CN112708562A

CN112708562A - Fixed bed type bioreactor

Info

Publication number: CN112708562A
Application number: CN202110028398.XA
Authority: CN
Inventors: 张超
Original assignee: Kunming Baite Biotechnology Co ltd
Current assignee: Kunming Baite Biotechnology Co ltd
Priority date: 2021-01-11
Filing date: 2021-01-11
Publication date: 2021-04-27

Abstract

The invention relates to the technical field of bioreactors for culturing adherent cells and suspension cells with low shear force, in particular to a fixed bed type bioreactor. The fixed bed bioreactor comprises a tank body and a carrier column, wherein the upper part of the tank body is hermetically connected with a tank cover, a magnetic stirring base is arranged below the tank body, the bottom of the magnetic stirring base is provided with a drainage hole, a magnetic stirrer for rotationally stirring water is arranged in the magnetic stirring base, the carrier column is detachably arranged above the magnetic stirring base, and a plurality of fibrous carrier sheets are filled in the carrier column; magnetic stirrers stir through the rotation, and the drive culture medium flows in from the drainage hole of bottom, passes the inside carrier piece of carrier post, flows out from the carrier post top at last, gets back to jar in the body, forms the liquid flow direction of a culture medium inner loop. Aiming at the defects of the prior art, the invention solves the problems that part of cells are difficult to grow in a bioreactor and part of suspended cells produce toxicity or have low protein expression in a common STR reactor.

Description

Fixed bed type bioreactor

Technical Field

The invention relates to the technical field of bioreactors for culturing adherent cells and suspension cells with low shear force, in particular to a fixed bed type bioreactor.

Background

In the traditional large-scale cell culture, an STR reactor (STR) is mostly adopted, i.e. cells are attached to microcarriers and fiber carrier sheets or are completely suspended, and are suspended and grown in a bioreactor under the stirring of a bioreactor stirring paddle. However, the shear force caused by this form of agitation can cause damage or death of the cells. Part of adherent cells which are already used for production are difficult to grow on the traditional STR reactor, only the traditional standing culture mode can be adopted, and the requirement of large-scale culture is difficult to adapt. Some of the fully suspended cells can grow, but the protein expression level is low under the action of shear force. Although the prior basket reactor has relatively fixed cells and liquid flows through the cells under the condition of self weight, the problem of larger shearing force is solved, but the flow rate is not easy to control, so that the cells are not uniformly distributed in the whole carrier cavity, and great difficulty is brought to process control.

Disclosure of Invention

The invention provides a fixed bed bioreactor with reliable performance aiming at the defects of the prior art, and solves the problems that part of cells are difficult to grow in the bioreactor and part of suspended cells produce toxicity or have low protein expression in a common STR reactor.

The technical scheme of the invention is realized as follows: this fixed bed formula bioreactor, including jar body and carrier post, jar body upper portion sealing connection has a cover, its characterized in that: a magnetic stirring base is arranged below the tank body, the bottom of the magnetic stirring base is provided with a drainage hole, a magnetic stirrer for rotationally stirring water is arranged in the magnetic stirring base, the carrier column is detachably inserted into a drainage cavity above the magnetic stirring base, and a plurality of fibrous carrier sheets are filled in the carrier column; magnetic stirrers stirs through the rotation, and the drive culture medium flows in the drainage chamber from the drainage hole of bottom, passes the inside carrier piece of carrier post, flows out from the carrier post top at last, gets back to jar body in, forms the liquid flow direction of a culture medium inner loop.

The carrier column comprises a cylindrical shell, a sealing ring and a clamping plate which is connected with the drainage cavity in a clamping manner are arranged below the side wall of the shell, a spoiler, a turbulent flow plate and a top baffle plate are sequentially nested in the shell from bottom to top, the spoiler is a circular thin plate, three turbulent flow grooves for enabling liquid to flow to be divided are uniformly distributed on the circumference of the spoiler, the lower turbulent flow plate is a circular thin plate with honeycomb turbulent flow holes, and fluid can uniformly and horizontally enter the column through the turbulent flow holes to prevent the formation of a central edge effect; the carrier sheet is filled between the turbulence plate and the top baffle.

The cover is provided with a GL45 sampling port for sampling and observing, a feed supplement port for adding culture medium, and a medium-length discharge pipe for controlling the liquid level of the culture medium.

The medium-length discharge pipe is externally connected with a peristaltic pump, and excess culture medium liquid is pumped out outwards under the action of the peristaltic pump or air pressure so as to ensure that the volume of the liquid in the tank is unchanged.

The tank body and the tank cover are connected in a sealing mode through the flange plate and the silica gel gasket.

The rotating speed of the magnetic stirrer is adjustable, and the fixed bed bioreactor accurately controls the linear flow rate of liquid passing through the carrier column and culture process parameters by adjusting the rotating speed of the magnetic stirrer.

The invention solves the defects in the background technology and has the following beneficial effects:

the invention has scientific and reasonable structural design, solves the problem of overlarge shearing force of the STR reactor, forcibly controls the liquid flow rate, solves the problem of uneven adherence of cells in the existing basket reactor, and enables adherence cells which are difficult to be cultured in a suspension way to be cultured in a bioreactor on a large scale. The liquid culture medium is driven to flow in from the drainage hole at the bottom of the magnetic stirring base by the rotation of the magnetic stirrer, passes through the carrier sheet in the carrier column, finally flows out from the top of the carrier column, and returns to the tank body of the reactor, so that a liquid flow direction circulating in the culture medium is formed. Meanwhile, the linear flow velocity of the liquid passing through the carrier column can be accurately controlled by adjusting the rotating speed of the magnetic stirrer, so that the cell suspension is forced to rapidly pass through the carrier pieces when the high flow velocity is realized, and the cells can be uniformly attached to each carrier piece in the carrier column; and after the adherence is finished, the solution is passed through the carrier plate at a low flow rate, so that the fresh culture medium continuously flows through the cells under the condition of extremely low shearing force, and necessary nutrient substances are provided for the growth of the cells. The flow rate required by culture strategies such as circulation or perfusion and the like can be accurately controlled by the peristaltic pump connected with the medium-length discharge pipe. By arranging the spoilers and the turbulent plates on the carrier columns, fluid can uniformly and horizontally enter the columns, so that the central edge effect is prevented from being formed, and cells are further uniformly attached to each carrier sheet.

Drawings

FIG. 1 is a schematic structural view of the present invention;

FIG. 2 is a schematic structural view of the magnetic stirring base of FIG. 1;

FIG. 3 is a schematic cross-sectional view of the magnetic stirring base of FIG. 2;

FIG. 4 is a schematic structural view of the support column of FIG. 1;

FIG. 5 is a schematic structural diagram of the spoiler shown in FIG. 4;

FIG. 6 is a schematic structural view of the turbulator of FIG. 4;

in the figure: 1-tank body, 2-tank cover, 3-carrier column, 4-magnetic stirring base, 5-feeding port, 6-middle-long discharging pipe, 7-GL45 sampling port, 8-magnetic stirrer, 9-culture medium, 10-buckle, 11-drainage hole, 12-drainage cavity, 31-shell, 32-spoiler, 33-spoiler, 34-top baffle, 35-buckle plate, 321-spoiler groove and 331-turbulence hole.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to the embodiment of the fixed bed bioreactor shown in fig. 1 to 6, the fixed bed bioreactor comprises a tank body 1 and a carrier column 3, wherein the upper part of the tank body 1 is hermetically connected with a tank cover 2, a magnetic stirring base 4 is arranged below the inside of the tank body 1, the bottom of the magnetic stirring base 4 is provided with a drainage hole 11, a magnetic stirrer 8 for rotationally stirring water is arranged in the magnetic stirring base, the carrier column 3 is detachably inserted in a drainage cavity 12 above the magnetic stirring base, and a plurality of fibrous carrier sheets are filled in the carrier column 3; the magnetic stirrer 8 drives the culture medium 9 to flow into the drainage cavity 12 from the drainage hole 11 at the bottom through the drainage holes by rotating and stirring, passes through the carrier sheets in the carrier column 3, finally flows out from the top of the carrier column 3 and returns to the tank body, and a liquid flow direction circulating in the culture medium 9 is formed. The rotating speed of the magnetic stirrer 8 is adjustable, and the fixed bed bioreactor accurately controls the linear flow rate of liquid passing through the carrier column 3 and culture process parameters by adjusting the rotating speed of the magnetic stirrer 8. The carrier column 3 comprises a cylindrical shell 31, a sealing ring and a buckle plate 35 connected with the buckle 10 in the drainage cavity 12 are arranged below the side wall of the shell 31, a spoiler 32, a turbulence plate 33 and a top baffle 34 are sequentially nested in the shell 31 from bottom to top, the spoiler 32 is a circular thin plate, three spoiler grooves 321 for enabling liquid to flow and split are uniformly distributed on the circumference of the spoiler, the lower turbulence plate 33 is a circular thin plate provided with honeycomb turbulence holes 331, and the turbulence holes can enable the fluid to uniformly and horizontally enter the column to prevent the formation of a central edge effect; the carrier sheet is filled between the turbulence plate 33 and the top baffle 34. The cover 2 is provided with a GL45 sampling port 7 for sampling and observation, a feed supplement port 5 for adding a culture medium 9, and a medium-length discharge pipe 6 for controlling the liquid level of the culture medium 9. The medium-length discharge pipe 6 is externally connected with a peristaltic pump, and excess liquid is pumped out through the action of the peristaltic pump or air pressure so as to ensure that the volume of the solution in the tank is unchanged. The tank body 1 and the tank cover 2 are hermetically connected through a flange plate and a silica gel gasket.

The working process of the invention is as follows: the carrier sheet is desirably loaded between the turbulence plate 33 and the top baffle 34 to ensure that the carrier sheet is present in a certain density in the column of the carrier, and then the lower turbulence plate 32 is installed and all the components are packaged and formed at once. When the carrier column 3 is used, the end part of the sealing ring is inserted into the magnetic stirring base 4 at the bottom of the tank body 1, and the clamping plate 35 and the upper clamping buckle 10 of the stirring base are clamped in an aligning manner in a rotating manner. And carrying out pressure maintaining test after the installation is finished, and ensuring the sealing of the bioreactor. After high-pressure sterilization, the bioreactor is connected with a bioreactor control cabinet or connected with a corresponding electrode transmitter for cell culture. The magnetic stirrer 8 is powered by a magnetic stirrer motor to rotate. Thereby drive liquid culture medium 9 and flow into in drainage chamber 12 from the bottom drainage hole 11 of magnetic stirring base 4, pass the inside carrier piece of carrier post, flow out from carrier post 3 top at last, get back to the reactor jar body in, flow down again to the bottom of

magnetic stirring base

4, 3 in the reentrant carrier post, form the liquid flow direction of a culture medium 9 inner loop. The cell culture mode is high-density culture, consumed nutrient substances need to be continuously supplemented in the culture process, in order to ensure that the total volume of nutrient solution in a tank body is unchanged, a medium-length discharging pipe 6 is arranged on a tank cover and is connected with a peristaltic pump through a silicone tube, the solution is pumped outwards all the time, and when the liquid level of a culture medium 9 is lower than the pipe orifice, the solution cannot be pumped out, so that the volume of the solution in the tank body is unchanged. The volume of culture medium ensures that the flow rate of liquid through the carrier sheet is constant when the magnetic stirrer is externally driven at a particular rotational speed. The flow rate of liquid through the support column 3 is accurately adjustable while ensuring this volume of medium. The flow rate is increased during the cell adherence process, and the cell suspension is forced to rapidly pass through the carrier plates, so that the cells can be uniformly attached to each carrier plate in the carrier column 3. After the adherence is finished or the suspension cells are positioned, the flow velocity can be adjusted to be low, so that the solution passes through the carrier sheet at a very low flow velocity, and the cells are continuously flowed through by fresh culture medium under the condition of very low shearing force, thereby providing necessary nutrient substances for the growth of the cells. Therefore, different culture modes such as circulation, perfusion and the like can be simply realized by setting the rotating speed of the peristaltic pump connected with the peristaltic pump to be slightly greater than that of the peristaltic pump with the feeding port so as to meet the requirements of different cell growth. The flow rate required by culture strategies such as circulation or perfusion and the like can be accurately controlled by the peristaltic pump connected with the medium-length discharge pipe 6. Meanwhile, the linear flow velocity of the liquid passing through the carrier column can be accurately controlled by adjusting the rotating speed of the magnetic stirrer 8, so that the culture process parameters can be conveniently controlled. According to the invention, the spoiler 32 is arranged, and the three spoiler grooves 321 are uniformly distributed on the circumference of the spoiler, so that the cell suspension can flow into the carrier column 3 from multiple directions from the circumferential edge of the carrier column 3; the honeycomb-shaped turbulence holes 331 of the turbulence plate 33 allow the fluid to uniformly and horizontally enter the column, preventing the formation of the center-edge effect, thereby uniformly attaching the cells to each carrier sheet. The low shear force allows the suspensible cells to grow and express on the carrier sheet with high efficiency. Meanwhile, the fibrous carrier sheet filled in the carrier column can provide a great culture area, so that high-density culture of cells is realized.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. The utility model provides a fixed bed formula bioreactor, includes jar body (1) and carrier column (3), jar body (1) upper portion sealing connection has cover (2), its characterized in that: a magnetic stirring base (4) is arranged below the tank body (1), a drainage hole (11) is formed in the bottom of the magnetic stirring base (4), a magnetic stirrer (8) for rotationally stirring water is arranged in the magnetic stirring base, the carrier column (3) is detachably inserted into a drainage cavity (12) above the magnetic stirring base, and a plurality of fibrous carrier sheets are filled in the carrier column (3); the magnetic stirrer (8) drives the culture medium (9) to flow into the drainage cavity (12) from the drainage hole (11) at the bottom through rotary stirring, pass through the carrier sheets in the carrier column (3), finally flow out from the top of the carrier column (3) and return to the tank body (1) to form a liquid flow direction of the internal circulation of the culture medium (9).

2. The fixed bed bioreactor as set forth in claim 1, wherein: the carrier column (3) comprises a cylindrical shell (31), a sealing ring and a clamping plate (35) connected with a clamp (10) in the drainage cavity (12) are arranged below the side wall of the shell (31), a spoiler (32), a turbulence plate (33) and a top baffle (34) are sequentially nested in the shell (31) from bottom to top, the spoiler (32) is a circular thin plate, three spoiler grooves (321) for shunting liquid flow are uniformly distributed on the circumference of the spoiler (32), the turbulence plate (33) is a circular thin plate provided with honeycomb turbulence holes (331), and the turbulence holes can enable fluid to uniformly and horizontally enter the column to prevent the formation of a central edge effect; the carrier sheet is filled between the turbulence plate (33) and the top baffle (34).

3. The fixed bed bioreactor as claimed in claim 1 or 2, wherein: be equipped with on cover (2) and be used for the sample and observe GL45 thief hatch (7), add feed supplement mouth (5) of culture medium (9), be used for medium-length discharge pipe (6) of culture medium (9) liquid level control.

4. The fixed bed bioreactor as set forth in claim 3, wherein: the medium-length discharge pipe (6) is externally connected with a peristaltic pump, and excess culture medium (9) liquid is pumped out through the action of the peristaltic pump or air pressure so as to ensure that the volume of the liquid in the tank is unchanged.

5. The fixed bed bioreactor as set forth in claim 1, wherein: the tank body (1) and the tank cover (2) are connected in a sealing mode through the flange plate and the silica gel gasket.

6. The fixed bed bioreactor as set forth in claim 1, wherein: the rotating speed of the magnetic stirrer (8) is adjustable, and the fixed bed bioreactor accurately controls the linear flow rate of liquid passing through the carrier column (3) and culture process parameters by adjusting the rotating speed of the magnetic stirrer (8).