CN113373038A - Preparation method of cell culture medium and automatic equipment - Google Patents
Preparation method of cell culture medium and automatic equipment Download PDFInfo
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- CN113373038A CN113373038A CN202110457733.8A CN202110457733A CN113373038A CN 113373038 A CN113373038 A CN 113373038A CN 202110457733 A CN202110457733 A CN 202110457733A CN 113373038 A CN113373038 A CN 113373038A
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- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
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Abstract
The invention provides a preparation method of a cell culture medium and automatic equipment, wherein the preparation method comprises the following steps: grouping culture medium components, performing high-temperature grouping sterilization, automatically mixing, and automatically packaging; wherein, the three steps of high-temperature grouping sterilization, automatic mixing and automatic packaging are automatically completed in the same independent closed system. The invention also provides an automatic device for the cell culture medium. The invention eliminates the main adverse reaction of high-temperature sterilization, has high automation degree and saves scientific research manpower.
Description
Technical Field
The invention belongs to the technical field of cell culture media, and particularly relates to a preparation method of a cell culture medium and automatic equipment.
Background
The cell culture medium simulates a natural nutritional environment, is the basis of artificial cell culture experiments, and is widely applied to the scientific research fields of medical health, life science, chemical pharmacy and the like; the sterilizer and other equipment of the culture medium preparation facility are also standard matching of relevant laboratories or industrial production links. Agar media are currently the most widely used type of media.
According to the components of the culture medium, the culture medium can be divided into a serum culture medium and a serum-free culture medium, and the essential components comprise: serum, inorganic salts, buffer systems, carbohydrates, amino acids, vitamins, proteins, peptides, fats, trace elements, special antibiotics. Among them, serum is a complex mixture of albumin, growth factors and growth inhibitors, and is the most important component in serum cell culture media; the inorganic salt is mainly sodium, potassium and calcium ions, establishes and maintains osmotic balance of cells and regulates membrane potential, and is a necessary coenzyme factor; a buffer system for maintaining a suitable pH of 7.2-7.4 for cell culture; carbohydrates, which are the main energy and carbon source; amino acids, proteins and peptides, a source of nutrients for proteins synthesized by cells; trace elements, essential trace elements including zinc, copper, selenium and tricarboxylic acid intermediates, etc., selenium is also an antioxidant, helping to scavenge oxygen radicals.
Any medium must be pre-treated aseptically before use. In the existing culture medium preparation technology, the processes generally comprise mixing, high-temperature sterilization and packaging in sequence; during high temperature sterilization, the components are subjected to chemical reaction, nutrition is lost, and the product can have biological toxicity. Sterilization treatment or anti-contamination techniques, are the main technical drawbacks of the preparation of culture media, specifically including:
1. the whole sterilization process is carried out by mixing the components, and the high-temperature steam sterilization technology is commonly adopted, wherein the sterilization temperature is usually 121 ℃ or 135 ℃, and a plurality of the components of the culture medium can be thermally decomposed.
2. At least for 2014, studies in a number of mainstream academic journals have indicated that exposure of culture media to temperatures in excess of 121 ℃ can lead to significant poor cell growth if not properly gelled (referred to as agar). The Japanese scientist Kamagata found: when the phosphate is sterilized together with agar, it reacts to form a large amount of H2O2It has strong toxic action on various groups of microorganisms. The Kamagata team published the above series of studies three times in 2014, 2017, 2018 in bioengineering TOP journal, Appl environ microbiol.
3. Regardless of the sterilization technique employed, the media configuration is time consuming for most laboratories performing cell culture; moreover, in order to eliminate the above-mentioned drawbacks, the improvement of the sterilization of the components in groups or the combination of multiple sterilization techniques also increases the number of operating steps and, ultimately, the risk of contamination.
Disclosure of Invention
Aiming at the technical problems in the prior art, the invention provides a preparation method of a cell culture medium and automatic equipment, which change the flow, eliminate the main adverse reaction, have high automation degree and liberate the scientific research manpower.
The invention provides a preparation method of a cell culture medium, which comprises the following steps: grouping culture medium components, performing high-temperature grouping sterilization, automatically mixing, and automatically packaging; wherein, the three steps of high-temperature grouping sterilization, automatic mixing and automatic packaging are automatically completed in the same independent closed system.
Preferably, in the step of grouping the medium components, when the separate containers are limited, the order of the separate priorities is: the first preferential separation reaction product has a component with biological toxicity; second preference for other reaction-known components; third, the animal and plant source components are placed separately, and no purification is included, so as to eliminate potential unknown reactions;
in the high-temperature grouping sterilization step, at least three high-temperature sterilization containers are utilized, a steam sterilization mode is preferentially adopted, the sterilization is carried out for 20min at the pressure of approximately 104-; the known heat-resistant reaction components are: maintaining at 120 deg.C for 15min, and thermally decomposing or thermally denaturing less than 30% of the components.
Preferably, in the grouping step, when the number of the separate containers is limited, the order of the separate priorities is as follows: first preferred separation of agar and phosphate components, broadly orthophosphate, phosphite, hypophosphite and condensed phosphate and derivatives; second preferentially separating the glycoaldehyde from the amino acid protein component; the third priority is to place the animal and plant derived components separately, excluding purifications, to exclude potential unknown reactions.
Preferably, in the automatic mixing step, after the sterilization step is completed, all the components (solution state) are transferred to the same container and stirred and mixed, and the triggering condition is that the high-temperature sterilization component is cooled to a preset temperature; the automatic packaging is automatic packaging or automatic subpackaging.
Preferably, in order to synchronize the heat transfer rates of the components in the high-temperature sterilization vessel according to the physical theory of heat exchange, in the high-temperature group sterilization step, the components in the high-temperature sterilization vessel are uniformly dispersed in a solvent, the specific surface areas Sv of the liquid-containing parts among at least three high-temperature sterilization vessels are the same, and Sv is the ratio of the area S of the heat exchange wall surface to the volume V of the liquid-containing part; for a given sterilizer, the Sv of its container or chamber is constant.
The invention also discloses an automatic device of the cell culture medium, which is an implementation device of the last three steps in the preparation method of the cell culture medium, the device is an independent closed system and comprises at least one subsystem, and the functions of high-temperature grouping sterilization, automatic mixing and automatic packaging are integrated; said subsystem comprising at least three separate containers or separate chambers, being removable and replaceable as a heat sterilization container for the dispensing of said preferentially dispensed media components, thereby providing said heat sterilization function; the independent containers or the independent cavities are communicated through pipelines, and at least one fluid machine, such as a high-temperature micro pump, is arranged on the pipeline.
Under empirical guidance, the number of individual containers or individual chambers ranges from 3 to 5. Of which 3 are theoretical values and 5 are empirical values. By way of example, the medium formulation of example 1: contains animal source components and chemical components, the reaction chain A-B-C-D-E-F-G is confirmed, and 3 independent containers can be completely separated; example 2 medium formulation: contains animal source components and chemical components, and the fact that the ABCD can carry out pairwise cross reaction is confirmed, namely 6 reactions A-B, A-C, A-D, B-C, B-D, C-D are carried out, and 5 independent containers are needed when the ABCD is completely separated. Therefore, 3-5 independent containers are generally suitable. The actual formulation of various culture mediums rarely has the complex reaction relation of example 2, and an independent container is not needed to be arranged.
Preferably, in the subsystem: in order to uniformly disperse the component systems in each container or cavity, an electromagnetic stirrer rotor is arranged in at least one independent container or independent cavity, and the influence of thermodynamic property difference of different components is reduced by convection-dominated heat exchange under stirring; the main body of the pipeline is a hard pipe, and a hose is arranged at the end close to the independent container or the independent cavity so as to allow the independent container or the independent cavity to move relative to the independent container or the independent cavity.
Preferably, in the subsystem: the independent container or the independent cavity is connected with the automatic subpackaging structure through a pipeline or the top of the independent container or the independent cavity is provided with an automatic packaging structure; the automatic subpackaging structure is an independent container or an independent cavity, and an automatic packaging structure is arranged at the top of the automatic subpackaging structure.
Preferably, the automatic packaging structure is a screw thread cover or a buckle cover or a hot-pressing film seal; the screw thread cover is screwed or manually screwed by a rotary mechanical arm (semi-automatic simplified structure), and the buckle cover is pressed and clamped or manually pressed and clamped by the mechanical arm (semi-automatic simplified structure); when manually tightened or pressed, the operator only touches the outside surfaces of the container and the lid.
According to the preferable scheme, a groove is formed in the outer wall of the top of the independent container or the independent cavity, a circular ring is sleeved on the groove, the circular ring has two degrees of freedom of vertical translation and horizontal plane rotation in the groove, the circular ring is connected with a screw thread cover or a buckle cover through a revolute pair, and the revolute pair has one degree of freedom of vertical plane rotation.
Compared with the prior art, the invention has the beneficial effects that: any media formulation, regardless of the sterilization technique used, is time consuming for most laboratories; high temperature sterilization can thermally decompose many nutrients in the medium and can also lead to chemical reactions that produce toxic products. In order to overcome the defects of the prior art, the invention provides an automatic preparation method of a cell culture medium and a high-temperature sterilizer, which change the process, eliminate the main adverse reaction of high-temperature sterilization, have high automation degree and save the manpower for scientific research.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a schematic flow chart of a method for preparing a cell culture medium according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing the structure of an automated apparatus for cell culture according to an embodiment of the present invention.
Fig. 3 is a schematic structural view of the main tank in the embodiment of the present invention.
Wherein, 1, controlling the display component; 2. a pressure-bearing housing; 3. a main vessel; 4. a sub-tank; 5. a communicating pipe; 6. a high temperature micro pump; 7. an electromagnetic stirring rotor; 31. a groove; 32. a circular ring; 33. a revolute pair; 34. a threaded cap; 51. a hose section.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The examples do not show the specific experimental steps or conditions, and can be performed according to the conventional experimental steps described in the literature in the field. The reagents or instruments used are not indicated by manufacturers, and are all conventional reagent products which can be obtained commercially.
Examples
FIG. 1 is a schematic flow chart of a method for preparing a cell culture medium according to the present embodiment, which includes the following steps: grouping culture medium components, performing high-temperature grouping sterilization, automatically mixing, and automatically packaging; wherein, the three steps of high-temperature grouping sterilization, automatic mixing and automatic packaging are automatically completed in the same independent closed system.
In the step of grouping the components of the culture medium, when the distribution containers are limited, the distribution priorities are as follows: the first priority is to divide the reaction product into components with biological toxicity, the second priority is to divide other components with known reaction, and the third priority is to independently divide the components with animal and plant sources to eliminate unknown reaction.
In the high-temperature grouping sterilization step, at least three high-temperature sterilization containers are utilized, known heat-resistant reaction components are arranged in a grouping manner, and animal and plant source components are arranged separately.
In the automatic mixing step, after the sterilization step is finished, all the components (solution state) are moved to the same container and stirred and mixed, and the triggering condition is that the high-temperature sterilization component is cooled to the preset temperature.
The automatic packaging is automatic packaging or automatic subpackaging.
Fig. 2-3 are schematic structural diagrams of an automated apparatus for cell culture medium according to the present embodiment, which is a pressure-bearing apparatus for high-temperature sterilization, and integrates the functions of high-temperature group sterilization, automatic mixing, and automatic packaging. It is an independent closed system, and the structure thereof comprises: the device comprises a man-machine interaction control display assembly 1 and a pressure-bearing shell 2 with a sealed cabin cover; the inside of pressure-bearing shell 2 is equipped with three independent sterilization container, including a main container 3 and two auxiliary container 4, the top of three sterilization container is linked together through communicating pipe 5, communicating pipe 5 inserts the bottom of two auxiliary container 4, installs a high temperature micropump 6 on communicating pipe 5, and communicating pipe 5 is whole can be gone up and down by mechanical structure, and 5 main parts of communicating pipe are the rigid pipe, are the hose near mechanical structure and sterilization container end to allow sterilization container and communicating pipe 5 to take place relative displacement.
Wherein, there are electromagnetic stirring rotors 7 in the main container 3, there are grooves 31 on the outer wall of the top of the main container 3, the groove 31 is fitted with the circular ring 32, the circular ring 32 has two degrees of freedom that translate up and down and rotate in the horizontal plane in the groove, the circular ring 32 is connected with screw thread cover 34 through revolute pair 33, revolute pair 33 has a degree of freedom that the vertical plane rotates, therefore the screw thread cover 34 has three degrees of freedom; after the high-temperature grouping sterilization and automatic mixing steps are finished, the communicating pipe 5 is mechanically lifted until the communicating pipe is completely separated from the main container 3, the screw thread cover 34 automatically falls back under the action of gravity, and the opening of the main container 3 is closed; finally, the threaded cap 34 is tightened, either mechanically automatically or manually, to achieve a seal.
Manually placing the cell culture medium components in the main container 3 and the two auxiliary containers 4 according to the placing priority, and then adopting the automatic equipment to operate, wherein the main working steps are as follows:
the first step is as follows: high-temperature high-pressure sterilization: sterilizing for 20min in steam sterilization mode at a pressure of 120kPa and a temperature of 123 ℃;
the second step is that: automatic mixing: after the sterilization step, when the temperature of the solution in each container is reduced to a preset value, the high-temperature micro pump 6 automatically starts to work, the solution in the auxiliary container 4 is pumped into the main container 3, and is fully stirred and fully mixed by the electromagnetic stirring rotor 7;
the third step: automatic packaging: after the automatic mixing step is completed, the communicating tube 5 is mechanically lifted until it is completely separated from the main container 3, the screw cap 34 automatically falls down due to gravity, the opening of the main container 3 is closed, and the screw cap 34 is mechanically and automatically screwed to achieve sealing.
The preparation method of the cell culture medium and the automation equipment in the embodiment change the process in the prior art, eliminate the main adverse reaction of high-temperature sterilization, have high automation degree, and save scientific research manpower.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures made by using the contents of the present specification and the drawings can be directly or indirectly applied to other related technical fields, and are within the scope of the present invention.
Claims (10)
1. A method of preparing a cell culture medium, comprising the steps of: grouping culture medium components, performing high-temperature grouping sterilization, automatically mixing, and automatically packaging; wherein, the three steps of high-temperature grouping sterilization, automatic mixing and automatic packaging are automatically completed in the same independent closed system.
2. The method for preparing a cell culture medium according to claim 1, wherein in the step of grouping medium components, when the number of separate containers is limited, the order of separate priorities is: the first is to divide the reaction product into components with biological toxicity, the second is to divide other components with known reaction, and the third is to divide the components with animal and plant sources into parts; in the high-temperature grouping sterilization step, at least three high-temperature sterilization containers are utilized, known heat-resistant reaction components are arranged in a grouping manner, and animal and plant source components are arranged separately.
3. The method for preparing a cell culture medium according to claim 1, wherein the medium is an agar medium; in the step of grouping the components of the agar culture medium, when the number of the containers to be placed is limited, the priority order of the placement is as follows: the first is to preferentially separate agar and phosphate components, the second is to preferentially separate sugar aldehyde and amino acid protein components, and the third is to preferentially and separately place animal and plant source components.
4. The method for preparing a cell culture medium according to claim 1, wherein in the automatic mixing step, after the sterilization step is completed, all the components are transferred to the same vessel and mixed with stirring under a trigger condition that the high-temperature sterilized components are cooled to a predetermined temperature; the automatic packaging is automatic packaging or automatic subpackaging.
5. The method for preparing a cell culture medium according to claim 2, wherein in the high-temperature group sterilization step, the components in the high-temperature sterilization vessel are uniformly dispersed in the solvent, and the specific surface areas Sv of the liquid-containing portions among at least three high-temperature sterilization vessels are the same, and Sv is the ratio of the area S of the heat exchange wall surface to the volume V of the liquid-containing portion.
6. An automated apparatus for the cell culture medium which performs the last three steps of the cell culture medium preparation process according to any one of claims 1 to 5, wherein the apparatus is a self-contained, closed system comprising at least one subsystem; the subsystem comprises at least three independent containers or independent cavities and is used for high-temperature sterilization; the independent containers or the independent cavities are communicated through pipelines, and at least one fluid machine is arranged on each pipeline.
7. The automated apparatus of cell culture media of claim 6, wherein in the subsystem: an electromagnetic stirrer rotor is arranged in at least one independent container or independent cavity; the main body of the pipeline is a hard pipe, and a hose is arranged at the end close to the independent container or the independent cavity.
8. The automated apparatus of cell culture media of claim 6, wherein in the subsystem: the independent container or the independent cavity is connected with the automatic subpackaging structure through a pipeline or the top of the independent container or the independent cavity is provided with an automatic packaging structure; the automatic subpackaging structure is an independent container or an independent cavity, and an automatic packaging structure is arranged at the top of the automatic subpackaging structure.
9. The automated apparatus of claim 8, wherein the automated packaging structure is a threaded cap or a snap cap or a heat and pressure film seal; the screw thread cover is screwed or manually screwed by a rotary mechanical arm, and the buckle cover is pressed and clamped or manually pressed and clamped by the mechanical arm.
10. The apparatus of claim 8 or 9, wherein the independent container or the independent chamber has a groove on the top outer wall, and the groove is covered with a ring, the ring has two degrees of freedom for translation up and down and rotation in the horizontal plane, the ring is connected with the screw cap or the snap cap through a revolute pair, and the revolute pair has one degree of freedom for rotation in the vertical plane.
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| CN202110457733.8A CN113373038A (en) | 2021-04-27 | 2021-04-27 | Preparation method of cell culture medium and automatic equipment |
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