Culture chip based on open chamber patterned array microfluid and automatic culture system

Abstract

The utility model belongs to the field of micro-fluidic chips, and discloses a culture chip and an automatic culture system based on open chamber patterned array microfluid. The culture chip comprises an array channel layer, an open channel region, a culture solution storage chamber, a hole array layer and a patterned array, wherein the open channel region is formed by arranging a plurality of runners side by side; the culture solution storage chamber is connected with an outlet of the open channel region; the hole array layer and the patterned array are sequentially bonded below the open channel region; holes in the hole array layer and patterns of the patterned array are in one-to-one correspondence to form an isolation chamber array; the open chamber cover plate covers the array channel, a hollow opening is formed in the middle of the open chamber cover plate, and a sample inlet connected with the open channel area and a sample outlet connected with the culture solution storage chamber are formed in the two ends of the open chamber cover plate. According to the chip, the open channel region, the holes below the open channel region and the patterned array are designed to form the isolation chamber array, and the culture solution storage chamber is combined, so that frequent solution change and cell impact and loss caused by solution change are avoided.

Description

Technical Field

[0001] The utility model belongs to the technical field related to microfluidic chips, and more specifically, relates to a culture chip and an automated culture system based on open chamber patterned array microfluidics. Background Art

[0002] Biomedical research is gradually shifting from traditional two-dimensional cell culture models to more biologically relevant three-dimensional culture methods. Three-dimensional cell culture technology can more realistically replicate the three-dimensional structure, biomechanical properties and cell-to-cell interactions of cells in the body, providing an important experimental platform for disease modeling, drug discovery and tissue engineering.

[0003] At present, conventional three-dimensional culture systems usually rely on manual operations, which is not only time-consuming and labor-intensive, but also has operational inconsistencies, which limits the application of three-dimensional cell culture in high-throughput analysis and industrial production. In addition, existing three-dimensional culture methods often require regular fluid replacement to maintain nutrition and remove metabolic waste, which increases the risk of cross-contamination and may disrupt the cell microenvironment. In the process of forming cell spheres, it is difficult for existing technologies to maintain the consistency and controllability of spheres, which poses an obstacle to standardized analysis and clinical applications.

[0004] In summary, existing three-dimensional culture technologies generally have problems such as low culture efficiency, complex operation, repeated fluid replacement, uncontrollable spheroid size, and difficulty in achieving high-throughput automated culture. Therefore, it is urgent to develop a simple patterned array microfluidic three-dimensional cell and organoid culture chip that is easy to culture for a long time. Utility Model Content

[0005] In response to the above defects or improvement needs of the prior art, the utility model provides a culture chip and an automated culture system based on open chamber patterned array microfluidics, the purpose of which is to form an isolation chamber array with separated chambers through an open channel area and the holes stacked and bonded thereunder and the patterned array at the bottom, and to form an array channel layer together with the culture fluid storage chamber horizontally connected to the open channel area, thereby solving technical problems such as frequent fluid changes in cells and avoiding cell shock and loss caused by fluid changes.

[0006] To achieve the above object, according to one aspect of the utility model, a culture chip based on open chamber patterned array microfluidics is provided, comprising an open chamber cover plate and an array channel layer stacked sequentially from top to bottom;

[0007] The array channel layer comprises: an open channel area composed of a plurality of flow channels arranged side by side and a culture liquid storage chamber connected to the outlet of the open channel area, and a hole array layer and a pattern array bonded in sequence below the open channel area; wherein the hole rows in the hole array layer and the pattern rows in the pattern array correspond directly below the flow channels, and the holes in the hole array layer and the patterns in the pattern array correspond one to one to form an isolated chamber array;

[0008] The open chamber cover has a hollow opening in the middle, and both ends of the open chamber cover have an inlet connected to the open channel area and an outlet connected to the culture fluid storage chamber; wherein the hollow opening is the same size as the open channel area.

[0009] As a preferred embodiment of the present invention, the capacity of each flow channel in the open channel area is the same, and the capacity of each isolation chamber in the isolation chamber array is the same.

[0010] As a preferred embodiment of the present invention, the array channel layer also includes a first diversion channel connected to the outlet of the injection port, which is used to transport the liquid introduced from the injection port to the flow channel in the open channel area through the first diversion channel; wherein the channel of the first diversion channel corresponds to the flow channel of the open channel area, and the path length of each channel of the first diversion channel is the same.

[0011] As a preferred embodiment of the present invention, the array channel layer further comprises a second shunt channel connected to the inlet of the sample outlet, for shunting the liquid in the culture fluid storage chamber to the sample outlet through the second shunt channel.

[0012] As a preferred embodiment of the present invention, the array channel layer further includes a flow rate stabilization chamber, specifically:

[0013] A first flow rate stabilization chamber is connected to the inlet of the open channel area, and a second flow rate stabilization chamber is connected to the outlet of the culture solution storage chamber.

[0014] As a preferred embodiment of the present invention, the first flow rate stabilization chamber is a channel with a storage space connected before the entrance of the open channel area;

[0015] The second flow rate stabilizing chamber is a channel with a decreasing spatial height along the outlet direction of the culture solution storage chamber.

[0016] As a preferred embodiment of the present invention, each pattern in the patterned array has a plurality of small holes, and the surface has a modified coating to prevent cell attachment.

[0017] As a preferred embodiment of the present invention, the bottom surface of the culture solution storage chamber is lower than the surface formed by the connection between the open channel area and the hole array layer.

[0018] To achieve the above-mentioned object, according to another aspect of the present invention, an automated culture system is provided, comprising a culture chip based on open chamber patterned array microfluidics as described in any one of the first aspects of the present invention.

[0019] As a preferred embodiment of the present invention, it also includes a control panel, a temperature control system, a carbon dioxide valve and an ultraviolet sterilization system, and the culture chip based on the open chamber patterned array microfluidic, the temperature control system, the carbon dioxide valve and the ultraviolet sterilization system are connected through the control panel.

[0020] In general, compared with the prior art, the above technical solutions conceived by the present invention have the following beneficial effects:

[0021] (1) The culture chip of the utility model forms an open isolation chamber array through an open channel area and a hole array layer and a patterned array below, which is connected to a culture medium storage chamber to form an open public culture environment. The effect of evenly distributing the culture is achieved by filling and emptying the open channel area. In addition, the nutrients consumed in the isolation chamber and the metabolic waste discharged are continuously exchanged with the culture medium in the culture medium storage chamber, thereby avoiding the problem of frequent liquid changes in traditional culture methods and improving culture efficiency and ease of operation.

[0022] For example, when in use, a cycle of fluid-free operation can be completed by manually filling up independent chambers; or after a peristaltic pump is connected to the inlet and outlet, the culture fluid storage chamber and the flow channel allow continuous flow of the culture fluid, thereby reducing cell shock and loss that may be caused during the fluid change process, reducing the complexity of cell culture and improving the quality of cells; or by filling up independent chambers in sequence through a timed manner, a gradient of cell spheroids can be formed, making the formation of cell spheroids more flexible.

[0023] (2) Preferably, the capacity of each flow channel in the open channel area of ​​the present invention is the same, and the capacity of each isolation chamber in the isolation chamber array is the same, ensuring that the flow rate of the fluid is the same and the culture environment of the cells in the same chip is consistent.

[0024] (3) As a preferred embodiment, the utility model uses the first flow channel connected to the outlet of the sample inlet to control the liquid distribution of each flow channel, ensuring that the cell suspension can flow evenly into the open channel, thereby facilitating the uniform distribution and aggregation of cells into spheres in the isolation chamber array, thereby improving the quality and consistency of the cell spheres. And the second flow channel at the inlet of the sample outlet is used to stabilize the liquid discharge.

[0025] (4) As a preferred embodiment, the steady flow chamber of the utility model ensures that the cell suspension can stably flow into and out of the open channel, thereby facilitating the uniform distribution and aggregation of cells into spheres within the isolation chamber array, which improves the quality and consistency of the cell spheres.

[0026] (5) Preferably, each pattern of the patterned array of the utility model comprises a plurality of small holes, which can realize batch culture of three-dimensional cells or organoids. The shape and size of the holes in the hole array layer and the shape and size of the patterns in the patterned array can be adjusted according to the shape and size of the cultured object, and can be applied to large-scale cell experiments and drug testing.

[0027] (6) As a preferred embodiment, the present invention has the potential for automated cultivation. The design of the chip has the potential to be combined with automated equipment and can be further developed into an automated cultivation system to improve experimental efficiency and reduce human resource consumption.

[0028] In summary, the utility model forms an open isolation chamber array by opening the channel area and the hole array layer and patterned array below, and the design connected to the culture medium storage chamber realizes the elimination of liquid change during cell culture, thereby avoiding the cell shock and loss problems caused by liquid change and improving the culture efficiency. Furthermore, it is applied to control the size of cell spheroids and culture three-dimensional cell spheroids or organoids in batches. At the same time, it can also be combined with equipment to realize automated culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a structural exploded diagram of a culture chip based on open chamber patterned array microfluidics according to an example of the present utility model;

[0030] Figure 2 It is a structural diagram and a partially enlarged schematic diagram of a culture chip based on an open chamber patterned array microfluidic according to an example of the present utility model;

[0031] Figure 3 It is a time series diagram of colorectal cancer organoid culture illustrated in Example 2 of the present utility model;

[0032] Figure 4 is a growth analysis diagram of colorectal cancer organoids illustrated in Example 2 of the present utility model, wherein Figure 4 a in the figure is the organoid size analysis, and b is the organoid live-dead analysis;

[0033] Figure 5 It is the MCF-7 cell sphere culture growth analysis diagram and time series diagram of Example 3 of the present utility model; wherein Figure 5 a is a growth analysis chart, and b is a time series chart;

[0034] Figure 6This is a live-dead staining analysis diagram of MCF-7 cell sphere culture illustrated in Example 3 of the present utility model, wherein Figure 6 a is the actual picture of the stained cell sphere, and b is the live-death analysis picture of the cell sphere;

[0035] Figure 7 This is a verification diagram of two spheroidization methods in MCF-7 cell spheroid culture as exemplified in Example 3 of the present utility model, wherein Figure 7 a is the data analysis of the gradient size of cell spheres formed during slow injection, b is the physical display of the gradient and uniformity of cell spheres, and c is the data analysis of the gradient uniformity of cells formed during fast injection;

[0036] Figure 8 4 is a diagram of a peristaltic pump access and flow culture method according to Example 4 of the present utility model, wherein Figure 8 Figure a is a schematic diagram of the peristaltic pump connection, and b is a diagram of the flow culture process.

[0037] Throughout the drawings, the same reference numerals are used to denote the same elements or structures, wherein:

[0038] 1-inlet, 2-outlet, 31-first diversion channel, 32-second diversion channel, 41-first flow rate stabilization chamber, 42-second flow rate stabilization chamber, 5-open channel, 6-culture fluid storage chamber, 7-hole array layer, 8-patterned array. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical solution and advantages of the utility model more clear, the utility model is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the utility model and are not used to limit the utility model. In addition, the technical features involved in each embodiment of the utility model described below can be combined with each other as long as they do not conflict with each other.

[0040] like Figure 1 As shown, the utility model provides a culture chip based on open chamber patterned array microfluidics, which integrates an open channel area, and an open channel area 5 and a hole array layer 7 and a patterned array 8 bonded thereunder to form an open array channel with independent isolation chambers (i.e., an isolation chamber array).

[0041] Specifically, the culture chip includes: an array channel and an open chamber cover plate covering the array channel;

[0042] like Figure 1 and Figure 2As shown, the culture liquid storage chamber 6 is horizontally connected to the open channel area 5; the chip bottom plate surface of the array channel is a patterned array 8, and each pattern in the patterned array 8 is composed of a number of small holes. Each hole in the hole array layer 7 corresponds to each pattern in the patterned array 8, forming an isolated chamber array. The number of flow channels in the open channel 5 is the same as the number of rows in the isolated chamber array, and the hole row in the bonding hole array layer 7 and the pattern row in the patterned array 8 are directly below a flow channel in the open channel 5.

[0043] The open chamber cover includes a Figure 1 The inlet 1 and outlet 2 shown at the two ends are respectively connected to the open channel area and the outlet 2 is connected to the culture medium storage chamber 6, which are used for inputting and outputting the culture medium; the area covered above the open channel area 5 adapted for press-drug administration is open (open or detachable and openable), and the hollow opening, the open channel area 5 and the isolation chamber array are of the same size to facilitate unobstructed subsequent operations and observations.

[0044] The patterned array arrangement in the utility model, the open channel area is laterally connected to the culture fluid storage chamber, the setting of this structure forms the free diffusion of lateral material density, realizes the delivery of nutrients and the discharge of metabolites, and allows a culture cycle without changing the liquid through manual addition, or after the inlet 1 and the outlet 2 are connected to the external peristaltic pump, on the one hand, it can continue to automatically culture without changing the liquid, so that cells can be evenly or gradiently distributed in the patterned micropores, and on the other hand, it is used to adjust the flow rate of the culture fluid or cell suspension in the chip to stabilize the flow culture environment and support the formation and growth of cell spheres. Or, by manual or automatic control of single-row injection, the number of independent chambers passed in a certain period of time forms a uniform cell spheroid or a cell spheroid with a size gradient. And the independent compartment array can be used to culture different cell types or mixed cultures, increasing the flexibility and diversity of experimental design.

[0045] In some embodiments, each flow path of the open channel area 5 serves as a flow splitting structure including but not limited to using the same flow channel capacity to evenly divide the fluid resistance.

[0046] In some embodiments, the array channel layer further includes a first shunt channel at the outlet of the injection port, which is used to transport the liquid introduced from the injection port 1 to the flow channel in the open channel area 5 through the first shunt channel. At the same time, the number of channels of the first shunt channel is the same as and corresponds to the number of flow channels in the open channel area 5, and is used to isolate and transport the culture fluid in each flow channel one by one. The channel design of the above-mentioned first shunt channel is based on the principle of fluid resistance, and the same path length of each channel ensures uniform distribution of the culture fluid in the microporous array, so as to ensure uniform cell injection and equal supply of culture fluid in each row of channels.

[0047] In some embodiments, the array channel layer also includes a second diversion channel at the entrance of the sample outlet 2, which is used to divert the liquid in the culture medium storage chamber 6 to the sample outlet 2 through the second diversion channel to make the fluid converge evenly, thereby avoiding overall imbalance caused by local poor circulation during the flow process.

[0048] In some embodiments, the array channel layer further comprises a flow rate stabilization chamber 4 for stabilizing the flow rate of the culture fluid to reduce fluid dynamic instability in the microwell array.

[0049] The flow rate stabilization chamber 4 includes: a first flow rate stabilization chamber 41 connected to the inlet of the open channel area 5, and a second flow rate stabilization chamber 42 connected to the outlet of the culture fluid storage chamber 6. The first flow rate stabilization chamber 41 is a channel with a storage space connected before the inlet of the open channel area 5, for example Figure 2 As shown in the partial enlarged view of 41, by setting a thickness above the channel space, and by setting a height barrier close to the open channel area 5, and reserving an opening leading to the flow channel in the open channel area 5, the liquid stored in the first flow rate stabilization chamber 41 is evenly transported to the flow channel in the open channel area 5; and the second flow rate stabilization chamber 42 is a channel with a reduced spatial height along the outlet direction of the culture liquid storage chamber 6, for example Figure 2 As shown in the partial enlarged view of FIG41 , a high barrier is provided near the second flow rate stabilizing chamber 41 , so that the culture fluid in the culture fluid storage chamber 6 can flow out through the second flow rate stabilizing chamber 41 slowly and steadily.

[0050] In some embodiments, a patterned array microfluidic chip is made, including but not limited to using polymethyl methacrylate relief to make a patterned array microfluidic chip or a gel mold. The pattern in the patterned array 8 has a number of micropores, and can realize batch culture of three-dimensional cells or organoids, wherein the hole shape and size of the hole array layer and the shape and size of the pattern of the patterned array can be adjusted according to the shape and size of the cultured object, and applied to large-scale cell experiments and drug testing. The pattern surface of the patterned array has a modified coating for preventing cell attachment to prevent cell attachment and promote the formation of cell spheroids. The modified coating with anti-cell attachment includes a surface active modification, a super hydrophilic layer or a super hydrophobic layer, for example, including but not limited to using a polyadduct of polypropylene glycol and ethylene oxide to modify the patterned array.

[0051] In some embodiments, the bottom surface of the culture fluid storage chamber 6 is lower than the surface formed by the connecting portion of the open channel area 5 and the hole array layer 7, so as to increase the capacity of the culture fluid storage chamber.

[0052] The utility model also provides an automated culture system, including a culture chip based on an open chamber patterned array microfluidic. It also includes a control panel, a temperature control system, a carbon dioxide valve, and a UV sterilization system. The chip can be integrated into the automated culture system, and has interoperability with components such as the control panel, the temperature control system, the carbon dioxide valve, and the UV sterilization system, to achieve fully automated cell or organoid culture.

[0053] The culture chip based on open chamber patterned array microfluidics provided in the above embodiment is used by directly adding culture suspension through the injection port 1. Under the action of the first diversion channel 31 and the first stabilization chamber 41 including but not limited to the overflow, the culture evenly enters the open channel area 5, but does not reach the culture fluid storage chamber 6. Then, the culture suspension is refluxed through the injection port 1, and the culture is evenly divided under the action of the hole array layer 7. After settling in the open channel area 5, the culture enters the isolation chamber to form cell spheroids.

[0054] Among them, the chip allows connection to an external micro peristaltic pump, and through the peristaltic pump body and its flow control system, it promotes the formation of uniform cell spheroids or cell spheroids with a size gradient, while realizing closed-loop flow of the liquid in the chip.

[0055] The culture chip based on open chamber patterned array microfluidics provided in the above embodiments is used in the culture of three-dimensional cell spheroids or organoids, for example, it is used to form cell spheroids in most cell lines, such as mcf-7, 1975, KYSE450 and HT22; and the stable growth of various organoids, such as normal or tumor organoids of intestine, stomach, breast, liver, lung, etc.

[0056] The manufacturing of the chip includes but is not limited to laser engraving, mold casting, soft lithography and other processes, and the materials used include but are not limited to high molecular polymers, inorganic glass, quartz, silicon wafers, polymethyl methacrylate, etc., which can adapt to different experimental designs and culture needs.

[0057] The following is an example of the preparation and application of a culture chip based on open chamber patterned array microfluidics.

[0058] Embodiment 1:

[0059] A three-dimensional cell and organoid culture device chip preparation based on an open chamber patterned array microfluidic chip:

[0060] (1) First, peel off the 1.5 mm polymethyl methacrylate upper and lower layers for later use;

[0061] (2) Use a laser engraving machine to emboss on polymethyl methacrylate such as Figure 2 The patterned array shown;

[0062] (3) Prepare another piece of polymethyl methacrylate, peel off the film on one side, stick on the special double-sided tape, and then use the laser engraving machine to engrave directly. Figure 2 The chip upper structure shown;

[0063] (4) peeling off the double-sided adhesive layer of the engraved patterned array microfluidic chip and the upper structure under the action of the fixed column to bond them together;

[0064] (5) Place the bonded chip into a hot press with the upper and lower pressing surfaces at 60°C for 20 minutes;

[0065] (6) After the temperature of the hot-pressed chip returns to room temperature, it is placed in pure water for 24 hours of ultrasound and then blown dry with a nitrogen gun;

[0066] (7) using a polypropylene glycol and ethylene oxide addition polymer, dripping the polypropylene glycol and ethylene oxide addition polymer on each macropore, sucking it out after completely covering the macropore, waiting for it to air dry to complete the modification, and putting it into a transparent plastic bag and sealing it for later use;

[0067] (8) Before the experiment, place the sealed chip in (7) in a UV sterilizer or clean bench for 30 minutes for subsequent experiments.

[0068] Example 2: The present invention describes the method of manually controlling the uniform sphering in combination with the growth of intestinal organoids.

[0069] The utility model provides a method for accurately controlling the spheroidization process of organoids by utilizing microfluidics technology and colorectal cancer organoid culture, thereby ensuring the growth uniformity of organoids and being suitable for high-throughput screening and drug testing of intestinal organoids.

[0070] (1) First, colorectal cancer organoids were digested into single-cell suspensions and the cell concentration was adjusted to 5×10 5 / ml, and 1ml of the cell suspension was inoculated into a patterned array microfluidic chip designed for organoid culture. The microfluidic chip has an optimized geometric design to support the independent and uniform growth of organoids.

[0071] (2) During inoculation, the cell suspension was mixed with PBS to a total volume of 4 ml, and the mixed liquid was quickly and evenly introduced into the culture channel of the microfluidic chip through the inlet of the open chamber.

[0072] (3) After a 20-minute sedimentation period, the cells begin to settle in their respective depressions in the microcavity. The upper PBS is then slowly aspirated and replaced with an appropriate organoid culture medium to supply the necessary nutrients and growth factors. After the organoids consume the nutrients in the culture medium, the attached storage chamber will automatically replenish fresh culture medium due to the change in the nutrient density gradient, thereby maintaining the continuity of nutrient supply.

[0073] (4) Figure 4 As shown in a, after 7 days of culture, colorectal cancer organoids showed good spheroid formation in each microchamber, which is consistent with Figure 3 The results are consistent with those shown in Figure 1, that is, through precise manual control and the assistance of microfluidics, the size distribution of organoid spheres showed a high degree of uniformity. Furthermore, the live and dead cells of organoid spheres cultured on day 7 were stained with Calcein AM and propidium iodide (PI) and observed using confocal microscopy imaging technology, as shown in Figure 1. Figure 4 As shown in b, the proportion of live cells inside the cell sphere is high, and there are very few apoptotic or dead cells, indicating that this culture method maintains the high activity and health of the organoids.

[0074] Through the above steps, the utility model not only significantly improves the quality and consistency of organoid spheroid formation, but also provides a reliable and standardized platform for subsequent drug screening or biological research. In addition, the high throughput and automatic replenishment characteristics of this method greatly improve experimental efficiency and reduce the common human errors and differences in organoid growth in manual culture.

[0075] Example 3: The present invention describes in detail the cell culture and manual formation of uniform or gradient cell spheres in combination with breast tumor sphere formation

[0076] In breast tumor research, the formation of cell spheres is a key step in studying tumor microenvironment and drug response. The utility model combines microfluidic technology to elaborate a method for forming a gradient of breast tumor cell spheres in a microfluidic chip by manual operation. The uniform formation of cell spheres has been described in Example 2 and will not be repeated here.

[0077] (1) First, breast tumor cells (e.g., MCF-7 cells) were prepared into a single-cell suspension by enzymatic digestion and the cell concentration was adjusted to 5 × 10 5 Then, 1 ml of the cell suspension was inoculated into a specially designed open chamber patterned array microfluidic chip, which facilitated the formation and maintenance of cell spheres through a sophisticated microchamber design.

[0078] (2) During the manual inoculation process, the time is precisely controlled to fill each independent chamber one by one in sequence. By controlling the filling sequence and speed, combined with the natural sedimentation of cells under the action of gravity, such as Figure 7 As shown in a and b in the figure, under the condition of slow injection with controlled time, the cell sizes formed in the front, middle and back micropores are significantly different, and present a gradient distribution, thus forming a gradient of cell concentration in different areas of the microfluidic chip. Figure 7As shown in b and c, under fast injection, there is no significant difference in the size of cells formed in the front and middle microwells, and there is a slight difference in the size of cells in the front and back to achieve uniform size of cell spheres.

[0079] (3) After the inoculation is completed, wait for 20 minutes for the cells to begin to settle in their respective depressions in the microcavity. Then, slowly aspirate the upper PBS and replace it with a suitable culture medium to supply the necessary nutrients and growth factors. After the cell spheres consume the nutrients in the culture medium, the attached storage chamber will automatically replenish fresh culture medium due to the change in the nutrient density gradient, thereby maintaining the continuity of nutrient supply.

[0080] (4) Figure 5 As shown in b, after 7 days of culture, the cells formed spherical structures in each microchamber. Figure 6 As shown in a, there are no obvious dead cells as shown by live-dead staining. Figure 6 Quantitative analysis in b shows that the live cell staining area is significantly higher than the dead cell staining area, indicating that the cell sphere is growing well. Figure 5 As shown in a, the size of the cell spheres in different chambers can present a predetermined gradient distribution or uniform distribution, demonstrating the controllable ability of the cell suspension inoculation amount and sedimentation time on the size of the cell spheres.

[0081] Through the above steps, this method not only improves the experimental efficiency and repeatability of the cell spheroid model in breast tumor research, but also creates conditions for further drug screening and cell biology research, especially for those applications that require high-throughput analysis of the effects of different cell spheroid sizes on drug screening.

[0082] Example 4: The utility model further illustrates the chip expansion application in combination with a peristaltic pump

[0083] 1. Use a peristaltic pump to form uniform cell spheroids: The remaining steps are the same as those in Example 1 and will not be described in detail. The main difference is that when using a peristaltic pump for inoculation, 4 ml of cell suspension is connected to the peristaltic pump and pumped into the pipeline, and the peristaltic pump speed is adjusted to the maximum to push the cell suspension to quickly flow into the entire channel;

[0084] 2. Use a peristaltic pump to form a cell spheroid with a size gradient: The remaining steps are the same as those in Example 2, and will not be described in detail again. The main difference is that when using a peristaltic pump for inoculation, 4 ml of cell suspension is connected to the peristaltic pump and pumped into the pipeline, the peristaltic pump speed is adjusted to the minimum (500 μ / ml), the cell suspension is pushed to slowly flow into the entire channel, and the peristaltic pump is stopped;

[0085] 3. Use a peristaltic pump for flow circulation culture: Change the peristaltic pump inlet to the chip outlet, and the pump outlet to the chip inlet. Figure 8 a in the figure is connected to a peristaltic pump for circulation culture. The culture process is as follows Figure 8 As shown in b.

[0086] Example 5: The utility model combines automated equipment to perform drug testing

[0087] (1) Cell inoculation: First, digest the colorectal cancer organoids into a single cell suspension and adjust the cell concentration to 5×10 5 / ml, and 1ml of the cell suspension was inoculated into the three-dimensional cell and organoid culture device chip of the open chamber patterned array microfluidic chip. During inoculation, the cell suspension was mixed with PBS to a total volume of 4ml, and the mixed liquid was quickly and evenly introduced into the culture channel of the microfluidic chip through the inlet of the open chamber; after a 20-minute sedimentation period, the cells began to settle in their respective depressions in the microcavity, and then the upper PBS was slowly aspirated and replaced with a suitable organoid culture medium to supply the necessary nutrients and growth factors.

[0088] (2) The inoculated chip is placed in an automated instrument. The inlet and outlet of the chip can be directly connected to the automated instrument. The chip is cultured in a circulating flow manner by setting a corresponding program inside the instrument until an independent operation stage is required.

[0089] (3) Through the program settings in the instrument, the outflow of culture fluid from each chamber is precisely controlled from the chip outlet, forming a height difference between the chambers and the liquid surface is concave, thereby achieving physical separation between adjacent chambers. This isolation method can prevent both the flow of liquids and the migration of cells, thereby avoiding cross contamination.

[0090] (4) A drug-sensitive plate is magnetically attached to the top of the device. The drug-sensitive plate has multiple drug probes. The probes on the drug-sensitive plate are pressed to contact the liquid surface of each independent chamber to achieve quantitative drug delivery.

[0091] (5) Use an automated system to monitor cell growth and biological responses after drug administration. Collect data on cells in each chamber in real time through imaging technology and biomarker detection. Summarize and analyze the growth data and biological responses of cells in all chambers to quantitatively evaluate the effects of each drug. Use statistical software to compare differences between different treatment groups to screen out effective drug candidate molecules. Confirm the toxicity and efficacy of candidate drugs on target cells for subsequent experimental verification and clinical research advancement.

[0092] It is easy for those skilled in the art to understand that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A culture chip based on open chamber patterned array microfluidics, characterized in that: It includes an open chamber cover plate and an array channel layer stacked in sequence from top to bottom; The array channel layer comprises: an open channel area (5) composed of a plurality of flow channels arranged side by side and a culture fluid storage chamber (6) connected to the outlet of the open channel area (5), and a hole array layer (7) and a pattern array (8) bonded in sequence below the open channel area (5); wherein the hole rows in the hole array layer (7) and the pattern rows in the pattern array (8) correspond directly below the flow channels, and the holes in the hole array layer (7) and the patterns in the pattern array (8) correspond one to one to form an isolated chamber array; The open chamber cover has a hollow opening in the middle, and both ends of the open chamber cover have an inlet (1) connected to the open channel area (5), and an outlet (2) connected to the culture fluid storage chamber (6); wherein the hollow opening is the same size as the open channel area (5).

2. The culture chip based on open chamber patterned array microfluidics according to claim 1, characterized in that: The capacity of each flow channel of the open channel area (5) is the same, and the capacity of each isolation chamber in the isolation chamber array is the same.

3. The culture chip based on open chamber patterned array microfluidics according to claim 1, characterized in that: The array channel layer further comprises a first shunt channel connected to the outlet of the injection port (1), for conveying the liquid introduced from the injection port (1) to the flow channel in the open channel area (5) through the first shunt channel; wherein the channel of the first shunt channel corresponds to the flow channel of the open channel area (5), and the path length of each channel of the first shunt channel is the same.

4. The culture chip based on open chamber patterned array microfluidics according to claim 1, characterized in that: The array channel layer further comprises a second shunt channel connected to the inlet of the sample outlet (2), for shunting the liquid in the culture liquid storage chamber (6) to the sample outlet (2) through the second shunt channel.

5. The culture chip based on open chamber patterned array microfluidics according to claim 1, characterized in that: The array channel layer further comprises a flow rate stabilization chamber (4), specifically: A first flow rate stabilization chamber (41) connected to the inlet of the open channel area (5), and a second flow rate stabilization chamber (42) connected to the outlet of the culture solution storage chamber (6).

6. The culture chip based on open chamber patterned array microfluidics according to claim 5, characterized in that: The first flow rate stabilization chamber (41) is a channel with a storage space connected before the entrance of the open channel area (5); The second flow rate stabilization chamber (42) is a channel with a decreasing spatial height along the outlet direction of the culture solution storage chamber (6).

7. The culture chip based on open chamber patterned array microfluidics according to claim 1, characterized in that: Each pattern in the patterned array (8) has a plurality of small holes, and the surface has a modified coating that prevents cell attachment.

8. The culture chip based on open chamber patterned array microfluidics according to claim 1, characterized in that: The bottom surface of the culture fluid storage chamber (6) is lower than the surface formed by the connection between the open channel area (5) and the hole array layer (7).

9. An automated culture system, characterized in that: It comprises a culture chip based on open chamber patterned array microfluidics as described in any one of claims 1 to 8.

10. The automated culture system according to claim 9, characterized in that: It also includes a control panel, a temperature control system, a carbon dioxide valve and an ultraviolet sterilization system, and the culture chip based on the open chamber patterned array microfluidic, the temperature control system, the carbon dioxide valve and the ultraviolet sterilization system are connected through the control panel.

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