WO2018137428A1 - 一种大规模细胞生产和培养的高效率培养系统及应用方法 - Google Patents

一种大规模细胞生产和培养的高效率培养系统及应用方法 Download PDF

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WO2018137428A1
WO2018137428A1 PCT/CN2017/115779 CN2017115779W WO2018137428A1 WO 2018137428 A1 WO2018137428 A1 WO 2018137428A1 CN 2017115779 W CN2017115779 W CN 2017115779W WO 2018137428 A1 WO2018137428 A1 WO 2018137428A1
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cell culture
bag
connecting tube
cell
culture medium
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PCT/CN2017/115779
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English (en)
French (fr)
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顾雨春
刘建强
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北京呈诺医学科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/14—Bags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps

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  • the invention relates to the technical field of cell industrial production, in particular to a high-efficiency culture system and application method for large-scale cell production and culture.
  • Cell culture is a culture process in which cells are cultured and propagated in an artificial environment using nutrients and the like, and used for various purposes. Cell culture is now widely used in research, biopharmaceutical, regenerative medicine, clinical applications and genetic engineering.
  • Cell therapy is a new disease treatment technology that has emerged in recent years. It refers to the use of certain cells with specific functions, bioengineering methods, and in vitro amplification and culture modification to enhance the immune killing ability of these cells. It specifically recognizes pathogens and tumor cells, promotes tissue and organ regeneration, and rejuvenates the body to achieve therapeutic goals.
  • This technology offers new options for the treatment of refractory diseases with its unique advantages such as good efficacy, low side effects and personalization.
  • cell therapy will play an important role in clinical treatment, and the 21st century will be an era in which cell therapy plays an important role.
  • Cell culture in vitro is the most critical and important part of cell therapy technology. It is the basis of cell therapy and is directly related to the effectiveness and safety of the treatment. In cell therapy, the number of cells returned to the patient is huge, and the quality requirements are extremely high. Therefore, it is necessary to carry out long-term cultivation in a very strict tens of thousands of benches in a 10,000-scale laboratory environment, and it is necessary to have a special experience in the cultivation process. Long-trained professional trainers operate. Since the cultured cells eventually need to be returned to the human body, it is necessary to ensure the safety of the cultured cells and to be absolutely free from contamination.
  • the invention relates to a high-efficiency culture system and application method for large-scale cell production and culture.
  • the invention adopts a cell culture bag capable of isolating the environment to carry out the whole process of cell culture, which can be simplified.
  • the step of cell culture reduces the stringent requirements of cell culture and saves costs.
  • the cell culture bag of the present invention cell culture can be carried out only in a clean environment.
  • the steps required in the cell culture process of the present invention are extremely simplified, and the technical requirements of the conventional cell culture are not required, and the cell culture personnel can operate without long-time professional training, and can reduce the difficulty of cell culture, and is suitable for cells. Large-scale cultivation.
  • 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 preservation bag;
  • the cell culture bag is used for cell culture, and the cell culture bag has different specifications, and the cell culture bag has a connection tube at a sealing edge at a lower end thereof, the connection tube includes a first connection tube, a second connection tube, and a third connecting tube, the first connecting tube is for infusion of the medium, the second connecting tube is for outputting the cells, and the third connecting tube is for inputting the cells;
  • the cell culture medium storage bag is used for storing the prepared cell culture medium, and the cell culture medium is infused into the cell culture medium of the cell culture bag, and the cell culture medium storage bag has different specifications, and the cell culture medium stores the lower end sealing edge of the bag.
  • a connecting tube is provided, the connecting tube includes a fourth connecting tube and a fifth connecting tube, and the fourth connecting tube and the fifth connecting tube are used for injecting the cell culture medium into the cell culture bag;
  • the nozzles of the first connecting pipe, the second connecting pipe, the third connecting pipe, the fourth connecting pipe and the fifth connecting pipe are sealed by heat sealing.
  • the cell culture bag comprises at least a first cell culture bag and a second cell culture bag, and the connecting tube of the first cell culture bag and the connecting tube of the second cell culture bag When connecting, use the take-over method to connect;
  • connection method When the connecting tube of the cell culture bag is connected to the connecting tube of the cell culture medium storage bag, the connection method is used,
  • connection method is connected, and the heat sealing connection or the heat cutting is performed by means of aseptic heat sealing, and the aseptic contamination is ensured by the high temperature instantaneous operation.
  • the first connecting tube of the cell culture bag When the first connecting tube of the cell culture bag is connected to the fourth connecting tube or the fifth connecting tube of the cell culture medium storage bag, it is connected by a pipe connection method.
  • the cell culture bag ensures that the concentration of the cells cultured in the bag is maintained within an effective range, and the cell culture bags are divided into different specifications from the volume, and the cell culture bags of different specifications constitute the complete culture system.
  • the size of the cell culture bag can be continuously extended depending on the number of cells actually needed.
  • the original cell culture bag is passed through the cell output connection tube and the first stage.
  • the cell culture bag of the specification is input into the connecting tube, and the transfer of the cells and the culture solution in different cell culture bags is completed by taking the heat sealing; after the transfer is completed, the two cell culture bags are separated by thermal cutting;
  • the medium infusion connection tube of the cell culture storage bag is connected with the medium input connection tube of the cell culture bag, and the cell culture medium is input into the cell culture bag by heat sealing, and after the infusion is completed, the cell culture bag and the cell culture are separated by thermal cutting.
  • the base holds the bag connection tube and is heat sealed.
  • the specifications of the cell culture bag include 50ml, 200ml, 2000ml, 5000ml
  • the specifications of the cell culture storage bag include 50ml, 100ml, and 200ml, in order to ensure a suitable cell culture concentration during cell culture, Design cell culture bags and cell culture medium storage bags of different specifications, and maintain cell culture medium by adding cell culture medium to cell culture medium storage bags or replacing larger cell culture bags during cell culture. Different cell planting densities are required, and cell culture bags and cell culture media storage bags of corresponding sizes are used.
  • the cell culture medium storage bag is provided with a scale to facilitate adjustment of the amount of the infusion medium.
  • the cell culture bag and the connecting tube are made of a highly gas permeable material conforming to the standard of the plastic water-soluble injection container.
  • the high gas permeability material may be selected from polyethylene, TPX or EVA.
  • the cell culture medium storage bag is made of a gas barrier material with high barrier property, and is made of a gas-impermeable material, which can effectively ensure a safe and sterile environment of the culture medium, and can be prepared in one batch. .
  • the method for applying the high-efficiency culture system of the large-scale cell production and culture comprises the following steps:
  • the cells to be cultured are injected into the cell culture bag through the third connecting tube, and the fourth or fifth connecting tube of the cell culture medium storage bag is passed through the sterile heat sealing tube machine and the cell culture bag by using the nozzle method.
  • the first connecting tube is heat-sealed, and the cell culture medium is injected into the cell culture bag by extrusion.
  • the fourth connecting tube or the fifth connecting tube and the first connection are instantaneously cut off by the high temperature of the aseptic heat sealing machine. And sealing the tube of the fourth connecting tube or the fifth connecting tube and the first connecting tube to separate the cell culture bag from the cell culture storage bag;
  • the cell culture concentration is determined by the turbidimetric method.
  • the cell culture medium needs to be added, the cell culture medium storage bag is taken, and the aseptic heat sealing tube machine is used.
  • the fourth connecting tube or the fifth connecting tube connected to the cell culture storage bag is heat-sealed to the first connecting tube connected to the cell culture bag, thereby effectively ensuring that the cell culture medium is preserved in the cell culture medium in the bag under aseptic conditions. Squeeze into a cell culture bag;
  • the fourth connecting tube or the fifth connecting tube and the first connecting tube are instantaneously cut off by the high temperature of the aseptic heat sealing machine, and the fourth connecting tube or the fifth connecting tube is connected with the first connecting tube.
  • the tube mouth is heat sealed to separate the cell culture bag from the cell culture medium storage bag;
  • the original cell culture bag and the current cell culture bag can be separated by a sterile heat sealing machine.
  • the cell culture medium includes a cell production factor, a serum-free medium, and a basal medium.
  • the cell culture cell species include suspension cells and adherent cells, the suspension cell species including immune cells, the adherent cell species including mesenchymal stem cells and IPS and induced differentiation stem cells;
  • Cell culture methods include suspension culture and adherent culture.
  • the cell culture concentration in the cell culture bag is determined by a turbidimetric method, and the cell culture concentration is determined by measuring the absorbance of the cell culture solution using a spectrophotometer and comparing the cell concentration standard curve.
  • the cell culture bag is cultured in a cell culture incubator, and the cell culture incubator is a CO 2 cell incubator.
  • the cell culture bag used in the cell culture system has high safety, simple structure, reasonable design and convenient use. It is extremely safe and convenient to use by setting the cell culture bag and the cell culture medium storage bag, and each cell culture bag is used.
  • the use of a sterile heat-sealing machine for heat-sealing when connecting is required, which reduces the risk of cell contamination and operator infection, improves the safety of operation, and improves the efficiency of cell culture, and can be dedicated to the cell culture incubator.
  • the cell culture bag of the system adopts the gas permeable material conforming to the standard of plastic water-soluble injection container, no dissolution material in the cell culture process, and no plasticizer or dissolution material which adversely affects the cells;
  • the polyethylene with high gas permeability , TPX or EVA, etc. can meet the gas concentration environmental requirements and pH requirements during cell culture; using a connecting tube connection, when injecting cell culture medium or sampling, through direct contact operation, improve the safety of operation;
  • the gas exchange area is large, which facilitates large-scale cultivation in a limited culture space, and can obtain a larger number of cells, and the closed culture reduces the probability of cell contamination and operator infection;
  • the cell culture bag is transparent and flexible. It is convenient to directly observe the state of the cells under the microscope.
  • the present invention is mainly a high-efficiency culture system for large-scale cell production and culture used in a conventional environment, including a cell culture bag and a cell culture medium preservation bag, each bag being independent of each other, and each bag has different specifications.
  • the cell culture bag adopts a gas permeable material conforming to the standard of the plastic water-soluble injection container, and is connected with three connecting pipes, which are a first connecting pipe, a second connecting pipe and a third connecting pipe respectively, and the cell culture storage bag is air-tight material.
  • the fourth connecting pipe and the fifth connecting pipe are connected.
  • the individual bags are usually in an independent state.
  • a sterile heat sealing machine is used to ensure the heat sealing connection and disconnection under aseptic conditions.
  • culture cells of different sizes are selected according to the culture density of different cells to ensure the optimal concentration of cell growth.
  • cell culture medium storage bag can be prepared in a large amount of cell culture medium, can be used within one week, no need to prepare at any time, there is a scale on the cell culture medium storage bag.
  • the cell types cultured by the cell culture system include suspended cells and adherent cells.
  • Figure 1 is a structural diagram of a cell culture bag.
  • Figure 2 is a structural diagram of a cell culture medium preservation bag.
  • 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 preservation bag;
  • the cell culture bag is used for cell culture, and the cell culture bag has different specifications.
  • the cell culture bag is provided with a connecting tube at a sealing edge of the lower end thereof, and the connecting tube includes a first connecting tube 1 and a second connecting tube. 2 and a third connecting tube 3, the first connecting tube 1 is for infusion of the medium, the second connecting tube 2 is for outputting of cells, and the third connecting tube 3 is for inputting of cells;
  • the cell culture medium storage bag is used for storing the prepared cell culture medium, and the cell culture medium is infused into the cell culture medium of the cell culture bag, and the cell culture medium storage bag has different specifications, and the cell culture medium stores the lower end sealing edge of the bag.
  • a connecting tube is provided, the connecting tube includes a fourth connecting tube 4 and a fifth connecting tube 5, and the fourth connecting tube 4 and the fifth connecting tube 5 are used for injecting a cell culture medium into the cell culture bag;
  • the nozzles 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 by heat sealing.
  • the cell culture bag comprises at least a first cell culture bag and a second cell culture bag, and the connecting tube of the first cell culture bag and the connecting tube of the second cell culture bag When connecting, use the take-over method to connect;
  • connection method When the connecting tube of the cell culture bag is connected to the connecting tube of the cell culture medium storage bag, the connection method is used,
  • connection method is connected, and the heat sealing connection or the heat cutting is performed by means of aseptic heat sealing, and the aseptic contamination is ensured by the high temperature instantaneous operation.
  • the connecting method is connected by using a pipe connection method
  • the specifications of the cell culture bag include 50ml, 200ml, 2000ml, 5000ml
  • the specifications of the cell culture storage bag include 50ml, 100ml, and 200ml, in order to ensure a suitable cell culture concentration during cell culture, Design cell culture bags and cell culture medium storage bags of different specifications, and maintain the appropriate cell culture concentration by adding medium to the cell culture storage bag or replacing larger cell culture bags during cell culture.
  • the cell planting density is required to use a cell culture bag of the corresponding size and a cell culture medium preservation bag.
  • the cell culture medium storage bag is provided with a scale to facilitate adjustment of the amount of the infusion medium.
  • the cell culture bag and the connecting tube are made of a highly gas permeable material conforming to the standard of the plastic water-soluble injection container.
  • the high gas permeability material may be selected from polyethylene, TPX or EVA.
  • the cell culture medium storage bag is made of a gas barrier material with high barrier property, and is made of a gas-impermeable material, which can effectively ensure a safe and sterile environment of the culture medium, and can be prepared in one batch. .
  • the method for applying the high-efficiency culture system of the large-scale cell production and culture comprises the following steps:
  • the cells to be cultured are injected into the cell culture bag through the third connecting tube 3, and the fourth connecting tube 4 or the fifth connecting tube 5 of the cell culture medium storage bag is passed through the sterile heat sealing tube machine and the cells by the nozzle method.
  • the first connecting tube 1 of the culture bag is heat-sealed, and the cell culture medium is injected into the cell culture bag by extrusion.
  • the fourth connecting tube 4 or the fifth connection is instantaneously cut off by the high temperature of the aseptic heat sealing machine.
  • the cell culture concentration is determined by the turbidimetric method.
  • the cell culture medium needs to be added, the cell culture medium storage bag is taken, and the aseptic heat sealing tube machine is used.
  • the fourth connecting tube 4 or the fifth connecting tube 5 connected to the cell culture storage bag is heat-sealed to the first connecting tube 1 connected to the cell culture bag, which can effectively ensure that the cell culture medium is stored in the bag under a sterile environment.
  • the cell culture medium is extruded into a cell culture bag;
  • the fourth connecting tube 4 or the fifth connecting tube 5 and the first connecting tube 1 are instantaneously cut off by the high temperature of the aseptic heat sealing machine, and the fourth connecting tube 4 or the fifth connecting tube is connected. 5 and the nozzle of the first connecting tube 1 is heat sealed to separate the cell culture bag from the cell culture medium storage bag;
  • the original cell culture bag and the current cell culture bag can be separated by a sterile heat sealing machine.
  • the cell culture medium includes a cell production factor, a serum-free medium, and a basal medium.
  • the cell culture cell species include suspension cells and adherent cells, and the suspension cell species include immune cells, and the adherent cell types include mesenchymal stem cells and IPS and induced differentiation stem cells.
  • the cell culture concentration in the cell culture bag is determined by a turbidimetric method, and the cell culture concentration is determined by measuring the absorbance of the cell culture solution using a spectrophotometer and comparing the cell concentration standard curve.
  • the cell culture bag is cultured in a cell culture incubator, and the cell culture incubator is a CO 2 cell incubator.
  • the concentration of the cultured cells is determined by the turbidimetric method.
  • the standard cell method has been used to draw the standard curve of the cell concentration, and the amount of the cell culture medium is obtained by the curve) to know the culture state of the cells, and after the culture state is determined, By adjusting the amount of cell culture medium (infusion of cell culture medium) and the size of the cell culture bag (replacement of cell culture bags), the cell culture concentration during the culture process is ensured, and the optimal culture effect is ensured.
  • a high-breathing plastic material suitable for cell culture prepare a cell culture bag according to the method of FIG. 1, and weld the connecting tube to the lower end of the cell culture bag by high-frequency heat sealing, wherein the second connecting tube 2 is used.
  • the output of the cells is collected for collection; the first connecting tube 1 is for infusion of the medium; and the third connecting tube 3 is for input of cells.
  • a high-barrier, air-tight plastic material prepare a cell culture storage bag according to the method of Figure 2, and weld the connecting tube to the lower end of the cell culture storage bag by high-frequency heat sealing, wherein the fourth connecting tube 4.
  • the fifth connecting tube 5 is used for infusing the cell culture medium into the cell culture bag; the cell culture medium storage bag is provided with a scale.
  • Fresh cell culture can be carried out by using a sterile heat sealing machine in an open environment, after the first connecting tube 1 of the cell culture bag is heat-sealed with the fourth connecting tube 4 or the fifth connecting tube 5 of the cell culture storage bag.
  • the base is injected into a cell culture bag to perform cell expansion culture.

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Abstract

一种大规模细胞生产和培养的高效率培养系统及应用方法，包括至少一个细胞培养袋和至少一个细胞培养基保存袋；所述细胞培养袋用于细胞培养，所述细胞培养基保存袋用于储存制备的细胞培养基，为细胞培养袋的细胞培养输注细胞培养基，细胞培养袋和细胞培养基保存袋均存在不同的规格，各个袋体之间平时为独立状态，需要进行细胞培养基输注时采用无菌热合接管机保证无菌状态下进行热合连接、断开。

Description

一种大规模细胞生产和培养的高效率培养系统及应用方法 技术领域
本发明涉及细胞工业化生产技术领域，尤其涉及一种大规模细胞生产和培养的高效率培养系统及应用方法。
背景技术
细胞培养是把细胞在人工环境下，利用营养物质等，使其繁育增殖，并用于各种目的的培养过程。细胞培养现已广泛应用于科研、生物制药、再生医学、临床应用和基因工程等各个领域。
随着社会的进步，科技的蓬勃发展，人类对生命质量和预期寿命也有了更高的期望。拥有一个健康、幸福、快乐的生命周期是每一个人的梦想。但如同肿瘤等顽疾，传统的治疗手段所带来的副作用也让很多的患者痛苦不堪。
细胞治疗是近几年兴起的疾病治疗新技术，是指利用某些具有特定功能的细胞的特性，采用生物工程方法获取和通过体外扩增、培养修饰后，使这些细胞具有增强免疫杀伤能力、特异性识别病原体和肿瘤细胞、促进组织器官再生和机体康复等治疗功效，从而达到治疗疾病的目的。
该技术以良好的疗效、低副作用和个性化等独特的优势，为一些难治性疾病的治疗，提供新的选择。在当前和今后很长的阶段，细胞治疗都将在临床治疗中担当重要的角色，二十一世纪将是细胞治疗发挥重要作用的时代。
细胞治疗技术中“细胞体外培养”是最为关键、重要的一环。它是细胞治疗的基础，直接关系到治疗的效果和安全性。而在细胞治疗中针对患者回输的细胞数量巨大，并且质量要求极高，因此需要在极其严格的万级实验室环境下的百级工作台进行长时间的培养，培养过程中需要有专门受过长时间培训的专业培养人员进行操作。因所培养 的细胞最终是需要回输人体，因此必须确保所培养细胞的安全性，绝对不受污染。因此在细胞培养过程中维持无菌环境占整个过程的大部分时间，所耗费的人力、物力和财力是不可估量的，但即使在设备完善的实验室内，若操作者技术操作不规范，也会导致污染，最终导致一系列的治疗方案的延迟，对患者的治疗造成危害。大批量，高要求的培养细胞作为细胞治疗的关键，仅仅依靠实验室环境下的专人培养已经无法满足日益增长的科研、生物制药、再生医学、临床应用和基因工程等需求，并且还存在很多缺点。因此亟需一种新的细胞生产系统及其相关配套部件。
发明内容
本发明为一种大规模细胞生产和培养的高效率培养系统及应用方法，相较于现有的细胞培养技术，本发明采用可以隔绝污染环境的细胞培养袋进行细胞培养的全过程，可以简化细胞培养的步骤，降低细胞培养的苛刻要求，节省成本。采用透气的细胞培养袋来进行细胞培养，仅需保证细胞培养袋内的环境是无菌环境即可，无需要求培养环境无菌的实验室和工作台，减少了实验室中采用手工消毒过程存在的污染风险，能够极大保证细胞培养的安全性，简化细胞培养步骤。使用本发明的细胞培养袋，只需在干净环境下即可进行细胞的培养。其次，本发明的细胞培养过程中所需步骤极为简化，无需传统细胞培养的各项技术要求，对于细胞培养人员无需进行长时间专业培训，即可进行操作，能够降低细胞培养的难度，适合细胞的大批量培养。
为达到以上目的，本发明采取的技术方案是：
一种大规模细胞生产和培养的高效率培养系统，包括至少一个细胞培养袋和至少一个细胞培养基保存袋；
所述细胞培养袋用于细胞培养，并且细胞培养袋存在不同的规格，所述细胞培养袋的下端密封边缘处间隔设有连接管，所述连接管包括第一连接管、第二连接管和第三连接管，第一连接管用于输注培 养基，第二连接管用于细胞的输出，第三连接管用于细胞的输入；
所述细胞培养基保存袋用于储存制备的细胞培养基，为细胞培养袋的细胞培养输注细胞培养基，细胞培养基保存袋存在不同的规格，所述细胞培养基保存袋的下端密封边缘处设有连接管，所述连接管包括第四连接管、第五连接管，第四连接管、第五连接管用于向细胞培养袋输注细胞培养基；
所述第一连接管、第二连接管、第三连接管、第四连接管、第五连接管的管口采用热合密封方式密封。
在上述方案的基础上，所述的细胞培养袋至少包括第一细胞培养袋和第二细胞培养袋，所述的第一细胞培养袋的连接管与所述的第二细胞培养袋的连接管连接时，采用接管法连接；
所述的细胞培养袋的连接管与所述的细胞培养基保存袋的连接管连接时，采用接管法连接，
所述的接管法连接，借助无菌热合方式进行热合连接或热切断开，通过高温瞬间操作保证无菌污染。
在上述方案的基础上，所述的第一细胞培养袋的第二连接管与所述的第二细胞培养袋的第三连接管连接时，采用接管法连接；
所述的细胞培养袋的第一连接管与所述的细胞培养基保存袋的第四连接管或第五连接管连接时，采用接管法连接。
所述的细胞培养袋为保证袋内培养的细胞浓度维持在一个有效区间内，细胞培养袋从体积上分为不同的规格，不同规格的细胞培养袋构成这个完整的培养体系。根据实际需求的细胞数量，细胞培养袋的规格可以不断上延。细胞培养时，根据细胞初始数量，一般从小规格细胞培养袋开始培养，到原规格细胞培养袋的培养液体积无法维持合适的细胞浓度，则将原细胞培养袋通过细胞输出连接管与大一级规格的细胞培养袋细胞输入连接管，通过接管热合完成细胞和培养液在不同细胞培养袋的转移；转移完成后，通过热切断，分离两个细胞培养袋；
为维持培养过程中的细胞密度，当细胞密度升高时，需要添加细 胞培养基。细胞培养基保存袋的培养基输注连接管和细胞培养袋的培养基输入连接管连接，通过热合完成细胞培养液输入细胞培养袋，输注完成后通过热切断，分离细胞培养袋和细胞培养基保存袋连接管，并热压密封。
在上述方案的基础上，所述细胞培养袋的规格包括50ml、200ml、2000ml、5000ml，细胞培养基保存袋的规格包括50ml、100ml、200ml，为保证细胞培养过程中维持适宜的细胞培养浓度，而设计不同规格细胞培养袋和细胞培养基保存袋，在细胞培养过程中，通过向细胞培养基保存袋中添加细胞培养基或更换更大规格的细胞培养袋来维持适宜的细胞培养浓度，根据不同的细胞种植密度的需求，采用相应规格大小的细胞培养袋和细胞培养基保存袋。
在上述方案的基础上，所述细胞培养基保存袋体上设有刻度，便于调整输注培养基量。
在上述方案的基础上，所述细胞培养袋和连接管的材质为符合塑料水溶性注射容器标准的高透气性材质。
在上述方案的基础上，所述高透气性材质可以选用聚乙烯、TPX或EVA等。
在上述方案的基础上，所述细胞培养基保存袋由高阻隔性的不透气材质制成，因采用不透气的材料制成，能够有效保证培养基的安全无菌环境，可一次性多量制备。
所述大规模细胞生产和培养的高效率培养系统的应用方法，包括以下步骤：
1、配制细胞培养基，将细胞培养基置于细胞培养基保存袋中，一次性制备多个细胞培养基保存袋，将细胞培养基保存袋放置于4℃环境中冷储待用；使用时将装有细胞培养基的细胞培养基保存袋升温至37℃；
2、将所需培养的细胞通过第三连接管注入细胞培养袋中，同时使用接管法将细胞培养基保存袋的第四连接管或第五连接管通过无菌热合接管机与细胞培养袋的第一连接管热合连接，通过挤压将细胞 培养基注入细胞培养袋，当细胞培养基输注完毕后，采用无菌热合接管机高温瞬间截断第四连接管或第五连接管和第一连接管，并将第四连接管或第五连接管和第一连接管的管口热合密封，使细胞培养袋与细胞培养基保存袋分离；
3、在细胞培养过程中，通过比浊法进行细胞培养浓度的测定，当细胞培养未在适宜培养浓度时，需要添注细胞培养基，取用细胞培养基保存袋，采用无菌热合接管机将细胞培养基保存袋连接的第四连接管或第五连接管与细胞培养袋连接的第一连接管热合连接，能够有效保证在无菌环境下，将细胞培养基保存袋中的细胞培养基挤压至细胞培养袋中；
4、当细胞培养基输注完毕后，采用无菌热合接管机高温瞬间截断第四连接管或第五连接管和第一连接管，并将第四连接管或第五连接管和第一连接管的管口热合密封，使细胞培养袋与细胞培养基保存袋分离；
5、在细胞培养过程中随着培养时间增长，当细胞培养袋无法保证细胞适宜培养浓度时，需要更换更大规格的细胞培养袋，通过无菌热合接管机将原细胞培养袋中培养的细胞通过第二连接管转移到规格更大的细胞培养袋中，继续进行培养；
6、原细胞培养袋中培养的细胞转移完成后，通过无菌热合接管机将原细胞培养袋和现细胞培养袋分离即可。
在上述方案的基础上，所述细胞培养基包括细胞生产因子、无血清培养基和基础培养基。
在上述方案的基础上，所述细胞培养的细胞种类包括悬浮细胞和贴壁细胞，所述悬浮细胞种类包括免疫细胞，贴壁细胞种类包括间充质干细胞和IPS及其诱导分化干细胞；所述细胞培养方式包括悬浮培养和贴壁培养。
在上述方案的基础上，所述细胞培养袋中的细胞培养浓度，采用比浊法进行确定，通过使用分光光度计测定细胞培养液吸光度，对比细胞浓度标准曲线来确定所述的细胞培养浓度。
在上述方案的基础上，所述的细胞培养袋培养于细胞培养箱中，所述的细胞培养箱为CO 2细胞培养箱。
有益效果：
1、本细胞培养系统中所采用的细胞培养袋，安全性高，结构简单、设计合理、使用方便，通过设置细胞培养袋和细胞培养基保存袋，使用时极为安全方便，各个细胞培养袋之间在需要进行连接时采用无菌热合接管机热合连接，降低了细胞污染和操作人员感染的几率，提高了操作的安全性，提高了细胞培养的工作效率，可专用于细胞培养箱中。
2、本系统的细胞培养袋采用符合塑料水溶性注射容器标准的透气材质，在细胞培养过程中无溶出物质，不含对细胞产生不良影响的可塑剂或溶出物质；采用高透气性的聚乙烯、TPX或EVA等，可以满足细胞培养时气体浓度环境要求和pH值的要求；采用连接管连接，在注入细胞培养基或者取样时，通过直接接触操作，提高了操作的安全性；与使用培养瓶相比，气体交换面积大，利于在有限的培养空间内大量培养，可获得更多数量的细胞，封闭式培养，降低了细胞污染和操作人员感染的几率；细胞培养袋的材质透明柔韧，便于在显微镜下直接观察细胞的状态。
该发明的技术关键点和欲保护点：
1、本发明主要为一种常规环境下使用的大规模细胞生产和培养的高效率培养系统，包括细胞培养袋和细胞培养基保存袋，各袋相互独立，且各袋存在不同的规格。
2、细胞培养袋采用符合塑料水溶性注射容器标准的透气材质，连接有三根连接管，分别为第一连接管、第二连接管和第三连接管，细胞培养基保存袋为不透气材质，连接第四连接管、第五连接管。
3、各个袋体之间平时为独立状态，需要进行培养液输注时采用无菌热合接管机保证无菌状态下进行热合连接、断开。
4、细胞培养中通过根据不同细胞的培养密度，选用不同大小的细胞培养袋进行培养，以保证细胞生长的最佳浓度。
5、细胞培养基保存袋可一次性多量制备细胞培养基，一周内用完即可，无需随时制备，细胞培养基保存袋体上存在刻度。
6、本细胞培养系统培养的细胞种类包括悬浮细胞和贴壁细胞。
附图说明
本发明有如下附图：
图1细胞培养袋的结构图。
图2细胞培养基保存袋的结构图。
具体实施方式
以下结合附图1和2对本发明作进一步详细说明。
一种大规模细胞生产和培养的高效率培养系统，包括至少一个细胞培养袋和至少一个细胞培养基保存袋；
所述细胞培养袋用于细胞培养，并且细胞培养袋存在不同的规格，所述细胞培养袋的下端密封边缘处间隔设有连接管，所述连接管包括第一连接管1、第二连接管2和第三连接管3，第一连接管1用于输注培养基，第二连接管2用于细胞的输出，第三连接管3用于细胞的输入；
所述细胞培养基保存袋用于储存制备的细胞培养基，为细胞培养袋的细胞培养输注细胞培养基，细胞培养基保存袋存在不同的规格，所述细胞培养基保存袋的下端密封边缘处设有连接管，所述连接管包括第四连接管4、第五连接管5，第四连接管4、第五连接管5用于向细胞培养袋输注细胞培养基；
所述第一连接管1、第二连接管2、第三连接管3、第四连接管4、第五连接管5的管口采用热合密封方式密封。
在上述方案的基础上，所述的细胞培养袋至少包括第一细胞培养袋和第二细胞培养袋，所述的第一细胞培养袋的连接管与所述的第二 细胞培养袋的连接管连接时，采用接管法连接；
所述的细胞培养袋的连接管与所述的细胞培养基保存袋的连接管连接时，采用接管法连接，
所述的接管法连接，借助无菌热合方式进行热合连接或热切断开，通过高温瞬间操作保证无菌污染。
在上述方案的基础上，所述的第一细胞培养袋的第二连接管2与所述的第二细胞培养袋的第三连接管3连接时，采用接管法连接；
所述的细胞培养袋的第一连接管1与所述的细胞培养基保存袋的第四连接管4或第五连接管5连接时，采用接管法连接。
在上述方案的基础上，所述细胞培养袋的规格包括50ml、200ml、2000ml、5000ml，细胞培养基保存袋的规格包括50ml、100ml、200ml，为保证细胞培养过程中维持适宜的细胞培养浓度，而设计不同规格细胞培养袋和细胞培养基保存袋，在细胞培养过程中，通过向细胞培养基保存袋中添加培养基或更换更大规格的细胞培养袋来维持适宜的细胞培养浓度，根据不同的细胞种植密度的需求，采用相应规格大小的细胞培养袋和细胞培养基保存袋。
在上述方案的基础上，所述细胞培养基保存袋体上设有刻度，便于调整输注培养基量。
在上述方案的基础上，所述细胞培养袋和连接管的材质为符合塑料水溶性注射容器标准的高透气性材质。
在上述方案的基础上，所述高透气性材质可以选用聚乙烯、TPX或EVA等。
在上述方案的基础上，所述细胞培养基保存袋由高阻隔性的不透气材质制成，因采用不透气的材料制成，能够有效保证培养基的安全无菌环境，可一次性多量制备。
所述大规模细胞生产和培养的高效率培养系统的应用方法，包括以下步骤：
1、配制细胞培养基，将细胞培养基置于细胞培养基保存袋中，一次性制备多个细胞培养基保存袋，将细胞培养基保存袋放置于4℃ 环境中冷储待用；使用时将装有细胞培养基的细胞培养基保存袋升温至37℃；
2、将所需培养的细胞通过第三连接管3注入细胞培养袋中，同时使用接管法将细胞培养基保存袋的第四连接管4或第五连接管5通过无菌热合接管机与细胞培养袋的第一连接管1热合连接，通过挤压将细胞培养基注入细胞培养袋，当细胞培养基输注完毕后，采用无菌热合接管机高温瞬间截断第四连接管4或第五连接管5和第一连接管1，并将第四连接管4或第五连接管5和第一连接管1的管口热合密封，使细胞培养袋与细胞培养基保存袋分离；
3、在细胞培养过程中，通过比浊法进行细胞培养浓度的测定，当细胞培养未在适宜培养浓度时，需要添注细胞培养基，取用细胞培养基保存袋，采用无菌热合接管机将细胞培养基保存袋连接的第四连接管4或第五连接管5与细胞培养袋连接的第一连接管1热合连接，能够有效保证在无菌环境下，将细胞培养基保存袋中的细胞培养基挤压至细胞培养袋中；
4、当细胞培养基输注完毕后，采用无菌热合接管机高温瞬间截断第四连接管4或第五连接管5和第一连接管1，并将第四连接管4或第五连接管5和第一连接管1的管口热合密封，使细胞培养袋与细胞培养基保存袋分离；
5、在细胞培养过程中随着培养时间增长，当细胞培养袋无法保证细胞适宜培养浓度时，需要更换更大规格的细胞培养袋，通过无菌热合接管机将原细胞培养袋中培养的细胞通过第二连接管2转移到规格更大的细胞培养袋中，继续进行培养；
6、原细胞培养袋中培养的细胞转移完成后，通过无菌热合接管机将原细胞培养袋和现细胞培养袋分离即可。
在上述方案的基础上，所述细胞培养基包括细胞生产因子、无血清培养基和基础培养基。
在上述方案的基础上，所述细胞培养的细胞种类包括悬浮细胞和贴壁细胞，所述悬浮细胞种类包括免疫细胞，贴壁细胞种类包括间充 质干细胞和IPS及其诱导分化干细胞。
在上述方案的基础上，所述细胞培养袋中的细胞培养浓度，采用比浊法进行确定，通过使用分光光度计测定细胞培养液吸光度，对比细胞浓度标准曲线来确定所述的细胞培养浓度。
在上述方案的基础上，所述的细胞培养袋培养于细胞培养箱中，所述的细胞培养箱为CO 2细胞培养箱。
细胞培养过程中通过比浊法来判断培养的细胞浓度，(前期采用标准的细胞方法已经绘制完成细胞浓度标准曲线，通过曲线获知细胞培养基添加量)获知细胞的培养状态，确定培养状态后，通过调节细胞培养基的多少(输注细胞培养基)和细胞培养袋的大小(更换细胞培养袋)来保证培养过程中细胞培养浓度，保证最优的培养效果。
实施例1：
选取适用于细胞培养的高透气性的塑料材质，按照图1的方式，制备为细胞培养袋，采用高频热合的方式，将连接管焊接于细胞培养袋的下端，其中第二连接管2用于细胞的输出以便收集；第一连接管1用于输注培养基；第三连接管3用于细胞的输入。
实施例2：
选取高阻隔性，不透气的塑料材质，按照图2的方式，制备为细胞培养基保存袋，采用高频热合的方式，将连接管焊接于细胞培养基保存袋的下端，其中第四连接管4、第五连接管5用于向细胞培养袋输注细胞培养基；细胞培养基保存袋体上设有刻度。
实施例3
可在开放环境下，使用无菌热合接管机，将细胞培养袋的第一连接管1与细胞培养基保存袋的第四连接管4或第五连接管5热合连接后，将新鲜的细胞培养基注入细胞培养袋，进行细胞扩增培养。
本说明书中未作详细描述的内容属于本领域专业技术人员公知的现有技术。

Claims (10)

  1. 一种大规模细胞生产和培养的高效率培养系统，其特征在于：包括至少一个细胞培养袋和至少一个细胞培养基保存袋；
    所述细胞培养袋用于细胞培养，所述细胞培养袋的下端密封边缘处间隔设有连接管，所述连接管包括第一连接管、第二连接管和第三连接管，第一连接管用于输注培养基，第二连接管用于细胞的输出，第三连接管用于细胞的输入；
    所述细胞培养基保存袋用于临时储存制备的细胞培养基，为细胞培养袋输注细胞培养基，所述细胞培养基保存袋的下端密封边缘处设有至少一个连接管，所述连接管包括第四连接管、第五连接管，第四连接管、第五连接管用于向细胞培养袋输注细胞培养基；
    所述第一连接管、第二连接管、第三连接管、第四连接管、第五连接管的管口采用热合密封方式密封。
  2. 如权利要求1所述的大规模细胞生产和培养的高效率培养系统，其特征在于：
    所述的细胞培养袋为保证袋内培养的细胞浓度维持在一个有效区间内，细胞培养袋从体积上分为不同的规格，不同规格的细胞培养袋构成这个完整的培养体系；根据实际需求的细胞数量，细胞培养袋的规格可以不断上延；细胞培养时，根据细胞初始数量，一般从小规格细胞培养袋开始培养，到原规格细胞培养袋的培养液体积无法维持合适的细胞浓度，则将原细胞培养袋通过细胞输出连接管与大一级规格的细胞培养袋细胞输入连接管，通过接管热合完成细胞和培养液在不同细胞培养袋的转移；转移完成后，通过热切断，分离两个细胞培养袋；
    为维持培养过程中的细胞密度，当细胞密度升高时，需要添加细胞培养基，细胞培养基保存袋的培养基输注连接管和细胞培养袋的培养基输入连接管连接，通过热合完成细胞培养液输入细胞培养袋，输注完成后通过热切断，分离细胞培养袋和细胞培养基保存袋连接管， 并热压密封。
  3. 如权利要求1所述的大规模细胞生产和培养的高效率培养系统，其特征在于：
    所述的细胞培养袋至少包括第一细胞培养袋和第二细胞培养袋，所述的第一细胞培养袋的连接管与所述的第二细胞培养袋的连接管连接时，采用接管法连接；所述的细胞培养袋的连接管与所述的细胞培养基保存袋的连接管连接时，采用接管法连接，所述的接管法连接，借助无菌热合方式进行热合连接或热切断开，通过高温瞬间操作保证无菌污染；或者，
    所述的第一细胞培养袋的第二连接管与所述的第二细胞培养袋的第三连接管连接时，采用接管法连接；所述的细胞培养袋的第一连接管与所述的细胞培养基保存袋的第四连接管或第五连接管连接时，采用接管法连接。
  4. 如权利要求1所述的大规模细胞生产和培养的高效率培养系统，其特征在于：所述的细胞培养袋的规格包括50ml、200ml、2000ml、5000ml，细胞培养基保存袋的规格包括50ml、100ml、200ml，细胞培养基保存袋体上设有刻度，便于调整输注培养基量；
    为保证细胞培养过程中维持适宜的细胞培养浓度，而设计不同规格细胞培养袋和细胞培养基保存袋，在细胞培养过程中，通过向细胞培养基保存袋中添加细胞培养基或更换更大规格的细胞培养袋来维持适宜的细胞培养浓度。
  5. 如权利要求1所述的大规模细胞生产和培养的高效率培养系统，其特征在于：所述细胞培养袋和连接管的材质为符合塑料水溶性注射容器标准的高透气性材质；所述高透气性材质包括聚乙烯、TPX和EVA；所述细胞培养基保存袋由高阻隔性的不透气材质制成。
  6. 如权利要求1-5任一权利要求所述的大规模细胞生产和培养的高效率培养系统的应用方法，其特征在于：包括以下步骤：
    (1)、配制细胞培养基，将细胞培养基置于细胞培养基保存袋中，一次性制备多个细胞培养基保存袋，将细胞培养基保存袋放置于4℃ 环境中冷储待用；使用时将装有细胞培养基的细胞培养基保存袋升温至37℃；
    (2)、将所需培养的细胞通过第三连接管注入细胞培养袋中，同时使用接管法将细胞培养基保存袋的第四连接管或第五连接管，通过无菌热合接管机与细胞培养袋的第一连接管热合连接，通过挤压将细胞培养基注入细胞培养袋，当细胞培养基输注完毕后，采用无菌热合接管机高温瞬间截断第四连接管或第五连接管和第一连接管，并将第四连接管或第五连接管和第一连接管的管口热合密封，使细胞培养袋与细胞培养基保存袋分离；
    (3)、在细胞培养过程中，借助分光光度计使用比浊法进行细胞培养浓度的测定，当细胞密度超过最佳生长密度时，需要添注细胞培养基，取用细胞培养基保存袋，采用无菌热合接管机将细胞培养基保存袋连接的第四连接管或第五连接管与细胞培养袋连接的第一连接管热合连接，将细胞培养基保存袋中的细胞培养基按照所需的培养基量挤压至细胞培养袋中；
    (4)、当细胞培养基输注完毕后，采用无菌热合接管机高温瞬间截断第四连接管或第五连接管和第一连接管，并将第四连接管或第五连接管和第一连接管的管口热合密封，使细胞培养袋与细胞培养基保存袋分离；
    (5)、在细胞培养过程中随着培养时间增长，当细胞培养袋中无法保证细胞适宜培养浓度时，需要更换更大规格的细胞培养袋，通过无菌热合接管机将原细胞培养袋中培养的细胞通过第二连接管转移到更大规格的细胞培养袋中，继续进行培养；
    (6)、原细胞培养袋中培养的细胞转移完成后，通过无菌热合接管机将原细胞培养袋和现细胞培养袋分离即可。
  7. 如权利要求6所述的大规模细胞生产和培养的高效率培养系统的应用方法，其特征在于：所述细胞培养基包括细胞生产因子、无血清培养基和基础培养基。
  8. 如权利要求6所述的大规模细胞生产和培养的高效率培养系 统的应用方法，其特征在于：所述的细胞培养袋培养于细胞培养箱中，所述的细胞培养箱为CO 2细胞培养箱。
  9. 如权利要求6所述的大规模细胞生产和培养的高效率培养系统的应用方法，其特征在于：所述细胞培养袋中的细胞培养浓度，采用比浊法进行确定，通过使用分光光度计测定细胞培养液吸光度，对比细胞浓度标准曲线来确定所述的细胞培养浓度。
  10. 如权利要求6所述的大规模细胞生产和培养的高效率培养系统的应用方法，其特征在于：所述细胞培养的细胞种类包括悬浮细胞和贴壁细胞，悬浮细胞种类包括免疫细胞，贴壁细胞种类包括间充质干细胞和IPS及其诱导分化干细胞。
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