CN108424855A - A plant cell and organ liquid culture device - Google Patents

Abstract

The invention provides a simple plant cell and organ liquid culture device, which comprises a hollow culture tank body, an inoculation port and an air inlet which are positioned at one end of the culture tank body, and an exhaust port which is positioned at the other end of the tank body, wherein one end of the tank body is a plane, the other end of the tank body is a cone, the top end of the cone is provided with the inoculation port, and the inoculation port is matched with a sealing cover.

Description

A plant cell and organ liquid culture device

technical field

The invention relates to the technical field of cell culture, in particular to a plant cell and organ liquid culture device suitable for large-scale production.

Background technique

With the rapid development of global society and economy, the sufficient supply of existing fossil resources such as petroleum and coal has become unsustainable, and it is difficult to support the high-level development goals of human society. The development of human society will change from relying on hydrocarbon resources such as fossil fuels to relying on starch, cellulose, polysaccharides, plants and microbial oils and other carbohydrate resources that can be continuously synthesized by solar energy. This transformation of energy and resource structure will bring great opportunities and challenges to the development of biotechnology and its industries.

In terms of available plant resources, my country is one of the countries with the richest medicinal plant resources in the world. With the extensive use of herbal medicines, the excavation and use of medicinal plants far exceeds the growth rate of plants. Medicinal plants Resources are becoming depleted. Moreover, the traditional field cultivation of medicinal plants is affected by "time and place", the yield and quality fluctuate greatly, and the problem of pesticide residue pollution is also relatively serious.

Therefore, with the help of various biological systems, carbohydrates are utilized for the large-scale production of various useful components such as plant cells, tissues and organs required by modern society. The biochemical reaction of material transformation in the biological system is carried out in the relatively closed niche of the bioreactor. Bioreactors provide controllable environmental conditions for biochemical reactions in biological systems to promote efficient biomass conversion processes, such as temperature, pH, dissolved oxygen, mixing, shearing, feeding, etc. Traditional bioreactors generally include suspension culture system reactors and immobilized culture system reactors. The former mainly includes stirred reactors and airlift reactors; the latter mainly includes membrane reactors and packed bed reactors.

At present, bioreactors for cultivating plant cells, tissues and organs have reached the stage of industrialized mass production. For example, air-lift bioreactors were used to cultivate vinca cells to produce stalinine [A.H.Scragg et al., Enzyme MicrobTechnol, 1987, 9: 619]; Korea S.H.Son et al. carried out reactor large-scale cultivation of yew cells, Using air-lift reactors from 100L to 500L [S.H.Son et al., Plant Cell Reports (2000) 19: 628-633]; a few years later, Kee-Yoeup Paek used a spherical bubble reactor to treat ginseng adventitious roots Ginsenosides with strong pharmacological activity were obtained through large-scale cultivation [Kee-Yoeup Paek et al., Adv Biochem Engin/Biotechnol (2009) 113:151-176], and adventitious roots of ginseng with relatively high growth were obtained. Meixian Meiyan Bioengineering Institute has used gas circulation bioreactor to produce yew cells and has reached the level of 500L [gas circulation plant cell reactor CN2510499Y]. Phyton, Inc of the United States uses a stirred bioreactor to cultivate yew cells to produce paclitaxel and has reached a scale of 7 tons. South Korea's Samyang Genex is also using stirred fermenters to grow yew cells to produce paclitaxel.

The stirred reactor is one of the most widely used industrial reactors. Due to the characteristics of large volume, brittle cell wall and large vacuoles, plant cells are very sensitive to shear stress, and the sensitivity depends on the type of cells. , state, shear force tolerance, shear rate and the structure of the reactor itself are determined by many factors. At present, the solution is usually to start from the cell line itself, to screen high-yield lines resistant to shear stress or to transform existing cell lines through cell engineering and genetic engineering to establish strains resistant to shear stress; The structure of the reactor is modified to have a moderate flow field and good mixing performance. However, there are still the following problems: 1) currently commonly used reactors are all fixed at a certain position, and open-air inoculation is required during inoculation, and the tank body needs to be opened twice when feeding at the same time, the probability of bacterial contamination will increase significantly, and the problem of bacterial contamination is The most difficult problem to solve in the plant tissue culture; 2) The reactor is mostly a tank made of stainless steel, so one or more transparent observation ports need to be set during production. There are still many dead angles that are difficult to observe during actual use; 3) Stirred bioreactors have a large operating range, and many industrialized productions have been realized, but the shear force generated during the stirring process is relatively large, which is easy to damage the isolated plant tissues. 4) Since the mechanical stirring device cannot be sterilized, external steam is required for sterilization of this type of equipment, and special attention needs to be paid to the danger and complexity of high-pressure steam, and steam sterilization Sterilization will also lead to an increase in the volume of the medium, and the increased quantity is unstable, and at the same time cause the volatile substances in the medium and substances whose gasification temperature is lower than water to be discharged together with the steam;

Relatively speaking, the shear force of the airlift reactor and the bubble reactor is small. At present, it is generally believed that the bubble reactor is a kind of bioreactor with a wide range of applications. The structural characteristics of this type of reactor are Bubbles rise up the liquid phase, agitating the liquid while reacting in contact with the liquid phase to increase the rate of mass transfer. However, the above are only theoretical advantages, and very few have actually realized industrialization. Most of the bubble reactors only remove the stirring equipment of the stirred reactor, so their application range is far lower than that of the stirred fermenter. The traditional Because there is no stirring device in the bubble reactor, it is prone to serious back-mixing, the bubbles are easy to coalesce, and the foam will entrain some volatile CO 2 substances. And aeration will cause a large amount of evaporation of the medium, which will have a bad effect on the growth of plant cells, and the efficiency is low. Although this effect can be weakened by adding a defoamer, adding a defoamer will have a negative impact on plant cells, tissues and organs. Growth and accumulation of secondary metabolites are poorly produced. At the same time, because the air intake does not increase the amount of dissolved oxygen in the medium, the problem of dissolved oxygen in the medium is more serious. At the same time, the excessive ventilation will cause some materials that need to be cultivated for a long time, and the cultivation will have to be terminated due to the evaporation of the medium in the later stage. Many devices of this type need to be sterilized with external high-pressure steam like stirred bioreactors, which increases the risk and complexity of operation, as well as the instability of medium capacity during sterilization.

The above-mentioned various types of reactors are all based on scale-up. Due to the need for step-by-step scale-up, errors in any step in the middle, such as bacterial contamination, browning, etc., will lead to the failure of the final culture, but the small and easy-to-operate reactors are It can solve the above problems very well.

Among the simple and easy-to-operate small bioreactors, the wave bioreactor produced by GE Healthcare Wave Products Group has been applied. The WAVE wave bioreactor has flexible culture volume range, simple operation, precise and reliable control, and easy process scale-up. However, the wave wave reactor uses composite materials. The plastic bag used as the fermentation tank is composed of three layers of plastics. The inner layer is LDPE, the middle is EVOH, and the outermost layer is EVA. It cannot be autoclaved. The culture bag needs to be sterilized by irradiation first, and then the culture medium is sterilized separately and then poured into the culture bag. If feeding is needed in the middle, the culture bag needs to be reopened for operation. Adventitious root cultures are difficult to cultivate in the wave reactor , and the culture bag cannot be reused, so it will cause environmental pollution. In addition, the oxygen of the wave culture bag medium is exchanged through the liquid surface gas, so the dissolved oxygen in the medium is likely to be insufficient, and the growth of the culture in the later stage will be severely restricted. This ultimately leads to problems with culture quality and yield.

In order to solve the problem that the equipment in the prior art is expensive and complicated to operate, the evaporation of the culture medium is large during the cultivation process, a large number of bubbles are generated during the cultivation process, the feeding equipment, the culture medium and the cultivation equipment are sterilized separately, and the problem of increasing bacterial contamination when feeding, The present invention intends to provide an incubator that can meet the demand for dissolved oxygen under low aeration, can sterilize feeding equipment together with the cultivation equipment, and has a lower bubble formation rate during the cultivation process.

Contents of the invention

The object of the present invention is to provide a plant cell and organ liquid culture device.

In order to solve the problems of the technologies described above, the present invention adopts the following technical solutions:

A plant cell and organ liquid culture device, comprising a hollow culture tank body, an inoculation port and an air inlet located at one end of the culture tank body, and an exhaust port located at the other end of the tank body; wherein, one end of the tank body is a plane , one end is cone-shaped, the top of the cone is provided with an inoculation port, and the inoculation port is matched with a sealing cover.

The biological component liquid culture device of the present invention is made of materials that can be sterilized at high temperatures, especially materials that can withstand high-pressure sterilization at 121°C, more preferably transparent materials, such as PC, PE, glass, etc., so that accessories and culture tanks can be Put the body directly into the autoclave for sterilization. Its use method is as follows: when inoculating, place it forward, the plane is located below, close the exhaust port, shake a certain amount of cells together with the culture medium, and then directly introduce it into the medium from the inoculation port; The tank body is turned upside down, so that the air inlet is located at the bottom, and the exhaust port is located at the top, and the gas is introduced, and the bubbles are fully contacted with the culture solution in the reaction tank by using the rising process of the bubbles.

Therefore, for the convenience of placement, the exhaust port is arranged on the side close to the plane of the bottom end thereof.

Further, the air inlet can be arranged on the inclined surface of the conical tank body, and the air inlet is connected with the air inlet pipe to introduce the gas required for cultivation into the culture tank body.

Further, the air inlet is arranged on the sealing cover, which can effectively reduce leakage and reduce sealing difficulty.

Furthermore, the air inlet is set in an inclined manner so that the discharged gas does not rise vertically;

Furthermore, the air inlet is also provided with an air distributor, so as to promote the state of gas circulation instead of vertical upward, and prolong the trajectory of the air bubbles in the culture tank. The air distributor can greatly reduce the diameter of the bubbles, preferably an air distributor that can make the size of the bubbles between 0.2mm-8mm.

Further, the air inlet of the air intake pipe is provided with gas filtration and sterilization equipment, preferably a 0.22um air filtration and sterilization equipment commonly used in the field, to prevent the miscellaneous bacteria in the air from entering the cultivation tank.

Further, the inoculation opening is a screw opening, and the sealing cover is a double-opening screw sealing cover, the upper opening of which is connected to the air intake pipe, and the lower opening is connected to the inoculation opening of the culture tank.

The actual opening size of the inoculation port depends on the state of the culture. Considering that it is mainly used for culturing plant cells and organs, the diameter of the inoculation port can be 2-15 cm.

Further, the included angle of the cone of the culture tank is between 60-150°C.

Further, the inoculation port is a cylinder with a height of no more than 20 cm.

During the inoculation process, the height of the medium in the culture bucket should be greater than 20cm; during the cultivation process, the distance between the liquid level of the medium and the exhaust port should be greater than 3cm.

The exhaust port can be used for feeding in the middle of the cultivation; or the exhaust port branches out to a branch pipe for feeding;

Furthermore, one or more openings are provided on the side of the culture tank close to the exhaust port, and the openings are used for feeding and connected to one or more feeding pumps.

After the cultivation, the culture can be poured out from the inoculation port;

Or further, a collection port is provided on the side of the culture tank. If you encounter cultures that are difficult to collect, such as agglomerates or balls that are difficult to pour out from the inoculation port, or large cultures such as adventitious roots, you can use the collection port to collect. In actual use, determine whether it is equipped according to the state of the culture. The collection port is sealed during both inoculation and incubation.

In addition, in order to prevent contamination by miscellaneous bacteria, the outlet of the exhaust port is provided with filter and sterilization components, such as simple gauze or cotton, to prevent external microorganisms from entering the cultivation equipment through the exhaust port.

When sterilizing, the culture tank is placed upright, the exhaust port at the bottom is closed with a water-stop clamp and other devices, the inoculation port is sealed with a sterile sealing film, the sealing cover of the inoculation port and the air intake pipe, the air distributor and the air sterilizer , connected together, and then sterilized.

When in use, pour the culture seeds together with part of the culture medium into the tank, seal the inoculation port with a sealed cover, place it upside down on a culture rack with holes, place the culture equipment on the culture rack, and make the inoculation port face down. The exhaust port is upward, connect the intake pipe and open the water-stop clip on the exhaust pipe to ventilate. When using it, adjust the ventilation volume according to your needs.

The capacity of the cultivation tank body of the present invention is 2-100L, which is very convenient to operate, and can also be directly put into a common sterilization pot for sterilization.

The purpose of the present invention is to provide a simple plant cell and organ liquid culture device, the main advantages are as follows:

1) The materials of the culture tank and main accessories are transparent materials that can withstand high temperatures, such as PC, PE, glass, etc., which can be sterilized directly. When sterilizing, they are placed in a commonly used sterilizer without steam, so The capacity of the culture medium is stable; the culture process is intuitively visible, and the culture can be clearly observed in any corner of the cylinder during the culture process. The growth status of the culture tank is clear at a glance. Early detection and treatment as soon as possible to reduce pollution to other cylinders;

2) The air inlet is equipped with an air distributor, and the air inlet adopts an inclined way to discharge gas, and implements rotary air intake. While the air bubbles drive the liquid to rise vertically, they can also drive the liquid to rotate along the wall of the barrel to promote the medium and The gas exchange between the bubbles can meet the needs of dissolved oxygen in the medium under low ventilation, so in the actual culture process, little or no antifoaming agent can be used; thereby reducing the impact of the bubbles formed on the surface of the culture during the aeration process on the culture. The damage caused can reduce the cost of air supply; the loss of medium caused by aeration and evaporation can also be greatly reduced, and the loss rate of medium can be as low as 10% or less;

3) The tank and feeding equipment can be sterilized at the same time, reducing the secondary operation of opening the tank when feeding, thereby reducing the pollution rate;

4) For the cultivation of organs that are difficult to collect, such as adventitious roots, the equipment is equipped with a special collection port, which greatly reduces the collection of cultures;

5) The volume is 2-100L, and the operation is simple. Place it on the aseptic operation table during inoculation, which greatly reduces the bacterial contamination rate during inoculation; the operation process is simple and convenient, with low risk, low bacterial contamination rate, and strong operation repeatability , low cost, can greatly reduce the production cost of the culture.

Description of drawings

Fig. 1 is the schematic diagram of plant cell and organ liquid culture device described in embodiment 1 of the present invention;

2 is a schematic diagram of the plant cell and organ liquid culture device described in Example 2 of the present invention;

Among them, 1. Culture tank; 2. Inoculation port; 3. Sealing cover; 4. Air inlet; 5. Exhaust port; 6. Feeding port; 7. Air distributor; 8. Air filter; 9. Filter cotton; 10, collection port.

Detailed ways

The plant cell and organ liquid culture device of the present invention will be further described below in conjunction with the accompanying drawings.

Example 1

A plant cell and organ liquid culture device, comprising a culture tank 1 (made of PC that can withstand high pressure sterilization at 121°C, with a capacity of 40L), an inoculation port 2 and an air inlet 4 located at one end of the culture tank 1 , the diameter of the inoculation port 2 is 10cm; and the exhaust port at the other end of the tank; wherein, the tank is cylindrical and conical, and the angle of the cone of the culture tank 1 is 64°C, and the top of the cone is set The inoculation port 2, the height of the inoculation port 2 is 12cm, and the inoculation port 2 cooperates with the sealing cover 3; the exhaust port is arranged on the side of the cylinder near its bottom surface; the air inlet 4 is arranged on the sealing cover 3, and the The inoculation port 2 is a screw port, and the sealing cover 3 is a double-port screw sealing cover, the upper port of which is connected to the air intake pipe, and the lower port is connected to the inoculation port 2 of the culture tank body 1 .

The air inlet 4 is arranged in an inclined manner, so that the discharged gas does not rise vertically; the air inlet 4 is also provided with an air distributor 7 to promote the gas to show a circular flow state instead of vertically upward, so as to prolong the air bubbles in the culture tank. The running track in body 1. The air distributor 7 makes the diameter of the air bubbles in the range of 0.2mm-8mm.

A 0.22um air filter 8 is provided at the gas inlet of the air intake pipe, and a filter cotton 9 is provided at the outlet of the exhaust port 5 to prevent bacteria from entering the culture tank 1 . In addition, the exhaust port 5 is branched out of a branch pipe as a feeding port 6 for feeding;

Its use method is as follows: when sterilizing, the culture tank body 1 is placed upright, the exhaust port at the bottom is closed with a device such as a water stop clip, the inoculation port 2 is sealed with a sterile sealing film, and the sealing cover 3 of the inoculation port 2 is connected with the air inlet pipe. , the air distributor 7 and the air sterilizer are connected together and then sterilized; after the sterilization is completed, the culture tank 1 is placed forward, the cylinder is located below, the exhaust port is closed, and the culture medium is injected into the culture tank 1, then shake a certain amount of cells together with the culture medium and then directly introduce them into the culture medium from the inoculation port 2; then seal the inoculation port 2, place it upside down on a culture rack with holes, and place the culture equipment on the culture rack to make the inoculation Port 2 is downward, and exhaust port 5 is upward. Connect the air inlet pipe and open the water-stop clamp on the exhaust pipe to ventilate. Use the rising process of the air bubbles to make the air bubbles fully contact with the culture medium in the reaction tank. Use according to your needs Adjust the ventilation volume. During the inoculation process, the height of the medium in the culture bucket should be greater than 20cm; during the cultivation process, the distance between the liquid level of the medium and the exhaust port should be greater than 3cm.

After the cultivation, the culture was poured out from the inoculation port 2 .

Example 2

A liquid culture device for plant cells and organs, comprising a culture tank 1 (made of PC that can withstand autoclaving at 121°C, with a capacity of 100L), an inoculation port 2 and an air inlet at one end of the culture tank 1 4. The diameter of the inoculation port 2 is 10 cm; and the exhaust port at the other end of the tank; wherein, the tank is cylindrical and conical, and the angle of the cone of the culture tank 1 is 120°C, and the top of the cone is The inoculation port 2 is set, and the height of the inoculation port 2 is 8cm, and the inoculation port 2 cooperates with the sealing cover 3; the exhaust port is arranged on the side of the cylinder near its bottom surface; the air inlet 4 is arranged on the inclined plane of the conical tank body Above, the air inlet 4 is connected to the air inlet pipe, and the gas required for cultivation is introduced into the cultivation tank body 1 .

The side of the cylinder of the culture tank 1 close to the exhaust port 2 is also provided with a feeding port 6 for connecting one or more feeding pumps for feeding during the cultivation process.

A collection port 10 with a diameter of 30 cm is also provided on the side of the culture tank 1 . If you encounter cultures that are difficult to collect, such as agglomerates or balls that are difficult to pour out from the inoculation port 2, or large cultures such as adventitious roots, you can use the collection port 10 to collect them. The collection port 10 is sealed during inoculation and cultivation.

The air inlet 4 is arranged in an inclined manner, so that the discharged gas does not rise vertically; the air inlet 4 is also provided with an air distributor 7 to promote the gas to show a circular flow state instead of vertically upward, so as to prolong the air bubbles in the culture tank. The running track in body 1. The air distributor 7 makes the diameter of the air bubbles in the range of 0.2mm-8mm.

A 0.22um air filter 8 is provided at the gas inlet of the air intake pipe, and a filter cotton 9 is provided at the outlet of the exhaust port 5 to prevent bacteria from entering the culture tank 1 .

The process of use is the same as that described in Example 1.

The volume of the culture tank of the present invention is 2-100L, the operation is simple, the operation repeatability is strong, the cost is low, the risk is small, and the bacterial contamination rate is low; Bacteria, the capacity of the medium is stable, and the cultivation process is intuitively visible; the air inlet is equipped with an air distributor and is tilted, and the rotary air intake is implemented, which can meet the needs of the dissolved oxygen of the medium under low ventilation, reducing the cost of air supply and The loss of medium; and for organs that are difficult to collect such as adventitious roots, the equipment is equipped with a special collection port.

Claims (10)

1. A plant cell and organ liquid culture device, is characterized in that, comprises hollow culture tank body, is positioned at the inoculation port and air inlet of one end of culture tank body, and is positioned at the exhaust port of tank body other end; Wherein, all One end of the tank body is flat, and the other end is conical. The top of the conical shape is provided with an inoculation port, and the inoculation port is fitted with a sealing cover. 2. The culture device according to claim 1, characterized in that, the culture device is made of materials that can withstand autoclaving at 121°C; preferably transparent materials that can withstand autoclaving at 121°C. 3. The culture device according to claim 1 or 2, wherein the exhaust port is arranged on the side near its plane bottom surface; the air inlet is arranged on the slope of the conical tank body or on the sealing surface. When the cover is closed, the air inlet is connected with an air inlet pipe, and the gas required for cultivation is introduced into the cultivation tank body. 4. The culture device according to claim 1 or 2, characterized in that the diameter of the inoculation port is 2-15 cm. 5 . The cultivation device according to claim 3 , wherein the air inlet is arranged in an inclined manner, and the air inlet is provided with an air distributor. 6 . 6. The cultivation device according to claim 3, wherein the air inlet is arranged on a sealing cover, the inoculation port is a screw port, and the sealing cover is a double-port screw sealing cover, and its upper port is connected to The air inlet pipe, the lower port is connected to the inoculation port of the culture tank body. 7. The culture device according to claim 1, characterized in that, the included angle of the taper of the culture tank is 60-150°C. 8. The culture device according to claim 1, characterized in that, one or more openings are provided on the side of the culture tank close to the exhaust port, and the openings are used for feeding, connecting one or one above the feed pump. 9. The culture device according to claim 1, characterized in that, a collection port is provided on the tank side of the culture tank. 10. The cultivation device according to claim 1, wherein a gas filtration and sterilization device is arranged at the gas inlet of the air intake pipe, and a filtration and sterilization component is arranged at the outlet of the exhaust port; the cultivation tank body The capacity is 2-100L.