Cell culture units, devices, applications and culture methods

Abstract

The application discloses a cell culture unit, a device, an application and a culture method, wherein the cell culture unit comprises a base body, a culture cavity and a liquid storage cavity are formed in the base body and are connected to each other, and the culture cavity and the liquid storage cavity are open at the top of the base body; the culture cavity comprises a first cavity bottom wall, and the liquid storage cavity comprises a second cavity bottom wall; in the height direction of the base body, the first cavity bottom wall and the second cavity bottom wall have a height difference, and the first cavity bottom wall is lower than the second cavity bottom wall. The technical scheme can reduce the risk that liquid gel flows to the flow channel cavity of the liquid storage cavity to cause the flow channel cavity to be blocked when a biological sample is injected into the culture cavity, so that the smoothness of the culture medium when the culture medium is filled back and forth between the culture cavity and the liquid storage cavity can be ensured, the biological sample in the culture cavity can be better exchanged with the culture medium, the growth environment of cells in the body can be more truly simulated, and the culture success rate of a cell model is improved.

Description

Technical Field

[0001] This application relates to the field of cell culture technology, and in particular to a cell culture unit, apparatus, application, and culture method. Background Technology

[0002] Three-dimensional cell models, such as organoids and in vitro three-dimensional organ models, can simulate biological tissues with certain functions in the human body, and have significant advantages in studies on disease drug response, molecular mechanisms of tissue function, and signaling pathways. However, in related technologies, the culture medium has poor culturing effect on the cultured subjects during the cell culture process, resulting in poor simulation effect of the final cell model. Summary of the Invention

[0003] This application provides a cell culture unit, device, application, and culture method that can achieve cell models with good simulation effects (such as organoids, in vitro three-dimensional organ models, etc.) and improve the culture success rate.

[0004] In a first aspect, embodiments of this application provide a cell culture unit, which includes a substrate and has a culture chamber and a liquid storage chamber that are connected to each other, and the culture chamber and the liquid storage chamber have openings at the top of the substrate;

[0005] The culture chamber includes a first bottom wall, and the liquid storage chamber includes a second bottom wall. In the height direction of the substrate, the first bottom wall and the second bottom wall have a height difference, and the first bottom wall is lower than the second bottom wall.

[0006] The cell culture unit based on this application embodiment employs a height difference between the bottom walls of the first and second cavities along the height direction of the substrate, with the bottom wall of the first cavity being lower than that of the second cavity. This results in a greater depression in the culture cavity compared to the reservoir cavity. Specifically, the sidewalls of the culture cavity prevent liquid gel from flowing into the reservoir cavity. This reduces the risk of excessive liquid gel being injected into the culture cavity, potentially causing blockage of the reservoir cavity. It ensures smooth flow of culture medium from the culture cavity to the liquid gel or from the culture cavity back to the reservoir cavity, allowing for better exchange of substances between the cells in the liquid gel and the culture medium, more realistically simulating the in vivo cell growth environment, and thus improving the success rate of cell model culture. Furthermore, it prevents the liquid gel from spreading out excessively within the culture cavity, resulting in a more three-dimensional liquid gel within the culture cavity with a certain depth, ultimately creating a three-dimensional cell model.

[0007] In some embodiments, the culture chamber further includes a cavity sidewall surrounding the outer side of the bottom wall of the first cavity, the cavity sidewall including a first cavity sidewall and a second cavity sidewall, the first cavity sidewall being connected between the first cavity bottom wall and the second cavity sidewall;

[0008] The first cavity bottom wall and the first cavity side wall intersect to form a first boundary edge, and the first cavity side wall and the second cavity side wall intersect to form a second boundary edge. The projection of the second boundary edge on the horizontal plane is located outside the projection of the first boundary edge on the horizontal plane.

[0009] In some embodiments, the second boundary edge is coplanar with the bottom wall of the second cavity.

[0010] In some embodiments, the first cavity sidewall is an arc surface, and the angle between the line connecting the intersection of the first boundary edge and the second boundary edge with the vertical tangent and the horizontal plane is greater than 0 degrees and less than 90 degrees.

[0011] In some embodiments, the first cavity sidewall is an inclined surface, and the angle between the first cavity sidewall and the horizontal plane is greater than 0 degrees and less than 90 degrees.

[0012] In some embodiments, a separator membrane is also included, which covers at least a portion of the second cavity sidewall.

[0013] In some embodiments, the liquid storage chamber includes a main liquid storage chamber and a flow channel chamber, with the two ends of the flow channel chamber respectively connected to the main liquid storage chamber and the culture chamber. The bottom wall of the second chamber includes a first sub-chamber bottom wall located in the main liquid storage chamber and a second sub-chamber bottom wall located in the flow channel chamber, with the first sub-chamber bottom wall and the second sub-chamber bottom wall being coplanar.

[0014] In some embodiments, in a horizontal direction perpendicular to the flow direction from the reservoir to the culture chamber, the width of the main reservoir is approximately equal to the width of the culture chamber, and the width of the flow channel is less than the width of the main reservoir.

[0015] In some embodiments, the width of the flow channel cavity is from 0.01 mm to 4.40 mm.

[0016] In some embodiments, the substrate includes a culture layer and a reservoir layer, the reservoir layer covering the culture layer;

[0017] The liquid storage layer has a first flow channel and a second flow channel with the opening respectively. The surface of the culture layer near the culture layer has a first groove and a second groove. The bottom wall of the first groove is formed as the bottom wall of the first cavity, and the bottom wall of the second groove is formed as the bottom wall of the second cavity.

[0018] The first flow channel is connected to the first groove to jointly form the culture chamber, and the second flow channel is connected to the second groove to jointly form the liquid storage chamber.

[0019] In some embodiments, there are two liquid storage chambers, which are symmetrically arranged in the flow direction from the liquid storage chamber to the culture chamber, and the culture chamber is located between the two liquid storage chambers.

[0020] Secondly, embodiments of this application provide a cell culture apparatus, including a plurality of cell culture units as described above, wherein the plurality of cell culture units are arranged in an array.

[0021] Thirdly, embodiments of this application provide a cell culture method applied to the cell culture apparatus described above. The cell culture method includes the following steps:

[0022] Biological samples are injected into the culture chamber of the cell culture unit;

[0023] Culture medium is added to the reservoir of the cell culture unit, and the culture medium is used to immerse the biological sample.

[0024] The cell culture device was oscillated and adjusted to dynamically culture the biological sample, thereby obtaining an in vitro three-dimensional biological model.

[0025] Fourthly, embodiments of this application provide an application of the cell culture device described above in cell model culture and drug analysis. Attached Figure Description

[0026] To more clearly illustrate the technical solutions 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 the structures shown in these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the structure of an embodiment of the cell culture device of this application;

[0028] Figure 2 for Figure 1 A schematic diagram of the structure of a cell culture unit in a medium-sized cell culture apparatus according to an embodiment;

[0029] Figure 3 for Figure 2 A schematic diagram of the cell culture unit;

[0030] Figure 4 for Figure 2Cross-sectional view of an embodiment of a cell culture unit;

[0031] Figure 5 for Figure 2 Cross-sectional view of another embodiment of the cell culture unit;

[0032] Figure 6 for Figure 2 A schematic diagram of the culture layer structure of the substrate in a medium-sized cell culture unit;

[0033] Figure 7 for Figure 6 A partial structural diagram of the reservoir layer of the substrate in a medium-sized cell culture unit;

[0034] Figure 8 for Figure 2 A schematic diagram of the reservoir layer of the substrate in a medium-sized cell culture unit;

[0035] Figure 9 This is a schematic flowchart of an embodiment of the cell culture method of this application.

[0036] Explanation of icon numbers:

[0037] 100. Cell culture unit; 10. Substrate; 10a. Culture chamber; 10b. Liquid reservoir; 10c. Main liquid reservoir chamber; 10d. Flow channel chamber; 10e. Fourth chamber sidewall; 11. Culture layer; 111. First groove; 113. Second groove; 11a. First chamber bottom wall; 11b. Chamber sidewall; 11c. First chamber sidewall; 11d. Second chamber sidewall; 11e. First sub-chamber sidewall; 11f. Second 11g, bottom wall of the first sub-cavity; 11h, bottom wall of the second sub-cavity; 11i, side wall of the third cavity; 11j, side wall of the second sub-cavity; 13, liquid reservoir; 13a, first flow channel; 13b, second flow channel; 15, first boundary edge; 17, second boundary edge; 30, septum; 300, cell culture device; 310, outer shell; 500, cell model; 600, vertical section; 700, connecting line.

[0038] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0039] 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.

[0040] Where the following description relates to the accompanying drawings, unless otherwise indicated, the same numbers in different drawings denote the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with this application. Rather, they are merely examples of apparatuses and methods consistent with some aspects of this application as detailed in the appended claims.

[0041] In the description of this application, it should be understood that the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances. Furthermore, in the description of this application, unless otherwise stated, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. The character " / " generally indicates that the preceding and following related objects are in an "or" relationship.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0043] Please see Figures 1 to 2 One aspect of this application provides a cell culture device 300, which includes a housing 310 and a plurality of cell culture units 100. A receiving cavity is formed inside the housing 310, and the plurality of cell culture units 100 are housed in the receiving cavity and arranged in an array.

[0044] The cell culture units 100 can be arranged in M ​​columns * N rows, where M represents the number of cell culture units 100 in each row, N represents the number of cell culture units 100 in each column, M ≥ 1, N ≥ 1, M and N are integers and M and N are not both 1. In the multiple cell culture units 100 located in the same row, the distance between the opening of one cell culture unit 100 and the opening of the next cell culture unit 100 can be the preset channel spacing of an automated pipette (e.g., 9 mm). In the multiple cell culture units 100 located in the same column, the distance between the two openings of adjacent cell culture units 100, or the distance between two openings, can also be the preset channel spacing of an automated pipette (e.g., 9 mm).

[0045] The cell culture apparatus 300 can contain, for example, 64, 80, 96, 112, or 128 cell culture units to achieve high throughput requirements. As an example, such as... Figure 1 As shown, the cell culture device 300 includes 128 cell culture units 100, arranged in a 16x8 grid. With 128 cell culture units 100, it can be adapted to most imaging devices and liquid control systems on the market, making the cell culture device 300 more versatile. Imaging devices are generally equipped with clamps to accommodate containers of different shapes. Traditional standard well plates are commonly used cell culture plates, and the clamps of general imaging devices are size-compatible with standard well plates. The cell culture unit 100 of this application has the same shape and size as a traditional standard well plate, thus it can be adapted to compatible clamps. The liquid control system can be used for automated pipetting of biological samples, for example, by automatically controlling a pipette. The biological sample can be an aqueous medium containing biological samples or a liquid gel containing biological samples; the aqueous medium can be a mixed suspension. For ease of explanation, the following description refers to a liquid gel containing biological samples.

[0046] To improve the simulation effect of the final cell model 500 and increase the success rate of culture, this application focuses on improving the cell culture unit 100. Please refer to [link / reference needed]. Figures 2 to 4 In this embodiment of the application, the cell culture unit 100 includes a substrate 10.

[0047] In one embodiment, the substrate 10 includes a culture layer 11 and a liquid storage layer 13, with the liquid storage layer 13 covering the culture layer 11. That is, the culture layer 11 and the liquid storage layer 13 can be connected by assembly methods such as hot pressing, ultrasonic waves, or lasers. This design can effectively reduce the processing difficulty of the substrate 10. Alternatively, the culture layer 11 and the liquid storage layer 13 of the substrate 10 can be integrally formed using 3D printing technology.

[0048] Please see Figures 6 to 7The reservoir layer 13 has a first flow channel 13a and a second flow channel 13b, each with an opening. The culture layer 11 has a first groove 111 and a second groove 113 recessed near its surface. The first flow channel 13a and the first groove 111 are connected to form a culture chamber 10a, and the second flow channel 13b and the second groove 113 are connected to form a reservoir chamber 10b. Thus, a biological sample can be injected through the opening of the first flow channel 13a, allowing the biological sample to enter the culture chamber 10a. Culture medium, drugs, or drug-added culture medium can be injected through the opening of the second flow channel 13b, flowing into the reservoir chamber 10b and then into the culture chamber 10a to immerse the biological sample, for example, immersing a solidified liquid gel. Specifically, the culture medium can be a nutrient matrix providing nutrients to the biological sample, and the drug can act on the biological sample. Biological samples can include, but are not limited to, cells, tumor tissues, and organoids. When organoids are used, they can specifically be organoids for colon cancer, lung cancer, gastric cancer, or breast cancer, etc. It should be noted that the liquid gel and culture medium can be added via pump-free gravity-driven methods, or by using external peristaltic pumps, syringe pumps, or other devices to drive the flow of the liquid gel and culture medium, in order to provide the necessary growth environment for the cells.

[0049] The materials used to prepare the reservoir layer 13 and culture layer 11 of the substrate 10 can be, but are not limited to, glass, plastic, or PDMS (polydimethylsiloxane). By rationally selecting the materials used to prepare the reservoir layer 13 and culture layer 11 of the substrate 10, the cell culture unit 100 can possess good biocompatibility. The specific shape of the reservoir layer 13 and culture layer 11 of the substrate 10 is not limited here; designers can design them rationally according to actual needs. For example, the reservoir layer 13 and culture layer 11 of the substrate 10 can be in the form of a rectangular plate structure.

[0050] Understandably, when the substrate 10 is configured as a two-part structure consisting of a culture layer 11 and a reservoir layer 13, it offers the advantage of being able to replace either the culture layer 11 or the reservoir layer 13. Of course, the substrate 10 in this application can also be configured as a single, integral plate-like structure, meaning the culture layer 11 and the reservoir layer 13 are a single unit, which simplifies the manufacturing process. The following content will further elaborate on how this application improves the simulation effect of the final cell model 500, using the substrate 10 configured as a two-part structure of a culture layer 11 and a reservoir layer 13.

[0051] The culture chamber 10a is formed by a first chamber bottom wall 11a and a chamber side wall 11b surrounding the outside of the first chamber bottom wall 11a. The chamber side wall 11b includes a first chamber side wall 11c and a second chamber side wall 11d. The second chamber side wall 11d includes a first sub-chamber side wall 11e and a second sub-chamber side wall 11j. The bottom wall of the first groove 111 is formed as the first chamber bottom wall 11a, and its side walls respectively form the first sub-chamber side wall 11e of the first chamber side wall 11c and the second chamber side wall 11d. The side wall of the first flow channel 13a of the liquid storage layer 13 forms the second sub-chamber side wall 11j of the second chamber side wall 11d. The liquid storage chamber 10b includes a main liquid storage chamber 10c and a flow channel chamber 10d, and is surrounded by a second chamber bottom wall 11f and a third chamber side wall 11i surrounding the outside of the second chamber bottom wall 11f. The second chamber bottom wall 11f includes a first sub-chamber bottom wall 11g located in the main liquid storage chamber 10c and a second sub-chamber bottom wall 11h located in the flow channel chamber 10d. The bottom wall of the second groove 113 is formed as the first sub-chamber bottom wall 11g of the second chamber bottom wall 11f and the second sub-chamber bottom wall 11h of the second chamber bottom wall 11f, respectively. The side wall of the second groove 113 and the side wall of the second flow channel 13b together form the third chamber side wall 11i. Specifically, the first cavity sidewall 11c and the second cavity bottom wall 11f can be arranged vertically or inclined. Furthermore, the shape of the cross-section of the culture cavity 10a in the height direction of the substrate 10 can be square, circular, or triangular, etc. The shape of the cross-section of the main storage cavity 10c of the storage cavity 10b in the height direction of the substrate 10 can also be square, circular, or triangular, etc., without limitation. When the cross-sectional shape of the main storage cavity 10c of the culture cavity 10a and the storage cavity 10b is circular, compared with a square shape, it can reduce the amount of culture medium remaining at its corners, so as to ensure the fluidity of the culture medium.

[0052] There are two reservoir chambers 10b, which are symmetrically arranged in the flow direction from the reservoir chamber 10b to the culture chamber 10a, with the culture chamber 10a located between the two reservoir chambers 10b. This arrangement allows the cell culture apparatus 300 to be oscillated, enabling the culture medium to move back and forth between the two reservoir chambers 10b. Dynamic culture is achieved by generating physicochemical stimuli such as fluid shear force, mechanical stress, and biochemical concentration gradients, thus exhibiting more realistic physiological functions. Furthermore, it facilitates better exchange of substances between the culture medium and the biological sample, and removes waste products from the biological sample in the liquid gel, further simulating a realistic cellular environment. Of course, depending on the specific needs of the culture, the number of reservoirs 10b can also be three, four, or five, etc. For example, when there are three reservoirs 10b, the culture medium can be moved back and forth between two of them, while the remaining reservoir 10b can be filled with a different type of culture medium than the two reservoirs 10b, to meet the nutrient requirements for cell growth in various types of culture media. The number of culture chambers 10a can also be two, three, or four, etc., and is not limited thereto.

[0053] In some embodiments, please refer to Figure 5 The cell culture unit 100 further includes a septum membrane 30, which covers at least a portion of the second cavity sidewall 11d of the culture chamber 10a. Specifically, the septum membrane 30 may cover either the first sub-cavity sidewall 11e or the second sub-cavity sidewall 11j of the second cavity sidewall 11d. Thus, the culture chamber 10a can be divided into at least two sub-culture chambers 10a by the septum membrane 30, allowing different liquid gels to be cultured in different sub-culture chambers 10a, thereby obtaining different cell models 500 to meet the requirements of multi-organ co-culture. For example, when the culture chamber 10a is divided into two sub-culture chambers 10a, one biological model may be supported by the first cavity bottom wall 11a, and the other biological model may be supported by the septum membrane 30. Furthermore, the separator 30 can be a porous membrane, wherein through holes are formed on the porous membrane to communicate with the liquid storage chamber 10b, so that substances in the culture medium can flow into the liquid gel carried by the separator 30, and / or substances discharged from the liquid gel carried by the separator 30 can flow into the culture medium, thereby playing a role in material exchange.

[0054] The technical solution of this application employs a height difference between the bottom wall 11a of the first cavity and the bottom wall 11f of the second cavity in the height direction of the substrate 10, with the bottom wall 11a of the first cavity being lower than the bottom wall 11f of the second cavity. Thus, the culture cavity 10a is more sunken than the storage cavity 10b, meaning the sidewall 11b of the culture cavity 10a can prevent the liquid gel in the culture cavity 10a from flowing into the storage cavity 10b. This reduces the risk of excessive liquid gel being injected into the culture cavity 10a, causing the liquid gel to flow into the flow channel cavity 10d of the storage cavity 10b and block the flow channel cavity 10d. This ensures the smooth flow of culture medium from the culture cavity 10a to the liquid gel or from the culture cavity 10a back to the storage cavity 10b, allowing the cells in the liquid gel of the culture cavity 10a to better exchange substances with the culture medium, more realistically simulating the cell growth environment in vivo, thereby improving the success rate of cell model 500 culture. In addition, it can prevent the liquid gel from spreading too much in the culture chamber 10a, making the liquid gel more three-dimensional in the culture chamber 10a with a certain depth, so that the final cell model 500 has a three-dimensional shape.

[0055] In one embodiment, the first cavity bottom wall 11a and the first cavity side wall 11c intersect to form a first boundary edge 15, and the first cavity side wall 11c and the second cavity side wall 11d intersect to form a second boundary edge 17. The projection of the second boundary edge 17 onto the horizontal plane is located outside the projection of the first boundary edge 15 onto the horizontal plane. Thus, the first cavity side wall 11c can be tilted relative to the horizontal plane. This prevents the risk of excessive liquid gel injection flowing into the reservoir cavity 10b and causing blockage of the flow channel cavity 10d. Simultaneously, the tilting reduces the flow rate of the culture medium between the culture cavity 10a and the reservoir cavity 10b, i.e., it slows down the flow rate of the culture medium towards the liquid gel, avoiding excessive flow that could impact the liquid gel and cause the cells within the liquid gel to be dispersed by the culture medium. It also slows down the flow rate of the culture medium returning to the reservoir cavity 10b, increasing the time for the culture medium to wet the liquid gel, thus ensuring sufficient wetting of the cultured organisms.

[0056] In the exemplary embodiment shown in the figure, the second boundary edge 17 is coplanar with the bottom wall 11f of the second cavity. With this configuration, since the second boundary edge 17 is higher than the bottom wall 11f of the second cavity, when the culture medium flows out from the flow channel 10d of the storage cavity 10b, it can flow more smoothly into the culture cavity 10a, reducing the resistance when the culture medium flows into the culture cavity 10a, thereby ensuring the fluidity of the culture medium and more realistically simulating the material exchange environment of cells in vivo.

[0057] In one structural configuration, the first cavity sidewall 11c is an arc surface. This arc surface design allows the culture medium to flow more smoothly through the first cavity sidewall 11c of the culture cavity 10a. Furthermore, the angle between the line connecting the intersection of the first boundary edge 15 and the second boundary edge 17 with the vertical tangent plane 600 and the horizontal plane is greater than 0 degrees and less than 90 degrees. When the angle between the line 700 connecting the intersection of the first boundary edge 15 and the second boundary edge 17 with the vertical tangent 600 and the horizontal plane is too small, the first cavity sidewall 11c of the cavity sidewall 11b has a poor blocking effect on the liquid gel. This results in a high probability of blockage of the flow channel cavity 10d of the liquid storage cavity 10b when too much liquid gel is injected, and the liquid gel is still easy to spread out, resulting in poor three-dimensionality of the final liquid gel. When the angle is too large, the flow rate of the culture medium between the culture cavity 10a and the liquid storage cavity 10b is too high, resulting in a large impact of the culture medium on the liquid gel and the culture medium cannot fully wet the liquid gel. Specifically, the angle between the line 700 connecting the intersection point of the first dividing edge 15 and the second dividing edge 17 with the vertical tangent plane 600 and the horizontal plane can be specifically set to 10 degrees, 20 degrees, 30 degrees, 40 degrees, 45 degrees, 50 degrees, 60 degrees, 70 degrees, or 80 degrees, etc., and is not limited thereto. It should be noted that, please refer to... Figure 7 To be clear, the line 700 connecting the intersection of the first boundary edge 15 and the second boundary edge 17 with the vertical sectional plane 600 refers to the intersection of the first boundary edge 15 and the second boundary edge 17 on the same side of the culture chamber 10a with the vertical sectional plane 600, and the line connecting the two is the aforementioned connecting line 700.

[0058] In another structural form, the first cavity sidewall 11c is an inclined surface, and the angle between the first cavity sidewall 11c and the horizontal plane is greater than 0 degrees and less than 90 degrees. When the angle between the first cavity sidewall 11c and the horizontal plane is too small, the first cavity sidewall 11c of the cavity sidewall 11b has a poor blocking effect on the liquid gel, resulting in a high possibility of clogging the flow channel cavity 10d of the liquid storage cavity 10b when too much liquid gel is injected, and the liquid gel is still easy to spread out, resulting in poor three-dimensionality of the final liquid gel. When the angle is too large, the flow velocity of the culture medium between the culture cavity 10a and the liquid storage cavity 10b is too high, resulting in a large impact of the culture medium on the liquid gel and the culture medium cannot fully wet the liquid gel. Specifically, the angle between the first cavity sidewall 11c and the horizontal plane can be specifically set to 10 degrees, 20 degrees, 30 degrees, 40 degrees, 45 degrees, 50 degrees, 60 degrees, 70 degrees, or 80 degrees, etc., without limitation.

[0059] To ensure smooth flow of the culture medium between the main reservoir 10c and the flow channel 10d in the reservoir 10b, further please refer to... Figures 4 to 6The bottom wall 11g of the first sub-cavity and the bottom wall 11h of the second sub-cavity are coplanar. With this arrangement, the bottom wall 11g of the first sub-cavity of the main liquid storage cavity 10c and the bottom wall 11h of the second sub-cavity of the flow channel cavity 10d do not have a height difference. When the culture medium flows between the main liquid storage cavity 10c and the flow channel cavity 10d, it can always maintain the same height, thereby ensuring the smooth flow of the culture medium and allowing for better exchange of substances between the culture medium and the liquid gel.

[0060] In one embodiment, please refer to Figures 4 to 8 In the horizontal direction perpendicular to the flow direction from the reservoir 10b to the culture chamber 10a, the width of the main reservoir 10c is approximately equal to the width of the culture chamber 10a, while the width of the channel cavity 10d is smaller than the width of the main reservoir 10c. Thus, when the width of the main reservoir 10c is approximately equal to the width of the culture chamber 10a, by reducing the width of the channel cavity 10d to be smaller than the width of the main reservoir 10c, when the culture medium flows from the main reservoir 10c to the culture chamber 10a, the reduced width of the channel cavity 10d decreases the flow velocity of the culture medium as it flows through it. This prevents excessively high flow rates from impacting the cells within the liquid gel and causing them to be dispersed by the culture medium.

[0061] Furthermore, the width of the flow channel 10d ranges from 0.01 mm to 4.40 mm. When the width of the flow channel 10d is too large, even if the culture medium flows through the flow channel 10d, which is narrower than the main reservoir 10c, it still cannot effectively reduce the flow rate of the culture medium, and may still cause impact on the cells within the liquid gel due to excessive flow rate. Conversely, when the width of the flow channel 10d is too small, the flow of the culture medium within the flow channel 10d will be less smooth. Specifically, the width of the flow channel 10d refers to the distance between the two opposite fourth cavity sidewalls 10e in the horizontal direction, and this width value can be specifically set to 0.01 mm, 1.00 mm, 2.00 mm, 3.00 mm, 4.00 mm, or 4.40 mm, etc., without limitation.

[0062] The above content details the advantages of the cell culture device 300 of this application in cultivating cell models 500 with better simulation effects, focusing on the structural form of the cell culture unit 100. Based on this, please refer to... Figure 9 Another aspect of this application discloses a cell culture method, wherein the cell culture method of the cell culture device 300 includes the following steps:

[0063] Step S10: Inject the biological sample into the culture chamber 10a of the cell culture unit 100. In this step, the biological sample can be an aqueous medium, such as a mixed suspension, or a liquid gel. The biological sample can be injected through the opening of the first flow channel 13a using a pipette in a pump-free gravity-driven manner. Under the influence of its own gravity, the biological sample can flow from top to bottom from the first flow channel 13a to the culture chamber 10a. Alternatively, the biological sample can be driven from the first flow channel 13a of the liquid storage layer 13 of the substrate 10 to the culture chamber 10a by using an external peristaltic pump, syringe pump, or other device. There is no limitation on this method. When the biological sample is a liquid gel, the cell culture device 300, after being injected with the liquid gel, can be inverted. Due to the adhesiveness of the liquid gel and the influence of gravity, the inverted liquid gel can adhere to the bottom wall 11a of the first cavity of the culture chamber 10a. At the same time, the biological sample cells in the liquid gel are deposited on the side away from the bottom wall 11a of the first cavity due to gravity. This allows the cells in the liquid gel to be in a three-dimensional culture environment and to have sufficient contact with the culture medium when culture medium is added later. Of course, the cell culture device 300 can also be placed in an incubator in a non-inverted state. When placed in an incubator at a preset temperature for a preset time, the liquid gel can solidify and form a solidified gel. For example, the inverted cell culture device 300 can be placed in an incubator at 37 degrees Celsius for a preset time to solidify and form a solidified gel.

[0064] Step S20: Add culture medium to the reservoir 10b of the cell culture unit 100, allowing the culture medium to saturate the biological sample. Similarly, in this step, the culture medium can be injected via a pump-free gravity-driven method through the opening of the second flow channel 13b using a pipette. Under its own gravity, the culture medium flows from top to bottom from the second flow channel 13b to the reservoir 10b, and then from the reservoir 10b into the culture chamber 10a to saturate the biological sample. Alternatively, an external peristaltic pump, syringe pump, or other device can be used to drive the culture medium from the second flow channel 13b of the reservoir layer 13 of the substrate 10 to the reservoir 10b; this is not limited. It should be noted that the culture medium saturates the biological sample in either a dynamic or static culture state.

[0065] Step S30: The cell culture device 300 is oscillated to dynamically culture the biological sample and obtain an in vitro three-dimensional biological model. Specifically, the cell culture device 300 can be placed on an oscillating device and oscillated back and forth at a preset angle, causing the culture medium to flow back and forth between the reservoir 10b and the culture chamber 10a. To achieve dynamic culture of the biological sample by the culture medium, the oscillating device drives the cell culture device 300 to oscillate back and forth at a preset angle, combined with... Figure 3As shown, this embodiment enables the culture medium to flow back and forth between the two reservoirs 10b and the culture chamber 10a, achieving dynamic culture and better simulating the cell growth environment. Specifically, during the oscillation process, one of the two reservoirs 10b will be at a higher height than the other. Under the influence of gravity, the culture medium in the higher reservoir 10b will flow to the culture chamber 10a and then back to the lower reservoir 10b. Thus, through the back-and-forth oscillation, the biological sample can be dynamically cultured by the back-and-forth flow of the culture medium between the two reservoirs 10b. It should be noted that the oscillation device can be a shaker or a device that drives the cell culture device 300 to oscillate; of course, it can also be manually oscillated by the operator, and there is no limitation thereto.

[0066] Step S30 includes: adjusting the cell culture device 300 by swinging it to dynamically culture biological samples; periodically replacing the culture medium in the reservoir 10b and finally obtaining the cell model 500.

[0067] In this step, due to the exchange of substances between the culture medium and the cells, the cell excretions within the liquid gel are carried away. Therefore, some excretions will remain in the culture medium. Consequently, the culture medium in the reservoir 10b needs to be replaced periodically to improve the cell culture effect of the liquid gel and better simulate the cell growth environment, thereby ultimately obtaining the desired cell model 500. A replacement cycle in the reservoir 10b can be 2-3 days, or any other time interval is acceptable; there are no restrictions on this.

[0068] The technical solution of this application, by adopting the above-mentioned cell culture method, can realize the dynamic culture of liquid gel by the culture medium. Compared with static culture, the cell culture method of this application can better simulate the cell growth environment, so as to obtain a cell model 500 with better simulation effect, and at the same time improve the success rate of culturing cell model 500.

[0069] Another aspect of this application proposes the application of the cell culture apparatus 300 described above in cell model culture and drug analysis.

[0070] In this invention, drugs can be added to the cell culture apparatus 300, for example, by mixing them with a culture medium and then flowing into a liquid gel to wet the gel. Thus, the cell culture apparatus 300 of this application allows for the study of the effects of drugs on cell spheres, which can then be used in pharmaceutical fields such as drug analysis.

[0071] In the accompanying drawings of this embodiment, the same or similar reference numerals correspond to the same or similar components. In the description of this application, it should be understood that if terms such as "upper," "lower," "left," and "right" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, they are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the terms used to describe positional relationships in the accompanying drawings are only for illustrative purposes and should not be construed as limiting this patent. For those skilled in the art, the specific meaning of the above terms can be understood according to the specific circumstances.

[0072] The above are merely preferred embodiments of this application and are not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A cell culture unit, characterized by include: The substrate has a culture chamber and a liquid storage chamber that are connected to each other, and the culture chamber and the liquid storage chamber are respectively open at the top of the substrate; The culture chamber includes a first chamber bottom wall, and the liquid storage chamber includes a second chamber bottom wall, wherein, in the height direction of the substrate, the first chamber bottom wall and the second chamber bottom wall have a height difference, and the first chamber bottom wall is lower than the second chamber bottom wall; The liquid storage chamber includes a main liquid storage chamber and a flow channel chamber. The two ends of the flow channel chamber are respectively connected to the main liquid storage chamber and the culture chamber. The bottom wall of the second chamber includes a first sub-chamber bottom wall located in the main liquid storage chamber and a second sub-chamber bottom wall located in the flow channel chamber. The first sub-chamber bottom wall and the second sub-chamber bottom wall are coplanar. In a horizontal direction perpendicular to the flow direction from the reservoir to the culture chamber, the width of the main reservoir is approximately equal to the width of the culture chamber, and the width of the flow channel is less than the width of the main reservoir.

2. The cell culture unit of claim 1, wherein The culture chamber further includes a cavity sidewall surrounding the bottom wall of the first cavity, the cavity sidewall including a first cavity sidewall and a second cavity sidewall, the first cavity sidewall being connected between the first cavity bottom wall and the second cavity sidewall; The first cavity bottom wall and the first cavity side wall intersect to form a first boundary edge, and the first cavity side wall and the second cavity side wall intersect to form a second boundary edge. The projection of the second boundary edge on the horizontal plane is located outside the projection of the first boundary edge on the horizontal plane.

3. The cell culture unit as described in claim 2, characterized in that, The second dividing edge is coplanar with the bottom wall of the second cavity.

4. The cell culture unit as described in claim 2, characterized in that, The first cavity sidewall is an arc surface, and the angle between the line connecting the intersection of the first boundary edge and the second boundary edge with the vertical tangent and the horizontal plane is greater than 0 degrees and less than 90 degrees.

5. The cell culture unit as described in claim 2, characterized in that, The first cavity sidewall is an inclined surface, and the angle between the first cavity sidewall and the horizontal plane is greater than 0 degrees and less than 90 degrees.

6. The cell culture unit as described in claim 2, characterized in that, It also includes a separator membrane that covers at least a portion of the second cavity sidewall.

7. The cell culture unit as described in claim 6, characterized in that, The width of the flow channel cavity is from 0.01 mm to 4.40 mm.

8. The cell culture unit according to any one of claims 1 to 6, characterized in that, The substrate includes a culture layer and a liquid storage layer, with the liquid storage layer covering the culture layer; The liquid storage layer has a first flow channel and a second flow channel with the opening respectively. The surface of the culture layer near the liquid storage layer has a first groove and a second groove. The bottom wall of the first groove is formed as the bottom wall of the first cavity, and the bottom wall of the second groove is formed as the bottom wall of the second cavity. The first flow channel is connected to the first groove to jointly form the culture chamber, and the second flow channel is connected to the second groove to jointly form the liquid storage chamber.

9. The cell culture unit according to any one of claims 1 to 6, characterized in that, The number of liquid storage chambers is two. The two liquid storage chambers are symmetrically arranged in the flow direction from the liquid storage chamber to the culture chamber, and the culture chamber is located between the two liquid storage chambers.

10. A cell culture device, characterized in that, It includes a plurality of cell culture units as described in any one of claims 1 to 9, wherein the plurality of cell culture units are arranged in an array.

11. A cell culture method, applied to the cell culture apparatus as described in claim 10, characterized in that, The cell culture method includes the following steps: Biological samples are injected into the culture chamber of the cell culture unit; Culture medium is added to the reservoir of the cell culture unit, and the culture medium is used to immerse the biological sample. The cell culture device was oscillated and adjusted to dynamically culture the biological sample, thereby obtaining an in vitro three-dimensional biological model.

12. The application of the cell culture device as described in claim 10 in cell model culture and drug analysis.

Images