CN111206014A - Bionic alveolar culture method and culture device - Google Patents
Bionic alveolar culture method and culture device Download PDFInfo
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Abstract
The invention provides a bionic alveolus culture method and a culture device, wherein the bionic alveolus culture device comprises: an incubator having a receiving chamber; the cell growth membrane is arranged in the accommodating cavity, an accommodating space for accommodating cell culture solution is formed between the cell growth membrane and the wall of the accommodating cavity, the cell growth membrane comprises a sphere and a tube body communicated with the sphere, the sphere accommodates cell suspension, and the tube body is fixedly and hermetically connected with the incubator; the first plugging piece plugs the pipe orifice of the pipe body; the bottom of the incubator is provided with a vertical structure convenient for vertical placement, the incubator is also provided with at least three flat side faces convenient for flat placement, and the vertical structure is combined with the flat side faces to ensure that the incubator is in a vertical posture and a horizontal posture with multiple angles in the circumferential direction, so that cells are evenly inoculated on the inner wall of the sphere. Has the advantages that: even if the influence of gravity factor exists, the cells can be intensively grown at different positions, and compared with the prior art, the method is more uniform, can simulate a more real three-dimensional structure of alveolar vesicle-like cells, and improves the accuracy of subsequent experimental results.
Description
Technical Field
The invention relates to a bionic alveolus culture method and a culture device.
Background
Mechanical ventilation is used as an intervention measure for remedying dyspnea caused by lung injury diseases, and can be clinically classified into invasive mechanical ventilation and non-invasive mechanical ventilation at present. Invasive mechanical ventilation, which is found to be an independent risk factor for the death of COPD patients admitted to an acute respiratory failure Intensive Care Unit (ICU), also causes adverse effects while treating some diseases. Studies have shown that the main cause of this is the tidal volume produced by traumatic mechanical ventilation acting on the alveoli by creating some mechanical force and causing lung injury.
Alveoli are vesicles composed of a monolayer of epithelial cells, located around the enlarged vesicles at the end of the bronchioles of the lung. As a basic unit of ventilation of the lungs, its biomechanical behavior plays an important role in the respiratory physiology of mammals. The alveoli account for more than 70% of the lung volume, and during inspiration the alveoli expand by stretching of the intrinsic stress to adapt to the inhaled air, and during expiration the air is expelled from the lungs by the change of the ambient pressure outside the alveoli and the elastic recoil of the alveoli. In such a ventilation cycle, the pulmonary alveoli undergo a change in dynamic stress state, which is crucial for better understanding of the lung respiratory function, gas exchange, pulmonary stability and mechanism of mechanical ventilation to the mechanism of pulmonary alveolar injury, and it is necessary to study key determinants of the pulmonary alveoli in the respiratory cycle, such as the change in the size and shape of the pulmonary alveoli caused by contraction and expansion of the pulmonary alveoli, the degree of stretching of the pulmonary alveoli and the change in the number of new characterized pulmonary alveoli.
In contrast, chinese patent No. CN104316661B discloses a lung tissue model for detecting biological toxicity and a biological toxicity detection method, where the lung tissue model includes trachea, blood vessels, and alveolus units, and both ends of the trachea are provided with air inlets and air outlets; a liquid inlet and a liquid outlet are arranged at two ends of the blood vessel; the alveolus unit is of a hollow sphere structure and is provided with a cavity, the wall of the alveolus unit is an elastic breathable porous membrane, the alveolus unit is arranged in the blood channel, the cavity is communicated with the gas channel, the alveolus unit can expand or contract along with the change of the gas pressure in the gas channel, and the gas in the cavity can exchange gas with the gas in the blood channel, so that the gas exchange function of simulating the pulmonary blood in vivo is realized. Meanwhile, cells are planted in the alveolus unit part, so that the bionics of the structure and the function of the alveolus in vivo is realized.
The final realization form of the lung tissue model is a chip consisting of a basal layer, a blood vessel layer, an air tube layer and a capping layer, wherein the blood vessel, the air tube and the alveolus unit are clamped in the chip, the alveolus unit is a cell growth membrane, and cells are planted in the cell growth membrane during experiments to form bionic alveolus. However, in practical application, due to the existence of gravity, cells usually grow intensively at the bottom of a cell growth membrane, the cells grow unevenly, the most real alveolar vesicle-like three-dimensional cell structure cannot be simulated, and the accuracy of subsequent experimental results is affected.
Disclosure of Invention
The invention aims to provide a bionic alveolus culture method which ensures that cells grow more uniformly on a cell growth membrane and form a vesicle-like membrane structure consisting of cell links; the invention also aims to provide a bionic alveolus culture device for implementing the bionic alveolus culture method.
In order to realize the purpose, the bionic alveolus culture method adopts the following technical scheme:
a bionic alveolus culture method is characterized in that a culture device which is fixed with a cell growth membrane, in which cell suspension is added and cell culture solution is arranged on the periphery of the cell growth membrane is maintained in a vertical posture and a horizontal posture at multiple angles in the circumferential direction according to a set sequence, so that cells are evenly inoculated on the inner wall of a sphere of the cell growth membrane.
The beneficial effects of the above technical scheme are that: because the incubator keeps the vertical posture and the horizontal posture of the multi-angle in the circumferential direction in the set sequence, even if the incubator is influenced by gravity factors, cells can intensively grow at different positions, and compared with the prior art, the incubator is more uniform, forms a three-dimensional distributed intercellular link mode, realizes a vesicle-like membrane structure formed by cell link, can simulate a more real three-dimensional structure of the alveolar vesicle, and improves the accuracy of subsequent experimental results.
Further, in order to make the cell growth more uniform and better, the cell suspension in the cell growth membrane and/or the cell culture solution in the incubator are replaced in the process that the incubator is switched from the vertical posture to the horizontal posture, from the horizontal posture to the vertical posture, or in the process of multi-angle horizontal posture switching.
In order to achieve the purpose, the bionic alveolar culture device for implementing the bionic alveolar culture method adopts the following technical scheme:
a biomimetic alveolar culture apparatus comprising:
an incubator having a receiving chamber;
the cell growth membrane is arranged in the accommodating cavity of the incubator, an accommodating space for accommodating cell culture solution is formed between the cell growth membrane and the wall of the accommodating cavity, the cell growth membrane comprises a sphere and a tube body communicated with the sphere, the sphere is used for accommodating cell suspension, and the tube body is fixedly and hermetically connected with the incubator;
the first blocking piece is used for blocking the pipe orifice of the pipe body so as to prevent the cell suspension from flowing out;
when the incubator is vertically placed, the tube body extends vertically, the sphere is positioned below the tube body, the bottom of the incubator is provided with a vertical placing structure which is convenient for the incubator to vertically place, the incubator is also provided with at least three horizontal side faces which are convenient for the incubator to horizontally place, and the vertical placing structure and the horizontal side faces are combined to enable the incubator to be in a vertical posture and a horizontal posture with multiple angles in the circumferential direction, so that cells are uniformly inoculated on the inner wall of the sphere; alternatively, the first and second electrodes may be,
the bionic alveolus culture device also comprises a bracket, wherein the bracket is used for being arranged below the incubator in a horizontal posture, supporting the incubator and switching the incubator among the horizontal postures in multiple angles in the circumferential direction; alternatively, the first and second electrodes may be,
the pipe body extends from top to bottom when the definition incubator is placed vertically, the ball body is located below the pipe body, the bionic alveolus culture device further comprises a support, and the support is used for being arranged below the incubator and supporting the incubator, so that the incubator can be switched between vertical postures and horizontal postures of multiple angles in the circumferential direction.
The beneficial effects of the above technical scheme are that: the accommodating cavity of the incubator is convenient for installing and fixing the cell growth membrane, and the space between the cell growth membrane and the cavity wall of the accommodating cavity is convenient for accommodating cell culture solution and providing a nutrient environment for cell growth; the sphere of the cell growth membrane conveniently contains cell suspension, so that cells are inoculated on the inner wall of the sphere; the pipe body of the cell growth membrane is convenient to be fixedly and hermetically connected with the incubator, and the first plugging piece can plug the pipe opening of the pipe body so as to avoid the outflow of cell suspension. Simultaneously, the incubator bottom is provided with puts the structure immediately, the incubator of being convenient for is put immediately, the incubator still has at least three side of keeping flat, the incubator of being convenient for is kept flat, to put the structure immediately like this and the side combination of keeping flat, just can make the incubator be in the horizontal gesture of multi-angle on vertical gesture and the circumferencial direction, even there is the influence of gravity factor, the cell can concentrate the growth in the position of difference, compare prior art, can be more even, form three-dimensional distribution's intercellular linking mode, realize the vesicle appearance membrane structure of constituteing by the cell link, can simulate more real alveolar vesicle appearance three-dimensional structure, improve the accuracy of follow-up experimental result.
In addition, as one of the parallel technical schemes, the bottom of the incubator is provided with a vertical structure, the bionic alveolar culture device further comprises a support, the support is arranged below the incubator in a horizontal posture and used for supporting the incubator, and the incubator can be switched between the horizontal postures at multiple angles in the circumferential direction, so that the vertical structure at the bottom of the incubator is combined with the support, the incubator is in the horizontal postures at multiple angles in the vertical posture and the circumferential direction, cells can also intensively grow at different positions even under the influence of gravity factors, and the bionic alveolar culture device is more uniform compared with the prior art.
In addition, as parallel technical scheme two, bionical alveolus culture apparatus still includes the support, and the support is used for setting up in the incubator below, supports the incubator to make the incubator can switch between the horizontal gesture of vertical gesture and multi-angle on the circumferencial direction, even there is the influence of gravity factor equally, the cell also can concentrate the growth in different positions, compares prior art more evenly.
Further, in order to facilitate the manufacture of the incubator, the incubator comprises a housing and a culture bottle fixed on the housing, and the accommodating chamber is formed by an inner cavity of the culture bottle.
Further, in order to add cell culture solution conveniently, the bottom of the culture bottle is provided with a communicating pipe communicated with the inner cavity of the culture bottle, the communicating pipe penetrates out of the shell and is connected with a second plugging piece at the penetrating end, and the second plugging piece is used for plugging the pipe orifice of the communicating pipe.
Furthermore, in order to add the cell culture solution when the incubator is horizontally arranged, the liquid adding operation is convenient, the communicating pipe is L-shaped and comprises a vertical part and a horizontal part, the vertical part penetrates out of the shell, and the second plugging piece is connected to the horizontal part.
Furthermore, in order to facilitate the growth of cells and save cell culture solution, the shape of the culture bottle is matched with that of the cell growth membrane.
Furthermore, in order to facilitate the fixed connection of the tube body and the incubator, the bottle mouth of the culture bottle protrudes out of the shell, and the end part of the tube body is turned outwards and sleeved and fixed on the bottle mouth of the culture bottle.
Furthermore, in order to facilitate the configuration and installation of the first blocking piece, the first blocking piece is a first blocking cover in threaded connection with the mouth of the culture bottle.
Furthermore, in order to facilitate the configuration and installation of the first blocking piece, the mouth of the culture bottle protrudes out of the shell, and the first blocking piece is a first blocking cover in threaded connection with the mouth of the culture bottle.
Drawings
FIG. 1 is a schematic front view of a bionic alveolar culture device according to example 1 of the present invention;
FIG. 2 is a state diagram (vertical posture) of the biomimetic alveolar culture apparatus in example 1 of the present invention in use;
FIG. 3 is a state diagram (horizontal posture) of the biomimetic alveolar culture apparatus in example 1 of the present invention in use;
FIG. 4 is a schematic perspective view of a bionic alveolar culture device according to example 2 of the present invention;
FIG. 5 is a schematic top view of the bionic alveolar culture device of example 2 of the present invention;
FIG. 6 is a schematic top view of the bionic alveolar culture device of example 3 of the present invention;
FIG. 7 is a schematic perspective view of a biomimetic alveolar culture apparatus in accordance with embodiment 4 of the present invention;
FIG. 8 is a schematic perspective view of a bionic alveolar culture device according to example 5 of the present invention;
FIG. 9 is a schematic perspective view of a bionic alveolar culture device according to example 6 of the present invention;
FIG. 10 is a schematic perspective view of a first blocking cap in example 7 of a biomimetic alveolar culture apparatus in accordance with the present invention.
In the figure: 1-a shell; 2-culture flask; 21-a spherical portion; 22-a tubular portion; 23-communicating tube; 3-a first plugging cover; 30-a first plugging cover; 31-a gas permeable membrane; 32-connecting column; 4-cell growth membrane; 41-sphere; 42-a tube body; 5-a support leg; 6-a second plugging cover; 7-cell culture fluid; 8-cell suspension; 9-a scaffold; 90-a scaffold; 10-a housing; 100-a housing; 1000-shell.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.
Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.
It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The features and properties of the present invention are described in further detail below with reference to examples.
An embodiment 1 of the bionic alveolar culture device of the invention is shown in fig. 1 and comprises an incubator, wherein the incubator is composed of a shell 1 and a culture bottle 2 fixed on the shell 1, a cavity is arranged in the shell 1, most of the culture bottle 2 is positioned in the shell 1, and the top end of the culture bottle 2 is positioned outside the shell 1, so that the bottle mouth of the culture bottle 2 protrudes out of the shell 1. The culture bottle 2 and the shell 1 can be adhered and fixed, or an external thread structure can be formed on the culture bottle to be connected with the shell 1 in a threaded mode.
The inner cavity of the culture bottle 2 forms a containing cavity, the bionic alveolar culture device further comprises a cell growth membrane 4 arranged in the containing cavity, the cell growth membrane 4 is an elastic breathable membrane and comprises a hollow sphere 41 and a tube body 42 communicated with the sphere 41, and the sphere 41 is used for containing cell suspension 8, as shown in fig. 2 and 3.
The shape of the flask 2 is adapted to the shape of the cell growth membrane 4, i.e. the flask 2 is formed by a spherical part 21 and a tubular part 22 communicating with the spherical part 21, the sphere 41 of the cell growth membrane being located in the spherical part 21 and the tubular body 42 being located in the tubular part 22. The cell growth membrane 4 and the culture bottle 2 are spaced apart from each other by a suitable distance so that a receiving space for receiving a cell culture solution 7 is formed between the cell growth membrane 4 and the inner wall of the culture bottle 2, as shown in fig. 2 and 3, it should be noted that the receiving space is not actually as large as that shown in the figures, and the structural arrangement is illustrated in the figures by enlarging the distance between the cell growth membrane 4 and the culture bottle 2 mainly for distinguishing the cell growth membrane 4 from the culture bottle 2.
The tube body 42 of the cell growth membrane 4 is fixedly and hermetically connected with the tubular part 22 of the culture bottle 2, specifically, the tube body 42 is longer than the tubular part 22, the grown part, namely the top end of the tube body 42, is turned outwards and is sleeved on the bottle mouth of the culture bottle through deformation, and in order to ensure the fixing and sealing effect, a rubber band is fixed outside the turned-out part. In order to block the orifice of the tube body 42 and prevent the cell suspension 8 from flowing out, the bionic alveolar culture device further comprises a first blocking piece, and specifically, the first blocking piece is a first blocking cover 3 which is in threaded connection with the bottleneck of the culture bottle 2. The first blocking cover 3 can be provided with air holes or completely closed, and when no air hole exists, the first blocking cover 3 needs to be unscrewed to facilitate air inlet during cell culture; when the air holes are arranged, the air holes can be directly screwed down during cell culture, and the air holes are attached with air-permeable films and only air-permeable and liquid-impermeable.
The culture bottle 2 further comprises a communicating pipe 23 which is arranged at the bottom of the culture bottle 2 and communicated with the inner cavity of the culture bottle 2, the communicating pipe 23 penetrates out of the shell 1 and is connected with a second plugging piece at the penetrating end, and the second plugging piece is used for plugging the pipe orifice of the communicating pipe 23. Specifically, communicating pipe 23 is L shape, and communicating pipe 23 includes vertical portion and horizontal part, and casing 1 is worn out to vertical portion, and the second shutoff piece is second shutoff lid 6, and 6 threaded connection of second shutoff lid are on the horizontal part.
In addition, the bottom of the casing 1 is provided with a vertical structure which is convenient for the casing 1 to be vertically placed, and specifically, the vertical structure is four support legs 5 fixed at the bottom of the casing 1. The housing 1 in this embodiment has four side faces, that is, the housing 1 is a rectangular parallelepiped structure, the four side faces form a flat side face convenient for the housing 1 to be flat, and the four flat side faces can make the housing 1 be in a horizontal posture at four angles in the circumferential direction.
An embodiment 2 of the bionic alveolar culture device disclosed by the invention is shown in fig. 4 and 5 and comprises an incubator, wherein the incubator consists of a shell 10 and a culture bottle 2 fixed on the shell 10, the bottom of the shell 10 is provided with a supporting leg 5, a cell growth membrane 4 is embedded and fixed in the culture bottle 2, and a first blocking cover 3 is in threaded connection with the pipe orifice of the culture bottle 2. This embodiment differs from embodiment 1 only in that: the number of the legs 5 and the shape of the housing 10, six legs 5 in this embodiment, and the housing 10 has a hexagonal prism shape, that is, the housing 10 has six flat sides, so that the incubator can be in a horizontal posture at six angles in the circumferential direction.
Fig. 6 shows an embodiment 3 of the bionic alveolar culture apparatus of the present invention, and the same points as those in embodiment 1 are not repeated, but mainly different therefrom: the housing 100 in this embodiment has an octagonal prism shape, that is, the housing 100 has eight flat sides, and the incubator can be placed in a horizontal posture at eight angles in the circumferential direction.
Fig. 7 shows an embodiment 4 of the bionic alveolar culture apparatus of the present invention, which is the same as embodiment 1 and is not repeated, but mainly differs therefrom in that: the housing 1000 in this embodiment is a dodecahedron, the bottom surface of the dodecahedron is a pentagonal plane, the bottom surface is fixed with support legs (not shown in the figure), the whole incubator can utilize five flat side surfaces of the lower circle of the dodecahedron to realize the switching of the horizontal postures of five angles in the circumferential direction, certainly, can also utilize five flat side surfaces of the upper circle of the dodecahedron to realize the switching of the horizontal postures of five angles in the circumferential direction, that is, ten angles can be switched, but the culture bottles 2 at the ten angles are slightly inclined, not standard horizontal, but inclined, and the switching of the ten angles can make the cell growth more uniform.
Fig. 8 shows an embodiment 5 of the bionic alveolar culture apparatus of the present invention, and the same points as those in embodiment 2 are not repeated, but mainly different therefrom: the bionic alveolar culture device in the embodiment further comprises a support 9, the support 9 is of a cuboid box-shaped structure or a cuboid frame structure, the support 9 is used for being arranged below the incubator in the horizontal posture and supporting the incubator, the incubator can be switched between the horizontal postures at multiple angles in the circumferential direction, one side edge of the hexagonal prism-shaped shell 10 sinks into the support 9, and therefore the support is combined with six square side surfaces of the hexagonal prism-shaped shell 10 per se, and the incubator can be switched between the horizontal postures at 12 angles in the circumferential direction. The support 9 not only increases the switching angle of the horizontal posture, but also can stably support the culture device, and especially can ensure the stable support and the stable placement of the culture device for the shell with more square side surfaces and smaller area of the square side surfaces.
Fig. 9 shows an embodiment 6 of the bionic alveolar culture apparatus of the present invention, and the same points as those in embodiment 4 are not repeated, but mainly different therefrom: do not set up the landing leg in this embodiment on dodecahedron's structure's the casing 1000, bionical alveolus culture apparatus still includes support 90, and support 90 is cylinder tubular structure, and support 90 is used for setting up in casing 1000 below, carries out stable support to casing 1000, makes the incubator can switch between the horizontal gesture of vertical gesture and multi-angle on the circumferencial direction.
As shown in fig. 10, in example 7 of the biomimetic alveolar culture apparatus according to the present invention, the first sealing cover 30 of the present example is provided with a gas permeable membrane 31, and the gas permeable membrane 31 is only gas permeable and liquid impermeable, which is different from the above examples: still be provided with spliced pole 32 on the first shutoff lid 30, spliced pole 32 is used for being connected with automatic arm (belonging to prior art), and automatic arm can the automatic posture of adjustment incubator, makes it switch between vertical gesture and the horizontal gesture of multi-angle on the circumferencial direction.
In other embodiments of the bionic alveolar culture device, the first blocking piece may not be a first blocking cover in threaded connection with the mouth of the culture bottle, for example, the first blocking piece may be a first blocking plug directly inserted into the tube body of the cell growth membrane, and at this time, the mouth of the culture bottle may or may not protrude from the housing, and no matter whether the mouth of the culture bottle protrudes from the housing, the end of the tube body of the cell growth membrane may be bonded, sealed and fixed with the inner wall of the mouth of the culture bottle.
In other embodiments of the biomimetic alveolar culture device, the shape of the culture flask may not be adapted to the shape of the cell growth membrane, for example, the shape of the culture flask is cylindrical.
In other embodiments of the biomimetic alveolar culture apparatus, the communicating tubes may not be L-shaped, but straight tubes.
In other embodiments of the bionic alveolar culture device, the second plug can also be a second plug plugged into the mouth of the communicating pipe.
In other embodiments of the bionic alveolar culture device, the bottom of the culture bottle may not be provided with a communicating pipe, and when the cell culture solution is replaced, the end of the tube body of the cell growth membrane needs to be released from the mouth of the culture bottle, and the cell culture solution is added from the mouth of the culture bottle.
In other embodiments of the biomimetic alveolar culture device, the incubator may not be composed of a housing and a culture flask fixed on the housing, for example, the incubator may be a solid material, a receiving cavity formed by removing material, or an integrally formed container having a receiving cavity.
In other embodiments of the biomimetic alveolar culture device, the standing structure may not be a leg but an annular support cylinder disposed at the bottom of the incubator; or the cubic structure is the bottom surface of the incubator.
In other embodiments of the bionic alveolar culture device, the number of the flat sides can be three, and the incubator has a triangular prism structure, but the number of the flat sides can be more.
The specific implementation process of the bionic alveolar culture method comprises the following steps: the bionic alveolar culture method is carried out by relying on the bionic alveolar culture device in any one of the above embodiments, or the bionic alveolar culture device in any one of the above embodiments is used for implementing the bionic alveolar culture method in the present invention, and the bionic alveolar culture method is described below only by using the cell membrane culture device in embodiment 1, and the specific implementation steps are:
firstly, the first blocking cover 3 is opened, the cell culture solution 7 is added into the culture bottle 2, then the cell growth membrane 2 is nested and fixed in the culture bottle 2, the first blocking cover 3 is connected, the culture device is kept in a vertical posture (as shown in figure 2, the tube body 42 extends up and down, and the ball body 41 is positioned below the tube body 42), and sterilization treatment is carried out. And opening the first blocking cover 3 after sterilization, adding the cell suspension 8 into the cell growth membrane 2, and adding the cell suspension 8 into the junction of the sphere 41 and the tube body 42 to ensure that cells are only inoculated on the inner wall of the sphere to form the required bionic alveolus. The level of the cell culture liquid 7 is here higher than the level of the cell suspension 8, ensuring that all cells are nourished.
Then the incubator is kept standing for a period of time in a vertical posture, and when the cells grow adherent to the inner wall of the sphere 41, the cells are concentrated at the bottom of the sphere 41 to grow due to the action of gravity. After a certain time, the first blocking cover 3 is opened, the liquid in the cell growth membrane 2 is removed, new cell suspension is added, the first blocking cover 3 is screwed, the adding amount of the new cell suspension is shown in figure 3, the incubator is placed on a certain flat side face, and the cell suspension is only positioned in the sphere 41 when the incubator is in the horizontal posture. In the process, the cell culture solution 7 can be discharged through the second blocking cover 6 according to actual needs, and new cell culture solution is added into the culture bottle 2 from the pipe orifice of the communicating pipe 23 for replacing the cell culture solution, and the liquid level of the cell culture solution still needs to be higher than that of the cell suspension.
When the incubator is placed on a certain flat side, the incubator is kept in a horizontal posture for a period of time, and when cells grow adherent to the inner wall of the sphere 41, the cells are concentrated at the bottom of the sphere 41 to grow due to the gravity. After a certain time, the cell suspension is replaced, the incubator is placed on the other flat side, and the rest is done until all the flat sides are used, the switching of the multi-angle horizontal postures in the circumferential direction is completed, at the moment, the cells are inoculated on the inner wall of the sphere in batches, the cells can grow in different positions in a concentrated mode, and compared with the prior art, the cell suspension is more uniform, a more real alveolar cell structure can be simulated, and the accuracy of a subsequent experiment result is improved.
In other embodiments of the biomimetic alveolar culture method, sterilization treatment may not be performed.
In other embodiments of the biomimetic alveolar culture method, the cell suspension can be added into the cell growth membrane, the first sealing cover is screwed down, then the second sealing cover is unscrewed, and the cell culture solution is added into the culture bottle from the communicating pipe.
In other embodiments of the biomimetic alveolar culture method, the cell suspension can be added in an amount that is more than the amount of the cell suspension on the inner wall of the tubular body.
In other embodiments of the biomimetic alveolar culture method, the cell culture solution may not be replaced during the cell culture process according to the actual situation.
In other embodiments of the biomimetic alveolar culture method, the cell suspension may not be replaced during the cell culture process according to the actual situation, for example, during the process of switching from the vertical posture to the horizontal posture, a part of the supernatant of the cell suspension may be removed, and the remaining cell suspension that has not adhered to the wall continues the subsequent experiment.
In other embodiments of the bionic alveolar culture method, switching of multi-angle horizontal postures in the circumferential direction can be performed first, and finally the culture device is in the vertical posture; of course, the vertical posture and the horizontal posture may be performed alternately.
The above description is only a preferred embodiment of the present invention, and not intended to limit the present invention, the scope of the present invention is defined by the appended claims, and all structural changes that can be made by using the contents of the description and the drawings of the present invention are intended to be embraced therein.
Claims (10)
1. A bionic alveolus culture method is characterized in that a culture device which is fixed with a cell growth membrane, in which cell suspension is added and cell culture solution is arranged on the periphery of the cell growth membrane is maintained in a vertical posture and a horizontal posture at multiple angles in the circumferential direction according to a set sequence, so that cells are evenly inoculated on the inner wall of a sphere of the cell growth membrane.
2. The biomimetic alveolar culture method according to claim 1, wherein the cell suspension in the cell growth membrane and/or the cell culture solution in the incubator is replaced during switching of the incubator from a vertical posture to a horizontal posture, from a horizontal posture to a vertical posture, or during switching of a multi-angle horizontal posture.
3. A biomimetic alveolar culture apparatus for performing the biomimetic alveolar culture method according to claim 1, comprising:
an incubator having a receiving chamber;
the cell growth membrane is arranged in the accommodating cavity of the incubator, an accommodating space for accommodating cell culture solution is formed between the cell growth membrane and the wall of the accommodating cavity, the cell growth membrane comprises a sphere and a tube body communicated with the sphere, the sphere is used for accommodating cell suspension, and the tube body is fixedly and hermetically connected with the incubator;
the first blocking piece is used for blocking the pipe orifice of the pipe body so as to prevent the cell suspension from flowing out;
when the incubator is vertically placed, the tube body extends vertically, the sphere is positioned below the tube body, the bottom of the incubator is provided with a vertical placing structure which is convenient for the incubator to vertically place, the incubator is also provided with at least three horizontal side faces which are convenient for the incubator to horizontally place, and the vertical placing structure and the horizontal side faces are combined to enable the incubator to be in a vertical posture and a horizontal posture with multiple angles in the circumferential direction, so that cells are uniformly inoculated on the inner wall of the sphere; alternatively, the first and second electrodes may be,
the bionic alveolus culture device also comprises a bracket, wherein the bracket is used for being arranged below the incubator in a horizontal posture, supporting the incubator and switching the incubator among the horizontal postures in multiple angles in the circumferential direction; alternatively, the first and second electrodes may be,
the pipe body extends from top to bottom when the definition incubator is placed vertically, the ball body is located below the pipe body, the bionic alveolus culture device further comprises a support, and the support is used for being arranged below the incubator and supporting the incubator, so that the incubator can be switched between vertical postures and horizontal postures of multiple angles in the circumferential direction.
4. The biomimetic alveolar culture device of claim 3, wherein the incubator comprises a housing and a culture flask fixed on the housing, and the accommodating cavity is formed by an inner cavity of the culture flask.
5. The bionic alveolar culture device of claim 4, wherein a communicating pipe communicated with the inner cavity of the culture bottle is arranged at the bottom of the culture bottle, the communicating pipe penetrates out of the shell, and a second plugging piece is connected to the penetrating end of the communicating pipe and is used for plugging the orifice of the communicating pipe.
6. The biomimetic alveolar culture device according to claim 5, wherein the communication pipe is L-shaped, the communication pipe comprises a vertical part and a horizontal part, the vertical part penetrates out of the housing, and the second blocking member is connected to the horizontal part.
7. The biomimetic alveolar culture device according to any one of claims 4-6, wherein the shape of the culture bottle is adapted to the shape of a cell growth membrane.
8. The bionic alveolar culture device according to any one of claims 4 to 6, wherein a mouth of the culture bottle protrudes out of the shell, and the end of the tube body is turned outwards and sleeved and fixed on the mouth of the culture bottle.
9. The biomimetic alveolar culture device of claim 8, wherein the first closure is a first closure cap in threaded connection with the mouth of the culture bottle.
10. The bionic alveolar culture device according to any one of claims 4 to 6, wherein the mouth of the culture bottle protrudes from the shell, and the first blocking piece is a first blocking cover in threaded connection with the mouth of the culture bottle.
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