CN211035948U - Modular three-dimensional perfusion cell culture system - Google Patents
Modular three-dimensional perfusion cell culture system Download PDFInfo
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- CN211035948U CN211035948U CN201921866429.3U CN201921866429U CN211035948U CN 211035948 U CN211035948 U CN 211035948U CN 201921866429 U CN201921866429 U CN 201921866429U CN 211035948 U CN211035948 U CN 211035948U
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Abstract
The utility model relates to a three-dimensional perfusion cell culture system of modularization. The system comprises a liquid changing module, a bioreactor module, an oxygenation module and a power module, wherein the bioreactor module comprises a bioreactor, an interface is arranged in the bioreactor, the interface is connected with a re-cell bracket, the liquid changing module is respectively communicated with the bioreactor and the power module through a three-way joint, the oxygenation module comprises a shell, the shell comprises a bubble filter, a membrane oxygenator and an air filter which are sequentially communicated, the interface is communicated with the bubble filter, and the membrane oxygenator and the air filter are respectively communicated with the power module. The utility model discloses easily preparation, rational in infrastructure, easy disinfection, pollution are few, the cost of manufacture is low, stability is high, easily extension and easy operation are worth using widely.
Description
Technical Field
The utility model belongs to the technical field of biotechnology and specifically relates to a three-dimensional cell culture system that fills of modularization.
Background
The current biological basic theory is mostly obtained by two-dimensional static culture cell experiments based on culture bottles or culture plates. A typical two-dimensional static culture will be performed by plating single or mixed cells from human or animal tissue sources on plastic or glass plates, soaking in nutrient rich media, and removing cellular metabolites by periodic medium changes, providing new nutrients. The two-dimensional static cell culture technology has been developed for many years and becomes one of the most important experimental methods in biology. However, the cells in the tissue and organ are three-dimensional and are continuously perfused by the blood circulation system. In the plate culture, the three-dimensional structure of the tissue, the connection between cells and the extracellular matrix and the connection between cells and the extracellular matrix are completely lost, so that many cell experiment results are difficult to reproduce in animals, and even if time and labor are wasted and confirmed in the animals, the related results are frequently deviated in human bodies due to species differences.
The three-dimensional perfusion culture method of introducing the perfusion oxygen-carrying culture medium on the basis of the three-dimensional static culture simulates a blood circulation system, can remove metabolites in time, provide fresh nutrients and maintain the steady state of a culture system (such as PH, oxygen, glucose and growth factors). The three-dimensional perfusion culture keeps the interconnection of cells similar to the cells in vivo, cells and an extracellular matrix, can better reflect the physiological state in vivo, the related experimental result can be consistent with the in vivo experimental result, and simultaneously, the intuition and condition controllability of the cell culture can be reflected. Perfusion culture can also provide controllable support strength, mechanical properties, shearing force and simulated fluid dynamics effect, can enhance cell function and effectively increase tissue diffusion capacity. In addition, the oxygen-carrying culture medium can eliminate the limitation of the size of the cultured tissue, expand the cell culture scale, provide a large amount of seed cells for the clinical application of regenerative medicine and simultaneously reduce the consumption of manpower, material resources and reagents.
At present, a three-dimensional perfusion culture system has no mature commodity, is designed by a laboratory, and is not integrated. Most of the devices are formed by simply assembling a peristaltic pump, an oxygenator, a culture bottle and a conveying pipeline. Many studies have designed bioreactors to be suitable for the culture of different tissue engineering scaffolds, such as hydrogels, microfluidics, bone scaffolds, polymer scaffolds, or some bioreactors that can provide biomechanical information, depending on experimental requirements, and some have designed the oxygenation part of the culture system. The three-dimensional perfusion culture systems have complex structures, multiple influencing factors, difficult disinfection and pollution, complex manufacturing process and difficult popularization, and greatly limit the application of three-dimensional perfusion culture.
SUMMERY OF THE UTILITY MODEL
The utility model discloses to the shortcoming of present three-dimensional culture system that fills, adopt integrate, the modularized design, divide into bioreactor module, oxygenation module, power module, trade the liquid module with the system. Each module can independently pack to use the ethylene oxide sterilization, when carrying out cell culture with each module simple connection can, reduce operating procedure and possible pollution link, the casing of each module all prints the preparation through 3D and forms, consequently the utility model discloses easily preparation, rational in infrastructure, easy disinfection, pollute less, the cost of manufacture is low, stability is high, easily extension and easy operation are worth using widely.
The technical solution of the utility model is as follows: the utility model relates to a three-dimensional perfusion cell culture system of modularization, its special character lies in: the culture system comprises a liquid changing module, a bioreactor module, an oxygenation module and a power module, wherein the bioreactor module comprises a bioreactor, an interface is arranged in the bioreactor, the interface is connected with a re-cell bracket, the liquid changing module is respectively communicated with the bioreactor and the power module through a three-way joint, the oxygenation module comprises a shell, the shell comprises a bubble filter, a membrane oxygenator and an air filter which are sequentially communicated, the interface is communicated with the bubble filter, and the membrane oxygenator and the air filter are respectively communicated with the power module.
Preferably, the liquid changing module is composed of a cuboid shell, the cuboid shell is internally inverted-cone-shaped, a deep ultraviolet L ED lamp is arranged in the center of the bottom, the liquid changing module is communicated with the bioreactor through a silica gel hose, one end of the silica gel hose is inserted into the liquid changing module through a silica gel hose joint, the other end of the silica gel hose is connected with a tee joint, and the other two ends of the tee joint are communicated with the bioreactor and a power module through the silica gel hose respectively.
Preferably, the interface is communicated with one end of the bubble filter through a silica gel hose, the other end of the bubble filter is communicated with the first interface of the membrane oxygenator, the second interface of the membrane oxygenator is communicated with the power module through an air filter, and the third interface of the membrane oxygenator is communicated with the power module through a silica gel hose.
Preferably, an air filter viewing window is provided on the housing of the oxygenation module.
Preferably, the power module consists of a peristaltic pump and an air pump.
Preferably, the bioreactor is an 80ml blue-mouthed bottle.
Preferably, the three-way joint is a three-way valve.
Preferably, the pipeline interfaces of the silica gel hose adopt standard 6% luer connectors.
The utility model has the advantages of it is following:
1. simple structure, pollution-free and easy disinfection, the utility model discloses a bioreactor module is as cell culture's container, loads culture medium and cell support compound, and oxygenation module loads diaphragm oxygenator, air cleaner, bubble filter etc, does the utility model discloses a core subassembly, power module is responsible for providing power to entire system's culture medium and gas, it comprises 3D printing cuboid casing and deep ultraviolet L ED lamp to trade the liquid module, can disinfect to trading the liquid interface, avoids trading the liquid process and arouse the pollution, consequently the utility model discloses a modular design, each module can carry out the ethylene oxide sterilization alone, and simple to operate is swift, only needs connect bioreactor module and oxygenation module during the use, has reduced the operation degree of difficulty, reduces the possible pollution link, avoids polluting in the at utmost, has solved the three-dimensional difficult problem of cultivateing that the system structure is complicated, difficult disinfection, easy pollution of pouring into of existing.
2. The temperature maintenance requirement is low. The power module often is the heat generating capital equipment, has brought the serious challenge to the temperature maintenance system of incubator, the utility model discloses a separation of power module has reduced the requirement to incubator temperature regulation system.
3. The manufacturing cost is low, and the popularization is convenient. The utility model discloses a bioreactor is formed by the common blue mouthful of bottle assembly in laboratory, and preparation simple process, and the casing of oxygenation module and liquid changing module is formed through the preparation of 3D printing technique, and the cost of manufacture is low, convenient popularization.
4. The application range is wide. The utility model discloses each pipeline interface all adopts standard 6% luer to connect, all can adopt to the mounting system that adopts standard luer.
5. The system stability is high. Change the culture medium process and be system pollution's main link, the utility model discloses carry out local ultraviolet irradiation sterilization to trading the liquid joint before changing the culture medium, reduced the pollution chance, improved the stability of system.
Drawings
Fig. 1 is a schematic structural diagram of the present invention.
The reference numbers are as follows:
1. three way connection, 2, a bubble filter, 3, a first interface, 4, a second interface, 5, an air filter, 6, a membrane oxygenator, 7, a power module, 8, a deep ultraviolet L ED lamp, 9, a silica gel hose connector, 10, a bioreactor module, 11, a recellularization bracket, 12, a bioreactor, 13, an oxygenation module, 14, a third interface, 15, an interface, 16 and a liquid changing module.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
Referring to fig. 1, the structure of the embodiment of the present invention includes a liquid changing module 16, a bioreactor module 10, an oxygenation module 13 and a power module 7, wherein:
The main body of the bioreactor module 10 is a bioreactor 12, the bioreactor 12 adopts an 80ml blue-mouth bottle, the bottle body is used as a cell culture container, the three-way joint 1 passes through a bottle cap to be connected with a perfusion pipeline in the bioreactor 12, and an interface 15 in the bioreactor 12 is connected with a recellularization bracket 11. One of the ports of the three-way joint 1 is connected with a liquid changing module 16, old culture medium is removed and new culture medium is added through the port, and the three-way joint 1 is a three-way valve.
The oxygenation module 13 comprises a shell, the shell is formed by 3D printing, a bubble filter 2, a membrane oxygenator 6 and an air filter 5 which are sequentially communicated are installed inside the shell, all the components are connected through silica gel hoses, an interface 15 is communicated with one end of the bubble filter 2 through the silica gel hoses, the other end of the bubble filter 2 is communicated with a first interface 3 of the membrane oxygenator 6, a second interface 4 of the membrane oxygenator 6 is communicated with a power module 7 through the air filter 5, and a third interface 14 of the membrane oxygenator 6 is communicated with the power module 7 through the silica gel hoses. An air filter observation window is reserved on the shell, the liquid level change of the oxygenation module 13 can be monitored, bubbles are prevented from entering the culture system, and luer connectors are adopted as the pipeline interfaces.
The power module 7 consists of a peristaltic pump and an air pump, is placed beside the incubator, is installed on a 30cm high-speed rail stand, and is powered by a 12v direct-current power supply. The perfusion speed of the peristaltic pump can be adjusted between 0 and 20ml/min, and the flow rate of the air pump can be adjusted between 0 and 300ml/min, so that circulating power is provided for the culture medium and the oxygen.
The utility model discloses use before the use to bioreactor module 10, oxygenation module 13 and trade liquid module 16 and use the ethylene oxide sterilization, add 50-60ml ordinary or contain the culture medium of carrying oxygen composition (such as fluorocarbon) in bioreactor 12, connect the system according to shown in figure 1. open peristaltic pump and air pump power, adjust bubble filter 2 liquid level height through adjusting oxygenation module 13 gesture, control liquid level height at the 2/3 position of pot, connect the recellularization support 11 that carries the cell support compound of waiting to cultivate subsequently at interface 15. adjust peristaltic pump and air pump speed, put bioreactor 12 and oxygenation module 13 into the incubator subsequently, power module 7 is placed aside the incubator, adjust peristaltic pump and air pump to suitable flow, regularly observe culture system operation condition daily, change the culture medium, open the switch of dark ultraviolet L ED lamp 8 in the trade liquid module 16 before changing the culture medium, the joint irradiation disinfection 2 hours of trading, close power module 7 power, connect disposable 50ml aseptic syringe, remove the culture medium in bioreactor module 12 and use the switch of opening of the culture medium again, the culture medium is connected and is done again to this culture medium connector.
The utility model discloses an experimental data as follows:
1. experimental materials:
experimental animals: healthy SD rats with the age of 3 months and the mass of 250-300 g are provided by the animal experiment center of the Sigan university of transportation. The feeding temperature is kept at 18-25 ℃, the standard feed is used for feeding, and the experimental process strictly conforms to the guidance opinions about animals to be tested, which are published by the national science and technology department in 2006.
Reagents and instruments:
the main reagents are as follows: DMEM medium, streptomycin (Hy-clone), calf serum (Gibco), trypsin (Biyunyan).
The main equipment comprises a membrane oxygenator (Xian Xijing medical supplies Co., Ltd., rat-shaped membrane lung), a CO2 constant-temperature incubator (Thermo Scientific, 3110), a 3D printer (extreme three-dimensional), a peristaltic pump (Lange constant flow pump Co., Ltd., BQ50-1J-A), an air pump (Mulberry electronic material, S L-300), a deep ultraviolet L ED lamp (Shenzhen Yongling technology Co., Ltd., TO39), an air filter (Mulberry electronic material, 8mm filter), a disposable infusion apparatus (Luzhou kang development Co., Ltd., IS-GA3, a luer connector (Mulberry electronic material, S L5/32-4.0 mm), and a disposable medical sterile three-way valve (Yangzhou Sheng medicine Co., Ltd., L JG-S).
2. The experimental method comprises the following steps:
a rat spleen acellular scaffold is prepared by taking a rat spleen through a spleen artery cannula, continuously perfusing the spleen with deionized water for 30min after the spleen is isolated, then immersing the spleen in PBS (phosphate buffer solution) for freezing and storing in a refrigerator at-80 ℃ for standby, repeatedly freezing and thawing the spleen for 2 times, then placing the spleen into a beaker, connecting a peristaltic pump, continuously perfusing at a perfusion speed of 4m L/min by using a 0.2% Sodium Dodecyl Sulfate (SDS) solution and a PBS solution through the spleen artery until spleen parenchymal cells are removed, washing the spleen with the PBS, then disinfecting the scaffold with 0.1% peracetic acid, and storing the scaffold in a 3% double-antibody PBS solution.
L02 cell culture, L02 cells were expanded to 4 × 107 cells in DMEM medium containing 10% calf serum, and the cells were resuspended in 4ml of medium after digestion and centrifugation.
And re-cellularizing the spleen decellularized scaffold by adopting a method of intermittent injection of a spleen artery. The recellularized spleen decellularized scaffolds were attached to the present invention according to the methods of use described in the previous section. The culture medium is cyclically perfused by using DMEM culture medium containing 10% calf serum, the perfusion speed of the culture medium is 1ml/min, the speed of an air pump is 10ml/min, and the culture medium is replaced at intervals of 3 days. Cultured recellularized scaffolds 11 were observed by SEM, HE staining and Ki67 immunofluorescence staining.
2. Results of the experiment
Use the utility model discloses carry out the three-dimensional cultivation of filling, the installation is simple and efficient, only need with the luer adapter connection between bioreactor module 10 and the oxygenation module 13 can. The ultraviolet lamp irradiation in the liquid changing process is combined to emit light, so that the pollution chance is greatly reduced, and the three-dimensional culture performed by using the system is not polluted.
The general observation shows that L02 cells grow in a lump in a spleen acellular scaffold, the cell lump volume is gradually increased, the SEM result of a re-cellularized spleen scaffold cultured for 1 week shows that the cells are tightly adhered to the scaffold, and the cells are distributed in the scaffold according to the structure of the lumen and the blood sinus in the spleen scaffold, the HE staining result shows that L02 cells present a typical hepatocyte shape, wherein the cell nucleus is blue-purple, round or oval, large and obvious, the cytoplasm is rich, and the cell activity is good, and the Ki67 positive rate is 38% +/-7% in immunofluorescence staining, which indicates that the cells in the scaffold are actively proliferated.
The above is only the specific embodiments of the present disclosure, but the scope of the present disclosure is not limited thereto, and the scope of the present disclosure should be subject to the protection scope of the claims.
Claims (8)
1. A modular three-dimensional perfusion cell culture system, characterized by: the culture system comprises a liquid changing module, a bioreactor module, an oxygenation module and a power module, wherein the bioreactor module comprises a bioreactor, an interface is arranged in the bioreactor and is connected with a recellularization support, the liquid changing module is respectively communicated with the bioreactor and the power module through a tee joint, the oxygenation module comprises a shell, the shell comprises a bubble filter, a membrane oxygenator and an air filter which are sequentially communicated, the interface is communicated with the bubble filter, and the oxygenation membrane and the air filter are respectively communicated with the power module.
2. The modular three-dimensional perfusion cell culture system according to claim 1, wherein the liquid changing module is composed of a cuboid shell, the inside of the cuboid shell is in an inverted cone shape, a deep ultraviolet L ED lamp is arranged in the center of the bottom of the cuboid shell, the liquid changing module is communicated with the bioreactor through a silica gel hose, one end of the silica gel hose is inserted into the liquid changing module through a silica gel hose joint, the other end of the silica gel hose is connected with a tee joint, and the other two ends of the tee joint are respectively communicated with the bioreactor and the power module through silica gel hoses.
3. The modular three-dimensional perfusion cell culture system of claim 2, wherein: the interface passes through silica gel hose and bubble filter one end intercommunication, the bubble filter other end communicates with the first interface of membrane oxygenator, the second interface of membrane oxygenator passes through air cleaner and power module intercommunication, the third interface of membrane oxygenator passes through silica gel hose and power module intercommunication.
4. The modular three-dimensional perfusion cell culture system of claim 3, wherein: an air filter observation window is arranged on the shell of the oxygenation module.
5. The modular three-dimensional perfusion cell culture system of claim 1 or 2 or 3 or 4, wherein: the power module consists of a peristaltic pump and an air pump.
6. The modular three-dimensional perfusion cell culture system of claim 5, wherein: the bioreactor is an 80ml blue-mouth bottle.
7. The modular three-dimensional perfusion cell culture system of claim 6, wherein: the three-way joint is a three-way valve.
8. The modular three-dimensional perfusion cell culture system of claim 3, wherein: and the pipeline interfaces of the silica gel hose are all standard 6% Ruhr joints.
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| CN110669669A (en) * | 2019-11-05 | 2020-01-10 | 西安交通大学医学院第一附属医院 | Modular three-dimensional perfusion cell culture system |
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| CN110669669A (en) * | 2019-11-05 | 2020-01-10 | 西安交通大学医学院第一附属医院 | Modular three-dimensional perfusion cell culture system |
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