Three-dimensional cell culture chip and load

Abstract

The utility model discloses a three -dimensional cell culture chip and load, wherein, three -dimensional cell culture chip, include: upper clamp, and the upper clamp is set up and is filled the entrance and the filling outlet of the current, lower clamp is detachably connected with upper clamp, chip is clamped between upper clamp and lower clamp, wherein, two first filling mouths are set up on the chip, and the filling entrance and the filling outlet are respectively with the coaxial heart communication of corresponding first filling mouth, and the chip includes a plurality of three -dimensional cell culture support, and the chip inside is provided with a plurality of installation cavities, and a plurality of three -dimensional cell culture supports are respectively detachably set up in corresponding installation cavity, and the chip inside is provided with micro -flow channel grid, and micro -flow channel grid is linked with a plurality of installation cavities and two first filling mouths respectively. Therefore, can realize more cell's high -efficient accommodation in limited space, and through the construction three -dimensional space culture environment, the natural survival state of cell in the body is restored to the maximum extent, thereby powerful guarantee cell culture effect.

Description

Technical Field

[0001] This utility model relates to the field of cell culture technology, and in particular to a three-dimensional cell culture chip and payload. Background Technology

[0002] In a ground-based laboratory setting, the core essence of cell culture technology lies in meticulously creating a suitable in vitro environment for cell survival. This environment must strictly meet aseptic conditions, while precisely controlling key parameters such as temperature, humidity, and pH, and providing sufficient and properly proportioned nutrients. Only in this way can cells achieve normal survival, continuous growth, effective reproduction, and maintain their critical physiological functions in the in vitro environment.

[0003] It is important to note that space cell culture must also adhere to these fundamental principles. However, to meet these stringent conditions, existing commercial instruments in ground-based laboratories are typically quite large, occupying significant space resources. Due to limitations in space adaptability and other factors, these commercially available instruments commonly used in ground-based laboratories cannot be directly applied to the space environment.

[0004] Therefore, when conducting life science and medical research using cell models in a space environment, traditional two-dimensional planar cell culture techniques are often the only option. However, in this technology, cells can only grow unidirectionally along the culture surface, which severely limits the number of cells that can be accommodated per unit area. Due to the extremely low cell culture density, it is difficult to fully utilize limited resources to culture more cells in the resource-scarce and precious environment of space, thus severely limiting the number of samples that can be obtained.

[0005] Furthermore, most cells in the human body exist within a complex three-dimensional spatial structure, tightly surrounded by other cells and the extracellular matrix. Two-dimensional planar cell culture technology, due to its inherent limitations, struggles to accurately simulate this complex three-dimensional structure, thus failing to realistically and comprehensively reproduce the interactions between cells and between cells and the matrix, ultimately having a significant negative impact on the effectiveness of cell culture. Utility Model Content

[0006] This utility model aims to at least partially solve one of the technical problems in the related art.

[0007] Therefore, one objective of this invention is to propose a three-dimensional cell culture chip and payload that can efficiently accommodate more cells in a limited space, and at the same time, by constructing a three-dimensional spatial culture environment, it can restore the natural survival state of cells in vivo to the greatest extent, thereby effectively ensuring the cell culture effect.

[0008] To achieve the above objectives, the first aspect of this utility model provides a three-dimensional cell culture chip, comprising:

[0009] The upper clamp is provided with an irrigation inlet and an irrigation outlet;

[0010] The lower clamp is detachably connected to the upper clamp;

[0011] The chip is held between the upper clamp and the lower clamp;

[0012] The chip has two first infusion ports, and the infusion inlet and the infusion outlet are coaxially connected to the corresponding first infusion ports.

[0013] The chip includes multiple three-dimensional cell culture scaffolds, and the chip has multiple mounting cavities inside, with each of the multiple three-dimensional cell culture scaffolds being detachably mounted in a corresponding mounting cavity;

[0014] The chip has a microfluidic channel grid inside, which is connected to multiple mounting cavities and two first infusion ports to form a fluid flow path.

[0015] This invention relates to a three-dimensional cell culture chip, which uses a tight fit between upper and lower clamps to secure the chip, ensuring its airtightness and stability after installation. It also allows for quick assembly and disassembly, facilitating cell inoculation and chip use. Furthermore, by constructing a microfluidic channel network within the chip connecting multiple mounting cavities, it enables simultaneous perfusion into multiple three-dimensional cell culture scaffolds, increasing the number of replicates and improving cell culture efficiency. This not only enhances cell culture efficiency but also allows for the placement of more cells in a smaller space, increasing cell density. The three-dimensional cell culture scaffold creates a three-dimensional cell culture environment, resulting in tissues that more closely resemble their in vivo state, maximally replicating the natural survival state of cells in vivo and thus effectively guaranteeing cell culture results.

[0016] In addition, the three-dimensional cell culture chip and payload proposed in the application may also have the following additional technical features:

[0017] Specifically, the chip includes a top mount, a base mount, and a sealing component sandwiched between the two. The sealing component has a first through hole and a plurality of second through holes on its surface.

[0018] The top seat is fitted with the upper clamp, and the base is fitted with the lower clamp;

[0019] The top surface of the top seat has two first infusion ports, the bottom surface of the top seat has multiple downward-opening first mounting cavities, and the top surface of the base has multiple upward-opening second mounting cavities. Each second mounting cavity, the second through hole, and the first mounting cavity are coaxially aligned in the vertical direction to form a closed mounting cavity.

[0020] Specifically, the microfluidic channel mesh includes:

[0021] A first microfluidic channel grid is formed on the lower surface of the top seat. The first microfluidic channel grid is connected to a plurality of first mounting cavities. The irrigation outlet is connected to the first microfluidic channel grid through a corresponding first irrigation port.

[0022] A second microfluidic channel grid is formed on the upper surface of the base. The second microfluidic channel grid is connected to multiple second mounting cavities. The top of another first infusion port is connected to the corresponding infusion inlet. The bottom of the first infusion port is connected to the second microfluidic channel grid through the first through hole.

[0023] Specifically, the chip further includes multiple support rings, each support ring having an open opening corresponding to the connection between the second microfluidic channel grid and the second mounting cavity. The support ring is placed in the second mounting cavity, and the top of the support ring is attached to the three-dimensional cell culture scaffold to lift the three-dimensional cell culture scaffold and form a gap between it and the bottom wall of the second mounting cavity.

[0024] Specifically, the three-dimensional cell culture scaffold is a three-dimensional porous scaffold.

[0025] Specifically, the upper clamp includes a first cover plate and a second cover plate, wherein,

[0026] The first cover plate, the second cover plate, and the top seat are attached to each other from top to bottom, and the first cover plate and the second cover plate are fixedly connected. The irrigation inlet and the irrigation outlet are opened on the first cover plate. The second cover plate has two second irrigation ports. One second irrigation port is connected to the irrigation inlet and the corresponding first irrigation port, and the other second irrigation port is connected to the irrigation outlet and the corresponding first irrigation port.

[0027] The second aspect of this invention provides a payload comprising a temperature control unit, a fluid control unit, a gas control unit, a control unit, an incubator, and the three-dimensional cell culture chip described in the first aspect, wherein...

[0028] The incubator has a removable sealing cover, and the incubator has an internal cavity in which the three-dimensional cell culture chip, temperature control unit, fluid control unit, gas control unit, and control unit are all located.

[0029] The temperature control unit, the fluid control unit, and the gas control unit are respectively connected to the control unit, and the fluid control unit is used to connect to the irrigation inlet and the irrigation outlet.

[0030] Specifically, the temperature control unit includes a temperature sensor and a semiconductor heating and cooling chip, wherein,

[0031] The temperature sensor is disposed in the cavity and is used to obtain the temperature inside the cavity;

[0032] The semiconductor heating and cooling chip is disposed in the cavity and is used to heat or cool the environment inside the cavity. The semiconductor heating and cooling chip and the temperature sensor are respectively connected to the control unit.

[0033] Specifically, the fluid control unit includes a reservoir, a fluid actuator, a first connecting pipe, two first connecting pipes, multiple first solenoid valves, a second connecting pipe, two Luer connectors, a collection bag, a multi-way pipe, and a second solenoid valve, wherein...

[0034] The liquid reservoir is provided with multiple areas, and each area is provided with a connecting port above it. Each connecting port is equipped with a first solenoid valve. One end of the multi-port pipe is connected to the first connecting pipe, and the other ports of the multi-port pipe are respectively connected to the corresponding first solenoid valve.

[0035] One end of the fluid actuator is connected to the first connecting pipe, and the other end of the fluid actuator is connected to the corresponding Luer connector through the first connecting pipe;

[0036] One end of the second solenoid valve is connected to the collection bag through the second connecting pipe, and the other end of the second solenoid valve is connected to the corresponding Luer connector through the first connecting pipe;

[0037] One of the Luer connectors is inserted into the irrigation outlet, and one end of the Luer connector extends through the second irrigation port, the first irrigation port and connects to the first microfluidic channel grid. The other Luer connector is inserted into the irrigation inlet, and one end of the Luer connector extends through the second irrigation port, the first irrigation port and the first through hole and connects to the second microfluidic channel grid. The first solenoid valve, the second solenoid valve and the fluid actuator are respectively connected to the control unit.

[0038] Specifically, the gas control unit includes a gas storage source, a concentration sensor, a second connecting pipe, and a third solenoid valve, wherein,

[0039] The second connecting pipe is used to connect the gas storage source and the third solenoid valve;

[0040] The concentration sensor is used to obtain the gas concentration in the cavity in real time, and the third solenoid valve and the concentration sensor are respectively connected to the control unit. Attached Figure Description

[0041] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the present invention and, together with the description, serve to explain the principles of the present invention.

[0042] To more clearly illustrate the technical solutions in the embodiments of this utility model or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0043] Figure 1 This is a schematic diagram of the structure of a three-dimensional cell culture chip according to an embodiment of the present invention;

[0044] Figure 2 This is an exploded view of a three-dimensional cell culture chip according to an embodiment of the present invention;

[0045] Figure 3 The images show the cell morphology of cells cultured using the three-dimensional cell culture chip of this invention at different time points.

[0046] Figure 4 This is a structural schematic diagram of the top seat according to one embodiment of the present invention from another perspective;

[0047] Figure 5 This is a cross-sectional structural diagram of a three-dimensional cell culture chip according to an embodiment of the present invention;

[0048] Figure 6 This is a structural block diagram of a load according to an embodiment of the present invention;

[0049] Figure 7 This is a schematic diagram of the structure of a fluid control unit according to an embodiment of the present invention;

[0050] Figure 8 This is a schematic diagram of the structure of a Luer connector according to an embodiment of the present invention;

[0051] Figure 9This is a schematic diagram of the structure of a gas control unit according to an embodiment of the present invention.

[0052] As shown in the figure:

[0053] 1. Three-dimensional cell culture chip; 10. Upper clamp; 11. Lower clamp; 12. Chip; 100. First cover plate; 101. Second cover plate; 120. Top seat; 121. Base; 122. Sealing component; 123. Three-dimensional cell culture scaffold; 124. Support ring; 1000. Irrigation inlet; 1001. Irrigation outlet; 1010. Second Irrigation port; 1200. First Irrigation port; 1201. First mounting cavity; 1202. First microfluidic channel mesh; 1210. Second mounting cavity; 1211. Second microfluidic channel mesh; 1220. First through hole; 1221. Second through hole;

[0054] 2. Temperature control unit;

[0055] 3. Fluid control unit; 30. Liquid reservoir; 31. Fluid actuator; 32. First connecting pipe; 33. First connecting pipe; 34. First solenoid valve; 35. Second connecting pipe; 36. Luer connector; 37. Collection bag; 38. Multi-port pipe; 39. Second solenoid valve;

[0056] 4. Gas control unit; 40. Gas storage source; 41. Concentration sensor; 42. Second connecting pipe; 43. Third solenoid valve;

[0057] 5. Control unit;

[0058] 6. Incubator. Detailed Implementation

[0059] To better understand the above-mentioned objectives, features, and advantages of this utility model, the solution of this utility model will be further described below. It should be noted that, unless otherwise specified, the embodiments and features of this utility model can be combined with each other.

[0060] Many specific details are set forth in the following description in order to provide a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; obviously, the embodiments in the specification are only some embodiments of the present invention, and not all embodiments.

[0061] The three-dimensional cell culture chip and payload of this utility model embodiment are described below with reference to the accompanying drawings.

[0062] like Figure 1 As shown, the three-dimensional cell culture chip of the first aspect embodiment of the present invention may include an upper clamp 10, a lower clamp 11 and a chip 12.

[0063] The upper clamp 10 has an inlet 1000 and an outlet 1001. The lower clamp 11 is detachably connected to the upper clamp 10. Specifically, the detachable connection between the lower clamp 11 and the upper clamp 10 can be achieved through several common methods: First, an elastic snap-fit ​​structure is designed, with a protruding snap head on the lower clamp 11 and a corresponding slot on the upper clamp 10, utilizing the elastic deformation characteristics of the material itself to achieve quick engagement and disengagement. Second, a bolt and nut connection method is preferred, with multiple connection holes on both the upper clamp 10 and the lower clamp 11, and the connection holes on the upper clamp 10 and the lower clamp 11 corresponding one-to-one. During installation, the bolts are passed through the corresponding connection holes and tightened with the nuts to achieve a stable connection between the upper clamp 10 and the lower clamp 11.

[0064] Furthermore, to optimize the clamping stability of chip 12, some of the holes in the multiple connection holes can be arranged in a close-fitting, surrounding manner along the outer edge of chip 12. This design allows for effective lateral restraint of chip 12 when bolts pass through these specific holes and are tightened, further enhancing the stability of chip 12 during clamping. Simultaneously, this layout provides clear positioning marks for the chip 12's installation location, facilitating quick and accurate assembly by operators.

[0065] It should be noted that both the upper clamp 10 and the lower clamp 11 are made of polymethyl methacrylate (PMMA) using laser engraving or 3D printing technology. This reduces costs and significantly decreases initial investment, simplifies the production process, and shortens the R&D cycle. The use of PMMA offers several advantages. First, it provides good optical transparency, allowing researchers to clearly observe the internal structure of the chip 12 during cell culture and other observation experiments without frequent disassembly of the upper and lower clamps 10 and 11, thus minimizing interference with the culture environment. Second, it is easy to process, has good biocompatibility, and has minimal impact on cell growth and metabolism. Furthermore, PMMA possesses a certain degree of hardness and rigidity, enabling the upper and lower clamps 10 and 11 to provide a relatively stable clamping force when they are in contact, ensuring the chip 12's airtightness.

[0066] The chip 12 is clamped between the upper clamp 10 and the lower clamp 11. The chip 12 has two first perfusion ports 1200. The perfusion inlet 1000 and the perfusion outlet 1001 are coaxially connected to the corresponding first perfusion ports 1200. The chip 12 includes multiple three-dimensional cell culture scaffolds 123. Multiple mounting cavities are provided inside the chip 12. The multiple three-dimensional cell culture scaffolds 123 are detachably installed in the corresponding mounting cavities. The three-dimensional cell culture scaffolds 123 are used to culture cells.

[0067] The chip 12 has a microfluidic channel grid inside, which is connected to multiple mounting cavities and two first perfusion ports 1200 to form a fluid flow path for synchronously perfusing multiple three-dimensional cell culture scaffolds 123 through the microfluidic channel grid.

[0068] Specifically, the chip 12 can be clamped and fixed by the tight cooperation of the upper clamp 10 and the lower clamp 11, which ensures the airtightness of the chip 12 and the stability after installation. At the same time, it can also enable quick assembly and disassembly of the chip 12, which is convenient for operators to inoculate cells and use the chip 12.

[0069] Meanwhile, by constructing a microfluidic channel mesh in chip 12 to connect multiple mounting cavities, it is possible to simultaneously perfuse multiple three-dimensional cell culture scaffolds 123 for cell culture, thereby increasing the number of cell culture replicates. This not only improves the efficiency of cell culture but also allows for more cells to be accommodated in a smaller space, increasing cell density. Furthermore, the three-dimensional cell culture scaffold 123 can create a three-dimensional cell culture environment, making the constructed tissue closer to its in vivo state and maximally restoring the natural survival state of cells in vivo, thus effectively ensuring the cell culture effect.

[0070] Specifically, U-87MG cells were cultured using the aforementioned chip 12, combined with Figure 3 The culture method is as follows: ① U-87MG cells are cultured in culture dishes or flasks according to conventional methods, and the cells are digested, centrifuged, and resuspended to obtain seed culture after conventional cell passage methods; ② The seed culture is dropped onto the three-dimensional cell culture scaffold 123, and after 6 hours of adhesion, it is placed in chip 12; ③ Finally, it is clamped using upper clamp 10 and lower clamp 11; ④ Every 24 hours, the culture medium is perfused into the microfluidic channel grid in chip 12 through the perfusion inlet 1000 at a flow rate of 3 mL / h, and the perfusion is 1 hour each time. The culture medium will flow into the corresponding mounting cavity through the microfluidic channel grid and enter the three-dimensional cell culture scaffold 123; ⑤ Culture for 72 hours, and observe and record under an inverted microscope every 24 hours during this period.

[0071] Furthermore, such as Figure 2 As shown, the chip 12 includes a top seat 120, a base 121 and a sealing component 122 sandwiched between the two. The sealing component 122 has a first through hole 1220 and a plurality of second through holes 1221 on its surface.

[0072] The top seat 120 is attached to the upper clamp 10, and the base 121 is attached to the lower clamp 11.

[0073] The top seat 120 has two first inlet ports 1200 on its upper surface and multiple downward-opening first mounting cavities 1201 on its lower surface. The base 121 has multiple upward-opening second mounting cavities 1210 on its upper surface. Each second mounting cavity 1210 and the second through hole 1221 are coaxially aligned with the first mounting cavity 1201 in the vertical direction, together forming a closed mounting cavity.

[0074] It should be noted that the top mount 120 and the base 121 are made of polydimethylsiloxane and are manufactured using laser engraving or 3D printing technology, which simplifies the manufacturing process and reduces production costs. This method can precisely control the size and shape of the chip 12, ensuring consistency between batches. At the same time, due to the use of polydimethylsiloxane, the permeability required for cell culture of the chip 12 is maintained. Moreover, compared with the traditional soft photolithography processing method, the aspect ratio of the chip 12 is effectively improved, the perfusion speed is increased, and the chip 12 can be installed in a commercial 3D cell culture scaffold 123.

[0075] Specifically, by disassembling the upper clamp 10 and the lower clamp 11, the restriction on the top seat 120 can be quickly released, and the sealing component 122 can be lifted to quickly remove the three-dimensional cell culture scaffold 123. The operation is convenient and quick.

[0076] The sealing component 122 not only increases the tightness between the top seat 120 and the base 121, but also divides the microfluidic channel grid and prevents the two fluids entering and exiting the chip 12 from mixing. At the same time, the second through hole 1221 can form a clearance space for the installation of the three-dimensional cell culture scaffold 123, so that the first mounting cavity 1201 and the second mounting cavity 1210 can meet the accommodating space of the three-dimensional cell culture scaffold 123, while providing a bottom-up flow channel to ensure that the liquid flows from the bottom layer to the top layer and passes through the three-dimensional cell culture scaffold 123.

[0077] Furthermore, such as Figure 2 , Figure 4 and Figure 5 As shown, the microfluidic channel grid includes: a first microfluidic channel grid 1202 formed on the lower surface of the top seat 120 and a second microfluidic channel grid 1211 formed on the upper surface of the base 121.

[0078] The first microfluidic channel grid 1202 is connected to multiple first mounting cavities 1201, the irrigation outlet 1001 is connected to the first microfluidic channel grid 1202 through the corresponding first irrigation port 1200, the second microfluidic channel grid 1211 is connected to multiple second mounting cavities 1210, the top of another first irrigation port 1200 is connected to the corresponding irrigation inlet 1000, and the bottom of the first irrigation port 1200 is connected to the second microfluidic channel grid 1211 through the first through hole 1220.

[0079] Specifically, during perfusion, the liquid first enters the second microfluidic channel grid 1211, then disperses from the second microfluidic channel grid 1211 into multiple second mounting cavities 1210, gradually spreading from bottom to top to the three-dimensional cell culture scaffold 123. From the three-dimensional cell culture scaffold 123, it enters the first mounting cavity 1201, and then converges from the first microfluidic channel grid 1202 and is discharged through the perfusion outlet 1001, forming a flow path of the three-dimensional cell culture scaffold 123 spreading from bottom to top through the second microfluidic channel grid 1211, the mounting cavity, and the first microfluidic channel grid 1202. This not only ensures that the three-dimensional cell culture scaffold 123 has more contact with the flowing liquid, but also carries away waste liquid to the greatest extent.

[0080] In one embodiment of this utility model, such as Figure 2 As shown, the chip 12 also includes multiple support rings 124. Each support ring 124 has an open opening, which corresponds to the connection between the second microfluidic channel grid 1211 and the second mounting cavity 1210. The support ring 124 is placed in the second mounting cavity 1210, and the top of the support ring 124 is attached to the three-dimensional cell culture scaffold 123 to lift the three-dimensional cell culture scaffold 123 and form a gap between it and the bottom wall of the second mounting cavity 1210.

[0081] It should be noted that the shape of the support ring 124 is adapted to the shape of the second mounting cavity 1210 to ensure that the support ring 124 can be stably engaged in the second mounting cavity 1210.

[0082] In the above design, the support ring 124 can lift the three-dimensional cell culture scaffold 123 to a certain height, creating a uniform gap between the three-dimensional cell culture scaffold 123 and the bottom wall of the second mounting cavity 1210. When liquid (fluid) flows in from the open end, it can smoothly penetrate to the lower region of the three-dimensional cell culture scaffold 123. Subsequently, the liquid flows through the three-dimensional cell culture scaffold 123 from bottom to top, allowing the liquid to flow through all parts of the three-dimensional cell culture scaffold 123 as comprehensively as possible. In this way, the cells attached to the surface of the three-dimensional cell culture scaffold 123 can more fully absorb nutrients from the liquid, while metabolic waste produced by the cells can also be carried away by the liquid in a timely manner, creating favorable conditions for healthy cell growth and normal metabolism.

[0083] From another perspective, without the support ring 124, the flow path of the liquid would be severely restricted. In this case, the liquid could only enter from the side of the three-dimensional cell culture scaffold 123 and flow out from the top. Under this flow pattern, only a portion of the three-dimensional cell culture scaffold 123 would be able to receive fluid, which would undoubtedly significantly reduce the efficiency of material exchange between the cells and the liquid, thereby affecting the cell growth status and culture results.

[0084] In one embodiment of this utility model, the three-dimensional cell culture scaffold 123 is a three-dimensional porous scaffold, that is, the three-dimensional cell culture scaffold 123 can be directly selected from commercially available three-dimensional cell culture scaffolds (three-dimensional porous scaffolds). The three-dimensional cell culture scaffold 123 uses polystyrene, and the channel and pore design inside the three-dimensional cell culture scaffold 123 helps to improve the transport efficiency of oxygen, nutrients and metabolic waste, ensuring that the cells receive sufficient nutrients and can effectively remove waste. At the same time, it can also ensure that liquid can pass through the three-dimensional cell culture scaffold 123 and enter the first mounting cavity 1201.

[0085] Compared to traditional hydrogels, which have a slower diffusion rate of substances and thus limit cell growth and survival, three-dimensional cell culture scaffolds typically have a robust physical structure, making them easy to handle and transfer, thus reducing operational difficulties. In contrast, traditional hydrogels are relatively soft, easily deformed, and more complex to handle. Furthermore, the cell attachment surface of the three-dimensional cell culture scaffold is made of polystyrene, which can accommodate the attachment requirements of different cells, reducing the adaptation cost of cell culture processes. Other methods for increasing cell culture density, such as hydrogels and suspension culture, all require exploring culture processes, while using three-dimensional porous scaffolds made of polystyrene can effectively avoid this problem.

[0086] Specifically, when the cells are cultured to a suitable level for research use using conventional methods, the seed culture solution is dropwise added to the three-dimensional cell culture scaffold 123. Since the three-dimensional cell culture scaffold 123 is a porous rigid material, its surface is suitable for adherent cell growth, allowing the cells to attach to the material surface. After the cells have attached to the surface of the three-dimensional cell culture scaffold 123, the three-dimensional cell culture scaffold 123 is assembled into the chip 12, and perfusion culture begins.

[0087] In one embodiment of this utility model, such as Figure 2 As shown, the upper clamp 10 includes a first cover plate 100 and a second cover plate 101, wherein,

[0088] The first cover plate 100, the second cover plate 101, and the top seat 120 are attached to each other from top to bottom, and the first cover plate 100 and the second cover plate 101 are fixedly connected. The irrigation inlet 1000 and the irrigation outlet 1001 are opened on the first cover plate 100. The second cover plate 101 has two second irrigation ports 1010. One second irrigation port 1010 is connected to the irrigation inlet 1000 and the corresponding first irrigation port 1200, and the other second irrigation port 1010 is connected to the irrigation outlet 1001 and the corresponding first irrigation port 1200.

[0089] It should be noted that the first cover plate 100 and the second cover plate 101 can be securely fixed together using epoxy resin. This connection method effectively ensures the tightness and reliability of their joint. Meanwhile, in terms of structural design, the orifice size of the second infusion port 1010 needs to be smaller than the orifice sizes of the infusion inlet 1000 and the infusion outlet 1001 to meet specific fluid infusion and subsequent connection requirements.

[0090] Considering the limitations of current laser engraving technology, which can only perform two-dimensional processing, conventional integrated processing methods are insufficient to achieve the design requirement that the diameter of the second inlet 1010 be smaller than that of the inlet 1000 and outlet 1001. Therefore, a solution is adopted that the upper clamp 10 is divided into two layers: a first cover plate 100 and a second cover plate 101. These two layers are first independently laser engraved to precisely control the diameter of each inlet, and then they are firmly bonded together with epoxy resin. This processing and assembly method provides a reliable guarantee for the subsequent successful insertion and installation of the Luer connector 36.

[0091] like Figure 6 As shown, the second aspect of this utility model provides a load including a temperature control unit 2, a fluid control unit 3, a gas control unit 4, a control unit 5, an incubator 6, and the three-dimensional cell culture chip 1 described in the first aspect above.

[0092] The incubator 6 has a removable sealing cover (not shown in the figure). Specifically, the incubator 6 has an opening at the top. The sealing cover can be fixed to the opening by directly covering or flipping and snapping, thus sealing the opening and the cavity. The connection method of the sealing cover is existing technology and will not be described in detail here. Opening the sealing cover makes it convenient to place or remove the three-dimensional cell culture chip 1 in the cavity.

[0093] The incubator 6 has an internal cavity, in which the three-dimensional cell culture chip 1, temperature control unit 2, fluid control unit 3, gas control unit 4 and control unit 5 are all located;

[0094] Temperature control unit 2, fluid control unit 3 and gas control unit 4 are respectively connected to control unit 5. Fluid control unit 3 is used to connect to irrigation inlet 1000 and irrigation outlet 1001.

[0095] It should be noted that the control unit 5 can use a low-power microcontroller to implement logic control and task processing. The microcontroller integrates a communication module and a standardized interface. The communication module supports multiple communication protocols, which can ensure a stable communication connection between the spacecraft and the ground laboratory, allowing remote monitoring and operation. The standardized interface is used to electrically connect with the temperature control unit 2, the fluid control unit 3, and the gas control unit 4.

[0096] In the above scheme, the temperature control unit 2 is set to change the temperature inside the cavity, and can also collect the temperature inside the cavity and send the collected temperature data to the control unit 5.

[0097] The fluid control unit 3 can be used to perfuse the perfusion inlet 1000, control the flow rate of the liquid during the cell culture and sample collection stages, and collect the sample or waste liquid flowing out of the perfusion outlet 1001.

[0098] The gas control unit 4 can change the concentration of carbon dioxide in the cavity, and can collect the concentration of carbon dioxide and transmit the collected concentration of carbon dioxide to the control unit 5.

[0099] Control unit 5 can intelligently control temperature control unit 2, fluid control unit 3 and gas control unit 4 to realize automated cell culture. At the same time, through modularity, it has high integration and small footprint, making it suitable for unmanned spacecraft and reducing experimental costs.

[0100] In addition, control unit 5, temperature control unit 2, fluid control unit 3 and gas control unit 4 can be directly connected to an external power source for power supply, or lithium batteries can be added in the incubator 6 for auxiliary power supply, so as to ensure that control unit 5, temperature control unit 2, fluid control unit 3 and gas control unit 4 can still operate in the event of main power failure or power outage.

[0101] In one embodiment of the present invention, the temperature control unit 2 includes a temperature sensor and a semiconductor heating and cooling chip.

[0102] The temperature sensor is installed in the cavity to obtain the temperature inside the cavity, and the semiconductor heating and cooling chip is installed in the cavity to heat or cool the environment inside the cavity. The semiconductor heating and cooling chip and the temperature sensor are respectively connected to the control unit 5.

[0103] Specifically, the semiconductor heating and cooling chip can be heated or cooled by forward or reverse power supply, and it is small in size and has low power consumption. The temperature sensor is used to obtain the temperature inside the cavity to ensure the temperature of the cell culture environment changes.

[0104] During the cell culture stage, the control unit 5 receives temperature data from the temperature sensor monitoring the current cell culture environment temperature (temperature data inside the cavity), and determines the magnitude of the current cell culture environment temperature data compared with the first preset temperature. Based on the determination result, it controls the semiconductor heating and cooling chip to heat or cool, so as to adjust the current cell culture environment temperature to the first preset temperature (e.g., 37 degrees Celsius).

[0105] Cell culture completion stage: Control unit 5 switches the semiconductor heating and cooling chip to cooling mode, gradually reducing the temperature until the second preset temperature (e.g., 4°C) is reached.

[0106] It should be understood that the first preset temperature and the second preset temperature can be flexibly set according to the actual situation, and are not intended to limit the scope of protection of this utility model.

[0107] Furthermore, thermal insulation material can be installed inside the cavity to reduce the impact of the external environment on the internal temperature of the cavity, while preventing heat loss or cold leakage.

[0108] In one embodiment of this utility model, such as Figure 7 and Figure 8 As shown, the fluid control unit 3 includes a reservoir 30, a fluid actuator 31, a first connecting pipe 32, two first connecting pipes 33, multiple first solenoid valves 34, a second connecting pipe 35, two Luer connectors 36, a collection bag 37, a multi-port pipe 38, and a second solenoid valve 39.

[0109] The reservoir 30 has multiple zones (not shown in the figure), and each zone has a connecting nozzle (not shown in the figure) above it.

[0110] It should be noted that the reservoir 30 is made of medical-grade polypropylene, polyethylene E, or other materials with good chemical stability and biocompatibility. It is used to store various biological reagents in multiple areas. For example, there are three areas, which store cell culture medium, cell lysis buffer, and cell fixative, respectively.

[0111] Each connecting port is equipped with a first solenoid valve 34. One end of the multi-port pipe 38 is connected to the first connecting pipe 32, and the other ports of the multi-port pipe 38 are respectively connected to the first solenoid valve 34. By controlling the opening or closing of different first solenoid valves 34, liquid in different areas or liquid in multiple areas can be selectively extracted.

[0112] One end of the fluid actuator 31 is connected to the first connecting pipe 32, and the other end of the fluid actuator 31 is connected to the corresponding Luer connector 36 through the first connecting pipe 33. The fluid actuator 31 can be a low-power micro pump such as a peristaltic pump or a syringe pump, used to drive the liquid in the reservoir 30 into the chip 12.

[0113] One end of the second solenoid valve 39 is connected to the collection bag 37 through the second connecting pipe 35.

[0114] The other end of the second solenoid valve 39 is connected to the corresponding Luer connector 36 through the first connecting pipe 33. One Luer connector 36 is inserted into the irrigation outlet 1001, and one end of the Luer connector 36 extends through the second irrigation port 1010, the first irrigation port 1200 and connects to the first microfluidic channel grid 1202. The other Luer connector 36 is inserted into the irrigation inlet 1000, and one end of the Luer connector 36 extends through the second irrigation port 1010, the first irrigation port 1200, the first through hole 1220 and connects to the second microfluidic channel grid 1211. The first solenoid valve 34, the second solenoid valve 39 and the fluid actuator 31 are respectively connected to the control unit 5.

[0115] It should be noted that the Luer connector 36 is existing technology, and its specific structure will not be described in detail here. Secondly, in order to ensure the sealing between the Luer connector 36 and the irrigation inlet 1000 or the irrigation outlet 1001, the tightness can be improved by injecting glue between the two.

[0116] Furthermore, the first connecting pipe 32, the first connecting pipe 33, and the second connecting pipe 35 can all be made of silicone or PVC flexible hoses, which are soft, flexible, and resistant to chemical corrosion, ensuring reliability during long-term use.

[0117] Specifically, in the experimental procedure, during the cell culture stage: the control unit 5 can precisely open the second solenoid valve 39 and the first solenoid valve 34 corresponding to the area storing cell culture medium according to the preset time and preset running time, and control the fluid driver 31 to draw cell culture medium from the reservoir 30 at a first flow rate range (0.1 mL / h-2 mL / h). The drawn cell culture medium will be transported sequentially through the second connecting tube 35 and Luer connector 36 to the second microfluidic channel grid 1211 for perfusion. During the perfusion process, the liquid will circulate along the flow path of the second microfluidic channel grid 1211, the mounting cavity, and the first microfluidic channel grid 1202, providing sufficient nutrients to the cells and promptly removing waste products generated by cell metabolism. Finally, the waste liquid will be discharged into the collection bag 37 through the Luer connector 36, the second solenoid valve 39, and the second connecting tube 35, completing the liquid circulation of the cell culture stage.

[0118] During the sample collection stage, the control unit 5 can also open the first solenoid valve 34 corresponding to the area storing cell fixative and cell lysis solution according to the preset timed operation and preset running time, and control the fluid driver 31 to deliver the cell fixative and cell lysis solution in the reservoir 30 sequentially through the second connecting tube 35 and Luer connector 36 to the second microfluidic channel grid 1211 for perfusion treatment in order to fix the cells in the three-dimensional cell culture scaffold 123.

[0119] It should be emphasized that the aforementioned first and second flow velocity ranges are not fixed, but can be flexibly adjusted and set according to actual experimental needs and operating conditions. Therefore, these two flow velocity ranges should not be used as a limitation on the scope of protection of this utility model.

[0120] In one embodiment of this utility model, such as Figure 9 As shown, the gas control unit 4 includes a gas storage source 40, a concentration sensor 41, a second connecting pipe 42, and a third solenoid valve 43.

[0121] The second connecting pipe 42 is used to connect the gas storage source 40 and the third solenoid valve 43, the concentration sensor 41 is used to obtain the gas concentration in the cavity in real time, and the third solenoid valve 43 and the concentration sensor 41 are respectively connected to the control unit 5.

[0122] It should be noted that the gas storage source 40 can use a medical-grade carbon dioxide cylinder or other pressure vessel as the main carbon dioxide source to ensure gas purity and stability. In addition, the gas storage source 40 is equipped with a safety valve and a pressure reducing valve to ensure operational safety. The concentration sensor 41 can be a high-precision sensor to ensure that the carbon dioxide concentration in the cell culture environment is stable at 0-10%, supporting rapid and accurate concentration monitoring and adjustment. The third solenoid valve 43 is a small solenoid valve with a high response speed, which can control the speed at which carbon dioxide enters the cavity and ensure precise flow control. The second connecting pipe 42 is made of corrosion-resistant and airtight materials, such as polytetrafluoroethylene or stainless steel pipe, to prevent carbon dioxide leakage.

[0123] Specifically, the concentration sensor 41 is used to acquire the carbon dioxide gas concentration in the cavity in real time and send the data to the control unit 5. The control unit 5 determines the difference between the current carbon dioxide concentration in the cell culture environment and the preset carbon dioxide concentration threshold.

[0124] Based on the judgment result, the control unit 5 determines whether to open or close the third solenoid valve 43 to adjust the carbon dioxide concentration to the preset carbon dioxide concentration threshold.

[0125] When the carbon dioxide concentration reaches the preset carbon dioxide concentration threshold, the control unit 5 enters steady-state mode and adjusts the opening and closing frequency of the third solenoid valve 43 through the PID control algorithm so that the preset carbon dioxide concentration threshold does not exceed the preset range of carbon dioxide concentration.

[0126] Specifically, the concentration sensor 41 monitors the carbon dioxide concentration in the current cell culture environment and determines whether the current carbon dioxide concentration in the cell culture environment is lower than the preset carbon dioxide concentration threshold. If it is lower than the preset carbon dioxide concentration threshold, the third solenoid valve 43 is opened to start aeration. If it is higher than the preset carbon dioxide concentration threshold, the third solenoid valve 43 is kept closed. Once the preset carbon dioxide concentration threshold is reached, the control unit 5 enters steady-state mode and adjusts the opening and closing frequency of the third solenoid valve 43 through a PID control algorithm so that the carbon dioxide concentration fluctuation does not exceed the preset carbon dioxide concentration threshold ±0.1%.

[0127] It should be understood that the preset carbon dioxide concentration threshold can be flexibly set according to the actual situation, and is not intended to limit the scope of protection of this utility model.

[0128] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0129] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not to be limited to the embodiments described herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A three-dimensional cell culture chip, characterized in that, include: The upper clamp is provided with an irrigation inlet and an irrigation outlet; The lower clamp is detachably connected to the upper clamp. The chip is held between the upper clamp and the lower clamp; The chip has two first infusion ports, and the infusion inlet and the infusion outlet are coaxially connected to the corresponding first infusion ports. The chip includes multiple three-dimensional cell culture scaffolds, and the chip has multiple mounting cavities inside, with each of the multiple three-dimensional cell culture scaffolds being detachably mounted in a corresponding mounting cavity; The chip has a microfluidic channel grid inside, which is connected to multiple mounting cavities and two first infusion ports to form a fluid flow path.

2. The three-dimensional cell culture chip according to claim 1, characterized in that, The chip includes a top mount, a base mount, and a sealing component sandwiched between the two. The sealing component has a first through hole and a plurality of second through holes on its surface. The top seat is fitted with the upper clamp, and the base is fitted with the lower clamp; The top surface of the top seat has two first infusion ports, the bottom surface of the top seat has multiple downward-opening first mounting cavities, and the top surface of the base has multiple upward-opening second mounting cavities. Each second mounting cavity, the second through hole, and the first mounting cavity are coaxially aligned in the vertical direction to form a closed mounting cavity.

3. The three-dimensional cell culture chip according to claim 2, characterized in that, The microfluidic channel mesh includes: A first microfluidic channel grid is formed on the lower surface of the top seat. The first microfluidic channel grid is connected to a plurality of first mounting cavities. The irrigation outlet is connected to the first microfluidic channel grid through a corresponding first irrigation port. A second microfluidic channel grid is formed on the upper surface of the base. The second microfluidic channel grid is connected to multiple second mounting cavities. The top of another first infusion port is connected to the corresponding infusion inlet. The bottom of the first infusion port is connected to the second microfluidic channel grid through the first through hole.

4. The three-dimensional cell culture chip according to claim 3, characterized in that, The chip also includes multiple support rings, each with an open opening corresponding to the connection between the second microfluidic channel grid and the second mounting cavity. The support ring is placed in the second mounting cavity, and the top of the support ring is attached to the three-dimensional cell culture scaffold to lift the three-dimensional cell culture scaffold and form a gap between it and the bottom wall of the second mounting cavity.

5. The three-dimensional cell culture chip according to claim 1, characterized in that, The three-dimensional cell culture scaffold is a three-dimensional porous scaffold.

6. The three-dimensional cell culture chip according to claim 2, characterized in that, The upper clamp includes a first cover plate and a second cover plate, wherein... The first cover plate, the second cover plate, and the top seat are attached to each other from top to bottom, and the first cover plate and the second cover plate are fixedly connected. The irrigation inlet and the irrigation outlet are opened on the first cover plate. The second cover plate has two second irrigation ports. One second irrigation port is connected to the irrigation inlet and the corresponding first irrigation port, and the other second irrigation port is connected to the irrigation outlet and the corresponding first irrigation port.

7. A load, characterized in that, Includes a temperature control unit, a fluid control unit, a gas control unit, a control unit, an incubator, and the three-dimensional cell culture chip according to any one of claims 1-6, wherein, The incubator has a removable sealing cover, and the incubator has an internal cavity in which the three-dimensional cell culture chip, temperature control unit, fluid control unit, gas control unit, and control unit are all located. The temperature control unit, the fluid control unit, and the gas control unit are respectively connected to the control unit, and the fluid control unit is used to connect to the irrigation inlet and the irrigation outlet.

8. The load according to claim 7, characterized in that, The temperature control unit includes a temperature sensor and a semiconductor heating / cooling element, wherein... The temperature sensor is disposed in the cavity and is used to obtain the temperature inside the cavity; The semiconductor heating and cooling chip is disposed in the cavity and is used to heat or cool the environment inside the cavity. The semiconductor heating and cooling chip and the temperature sensor are respectively connected to the control unit.

9. The load according to claim 7, characterized in that, The fluid control unit includes a reservoir, a fluid actuator, a first connecting pipe, two first connecting pipes, multiple first solenoid valves, a second connecting pipe, two Luer connectors, a collection bag, a multi-way pipe, and a second solenoid valve. The liquid reservoir is provided with multiple areas, and each area is provided with a connecting port above it. Each connecting port is equipped with a first solenoid valve. One end of the multi-port pipe is connected to the first connecting pipe, and the other ports of the multi-port pipe are respectively connected to the corresponding first solenoid valve. One end of the fluid actuator is connected to the first connecting pipe, and the other end of the fluid actuator is connected to the corresponding Luer connector through the first connecting pipe; One end of the second solenoid valve is connected to the collection bag through the second connecting pipe, and the other end of the second solenoid valve is connected to the corresponding Luer connector through the first connecting pipe; One of the Luer connectors is inserted into the irrigation outlet, and one end of the Luer connector extends through the second irrigation port, the first irrigation port and connects to the first microfluidic channel grid. The other Luer connector is inserted into the irrigation inlet, and one end of the Luer connector extends through the second irrigation port, the first irrigation port and the first through hole and connects to the second microfluidic channel grid. The first solenoid valve, the second solenoid valve and the fluid actuator are respectively connected to the control unit.

10. The load according to claim 7, characterized in that, The gas control unit includes a gas storage source, a concentration sensor, a second connecting pipe, and a third solenoid valve, wherein... The second connecting pipe is used to connect the gas storage source and the third solenoid valve; The concentration sensor is used to obtain the gas concentration in the cavity in real time, and the third solenoid valve and the concentration sensor are respectively connected to the control unit.

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