WO2019214465A1

WO2019214465A1 - Fluidized bed cell culture disposable membrane device filled with flaky carriers

Info

Publication number: WO2019214465A1
Application number: PCT/CN2019/084600
Authority: WO
Inventors: 甄宝贵; 李驰; 吴菲菲; 李昌武; 王树华
Original assignee: 武汉赛科成科技有限公司
Priority date: 2018-05-09
Filing date: 2019-04-26
Publication date: 2019-11-14
Also published as: CN108315259A; JP3233421U; CN108315259B

Abstract

The present invention provides a fluidized bed cell culture disposable membrane device filled with flaky carriers, comprising a closed bag. The interior of the closed bag is filled with several flaky carriers; either side of the exterior of the closed bag is provided with an extended side seal; the closed bag is fixed in a biological cell culture shaker by means of fixing mechanisms provided on the extended side seals on the two sides; the top surface of the closed bag is provided with an upper air inlet, an air outlet, a sampling port, and at least one liquid inlet and liquid outlet; the bottom surface of the closed bag is provided with a lower air inlet; the upper air inlet, the air outlet, the sampling port, the liquid inlet, the liquid outlet, and the lower air inlet each are connected to a sterile quick joint; a separation device is further provided in each liquid outlet; the closed bag withholds the flaky carriers in the closed bag by means of the separation device provided at the liquid outlet so as to separate cells from the flaky carriers. The present invention has advantages such as a good oxygen dissolution effect, a large cell culture final density, and convenience for industrial large scale of adherent cells.

Description

Fluidized bed cell culture disposable membrane device filled with sheet carrier

Technical field

The invention relates to the technical field of cell culture, in particular to a fluidized bed cell culture disposable membrane device filled with a sheet carrier.

Background technique

The bioreactor currently undergoing cell culture is mainly divided into a fluidized bed bioreactor and a fixed bed bioreactor. A fixed bed bioreactor is a cell adherent culture by loading a filler in the middle of a fixed container, the filler is composed of a cell culture carrier, and the cell culture carrier is usually a sheet fiber carrier having a diameter of 2 to 11 mm. Cell-attached on a sheet-shaped carrier for fixed-bed culture was first established by NBS, USA, but its sheet-like carrier is usually placed in a bioreactor tank by wire mesh, and the cells are in a static culture for a long period of time. The sheet-shaped carrier cannot be suspended, and after a long period of time, metabolites (hazardous substances) produced by the cells are aggregated in the sheet-like carrier and cannot be discharged, and it is difficult to achieve large-scale culture of the adherent cells, and it is difficult to directly perform cell sampling.

A fluidized bed reactor is a reactor that uses a gas or liquid to pass a granular solid layer to cause a solid particle to be in a suspended motion state, and performs a gas solid phase reaction process or a liquid solid phase reaction process. The purpose of the fluidized bed is to make a fluidized bed Large bubble breakage, improve the contact of gas-solid phase, reduce back-mixing, improve the reaction effect, but when the cells are cultured, the cells are easily damaged by large bubbles, the ventilation distribution is uneven, and the mass transfer and oxygen transmission are poor, resulting in cells. The growth state is not good.

In addition, most of the fixed-bed bioreactors currently on the market are mostly non-sterile products, with high levels of related components such as microorganisms and endotoxin, which have certain effects on subsequent sterilization and production processes and products; Most of the bioreactors are made of glass stainless steel and repeatedly used. It is easy to cause cross-contamination of different batches. It requires a lot of verification work, and the relative cost is high. At the same time, after each batch of cultivation, the parts are separated and cleaned. It is inconvenient and requires a lot of labor time.

Existing fluidized bed bioreactors and fixed bed bioreactors are inconvenient for industrial amplification of adherent cells, and the culture density of suspended cells is relatively low. For those skilled in the art, the conventional method for increasing the culture density of suspended cells is to use a cell culture device such as "a cell culture bag" of Patent No. 201521086949.4, but no technician has thought of a cell culture bag. The combination with the flaky carrier is used for adherent cell culture, and the final production of the cells is increased by increasing the production density of the adherent cells, thereby overcoming the technical problem that the conventional fixed-bed bioreactor is difficult to achieve large-scale cultivation of adherent cells.

Summary of the invention

The object of the present invention is to provide a fluidized bed cell culture disposable membrane device filled with a sheet carrier, which can remove metabolites in time, improve the cell culture environment on the sheet carrier, promote cell growth, and allow the adherent cells to obtain suspension. Cultivate a similar culture mechanism while maintaining the excellent specific effects of adherent cells and facilitating industrial amplification of adherent cells.

In order to achieve the above object, the technical solution adopted by the present invention is: a fluidized bed cell culture disposable membrane device filled with a sheet carrier, comprising a closed bag body, and the closed bag body is filled with a plurality of sheet carriers An extended side seal is disposed on both sides of the outer side of the closed bag body, and each of the extended side seals is provided with a fixing mechanism, and the closed bag body is sealed by two sides A fixing mechanism is fixed in the biological cell culture shaker, and an upper air inlet, an air outlet, a sampling port and at least one liquid inlet and the liquid inlet are provided on the top surface of the closed bag body, in the closed a bottom air inlet is provided on the bottom surface of the bag body, and a sterile quick connector is connected to the upper air inlet, the air outlet, the sampling port, the liquid inlet, the liquid outlet and the lower air inlet, in each of the A partitioning device is further disposed in the liquid outlet, and the closed bag body intercepts the sheet carrier in the closed bag through a partition device disposed at the liquid inlet and outlet, thereby realizing separation of the cells from the sheet carrier.

In the above technical solution, the closed bag body is composed of an upper bag piece and a lower bag piece.

Further, one or more sensor connectors are further disposed on the bottom surface of the closed bag body; preferably, two sensor connectors are disposed on the bottom surface of the closed bag body, one of which is for accessing the PH sensor probe The sensor connector and the other sensor connector for accessing the dissolved oxygen sensor probe.

Further, the contact sensor connector includes a welding port, a buckle and a valve block, the welding port is welded to the surface of the closed bag body, and the buckle is engaged with the welding port, the valve block The partition is disposed at a contact portion of the welding port and the closed bag body, wherein the buckle is used for fixing the sensor probe in the welding port, and the sensor probe is directly in contact with the culture liquid in the closed bag body through the block partition.

Further, the fixing mechanism is a fiberglass rod enclosed in the extended side seal or a fixing hole formed on the extended side seal.

Further, the separation device is a medical grade plastic separator.

Further, the biological cell culture shaker is composed of a shaker control box, a tray, and a shaker seat that can be rocked up and down or shaken left and right, the shaker seat is fixed on the top of the shaker control box, and the tray is set at The top of the shaker seat is respectively fixed with a locking device on both sides of the bottom wall of the tray.

Further, the closed bag body, the extended side seal and the aseptic quick joint are all made of medical grade plastic material.

Further, each of the sheet carriers comprises a plurality of sheet-like fiber blades which are arc-connected and made of polyester fiber and/or polypropylene material, and the plurality of sheets which are connected in an arc shape The fiber-like blades are flared outwardly.

Further, the sheet carrier has a loading of 1 g/L to 100 g/L.

Compared with the prior art, the advantages of the present invention are as follows: (1) by filling a sheet-shaped carrier in a closed bag, attaching the cells to the sheet-shaped carrier, and introducing the cell culture solution into the closed bag to make the tablet The carrier is suspended together with the cells adherent on the sheet carrier, and both the cell adherent culture and the suspension culture are used to obtain higher cell culture density, and the expanded culture of the adherent cells is also facilitated; (2) The ventilation method combining bottom ventilation and surface gas phase combination increases the traveling path of oxygen in the liquid and increases the dissolved oxygen amount; (3) the sheet carrier can act like a baffle and can pass The bubbles generated by the gas into the liquid are cut off, effectively preventing the bubbles from directly rupturing in the liquid, the risk of injuring the cells, and reducing the damage to the cells; (4) filling the sheet carrier by filling the closed bag body After the sheet-shaped carrier, the oxygen transfer and mass transfer path of the cell culture liquid is increased, so that the nutrients in the liquid can be better and more fully utilized by the cells; (5) one-time use, no assembly, no disassembly No need to clean, reduce the risk of cross-contamination and the cost of verification, shorten the production or development cycle; (6) can remove metabolites in time, improve the cell culture environment on the sheet carrier, promote cell growth; (7) use closed The culture method of the bag body and the shaker greatly reduces the shear damage caused to the cells; (8) The closed bag itself and its connecting parts are made of medical grade materials, and are sterile and microbial-free. The relative content of endotoxin and other related components is relatively low, which makes the cell storage time relatively long, greatly reducing the impact on subsequent production processes.

DRAWINGS

Figure 1 is a top plan view of a bag body according to a first embodiment of the present invention;

2 is a schematic bottom view of a bag body according to a second embodiment of the present invention;

3 is a perspective view of a bag body according to a first embodiment of the present invention;

4 is a perspective view of a bag body and a shaker according to a first embodiment of the present invention;

Figure 5 is a top plan view of a second embodiment of the present invention;

Figure 6 is a schematic bottom view of a second embodiment of the present invention;

Figure 7 is a perspective view of a bag body according to a second embodiment of the present invention;

Figure 8 is a perspective view of a bag body and a shaker according to a second embodiment of the present invention;

Figure 9 is a schematic view showing the connection pipe of the top surface of the bag body in the state of use according to the first embodiment of the present invention;

Figure 10 is a schematic view showing the connecting line of the bottom surface of the bag body in the state of use according to the first embodiment of the present invention;

Figure 11 is a cross-sectional view showing a sensor connector in accordance with a preferred embodiment of the present invention;

Figure 12 is a schematic view showing the structure of a sheet carrier in the present invention;

In the figure: 1, closed bag body; 2, sheet carrier; 3, extended edge seal; 4, fiberglass rod; 5, fixed hole; 6, upper air inlet; 7, air outlet; 8, sampling port; 9, liquid inlet; 10, inlet and outlet; 11, lower air inlet; 12, sterile quick connector; 13, separation device; 14, sensor connector; 141, sensor connector for accessing PH sensor probe 142, sensor connector for accessing the dissolved oxygen sensor probe; 1401, welding port; 1402, buckle; 1403, valve block; 15, shaker; 151, shaker control box; 152, shaker seat; , tray; 154, locking device; 16, cell culture solution; 17, cable tie; 18, medical silicone tube; 19, y-clip; 20, air filter; 21, Luer connector sampling head; Shrink tube; 23, blind block.

detailed description

In order to make the technical means, the creative features, the achievement of the object and the effect of the present invention easy to understand, the present invention will be further explained in conjunction with the accompanying drawings and specific embodiments.

As shown in FIG. 1 to FIG. 4 , a fluidized bed cell culture disposable membrane device filled with a sheet carrier comprises a closed bag body 1 filled with a plurality of pieces inside the closed bag body 1 . The carrier 2 is provided with an extended side seal 3 on both sides of the outer side of the closed bag body 1, and a fixing mechanism is arranged on each of the extended side seals 3, and the closed bag body 1 is extended by the side edges. The fixing mechanism provided is fixed in the biological cell culture shaker 15, and an upper air inlet 6, an air outlet 7, a sampling port 8, a liquid inlet 9 and a top are arranged on the top surface of the closed bag body 1. The liquid inlet port 10 is provided with a lower air inlet 11 on the bottom surface of the closed bag body 1, and the upper air inlet port 6, the air outlet port 7, the sampling port 8, the liquid inlet port 9, the liquid outlet port 10 and the lower air inlet. The mouth 11 is connected with a sterile quick joint 12, and a partitioning device 13 is further disposed in the liquid outlet 10; the closed bag body 1 encloses the sheet carrier 2 in a closed state by a partitioning device 13 disposed in the liquid outlet 10 In the bag body 1, the separation of the digested cells and the sheet carrier 2 is achieved, that is, the cells are detached from the sheet carrier 2 during digestion, and the cells are discharged. When the mouth 10 is output, the sheet carrier 2 is trapped in the closed bag 1 through the partitioning means 13 in the liquid outlet 10, and the cells are discharged from the liquid outlet 10 together with the cell culture liquid 16 to a larger organism. In the cell culture tank, industrialized amplification production of adherent cells is realized.

The upper air inlet 6 and the lower air inlet 11 are for satisfying the gas (such as oxygen) required for adding cells to the bag body 1 during cell culture, and the gas outlet 7 is for satisfying the cell culture process. The bag body 1 is discharged by a gas (such as carbon dioxide) discharged from the cells, and the liquid inlet port 9 is for satisfying the nutrient solution or other liquid required to add cells to the bag body 1 during the cell culture process, and the liquid outlet port 10 is for The digested adherent cells are discharged into the larger bioreactor with the cell culture fluid 16 to achieve industrialized scale-up production of the adherent cells.

Among them, the aseptic quick connector 12 is a conventional medical grade aseptic quick connector which can be obtained by outsourcing, and the aseptic quick connector 12 is directly welded to the closed bag body 1.

Wherein, the partitioning device 13 is a medical grade plastic separating net, and the mesh size of the dividing net is such that the cells can be allowed to pass, and the sheet carrier 2 is not allowed to pass.

Among them, the closed bag body 1 is formed by welding two upper and lower pocket pieces.

In order to monitor the relevant indicators of cell culture in the closed bag, one or more sensor connectors 14 may be provided on the bottom surface of the closed bag body 1 according to the actual cell culture environment requirements, or the sensor connector 14 may not be provided, for example, When the disposable membrane device provided by the invention is placed in a constant carbon dioxide incubator for small-scale cell culture, the sensor connector 14 can be omitted.

When it is required to provide the sensor connector 14 on the bottom surface of the closed bag body 1, it is preferable to provide two sensor connectors as shown in FIG. 2 or FIG. 6, one of which is a sensor connector for accessing the pH sensor probe. 141, the other is a sensor connector 142 for accessing the oxygen sensor probe.

In the present invention, the sensor connector 14 is a contact sensor connector. As shown in FIG. 10, the contact sensor connector includes a soldering port 1401, a buckle 1402, and a vane partition 1403. The soldering port 1401 is directly soldered to the surface of the closed bag body 1. The corresponding sensor probe is fixedly disposed in the buckle 1402, and one end extends into the welding port 1401 and passes through the valve partition 1403 to directly contact the cell culture liquid in the closed bag body 1 and the other end passes through the buckle 1402. It is engaged with the welding port 1401.

As shown in FIG. 1 to FIG. 4, the fixing mechanism may be a fiberglass rod 4 enclosed in the extended side seal 3. As shown in FIG. 5 to FIG. 7, the fixing mechanism may also be a fixing hole formed on the extended side seal 3. 5.

As shown in FIG. 4 or FIG. 8, the biological cell culture shaker 15 is composed of a shaker control box 151, a tray 153, and a shaker seat 152 which can be rocked up and down or left and right, and the shaker seat 152 is fixed to the shaker control. At the top of the box 151, the tray 153 is disposed at the top of the rocker seat 152, and a locking device 154 is respectively fixed on both sides of the bottom wall of the tray 151. As shown in FIG. 4 and FIG. 8, the locking device 154 is according to actual needs. It can be designed as a locking clip or a locking screw or any other structure having a locking and fixing function; when the cell is cultured, the closed bag body 1 is fixed by the locking device 154 provided on both sides of the inner bottom wall of the tray 153. On the biological cell culture shaker 15, then the shaker seat 152 is controlled to swing up and down or left and right by the shaker control box 151 to drive the tray 153 to shake with the closed bag 1 fixed on the tray 153, so that the cell culture solution 13 is The sheet carrier 2 is suspended in the closed bag body 1 to sufficiently contact the cell culture solution 13 with the cells attached to the sheet carrier 2 and to reduce the accumulation of metabolites (hazardous substances) produced by the cells on the sheet carrier 2. , thereby improving the sheet carrier 2 Amplifying cell culture environment, promote the growth of cells, adherent cells so obtained suspension culture cultured similar mechanism, while maintaining excellent effects related adherent cells, adherent cells facilitates industry.

Among them, the closed bag body 1, the extended side seal 3 and the aseptic quick connector 12 are all made of medical grade plastic materials, such as non-toxic plastic materials such as PE and PP.

Wherein, as shown in FIG. 12, each of the sheet-like carriers 2 comprises a plurality of sheet-like fiber blades 2a which are arc-connected and made of polyester fiber and/or polypropylene material, and some of which are curved. The connected sheet-like fiber blades 2a are arranged to be flared outward.

The loading of the sheet-like carrier 2 in the closed bag body 1 can be set according to the capacity requirement of the closed bag body 1, and the loading range thereof is specifically 1 g/L-100 g/L.

In practical applications, according to actual design requirements, a plurality of liquid inlets 9 and a plurality of liquid outlets 10 may be disposed on the top and bottom surfaces of the closed bag body 1, as shown in FIG.

The fluidized bed cell culture disposable membrane device for filling the sheet-shaped carrier provided by the present invention can be fixed on the biological cell culture shaker 15 by a fixing mechanism provided on the side extension edge seal 3 in practical application, and then The upper air inlet 6, the air outlet 7, the sampling port 8, the liquid inlet 9, the liquid outlet 10 and the lower air inlet 11 of the closed bag body 1 are passed through the aseptic quick joint 12 through the pipeline and the external liquid respectively. , gas or recovery liquid, gas device connection, the sensor connector 14 is plugged into the corresponding sensor probe, and in the process of culturing the cell, at any time, as needed, add external liquid, change the culture solution and provide appropriate cell culture gas.

Specifically, the upper air inlet 6 can be quickly connected to the external air intake pipeline through the aseptic quick connector 12 as shown in FIG. 9, and the external air intake pipeline can sequentially include a medical silicone tube 18, an air filter 20, and then connect the external air. Supply device (not shown);

The air outlet 7 can be quickly connected to the external exhaust line via the aseptic quick connector 12 as shown in FIG. 9. The external exhaust line can sequentially include a medical silicone tube 18, an air filter 20, and then connected to the outside atmosphere or gas collection. Device (not shown);

The liquid inlet port 9 can be quickly butted to the external liquid inlet pipe through the aseptic quick connector 12 as shown in FIG. 9. The external liquid inlet pipe can sequentially include a medical silicone tube 18, a Luer connector sampling head 21, and then an external liquid. Supply device (not shown);

The liquid outlet 10 can be quickly butted to the external liquid outlet pipe through the aseptic quick connector 12 as shown in FIG. 9, and the external liquid inlet pipe can include the medical heat shrinkable tube 22, the blind plug 23, and then the external liquid collecting device. (not shown in the figure);

The lower air inlet 11 can be quickly butted to the external air intake pipe via the aseptic quick connector 12 as shown in FIG. 10, and the external air intake pipe can sequentially include a medical silicone tube 18, an air filter 20, and then connected to an external gas supply device ( Not shown in the figure).

As shown in Fig. 9 and Fig. 10, the medical silicone tube 18 and the medical heat-shrinkable tube 22 on each of the tubes are provided with a Y-shaped clip 19 for controlling the ingress and egress of liquid and gas, and the respective components of the tube are sealedly connected by a cable tie 17.

In the following, the above description is only the embodiment of the present invention, and thus does not limit the scope of the patent of the present invention, and the equivalent structure or equivalent flow transformation made by using the description of the present invention and the contents of the drawings, or directly or indirectly applied to other The related technical fields are all included in the scope of patent protection of the present invention.

Claims

A fluidized bed cell culture disposable membrane device filled with a sheet carrier, comprising a closed bag body (1), characterized in that: the closed bag body (1) is filled with a plurality of sheet carriers (2) An extended side seal (3) is disposed on both sides of the outside of the closed bag body (1), and a fixing mechanism is disposed on each of the extended side seals (3), the closed bag The body (1) is fixed in the biological cell culture shaker (15) by a fixing mechanism provided on the side extension edge seals (3), and an upper air inlet is arranged on the top surface of the closed bag body (1) ( 6), an air outlet (7), a sampling port (8), and at least one liquid inlet (9) and a liquid inlet (10), and a lower air inlet is provided on a bottom surface of the closed bag body (1) 11), connected to the upper air inlet (6), the air outlet (7), the sampling port (8), the liquid inlet (9), the liquid outlet (10) and the lower air inlet (11) There is a sterile quick connector (12), and a partitioning device (13) is further disposed in each of the liquid outlets (10), and the closed bag body (1) is disposed in the liquid outlet (10) The separating device (13) traps the sheet-like carrier (2) in the closed bag (1) to effect separation of the cells from the sheet carrier (2).
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, wherein the closed bag body (1) is composed of an upper bag piece and a lower bag piece.
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, characterized in that one or more sensor joints (14) are further provided on the bottom surface of the closed bag body (1).
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, wherein the sensor connector (14) is a contact sensor connector, and the contact sensor connector comprises a solder joint (1401) ), a buckle (1402) and a flap partition (1403), the welded joint (1401) is welded to the surface of the closed bag body (1), the buckle (1402) and the welded joint (1401) The snap-fit connection, the vane partition (1403) is disposed at a contact portion of the weld port (1401) and the closed bag body (1).
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, wherein the fixing mechanism (4) is a fiberglass rod encapsulated in the extended side seal (3) ( 4) or a fixing hole (5) opened on the extended side seal (3).
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, wherein the partitioning device (13) is a medical grade plastic separator.
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, wherein: the biological cell culture shaker (15) comprises a shaker control box (151), a tray (153), and The rocker seat (152) can be rocked up and down or rocked up and down, the rocker seat (152) is fixed on the top of the shaker control box (151), and the tray (153) is disposed on the shaker seat At the top of (152), a locking device (154) is respectively fixed on both sides of the inner wall of the tray (151).
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, wherein the closed bag body (1), the extended side seal (3) and the aseptic quick joint (12) They are all made of medical grade plastic materials.
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, wherein each of the sheet carriers (2) comprises a plurality of arc-shaped connections and is assembled by A sheet-like fiber blade (2a) made of an ester fiber and/or a polypropylene material, the plurality of blade-shaped fiber blades (2a) connected in an arc shape are outwardly flared.
The fluidized bed cell culture disposable membrane device for filling a sheet-shaped carrier according to claim 1, wherein the sheet carrier (2) is loaded in an amount of from 1 g/L to 100 g/L.