Cell culture unit, device, application and culture method

Abstract

The present application discloses a cell culture unit, device, application and culture method, wherein the cell culture unit includes a substrate, a culture chamber, a liquid storage chamber and a flow channel chamber are formed in the substrate, and the culture chamber and the liquid storage chamber have an opening at the top of the substrate, and the two ends of the flow channel chamber are connected to the culture chamber and the liquid storage chamber respectively, and the flow channel chamber includes a first port portion connected to the liquid storage chamber and a second port portion connected to the culture chamber, and the flow channel chamber is also formed with a constriction portion between the first port portion and the second port portion, and the opening area of ​​the constriction portion is smaller than the opening area of ​​the first port portion and the second port portion. The technical solution of the present application can avoid the impact on the biological sample caused by too fast flow rate, resulting in the cells in the liquid gel being dispersed by the culture medium, and increase the time for the culture medium to infiltrate the biological sample to fully infiltrate the biological sample, thereby being able to improve the simulation degree of the cell model finally obtained and improve the success rate of culture.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to a cell culture unit, device, application and culture method. Background Art

[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 great advantages in the study of disease drug response, molecular mechanisms of tissue function, signal pathways, etc. Compared with traditional two-dimensional cell culture methods, the cultivation of three-dimensional cell models can better simulate the functional characteristics of organs in the body. However, in related technologies, during the cell culture process, the culture medium has a poor culture effect on the cultured object, resulting in a poor simulation effect of the final cell model. Summary of the invention

[0003] The embodiments of the present application provide a cell culture unit, device, application and culture method, which can achieve a cell model with better simulation effect (such as organoids, in vitro three-dimensional organ models, etc.) and improve the culture success rate.

[0004] In a first aspect, an embodiment of the present application provides a cell culture unit, which includes a substrate, in which a culture cavity, a liquid storage cavity and a flow channel cavity are formed, and the culture cavity and the liquid storage cavity have openings at the top of the substrate, and two ends of the flow channel cavity are connected to the culture cavity and the liquid storage cavity respectively;

[0005] The flow channel cavity includes a first port portion connected to the liquid storage cavity and a second port portion connected to the culture cavity. The flow channel cavity also forms a constricted portion between the first port portion and the second port portion, and the opening area of ​​the constricted portion is smaller than the opening area of ​​the first port portion and the second port portion.

[0006] Based on the cell culture unit of the embodiment of the present application, a necking portion is formed between the first port portion and the second port portion in the flow channel cavity, and the opening area of ​​the necking portion is smaller than the opening area of ​​the first port portion and the second port portion, so that after the same volume of culture medium flows through the necking portion of the flow channel cavity, its own flow velocity can be reduced and slowed down, thereby avoiding the impact of excessive flow rate on the biological sample, causing the cells in the liquid gel to be dispersed by the culture medium, and increasing the time for the culture medium to infiltrate the biological sample to fully infiltrate the biological sample, thereby improving the simulation degree of the final cell model and improving the success rate of culture.

[0007] In some embodiments, in the horizontal direction, the width of the necked portion is smaller than the width of the first port portion and the second port portion.

[0008] In some embodiments, the flow channel cavity includes a first cavity section, and the first cavity section communicates with the first port portion and the constricted portion;

[0009] In the direction from the liquid storage cavity to the flow channel cavity, the width of the first cavity section in the horizontal direction gradually decreases.

[0010] In some embodiments, the flow channel cavity further includes a second cavity section, and the second cavity section communicates with the constricted portion and the second end portion;

[0011] In the direction from the liquid storage cavity to the flow channel cavity, the width of the second cavity section in the horizontal direction gradually increases.

[0012] In some embodiments, the culture chamber includes a first chamber bottom wall, and the flow channel chamber includes a second chamber bottom wall extending from the first port portion to the second port portion, 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.

[0013] In some embodiments, the substrate includes a culture layer and a liquid storage layer, the liquid storage layer is covered on the culture layer, and the liquid storage layer is formed with a first flow channel and a second flow channel having the openings respectively;

[0014] The surface of the culture layer near the liquid storage layer is concave to form a first groove, a second groove and a third groove that are connected to each other, the first flow channel is connected to the first groove to form the culture cavity, the second flow channel is connected to the second groove to form the liquid storage cavity, the lower surface of the liquid storage layer cooperates with the third groove to form the flow channel cavity, the bottom wall of the first groove is formed as the first cavity bottom wall, and the bottom wall of the third groove is formed as the second cavity bottom wall;

[0015] The first groove also includes a groove side wall, which surrounds the outside of the first cavity bottom wall and has a lower edge intersecting with the first cavity bottom wall and an upper edge intersecting with the second cavity bottom wall, the upper edge is coplanar with the second cavity bottom wall, and the intersection portion of the upper edge and the second cavity bottom wall is an arc structure.

[0016] In some embodiments, a projection of the upper edge on the horizontal plane is located outside a projection of the lower edge on the horizontal plane.

[0017] In some embodiments, the groove sidewall is an arc surface, and the angle between the connecting line of the intersection points of the upper edge and the lower edge with the vertical tangent plane and the horizontal plane is greater than 0 degrees and less than 90 degrees;

[0018] Alternatively, the groove side wall is an inclined surface, and the angle between the groove side wall and the horizontal plane is greater than 0 degree and less than 90 degrees.

[0019] In some embodiments, the cell culture unit further includes a separation membrane, and the separation membrane covers at least a portion of a side wall of the first flow channel.

[0020] In some embodiments, the liquid storage chamber includes a third chamber bottom wall, and the third chamber bottom wall is coplanar with the second chamber bottom wall.

[0021] In some embodiments, the number of the liquid storage chambers is two, the number of the flow channel chambers is two, and in the flow direction from the liquid storage chamber to the culture chamber, the two liquid storage chambers are symmetrically arranged, and the culture chamber is located between the two liquid storage chambers, and the two liquid storage chambers correspond one-to-one to the two flow channel chambers.

[0022] In a second aspect, an embodiment of the present application provides a cell culture device, comprising a plurality of cell culture units as described above, wherein the plurality of cell culture units are arranged in an array.

[0023] In a third aspect, an embodiment of the present application provides a cell culture method, which is applied to the cell culture device as described above, comprising the following steps:

[0024] injecting the biological sample into the culture chamber of the cell culture unit;

[0025] Adding culture medium to the liquid storage chamber of the cell culture unit, and allowing the culture medium to infiltrate the biological sample;

[0026] The cell culture device is swung and adjusted to dynamically culture the biological sample to obtain an in vitro three-dimensional biological model.

[0027] In a fourth aspect, an embodiment of the present application provides an application of the cell culture device as described above in cell model culture and drug analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0029] Figure 1 This is a schematic structural diagram of an embodiment of a cell culture device of the present application;

[0030] Figure 2 for Figure 1A schematic structural diagram of an embodiment of a cell culture unit of a cell culture device;

[0031] Figure 3 for Figure 2 Schematic diagram of the cell culture unit in Figure 1;

[0032] Figure 4 for Figure 3 A schematic diagram of the exploded culture layer of the matrix of the cell culture unit;

[0033] Figure 5 for Figure 4 A partial enlarged view of point A shown;

[0034] Figure 6 for Figure 2 A cross-sectional view of an embodiment of a cell culture unit;

[0035] Figure 7 for Figure 2 A cross-sectional view of another embodiment of a cell culture unit;

[0036] Figure 8 for Figure 4 A partial structural diagram of the culture chamber of the cell culture unit;

[0037] Fig. 9 This is a schematic diagram of a process of an embodiment of the cell culture method of the present application.

[0038] Description of Figure Numbers:

[0039] 100, cell culture unit; 10, matrix; 10a, culture chamber; 10b, liquid storage chamber; 10c, flow channel chamber; 10d, first port portion; 10e, second port portion; 10f, constriction portion; 10g, first cavity section; 10h, second cavity section; 11, culture layer; 11a, first groove; 11b, first cavity bottom wall; 11c, groove side wall; 11d, upper edge; 11e, lower edge; 11f, second groove; 11g, third cavity bottom wall; 11h, third groove; 11i, second cavity bottom wall; 13, liquid storage layer; 13a, first flow channel; 13b, second flow channel; 30, separation membrane; 300, cell culture device; 500, cell model; 600, vertical section; 700, connecting line.

[0040] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0041] In order to make the objectives, technical solutions and advantages of the present application clearer, the following part will further describe the embodiments of the present application in detail in conjunction with the accompanying drawings.

[0042] When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present application. Instead, they are only examples of devices and methods consistent with some aspects of the present application as detailed in the attached claims.

[0043] In the description of the present application, it should be understood that the terms "first", "second", etc. are used for descriptive purposes only and should not be understood as indicating or implying relative importance. For those of ordinary skill in the art, the specific meanings of the above terms in the present application can be understood according to the specific circumstances. In addition, in the description of the present application, unless otherwise specified, "multiple" refers to two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the objects associated before and after are in an "or" relationship.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used in this specification are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0045] See also Figure 1 to Figure 2 In one aspect, the present application provides a cell culture device 300, which includes a shell and a plurality of cell culture units 100. A receiving cavity is formed in the shell, and the plurality of cell culture units 100 are received in the receiving cavity and arranged in an array.

[0046] The multiple cell culture units 100 may be arranged in M ​​columns and N rows, wherein 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 1 at the same time. In the multiple cell culture units 100 located in the same row, the spacing between the opening of the previous cell culture unit 100 and the opening of the next cell culture unit 100 may adopt the preset channel spacing (e.g., 9 mm) of the automatic pipette, and in the multiple cell culture units 100 located in the same column, the spacing between two openings of adjacent cell culture units 100 or the spacing between two openings may also adopt the preset channel spacing (e.g., 9 mm) of the automatic pipette.

[0047] The number of cell culture units 100 in the cell culture device 300 may be, for example, 64, 80, 96, 112 or 128, etc., to achieve high throughput requirements. Figure 1 As shown, the cell culture device 300 includes 128 cell culture units 100, which are arranged in 16 rows * 8 columns, and when the number of cell culture units 100 is 128, it can adapt 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 fixtures to adapt to containers of different shapes to be observed. Traditional standard well plates are commonly used cell culture plates, and the fixtures of general imaging devices are adapted to the size of standard well plates. The shape of the cell culture unit 100 of the present application is consistent with the size of the traditional standard well plate, so it can be adapted to the fixture adapted thereto, that is. Among them, the liquid control system can be used for automatic pipetting of biological samples, for example, by automatic control of a pipette gun to achieve pipetting, wherein the biological sample can be an aqueous medium containing the biological sample or a liquid gel containing the biological sample. For the convenience of explanation, the biological sample is described as a liquid gel containing the biological sample below.

[0048] In order to obtain a better simulation effect of the cell model 500 and improve the success of the culture, the present application focuses on improving the cell culture unit 100. Figures 3 to 5 In the embodiment of the present application, the cell culture unit 100 includes a substrate 10 .

[0049] In one embodiment, the substrate 10 includes a culture layer 11 and a liquid storage layer 13, and the liquid storage layer 13 is covered on 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, ultrasound, laser, etc., and such a 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 by 3D printing technology.

[0050] See also Figures 3 to 5, a first flow channel 13a and a second flow channel 13b with openings are formed in the liquid storage layer 13, and a first groove 11a, a second groove 11f and a third groove 11h are formed in the surface depression near the culture layer 11, the first flow channel 13a is connected with the first groove 11a to form a culture chamber 10a, the second flow channel 13b is connected with the second groove 11f to form a liquid storage chamber 10b, and the lower surface of the liquid storage layer 13 cooperates with the third groove 11h to form a flow channel chamber 10c. In this way, a liquid gel containing a biological sample can be injected through the opening of the first flow channel 13a so that the liquid gel enters the culture chamber 10a, and a culture medium, a drug or a culture medium with a drug added can be injected through the opening of the second flow channel 13b so that it flows to the liquid storage chamber 10b and then flows into the culture chamber 10a through the flow channel chamber 10c to immerse the biological sample (aqueous medium containing a biological sample or a liquid gel containing a biological sample). Specifically, the culture medium can be a nutrient matrix that provides nutrients for the biological sample, and the drug can act on the biological sample. Among them, the biological sample can include but is not limited to cells, tumor tissues, organoids, etc. When it is an organoid, it can be specifically colon cancer organoids, lung cancer organoids, gastric cancer organoids or breast cancer organoids, etc. It should be noted that the addition method of the liquid gel and the culture medium can be pumpless gravity drive, or an external peristaltic pump, injection pump or other device can be used to drive the flow of liquid gel and culture medium to provide the growth environment required by the cells.

[0051] The preparation materials of the liquid storage layer 13 and the culture layer 11 of the substrate 10 may include, but are not limited to, glass, plastic or PDMS (polydimethylsiloxane). By reasonably selecting the preparation materials of the liquid storage layer 13 and the culture layer 11 of the substrate 10, the cell culture unit 100 can have good bioaffinity. The specific shapes of the liquid storage layer 13 and the culture layer 11 of the substrate 10 are not limited here, and the designer can make reasonable designs according to actual needs. For example, the liquid storage layer 13 and the culture layer 11 of the substrate 10 can be rectangular plate-shaped structures.

[0052] It can be understood that when the substrate 10 is configured as a two-part structure of the culture layer 11 and the liquid storage layer 13, it has the advantage of being able to replace either the culture layer 11 or the liquid storage layer 13. Of course, the substrate 10 of the present application can also be configured as an integral plate-like structure, that is, the culture layer 11 and the liquid storage layer 13 are in an integral form, so that the manufacturing process can be simplified. The following content is still in the form of the substrate 10 configured as a two-part structure of the culture layer 11 and the liquid storage layer 13, and further explains how the present application can improve the simulation effect of the finally obtained cell model 500.

[0053] The culture chamber 10a is surrounded by the first chamber bottom wall 11b, the chamber side wall 11c of the first groove 11a, and the side wall of the first flow channel 13a, and the groove bottom wall of the first groove 11a is formed as the first chamber bottom wall 11b, the first groove 11a is surrounded by the groove side wall 11c and the groove bottom wall, and the groove side wall 11c surrounds the outside of the first chamber bottom wall 11b, the flow channel chamber 10c is surrounded by its own chamber side wall and the second chamber bottom wall 11i, and the liquid storage chamber 10b is surrounded by its own chamber side wall and the third chamber bottom wall 11g. Among them, the groove side wall 11c has a lower edge 11e intersecting with the first chamber bottom wall 11b and an upper edge 11d intersecting with the second chamber bottom wall 11i. The groove bottom wall of the third groove 11h is formed as the second chamber bottom wall 11i. The groove bottom wall of the second groove 11f is formed as the third chamber bottom wall 11g. Specifically, the shape of the cross section of the culture chamber 10a in the height direction of the substrate 10 can be square, circular, triangular, etc., and the shape of the cross section of the main liquid storage cavity of the liquid storage cavity 10b in the height direction of the substrate 10 can also be square, circular, triangular, etc., without limitation. When the cross-sectional shape of the culture chamber 10a and the main liquid storage cavity of the liquid storage cavity 10b is circular, compared with a square shape, the culture medium remaining at its own corner can be reduced to ensure the fluidity of the culture medium.

[0054] There are two liquid storage chambers 10b and two flow channel chambers 10c. In the flow direction from the liquid storage chamber 10b to the culture chamber 10a, the two liquid storage chambers 10b are symmetrically arranged, and the culture chamber 10a is located between the two liquid storage chambers 10b, and the two liquid storage chambers 10b correspond to the two flow channel chambers 10c one by one. With this arrangement, the cell culture device 300 can be swung, and the culture medium can move back and forth between the two liquid storage chambers 10b under the swing, and dynamic culture can be achieved by generating fluid shear force, mechanical stress, biochemical concentration gradient and other physical and chemical stimuli, while showing more realistic physiological functions; the material exchange between the culture medium and the liquid gel can be better realized, and the excretions of the biological sample in the liquid gel can be taken away, which can better simulate the real cell environment. Of course, according to the specific needs of the culture, the number of liquid storage chambers 10b can also be three, four or five, etc. For example, when the number of liquid storage chambers 10b is three, two of the liquid storage chambers 10b can be used to realize the form of moving the culture medium back and forth between the two liquid storage chambers 10b, and the remaining liquid storage chamber 10b can be added with culture medium of a different type from the culture medium in the above two liquid storage chambers 10b to meet the needs of various types of culture media for nutrients required for cell growth. The number of culture chambers 10a can also be two, three or four, etc., and there is no limitation on this.

[0055] In one embodiment, the cell culture unit 100 further includes a separator 30, which covers at least a portion of the side wall of the first flow channel 13a. In this way, the culture chamber 10a can be divided into at least two sub-culture chambers 10a by the separator 30, so that different liquid gels can be cultured through different sub-culture chambers 10a to obtain different cell models 500, so as 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 liquid gel can be carried by the first chamber bottom wall 11b, and the other liquid gel can be carried by the separator 30. Further, the separator 30 can be a porous membrane, wherein a through hole connected to the liquid storage chamber 10b is formed on the porous membrane, so that the culture medium can flow into the liquid gel carried by the separator 30 through the through hole, and / or the discharged substances in the liquid gel carried by the separator 30 can flow into the culture medium to play a role in material exchange.

[0056] The flow channel cavity 10c includes a first port portion 10d, a second port portion 10e, a first cavity section 10g and a second cavity section 10h, wherein the flow channel cavity 10c also forms a constriction portion 10f between the first port portion 10d and the second port portion 10e, the first cavity section 10g communicates with the first port portion 10d and the constriction portion 10f, and the second cavity section 10h communicates with the constriction portion 10f and the second port portion 10e. It can be understood that the first port portion 10d of the flow channel cavity 10c can be a portion at an inlet position of the flow channel cavity 10c, and the second port portion 10e can be a portion at an outlet position of the flow channel cavity 10c, and the position of the constriction portion 10f can be set at any position between the first port portion 10d and the second port portion 10e, for example, it can be set at a position close to the first port portion 10d, or at a position close to the second port portion 10e, or at a position between the first port portion 10d and the second port portion 10e, and this is not limited.

[0057] Among them, the opening area of ​​the constricted portion 10f is smaller than the opening area of ​​the first port portion 10d and the second port portion 10e, that is, when the culture medium passes through the constricted portion 10f, since the opening volume of the constricted portion 10f is smaller than the opening area of ​​the first port portion 10d and the second port portion 10e, the same volume of culture medium can reduce its own flow rate after flowing through the constricted portion 10f of the flow channel cavity 10c. Specifically, the opening area of ​​the constricted portion 10f can be reduced by reducing the width of the constricted portion 10f in the horizontal direction, or by reducing the length of the constricted portion 10f in the height direction, or by reducing the width of the constricted portion 10f in the horizontal direction and the length of the constricted portion 10f in the height direction at the same time.

[0058] The technical solution of the present application forms a constriction portion 10f between the first port portion 10d and the second port portion 10e in the flow channel cavity 10c, and the opening area of ​​the constriction portion 10f is smaller than the opening area of ​​the first port portion 10d and the second port portion 10e, so that after the same volume of culture medium flows through the constriction portion 10f of the flow channel cavity 10c, its own flow rate can be reduced and slowed down, thereby avoiding the impact of excessive flow rate on the cells in the liquid gel, causing the biological sample (solidified liquid gel or aqueous medium) to be dispersed by the culture medium, and increasing the time for the culture medium to infiltrate the liquid gel to fully infiltrate the culture object, thereby improving the simulation degree of the final cell model 500 and improving the success rate of culture.

[0059] In one embodiment, see Figure 4 , in the horizontal direction, the width of the constricted portion 10f is smaller than the width of the first port portion 10d and the second port portion 10e. In this way, by reducing the width of the constricted portion 10f in the horizontal direction to a width smaller than the width of the first port portion 10d and the second port portion 10e, the same volume of culture medium can flow through the constricted portion 10f of the flow channel cavity 10c. The flow rate of the culture medium can be reduced, so that the culture medium can flow to the liquid gel in the culture cavity 10a at a relatively low speed, thereby avoiding the flow rate being too fast to cause a large impact on the cells in the liquid gel so that the cells in the liquid gel are dispersed by the culture medium, and the flow time of the culture medium in the culture cavity 10a can be increased, so as to increase the time for the culture medium to infiltrate the liquid gel, so that the culture object can be fully infiltrated.

[0060] In order to further slow down the flow rate of the culture medium, please refer to Figure 4 In the direction from the liquid storage chamber 10b to the flow channel chamber 10c, the width of the first cavity section 10g in the horizontal direction gradually decreases. In this way, when the culture medium begins to enter the flow channel chamber 10c from the liquid storage chamber 10b, the flow rate of the culture medium can be gradually reduced through the first cavity section 10g to avoid turbulence inside the culture medium, so that the culture medium flows more smoothly.

[0061] In the direction from the liquid storage chamber 10b to the flow channel chamber 10c, the width of the second cavity section 10h in the horizontal direction gradually increases. In this way, when the culture medium has a reduced flow rate after passing through the first cavity section 10g, it can flow into the culture chamber 10a through the second cavity section 10h whose width gradually increases in the horizontal direction. Compared with the second cavity section 10h, whose horizontal width is the same as the horizontal width of the constricted portion 10f, the culture medium can increase the range of infiltration of the liquid gel and improve the utilization rate of the culture medium. It should be noted that the above content refers to the direction from the liquid storage chamber 10b to the flow channel chamber 10c. Figure 3 The flow direction in .

[0062] The above content specifically introduces the advantages of the cell culture device 300 of the present application in that it can culture a cell model 500 with a better simulation effect from the structural form of the flow channel cavity 10c of the cell culture unit 100. On this basis, please refer to Figures 4 to 7 In the height direction of the substrate 10, the first cavity bottom wall 11b and the second cavity bottom wall 11i have a height difference, and the first cavity bottom wall 11b is lower than the second cavity bottom wall 11i. Under such a setting, the culture cavity 10a will sink more than the liquid storage cavity 10b, that is, the groove side wall 11c of the first groove 11a of the culture cavity 10a can prevent the liquid gel in the culture cavity 10a from flowing to the flow channel cavity 10c, so that when the liquid gel is injected into the culture cavity 10a, the risk of excessive injection of liquid gel causing the liquid gel to flow to the flow channel cavity 10c of the liquid storage cavity 10b and causing blockage of the flow channel cavity 10c is reduced, thereby ensuring the smoothness of the culture medium when it flows to the liquid gel or when the culture medium flows from the culture cavity 10a back to the liquid storage cavity 10b, so that the cells in the liquid gel in the culture cavity 10a can better exchange substances with the culture medium, more realistically simulate the growth environment of the cells in the body, thereby improving the culture success rate of the cell model 500. In addition, it can prevent the liquid gel from spreading too much in the culture chamber 10a, so that the liquid gel is more three-dimensional in the culture chamber 10a with a certain recessed depth, so that the finally formed cell model 500 has three-dimensionality.

[0063] In the scheme shown in the figure, see Figure 4 , the upper edge 11d is coplanar with the second cavity bottom wall 11i. In this way, there is no height difference between the second cavity bottom wall 11i and the upper edge 11d of the flow channel cavity 10c, and the culture medium can always flow at the same height when flowing between the flow channel cavity 10c and the upper edge 11d, thereby ensuring the smooth flow of the culture medium, so that the culture medium and the liquid gel can better exchange substances.

[0064] In order to achieve that when the culture medium flows to the liquid gel, the biological sample (solidified liquid gel) can be infiltrated in the annular direction as much as possible, in one structural form, the intersection of the upper edge 11d and the second cavity bottom wall 11i is an arc-shaped structure. Under the arc-shaped structure, that is, part of the second cavity bottom wall 11i can extend to both sides of the upper edge 11d in the horizontal direction to support the culture medium, so that part of the culture medium will flow down from the middle of the second cavity bottom wall 11i, and the rest of the culture medium is supported by the part of the second cavity bottom wall 11i extending to both sides of the upper edge 11d in the horizontal direction to continue to flow, so as to flow down on both sides of the upper edge 11d in the horizontal direction, so as to form a circular infiltration effect, so that the cells in the biological sample can better exchange substances with the culture medium.

[0065] For further information, see Figure 4, the projection of the upper edge 11d on the horizontal plane is located outside the projection of the lower edge 11e on the horizontal plane. With this arrangement, the groove sidewall 11c can be tilted relative to the horizontal plane, so that while preventing the risk of excessive injection of liquid gel to flow into the flow channel cavity 10c and causing blockage of the flow channel cavity 10c, the inclination can be used to slow down the flow rate of the culture medium between the culture cavity 10a and the flow channel cavity 10c, that is, the flow rate of the culture medium flowing into the liquid gel can be slowed down to avoid the impact of the liquid gel caused by the excessive flow rate, resulting in the cells in the liquid gel being dispersed by the culture medium, and the flow rate of the culture medium flowing back to the flow channel cavity 10c can be slowed down to increase the time for the culture medium to infiltrate the liquid gel, so as to more fully infiltrate the culture object.

[0066] In one structural form, the groove side wall 11c is an arc surface, and the arc surface setting can make the culture medium flow more smoothly on the groove side wall 11c of the culture chamber 10a. In addition, the angle between the connecting line 700 of the intersection point where the upper edge 11d and the lower edge 11e intersect with the vertical section 600 and the horizontal plane is greater than 0 degrees and less than 90 degrees. When the angle between the connecting line 700 of the intersection point where the upper edge 11d and the lower edge 11e intersect with the vertical section 600 and the horizontal plane is too small, the blocking effect of the cavity side wall on the liquid gel is poor, resulting in a high possibility of clogging the flow channel cavity 10c when too much liquid gel is injected, and the liquid gel is still easy to spread, resulting in a poor degree of three-dimensionality of the final formed liquid gel, and when the angle is too large, the flow velocity of the culture medium flowing between the culture chamber 10a and the flow channel cavity 10c is too large, resulting in a large impact of the culture medium on the biological sample (for example, the solidified liquid gel) and the culture medium cannot fully infiltrate the biological sample. Specifically, the angle between the connecting line 700 of the intersection point where the upper edge 11d and the lower edge 11e intersect the vertical section 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., without limitation. Figure 8 It should be understood that the connecting line 700 of the intersection of the upper edge 11d and the lower edge 11e with the vertical section 600 referred to here refers to the intersection of the upper edge 11d and the lower edge 11e located on the same side of the culture chamber 10a and the intersecting point with the vertical section 600, and the line connecting the two is the connecting line 700 mentioned above.

[0067] In another structural form, the groove side wall 11c is an inclined surface, and the angle between the groove side wall 11c and the horizontal plane is greater than 0 degrees and less than 90 degrees. When the angle between the groove side wall 11c and the horizontal plane is too small, the groove side wall 11c has a poor blocking effect on the liquid gel, resulting in a large possibility of clogging the flow channel cavity 10c when too much liquid gel is injected, and the liquid gel is still easy to spread, so that the three-dimensional degree of the final liquid gel is poor. When the angle is too large, the flow rate of the culture medium between the culture cavity 10a and the flow cavity is too large, resulting in a large impact of the culture medium on the biological sample (for example, the solidified liquid gel) and the culture medium cannot fully infiltrate the biological sample. Specifically, the angle between the groove side wall 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.

[0068] In the exemplary solution shown in the figure, the liquid storage chamber 10b includes a third chamber bottom wall 11g, and the third chamber bottom wall 11g is arranged coplanar with the second chamber bottom wall 11i. In this arrangement, the third chamber bottom wall 11g of the liquid storage chamber 10b and the second chamber bottom wall 11i of the flow channel chamber 10c have no height difference, and the culture medium can always flow at the same height when flowing between the liquid storage chamber 10b and the flow channel chamber 10c, thereby ensuring the smooth flow of the culture medium, so that the culture medium and the liquid gel can better exchange substances.

[0069] The above content specifically introduces the advantages of the cell culture device 300 of the present application in that it can culture a cell model 500 with a better simulation effect from the perspective of the overall structure of the cell culture unit 100 of the cell culture device 300. On this basis, please refer to Fig. 9 Another aspect of the present application provides a cell culture method. The cell culture method of the cell culture device 300 includes the following steps:

[0070] Step S10: injecting 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, and the biological sample can be injected into the biological sample through the open opening of the first flow channel 13a by a pipette gun in a pumpless gravity-driven manner. Under the influence of its own gravity, the biological sample can flow from the first flow channel 13a to the culture chamber 10a from top to bottom. Of course, the biological sample can also be driven by using an external peristaltic pump, a syringe pump or other device to flow from the first flow channel 13a of the liquid storage layer 13 of the matrix 10 to the culture chamber 10a, which is not limited to this. When the biological sample is a liquid gel, the cell culture device 300 after the liquid gel is injected can be inverted. Due to the adhesion of the liquid gel itself and the influence of gravity, the liquid gel can be adsorbed on the first cavity bottom wall 11b of the culture cavity 10a when inverted. At the same time, the biological sample cells contained in the liquid gel are deposited on the side of the liquid gel away from the first cavity bottom wall 11b under the influence of gravity, so that when the culture medium is added later, the cells in the liquid gel are in a three-dimensional culture environment and can fully contact with the culture medium. 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. For example, the inverted cell culture device 300 is placed in an incubator at 37 degrees Celsius for a preset time to solidify and form a solidified gel.

[0071] Step S20: Add culture medium to the liquid storage chamber 10b of the cell culture unit 100, and allow the culture medium to infiltrate the biological sample. Similarly, in this step, the culture medium can be injected into the open opening of the second flow channel 13b by a pipette through a pumpless gravity-driven method. Under the influence of its own gravity, the culture medium can flow from the second flow channel 13b to the liquid storage chamber 10b from top to bottom, and then flow from the liquid storage chamber 10b into the culture chamber 10a to infiltrate the biological sample. Of course, it is also possible to drive the culture medium to flow from the second flow channel 13b of the liquid storage layer 13 of the matrix 10 to the liquid storage chamber 10b by using an external peristaltic pump, a syringe pump or the like, which is not limited to this. It should be noted that the culture medium infiltrating the biological sample can be infiltrating the biological sample in a dynamic culture state or in a static culture state.

[0072] Step S30: Swing the cell culture device 300 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 the swing device and swung back and forth at a preset angle, so that the culture medium flows back and forth between the liquid storage chamber 10b and the culture chamber 10a. In order to achieve dynamic culture of the biological sample by the culture medium, the cell culture device 300 is driven by the swing device to swing back and forth at a preset angle, combined with Figure 3As shown, under this embodiment, the culture medium can flow back and forth between the two liquid storage chambers 10b and the culture chamber 10a to achieve dynamic culture and better simulate the growth environment of cells. Specifically, during the swinging process, one of the two liquid storage chambers 10b will be higher and the other lower. Under the action of gravity, the culture medium in the higher liquid storage chamber 10b will flow to the culture chamber 10a and then to the lower liquid storage chamber 10b. In this way, under the back and forth swinging, the dynamic culture of the biological sample can be achieved by the back and forth flow of the culture medium in the two liquid storage chambers 10b. It should be noted that the swinging device can be a shaker or a device that drives the cell culture device 300 to swing. Of course, it can also be swung manually by the staff, and there is no limitation on this.

[0073] Step S30 includes: swinging and adjusting the cell culture device 300 to dynamically culture the biological sample; periodically replacing the culture medium in the liquid storage chamber 10 b, and finally obtaining the cell model 500 .

[0074] In this step, as the culture medium and cells exchange substances, the excreta of the cells in the liquid gel are taken away, so there will be excreta in part of the culture medium, so it is necessary to periodically replace the culture medium in the liquid storage chamber 10b, so that the culture medium has a better cell culture effect on the liquid gel and better simulates the growth environment of the cells, so as to finally obtain the desired cell model 500. Among them, 2-3 days can be used as a cycle for replacing the culture medium in the liquid storage chamber 10b, of course, it can also be other time periods, which are not limited to this.

[0075] The technical solution of the present application can realize dynamic cultivation of liquid gel by culture medium by adopting the above-mentioned cell culture method. Compared with static culture, the cell culture method of the present application can better simulate the growth environment of cells, so as to finally obtain a cell model 500 with better simulation effect, and at the same time can improve the success rate of culturing cell model 500.

[0076] The technical solution of the present application can realize dynamic cultivation of liquid gel by culture medium by adopting the above-mentioned cell culture method. Compared with static culture, the cell culture method of the present application can better simulate the growth environment of cells, so as to finally obtain a cell model 500 with better simulation effect, and at the same time can improve the success rate of culturing cell model 500.

[0077] Another aspect of the present application provides an application of the cell culture device 300 as described above in cell model culture and drug analysis.

[0078] The drug can be added into the cell culture device 300, for example, it can be mixed with the culture medium and then flowed to the liquid gel to infiltrate the liquid gel. In this way, the cell culture device 300 of the present application can be used to study the effect of the drug on the cell sphere, so as to be used in the pharmaceutical field such as drug analysis.

[0079] The same or similar numbers in the drawings of this embodiment correspond to the same or similar parts; in the description of this application, it should be understood that if the terms "upper", "lower", "left", "right", etc. indicate an orientation or position relationship based on the orientation or position relationship shown in the drawings, it is only for the convenience of describing this application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as a limitation on this patent. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0080] The above are only preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A cell culture unit, characterized in that include: A substrate having a culture cavity, a liquid storage cavity and a flow channel cavity formed therein, and the culture cavity and the liquid storage cavity have openings at the top of the substrate, and the openings of the culture cavities are used to allow the liquid gel containing the biological sample to enter the culture cavities; The two ends of the flow channel cavity are respectively connected to the culture cavity and the liquid storage cavity; The flow channel cavity comprises a first port portion communicating with the liquid storage cavity and a second port portion communicating with the culture cavity, and the flow channel cavity further forms a constricted portion between the first port portion and the second port portion, and the opening area of ​​the constricted portion is smaller than the opening area of ​​the first port portion and the second port portion; In the horizontal direction, the width of the necking portion is smaller than the width of the first port portion and the second port portion, so that the flow rate of the culture medium can be reduced after the culture medium flows through the necking portion.

2. The cell culture unit according to claim 1, characterized in that The flow channel cavity comprises a first channel section, and the first channel section is connected with the first port portion and the constricted portion; In the direction from the liquid storage cavity to the flow channel cavity, the width of the first cavity section in the horizontal direction gradually decreases.

3. The cell culture unit according to claim 2, characterized in that The flow channel cavity further includes a second cavity section, and the second cavity section communicates with the constricted portion and the second port portion; In the direction from the liquid storage cavity to the flow channel cavity, the width of the second cavity section in the horizontal direction gradually increases.

4. The cell culture unit according to claim 1, characterized in that The culture chamber includes a first chamber bottom wall, and the flow channel chamber includes a second chamber bottom wall extending from the first port portion to the second port portion, 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.

5. The cell culture unit according to claim 4, characterized in that The substrate comprises a culture layer and a liquid storage layer, wherein the liquid storage layer is covered on the culture layer, and a first flow channel and a second flow channel having the openings are formed in the liquid storage layer; The surface of the culture layer near the liquid storage layer is concave to form a first groove, a second groove and a third groove that are connected to each other, the first flow channel is connected to the first groove to form the culture cavity, the second flow channel is connected to the second groove to form the liquid storage cavity, the lower surface of the liquid storage layer cooperates with the third groove to form the flow channel cavity, the bottom wall of the first groove is formed as the first cavity bottom wall, and the bottom wall of the third groove is formed as the second cavity bottom wall; The first groove also includes a groove side wall, which surrounds the outside of the first cavity bottom wall and has a lower edge intersecting with the first cavity bottom wall and an upper edge intersecting with the second cavity bottom wall, the upper edge is coplanar with the second cavity bottom wall, and the intersection portion of the upper edge and the second cavity bottom wall is an arc structure.

6. The cell culture unit according to claim 5, characterized in that The projection of the upper edge on the horizontal plane is located outside the projection of the lower edge on the horizontal plane.

7. The cell culture unit according to claim 6, characterized in that The groove sidewall is an arc surface, and the angle between the connecting line of the intersection points of the upper edge and the lower edge with the vertical section and the horizontal plane is greater than 0 degrees and less than 90 degrees; Alternatively, the groove side wall is an inclined surface, and the angle between the groove side wall and the horizontal plane is greater than 0 degree and less than 90 degrees.

8. The cell culture unit according to claim 5, characterized in that The cell culture unit further includes a separation membrane, and the separation membrane covers at least a portion of a side wall of the first flow channel.

9. The cell culture unit according to claim 4, characterized in that: The liquid storage cavity comprises a third cavity bottom wall, and the third cavity bottom wall is arranged coplanar with the second cavity bottom wall.

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

11. A cell culture device, characterized in that: The method comprises a plurality of cell culture units according to any one of claims 1 to 10, wherein the plurality of cell culture units are arranged in an array.

12. A cell culture method, applied to the cell culture device according to claim 11, characterized in that: The following steps are involved: injecting the biological sample into the culture chamber of the cell culture unit; Adding culture medium to the liquid storage chamber of the cell culture unit, and allowing the culture medium to infiltrate the biological sample; The cell culture device is swung and adjusted to dynamically culture the biological sample to obtain an in vitro three-dimensional biological model.

13. Use of the cell culture device according to claim 11 in cell model culture and drug analysis.

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