High-efficiency culture system for large-scale cell production and culture
Technical Field
The utility model relates to a cell industrial production technical field especially relates to a high efficiency culture system of large-scale cell production and cultivation.
Background
Cell culture is a culture process in which cells are bred and proliferated using nutrients and the like in an artificial environment and used for various purposes. Cell culture is widely used in various fields such as scientific research, biopharmaceuticals, regenerative medicine, clinical applications, genetic engineering, and the like.
With the progress of society and the vigorous development of science and technology, human beings have higher expectations on life quality and life expectancy. Having a healthy, happy and happy life cycle is a dream for everyone. However, like the stubborn diseases such as tumor, the side effects of the traditional treatment means can cause much pain to the patients.
The cell therapy is a new disease treatment technology which is emerging in recent years, and means that the characteristics of certain cells with specific functions are utilized, and the cells are obtained by adopting a biological engineering method, are amplified in vitro, cultured and modified, and have the treatment effects of enhancing the immune killing capacity, specifically identifying pathogens and tumor cells, promoting the regeneration of tissues and organs, recovering organisms and the like, so that the aim of treating diseases is fulfilled.
The technology provides a new choice for treating certain intractable diseases by using unique advantages of good curative effect, low side effect, individuation and the like. At present and in the long future, cell therapy will play an important role in clinical treatment, and the twenty-first century will be an age in which cell therapy plays an important role.
The "in vitro culture of cells" is the most critical and important part of the cell therapy technology. It is the basis of cell therapy and is directly related to the effect and safety of the therapy. In cell therapy, the number of cells to be returned to a patient is huge, and the quality requirement is extremely high, so that a hundred-grade workbench in an extremely strict ten-thousand-grade laboratory environment needs to be cultured for a long time, and a professional culture worker specially trained for a long time needs to operate in the culture process. Since the cultured cells are eventually to be returned to the human body, the safety of the cultured cells must be ensured, absolutely free from contamination. Therefore, maintaining a sterile environment in the cell culture process takes a large part of the whole process, and the consumed manpower, material resources and financial resources are immeasurable, but even in a laboratory with perfect equipment, if the technical operation of an operator is not standard, pollution can be caused, and finally a series of treatment schemes are delayed, so that the treatment of patients is damaged. The mass culture of cells with high requirements as the key of cell therapy cannot meet the increasing demands of scientific research, biological pharmacy, regenerative medicine, clinical application, genetic engineering and the like only by depending on the culture of specially-assigned people in the laboratory environment, and has a plurality of defects. There is therefore a need for a new cell production system and its associated kit of parts.
SUMMERY OF THE UTILITY MODEL
The utility model relates to a high efficiency culture system of extensive cell production and cultivation compares in current cell culture technique, the utility model discloses a cell culture bag that can completely cut off polluted environment carries out cell culture's overall process, can simplify cell culture's step, reduces cell culture's harsh requirement, saves the cost. Adopt ventilative cell culture bag to carry out cell culture, only need guarantee that the environment in the cell culture bag is sterile environment can, need not to require the aseptic laboratory of culture environment and workstation, reduced the pollution risk that adopts manual disinfection process to exist in the laboratory, can greatly guarantee cell culture's security, simplify the cell culture step. Use the cell culture bag of the utility model, only need can carry out the cultivation of cell under clean environment. Secondly, the utility model discloses an among the cell culture process required step very simplify, need not each item technical requirement of traditional cell culture, need not to carry out long-time professional training to the cell culture personnel, can operate, can reduce the degree of difficulty of cell culture, is fit for the big batch culture of cell.
In order to achieve the above purpose, the utility model adopts the technical proposal that:
a high efficiency culture system for large scale cell production and culture comprising at least one cell culture bag and at least one cell culture medium storage bag;
the cell culture bag is used for cell culture, different specifications exist in the cell culture bag, connecting pipes are arranged at the sealed edge of the lower end of the cell culture bag at intervals, each connecting pipe comprises a first connecting pipe, a second connecting pipe and a third connecting pipe, the first connecting pipe is used for infusing culture medium, the second connecting pipe is used for outputting cells, and the third connecting pipe is used for inputting cells;
the cell culture medium storage bag is used for storing prepared cell culture medium and infusing the cell culture medium for cell culture of the cell culture bag, the cell culture medium storage bag has different specifications, a connecting pipe is arranged at the sealing edge of the lower end of the cell culture medium storage bag, the connecting pipe comprises a fourth connecting pipe and a fifth connecting pipe, and the fourth connecting pipe and the fifth connecting pipe are used for infusing the cell culture medium into the cell culture bag;
and the pipe orifices of the first connecting pipe, the second connecting pipe, the third connecting pipe, the fourth connecting pipe and the fifth connecting pipe are sealed in a heat seal mode.
On the basis of the scheme, the cell culture bag at least comprises a first cell culture bag and a second cell culture bag, and when the connecting pipe of the first cell culture bag is connected with the connecting pipe of the second cell culture bag, the first cell culture bag is connected with the second cell culture bag by adopting a pipe connection method;
when the connecting pipe of the cell culture bag is connected with the connecting pipe of the cell culture medium preservation bag, the connecting pipes are connected by adopting a pipe connection method,
the pipe connection method is characterized in that heat sealing connection or heat cutting disconnection is carried out in an aseptic heat sealing mode, and aseptic pollution is guaranteed through high-temperature instant operation.
On the basis of the scheme, when the second connecting pipe of the first cell culture bag is connected with the third connecting pipe of the second cell culture bag, the second connecting pipe of the first cell culture bag is connected with the third connecting pipe of the second cell culture bag by adopting a pipe connection method;
when the first connecting pipe of the cell culture bag is connected with the fourth connecting pipe or the fifth connecting pipe of the cell culture medium storage bag, the first connecting pipe and the fifth connecting pipe are connected by adopting a pipe connection method.
The cell culture bags are divided into different specifications in terms of volume in order to ensure that the concentration of cells cultured in the bags is maintained in an effective interval, and the cell culture bags of different specifications form the complete culture system. The specifications of cell culture bags can be continuously extended according to the number of cells actually required. During cell culture, according to the initial number of cells, generally, the cell culture is started from a small cell culture bag, and the proper cell concentration cannot be maintained in the volume of the culture solution of the original cell culture bag, then the original cell culture bag is connected with a cell input connecting pipe of a cell culture bag with a first-grade specification through a cell output connecting pipe, and the cells and the culture solution are transferred in different cell culture bags through heat sealing of a connecting pipe; after the transfer is completed, separating the two cell culture bags by thermal cutting;
to maintain cell density during culture, it is necessary to add cell culture medium as the cell density increases. The culture medium infusion connecting pipe of the cell culture medium storage bag is connected with the culture medium input connecting pipe of the cell culture bag, the cell culture solution is input into the cell culture bag through heat sealing, the cell culture bag and the cell culture medium storage bag connecting pipe are separated through heat cutting after infusion is finished, and hot-pressing sealing is carried out.
On the basis of the scheme, the specifications of the cell culture bags comprise 50ml, 200ml, 2000ml and 5000ml, the specifications of the cell culture medium storage bags comprise 50ml, 100ml and 200ml, cell culture bags and cell culture medium storage bags of different specifications are designed for ensuring that the proper cell culture concentration is maintained in the cell culture process, the proper cell culture concentration is maintained by adding cell culture medium into the cell culture medium storage bags or replacing the cell culture bags of larger specifications in the cell culture process, and the cell culture bags and the cell culture medium storage bags of corresponding specifications are adopted according to the requirements of different cell planting densities.
On the basis of the scheme, scales are arranged on the cell culture medium storage bag body, so that the volume of the infusion culture medium can be conveniently adjusted.
On the basis of the scheme, the cell culture bag and the connecting pipe are made of high-air-permeability materials which meet the standard of a plastic water-soluble injection container.
On the basis of the scheme, the high-air-permeability material can be selected from polyethylene, TPX, EVA and the like.
On the basis of the scheme, the cell culture medium storage bag is made of an airtight material with high barrier property, and the airtight material is adopted, so that the safe and sterile environment of the culture medium can be effectively guaranteed, and the cell culture medium storage bag can be prepared in a large amount at one time.
The application method of the high-efficiency culture system for large-scale cell production and culture comprises the following steps:
1. preparing a cell culture medium, placing the cell culture medium in cell culture medium storage bags, preparing a plurality of cell culture medium storage bags at one time, and placing the cell culture medium storage bags in an environment of 4 ℃ for cold storage for later use; when in use, the cell culture medium storage bag filled with the cell culture medium is heated to 37 ℃;
2. injecting cells to be cultured into the cell culture bag through a third connecting pipe, simultaneously using a pipe connection method to thermally connect a fourth connecting pipe or a fifth connecting pipe of the cell culture medium storage bag with a first connecting pipe of the cell culture bag through an aseptic heat-seal pipe connector, injecting the cell culture medium into the cell culture bag through extrusion, cutting off the fourth connecting pipe or the fifth connecting pipe and the first connecting pipe at high temperature in an instant by using the aseptic heat-seal pipe connector after the cell culture medium is completely infused, and thermally sealing the pipe orifices of the fourth connecting pipe or the fifth connecting pipe and the first connecting pipe to separate the cell culture bag from the cell culture medium storage bag;
3. in the cell culture process, the cell culture concentration is measured by a turbidimetric method, when the cell culture is not at the proper culture concentration, a cell culture medium needs to be injected, a cell culture medium storage bag is taken, and a fourth connecting pipe or a fifth connecting pipe connected with the cell culture medium storage bag is thermally connected with a first connecting pipe connected with the cell culture bag by adopting an aseptic heat-sealing pipe connecting machine, so that the cell culture medium in the cell culture medium storage bag can be effectively extruded into the cell culture bag in an aseptic environment;
4. after the cell culture medium is infused, the fourth connecting pipe or the fifth connecting pipe and the first connecting pipe are cut off instantly at high temperature by adopting an aseptic heat sealing pipe connecting machine, and the pipe orifices of the fourth connecting pipe or the fifth connecting pipe and the first connecting pipe are sealed in a heat sealing way, so that the cell culture bag is separated from the cell culture medium storage bag;
5. along with the increase of the culture time in the cell culture process, when the cell culture bag can not ensure the proper culture concentration of the cells, the cell culture bag with a larger size needs to be replaced, the cells cultured in the original cell culture bag are transferred into the cell culture bag with a larger size through the second connecting pipe by the sterile heat-sealing pipe connecting machine, and the cells are continuously cultured;
6. after the cells cultured in the original cell culture bag are transferred, the original cell culture bag and the existing cell culture bag are separated by a sterile heat sealing pipe connecting machine.
On the basis of the scheme, the cell culture medium comprises cell production factors, a serum-free culture medium and a basic culture medium.
On the basis of the scheme, cell types cultured by the cells comprise suspension cells and adherent cells, the suspension cell types comprise immune cells, and the adherent cell types comprise mesenchymal stem cells, IPS (in-plane switching) and induced differentiation stem cells thereof; the cell culture mode comprises suspension culture and adherent culture.
On the basis of the scheme, the cell culture concentration in the cell culture bag is determined by adopting a turbidimetric method, and the cell culture concentration is determined by measuring the absorbance of a cell culture solution by using a spectrophotometer and comparing with a cell concentration standard curve.
On the basis of the scheme, the cell culture bag is cultured in a cell culture box, and the cell culture box is CO2A cell culture box.
Has the advantages that:
1. the cell culture bag that adopts among this cell culture system, the security is high, simple structure, reasonable in design, convenient to use, preserve the bag through setting up cell culture bag and cell culture medium, it is very safe convenient during the use, adopt aseptic heat seal pipe connecting machine heat seal to connect when needs connect between each cell culture bag, reduced the probability that cell pollution and operating personnel infected, improved the security of operation, improved cell culture's work efficiency, can be exclusively used in the cell culture case.
2. The cell culture bag of the system adopts a breathable material which meets the standard of a plastic water-soluble injection container, and has no dissolved substance in the cell culture process and no plasticizer or dissolved substance which has adverse effect on cells; high-permeability polyethylene, TPX or EVA and the like are adopted, so that the requirements of gas concentration environment and pH value during cell culture can be met; the connecting pipes are adopted for connection, and the operation safety is improved through direct contact operation when a cell culture medium is injected or a sample is taken; compared with a culture bottle, the gas exchange area is large, the culture in a large amount in a limited culture space is facilitated, a larger number of cells can be obtained, the closed culture is realized, and the probability of cell pollution and operator infection is reduced; the cell culture bag is made of transparent and flexible materials, so that the state of cells can be directly observed under a microscope conveniently.
The utility model discloses a technical key point with want the guard point:
1. the utility model discloses mainly be a large-scale cell production that uses under the conventional environment and cultivate high efficiency culture system with cultivateing, preserve the bag including cell culture bag and cell culture medium, each bag mutual independence, and each bag has different specifications.
2. The cell culture bag adopts the ventilative material that accords with plastics water-soluble injection container standard, is connected with three connecting pipes, is first connecting pipe, second connecting pipe and third connecting pipe respectively, and the cell culture medium is preserved the bag and is ventilative material, connects fourth connecting pipe, fifth connecting pipe.
3. The bag bodies are in an independent state at ordinary times, and when culture fluid needs to be infused, a sterile heat sealing pipe connecting machine is adopted to ensure that heat sealing connection and disconnection are carried out under a sterile state.
4. In the cell culture, cell culture bags with different sizes are selected for culture according to the culture density of different cells so as to ensure the optimal concentration of cell growth.
5. The cell culture medium storage bag can be used for preparing a large amount of cell culture medium at one time, the cell culture medium can be used up in one week, preparation at any time is not needed, and scales are arranged on the cell culture medium storage bag body.
6 the cell culture system can culture cell types including suspension cells and adherent cells.
Drawings
The utility model discloses there is following figure:
FIG. 1 is a schematic diagram of a cell culture bag.
FIG. 2 is a schematic diagram of a cell culture medium storage bag.
Detailed Description
The present invention will be described in further detail with reference to fig. 1 and 2.
A high efficiency culture system for large scale cell production and culture comprising at least one cell culture bag and at least one cell culture medium storage bag;
the cell culture bag is used for cell culture, different specifications exist in the cell culture bag, connecting pipes are arranged at the sealed edge of the lower end of the cell culture bag at intervals, the connecting pipes comprise a first connecting pipe 1, a second connecting pipe 2 and a third connecting pipe 3, the first connecting pipe 1 is used for infusing a culture medium, the second connecting pipe 2 is used for outputting cells, and the third connecting pipe 3 is used for inputting cells;
the cell culture medium storage bag is used for storing prepared cell culture medium and infusing the cell culture medium for cell culture of the cell culture bag, the cell culture medium storage bag has different specifications, a connecting pipe is arranged at the sealing edge of the lower end of the cell culture medium storage bag, the connecting pipe comprises a fourth connecting pipe 4 and a fifth connecting pipe 5, and the fourth connecting pipe 4 and the fifth connecting pipe 5 are used for infusing the cell culture medium into the cell culture bag;
the pipe orifices of the first connecting pipe 1, the second connecting pipe 2, the third connecting pipe 3, the fourth connecting pipe 4 and the fifth connecting pipe 5 are sealed in a heat seal mode.
On the basis of the scheme, the cell culture bag at least comprises a first cell culture bag and a second cell culture bag, and when the connecting pipe of the first cell culture bag is connected with the connecting pipe of the second cell culture bag, the first cell culture bag is connected with the second cell culture bag by adopting a pipe connection method;
when the connecting pipe of the cell culture bag is connected with the connecting pipe of the cell culture medium preservation bag, the connecting pipes are connected by adopting a pipe connection method,
the pipe connection method is characterized in that heat sealing connection or heat cutting disconnection is carried out in an aseptic heat sealing mode, and aseptic pollution is guaranteed through high-temperature instant operation.
On the basis of the scheme, when the second connecting pipe 2 of the first cell culture bag is connected with the third connecting pipe 3 of the second cell culture bag, the connection is carried out by adopting a pipe connection method;
when the first connecting pipe 1 of the cell culture bag is connected with the fourth connecting pipe 4 or the fifth connecting pipe 5 of the cell culture medium storage bag, the first connecting pipe and the fifth connecting pipe are connected by adopting a pipe connection method.
On the basis of the scheme, the specifications of the cell culture bags comprise 50ml, 200ml, 2000ml and 5000ml, the specifications of the cell culture medium storage bags comprise 50ml, 100ml and 200ml, the cell culture bags and the cell culture medium storage bags with different specifications are designed for ensuring that the proper cell culture concentration is maintained in the cell culture process, the proper cell culture concentration is maintained by adding culture media into the cell culture medium storage bags or replacing the cell culture bags with larger specifications in the cell culture process, and the cell culture bags and the cell culture medium storage bags with corresponding specifications are adopted according to the requirements of different cell planting densities.
On the basis of the scheme, scales are arranged on the cell culture medium storage bag body, so that the volume of the infusion culture medium can be conveniently adjusted.
On the basis of the scheme, the cell culture bag and the connecting pipe are made of high-air-permeability materials which meet the standard of a plastic water-soluble injection container.
On the basis of the scheme, the high-air-permeability material can be selected from polyethylene, TPX, EVA and the like.
On the basis of the scheme, the cell culture medium storage bag is made of an airtight material with high barrier property, and the airtight material is adopted, so that the safe and sterile environment of the culture medium can be effectively guaranteed, and the cell culture medium storage bag can be prepared in a large amount at one time.
The application method of the high-efficiency culture system for large-scale cell production and culture comprises the following steps:
1. preparing a cell culture medium, placing the cell culture medium in cell culture medium storage bags, preparing a plurality of cell culture medium storage bags at one time, and placing the cell culture medium storage bags in an environment of 4 ℃ for cold storage for later use; when in use, the cell culture medium storage bag filled with the cell culture medium is heated to 37 ℃;
2. injecting cells to be cultured into the cell culture bag through the third connecting pipe 3, simultaneously thermally connecting the fourth connecting pipe 4 or the fifth connecting pipe 5 of the cell culture medium storage bag with the first connecting pipe 1 of the cell culture bag through an aseptic heat-seal connecting pipe machine by using a pipe connection method, injecting the cell culture medium into the cell culture bag through extrusion, cutting off the fourth connecting pipe 4 or the fifth connecting pipe 5 and the first connecting pipe 1 instantly at high temperature by using the aseptic heat-seal connecting pipe machine after the cell culture medium is completely infused, and thermally sealing the pipe orifices of the fourth connecting pipe 4 or the fifth connecting pipe 5 and the first connecting pipe 1 to separate the cell culture bag from the cell culture medium storage bag;
3. in the cell culture process, the cell culture concentration is measured by a turbidimetric method, when the cell culture is not at the proper culture concentration, a cell culture medium needs to be injected, a cell culture medium storage bag is taken, and a fourth connecting pipe 4 or a fifth connecting pipe 5 connected with the cell culture medium storage bag is connected with a first connecting pipe 1 connected with the cell culture bag in a heat sealing way by adopting an aseptic heat sealing pipe connecting machine, so that the cell culture medium in the cell culture medium storage bag can be effectively extruded into the cell culture bag in an aseptic environment;
4. after the cell culture medium is infused, the fourth connecting pipe 4 or the fifth connecting pipe 5 and the first connecting pipe 1 are cut off instantly at high temperature by adopting an aseptic heat sealing pipe connecting machine, and the pipe orifices of the fourth connecting pipe 4 or the fifth connecting pipe 5 and the first connecting pipe 1 are sealed in a heat sealing way, so that the cell culture bag is separated from the cell culture medium storage bag;
5. along with the increase of the culture time in the cell culture process, when the cell culture bag can not ensure the proper culture concentration of the cells, the cell culture bag with a larger size needs to be replaced, the cells cultured in the original cell culture bag are transferred into the cell culture bag with a larger size through the second connecting pipe 2 by the sterile heat-sealing pipe connecting machine, and the culture is continued;
6. after the cells cultured in the original cell culture bag are transferred, the original cell culture bag and the existing cell culture bag are separated by a sterile heat sealing pipe connecting machine.
On the basis of the scheme, the cell culture medium comprises cell production factors, a serum-free culture medium and a basic culture medium.
On the basis of the scheme, the cell types cultured by the cell comprise suspension cells and adherent cells, the suspension cell types comprise immune cells, and the adherent cell types comprise mesenchymal stem cells, IPS (in-plane switching) and induced differentiation stem cells thereof.
On the basis of the scheme, the cell culture concentration in the cell culture bag is determined by adopting a turbidimetric method, and the cell culture concentration is determined by measuring the absorbance of a cell culture solution by using a spectrophotometer and comparing with a cell concentration standard curve.
On the basis of the scheme, the cell culture bag is cultured in a cell culture box, and the cell culture box is CO2A cell culture box.
The cell culture concentration in the cell culture process is judged by a turbidimetry method, (a standard cell concentration curve is drawn by adopting a standard cell method in the early stage, the addition amount of a cell culture medium is obtained by the curve) to obtain the culture state of the cells, and after the culture state is determined, the cell culture concentration in the culture process is ensured by adjusting the amount of the cell culture medium (infusing the cell culture medium) and the size of a cell culture bag (replacing the cell culture bag), so that the optimal culture effect is ensured.
Example 1:
selecting a high-permeability plastic material suitable for cell culture, preparing a cell culture bag in a mode of a figure 1, welding a connecting pipe at the lower end of the cell culture bag in a high-frequency heat sealing mode, wherein the second connecting pipe 2 is used for outputting cells so as to be collected; the first connection tube 1 is used for the infusion of culture medium; the third connecting tube 3 is used for the input of cells.
Example 2:
selecting a high-barrier airtight plastic material, preparing the plastic material into a cell culture medium storage bag in a mode shown in figure 2, welding a connecting pipe at the lower end of the cell culture medium storage bag in a high-frequency heat sealing mode, wherein a fourth connecting pipe 4 and a fifth connecting pipe 5 are used for infusing a cell culture medium into the cell culture bag; the cell culture medium preservation bag body is provided with scales.
Example 3
In an open environment, after the first connecting pipe 1 of the cell culture bag is thermally connected with the fourth connecting pipe 4 or the fifth connecting pipe 5 of the cell culture medium storage bag by using an aseptic heat-sealing pipe connecting machine, fresh cell culture medium is injected into the cell culture bag for cell amplification culture.
Those not described in detail in this specification are within the skill of the art.