CN210237641U

CN210237641U - Basket type bioreactor

Info

Publication number: CN210237641U
Application number: CN201822085800.4U
Authority: CN
Inventors: Xianshao Xiang; 项宪绍
Original assignee: Shanghai Naili Fluid Equipment Co ltd
Current assignee: Shanghai Naili Fluid Equipment Co ltd
Priority date: 2018-12-12
Filing date: 2018-12-12
Publication date: 2020-04-03
Anticipated expiration: 2028-12-12

Abstract

The utility model discloses a basket bioreactor in the field of cell culture of biotechnology, including a jar body, place in jar internal basket, install in the agitator and the tank bottoms valve of jar body bottom. The tank body comprises an upper end enclosure, a cylinder body, a lower end enclosure, a jacket and a tank body heat-insulating layer. The basket consists of a defoaming cover at the upper part, a guide cylinder at the middle part and a conical seal head at the lower part. The defoaming hood consists of a top circular sieve plate, a middle cylindrical sieve plate, a bottom outer flange, a circular sieve plate and an inner flange. And a vent pipe, a material supplementing pipe, an alkali supplementing pipe and a pipette are vertically arranged on the circular sieve plate at the top of the defoaming cover. The stirrer is positioned in the center of the lower end socket of the tank body and is coaxial with the guide cylinder of the basket, and the stirring paddle of the stirrer is divided into an upper layer and a lower layer. The stirrer is driven by a frequency converter to regulate the speed, and a stirring paddle of the stirrer is of a turbine type or a propulsion type. The technical advantages are as follows: the culture medium has high dissolved oxygen, small shearing force and mechanical defoaming.

Description

Basket type bioreactor

Technical Field

The utility model relates to a basket bioreactor, belonging to the field of cell culture of biotechnology.

Background

With the continuous development of biotechnology, cell culture technology is not only used for researching life sciences, but also becomes a research means of subjects such as bioengineering, genetic engineering, tissue engineering and the like; in particular, in the field of medicine, cell culture has been developed to have a more specific effect and value, such as vaccines produced by animal cell culture, monoclonal antibodies, various biologically active proteins for therapeutic use, and the like. In summary, cell culture plays a critical role in the development of the entire biotechnology industry.

① because cell culture medium contains abundant protein, a great amount of foam is generated in the process of using the fixed bed bioreactors for cell culture, but no defoaming measure is provided, so that the foam is easy to overflow into a tail gas filter to cause contamination and culture failure, ② because the fixed bed bioreactors adopt single-layer stirring paddles, the gas-liquid mixing and the culture medium flowability are not good.

Disclosure of Invention

The main object of the present invention is to provide a basket bioreactor to overcome the disadvantages of the prior art.

One technical scheme for achieving the above purpose is as follows: a basket type bioreactor comprises a tank body, a basket placed in the tank body, a stirrer installed at the bottom of the tank body and a tank bottom valve.

The tank body comprises an upper end enclosure, a cylinder body, a lower end enclosure, a jacket and a tank body heat-insulating layer.

Furthermore, a CIP port, an exhaust port, a feed supplement port and a standby port are arranged on the upper end enclosure.

Furthermore, the upper part of the cylinder body is provided with a material supplementing hole, an alkali supplementing hole, a liquid moving hole, a deep layer air inlet, a surface layer air inlet, a rupture disk mounting hole, a sight lamp and an upper sight glass; the lower part of the cylinder body is provided with a sampling valve interface, a thermometer interface, a DO electrode interface, a PH electrode interface and a lower sight glass.

The basket consists of a defoaming cover at the top, a guide cylinder in the middle and a conical seal head at the bottom; the space formed by the basket and the inner wall of the tank body is a chamber for placing an adherent cell culture carrier.

Furthermore, the defoaming cover comprises a top circular sieve plate, a middle cylindrical sieve plate, and a bottom outer flange, a circular sieve plate and an inner flange which are fixed by welding or screws.

Furthermore, the diameter of the circular sieve plate at the top of the defoaming cover is 0.5-0.9 times of the inner diameter of the cylinder body of the tank body, the shape of an opening of the circular sieve plate at the top of the defoaming cover is circular or regular polygon, the aperture is 0.1-6 mm, and the opening rate is 10% -80%; and a vent pipe, a material supplementing pipe, an alkali supplementing pipe and a pipette are vertically arranged on the circular sieve plate at the top of the defoaming cover.

Furthermore, the top of the vent pipe, the material supplementing pipe, the alkali supplementing pipe and the pipette is 2-10 cm higher than the circular sieve plate at the top of the defoaming cover, the length of the bottom of the vent pipe and the bottom of the material supplementing pipe extending into the guide cylinder is 0.5-0.8 time of the length of the guide cylinder, the length of the bottom of the alkali supplementing pipe extending into the guide cylinder is 0.1-0.5 time of the length of the guide cylinder, and the distance between the bottom of the pipette and the circular sieve plate at the bottom of the defoaming cover is 1-5 cm; and the bottom of the vent pipe is provided with a microporous titanium rod filter.

Furthermore, the outer diameter of the cylindrical screen in the middle of the defoaming hood is the same as that of the circular screen plate at the top of the defoaming hood, and the aperture of the screen is 0.1-6 mm.

Furthermore, the outer ring of the outer flange at the bottom of the defoaming cover forms a seal with the inner wall of the tank body cylinder body through a gasket; the sieve plate at the bottom of the defoaming cover is circular or regular polygonal, the aperture is 0.1-6 mm, and the aperture ratio is 10-80%.

Furthermore, the guide shell in the middle is a hollow cylinder with the same upper and lower outer diameters; the upper part of the guide shell is inserted into the inner ring of the inner flange at the bottom of the defoaming cover and is connected in a detachable mode.

Furthermore, the vertex angle of the conical seal head is 90-170 degrees, the outer diameter of the small end of the conical seal head is the same as the outer diameter of the lower end of the guide cylinder and is fixed with the lower end of the guide cylinder in a welding mode, and the large end of the conical seal head forms a sealing chamber with the inner wall of the lower seal head of the tank body through a gasket; the surface of the conical end socket is provided with a through hole with the diameter of 1-5 mm, and the aperture ratio is 10% -80%.

The stirrer is positioned in the center of the lower end socket of the tank body and is coaxial with the guide cylinder of the basket; the stirring paddle of the stirrer is divided into an upper layer and a lower layer, the upper stirring paddle wholly or partially extends into the guide cylinder, and the lower stirring paddle is positioned in a cavity formed by the conical seal head at the lower part of the basket and the inner wall of the lower seal head of the tank body.

Furthermore, the stirrer is driven by a frequency converter to regulate the speed, and an upper stirring paddle and a lower stirring paddle of the stirrer are of a turbine type or a propulsion type.

Compared with the prior art, the utility model has the advantages as follows:

the utility model discloses a gas-liquid exchange goes on in the draft tube to gaseous titanium stick filter dispersion has not only improved the solubility of oxygen in the culture medium, has avoided the bubble to get into the carrier moreover and has harmd the cell.

The utility model discloses a double-deck lower magnetic stirrers compares with the lower magnetic stirrers of individual layer, requires under the same condition at the air feed with dissolved oxygen, can reduce the rotational speed of stirring to reduce the damage of shearing force to the cell, can improve the dissolved oxygen and the mobility of culture medium moreover, be favorable to the growth of adherence cell.

The utility model discloses a basket has the fire fighting equipment, can pass through mechanical mode breakage with the foam that produces among the cell culture process, has solved the culture in-process, and the foam is excessive, causes the contamination, leads to the problem of cell culture failure.

Drawings

FIG. 1 is a schematic structural diagram of a basket bioreactor of the present invention.

Detailed Description

In order to clearly illustrate the technical features of the present invention, the following detailed description of the present invention is made with reference to fig. 1.

Referring to fig. 1, the present invention provides a basket bioreactor, which comprises a tank 1, a basket 2 disposed in the tank 1, a stirrer 3 mounted at the bottom of the tank 1, and a tank bottom valve 4.

The tank body 1 comprises an upper end enclosure 11, a cylinder body 12, a lower end enclosure 13, a jacket 14 and a tank body heat-insulating layer 15.

The upper end enclosure 11 is provided with a CIP port 111, an exhaust port 112, a feeding port 113 and a spare port 114.

The upper part of the cylinder body 12 is provided with a feed supplement port 1201, an alkali supplement port 1202, a liquid transfer port 1203, a deep layer air inlet 1204, a surface layer air inlet 1205, a rupture disc mounting port 1206, a sight lamp 1207 and an upper view mirror 1208; the lower part of the cylinder body 12 is provided with a sampling valve interface 1210, a thermometer interface 1211, a DO electrode interface 1212, a PH electrode interface 1213 and a lower view mirror 1209.

The basket 2 consists of a defoaming cover 21 at the upper part, a guide cylinder 22 at the middle part and a conical seal head 23 at the lower part; the space formed by the basket 2 and the inner wall of the tank body 1 is a chamber 5 for placing an adherent cell culture carrier 7.

The defoaming cover 21 is composed of a top circular sieve plate 211, a middle cylindrical sieve 212, a bottom outer flange 213, a circular sieve plate 214 and an inner flange 215, which are fixed by welding or screws.

The diameter of the circular sieve plate 211 at the top of the defoaming cover 21 is 0.5-0.9 times of the inner diameter of the cylinder 12 of the tank body 1, and is 0.8 times in the embodiment; the opening shape of the circular sieve plate 211 at the top of the defoaming cover 21 is circular or regular polygon, the aperture is 0.1-6 mm, the aperture ratio is 10% -80%, in the embodiment, the opening is circular, the aperture is 2mm, and the aperture ratio is 35%; and a vent pipe 217, a material supplementing pipe 216, an alkali supplementing pipe 218 and a pipette 210 are vertically arranged on the circular sieve plate 211 at the top of the defoaming hood 21.

The distance that the top of the vent pipe 217, the feed supplement pipe 216, the alkali supplement pipe 218 and the pipette 210 is higher than the top of the circular sieve plate 211 of the bubble eliminating cap 21 is 2-10 cm, which is 3 cm in the embodiment; the length of the vent pipe 217 and the feed supplement pipe 216 extending into the guide shell 22 is 0.5-0.8 times of the length of the guide shell 22, and in the embodiment, the length is 0.6 times; the length of the alkali supplementing pipe 218 extending into the guide shell 22 is 0.1-0.5 times of the length of the guide shell 22, and is 0.2 times in the embodiment; the distance between the bottom of the pipette 210 and the circular sieve plate 214 at the bottom of the defoaming hood 21 is 1-5 cm, and in the embodiment, 2 cm is taken; and a microporous titanium rod filter 219 is installed at the bottom of the breather pipe 217.

The outer diameter of the cylindrical screen 212 in the middle of the defoaming cap 21 is the same as that of the circular sieve plate 211 at the top of the defoaming cap 21, and the aperture of the screen is 0.1-6 mm, which is 2mm in the embodiment.

The outer ring of the outer flange 213 at the bottom of the defoaming cover 21 forms a seal with the inner wall of the cylinder 12 of the tank body 1 through a gasket; the opening shape of the circular sieve plate 214 at the bottom of the defoaming cover 21 is circular or regular polygon, the aperture is 0.1-6 mm, the opening rate is 10% -80%, in the embodiment, the opening shape is circular, the aperture is 3.5 mm, and the opening rate is 31%.

The guide shell 22 in the middle is a hollow cylinder with the same upper and lower outer diameters; the upper part of the guide shell 22 is inserted into the inner ring of the inner flange 215 at the bottom of the defoaming cover 21 and is connected in a detachable mode.

The vertex angle of the conical seal head 23 is 90-170 degrees, in the embodiment, the vertex angle is 150 degrees, the outer diameter of the small end of the conical seal head 23 is the same as the outer diameter of the lower end of the guide shell 22 and is fixed with the lower end of the guide shell 22 in a welding mode, and the large end of the conical seal head 23 and the inner wall of the lower seal head 13 of the tank body 1 form a sealing chamber 6 through a gasket; the surface of the conical end socket 23 is provided with a through hole with the diameter of 1-5 mm, the aperture ratio is 10% -80%, in the embodiment, the diameter of the through hole is 3.5 mm, and the aperture ratio is 35%.

The stirrer 3 is positioned in the center of the lower end enclosure 13 of the tank body 1 and is coaxial with the guide cylinder 22 of the basket 2; the stirring paddles of the stirrer 3 are divided into an upper layer and a lower layer, the whole or part of the upper stirring paddle 31 extends into the guide cylinder 22, and the lower stirring paddle 32 is positioned in a cavity 6 formed by the conical seal head 23 at the lower part of the basket 2 and the inner wall of the lower seal head 13 of the tank body 1.

The stirrer 3 is driven by a frequency converter to regulate the speed, and an upper stirring paddle 31 and a lower stirring paddle 32 of the stirrer 3 are of a turbine type or a propulsion type. The stirrer 3 drives the culture medium to flow down the guide cylinder 22.

The operation process is as follows:

before cell culture, the processed carrier 7 is loaded into a chamber 5 formed by a cleaned basket 2 and a tank body 1, corrected instruments are installed, PBS is added into the tank body 1, after pressure maintaining is qualified, the PBS is discharged through a tank bottom valve 4 after sterilization and cooling, and then a sterile culture medium and cells enter the tank body 1 through a feeding port 1201 on a cylinder body 12, a silica gel hose (not marked in the figure) and a feeding pipe 216 on a defoaming cover 21.

During the cell culture process, one path of deep intake air is sterilized by an air filter and enters the draft tube 22 through a deep intake 1204, a silica gel hose (not shown), the vent pipe 217 and the microporous titanium rod filter 219; at the moment, the gas and the culture medium are mixed by the upper layer stirring paddle 31 of the stirrer 3, the culture medium dissolved with the gas moves downwards by the lower stirring paddle 32 of the stirrer 3, the culture medium contacts the lower end socket 13 of the tank body 1 and flows upwards, passes through the conical end socket 23 with holes, enters the carrier 7 for cell culture, flows through the bottom circular sieve plate 214 of the defoaming cover 21 and then returns to the guide shell 22; the medium is continuously circulated by the drive of the stirrer 3. The amount of ventilation is controlled by the mass flow meter and the DO electrode. Undissolved bubbles move upwards in the guide shell 22, generated foam enters the defoaming cap 21, and along with the continuous increase of the foam, the foam is crushed by the gas carried by the foam through the top circular sieve plate 211 and the middle cylindrical sieve 212, so that the physical defoaming effect is achieved. The other path of surface air intake passes through an air filter for degerming, enters the tank body 1 through a surface air inlet 1205, and the flow rate of the surface air intake is controlled by a mass flow meter. The gas in the tank body is exhausted through an exhaust port 112 of the upper end enclosure 11, a condenser (not shown in the figure), a tail gas filter (not shown in the figure) and a pneumatic proportional control valve (not shown in the figure); the pneumatic proportional control valve (not shown) is used for controlling the pressure in the tank 1 and ensuring that the pressure in the tank 1 is always in positive pressure. The pH of the culture medium is controlled by a pH electrode of the pH electrode interface 1213, and when the pH value is lower than a set value, the sterile alkali liquor enters the guide cylinder 22 through the alkali supplement port 1202 on the cylinder 12, a silica gel hose (not marked in the figure) and the alkali supplement pipe 218 on the defoaming cap 21, is mixed with the culture medium, and then enters the carrier 7 in the basket 2. The temperature of the medium in the tank 1 is controlled by a temperature probe attached to the thermometer interface 1211 and a jacket 14.

When perfusion is needed in the cell culture process, the sterile culture medium enters the bottom of the guide cylinder 22 through the feeding port 1201 on the cylinder 12, the silica gel hose (not marked in the figure) and the feeding pipe 216 on the defoaming cover 21, is mixed by the stirrer 3 and then enters the carrier 7 in the basket 2; at this time, the culture medium in the tank 1 is discharged out of the tank 1 through the pipette 210 on the pipette tip 21, a silicone tube (not shown), and a pipetting port 1203 on the cylinder 12. By continuously supplementing fresh culture medium and discharging culture medium containing harmful products, cells are stably positioned in a better nutrient environment, the concentration accumulation of harmful metabolic waste is lower, and higher cell density is obtained.

During the cell culture, the viewing lamp 1207 on the cylinder 12 can be turned on, and the growth of the cells in the tank can be observed through the upper view mirror 1208 and the lower view mirror 1209.

In the whole operation process of the tank body, if the pressure in the tank body 1 exceeds the working pressure of the container, the rupture disk arranged on the rupture disk mounting port 1206 is broken, the pressure in the tank is released, and the operation safety is ensured.

Example 1

A50L basket bioreactor is adopted to culture Vero cells, and the specific implementation process is as follows:

preparation of a reactor: cleaning a reactor, loading into a calibrated PH electrode and a DO electrode, adding a treated carrier (30 g/L), supplementing PBS to 50L, performing a tank body pressure maintaining test (0.2 MPa) for 30min, and sterilizing at 121 ℃ for 45min after the reactor is qualified.

Pre-culturing: and (3) after the reactor is cooled, discharging PBS, adding 50L DMEM solution containing 8% bovine serum, culturing for 3-4 days at the rotating speed of 80 r/min, and observing DO and PH values.

Cell culture: after 3-4 days of pre-culture, after the sterility in the tank is qualified, inoculating the digested Vero cell suspension to ensure that the final cell density in the tank is 2.0-3.0 multiplied by 105 cells/ml, the set rotating speed is 60 r/min, the set pH value is 7.2, the set DO is 50%, and the temperature in the tank is 37 ℃. After 8 hours, perfusion culture was performed. Sampling in the morning and afternoon every day after perfusion, detecting the sugar content, determining the perfusion volume according to the sugar content of the culture solution in the tank, and keeping the sugar content in the culture medium at 2-3 mmol/L.

Cell digestion: after culturing for 120 hours, the cells were subjected to digestion treatment. The medium was counted and the average cell density in the canister was 5.6X 106 cells/ml by counting 3 samples.

The present invention is not limited to the above embodiments, and variations, modifications, additions and substitutions which can be made by a person skilled in the art within the scope of the invention shall fall within the protection scope of the invention.

Claims

1. A basket type bioreactor comprises a tank body, a basket placed in the tank body, a stirrer installed at the bottom of the tank body and a tank bottom valve; the method is characterized in that: the basket consists of a defoaming cover at the top, a guide cylinder in the middle and a conical seal head at the bottom; the space formed by the basket and the inner wall of the tank body is a chamber for placing an adherent cell culture carrier.

2. The basket bioreactor of claim 1, wherein:

the tank body comprises an upper end enclosure, a cylinder body, a lower end enclosure, a jacket and a tank body heat-insulating layer, wherein the upper end enclosure is provided with a CIP port, an exhaust port, a replenishing port and a standby port, the upper part of the cylinder body is provided with a replenishing port, an alkali replenishing port, a liquid moving port, a deep air inlet, a surface layer air inlet, a rupture disk mounting port, a sight lamp and an upper sight glass, and the lower part of the cylinder body is provided with a sampling valve port, a thermometer port, a DO electrode port, a PH electrode port and a lower sight glass.

3. The basket bioreactor of claim 1, wherein:

the defoaming cover consists of a top circular sieve plate, a middle cylindrical sieve plate, a bottom outer flange, a circular sieve plate and an inner flange which are fixed by welding or screws, the diameter of the top circular sieve plate of the defoaming cover is 0.5-0.9 times of the inner diameter of the cylinder body of the tank body, the opening of the top circular sieve plate of the defoaming cover is circular or regular polygon, the aperture is 0.1-6 mm, the opening rate is 10% -80%, a vent pipe, a material supplementing pipe, an alkali supplementing pipe and a pipette are vertically arranged on the top circular sieve plate of the defoaming cover, the outer diameter of the middle cylindrical sieve plate of the defoaming cover is the same as the outer diameter of the top circular sieve plate of the defoaming cover, the aperture of the sieve plate is 0.1-6 mm, the outer ring of the outer flange at the bottom of the defoaming cover forms a seal with the inner wall of the cylinder body of the tank body through a gasket, and the opening of the sieve plate at the bottom, the aperture is 0.1-6 mm, and the aperture ratio is 10-80%.

4. The basket bioreactor of claim 3, wherein: the top of the vent pipe, the top of the feed supplement pipe, the top of the alkali supplement pipe and the top of the pipette are 2-10 cm higher than the circular sieve plate at the top of the defoaming cover, the length of the bottom of the vent pipe and the bottom of the feed supplement pipe extending into the guide cylinder is 0.5-0.8 time of the length of the guide cylinder, the length of the bottom of the alkali supplement pipe extending into the guide cylinder is 0.1-0.5 time of the length of the guide cylinder, and the distance between the bottom of the pipette and the circular sieve plate at the bottom of the defoaming cover is 1-5 cm; and the bottom of the vent pipe is provided with a microporous titanium rod filter.

5. A basket bioreactor as claimed in claim 1 or 3, wherein: the middle guide shell is a hollow cylinder with the same upper and lower outer diameters; the upper part of the guide shell is inserted into the inner ring of the inner flange at the bottom of the defoaming cover and is connected in a detachable mode.

6. The basket bioreactor of claim 1, wherein:

the apex angle of the conical seal head is 90-170 degrees, the outer diameter of the small end of the conical seal head is the same as the outer diameter of the lower end of the guide cylinder and is fixed with the lower end of the guide cylinder in a welding mode, and the large end of the conical seal head forms a sealing cavity with the inner wall of the lower seal head of the tank body through a gasket; the surface of the conical end socket is provided with a through hole with the diameter of 1-5 mm, and the aperture ratio is 10% -80%.

7. The basket bioreactor of claim 1, wherein:

8. The basket bioreactor of claim 7, wherein: the stirrer is driven by a frequency converter to regulate the speed, and an upper stirring paddle and a lower stirring paddle of the stirrer are of a turbine type or a propulsion type.