersion layer was photographed using a fluorescence microscope. The fluorescence images were then analyzed quantitatively using ImageJ software, and a concentration curve of the concentration dispersion layer was plotted. Figure 3 It can be seen that the optical density of FITC in multiple concentration dispersion layers can form a straight line, exhibiting a good linear relationship, with the linearity coefficient R0. 2 The value is 0.9421. This indicates that the concentration dispersion structure of cell sphere culture organ-on-a-chip has good dispersibility and can form a uniform concentration gradient. In the drug screening process, it can eliminate the need to prepare drugs of different concentrations and achieve high-throughput screening.

[0079] Figure 4This is a schematic diagram illustrating the injection of a cell suspension to be cultured, provided in an embodiment of this application. A 20 μL pipette is used to slowly and uniformly inject the cell suspension into the second flow channel through the first inlet / outlet. Due to gravity, the cells settle into the cell culture chamber array at the bottom of the second chip structure, specifically within the U-shaped grooves of the second chip structure. Repeating the above steps three times maintains a stable flow rate of the cell suspension during injection, ensuring the uniformity of cell settling velocity in the flow channel and the number of cells settling in each U-shaped groove.

[0080] Using the cell sphere culture organ-on-a-chip provided in this application, a uniform concentration gradient can be formed by setting a concentration dispersion structure between the drug inlet and the first flow channel array. In the drug screening process, the preparation of drugs of different concentrations can be eliminated, the effects of drug pairs of different concentrations can be detected simultaneously, high-throughput screening can be achieved, and the efficiency of drug detection can be improved.

[0081] The following describes a specific embodiment of the method for preparing a cell sphere culture organ-on-a-chip according to this application. Figure 5 This is a schematic flowchart illustrating a method for preparing a cell sphere culture organ-on-a-chip according to an embodiment of this application. This specification provides method steps as shown in the embodiments or accompanying drawings, but based on conventional or non-inventive labor, more or fewer steps may be included. The method steps listed in the embodiments are merely one of many methods and do not represent the only method steps. In actual implementation, the method steps shown in the embodiments or accompanying drawings can be followed.

[0082] Specifically, such as Figure 5 As shown, the preparation method of cell sphere culture organ-on-a-chip may include:

[0083] S501: Obtain the chip to be processed and the mold; the mold has a channel array region to be prepared and an injection groove set in each channel to be prepared in the channel array to be prepared.

[0084] In this embodiment, a silicon wafer can be used as the chip to be processed, and the silicon wafer can be pre-treated. Specifically, after the silicon wafer is removed from the wafer cassette, its surface can be gently blown with nitrogen gas to remove dust and other substances adhering to the surface of the silicon wafer. Then, the silicon wafer can be placed in a plasma cleaning device for cleaning to remove organic matter adhering to the surface of the silicon wafer, so that the photoresist subsequently coated on the silicon wafer can adhere well to the surface of the silicon wafer.

[0085] S503: Perform photolithography on the chip to be processed to fabricate a drug inlet, a concentration dispersion structure and a first flow channel array on the chip to be processed, thereby obtaining a first chip structure.

[0086] In this embodiment, after preprocessing the chip to be processed, a photoresist coating process can be performed. Before coating, a coin-sized amount of photoresist can be poured onto the center of the silicon wafer, and the wafer can be manually rotated to spread the photoresist as much as possible on the wafer. Then, the chip to be processed can undergo pre-baking, photolithography, post-baking, and development processes to form a drug inlet, a concentration dispersion structure, and a first flow channel array on the chip, resulting in a first chip structure.

[0087] Figure 6 This is a schematic diagram illustrating the fabrication of a first chip structure according to an embodiment of this application. In actual operation, since the channel height of the chip to be processed is required to be within the range [100μm, 150μm], the spin coater parameters can be set to an initial rotation speed of 500 rpm. After 15 seconds, the spin coater speed can be set to 1000 rpm and maintained for 30 seconds. Then, the silicon wafer can be placed in the center of the spin coater turntable, and the wafer can be sucked in. Simultaneously, SU-8 3050 photoresist is applied to the center of the silicon wafer for spin coating. Care should be taken to avoid the formation of air bubbles during the operation. After completion, air bubbles can be carefully removed, and the silicon wafer can be covered with an aluminum foil cover (using a petri dish lid) to block light and left overnight. Then, the hot plate can be leveled, and the silicon wafer can be transferred to the hot plate. The temperature of the hot plate can be set to 65°C, heated for 10 minutes to increase the temperature to 95°C, and then heated for another 30 minutes to allow the organic solvent in the photoresist to evaporate. Finally, the hot plate can be turned off, allowing the silicon wafer to cool naturally to room temperature. After pre-baking the chip, the cooled silicon wafer can be fixed on the photolithography platform, and the photomask can be aligned and covered. Exposure is then performed at a wavelength of 300-350nm for a period of time, maintaining the exposure energy within the range of 150mJ·cm⁻¹. -2 -250mJ·cm -2 The exposure time can be determined by the intensity of the mercury lamp and the thickness of the photoresist. The specific calculation formula is as follows:

[0088]

[0089] Where t represents the exposure time in seconds, Δx represents the photoresist thickness in micrometers, and E represents the ultraviolet light intensity of the mercury lamp, which can be measured by a specific light intensity measuring instrument.

[0090] After photolithography, the chip can undergo post-baking. The UV-exposed silicon wafer is placed on a thermal baking plate, set to 65°C, heated for 1 minute, then increased to 95°C, and heated for another 10 minutes to allow the photoresist in the exposed areas to cross-link. The thermal plate is then turned off, allowing the wafer to cool naturally to room temperature. Next, the chip can be developed. Propylene glycol methyl ether acetate (PGMEA) developer is poured into a glass dish, and the post-baked silicon wafer is placed inside, ensuring the liquid level covers the wafer's surface. The dish is gently agitated to dissolve the unexposed photoresist. This cleaning process takes approximately 10-25 minutes. The cleaning progress needs to be continuously monitored. Once all unexposed areas have dissolved, the wafer can be removed with tweezers, taking care not to touch the patterned areas. Finally, nitrogen gas can be used to gently blow nitrogen onto the wafer's surface to remove any remaining developer. After drying, the silicon wafer can be placed in fresh PGMEA for approximately 10 seconds, then removed. The upper surface of the wafer is then gently blew with nitrogen gas, and this cleaning and drying process is repeated until the upper surface is dry and free of residual photoresist. Next, the wafer can be flipped over, and the lower surface rinsed with fresh PGMEA and gently blew with nitrogen gas. This cleaning and drying process is repeated until the lower surface is dry and free of residual photoresist, yielding the first chip structure. A drop of PGMEA can then be placed in the center of a petri dish, and the first chip structure placed in the dish to adhere to it.

[0091] After developing the chip, a hardening and surface hydrophobic treatment can be performed. Specifically, the developed silicon wafer can be placed on a hot baking plate and baked at 180°C for 2 hours to evaporate any residual solvent in the photoresist film. Hardening the chip allows the modified photoresist to bond more firmly to the silicon wafer, enhancing the etching resistance of the first chip structure. During the surface hydrophobic treatment, a drop of tridecafluorooctylsilane can be dropped onto a glass slide, and the slide and silicon wafer can be placed together in a vacuum chamber. The vacuum chamber can then be evacuated and maintained for 30 minutes, allowing the tridecafluorooctylsilane to adhere to the surface of the first chip structure. Multiple first sample inlet / outlet groups can then be formed on the first chip structure. For example, a first sample inlet / outlet can be formed at one end of any first channel in the first channel array, and a second sample inlet / outlet can be formed on the other side of that first channel. The diameter of the first inlet / outlet can be smaller than the diameter of the second inlet / outlet. The diameter of the first inlet / outlet can be within the range of [1.5mm, 2.5mm], and the diameter of the second inlet / outlet can be within the range of [3.5mm, 4.5mm]. Optionally, the diameter of the first inlet / outlet can be 2mm, and the diameter of the second inlet / outlet can be 4mm.

[0092] S505: Polydimethylsiloxane is cured in a mold to obtain a second chip structure; the second chip structure includes a second flow channel array and a cell culture chamber array, the cell culture chamber array being distributed within the second flow channel of the second flow channel array.

[0093] In this embodiment, PDMS A solution and PDMS B solution are mixed at a ratio of 10:1 and stored in a clean disposable cup. The higher the proportion of PDMS B solution, the greater the mechanical strength of the solidified substance. After thorough mixing, the mixture is placed in a vacuum chamber for 30 minutes to remove air bubbles. After removing the air bubbles, the mixture is poured into a culture dish containing a silicon wafer and subjected to vacuum treatment again to remove air bubbles. The culture dish is then placed on a hot plate and heated at 65°C for 4 hours to cure the PDMS.

[0094] Figure 7 This is a schematic diagram illustrating the fabrication of a second chip structure according to an embodiment of this application. The second chip structure can be created using Computer-Aided Design (CAD) technology, resulting in a 3D model. This model is then sent to a 3D printing company for high-precision 3D printing to obtain a plastic printing mold. This mold can have a channel array region to be fabricated and injection grooves set within each channel in the channel array. The mold can then be fixed in a petri dish, and the PDMS curing step can be repeated. Figure 8 This is a cross-sectional schematic diagram of a groove to be injected before processing, provided in an embodiment of this application. Figure 9This is a cross-sectional schematic diagram of a groove to be injected after processing, provided in an embodiment of this application. Since the walls of the groove are not smooth, which is not conducive to cell spheroid formation, uncured PDMS can be dropped onto the groove, and a vacuum process can be performed to allow the uncured PDMS to enter the groove. Then, excess PDMS can be scraped off with a coverslip. Based on the surface tension of PDMS, some PDMS will adhere to the sidewalls and bottom of the groove, forming a U-shaped groove, i.e., a cell spheroid culture chamber array. Then, in a clean bench, the solidified PDMS adhesive can be carefully cut along the border using a cutter, and wrapped with a clean plastic film. Care should be taken not to scratch the silicon wafer surface or damage the shape of the channels within the chip during cutting. Then, holes are punched according to the designed dimensions using a punching pen and puncher to create the second inlet / outlet group, obtaining the second chip structure. Specifically, a third inlet / outlet can be opened at one end of any second channel in the second channel array, and a fourth inlet / outlet can be opened at the other end of the second channel. The diameter of the third inlet / outlet can be smaller than the diameter of the fourth inlet / outlet. The diameter of the third inlet / outlet can be within the range of [1.5mm, 2.5mm], and the diameter of the fourth inlet / outlet can be within the range of [3.5mm, 4.5mm]. Optionally, the diameter of the third inlet / outlet can be 2mm, and the diameter of the fourth inlet / outlet can be 4mm. The third inlet / outlet can be connected to one end of any of the second channels. The third inlet / outlet can be used to inject the cultured cell suspension into the second channel, and it can also be used to remove the cultured cell suspension from the second channel. The fourth inlet / outlet can be connected to the other end of any of the second channels. The fourth inlet / outlet can be used to inject culture medium into the second channel, and it can also be used to remove culture medium from the second channel.

[0095] S507: The first chip structure and the second chip structure are bonded together to obtain a cell sphere culture organ chip.

[0096] In this embodiment, before bonding, the inner cavity of the plasma cleaning equipment can be cleaned with 75% ethanol. After cleaning and drying, the patterned sides of the first and second chip structures are placed in the inner cavity of the plasma cleaning equipment. The vacuum pump is turned on, the chamber door of the plasma cleaning equipment is closed, and the three-way valve is rotated to connect the chamber to the pump. Vacuuming is performed for 1-2 minutes. At the same time, the Power button is pressed and the setting is adjusted to High. When glowing is observed in the inner cavity (i.e., gray or purplish-red light appears in the cavity), the timer is set for 1 minute. After 1 minute, the setting is turned back to Off, and the Power button is turned off. The three-way valve is rotated to turn off the vacuum pump and connect the chamber to the atmosphere. The first and second chip structures are removed, the first and second flow channel arrays are aligned, and the first and second inlet / outlet groups are aligned and pressed. After two minutes, 0.5% polyether F127 is dripped into the well on one side of the cell culture organ chip to maintain the hydrophilicity of the chip, and the chip is placed in a 4°C refrigerator overnight.

[0097] During the experiment, to ensure that cells grow as spheres in the grooves rather than adhering to the wall, the cell sphere culture organochip needs to be modified. After the cell sphere culture organochip is bonded, 0.5% polyether F127 can be added to the first inlet / outlet on one side of the chip. Due to gravity, F127 can flow from the first inlet / outlet on one side through the third inlet / outlet, into the second channel, and then through the fourth inlet / outlet, exiting from the second inlet / outlet on the other side. However, due to surface tension, the gas in the cell culture chamber cannot be completely expelled in time. To remove the air bubbles, the chip filled with F127 can be placed in a vacuum chamber and evacuated for 30 minutes to allow the gas in the cell culture chamber to be completely expelled. Then, the chip can be placed in a 4°C refrigerator overnight. The next day, F127 can be aspirated, Thermo Fisher PBS solution can be added, and PBS can be aspirated from the first and second inlets / outlets until the PBS in the first and second inlets / outlets is basically dried out. During this process, the second channel needs to be kept full of PBS liquid. Then, culture medium can be added to the second channel through the second inlet and outlet. Similarly, about 60 μL of culture medium is aspirated from the second channel through the first inlet and outlet so that the PBS liquid in the second channel is aspirated and placed in an incubator for later use.

[0098] Then, cell seeding can be performed on the cell-sphere culture organ-on-a-chip. Cell counts are performed on suspensions of human umbilical vein endothelial cells (HUVECs) and human liver cancer cells (HepaRGs). The counted cells are then mixed at a 1:3 ratio, and the cell density is adjusted. The cells are then thoroughly mixed by pipetting. Using a 20mm pipette, the mixed cell suspension is drawn up and injected at a constant speed into the six secondary channels of the secondary channel array through the smaller diameter first inlet / outlet. After all six channels are filled, the cells are allowed to settle naturally to the bottom of the cell culture chamber. This process of injecting 20mm cell suspensions is repeated three times. Once all cells have settled, the cell-sphere culture organ-on-a-chip is placed in an incubator. After 2 hours of culture, the culture medium is aspirated through the larger diameter second inlet / outlet, and fresh culture medium is added. The culture medium is then changed daily. After 8 days of culture, liver-related functional protein detection and drug toxicity testing experiments can be performed.

[0099] After the cell suspension is added to the cell spheres for organ-on-a-chip culture, the cells can settle and aggregate in the cell culture chamber. After about 3 days of culture, the cells can form dense cell spheres, and the size of the cell spheres shrinks as the cell clusters become more compact. Figure 10 This is a bright-field image of cell culture after 7 days, provided in an embodiment of this application. It shows that cell growth tends to stabilize after 5-7 days, maintaining a value of approximately 110. After 8 days of cell growth, a large-scale image can be taken using a microscope, capturing the entire flow channel and stitching the images together. Then, the diameter of the cell spheroids in the flow channels is measured using the microscope's built-in software, and the data is statistically analyzed and plotted. Figure 11 This is a distribution diagram of cell spheroid diameter provided in an embodiment of this application. Cells can form uniformly sized cell spheroids in the cell culture chamber, with the diameter of the cell spheroids distributed at approximately 120 μm. Many studies have confirmed that when the diameter of the cell spheroids exceeds 200 μm, due to the lack of vascular structures, the cells in the central part of the cell spheroid cannot absorb nutrients, thus leading to necrosis. Therefore, maintaining a cell size of approximately 110 μm is considered ideal.

[0100] It should be noted that the order of the embodiments described above is merely for descriptive purposes and does not represent the superiority or inferiority of the embodiments. Furthermore, while this specification describes specific embodiments, other embodiments are also within the scope of the appended claims. In some cases, the actions or steps described in the claims can be performed in the order shown in different embodiments and still achieve the desired results. Additionally, the processes depicted in the drawings do not necessarily require a specific order or sequence of connections to achieve the desired results; in some implementations, parallel processing of multiple tasks is possible or may be advantageous.

[0101] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to mutually. Each embodiment focuses on describing the differences from other embodiments. In particular, embodiments of apparatus and electronic devices are described simply because they are based on similar method embodiments; relevant parts can be referred to the descriptions of the method embodiments.

[0102] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A cell sphere culture organ-on-a-chip, characterized in that, include: First chip structure and second chip structure; The first chip structure is disposed on top of the second chip structure; The first chip structure is provided with a drug inlet, a concentration dispersion structure, and a first flow channel array; The drug inlet is connected to one end of the concentration dispersion structure, and the other end of the concentration dispersion structure is connected to the first flow channel array; The first chip structure is provided with multiple first sample inlet / outlet groups; Each of the multiple first inlet / outlet groups includes a first inlet / outlet and a second inlet / outlet; For any one of the first channels in the first channel array, the first sample inlet / outlet is located at one end of any one of the first channels; The second sample inlet / outlet is located at the other end of any one of the first flow channels; The first and second sample inlets / outlets are disposed through the first chip structure; The second chip structure is provided with a second flow channel array and a cell culture chamber array; The cell culture chamber array is distributed within the second channel of the second channel array; The second chip structure is provided with multiple second sample inlet / outlet groups; Each of the multiple second inlet / outlet groups includes a third inlet / outlet and a fourth inlet / outlet; The first inlet / outlet in the first inlet / outlet group corresponds one-to-one with the third inlet / outlet in the second inlet / outlet group, and the second inlet / outlet in the first inlet / outlet group corresponds one-to-one with the fourth inlet / outlet in the second inlet / outlet group. The position of the third sample inlet / outlet on the second chip structure corresponds to the position of the first sample inlet / outlet on the first chip structure, and the position of the fourth sample inlet / outlet on the second chip structure corresponds to the position of the second sample inlet / outlet on the second chip structure. For any one of the second channels in the second channel array, the third inlet / outlet is connected to one end of the second channel and is used to inject or export the cell suspension to be cultured into or out of the second channel. The fourth inlet / outlet is connected to the other end of the second flow channel, and the fourth inlet / outlet is used to inject or discharge culture medium into the second flow channel. The position of the first channel array in the first chip structure corresponds to the position of the second channel array in the second chip structure; The first flow channel in the first flow channel array corresponds one-to-one with the second flow channel in the second flow channel array; The first channel spacing in the first channel array is disposed on the first chip structure; The second channel spacing in the second channel array is disposed on the second chip structure; The multiple first channels in the first channel array are isolated from each other, and the corresponding first channels are connected to the second channels. The positions of the first flow channel on the first chip structure and the positions of the second flow channel on the second chip structure are correspondingly coincident.

2. The chip according to claim 1, characterized in that, The concentration dispersion structure includes multiple concentration dispersion layers; Each of the multiple concentration dispersion layers corresponds one-to-one with a first flow channel in the first flow channel array. The concentration dispersion layer, which corresponds to the first flow channel, is connected to it. The concentration of the drug flowing through each of the multiple concentration dispersion layers is different.

3. The chip according to claim 1, characterized in that, The diameter of the first inlet / outlet is smaller than the diameter of the second inlet / outlet; The diameter of the first inlet / outlet is within the range [1.5mm, 2.5mm]; The diameter of the second inlet / outlet is within the range of [3.5 mm, 4.5 mm].

4. The chip according to claim 1, characterized in that, The cell culture chamber array is a U-shaped groove with the opening facing upwards; The diameter of the cell culture chamber is within the range [550 μm, 650 μm]; The depth of the cell culture chamber is within the range of [550 μm, 650 μm].

5. A method for preparing a cell sphere culture organ-on-a-chip, characterized in that, The method for preparing cell spheroid culture organ microarrays is used to prepare cell spheroid culture organ microarrays as described in any one of claims 1-4, the method comprising: Obtain the chip to be processed and the mold; the mold has a channel array region to be prepared and an injection groove disposed in each channel to be prepared in the channel array to be prepared; The chip to be processed is subjected to photolithography to fabricate a drug inlet, a concentration dispersion structure and a first flow channel array on the chip to be processed, thereby obtaining a first chip structure; Polydimethylsiloxane is cured within the mold to obtain a second chip structure; the second chip structure includes a second flow channel array and a cell culture chamber array, wherein the cell culture chamber array is distributed within the second flow channel of the second flow channel array; The first chip structure and the second chip structure are bonded together to obtain a cell sphere culture organ chip.

6. The method according to claim 5, characterized in that, The polydimethylsiloxane is cured within the mold to obtain a second chip structure, including: Polydimethylsiloxane is injected into the mold and left to stand until the polydimethylsiloxane solidifies to obtain the chip structure to be prepared. Polydimethylsiloxane is injected into the groove to be injected in the chip structure to be prepared. Excess polydimethylsiloxane is removed. Surface tension is used to make the polydimethylsiloxane adhere to the sidewalls and bottom of the groove to be injected, forming a U-shaped groove, thus obtaining the second chip structure.

7. The method according to claim 5, characterized in that, After performing photolithography on the chip to be processed to fabricate a drug inlet, a concentration dispersion structure, and a first flow channel array on the chip to obtain the first chip structure, the process further includes: Drop tridecylfluorooctyltriethoxyalkylene onto a glass slide; The glass slide and the first chip structure are placed in a vacuum for vacuum treatment, so that the tridecafluorooctyltriethoxyalkylene adheres to the surface of the first chip structure.

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