A multilayer synchronous uniform medium exchange device for cell culture and its usage method

Abstract

This invention belongs to the field of biotechnology and medical device technology, and discloses a multi-layer synchronous uniform fluid exchange device for cell culture, including a base and a top plate. From top to bottom, partitions and hollow structural plates are staggered between the base and the top plate. Each hollow structural plate contains a cell culture chamber. Each partition has an inlet buffer chamber and an outlet buffer chamber symmetrically arranged at both ends. The top plate has inlet and outlet holes corresponding to the inlet and outlet buffer chambers. Through a buffer chamber design of "full-width butt joint + ultra-high flow resistance ratio," the fluid is fully pressure-equalized before entering each layer of parallel cell culture chambers, achieving a high degree of synchronization and uniformity of flow velocity in each cell culture chamber, providing a consistent fluid shear force and nutrient exchange environment for each cell layer.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and medical device technology, specifically to a multilayer synchronous uniform medium exchange device for cell culture and its usage method. Background Technology

[0002] In biomedical research, drug screening, and tissue engineering, multilayer cell co-culture or the construction of three-dimensional tissue models has become an important research method. In such experiments, providing a highly homogeneous and controllable fluid microenvironment for cells at different layers is the core challenge in ensuring experimental reliability and reproducibility.

[0003] Currently, common multilayer culture techniques have the following limitations:

[0004] First, inconsistent manual operation: using traditional multi-well plates for layered liquid exchange relies on manual operation, which is inefficient and makes it difficult to ensure the synchronization between wells and the consistency of reagent dosage. Human error is large and it is difficult to meet the requirements of high-throughput and standardized experiments.

[0005] Second, uneven fluid distribution: Some existing integrated multilayer perfusion systems have complex channel network designs. Uneven inlet pressure or mismatched flow resistance between channels often leads to fluid preferentially flowing through paths with lower resistance, resulting in significant differences in flow velocity and asynchronous medium exchange between culture chambers. This unevenness causes inconsistencies in the time, concentration, and shear stress of reagent exposure for cells in different layers, severely affecting the accuracy and reproducibility of experimental results. Studies have shown that differences in shear stress caused by interlayer flow velocity differences directly lead to differences in cell responses.

[0006] Third, the system is complex and costly: In order to achieve uniform distribution, some solutions use multi-channel independent pump control or integrated complex micro-valve systems for flow regulation. This not only greatly increases the cost and complexity of the equipment, but also reduces the stability and convenience of operation, and significantly increases the failure rate.

[0007] Therefore, there is a need in the field for a novel device that is simple in structure and does not rely on complex active control, but can ensure, in principle, the simultaneous and uniform distribution of fluid in each culture chamber layer, in order to overcome the above-mentioned technical defects. Summary of the Invention

[0008] The purpose of this invention is to provide a multilayer synchronous uniform medium exchange device and its usage method for cell culture, so as to solve the problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: A multilayer synchronous uniform medium exchange device for cell culture includes a base and a top plate. The base and the top plate are staggered from top to bottom with partitions and hollow structural plates. Each hollow structural plate is provided with a cell culture chamber. Each partition is symmetrically provided with an inlet buffer chamber and an outlet buffer chamber at both ends. The top plate is provided with a liquid inlet hole and a liquid outlet hole at the positions corresponding to the inlet buffer chamber and the outlet buffer chamber. The inlet buffer chamber and the outlet buffer chamber are provided with channels along the width direction on the side that are close to each other, which are directly connected to and communicate with the inlet and outlet ends of each cell culture chamber in the whole width. The characteristic height H of the inlet buffer chamber and outlet buffer chamber on the horizontal cross section perpendicular to the flow direction is greater than the chamber height h of the single-layer cell culture chamber, so that the flow resistance of the inlet buffer chamber and outlet buffer chamber regions is less than the flow resistance of the cell culture chamber region.

[0010] More preferably, the base and the hollow structural plate, the top plate and the partition, and the partition and the hollow structural plate are all stacked by adhesive bonding. The multi-layer synchronous uniform liquid exchange device for cell culture forms a closed flow channel inside, in which liquid enters through the inlet hole, passes through the inlet buffer chamber, each layer of cell culture chamber and the outlet buffer chamber in sequence, and finally exits through the outlet hole.

[0011] More preferably, the inlet and outlet holes are respectively disposed on the top plate surface at the center of the inlet buffer chamber and the outlet buffer chamber, and quick connectors are respectively connected to the top of the inlet and outlet holes, the quick connectors being used to connect to external pump pipes.

[0012] More preferably, the partition and the hollow structural plate are provided with at least three layers, and the thickness of the partition adjacent to the bottom surface of the top plate is greater than the thickness of the other partitions.

[0013] More preferably, the ratio H / h of the characteristic height H of the inlet buffer chamber and the outlet buffer chamber on the horizontal cross section perpendicular to the flow direction to the chamber height h of the single-layer cell culture chamber is ≥8.

[0014] More preferably, the flow resistance ratio of the inlet buffer chamber and the outlet buffer chamber to the cell culture chamber satisfies the following relationship: R_plenum / R_channel ≤1 / 1331 Where R_plenum is the flow resistance of the inlet buffer chamber and the outlet buffer chamber, and R_channel is the flow resistance of the single-layer cell culture chamber.

[0015] The present invention also provides a technical solution, a method for using a multilayer synchronous uniform medium exchange device for cell culture, comprising the following steps: Step 1: Before using the device, the cell culture chamber is filled with the culture medium to be replaced and the cells are seeded. Connect both the inlet and outlet to the liquid inlet pipes, and connect the outlet pipe to the centrifuge tube. Connect the inlet to the external syringe pump to provide power. At the same time, prepare PBS solution, EDTA solution, and DMEM solution. Step 2: Use a syringe pump to introduce PBS solution through the inlet hole to drain the old DMEM culture medium from the outlet hole in the cell culture chamber. After observing that the remaining old culture medium in the cell culture chamber has been flushed out, continue to introduce PBS solution to ensure that the cell culture chamber is completely filled with PBS solution. Step 3: Use a syringe pump to introduce EDTA solution through the inlet hole, and drain the PBS solution in the cell culture chamber through the outlet hole. After observing that the remaining PBS solution in the cell culture chamber has been flushed out, continue to introduce EDTA solution to ensure that the chamber is completely filled with EDTA solution. Then let it stand for 3-5 minutes to allow the cells to detach from the cell wall. Step 4: After observing the formation of a large number of cell clumps in the cell culture chamber, DMEM solution is introduced through the inlet using a syringe pump. The EDTA solution and cell clumps in the cell culture chamber are then discharged into a centrifuge tube through the outlet for collection. After observing that the remaining EDTA solution in the cell culture chamber has been flushed out, DMEM solution is continued to be introduced to ensure that the chamber is completely filled with DMEM solution. The syringe pump is then turned off, and a portion of the collected cell clump solution is extracted and transferred to a new centrifuge tube for later use. Step 5: Under sterile conditions, transfer the solution containing cell clusters collected in the new centrifuge tube to the syringe of the syringe pump using a pipette. Start the syringe pump and introduce the solution into the device through the inlet hole. After being pressurized by the inlet buffer chamber, the solution is evenly distributed among the cell culture chambers. The EDTA in the solution is neutralized by the DMEM solution in the chamber, completing the medium change operation of the cell culture device.

[0016] More preferably, the centrifuge tube has two pipes connected to its cap: one is an outlet pipe connected to the outlet hole of the cavity and its end is close to the bottom of the centrifuge tube, and the other is a waste liquid discharge pipe and its end is close to the cap of the centrifuge tube.

[0017] More preferably, the flow rate of the solution introduced in steps 2, 3, and 4 is 5 ml / min, and after observing that the remaining old solution has been flushed out, the new solution is continued to be introduced for at least 2 minutes to ensure that the cell culture chamber is completely filled with the new solution.

[0018] Compared with the prior art, the beneficial effects of the present invention are: Excellent synchronization and uniformity: Through the buffer cavity design of "full width docking + ultra-large flow resistance ratio", the fluid is fully pressure-equalized before entering each layer of parallel cell culture chambers, which can achieve a high degree of synchronization and uniformity of flow velocity in each layer of cell culture chambers, providing a consistent fluid shear force and nutrient exchange environment for each layer of cells.

[0019] Simple and efficient operation: The traditional cumbersome operation that requires stratification is integrated into a one-time perfusion and liquid replacement of the entire device, which significantly improves the liquid replacement efficiency and reduces human error and the risk of contamination.

[0020] Minimal cell damage: Uniform fluid distribution avoids harmful shear forces caused by excessively high local flow rates. Combined with optimized fluid exchange procedures (such as using EDTA for gentle cell detachment), cell clusters can be effectively collected and replanted, maintaining good cell viability.

[0021] Compact structure and good compatibility: The device adopts a flat stacked packaging, which is simple and reliable in structure, small in size, and compatible with standard laboratory equipment (such as syringe pumps and microscopes), making it easy to integrate and automate. Attached Figure Description

[0022] Figure 1 This is a three-dimensional exploded structure schematic diagram of Embodiment 1 of the present invention; Figure 2 This is a top cross-sectional view of Embodiment 1 of the present invention; Figure 3 yes Figure 2 A sectional view along the central axis; Figure 4 This is a schematic diagram of the longitudinal two-dimensional pressure change of the present invention; Figure 5 This is the pressure distribution diagram corresponding to the COMSOL of this invention; Figure 6 This is a simulation result diagram of COMSOL Multiphysics in Embodiment 1 of the present invention, specifically a concentration distribution cloud map of each cell culture chamber at t=277s during the PBS medium change process; Figure 7 This is a simulation result diagram of COMSOL Multiphysics in Embodiment 1 of the present invention, specifically a cloud map of the concentration distribution in each cell culture chamber at t=435s during the PBS medium change process; Figure 8 This is a simulation result diagram of COMSOL Multiphysics in Embodiment 1 of the present invention, specifically a concentration distribution cloud map of each cell culture chamber at t=633s during the PBS medium change process; Figure 9 This is a simulation result diagram of COMSOL Multiphysics in Embodiment 1 of the present invention, specifically a concentration distribution cloud map of each cell culture chamber at t=950s during the PBS medium change process; Figure 10 This is a graph showing the concentration change at the center of the PBS cross section during the PBS replacement process in Example 1 of this invention. Figure 11 This is a graph showing the concentration change at the center of the PBS cross section during the PBS replacement process in Example 2 of this invention.

[0023] In the diagram: 1. Inlet port; 2. Outlet port; 3. First partition; 4. First inlet buffer chamber; 5. First outlet buffer chamber; 6. First cell culture chamber; 7. Second partition; 8. Second inlet buffer chamber; 9. Second outlet buffer chamber; 10. Second cell culture chamber; 11. Third partition; 12. Third inlet buffer chamber; 13. Third outlet buffer chamber; 14. Third cell culture chamber; 15. Base; 16. Inlet buffer chamber; 17. Outlet buffer chamber. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Please see Figures 1-3 The present invention provides a technical solution: A multilayer synchronous uniform medium exchange device for cell culture includes a base 15 and a top plate. The base 15 and the top plate are alternately arranged with partitions and hollow structural plates from top to bottom. Each hollow structural plate is provided with a cell culture chamber. Each partition is symmetrically provided with an inlet buffer chamber 16 and an outlet buffer chamber 17 at both ends. The top plate is provided with a liquid inlet hole 1 and a liquid outlet hole 2 at the positions corresponding to the inlet buffer chamber 16 and the outlet buffer chamber 17. The inlet buffer chamber 16 and the outlet buffer chamber 17 are provided with channels along the width direction on the side that are close to each other, which are directly connected to and communicate with the inlet and outlet ends of each layer of cell culture chambers. The characteristic height H of the inlet buffer chamber 16 and the outlet buffer chamber 17 on the horizontal cross section perpendicular to the flow direction is greater than the chamber height h of the single-layer cell culture chamber, so that the flow resistance in the inlet buffer chamber 16 and the outlet buffer chamber 17 region is less than the flow resistance in the cell culture chamber region.

[0026] In this invention, the base 15 and the hollow structural plate, the top plate and the partition, and the partition and the hollow structural plate are all stacked by adhesive bonding. The multi-layer synchronous uniform medium exchange device for cell culture forms a closed flow channel inside, with liquid entering through the inlet hole 1, passing sequentially through the inlet buffer chamber 16, each layer of cell culture chambers, and the outlet buffer chamber 17, and finally exiting through the outlet hole 2. The base 15, the hollow structural plate, the top plate, and the partition are all made of acrylic sheets, and the bonding method is acrylic adhesive. The overall dimensions of the device are a cuboid structure with a length of 128mm, a width of 85mm, and a height of 13mm, which conforms to the size design of a 96-well plate, facilitating the observation of cell growth using a standard microscope.

[0027] In this invention, the liquid inlet 1 and the liquid outlet 2 are respectively disposed on the top plate surface at the center of the inlet buffer chamber 16 and the outlet buffer chamber 17, and quick connectors are respectively connected to the top of the liquid inlet 1 and the liquid outlet 2. The quick connectors are used to connect to the external pump pipe.

[0028] In this invention, the partition and the hollow structural plate are provided with at least three layers, and the thickness of the partition adjacent to the bottom surface of the top plate is greater than the thickness of the other partitions.

[0029] In this invention, the ratio H / h of the characteristic height H of the inlet buffer chamber 16 and the outlet buffer chamber 17 on the horizontal cross section perpendicular to the flow direction to the chamber height h of the single-layer cell culture chamber is ≥8.

[0030] In this invention, the flow resistance ratios of the inlet buffer chamber 16 and the outlet buffer chamber 17 to the cell culture chamber satisfy the following relationship: R_plenum / R_channel ≤1 / 1331 Where R_plenum is the flow resistance of the inlet buffer chamber 16 and the outlet buffer chamber 17, and R_channel is the flow resistance of the single-layer cell culture chamber.

[0031] The present invention also provides a technical solution, a method for using a multilayer synchronous uniform medium exchange device for cell culture, comprising the following steps: Step 1: Before using the device, the cell culture chamber is filled with the culture medium to be replaced and the cells are seeded. Connect both the inlet port 1 and the outlet port 2 to the liquid flow pipe. Connect the external pipe of the outlet port 2 to the centrifuge tube. Connect the inlet port 1 to the external syringe pump to provide power. At the same time, prepare PBS solution, EDTA solution and DMEM solution. Step 2: Use a syringe pump to introduce PBS solution through inlet 1 to drain the old DMEM culture medium in the cell culture chamber through outlet 2. After observing that the remaining old culture medium in the cell culture chamber has been flushed out, continue to introduce PBS solution to ensure that the cell culture chamber is completely filled with PBS solution. Step 3: Inject EDTA solution through inlet 1 using a syringe pump, and drain the PBS solution in the cell culture chamber through outlet 2. After observing that the remaining PBS solution in the cell culture chamber has been flushed out, continue to inject EDTA solution to ensure that the chamber is completely filled with EDTA solution. Then let it stand for 3-5 minutes to allow the cells to detach from the cell wall. Step 4: After observing the formation of a large number of cell clumps in the cell culture chamber, DMEM solution is introduced through inlet 1 using a syringe pump. The EDTA solution and cell clumps in the cell culture chamber are discharged into a centrifuge tube through outlet 2 for collection. After observing that the remaining EDTA solution in the cell culture chamber has been flushed out, DMEM solution is continued to be introduced to ensure that the chamber is completely filled with DMEM solution. The syringe pump is then turned off, and a portion of the collected cell clump solution is extracted and transferred to a new centrifuge tube for later use. Step 5: Under sterile conditions, transfer the solution containing cell clusters collected in the new centrifuge tube to the syringe of the syringe pump using a pipette. Start the syringe pump and introduce the solution into the device through the inlet port 1. After being pressurized by the inlet buffer chamber 16, the solution is evenly distributed among the cell culture chambers. The EDTA in the solution is neutralized by the DMEM solution in the chamber, thus completing the medium change operation of the cell culture device.

[0032] In this invention, the cap of the centrifuge tube is connected to two pipes: one is an outlet pipe connected to the outlet hole of the cavity and its end is close to the bottom of the centrifuge tube, and the other is a waste liquid discharge pipe and its end is close to the cap of the centrifuge tube.

[0033] In this invention, the flow rate of the solution introduced in steps 2, 3, and 4 is 5 ml / min, and after observing that the remaining old solution has been flushed out, the new solution is continued to be introduced for at least 2 minutes to ensure that the cell culture chamber is completely filled with the new solution.

[0034] Example 1: This example provides a three-layer cell culture synchronous uniform medium exchange device. The overall dimensions of the device are a cuboid with a length of 128mm, a width of 85mm, and a height of 13mm. It is made of eight layers of acrylic sheets of different thicknesses, precisely bonded and encapsulated with acrylic adhesive.

[0035] The specific layered structure is as follows: First layer: A top plate with a thickness of 1mm. The top of the left inlet buffer chamber 16 has a liquid inlet hole 1 with a diameter of 3mm, and the top of the right outlet buffer chamber 17 has a liquid outlet hole 2 with a diameter of 3mm.

[0036] The second layer: a first partition 3 with a thickness of 6mm, which serves as the cover plate for the first cell culture chamber 6; The third layer: a hollow structural plate with a thickness of 1 mm, which constitutes the first cell culture chamber 6 (128 mm long × 85 mm wide × 1 mm high). Fourth layer: a second partition 7 with a thickness of 1 mm, which serves as the bottom plate of the first cell culture chamber 6 and the cover plate of the second cell culture chamber 10; Fifth layer: A hollow structural plate with a thickness of 1 mm, forming the second cell culture chamber 10 (128 mm long × 85 mm wide × 1 mm high). The sixth layer: a 1mm thick intermediate partition plate, which serves as the bottom plate of the second cell culture chamber 10 and the cover plate of the third cell culture chamber 14; The seventh layer: a hollow structural plate with a thickness of 1 mm, forming the third cell culture chamber 14 (128 mm long × 85 mm wide × 1 mm high). The eighth layer: a 1mm thick base 15, which serves as the base plate of the third cell culture chamber 14 and encapsulates the entire chamber structure.

[0037] The left inlet buffer chamber 16 (occupying 14mm of the left side of the device) has a height of 11mm; the right outlet buffer chamber 17 (occupying 14mm of the right side of the device) also has a height of 11mm. The inlet buffer chamber 16 and the outlet buffer chamber 17 are directly connected to the inlet and outlet ends of each cell culture chamber through a vertical channel with a width of 85mm, achieving full-width connectivity.

[0038] This embodiment also discloses a method of using the above-described device, including the following steps: Step 1: The cell culture chamber is filled with the culture medium to be replaced and is seeded with cells. Both inlet 1 and outlet 2 are connected to tubing for fluid circulation. A centrifuge tube is connected to the tubing at outlet 2. One end of the centrifuge tube cap connects to the outlet tubing of the chamber, and the other end connects to the waste disposal tubing. The former is closer to the bottom of the centrifuge tube, and the latter is closer to the cap, facilitating subsequent cell collection. A syringe pump is connected to inlet 1 to provide power. Prepare the solutions needed for the experiment: PBS, EDTA, and DMEM.

[0039] Step 2: Inject PBS solution at a flow rate of 5 ml / min through inlet 1 using a syringe pump. The DMEM culture medium solution with insufficient nutrients in the first cell culture chamber 6, the second cell culture chamber 10, and the third cell culture chamber 14 will leave through outlet 2. After observing that the remaining culture medium solution has been flushed out, continue to inject PBS solution for 2 minutes to ensure that the cell culture chambers are completely filled with PBS solution.

[0040] Step 3: Using a syringe pump, inject EDTA solution at a flow rate of 5 ml / min through inlet 1. PBS solution in the first cell culture chamber 6, the second cell culture chamber 10, and the third cell culture chamber 14 exits through outlet 2. Observe that the remaining PBS solution has been completely flushed out, then continue injecting EDTA solution for 2 minutes. After ensuring the cell culture chambers are completely filled with EDTA solution, let them stand for 3-5 minutes to allow sufficient time for trypsin to react and for the cells to detach from the cell wall.

[0041] Step 4: After observing the formation of a large amount of turbid, detached cell clusters in the cell culture chambers, DMEM solution is introduced through the inlet port 1 at a flow rate of 5 ml / min using a syringe pump. The EDTA solution and cell clusters in the first cell culture chamber 6, the second cell culture chamber 10, and the third cell culture chamber 14 exit through the outlet port 2. The cell clusters are collected in centrifuge tubes. The syringe pump is turned off, and a portion of the collected cell cluster solution is extracted and introduced into a new centrifuge tube for later use. After observing that the remaining EDTA solution has been flushed out, DMEM solution is introduced again for 2 minutes. This ensures that the cell culture chambers are completely filled with DMEM solution.

[0042] Step 5: Under sterile conditions, transfer the EDTA solution containing cell clusters to the syringe of the syringe pump using a pipette. Start the syringe pump, and let the inlet port 1 flow with the portion of the EDTA solution containing cell clusters collected from the centrifuge tube. During this process, the solution will be evenly distributed among the first cell culture chamber 6, the second cell culture chamber 10, and the third cell culture chamber 14. The EDTA solution will be neutralized by a large amount of DMEM solution. This step completes the medium exchange operation of the cell culture device.

[0043] The transient fluid dynamics simulation results of this embodiment using COMSOL Multiphysics 6.3 software are illustrated in the figure below. Figures 4-10 The simulation setup for the fluid exchange process was as follows: first, PBS solution was introduced for 0-300s, then EDTA solution was introduced for 300-600s, and finally DMEM solution was introduced for 600-900s. Fluid parameters were set as follows: density 1000 kg / m³, viscosity 0.001 Pa·s, and inlet liquid flow rate 5 ml / min.

[0044] Taking PBS solution as an example, the simulation results show: At four monitoring times (t=277s, 435s, 633s, and 950s), the flow fronts of the three liquids in each culture chamber remained highly parallel, with little variation in the position of the flow fronts between layers. The cross-sectional concentration distribution plots showed that the concentration curves at the centerline of each layer over time were basically identical. Quantitative analysis indicated that the average concentrations in the three culture chambers at 435s were 0.9578 mol / m³, 0.9570 mol / m³, and 0.9544 mol / m³, respectively, with a concentration difference of less than 5% between layers.

[0045] Working principle: During media change, liquid such as PBS buffer is pumped into the tall inlet buffer chamber 16 on the left side through inlet 1 via an external syringe pump at a flow rate of 5 mL / min. Since the cross-sectional area of ​​the inlet buffer chamber 16 (85 mm × 11 mm) is much larger than that of the cell culture chamber (85 mm × 1 mm), the fluid velocity drops sharply after entering from the inlet, and kinetic energy is effectively converted into static pressure energy.

[0046] According to the lubrication approximation theory of laminar flow at low Reynolds numbers, the hydraulic resistance R of the parallel plate channel can be expressed as: R = (12μL) / (wh³), where μ is the fluid viscosity, L is the channel length, w is the width, and h is the height. It can be seen that the flow resistance is inversely proportional to the cube of the channel height h. Therefore, the flow resistance along the width direction of the inlet buffer chamber 16 with a height of 11mm is only about 1 / 1331 (1 / 11)³ of that of the cell culture chamber with a height of 1mm, and the logic for the outlet buffer chamber 17 is similar.

[0047] This extreme difference in flow resistance means that the inlet buffer chamber 16 and the outlet buffer chamber 17 can be considered as a nearly isobaric chamber.

[0048] Subsequently, driven by a nearly constant inlet pressure, the fluid simultaneously enters the first cell culture chamber 6, the second cell culture chamber 10, and the third cell culture chamber 14 through the inlet buffer chamber 16. For each cell culture chamber, due to its uniform inlet pressure, a uniform flow distribution in the width direction will be automatically generated according to the parallel plate flow formula q(y)∝[p_in(y)-p_out]. Finally, the fluids in each chamber advance in parallel, converge in the right-side outlet buffer chamber 17, and are discharged from the outlet 2.

[0049] Example 2: Based on the pressure equalization principle of Example 1, this example provides a five-layer synchronous uniform liquid exchange device. The structure is similar to Example 1, but two additional partitions and two hollow structural plates are added to form five cell culture chambers. The height H=11mm of the inlet buffer chamber 16 and the outlet buffer chamber 17 remains unchanged. The height h of each cell culture chamber is changed to 0.5mm, and the partition thickness remains 1mm, H / h=22. The thickness of the first partition 3 can be flexibly adjusted; in this example, its thickness is 3.5mm. The total thickness of the remaining partitions and cell culture chambers is 7.5mm. Figure 11 The five-layer concentration curves show that, after expansion to five layers, as long as the inlet buffer chamber 16 and the outlet buffer chamber 17 are kept at a certain height, the difference in concentration variation of each layer can still be controlled within the expected range, proving the excellent scalability of the present invention.

[0050] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered illustrative and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention, and no reference numerals in the claims should be construed as limiting the scope of the claims.

[0051] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A multilayer synchronous uniform medium exchange device for cell culture, characterized in that, The device includes a base (15) and a top plate. The base (15) and the top plate are alternately arranged with partitions and hollow structural plates from top to bottom. Each hollow structural plate is provided with a cell culture chamber. Each partition is symmetrically provided with an inlet buffer chamber (16) and an outlet buffer chamber (17) at both ends. The top plate is provided with an inlet hole (1) and an outlet hole (2) at the positions corresponding to the inlet buffer chamber (16) and the outlet buffer chamber (17). Among them, the inlet buffer chamber (16) and the outlet buffer chamber (17) are provided with channels along the width direction on the side that are close to each other, which are directly connected and communicate with the inlet end and outlet end of each layer of cell culture chamber. The characteristic height H of the inlet buffer chamber (16) and outlet buffer chamber (17) on the horizontal cross section perpendicular to the flow direction is greater than the chamber height h of the single-layer cell culture chamber, so that the flow resistance of the inlet buffer chamber (16) and outlet buffer chamber (17) region is less than the flow resistance of the cell culture chamber region.

2. The multilayer synchronous uniform medium exchange device for cell culture according to claim 1, characterized in that: The base (15) and the hollow structure plate, the top plate and the partition plate, and the partition plate and the hollow structure plate are all stacked by adhesive bonding. The multi-layer synchronous uniform liquid exchange device for cell culture forms a closed flow channel inside which liquid enters through the inlet hole (1), passes through the inlet buffer chamber (16), each layer of cell culture chamber and the outlet buffer chamber (17) in sequence, and finally exits through the outlet hole (2).

3. The multilayer synchronous uniform medium exchange device for cell culture according to claim 1, characterized in that: The inlet hole (1) and outlet hole (2) are respectively located at the center of the inlet buffer chamber (16) and the outlet buffer chamber (17) on the top plate surface, and quick connectors are respectively connected to the top of the inlet hole (1) and the outlet hole (2), which are used to connect to the external pump pipe.

4. The multilayer synchronous uniform medium exchange device for cell culture according to claim 1, characterized in that: The partition and hollow structural plate are provided with at least three layers, and the thickness of the partition adjacent to the bottom surface of the top plate is greater than the thickness of the other partitions.

5. A multilayer synchronous uniform medium exchange device for cell culture according to claim 1, characterized in that: The ratio of the characteristic height H of the inlet buffer chamber (16) and the outlet buffer chamber (17) on the horizontal cross section perpendicular to the flow direction to the chamber height h of the single-layer cell culture chamber is H / h≥8.

6. The multilayer synchronous uniform medium exchange device for cell culture according to claim 1, characterized in that: The flow resistance ratios of the inlet buffer chamber (16) and the outlet buffer chamber (17) to the cell culture chamber satisfy the following relationship: R_plenum / R_channel ≤1 / 1331 Where R_plenum is the flow resistance of the inlet buffer chamber (16) and the outlet buffer chamber (17), and R_channel is the flow resistance of the single-layer cell culture chamber.

7. The method of using a multilayer synchronous uniform medium exchange device for cell culture according to claims 1-6, characterized in that, Includes the following steps: Step 1: Before using the device, the cell culture chamber is filled with the culture medium to be replaced and the cells are seeded. Connect the inlet (1) and outlet (2) to the liquid inlet pipe. Connect the outlet (2) to the centrifuge tube. Connect the inlet (1) to the external power pump. Prepare PBS solution, EDTA solution and DMEM solution. Step 2: PBS solution is introduced through the inlet hole (1) by the injection pump, and the old DMEM culture medium in the cell culture chamber is discharged from the outlet hole (2). After the old culture medium remaining in the cell culture chamber is flushed out, PBS solution is continued to be introduced to ensure that the cell culture chamber is completely filled with PBS solution. Step 3: Use a syringe pump to introduce EDTA solution through the inlet (1) and drain the PBS solution in the cell culture chamber through the outlet (2). After observing that the remaining PBS solution in the cell culture chamber has been flushed out, continue to introduce EDTA solution to ensure that the chamber is completely filled with EDTA solution. Then let it stand for 3-5 minutes to allow the cells to detach from the cell wall. Step 4: After observing the formation of a large number of cell clumps in the cell culture chamber, DMEM solution is introduced through the inlet (1) using a syringe pump. The EDTA solution and cell clumps in the cell culture chamber are discharged into a centrifuge tube through the outlet (2) for collection. After observing that the remaining EDTA solution in the cell culture chamber has been flushed out, DMEM solution is continued to be introduced to ensure that the chamber is completely filled with DMEM solution. The syringe pump is then turned off, and a portion of the collected cell clump solution is extracted and transferred to a new centrifuge tube for later use. Step 5: Under sterile conditions, transfer the solution containing cell clusters collected in the new centrifuge tube to the syringe of the injection pump using a pipette. Start the injection pump and pass the solution into the device through the inlet hole (1). After the solution is pressurized by the inlet buffer chamber (16), it is evenly distributed among the cell culture chambers. The EDTA in the solution is neutralized by the DMEM solution in the chamber, thus completing the medium change operation of the cell culture device.

8. The method of using a multilayer synchronous uniform medium exchange device for cell culture according to claim 7, characterized in that: The centrifuge tube has two pipes connected to its cap: one is an outlet pipe connected to the outlet hole of the cavity with its end close to the bottom of the centrifuge tube, and the other is a waste liquid discharge pipe with its end close to the cap of the centrifuge tube.

9. The method of using a multilayer synchronous uniform medium exchange device for cell culture according to claim 7, characterized in that: In steps 2, 3, and 4, the flow rate of the solution introduced is 5 ml / min. After observing that the remaining old solution has been flushed out, the new solution is continued to be introduced for at least 2 minutes to ensure that the cell culture chamber is completely filled with the new solution.

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