A bioreactor

Abstract

The present invention provides a bioreactor, comprising a shell, a top cover and a plurality of cell culture plates, wherein the shell has an open upper end and a closed lower end, wherein the lower end is provided with an inlet connected to the fluid inside the shell; the top cover covers the open upper end of the shell and is provided with an outlet connected to the fluid inside the shell; the plurality of cell culture plates are spaced apart from each other and arranged in parallel inside the shell, each cell culture plate is provided with a through hole located at the center and a plurality of small holes distributed around the through hole, and the through holes of the plurality of cell culture plates are aligned with each other to form a central channel. The bioreactor of the present invention is more conducive to the plasma to be purified to diffuse around the cell culture plate and to fully exchange substances with the cells.

Description

Technical Field

[0001] The invention relates to medical equipment, in particular to a bioreactor. Background Art

[0002] The biological blood purification system is an artificial liver support system that is closest to a normal liver. By combining hepatocyte culture technology with blood purification technology, it can more comprehensively replace the liver's detoxification, biosynthesis, secretion and metabolism functions, and is expected to become the most innovative and challenging treatment measure for patients with severe hepatitis and liver failure.

[0003] The basic principle of the biological blood purification system is to place the in vitro cultured and proliferated hepatocytes in a special bioreactor, and to perform the biological blood purification function by exchanging substances and biological action with the hepatocytes, utilizing the hepatocytes to secrete endogenous active substances and transform exogenous toxins, wherein the endogenous active substances include various proteins, metabolic enzymes and active factors, etc. The bioreactor and hepatocytes are the core parts of the biological blood purification system, and their performance is directly related to the efficiency and effect of artificial liver support.

[0004] The main types of bioreactors currently under research and application are as follows: (1) Hollow fiber bioreactor: Hollow fiber reactors have an inner cavity and an outer cavity. Liver cells are often adhered to the outer cavity of the hollow fiber. Biofilms with appropriate molecular weight cutoffs are selected according to the different sources of cells to avoid immune responses caused by xenogeneic cell products. However, liver cells are unevenly distributed in this bioreactor, which can easily lead to decreased cell viability. (2) Flat monolayer bioreactor: Cells are planted on a flat plate for culture. Its advantages are uniform cell distribution and consistent microenvironment, but the ratio of surface area to volume decreases. (3) Perfusion bed or scaffold bioreactor: This bioreactor is a bioreactor in which liver cells are planted on a perfusion bed or scaffold. Its advantages are direct contact with cells, which increases the transport of substances and promotes the formation of three-dimensional structures. It is also easy to expand cell capacity. Its disadvantages are uneven perfusion and easy clogging. (4) Encapsulated suspension bioreactor: This bioreactor is a bioreactor in which liver cells are wrapped with materials to make porous microcapsules, and then perfused for culture. The advantage is that all cells have the same microenvironment, there is a lot of space for cell culture, and the occurrence of immune response is reduced; the disadvantage is that the cells have poor stability and limited material exchange capacity. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a new type of bioreactor for use in a biological blood purification system.

[0006] A bioreactor comprises a shell, a top cover and a plurality of cell culture plates, wherein:

[0007] The housing has an open upper end and a closed lower end, wherein the lower end is provided with an inlet in fluid communication with the interior of the housing;

[0008] The top cover covers the open upper end of the housing and is provided with an outlet in fluid communication with the interior of the housing;

[0009] The multiple cell culture plates are spaced apart from each other and arranged in parallel inside the shell, each cell culture plate is provided with a through hole located at the center and multiple small holes distributed around the through hole, and the through holes of the multiple cell culture plates are aligned with each other to form a channel.

[0010] Preferably, in the bioreactor provided by the present invention, the through-holes of the top cell culture plate closest to the top cover are closed, thereby sealing the upper end of the channel.

[0011] Preferably, in the bioreactor provided by the present invention, the through hole has a first end on one side of the cell culture plate, an annular mounting belt is arranged around the first end, a limiting base protruding from the annular mounting belt is arranged on the annular mounting belt, and a buckle protruding from the limiting base is arranged on the limiting base; the through hole has a second end on the other side of the cell culture plate, an annular base is arranged around the second end, and a concave buckle groove is arranged on the annular base.

[0012] Furthermore, in the bioreactor provided by the present invention, the buckles on the cell culture plate are arranged in buckle grooves on another adjacent cell culture plate, thereby allowing a plurality of cell culture plates to be spaced apart from each other and connected together in parallel inside the housing.

[0013] By the above arrangement, in the bioreactor of the present invention, multiple cell culture plates can be connected to each other through the components arranged around the through hole at the center thereof, thereby being stacked inside the shell, and the through holes of the multiple cell culture plates are aligned with each other to form a channel.

[0014] Preferably, in the bioreactor provided by the present invention, the limiting bases and the buckles on one side of the cell culture plate are the same number as the buckle grooves on the other side, and the multiple limiting bases are evenly spaced apart from each other on the annular mounting belt, and correspondingly, the multiple buckle grooves are evenly spaced apart from each other on the annular base.

[0015] In addition, the limiting base on the cell culture plate fits with the annular base on another adjacent cell culture plate.

[0016] Preferably, a plurality of gaps are provided around the annular base on the cell culture plate. The gaps can be used as channels for the hepatocyte suspension or the plasma to be purified to enter the culture plate. The gaps are, for example, slits around the annular base.

[0017] Preferably, in the bioreactor provided by the present invention, a plurality of small holes of one cell culture plate are aligned with a plurality of small holes of another adjacent cell culture plate to form a plurality of peripheral channels.

[0018] By the above arrangement, in the bioreactor of the present invention, a plurality of cell culture plates are stacked inside the housing, and a channel is formed by the through hole at the center of each cell culture plate, and the channel extends in the vertical direction of the bioreactor, but is closed at the top cell culture plate. In addition, the channel can be optionally aligned with the outlet and the inlet in the vertical direction. The peripheral channel formed by the small hole extends in the vertical direction of the bioreactor. The inlet, outlet, channel and peripheral channel are in fluid communication with each other.

[0019] Preferably, in the bioreactor provided by the present invention, the through hole of each cell culture plate is circular, the multiple small holes are radially distributed relative to the through hole on the cell culture plate, and the multiple small holes are spaced apart from each other at the same distance to form one or more rings around the through hole.

[0020] Preferably, the cell culture plate and the small hole are circular in shape.

[0021] Preferably, the ratio of the distance between the small hole closest to the through hole and the through hole (between the centers of the circles) to the radius of the cell culture plate is 1:2-3, preferably 1:2.7.

[0022] Preferably, the ratio of the diameter of the small hole to the diameter of the through hole is 1:3-5, preferably 1:4;

[0023] Preferably, the ratio of the diameter of the small hole to the diameter of the cell culture plate is 1:80-100, preferably 1:90.7;

[0024] Preferably, the ratio of the diameter of the through hole to the diameter of the cell culture plate is 1:20-30, preferably 1:22.7.

[0025] According to a specific embodiment of the present invention, the shell is a cylinder, and 66 cell culture plates with a thickness of 1 mm are stacked vertically inside. The cell culture plates are connected together by 4 clips and 4 clip grooves on both sides of the through hole, and the maximum interval between adjacent cell culture plates is 1 mm; in addition, the diameter of each cell culture plate is 136 mm, the diameter of the through hole is 6 mm, the diameter of the small hole is 1.5 mm, the distance between the small hole closest to the through hole is 25 mm, and the distance between adjacent small holes is 10 mm; each cell culture plate has 112 small holes.

[0026] The bioreactor in the biological blood purification system purifies the plasma entering the bioreactor through the pre-cultured hepatocytes therein. The plasma is usually obtained from patients with severe hepatitis, liver failure, etc. after separation of the patient's blood. The plasma purified by the hepatocytes will circulate back into the patient's body. Compared with the prior art, the bioreactor of the present invention has the following characteristics:

[0027] First, the bioreactor of the present invention sets the inlet at the lower end of the shell, so that the plasma to be purified enters the reactor from the lower end of the shell, and the through-hole of the top cell culture plate closest to the top cover is closed, thereby forming a plasma channel from bottom to top and closed at the top. It is found that through such a setting, the plasma to be purified flowing in from the lower inlet will be blocked at the top outlet, and can only flow laterally toward the two ends of the culture plates through the gaps between adjacent cell culture plates, ensuring the flow of liquid in the gaps between the cell culture plates, minimizing the shear force, and facilitating convection exchange.

[0028] Second, in the bioreactor of the present invention, the cell culture plates are not directly fixed in the bioreactor or any component therein, but buckles and buckle grooves are arranged around the through holes of the cell culture plates, and multiple cell culture plates are connected and fixed together through the cooperation of the buckles and buckle grooves. This arrangement makes the installation of cell culture plates more flexible, and can easily adjust the number of cell culture plates in the bioreactor, thereby adjusting the actual effective working area of ​​the bioreactor, the load of cells on the cell culture plates, and the effective exchange area between cells and the plasma to be purified; at the same time, it reduces and simplifies the components of the bioreactor, reducing the manufacturing cost.

[0029] Third, on the cell culture plate of the bioreactor of the present invention, the small holes are positioned at a certain distance from or outside the through hole, and are radially distributed in a radial direction relative to the through hole; in other words, the plurality of small holes form a pattern of a plurality of rings around the through hole relative to the through hole, and the radius of the ring closest to the through hole with the through hole as the center is increased. By such an arrangement, the liquid between the cell culture plates is more likely to flow to both ends, which is beneficial to the convection exchange between the plasma to be purified and the liver cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0031] Figure 1 Schematic diagram of the bioreactor structure in which no through hole is set in the center of the top cell culture plate when the distance between adjacent cell culture plates is 0.5 mm, and the liquid enters from the bottom.

[0032] Figure 2 Schematic diagram of a bioreactor in which the distance between adjacent cell culture plates is 0.5 mm and no through hole is set in the center of the bottom cell culture plate, wherein the liquid enters from the upper end.

[0033] Figure 3 Schematic diagram of a bioreactor in which no through-hole is set in the center of the top cell culture plate when the distance between adjacent cell culture plates is 2 mm, and the liquid enters from the bottom.

[0034] Figure 4Schematic diagram of a bioreactor in which the distance between adjacent cell culture plates is 2 mm and no through hole is set in the center of the bottom cell culture plate, wherein the liquid enters from the upper end.

[0035] Figure 5 for Figure 1 Schematic diagram of velocity field calculation results for the bioreactor structure shown.

[0036] Figure 6 for Figure 2 Schematic diagram of velocity field calculation results for the bioreactor structure shown.

[0037] Figure 7 for Figure 3 Schematic diagram of velocity field calculation results for the bioreactor structure shown.

[0038] Figure 8 for Figure 4 Schematic diagram of velocity field calculation results for the bioreactor structure shown.

[0039] Fig. 9 This is a schematic diagram of the velocity field calculation results when a small hole is set near the through hole in the center of the cell culture plate.

[0040] Fig.10 This is a schematic diagram of the velocity field calculation results when one less circle of small holes is set near the through hole in the center of the cell culture plate.

[0041] Fig.11 Schematic diagram of the internal structure of the bioreactor.

[0042] Fig.12 Schematic diagram of the external structure of the bioreactor.

[0043] Fig.13 Schematic diagram of the cell culture plate.

[0044] Fig.14 Schematic diagram of one side of a cell culture plate.

[0045] Fig.15 This is a schematic diagram of the other side of the cell culture plate. DETAILED DESCRIPTION

[0046] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0047] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0048] Example 1 Design of the bioreactor of the present invention

[0049] (I) Inlet position, plate spacing and flow field analysis

[0050] The through holes at the center of the top and bottom cell culture plates in the bioreactor were closed to prevent the liquid from flowing directly through. At the same time, the cell culture plates were set to two spacings of 0.5 mm and 2 mm to investigate the flow of the liquid. Figures 1 to 4 shown.

[0051] (1) Closure of through-holes on the bottom culture plate VS Closure of through-holes on the top culture plate

[0052] From the perspective of liquid flow, after the through-holes of the bottom cell culture plate are closed, the liquid flowing in from the upper inlet will be forced to flow to both ends at the bottom layer, and then continue to flow vertically upward through the small holes on the bottom cell culture plate. In this case, each small hole on the bottom cell culture plate is equivalent to an independent liquid inlet, providing vertical upward flow. In this way, the lateral flow of liquid between all the cell culture plates above is very weak, and there is a lack of corresponding convection exchange.

[0053] In contrast, when the through-holes of the top cell culture plate are closed, the liquid flowing into the lower inlet is blocked at the top outlet and can only flow laterally to both ends through the gaps between the layers, ensuring the flow of liquid in the gaps between the cell culture plates and facilitating convection exchange.

[0054] Therefore, the bioreactor of the present invention chooses to close the through-holes of the top cell culture plate closest to the top cover, and at the same time, the inlet is arranged at the lower end of the shell.

[0055] (2) 0.5mm pitch vs. 2.0mm pitch

[0056] From the perspective of liquid flow, when the distance between adjacent cell culture plates is large, the liquid flows faster in the gap between the cell culture plates, but this will sacrifice the cell carrying capacity of the entire device. It is necessary to establish a balance between the spacing between the cell culture plates in the reactor, the cell carrying capacity of the reactor, and the material exchange between the cells and the blood to be purified.

[0057] Therefore, the bioreactor of the present invention chooses to set the spacing between adjacent cell culture plates to 1.0 mm.

[0058] The velocity field calculation results are as follows: Figures 5 to 8 As shown, the scale units of the thermal diagrams on the left side of each figure are all m / s.

[0059] (II) Small hole position and flow field analysis

[0060] On the cell culture plate, multiple circles of small holes are arranged around the through hole. When the small holes and other settings are the same, the flow of liquid is investigated when one less circle of small holes is arranged around the through hole.

[0061] From the perspective of liquid flow, when there is one less circle of holes, the liquid between the cell culture plates is more likely to flow to both ends, which is conducive to convection exchange. Fig. 9 and Fig.10 shown.

[0062] Example 2 Bioreactor of the present invention

[0063] Figures 11 to 15 Shown is a bioreactor comprising a shell 1, a top cover 2, and a plurality of cell culture plates 6, wherein the shell 1 has an open upper end and a closed lower end, wherein the lower end is provided with an inlet 3 connected to the internal fluid of the shell 1, the top cover 2 covers the open upper end of the shell 1 and is provided with an outlet 4 connected to the internal fluid of the shell 1, the plurality of cell culture plates 6 are spaced apart from each other and arranged in parallel inside the shell 1, each cell culture plate 6 is provided with a through hole 51 located at the center and a plurality of small holes 61 distributed around the through hole 51, and the through holes 51 of the plurality of cell culture plates are aligned with each other to form a channel 5.

[0064] The through hole 51 of the top cell culture plate 62 closest to the top cover 2 is closed, thereby sealing the upper end of the channel 5 .

[0065] The through hole 51 has a first end on one side of the cell culture plate 6, and an annular mounting belt 601 is arranged around the first end. A limiting base 602 protruding from the annular mounting belt 601 is arranged on the annular mounting belt 601, and a buckle 603 protruding from the limiting base 602 is arranged on the limiting base 602; the through hole 51 has a second end on the other side of the cell culture plate 6, and an annular base 605 is arranged around the second end, and a concave buckle groove 606 is arranged on the annular base 605.

[0066] The buckle 603 on the cell culture plate 6 can be disposed in the buckle groove 606 on another adjacent cell culture plate 6 , thereby allowing a plurality of cell culture plates 6 to be spaced apart from each other and connected together in parallel inside the housing 1 .

[0067] The limiting base 602 and the buckle 603 on one side of the cell culture plate 6 are the same in number as the buckle groove 606 on the other side (4 are shown in the figure), and the multiple limiting bases 602 are evenly spaced apart from each other on the annular mounting belt 601, and correspondingly, the multiple buckle grooves 606 are evenly spaced apart from each other on the annular base 605.

[0068] The limiting base 602 on the cell culture plate 6 is fitted with the annular base 605 on another adjacent cell culture plate 6. A plurality of notches 52 (four in the figure) are arranged around the annular base 605 on the cell culture plate 6.

[0069] The multiple small holes 61 of one cell culture plate 6 are aligned with the multiple small holes 61 of another adjacent cell culture plate 6 to form a plurality of peripheral channels 7 .

[0070] By the above arrangement, in the bioreactor of the present invention, a plurality of cell culture plates 6 are stacked inside the housing 1, and a channel 5 is formed by a through hole 51 at the center of each cell culture plate 6, and the channel 5 extends in the vertical direction of the bioreactor, but is closed at the top cell culture plate 62. In addition, the channel 5 can be optionally aligned with the outlet 4 and the inlet 3 in the vertical direction. The peripheral channel 7 formed by the small hole 61 extends in the vertical direction of the bioreactor. The inlet 3, the outlet 4, the channel 5 and the peripheral channel 7 are in fluid communication with each other.

[0071] The through hole 51 of each cell culture plate 6 is circular, and a plurality of small holes 61 are radially distributed relative to the through hole 51 on the cell culture plate 6 and are spaced apart from each other at the same distance to form one or more rings (four are shown in the figure) surrounding the through hole 51 .

[0072] Furthermore, the shell 1 is a cylinder, and 66 cell culture plates 6 with a thickness of 1 mm are stacked vertically inside. The cell culture plates 6 are connected together by the buckles 603 and the buckle grooves 606 on both sides of the through hole 51, and the maximum interval between adjacent cell culture plates 6 is 1 mm; in addition, the diameter of each cell culture plate 6 is 136 mm, the diameter of the through hole 51 is 6 mm, the diameter of the small hole 61 is 1.5 mm, the distance between the small hole 61 closest to the through hole 51 is 25 mm, and the distance between adjacent small holes 61 is 10 mm; there are 112 small holes 61 on each cell culture plate.

[0073] Example 3 Use of the bioreactor of the present invention

[0074] The bioreactor of Example 2 was used.

[0075] Open the outlet 4 on the top cover 2, input the liver cell suspension from the inlet 3 at the lower end of the shell 1, and after the suspension basically fills the entire bioreactor, close the inlet 3 and let it stand for 3-4 hours to allow the cells to grow on the surface of the cell culture plate.

[0076] The bioreactor is connected to the human body through other components of the biological blood purification system to start the circulation of the plasma to be purified.

[0077] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the claims attached to the present invention.

Claims

1. A bioreactor, comprising a shell, a top cover and a plurality of cell culture plates, characterized in that: The housing has an open upper end and a closed lower end, wherein the lower end is provided with an inlet in fluid communication with the interior of the housing; The top cover covers the open upper end of the housing and is provided with an outlet in fluid communication with the interior of the housing; The multiple cell culture plates are spaced apart from each other and arranged in parallel inside the shell, each cell culture plate is provided with a through hole located at the center and a plurality of small holes distributed around the through hole, and the through holes of the multiple cell culture plates are aligned with each other to form a central channel, and the through hole of the top cell culture plate closest to the top cover is closed, thereby sealing the upper end of the channel; and the cell culture plates and the small holes are respectively circular, the through hole of each cell culture plate is circular, the multiple small holes are radially distributed on the cell culture plate relative to the through hole, and the multiple small holes are spaced apart from each other at the same distance to form one or more rings around the through hole, and the ratio of the distance between the small hole closest to the through hole and the through hole to the radius of the cell culture plate is 1:2 to 3.

2. The bioreactor according to claim 1, characterized in that The through hole has a first end on one side of the cell culture plate, an annular mounting belt is arranged around the first end, a limiting base protruding from the annular mounting belt is arranged on the annular mounting belt, and a buckle protruding from the limiting base is arranged on the limiting base; the through hole has a second end on the other side of the cell culture plate, an annular base is arranged around the second end, and a concave buckle groove is arranged on the annular base.

3. The bioreactor according to claim 2, characterized in that The buckles on the cell culture plate are arranged in the buckle grooves on another adjacent cell culture plate, thereby making the multiple cell culture plates spaced apart from each other and connected together in parallel inside the housing.

4. The bioreactor according to claim 2 or 3, characterized in that The limiting bases and buckles on one side of the cell culture plate are the same in number as the buckle grooves on the other side, and the multiple limiting bases are evenly spaced apart from each other on the annular mounting belt, and correspondingly, the multiple buckle grooves are evenly spaced apart from each other on the annular base.

5. The bioreactor according to claim 2 or 3, characterized in that: The limiting base on the cell culture plate fits with the annular base on another adjacent cell culture plate.

6. The bioreactor according to claim 2 or 3, characterized in that: A plurality of notches are arranged around the annular base on the cell culture plate.

7. The bioreactor according to claim 1 or 2, characterized in that: A plurality of small holes of one cell culture plate are aligned with a plurality of small holes of another adjacent cell culture plate to form a plurality of peripheral channels.

8. The bioreactor according to claim 1 or 2, characterized in that: The ratio of the distance between the small hole closest to the through hole and the through hole to the radius of the cell culture plate is 1:2.

7.

9. The bioreactor according to claim 1 or 2, characterized in that: The ratio of the diameter of the small hole to the diameter of the through hole is 1:3-5.

10. The bioreactor according to claim 1 or 2, characterized in that: The ratio of the diameter of the small hole to the diameter of the through hole is 1:

4.

11. The bioreactor according to claim 1 or 2, characterized in that: The ratio of the diameter of the small hole to the diameter of the cell culture plate is 1:80-100.

12. The bioreactor according to claim 1 or 2, characterized in that: The ratio of the diameter of the small hole to the diameter of the cell culture plate is 1:90.

7.

13. The bioreactor according to claim 1 or 2, characterized in that: The ratio of the diameter of the through hole to the diameter of the cell culture plate is 1:20-30.

14. The bioreactor according to claim 1 or 2, characterized in that: The ratio of the diameter of the through hole to the diameter of the cell culture plate is 1:22.7.

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