A double-layer hard plastic fluidized bed cell bioreactor with four-rocker shaker

Abstract

The present invention relates to the technical field of cell bioreactors, and in particular to a double-layer hard plastic fluidized bed cell bioreactor with a four-rocker shaker. The reactor includes a polygonal insulation tank body, a double-layer cell reaction tank and a four-rocker shaker; the four-rocker shaker of the present invention adopts a cross-axis structure with the upper and lower ends of the rocker, and its vertical load is directly guided to the ground through the rocker, which can eliminate the overturning moment acting on the shaker and the ground, reduce the vibration and noise generated during the entire culture process, and greatly reduce the manufacturing cost and weight of the shaker, and this shaker can withstand a large load; the double-layer reaction tank of the present invention and the filter screen thereon can be used to culture cells by perfusion process when high-density microcarriers are used to culture adherent cells, and the cells grow in a true microgravity environment, so that the cells grow in three dimensions and maintain a high differentiation rate and amplification. The reaction tank is particularly suitable for culturing mesenchymal stem cells, which is conducive to promotion and application.

Description

Technical Field

[0001] The invention relates to the technical field of cell bioreactors, in particular to a double-layer hard plastic fluidized bed cell bioreactor of a four-rocker shaker. Background Art

[0002] At present, the world's biotechnology pharmaceutical industry is developing rapidly and has achieved great success. The expression and production of various types of diagnostic and therapeutic monoclonal antibodies, vaccines, growth factors and other biologically active proteins through in vitro culture of animal cells have very broad market application prospects. Therefore, disposable cell bioreactors used for large-scale high-density culture of animal cells have become the most important equipment in the biotechnology pharmaceutical industry, which largely determines the cost, production scale and product types of biopharmaceutical companies.

[0003] When the working volume of the existing rocking cell reactor using circular oscillation is large, such as more than 50 liters, it will generate a large overturning moment on the ground, and the entire gravity of the reactor tank, the rocking plate and the culture medium in the reactor tank acts on the rocking plate. In this way, the rocking table not only has to bear the entire gravity of the tank body, but also has to apply torque to shake it. As a result, under the action of the overturning moment, the entire reaction process will generate a lot of vibration and noise. Due to the huge gravity acting on the rocking table, the size, strength and weight of each part of the rocking table will inevitably increase, which also greatly increases the cost and space occupied by the rocking table.

[0004] In addition, the culture bag of the existing tank external circulation reactor is made of a plastic film and a hard plastic support. The centroid and the center of gravity do not coincide, resulting in a center of gravity offset, unstable shaking, and large centrifugal force and vibration. In addition, there is only one water inlet for the guide tube. Every time the shaker rotates, water enters the water inlet of the guide tube, which makes the circulation speed of the culture medium low, and it is difficult to achieve high efficiency in culturing cells.

[0005] The information disclosed in this background technology section is only intended to deepen the understanding of the overall background technology of the present invention, and should not be regarded as acknowledging or suggesting in any form that the information constitutes the prior art already known to those skilled in the art. Summary of the invention

[0006] The purpose of the present invention is to provide a double-layer hard plastic fluidized bed cell bioreactor with a four-rocker shaker to solve the technical problems existing in the prior art.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a double-layer hard plastic fluidized bed cell bioreactor with a four-rocker shaker, comprising a polygonal heat-insulating tank body, which also includes: a double-layer cell reaction tank and a four-rocker shaker;

[0009] The double-layer cell reaction tank comprises: a tank cover, an outer tank, an inner tank and a filter; the tank cover is installed on the top of the outer tank; the inner tank is installed inside the outer tank, and the filter is installed above the waist of the inner tank; two water inlet convex parts are symmetrically arranged on both sides of the upper end of the outer tank, and two water outlets are symmetrically arranged on both sides of the upper end of the inner tank, the water inlet convex parts on the upper end of the outer tank are arranged one-to-one with the water outlets on the upper end of the inner tank, and a water return port is arranged at the bottom end of the inner tank, and a sandwich space is formed between the outer tank and the inner tank, and the outer tank and the inner tank are kept in a connected state through the water outlet of the inner tank, the water inlet convex parts of the outer tank, the sandwich space, the water return port at the bottom end of the inner tank and the filter;

[0010] The polygonal heat-insulating tank body is used to carry the double-layer cell reaction tank;

[0011] The polygonal heat-insulating tank body is installed on the four-rocker rocking bed.

[0012] As a further technical solution, the four-rocker rocker comprises: a rocker assembly of a cross-axis structure, a rocker plate assembly and a rocker drive box;

[0013] The rocking table drive box is connected to the rocking table assembly;

[0014] The polygonal heat-insulating tank body is mounted on the rocking plate assembly;

[0015] The double-layer cell reaction tank is installed on the polygonal heat preservation tank body;

[0016] The rocker assembly is provided with fixed sleeves with openings facing downwards at the four corners of the rocker, and the rocker assembly with a cross-axis structure includes four rockers with a cross-axis structure;

[0017] The top of the rocker with the cross structure is installed on the top of the fixed sleeve through an upper support seat, and the bottom of the rocker with the cross-axis structure is installed on the driving box chassis of the rocking bed driving box through a lower supporting channel steel.

[0018] As a further technical solution, the rocker of the cross-axis structure includes: an upper support seat, a cross-axis, a sliding bearing, a lower support seat, a short shaft at the upper end of the rocker, a hollow sleeve of the rocker, a short shaft at the lower end of the rocker, an upper swing channel steel, an upper limit shaft, an upper support shaft, an upper support channel steel, a lower swing channel steel, a lower support shaft, a lower limit shaft and a lower support channel steel;

[0019] The two shaft ends of the cross shaft that are opposite to each other are connected to the upper support seat through the sliding bearings, and the other two shaft ends of the cross shaft that are opposite to each other are connected to the lower support seat through the sliding bearings; the upper support seat, the cross shaft, the sliding bearings and the lower support seat together constitute a universal joint structure at the top end of the rocker of the cross shaft structure; the upper part of the short shaft at the upper end of the rocker is welded to the bottom of the lower support seat; the upper end of the hollow shaft sleeve of the rocker and the lower end of the short shaft at the upper end of the rocker are fitted together and spot-welded to achieve coaxial connection;

[0020] The upper end of the upper swing channel steel and the lower end of the short shaft at the lower end of the rocker are welded together; the bottom of the upper support channel steel and the upper part of the lower swing channel steel are welded together, and the groove angle between the two is 90 degrees; the upper swing channel steel (which has been welded together with the short shaft at the lower end of the rocker) uses two upper limit shafts and one upper support shaft to install the upper support channel steel (which has been welded together with the lower swing channel steel) through the shaft; the lower swing channel steel uses one lower support shaft and two lower limit shafts to install the lower support channel steel through the shaft; the upper end of the short shaft at the lower end of the rocker is concentrically installed on the lower end of the hollow shaft sleeve of the rocker and spot welded.

[0021] As a further technical solution, the two upper limit axes are symmetrically arranged on both sides above the upper support; the two lower limit axes are symmetrically arranged on both sides below the lower support axis.

[0022] As a further technical solution, a driving motor is installed in the rocking table driving box;

[0023] A small pulley is installed on the power output shaft of the driving motor;

[0024] The small pulley is connected to the large pulley through a belt tensioning connection;

[0025] The large pulley is mounted on the active eccentric shaft;

[0026] The upper end of the active eccentric shaft is connected to the shaking plate;

[0027] The upper shaking plate is connected to four sets of driven shafts;

[0028] The upper rocking plate is connected to the rocking plate by bolts;

[0029] The polygonal heat-insulating tank body is connected and installed on the rocking plate through the tank body bottom plate by bolts.

[0030] As a further technical solution, the polygonal heat-insulating tank body has 6-12 sides.

[0031] As a further technical solution, the flange on the top of the outer tank is connected to the flange of the tank cover by bolts, and the two are sealed with a sealing rubber ring; the flange on the top of the inner tank is welded to the inner ring of the flange of the outer tank; the top ring of the filter is welded to the flange of the inner tank, and the bottom ring of the filter is connected to the waist position of the inner tank by a clamping ring.

[0032] As a further technical solution, four supporting columns are arranged at the bottom of the outer tank; and four mounting holes matching the supporting columns are arranged at the bottom of the inner tank.

[0033] As a further technical solution, the lower part of the inner tank is cylindrical, the upper part of the inner tank is conical, the water outlet of the inner tank is symmetrically arranged on the conical tank wall at the upper part of the inner tank, and the water outlet is provided with a tangential flow baffle on its clockwise side, and a horizontal baffle is provided at the bottom of the tangential flow baffle.

[0034] As a further technical solution, the lower part of the outer tank is cylindrical, the upper part of the outer tank is conical, and the water inlet convex part of the outer tank is arranged to expand outward on the conical wall.

[0035] By adopting the above technical solution, the present invention has the following beneficial effects:

[0036] The double-layer hard plastic fluidized bed cell bioreactor of the four-rocker shaker provided by the present invention is

[0037] The four-rocker shaker of the present invention adopts the upper and lower ends of the rockers with a cross-axis structure, and the vertical load is directly guided to the ground through the rockers, which can eliminate the overturning moment acting on the shaker and the ground, reduce the vibration and noise generated during the entire culture process, greatly reduce the manufacturing cost and weight of the shaker, and the shaker can withstand a large load;

[0038] The double-layer reaction tank and the filter screen thereon of the present invention can be used to culture cells by perfusion process when high-density microcarriers are used to culture adherent cells, and the cells grow in a true microgravity environment. The cells grow three-dimensionally and maintain a high differentiation rate. The reaction tank is particularly suitable for culturing mesenchymal stem cells, which is conducive to promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0040] Figure 1 A front view of a double-layer hard plastic fluidized bed cell bioreactor of a four-rocker shaker provided in an embodiment of the present invention;

[0041] Figure 2 for Figure 1 A side view of a double-layer hard plastic fluidized bed cell bioreactor with a four-rocker shaker is shown;

[0042] Figure 3 for Figure 1 A top view of a double-layer hard plastic fluidized bed cell bioreactor with a four-rocker shaker is shown;

[0043] Figure 4 A schematic diagram of the structure of a double-layer cell reaction tank provided in an embodiment of the present invention;

[0044] Figure 5 A top view of a double-layer cell reaction tank provided in an embodiment of the present invention;

[0045] Figure 6 A schematic diagram of the structure of a can cover provided by an embodiment of the present invention;

[0046] Figure 7 A top view of a can cover provided by an embodiment of the present invention;

[0047] Figure 8 A schematic diagram of the structure of an outer tank provided in an embodiment of the present invention;

[0048] Fig. 9 A top view of an outer tank provided by an embodiment of the present invention;

[0049] Fig.10 A schematic diagram of the structure of an inner tank provided in an embodiment of the present invention;

[0050] Fig.11 A top view of an inner tank provided by an embodiment of the present invention;

[0051] Fig.12 A schematic diagram of the structure of a filter screen provided in an embodiment of the present invention;

[0052] Fig.13 A top view of a filter screen provided by an embodiment of the present invention;

[0053] Fig.14 A top view of a clamp ring provided in an embodiment of the present invention;

[0054] Fig.15 A front view of a rocker assembly with a cross-axis structure provided by an embodiment of the present invention;

[0055] Fig.16 A side view of a rocker assembly with a cross-axis structure provided in an embodiment of the present invention.

[0056] Icons: 202-tank cover; 205-inner tank; 208-filter screen; 209-clamp ring; 210-water outlet; 211-tangential flow baffle; 212-horizontal baffle; 214-outer tank; 217-water return port; 222-water inlet convex part; 223-sealing rubber ring; 225-support column; 300-rocker assembly of cross-axis structure; 302-upper support seat; 305-cross shaft; 308-sliding bearing; 310-lower support seat; 312-short shaft at the upper end of the rocker; 313-hollow sleeve of the rocker; 314-short shaft at the lower end of the rocker; 316-upper limit shaft; 320-upper support shaft; 322-lower support shaft; 326-lower limit shaft; 330-lower support channel steel; 333-lower swing channel steel; 334-upper support channel steel; 338-upper swing channel steel; 516-polygonal insulation tank body; 518-rocking table assembly; 520-drive box chassis; 530-rocking table drive box. DETAILED DESCRIPTION

[0057] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance.

[0059] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0060] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0061] Combination Figures 1 to 16 As shown, this embodiment provides a double-layer hard plastic fluidized bed cell bioreactor with a four-rocker shaker, including a polygonal heat-insulating tank body 516, which also includes: a double-layer cell reaction tank and a four-rocker shaker; the double-layer cell reaction tank includes: a tank cover 202, an outer tank 214, an inner tank 205 and a filter screen 208; the tank cover 202 is installed on the top of the outer tank 214; the inner tank 205 is installed inside the outer tank 214, and the filter screen 208 is installed above the waist of the inner tank 205; two water inlet convex parts 222 are symmetrically arranged on both sides of the upper end of the outer tank 214, and two water outlets 210 are symmetrically arranged on both sides of the upper end of the inner tank 205. The water inlet protrusion 222 at the upper end of the outer tank 214 is arranged in a one-to-one correspondence with the water outlet 210 at the upper end of the inner tank 205, and a return water port 217 is arranged at the bottom end of the inner tank 205. An interlayer space is formed between the outer tank 214 and the inner tank 205. The outer tank 214 and the inner tank 205 are connected through the water outlet 210 of the inner tank 205, the water inlet protrusion 222 of the outer tank 214, the interlayer space, the return water port 217 at the bottom end of the inner tank 205 and the filter 208; the polygonal insulation tank body 516 is used to carry the double-layer cell reaction tank; the polygonal insulation tank body 516 is installed on the four-rocker shaker.

[0062] In this embodiment, after the equipment is installed, the shaking table is not started first, and the double-layer reaction tank is installed in the polygonal heat preservation tank body 516, and the culture solution is added and heated, and then the sterilized microcarriers are placed in proportion and the shaking is started. When the temperature reaches the range of 35.5-37 degrees Celsius, the seed cells are injected. The cells are first allowed to adhere to the wall at a lower speed, that is, to the surface of the microcarrier.

[0063] After the cells are attached to the wall, they need to be cultured at a faster speed to grow, differentiate and expand. When the density is reached, the cells are washed off the surface of the microcarriers with enzymes and harvested. If the culture is to be expanded, the cans can be inverted for expansion. That is, the next reactor with a larger volume is used, and the above operation is repeated to complete the next level of inverted can expansion.

[0064] The perfusion process is when the nutrients in the culture medium in the reaction tank are exhausted, but the cultured cells have not yet reached the required density. If you want to continue the culture, you can extract the supernatant from the tank at the same flow rate while adding fresh culture medium to the tank to continue culturing and expanding the cells to increase the cell density. One point to note is that only the supernatant can be extracted, and the cells cannot be extracted at the same time. The cells must be retained in the tank. Continue to culture and expand until the density requirement is reached.

[0065] In this embodiment, preferably, the double-layer cell reaction tank can be made of double-layer PVC material, which is suitable for small working volume and high-density microcarrier culture of adherent cells. This embodiment is suitable for the culture of mesenchymal stem cells. When the volume is enlarged, the cell reaction tank can be made of a single-layer plastic film and a hard plastic frame.

[0066] In this embodiment, as a further technical solution, the four-rocker shaker includes: a rocker assembly 300 with a cross-axis structure, a rocker plate assembly 518 and a shaker drive box 530; the shaker drive box 530 is connected to the rocker plate assembly 518; the polygonal insulation tank body 516 is installed on the rocker plate assembly 518; the double-layer cell reaction tank is installed on the polygonal insulation tank body 516; the rocker plate assembly 518 is respectively provided with fixed sleeves with openings facing downwards at the four corners of the rocker plate, and the rocker assembly 300 with a cross-axis structure includes four rockers with a cross-axis structure; the top of the rocker with the cross structure is installed on the top of the fixed sleeve through the upper support seat 302, and the bottom of the rocker with the cross-axis structure is installed on the drive box chassis 520 of the shaker drive box 530 through the lower support channel steel 330.

[0067] In this embodiment, specifically, the rocker of the cross-axis structure includes: an upper support seat 302, a cross shaft 305, a sliding bearing 308, a lower support seat 310, a short shaft 312 at the upper end of the rocker, a hollow sleeve 313 of the rocker, a short shaft 314 at the lower end of the rocker, an upper swing channel steel 338, an upper limit shaft 316, an upper support shaft 320, an upper support channel steel 334, a lower swing channel steel 333, a lower support shaft 322, a lower limit shaft 326 and a lower support channel steel 330; the two shaft ends of the cross shaft that are oppositely arranged are connected to the upper support seat 302 through the sliding bearing 308, and the other two shaft ends of the cross shaft that are oppositely arranged are connected to the lower support seat 310 through the sliding bearing 308; the upper support seat 302, the cross shaft, the sliding bearing 308 and the lower support seat 310 together constitute a universal joint structure at the top end of the rocker of the cross-axis structure; the upper part of the short shaft 312 at the upper end of the rocker is welded to the lower support seat 3 10; the upper end of the rocker hollow shaft sleeve and the lower end of the rocker upper short shaft 312 are sleeved together and spot welded to achieve coaxial connection; the upper end of the upper swing channel steel 338 and the lower end of the rocker lower short shaft 314 are welded together; the bottom of the upper support channel steel and the upper part of the lower swing channel steel 333 are welded together, and the groove angle between the two is 90 degrees; the upper swing channel steel 338 (which has been welded to the rocker lower short shaft 314 The upper supporting channel steel 334 (which has been welded into one piece with the lower swing channel steel 333) is installed together with two upper limit shafts 316 and one upper support shaft 320; the lower swing channel steel 333 is installed together with the lower support channel steel 330 by passing through the lower support shaft 322 and two lower limit shafts 326; the upper end of the short shaft 314 at the lower end of the rocker is coaxially installed on the lower end of the hollow shaft sleeve of the rocker and spot welded.

[0068] In this embodiment, the upper and lower ends of the rocker adopt the cross-axis structure principle of the universal joint, and its longitudinal load is directly guided to the ground through the rocker. The function of the limit axis at the lower end of the rocker is to limit the rotation angle of the rocker in all directions within a preset angle, and the specific angle setting can be flexibly set according to actual needs.

[0069] In this embodiment, as a further technical solution, the two upper limit axes 316 are symmetrically arranged on both sides above the upper support; the two lower limit axes 326 are symmetrically arranged on both sides below the lower support axis 322.

[0070] In this embodiment, as a further technical solution, a driving motor is installed in the rocking bed driving box 530; a small pulley is installed on the power output shaft of the driving motor; the small pulley is connected to the large pulley by a belt tensioning connection; the large pulley is installed on the active eccentric shaft; the upper end of the active eccentric shaft is connected to the upper rocking plate; the upper rocking plate is connected to four sets of driven shafts; the upper rocking plate is connected to the rocking plate by bolts; the polygonal insulation tank body 516 is installed on the rocking plate by bolts through the tank body bottom plate. In this embodiment, a mechanical eccentric shaft is used to output torque to make the rocking plate perform circular oscillation motion. This rocking bed does not generate a tipping moment, and its support point can be higher than the center of gravity of the tank body. The vertical load of this rocking bed is directly transmitted to the ground through the rocking arm.

[0071] In this embodiment, as a further technical solution, the polygonal heat-insulating tank body 516 has 6-12 sides.

[0072] In this embodiment, as a further technical solution, the flange on the top of the outer tank 214 is connected to the flange of the tank cover 202 by bolts, and the two are sealed with a sealing rubber ring 223; the flange on the top of the inner tank 205 is welded to the inner ring of the flange of the outer tank 214; the top ring of the filter 208 is welded to the flange of the inner tank 205, and the bottom ring of the filter 208 is connected to the waist position of the inner tank 205 by a clamping ring 209.

[0073] In this embodiment, as a further technical solution, four support columns 225 are provided at the bottom of the outer tank 214 ; and four mounting holes matched with the support columns 225 are provided at the bottom of the inner tank 205 .

[0074] In this embodiment, as a further technical solution, the lower part of the inner tank 205 is cylindrical, the upper part of the inner tank 205 is conical, the water outlet 210 of the inner tank 205 is symmetrically arranged on the conical tank wall at the upper part of the inner tank 205, and the water outlet 210 is provided with a tangential flow baffle 211 on its clockwise side, and a horizontal baffle 212 is provided at the bottom of the tangential flow baffle 211.

[0075] In this embodiment, as a further technical solution, the lower portion of the outer tank 214 is cylindrical, the upper portion of the outer tank 214 is conical, and the water inlet convex portion 222 of the outer tank 214 is arranged to expand outward on the conical wall.

[0076] The tank body of this embodiment is a double-layer tank, preferably made of PC material, with a working volume of less than 50 liters; the tank cover and the tank body are sealed with a sealing rubber ring and bolts. The shape of the filter can be adjusted. The tank is particularly suitable for in vitro expansion and culture of mesenchymal stem cells; when using high-density microcarriers to culture adherent cells, the cells grow in a microgravity environment, the cells can grow three-dimensionally, and maintain a high differentiation rate and expansion; the structure of the reaction tank is designed according to the clockwise shaking of the shaker, and the shaker must shake clockwise during operation.

[0077] It is worth noting that the tank cover in this embodiment can integrate the structural characteristics of the multifunctional disk, for example, the tank cover has an air inlet, an air outlet, a liquid inlet, a liquid outlet and a sampling port, etc. It also has several sensor components including a dissolved oxygen sensor 201, a pH sensor and the like.

[0078] The working principle of the double-layer hard plastic fluidized bed cell bioreactor of the rocker shaker in the fourth embodiment is:

[0079] After the entire reactor is installed, the culture medium is first added to the double-layer reaction tank to the static liquid level, then heated to the insulation temperature through the double-layer cell reaction tank and kept warm, microcarriers are added in proportion, and then the shaker is turned on to start shaking, and then the seed cells are injected. At the beginning, the shaker is shaken at a low speed to allow the cells to adhere to the wall. After the adhesion is completed, the speed is increased to expand the cultured cells; the shaker is shaken clockwise, and after shaking, the culture liquid level is a dynamic liquid level rotation with one side high and the other side low. Each time the double-layer cell reaction tank rotates one circle, the culture solution with the higher liquid level enters the two water outlets 210 one after another, flows out of the inner tank 205, and enters the interlayer space between the inner tank 205 and the outer tank 214. The water inlet convex portion 222 of the outer tank 214 creates a larger gas-liquid mixing space for the culture solution flowing in. At this time, a large number of water splashes and water droplets are generated at the interlayer, thereby increasing the surface area of ​​the gas-liquid ratio, which is conducive to the rapid dissolution of oxygen and harmful gases from the water droplets and water splashes of the culture solution to escape into the air, and then be discharged out of the reactor; as the shaking table shakes, the culture solution entering the outer tank 214 flows downward in a spiral in the interlayer space, reaches the bottom, flows into the inner tank 205 from the water return port 217 of the inner tank 205, and then flows upward in a spiral until the water surface of the inner tank 205 flows into the interlayer space outside the inner tank 205 from the two water outlets 210 of the inner tank 205, and circulates over and over again.

[0080] At the same time, due to the effect of the filter 208, the high-density microcarriers with adherent cells in the culture medium are retained in the filter 208, and only the culture medium flows through the filter 208 for circulation.

[0081] At this time, the culture fluid in the inner tank 205 flows upward in a spiral from the bottom to the top. The cells on the microcarriers in this space are pushed upward, thereby overcoming their own gravity. Therefore, the cells grow in a microgravity environment, and the cells can grow three-dimensionally, and the cells can also be amplified with a high differentiation rate and good activity; the inner tank 205 and the outer tank 214 jacket top of the reaction tank are connected to the top of the inner tank 205. The flange of the tank cover 202 and the flange of the outer tank 214 are clamped and sealed with bolts and sealing rings.

[0082] Since the density of microcarriers is high, the sedimentation rate is high. The microcarriers in the inner tank 205 are lifted up by the culture solution flowing upward in a spiral. However, when they reach the upper truncated cone of the inner tank 205, the flow rate of the culture solution flowing upward decreases due to the gradual increase in the cross-sectional area. When they are close to the top, the upward thrust of the culture solution and the downward gravity of the microcarriers reach a balance. The microcarriers cannot reach the water outlet 210 of the inner tank 205. At this time, the microcarriers can only float up and down in the inner tank 205. Even if some microcarriers are rushed to the water outlet 210, they are also trapped in the filter screen 208.

[0083] By adopting the above technical solution, the present invention has the following beneficial effects:

[0084] The four-rocker shaker of the present invention adopts the upper and lower ends of the rockers with a cross-axis structure, and the vertical load is directly guided to the ground through the rockers, which can eliminate the overturning moment acting on the shaker and the ground, reduce the vibration and noise generated during the entire culture process, greatly reduce the manufacturing cost and weight of the shaker, and the shaker can withstand a large load;

[0085] The double-layer reaction tank of the present invention and the filter 208 thereon can be used to culture cells by perfusion process when high-density microcarriers are used to culture adherent cells, and the cells grow in a true microgravity environment. The cells grow three-dimensionally and maintain a high differentiation rate. The reaction tank is particularly suitable for culturing mesenchymal stem cells, which is conducive to promotion and application.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A double-layer hard plastic fluidized bed cell bioreactor with four rockers, comprising a polygonal insulation tank, characterized in that: Also includes: Double-layer cell reaction tank and four-shaker shaker; The double-layer cell reaction tank comprises: a tank cover, an outer tank, an inner tank and a filter; the tank cover is installed on the top of the outer tank; the inner tank is installed inside the outer tank, and the filter is installed above the waist of the inner tank; two water inlet convex parts are symmetrically arranged on both sides of the upper end of the outer tank, and two water outlets are symmetrically arranged on both sides of the upper end of the inner tank, the water inlet convex parts on the upper end of the outer tank are arranged one-to-one with the water outlets on the upper end of the inner tank, and a water return port is arranged at the bottom end of the inner tank, and a sandwich space is formed between the outer tank and the inner tank. The inner tank is connected to the outer tank through the water outlet of the inner tank, the water inlet convex part of the outer tank, the interlayer space, the water return port at the bottom of the inner tank and the filter screen; the lower part of the inner tank is cylindrical, the upper part of the inner tank is conical, the water outlet of the inner tank is symmetrically arranged on the conical tank wall at the upper part of the inner tank, the water outlet is provided with a tangential flow baffle on the clockwise side, and a horizontal baffle is provided at the bottom of the tangential flow baffle; the lower part of the outer tank is cylindrical, the upper part of the outer tank is conical, and the water inlet convex part of the outer tank is expanded outward on the conical tank wall; The polygonal heat-insulating tank body is used to carry the double-layer cell reaction tank; The polygonal heat-insulating tank body is installed on the four-rocker rocking bed.

2. The double-layer hard plastic fluidized bed cell bioreactor of the four-rocker shaker according to claim 1, characterized in that: The four-rocker rocker comprises: a rocker assembly of a cross-axis structure, a rocker plate assembly and a rocker drive box; The rocking table drive box is connected to the rocking table assembly; The polygonal heat-insulating tank body is mounted on the rocking plate assembly; The double-layer cell reaction tank is installed on the polygonal heat preservation tank body; The rocking plate assembly is provided with fixed sleeves with openings facing downwards at the four corners of the rocking plate. The cross-axis structure rocker assembly includes four cross-axis structure rockers; The top of the rocker of the cross-axis structure is installed on the top of the fixed sleeve through an upper support seat, and the bottom of the rocker of the cross-axis structure is installed on the driving box chassis of the rocking bed driving box through a lower supporting channel steel.

3. The double-layer hard plastic fluidized bed cell bioreactor of the four-rocker shaker according to claim 2, characterized in that: The rocker of the cross-axis structure comprises: an upper support seat, a cross-axis, a sliding bearing, a lower support seat, a short shaft at the upper end of the rocker, a hollow sleeve of the rocker, a short shaft at the lower end of the rocker, an upper swing channel steel, an upper limit shaft, an upper support shaft, an upper support channel steel, a lower swing channel steel, a lower support shaft, a lower limit shaft and a lower support channel steel; The two shaft ends of the cross shaft that are opposite to each other are connected to the upper support seat through the sliding bearings, and the other two shaft ends of the cross shaft that are opposite to each other are connected to the lower support seat through the sliding bearings; the upper support seat, the cross shaft, the sliding bearings and the lower support seat together constitute a universal joint structure at the top end of the rocker of the cross shaft structure; the upper part of the short shaft at the upper end of the rocker is welded to the bottom of the lower support seat; the upper end of the hollow sleeve of the rocker and the lower end of the short shaft at the upper end of the rocker are sheathed together and spot-welded to achieve coaxial connection; The upper end of the upper swing channel steel and the lower end of the short shaft at the lower end of the rocker are welded together; the bottom of the upper support channel steel and the upper part of the lower swing channel steel are welded together, and the groove angle between the two is 90 degrees; the upper swing channel steel uses two upper limit shafts and one upper support shaft to install the upper support channel steel through the shaft; the lower swing channel steel uses one lower support shaft and two lower limit shafts to install the lower support channel steel through the shaft; the upper end of the short shaft at the lower end of the rocker is concentrically installed on the lower end of the hollow sleeve of the rocker and spot welded.

4. The double-layer hard plastic fluidized bed cell bioreactor of the four-rocker shaker according to claim 3 is characterized in that: The two upper limit axes are symmetrically arranged on both sides above the upper support; The two lower limit shafts are symmetrically arranged on both sides below the lower support shaft.

5. The double-layer hard plastic fluidized bed cell bioreactor of the four-rocker shaker according to claim 2, characterized in that: A driving motor is installed in the shaking table driving box; A small pulley is installed on the power output shaft of the driving motor; The small pulley is connected to the large pulley through a belt tensioning connection; The large pulley is mounted on the active eccentric shaft; The upper end of the active eccentric shaft is connected to the shaking plate; The upper shaking plate is connected to four sets of driven shafts; The upper rocking plate is connected to the rocking plate by bolts; The polygonal heat-insulating tank body is connected and installed on the rocking plate through the tank body bottom plate by bolts.

6. The double-layer hard plastic fluidized bed cell bioreactor of the four-rocker shaker according to claim 5, characterized in that: The polygonal heat-insulating tank body has 6 to 12 sides.

7. The double-layer hard plastic fluidized bed cell bioreactor of the four-rocker shaker according to claim 3, characterized in that: The flange on the top of the outer tank is connected to the flange of the tank cover by bolts, and both are sealed by a sealing rubber ring; The flange on the top of the inner tank is welded to the inner ring of the flange of the outer tank; The top ring of the filter screen is welded to the flange of the inner tank, and the bottom ring of the filter screen is connected to the waist position of the inner tank through a clamping ring.

8. The double-layer hard plastic fluidized bed cell bioreactor of the four-rocker shaker according to claim 3, characterized in that: Four support columns are provided at the bottom of the outer tank; The bottom of the inner tank is provided with four mounting holes matched with the supporting columns.

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