CN101899394B

CN101899394B - External circulation animal cell culture bioreactor

Info

Publication number: CN101899394B
Application number: CN 201010242025
Authority: CN
Inventors: 罗火生
Original assignee: Individual
Current assignee: Individual
Priority date: 2010-07-29
Filing date: 2010-07-29
Publication date: 2012-09-26
Anticipated expiration: 2030-07-29
Also published as: CN101899394A

Abstract

The invention relates to an external circulation animal cell culture bioreactor which comprises a tank body, an external circulation pipeline, a heater, a peristaltic pump and a control system, wherein the tank body comprises a large tank and a small tank; the small tank is sheathed in the large tank and is clamped and fixed with the large tank respectively through ducts D, A and G; the external circulation pipeline comprises a tank external circulation pipeline and ducts A, B, E and D; on the upper part of the outside of the tank body, the ducts A and B are connected with the tank external circulation pipeline through a tee joint; on the lower part of the outside of the tank body, the ducts E and D are connected with the tank external circulation pipeline through a tee joint; the tank external circulation pipeline penetrates through the peristaltic pump; and the lowest position of the duct D is connected with a drainage pipe. The invention fully displays the characteristics of the animal cell large-scale culture technique, has the characteristics of thorough sterilization, simple operation, low pollution rate of cell culture, good quality and high yield, has favorable functionality and economical efficiency, and can be widely used for animal cell microcarrier suspension culture and animal cell suspension culture.

Description

External circulation animal cell culture bioreactor

Technical Field

The invention relates to a device for culturing animal cells by adopting a suspension method, in particular to an external circulation animal cell culture bioreactor.

Background

A bioreactor refers to a device which takes living cells or enzyme as a biocatalyst to carry out cell proliferation or biochemical reaction and provides a suitable environment, and is a key device in the biological reaction process. The structure, operation mode and operation condition of the bioreactor are closely related to the quality, yield (conversion rate) and energy consumption of the biochemical products.

With the continuous development of biotechnology, large-scale animal cell bioreactor culture technology is gradually replacing glass bottle static culture and rotary culture technology, because it can provide the best living environment for cells and viruses in time, thereby improving the yield and quality of products with the highest efficiency, it should be said that this is a qualitative leap in animal cell culture history, and is also a major breakthrough of biopharmaceutical technology, it not only simplifies the process, but also can produce high-quality products, at present, there are many bioreactors in the market, including domestic and imported, each has advantages and defects, some even have inevitable hard injuries, thereby limiting the maximum exertion of its advantages.

The following will compare and analyze the designs of several existing animal cell culture bioreactors with respect to the stirring mode, monitoring system, sterilization mode, aeration mode, blowdown mode, etc., and the details are shown in the following table:

disclosure of Invention

In view of the above problems of the existing bioreactor for animal cell culture and the influence thereof on the quality and yield of cell culture, the present invention discloses an external circulation bioreactor for animal cell culture, which can effectively ensure the effect of cell culture and embody good economy and functionality.

The technical solution of the invention is realized as follows:

the utility model provides an extrinsic cycle animal cell culture bioreactor, includes jar body and extrinsic cycle pipeline, heater, peristaltic pump and control system, its characterized in that:

the tank body comprises a large tank and a small tank; the large tank comprises a large tank body, a water bath interlayer and a tank cover, and the tank cover is provided with an air vent; the small tank comprises an upper end enclosure, a lower end enclosure and a middle tank body, and the upper end enclosure and the lower end enclosure are respectively connected with the middle tank body in a sealing manner; the small tank is divided into an upper space, a middle space and a lower space through an upper stainless steel screen plate and a lower stainless steel screen plate which are arranged at the connecting positions of an upper end socket and a lower end socket in the small tank and the middle tank body and are vertical to the central axis of the small tank;

the small tank is sleeved inside the large tank and is respectively fixed with the large tank by a pipeline D, A, G, namely: one end of the pipeline D is fixed and communicated to the lowest position in the lower seal head of the small tank, the other end of the pipeline D is connected to the outside of the large tank from the bottom of the large tank and is respectively communicated with the I section of the circulating pipeline outside the tank and the pipeline E through a tee joint, and the other end of the pipeline E is connected into the large tank from the lower part of the large tank; one end of the pipeline A is fixed at the highest position of the upper end enclosure of the small tank and is communicated into the small tank from the highest position, the other end of the pipeline A is connected out of the tank body from the upper part of the wall of the large tank body and is communicated with the section II of the circulating pipeline outside the tank body and the pipeline B through a tee joint, the end part of the pipeline B is provided with a diaphragm valve, and the other end of the pipeline B is connected into the large tank body from the middle upper part of the wall of the; one end of the pipeline G is fixed on the upper end enclosure of the small tank and sequentially passes through the upper end enclosure and the upper net plate of the small tank to be connected to the middle tank body of the small tank, and the other end of the pipeline G is communicated with the outside of the large tank body;

the external circulation pipeline comprises an external circulation pipeline, a pipeline A and a pipeline B, the upper part outside the tank body is connected with the section II of the external circulation pipeline through a tee joint, and a pipeline E and a pipeline D, the lower part outside the tank body is connected with the section I of the external circulation pipeline through a tee joint; the tank external circulation pipeline passes through the peristaltic pump; the peristaltic pump adopts stepless speed change;

the tank external circulation pipeline is also respectively connected with a liquid supplement pipeline, a gas supplement pipeline and a liquid receiving pipeline;

the pipeline D is connected with a sewage draining pipe at the lowest position.

Further, the monitoring probe of pH control and dissolved oxygen control has still been connected on the II section of jar extrinsic cycle pipeline, temperature control's monitoring probe has been placed to big jar internal, and above-mentioned monitoring probe is connected with the switch board through the wire, the switch board includes display, dissolved oxygen controller, pH controller, temperature controller, data record appearance.

Furthermore, the pipeline E is tangentially connected into the large tank from the lower part of the large tank body along the wall of the large tank body.

Furthermore, the upper and lower end enclosures are respectively connected with the intermediate tank body through external hoops in a sealing manner.

Compared with the prior art, the design of the external circulation animal cell culture bioreactor fully embodies the characteristics of the large-scale animal cell culture technology, and the pipeline layout is simple; the method has good functionality, reliability and convenience, and is specifically described as follows:

(1) stirring and sealing: the culture solution and the cells are stirred by a peristaltic pump through circulation of the liquid from bottom to top, and a completely closed loop ensures that the pH, the temperature, dissolved oxygen and the like of the liquid are sufficiently and uniformly distributed, and the shearing force on the cells tends to zero; meanwhile, the problem of sealing requirements of other forms of stirring on the tank body is solved;

(2) and (3) ventilation mode: the deep layer is directly ventilated, and the bubbles are not contacted with the cells completely, so that the damage of the bubbles to the cells can be completely avoided without a bubble selling device;

(3) and (3) sterilization mode: the structure design is reasonable, the pipeline layout is simple, sterilization is carried out in the damp and hot sterilization cabinet, the operation is simple, no sterilization dead angle exists, the maintenance and management of a container are not required by pressing force, and the safety is higher; meanwhile, the production preparation period is greatly shortened;

(4) a pollution discharge mode: the falling and apoptotic cells and the broken microcarrier can be discharged at any time through the discharge pipeline at the lowest end, so that the pollution rate is reduced;

(5) the monitoring system comprises: the monitoring probes for PH, temperature, dissolved oxygen and the like are arranged on the external circulation loop, and the external circulation enables the liquid to be fully and uniformly mixed, so that the monitoring data is more objective, accurate and representative.

In conclusion, the invention has scientific structural design and simple pipeline layout, skillfully isolates cells from a culture solution, can provide an optimal living environment for the cells and viruses in time, structurally avoids a plurality of hard injuries and defects existing in the prior equipment, fully exerts the characteristics of the large-scale culture technology of the animal cells, has the remarkable characteristics of thorough sterilization, simple operation, low cell culture pollution rate, good quality and high yield, has good functionality and economy, and can be widely applied to the suspension culture of animal cell microcarriers and the suspension culture of the animal cells.

Drawings

FIG. 1 is a schematic structural diagram of an embodiment of the present invention;

fig. 2 is a top view of fig. 1.

Detailed Description

The present invention will now be described in further detail with reference to the accompanying drawings.

The external circulation animal cell culture bioreactor comprises a tank body, an external circulation pipeline, a heater, a peristaltic pump and a control system, wherein the heater, the peristaltic pump and the control system adopt the existing forming equipment, and the structure of the tank body and the external circulation pipeline are greatly innovated, as shown in figures 1 and 2:

the tank body comprises a large tank 1 and a small tank 2; the large tank 1 comprises a large tank body 11, a water bath interlayer 12 and a tank cover 13, wherein a hot water tank inlet pipeline F is arranged at the lower part of one side of the water bath interlayer 12, a hot water tank outlet pipeline C is arranged at the upper part of the opposite side of the water bath interlayer 12, the hot water tank inlet pipeline F and the hot water tank outlet pipeline C are respectively connected with a circulating pipeline of a heater, and a vent hole connecting ventilation pipeline H, K is arranged on the tank cover 13; the small tank 2 comprises upper and

lower end enclosures

21, 23 and a middle tank body 22, and the upper and lower end enclosures are respectively connected with the middle tank body 22 in a sealing way through

external hoops

24, 25; the small tank 2 is divided into an upper space, a middle space and a lower space through upper and lower stainless

steel mesh plates

26 and 27 which are arranged at the connecting positions of upper and lower end enclosures and the middle tank body in the small tank 2 and are vertical to the central axis of the small tank;

the small tank 2 is sleeved inside the large tank 1 and is respectively fixed with the large tank 2 by a pipeline D, A, G in a clamping way, namely: one end of the pipeline D is fixed and communicated to the lowest position in the lower end enclosure 23 of the small tank through a clamping sleeve, the other end of the pipeline D is connected to the outside of the large tank from the bottom of the large tank 1 and is respectively communicated with the I section of the circulating pipeline outside the tank and the pipeline E through a tee joint, and the other end of the pipeline E is connected into the large tank 1 from the lower part of the large tank along the tangential direction of the wall of the large tank; one end of the pipeline A is fixed through a clamping sleeve and communicated into the small tank 2 from the highest position of an upper end enclosure 21 of the small tank, the other end of the pipeline A is connected out of the tank body from the upper part of the tank body wall of the large tank and communicated with the section II of the tank external circulation pipeline and the pipeline B through a tee joint, the end part of the pipeline B is provided with a diaphragm valve 6, and the other end of the pipeline B is connected into the large tank 1 from the middle upper part of the tank body wall of the large tank; one end of the pipeline G is fixed on the upper end enclosure 21 of the small tank through a clamping sleeve and sequentially penetrates through the upper end enclosure 21 of the small tank and the upper net plate 26 to be connected to the middle tank body 22 of the small tank, and the other end of the pipeline G is communicated with the outside of the tank body;

the external circulation pipeline comprises an external circulation pipeline, a pipeline A and a pipeline B which are connected with the external circulation pipeline through a tee joint at the upper part outside the tank body, and a pipeline E and a pipeline D which are connected with the external circulation pipeline through a tee joint at the lower part outside the tank body; the tank external circulation pipeline passes through the peristaltic pump;

the tank external circulation pipeline connected with the pipeline A and the pipeline B through the tee joint before entering the peristaltic pump is called as a section II of the tank external circulation pipeline; the other corresponding section of the external tank circulation pipeline which is taken out from the peristaltic pump and is connected with the pipeline E and the pipeline D through a tee joint is called as the section I of the external tank circulation pipeline; the section II of the tank external circulation pipeline is also respectively connected with an automatic control probe, a liquid supplement pipeline 71, a gas supplement pipeline 72 and a liquid receiving pipeline 73, the automatic control probe comprises a pH control monitoring probe 31 and a dissolved oxygen control monitoring probe 32, a temperature control monitoring probe 33 is placed in the large tank body, and the monitoring probes are connected with the control cabinet through leads.

The pipe D is connected to the drain pipe 5 at its lowest position.

The material of the tank body is 316L stainless steel, the water bath interlayer is 304 stainless steel, and the water bath interlayer is provided with a heat insulation layer. The top of the tank body is provided with a lamp mirror and a sight glass for observing the inside of the reactor.

When the tank body is assembled, the upper and

lower end enclosures

21 and 23 of the small tank are respectively connected with the intermediate tank body 22 through the

hoops

24 and 25, the intermediate tank body 22 is isolated from the upper and

lower end enclosures

21 and 23 through the stainless

steel mesh plates

26 and 27, the lower end enclosure 23 is connected with the pipeline D in a clamping sleeve mode, the upper end enclosure is connected with the pipeline A, G through the clamping sleeve, the tank cover 13 of the large tank is covered after the small tank 2 is fixed, and the whole tank body is assembled.

The connection of the tank body pipelines comprises that pipelines C, F are respectively connected with a heater to form a water bath heating loop of a large tank; a pipeline A, B (a connecting diaphragm valve 6) is connected with the II section of the tank external circulation pipeline through a stainless steel tee joint, and the II section of the pipeline is also respectively connected with a liquid supplement pipeline, a gas supplement pipeline, a liquid receiving pipeline and a monitoring probe comprising pH control and dissolved oxygen control; the pipeline E, D is connected with the I section of the circulating pipeline outside the tank through a stainless steel tee; the tank external circulation pipeline passes through the peristaltic pump; the pipe H, K is connected to the vent of the lid 13 of the large tank, and the pipe G is a passage for connecting the small tank 2 to the outside of the tank body.

And (3) sterilization treatment:

after the tank body assembly and the pipeline connection are completed, the whole large tank is directly placed into a damp and hot sterilization cabinet for sterilization treatment, so that complex online cleaning and online cleaning verification are not needed, more importantly, sterilization dead angles cannot be left due to online sterilization, the pollution rate is greatly reduced, and a pressing force container is not needed for use and management. The time required by the sterilization treatment link is about 1 day, the operation is simple, and the sterilization is thorough; the time required by the online steam sterilization treatment link of the existing equipment is about 7 days on average, the operation is complex, sterilization dead corners are easy to remain, pollution is caused, and the container must be maintained and managed by pressing force.

Stirring control:

the liquid circulation stirring is realized by a peristaltic pump. The microcarriers and the cells are sent into the middle tank body 22 of the small tank through a pipeline G, and the microcarriers and the cells are isolated from the liquid of the large tank through the

mesh plates

26 and 27; under the action of the peristaltic pump, the liquid in the small tank flows from bottom to top, the liquid in the large tank rotates to generate vortex, the liquid is fully stirred, parameters such as liquid temperature, pH value and dissolved oxygen are completely consistent, and the circulating pump adopts stepless speed change and operates stably.

The control system comprises:

the control function of the reactor is realized by a control cabinet, and the reactor comprises a display screen, a dissolved oxygen controller, a PH controller, a temperature controller and a data recorder. Wherein,

temperature control:

the reactor was temperature controlled using a water bath sandwich. The water bath interlayer is provided with a water bath heater which improves the sensitivity and stability of the temperature control of the reactor. The reactor temperature control system adopts a PID control mode based on the tank body temperature, the temperature compensation function enables the temperature control to be more stable, and the temperature control precision is +/-0.3 ℃.

And (3) pH control:

and an independent module control mode is adopted, and the pH controller is independently arranged. The PID control mode is used for realizing automatic control, has a self-adaptive PID control and adjustment function, and can automatically set the optimal PID parameters. The pH control is associated with a carbon dioxide ventilation control valve and an acid and alkali peristaltic pump, and the automatic control of the pH is realized by controlling the on-off of the carbon dioxide, the acid liquid and the alkali liquid. When the pH value is lower than the set value, the controller starts the alkali liquor peristaltic pump, and the alkali is supplemented to increase the pH value to the set value; when the pH value is higher than the set value, the controller starts a carbon dioxide gas electromagnetic valve and an acid liquid peristaltic pump to reduce the pH value to the set value. The flow of the carbon dioxide can be adjusted by a rotameter, and the flow of the acid liquid and alkali liquid peristaltic pump can be adjusted by a pump speed regulating valve, so that the sensitivity of pH control is further improved, and the pH control response deviation is +/-0.01.

Controlling dissolved oxygen:

and an independent module control mode is adopted, and the dissolved oxygen controller is independently arranged. The PID control mode is used for realizing automatic control, has a self-adaptive PID control and adjustment function, and can automatically set the optimal PID parameters. The dissolved oxygen control is associated with an oxygen ventilation control valve, and the automatic control of the dissolved oxygen is realized through the opening and closing of the control valve. When the dissolved oxygen is lower than the set value, the controller starts the control valve to introduce oxygen into the reactor to raise the dissolved oxygen to the set value. The oxygen flow can be adjusted by the rotameter, the sensitivity of dissolved oxygen control is further improved, and the dissolved oxygen response deviation is 0.5%.

A process pipeline system:

the gas system comprises a compressed air gas pipeline, an oxygen gas pipeline, a carbon dioxide gas pipeline, a gas pressure reducing valve and a pneumatic diaphragm valve which are automatically controlled, are respectively connected with the external circulation pipeline of the tank through a gas supplementing pipeline and are input into the tank under the action of a peristaltic pump.

The liquid system comprises a liquid supplementing pipeline and a liquid collecting pipeline which are respectively connected with the circulating pipeline outside the tank, and the liquid supplementing pipeline supplements the liquid into the tank body or collects the liquid from the tank body under the action of a peristaltic pump.

Process flow of implementation

Cell culture:

connecting all pipelines according to the aseptic requirement, injecting about 25L of cell culture solution into the big tank through the peristaltic pump 4, then preheating the big tank to 25-28 ℃ in a water bath, and directly injecting cells and microcarriers into the small built-in tank through the pipeline G, wherein the total liquid in the whole tank is 30-32L. The pipelines B and E are closed, the peristaltic pump 4 is started at a low speed, the cells are circulated in the built-in small tank for a plurality of hours until the cells are fully attached to the microcarriers (the cells can be attached outside the tank and then injected into the built-in small tank), meanwhile, the temperature is slowly increased to 37 +/-0.3 ℃, the unattached cells are discharged outside the tank through the pipeline D after being circulated for a plurality of hours, then the pipelines B and E are opened, the liquid in the large tank also participates in the circulation of the small tank, the liquid circulation process in the small tank is also the process of fully stirring the cells in the tank, the slowly flowing liquid can not cause damage to the cells, and the liquid flows from bottom to top, so that dead corners do not exist, and the stirring is uniform. According to the data that dissolved oxygen and pH probe detected, can close pipeline A, D earlier, inject oxygen and sodium bicarbonate to big jar circulation through the peristaltic pump, gaseous like this and sodium bicarbonate are not direct with the cell contact, can avoid bubble and local PH to cause the injury to the cell too high, transfer the back, open pipeline A, D and circulate to let the physical and chemical index in big jar unanimous.

Washing cells:

the liquid in the tank is discharged through the pipeline E, D, then the washing liquid is filled by the peristaltic pump for circulation, and the liquid in the tank is discharged through the pipeline E, D after a plurality of times, so that the washing can be repeated for a plurality of times according to specific conditions.

And (3) virus culture:

about 25L of virus culture solution is injected into the big tank through a peristaltic pump, and the virus seeds are directly injected into the built-in small tank through a pipeline G, wherein the total liquid in the whole tank is 30L-32L. The pipelines B and E are closed, the peristaltic pump is started at a low speed, liquid circulates in the built-in small tank for a plurality of hours to enable the virus to fully infect cells, the temperature is kept at 35 +/-0.3 ℃, the fallen cells are discharged out of the tank through the pipeline D after the liquid circulates for a plurality of hours, then the pipelines B and E are opened, the liquid in the large tank also participates in the circulation of the small tank, the pipeline A, D can be closed firstly according to the data detected by the dissolved oxygen and the PH probe, oxygen and sodium bicarbonate are injected into the large tank through the peristaltic pump to circulate, so that the gas and the sodium bicarbonate are not directly contacted with the cells, the cells can be prevented from being damaged by bubbles and local PH being too high, and after the regulation is completed, the pipeline A, D is opened for circulation, so that the physicochemical indexes in the large tank and the. After several hours of culture, virus-infected cells are harvested according to different products (one-time harvest), or virus culture fluid is collected through the pipe E, D (virus culture fluid can be harvested repeatedly many times according to cell maintenance), thus completing a production cycle.

The invention can provide an external circulation animal cell culture bioreactor with a working volume of 30-150L.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.

Claims

1. The utility model provides an extrinsic cycle animal cell culture bioreactor, includes jar body and extrinsic cycle pipeline, heater, peristaltic pump and control system, its characterized in that:

the external circulation pipeline comprises an external circulation pipeline, a pipeline A and a pipeline B which are connected with the external circulation pipeline through a tee joint at the upper part outside the tank body, and a pipeline E and a pipeline D which are connected with the external circulation pipeline through a tee joint at the lower part outside the tank body; the tank external circulation pipeline passes through the peristaltic pump; the peristaltic pump adopts stepless speed change;

the pipeline D is connected with a sewage discharge pipe at the lowest position;

the utility model discloses a temperature monitoring device, including jar outer circulation pipeline, big jar internal temperature control's monitor probe has been put to big jar, the monitor probe who has still connected pH control and dissolved oxygen control on the II section of jar outer circulation pipeline, the internal temperature control's of placing of big jar, above-mentioned monitor probe is connected with the switch board through the wire, the switch board includes display, dissolved oxygen controller, pH controller, temperature controller, data record appearance.

2. The external circulation animal cell culture bioreactor according to claim 1, wherein:

and the pipeline E is tangentially connected into the large tank from the lower part of the large tank along the wall of the large tank.

3. The external circulation animal cell culture bioreactor according to claim 2, wherein:

the upper and lower seal heads are respectively connected with the intermediate tank body through external clamps in a sealing manner.