CN110734847B - Cell microcarrier separation device - Google Patents
Cell microcarrier separation device Download PDFInfo
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- CN110734847B CN110734847B CN201911221057.3A CN201911221057A CN110734847B CN 110734847 B CN110734847 B CN 110734847B CN 201911221057 A CN201911221057 A CN 201911221057A CN 110734847 B CN110734847 B CN 110734847B
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- 239000012528 membrane Substances 0.000 claims abstract description 152
- 239000007788 liquid Substances 0.000 claims abstract description 89
- 238000003756 stirring Methods 0.000 claims abstract description 76
- 239000002699 waste material Substances 0.000 claims description 22
- 238000004113 cell culture Methods 0.000 claims description 8
- 238000007599 discharging Methods 0.000 claims description 3
- 238000013019 agitation Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 abstract description 9
- 239000011259 mixed solution Substances 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 103
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- 238000002659 cell therapy Methods 0.000 description 2
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Classifications
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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
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
-
- 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
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
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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
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
Abstract
The invention provides a cell microcarrier separation device, which comprises a tank body, a filter, a stirring system and a liquid inlet and outlet. Wherein, the filter is arranged in the tank body, and the filter can be fixed with a filter membrane; the stirring system is used to create a swirling flow field in the liquid with its center of swirl located inside the filter. After the stirring system is started, microcarriers and cells outside the filter flow to the vortex center in the filter, and when flowing to the filter membrane on the filter, the cells with the size smaller than the aperture of the filter membrane can normally pass through, and the microcarriers with the size larger than the aperture of the filter membrane are trapped, so that microcarriers and cells in the microcarrier cell mixed solution are separated. The cell microcarrier separation device provided by the invention utilizes the swirling flow field and the filter membrane to separate microcarriers and cells, has the advantages of quick operation time and high flow dividing efficiency, and is suitable for treating cell fluids in various volume scales.
Description
Technical Field
The invention belongs to the technical field of biological cell engineering, and particularly relates to a cell microcarrier separation device.
Background
Microcarrier culture is a common technology in the current cell culture field, and is a cell culture technology which adopts fine particles as a carrier (about 100 μm in size) and cells (about 10 μm in size) suspension to be fully mixed, and then the cells can be attached to the surface of the carrier for culture and proliferation. The microcarrier culture has the advantages of common monolayer culture and suspension culture, and has the advantages of high cell proliferation speed, wide application range and the like. In the fields of vaccine production, virus production or partial immune cell culture, microcarrier culture can be widely applied to culture of some adherent cells, culture domestication is not needed, the adherent characteristics of the cells are maintained, and cell culture solution can be fully utilized for rapid expansion.
In general, in microcarrier culture of adherent cells, harvesting of the cells of interest is a final critical process therein. The general procedure is to separate the cells from the microcarriers by adding a lytic enzyme after the culture is completed and to collect them by centrifugation or filtration means.
Cell therapy is taken as an accurate medical means, at present, a slow virus vector is mostly adopted to transfect exogenous genes into immune cells, an adherent cell is used in a virus production process, the cell therapy is far from a large-batch kiloliter-level production-level suspension cell bioreactor used by some conventional production enterprises, and a multi-layer cell factory or a micro cell factory is mostly adoptedCarrier production process. Because different products can not be obtained by adopting a filtering and clarifying method in an antibody production process, a centrifugal method can be adopted when only collecting cells, but when the cells are separated after being co-cultured with microcarriers, the ideal separation effect can not be achieved by adopting a simple centrifugal method, the complexity of a process flow is increased, and in addition, the density of the cells co-cultured with microcarriers is higher, and can reach 1 multiplied by 10 7 Above/ml, clogging of the separation membrane is likely to occur when passing through the membrane by the filtration method.
Thus, there remains a need in the art for a more efficient cell microcarrier separation apparatus suitable for separating a mixture of cell microcarriers at various volumetric scales.
Disclosure of Invention
The present invention provides a filter comprising a container frame structure and one or more stopper supports; the filter membrane fixing piece is connected with the structure forming the upper opening of the container and is formed around the container; the baffle support piece is arranged outside the container frame structure and fixed on the filter membrane fixing piece, and an included angle of 10-30 degrees is formed between the baffle support piece and the normal line at the connecting point of the baffle support piece and the filter membrane fixing piece.
In one or more embodiments, the filter membrane fixing member has a hollowed-out structure for fixing the filter membrane; the baffle support piece is provided with a hollow structure and is used for fixing the filter membrane.
In one or more embodiments, the filter further comprises a first filter membrane secured to the filter membrane mount and a second filter membrane secured to the baffle support; the mesh size of the first filter membrane and the second filter membrane is between the size of the microcarrier used for cell culture and the cell size, and is used for intercepting the microcarrier.
In one or more embodiments, the filter further comprises an end cap secured to the structure forming the upper opening of the container and sealing the upper opening, the end cap having two through holes.
The invention also provides a cell microcarrier separation device, comprising:
a tank body;
a filter disposed inside the tank; and
the stirring device is arranged in the stirring system inside the filter;
the filter comprises a container frame structure, wherein the container frame structure comprises a structure for forming an upper opening of a container, a filter membrane fixing piece which is connected with the structure for forming the upper opening of the container and is formed around the container and used for fixing a filter membrane, and a bottom container connected with the filter membrane fixing piece.
In one or more embodiments, the filter is a filter according to any one of the embodiments of the present invention.
In one or more embodiments, the separation device further comprises a liquid inlet disposed at a top or upper sidewall of the tank and a waste outlet disposed at a lower sidewall or bottom of the tank.
In one or more embodiments, the cell microcarrier separation apparatus further comprises an end cap for sealing the tank to form a closed space inside the tank, the end cap having two through holes communicating between the outside of the tank and the inside of the filter, wherein one through hole is for installing the agitation system and the other through hole is for discharging cells.
In one or more embodiments, the stirring system includes a motor, a stirring shaft, and a stirring paddle, the stirring shaft connecting the stirring paddle with a rotating shaft of the motor and extending into the filter.
The invention also provides a cell microcarrier separation device, comprising:
a tank body;
an end cap sealably connected to the can body;
a filter disposed inside the tank; and
the stirring device is arranged in the stirring system inside the filter;
wherein, a liquid inlet is arranged on the top or the upper side wall of the tank body, and a waste liquid outlet is arranged on the lower side wall or the bottom of the tank body;
the end cover is provided with a first through hole and a second through hole which are communicated with the outside of the tank body and the inside of the filter, the first through hole is used for installing the stirring system, and the second through hole is used for discharging cells;
the filter includes a container frame structure and one or more stopper supports; the filter membrane fixing piece is connected with the structure forming the upper opening of the container and is formed around the container; the baffle support piece is arranged outside the container frame structure and fixed on the filter membrane fixing piece, and an included angle of 10-30 degrees is formed between the baffle support piece and the normal line at the connecting point of the baffle support piece and the filter membrane fixing piece; the filter membrane fixing piece is provided with a hollowed-out structure and is used for fixing the filter membrane; the baffle support piece is provided with a hollowed-out structure and is used for fixing the filter membrane; the filter further comprises a first filter membrane and a second filter membrane, wherein the first filter membrane is fixed on the filter membrane fixing piece, and the second filter membrane is fixed on the baffle support piece; the mesh size of the first filter membrane and the second filter membrane is between the size of the microcarrier used for cell culture and the cell size, and is used for intercepting the microcarrier.
Drawings
FIG. 1 is a schematic diagram showing the structure of a cell microcarrier separation apparatus according to the present invention.
FIG. 2 is a schematic cross-sectional view of a filter, a stirring shaft and a stirring paddle of the cell microcarrier separation apparatus of the present invention.
The reference numerals are explained as follows:
1. a tank body; 2. a filter; 3. a stopper support; 4. a filter membrane fixing member; 5. a motor; 6. a stirring shaft; 7. stirring paddles; 8. a liquid inlet; 9. a cell fluid outlet; 10. a waste liquid outlet; 11. an end cap; 111. a first through hole; 112. and a second through hole.
Detailed Description
So that those skilled in the art can appreciate the features and effects of the present invention, a general description and definition of the terms and expressions set forth in the specification and claims follows. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and in the event of a conflict, the present specification shall control.
The cell microcarrier separation device of the invention can be suitable for treating cell fluids with various volume scales, and is especially suitable for treating cell fluids with volume scales of about 1L to 50L in small and medium sizes.
The cell microcarrier separation device comprises a tank body, a filter and a stirring system. Wherein, the filter is arranged in the tank body, and the filter can be fixed with a filter membrane; the stirring system is used to create a swirling flow field in the liquid with its center of swirl located inside the filter. After the stirring system is started, microcarriers and cells outside the filter flow to the vortex center in the filter, flow to the filter membrane on the filter, and the cells with the size smaller than the aperture of the filter membrane can normally pass through, and the microcarriers with the size larger than the aperture of the filter membrane are trapped, so that microcarriers and cells in the microcarrier cell mixed solution are separated.
In the present invention, the shape of the can body is not particularly limited, and may be, for example, a cylindrical shape, a rectangular parallelepiped shape, or the like.
The cell microcarrier separation apparatus can further include an end cap that can be sealingly connected to the tank. In certain embodiments, the filter is mounted inside the canister and sealingly secured to the end cap. The end cover is provided with a first through hole which is communicated with the outside of the tank body and the inside of the filter. The stirring system comprises a motor, a stirring shaft and stirring paddles. The motor is mounted on the end cap. The stirring shaft connected with the rotating shaft of the motor stretches into the filter through the first through hole on the end cover. The stirring shaft is fixedly provided with a stirring paddle. The stirring device of the stirring system comprises a stirring shaft and a stirring paddle, and is arranged inside the filter. In certain embodiments, the filters of the present invention are cylindrical.
The cell microcarrier separation device of the invention can also comprise a liquid inlet and a liquid outlet. The liquid inlet and outlet comprises a liquid inlet, a cell liquid outlet and a waste liquid outlet. The liquid inlet is positioned on the side wall or the end cover at the upper part of the tank body and is used for communicating the outside of the tank body and the space outside the filter in the tank body. The end cover is provided with a second through hole which is communicated with the outside of the tank body and the inside of the filter, and the cell liquid outlet is formed at the second through hole. The waste liquid outlet is positioned on the side wall or the bottom of the lower part of the tank body and is used for communicating the outside of the tank body and the outside part of the filter in the tank body.
The filter of the present invention comprises a vessel frame structure and optionally a first filter membrane. The container frame structure comprises a structure for forming an upper opening of the container, a filter membrane fixing piece which is connected with the structure for forming the upper opening of the container and is formed around the container for fixing a filter membrane, and a bottom container connected with the filter membrane fixing piece. The container frame structure forms a structure with one end being open and the other end being sealed. The open end is referred to herein as the top of the filter and the closed end is referred to as the bottom of the filter. The filter holder is preferably cylindrical. The filter membrane fixing piece is provided with a hollow structure. The first filter membrane is detachably fixed to the filter membrane holder, and thus, the first filter membrane may be additionally provided in the form of a consumable. The mesh size (pore size) of the first filter membrane is set to allow the cells to pass therethrough while entrapping the microcarriers, usually not more than 100 μm, and may be, for example, not more than 70 μm, 50 to 70 μm, or the like. Generally, the area of the hollowed-out part on the filter membrane fixing piece occupies most of the total area of the filter membrane fixing piece so as to fully exert the filtering function of the first filter membrane. In certain embodiments, the hollowed-out portion of the filter holder comprises more than 50% of its total area, such as more than 60%, more than 70%, more than 80%, more than 90%, etc. The manner of fixing the first filter to the filter holder is not particularly limited, and for example, the filter holder may have a slot corresponding to the shape and size of the first filter, and the first filter may be inserted into the slot to be fixed to the filter holder. In some embodiments, the filter holder has a double-layered frame structure, and the first filter is placed and sandwiched between the two layers of frames. The bottom container connected to the filter holder preferably has an outwardly protruding shape, such as an outwardly protruding shallow conical shape, an outwardly protruding shallow circular dish shape, an outwardly protruding spherical shape, an outwardly protruding truncated conical shape, etc., to reduce the liquid flow resistance and the shear force to the cells. Typically, the filter is sealingly secured to the end cap by a structure that forms an upper opening of the container. The filter may be sealingly secured to the end cap using methods conventional in the art. In some embodiments, the edge of the structure forming the upper opening of the container has a protrusion, the end cap has a fitting portion thereon that mates with the protrusion, and the filter is secured to the end cap by fitting the protrusion into the fitting portion on the end cap; preferably, a rubber sealing ring can be arranged in the embedded part on the end cover, so that the sealing connection between the filter and the end cover is ensured.
In certain embodiments, the filters of the present invention further comprise one or more baffle supports and optionally a second filter membrane. The baffle support piece is provided with a flat plate-shaped appearance and a hollowed-out structure. The second filter membrane is detachably fixed to the stopper support, and thus, the second filter membrane may be additionally provided in the form of a consumable. The baffle support and the second filter membrane form a baffle. The mesh size (pore size) of the second filter is set to allow cells to pass through the filter while entrapping microcarriers, typically not greater than 100 μm, and may be, for example, not greater than 70 μm, 50 to 70 μm, etc. Generally, the area of the hollowed-out part on the baffle support piece occupies most of the total surface area so as to fully play the filtering function of the second filter membrane. In certain embodiments, the hollowed-out portion of the baffle support comprises more than 50% of the total surface area, such as more than 60%, more than 70%, more than 80%, more than 90%, etc. The manner of fixing the second filter membrane to the stopper support is not particularly limited, and for example, the stopper support may have a slot corresponding to the shape and size of the second filter membrane, and the second filter membrane may be fixed to the stopper support by being inserted into the slot. In some embodiments, the baffle support has an elongated double-layered frame structure with the first filter membrane being sandwiched between the two layers of frames. The baffle support piece is arranged outside the container frame structure and is fixed on the filter membrane fixing piece; preferably, the stopper support is disposed parallel to the axial direction of the filter membrane holder. The filter of the present invention may include a plurality of baffle supports. In embodiments where the filter comprises a plurality of baffle supports, the plurality of baffle supports are preferably fixed to the filter membrane holder at equal intervals or symmetrically around the filter membrane holder.
In some embodiments, as shown in fig. 2, the baffle support is configured to tilt toward the rotational direction of the stirring shaft, such that the baffle is capable of retaining the microcarriers in the swirling flow field while ensuring that the microcarriers have a certain flowability, avoiding clogging of the second filter membrane. Preferably, the baffle support forms an angle of 10-30 degrees with the normal line at the connection point A of the baffle support and the filter membrane fixing piece (shown as the angle alpha in figure 2).
The length of the stopper support (axial direction of the filter) is not particularly limited and may be the same as or smaller than the height of the filter holder, and may be, for example, 1/5 to 4/5 of the height of the filter holder or 1/2 to 4/5 of the height of the filter holder. The width of the stopper support is also not particularly limited, and may be, for example, 1/10 to 1/2 of the inner diameter of the filter, or 1/5 to 1/3 of the inner diameter of the filter. It will be appreciated that the widthwise extent of the flight support should not touch the inner wall of the can. When the length of the stopper support is shorter than the height of the filter membrane holder, the placement position of the stopper support in the axial direction of the filter is not particularly limited, and for example, the stopper support may be placed at a center position outside the filter membrane holder (i.e., the distance from the upper end of the stopper support to the upper end of the filter membrane holder is the same as the distance from the lower end of the stopper support to the lower end of the filter membrane holder), or the lower end of the stopper support may be made flush with the lower end of the filter membrane holder (i.e., the connection between the filter membrane holder and the bottom container).
The present invention may employ a conventional attachment mechanism in the art, such as a shaft seal, for attaching the shaft of the motor to the stirring shaft. Typically, the stirring shaft is rotatably sealed through the first through hole, e.g. the connection of the stirring shaft and the first through hole may be provided with a rubber sealing ring or the like. In certain embodiments, the cell microcarrier separation apparatus of the invention further comprises a controller that is electrically connected to the motor for controlling the operation of the motor. The first through hole is preferably located on the axis of the filter fixed to the end cap. Preferably, the first through hole, the axis of the stirring shaft and the axis of the filter fixed to the end cover are positioned on the same straight line. The stirring paddle on the stirring shaft is driven by the motor to rotate so as to stir the liquid in the filter. The paddles may be known paddles capable of generating a spinning field, typically having 2-5 blades, for example, a 3-blade propeller. The stirring paddle can form a vortex flow field in the liquid in the rotating process, so that microcarriers and cells outside the filter flow towards the center of the vortex flow. Paddles typically have large diameter, high torque, low shear characteristics. Preferably, the blade edges are rounded and smooth so that the stirring paddle will not cause damage to the cells during rotation.
In the invention, the liquid inlet is the liquid inlet of the microcarrier cell mixed liquid and enzyme liquid to be separated, the cell liquid outlet is the liquid outlet of the target cell, and the waste liquid outlet is the liquid outlet of the waste liquid in the tank body. The liquid inlet is preferably arranged on the end cover or on the side wall of the tank body at a position close to the end cover. The waste liquid outlet is preferably arranged at the bottom of the tank body or on the side wall of the tank body at a position close to the bottom of the tank body. The liquid inlet, the cell liquid outlet and/or the waste liquid outlet are/is optionally provided with a liquid inlet pipeline, a cell liquid pipeline or a waste liquid pipeline in a sealing way, and each pipeline can be connected with a corresponding liquid storage device. In some embodiments, the cell fluid line on the cell fluid outlet extends into the interior of the filter to facilitate removal of the cells of interest. The liquid inlet pipeline, the cell liquid pipeline and/or the waste liquid pipeline can be provided with a switch for controlling the corresponding pipeline to be opened and closed. The liquid inlet pipeline, the cell liquid pipeline and/or the waste liquid pipeline can be further provided with a driving mechanism for driving the liquid in the pipeline to flow, and the driving mechanism can be various types of pumps.
The cell microcarrier separation apparatus of the present invention further comprises a controller electrically coupled to one or more of the switch and/or the drive mechanism for controlling operation of the switch and/or the drive mechanism.
In the invention, the tank body, the filter membrane fixing piece and the baffle support piece are made of stainless steel which is easy to clean and sterilize. The first filter membrane and the second filter membrane can be made of polymer, metal, ceramic, composite material and the like.
When the cell microcarrier separation device is used for separating cells and microcarriers in microcarrier cell mixed liquor, firstly adding the microcarrier cell mixed liquor to be separated into a tank body; the cells in the microcarrier cell mixture can be attached to the microcarrier or detached from the microcarrier; if the cells in the added microcarrier cell mixture are attached to the microcarriers, the enzyme separating liquid is needed to be added and fully mixed; placing a corresponding first filter membrane and an optional second filter membrane within the filter membrane holder and optional stopper support of the filter to secure the filter to the end cap; and (3) covering an end cover, starting the stirring system, and driving the stirring paddle to rotate by the motor through the stirring shaft to form a vortex flow field in the liquid. At this time, both the microcarrier and the cells outside the filter flow toward the center of the vortex, and when flowing to the first filter membrane on the filter membrane holder of the filter, the cells can normally pass through the filter membrane to enter the inside of the filter due to the difference in particle size, while the microcarrier is blocked outside the filter.
In embodiments where the filter includes a baffle support, a second filter membrane disposed on the baffle support on the filter membrane mount preferentially intercepts microcarriers in the liquid vortex, prevents the liquid vortex from passing through a first filter membrane on the filter membrane mount too quickly to cause clogging, and ensures that more cells smoothly pass through the first filter membrane on the filter membrane mount into the filter. In some embodiments, the baffle support is arranged to be inclined at a certain angle along the rotation direction of the stirring shaft, so that the baffle can intercept microcarriers in the vortex field, and can ensure that the intercepted microcarriers have certain fluidity, and avoid the loss of cells caused by direct blockage of the microcarriers after the second filter membrane.
After the stirring system rotates for a period of time, the stable stirring speed is maintained, the target cells are mostly positioned at the center of the vortex field, and at the moment, a pipeline switch of a cell liquid outlet and a driving mechanism for driving liquid in a pipeline to flow are opened, so that the collection of the target cell liquid at the center of the vortex field can be completed. During the collection, buffer solution can be added from the liquid inlet as appropriate to clean the filter membrane on the filter, so as to increase the recovery rate of cells. After the separation is completed, the stirring system stops rotating, and the end cover is opened to collect a small amount of cell liquid at the bottom of the filter. Opening the waste outlet can recover the microcarriers trapped outside the filter.
The cell microcarrier separation device is suitable for separating microcarriers and cells in various volume scales, and has the following beneficial effects:
1. the cell microcarrier separation device provided by the invention utilizes the swirling flow field and the filter membrane to separate microcarriers and cells, and has the advantages of quick operation time and high flow dividing efficiency;
2. in order to avoid that the microcarrier directly blocks the filter membrane of the filter in the vortex field in the centrifugal process, the filter is provided with the baffle piece, and the baffle piece is arranged to be inclined by a certain angle along the rotation direction of the stirring motor, so that the microcarrier can be intercepted in the vortex field, certain fluidity of the microcarrier can be ensured, and the loss of cells caused by directly blocking the filter membrane of the baffle piece is avoided;
3. the first filter membrane and the second filter membrane on the filter are respectively and detachably fixed on the filter membrane fixing piece and the baffle piece supporting piece, so that the filter membrane can be conveniently removed from the filter membrane fixing piece and the baffle piece supporting piece for cleaning or replacement, different filter membranes can be selected and used according to actual requirements, the production efficiency is improved, and the equipment configuration and maintenance cost is reduced;
4. after the separation of the cell microcarrier separation device is completed, the target cell liquid can be directly taken out from the cell liquid outlet, the operation is convenient, and the experimental operation is simplified.
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are provided for the purpose of illustration only and are not intended to limit the scope of the invention, as any devices and constructions similar or equivalent to those described herein may be employed in the present invention.
Example 1
This example describes a microcarrier separation apparatus according to the invention in combination with the accompanying drawings.
As shown in fig. 1, the separation device comprises a tank 1, a filter 2, a stirring system and a liquid inlet and outlet. The tank 1 can contain a microcarrier cell mixture to be separated. The can 1 has an end cap 11 for sealing the can 1. The material of the tank body 1 is stainless steel.
The filter 2 comprises a baffle support 3 and a vessel frame structure, optionally also a first filter membrane and a second filter membrane. The container frame structure comprises a structure for forming an upper opening of the container, a filter membrane fixing piece 4 which is connected with the structure for forming the upper opening of the container and is formed around the container and used for fixing the filter membrane, and a bottom container connected with the fixing piece. The baffle support 3 is made of stainless steel. The baffle support 3 has a strip-shaped double-layer frame structure, and a second filter membrane can be placed in the middle. The stopper support 3 and the second filter membrane constitute a stopper. The filter membrane fixing piece 4 is made of stainless steel. The filter fixing member 4 has a cylindrical shape. The bottom container connected to the filter holder 4 has a shallow conical shape protruding downward. The filter membrane fixing member 4 has a double-layer frame structure, and a first filter membrane can be placed in the middle. The first filter is fixed on the filter holder 4. The first filter membrane and the second filter membrane are made of polymers. The mesh size of the first filter and the second filter is set to allow cells to pass through the filters and entrap microcarriers, typically no greater than 100 μm. The baffle support 3 is fixed on the filter membrane fixing member 4, and the length direction of the baffle support 3 is parallel to the axial direction of the filter membrane fixing member 4. The filter 2 is positioned inside the tank 1, and the open end of the filter 2 is sealed and fixed on the end cover 11.
The stirring system comprises a motor 5, a stirring shaft 6 and a stirring paddle 7. The motor 5 is fixed to the end cap 11. The stirring shaft 6 is connected with the rotating shaft of the motor 5. The stirring shaft 6 passes through a first through hole 111 in the end cap 11 and enters the interior of the filter 2. The stirring paddle 7 is fixed on the stirring shaft 6. The stirring paddle 7 rotates under the drive of the motor 5, so as to stir the liquid in the filter 2, and a swirling flow field is formed in the liquid.
As shown in fig. 2, the stopper support 3 is provided to be inclined toward the rotation direction of the stirring shaft 6. The baffle support 3 forms an angle of 10-30 deg. (i.e. < alpha > in figure 2) with the normal line at the connection point a of the baffle support 3 and the filter membrane holder 4. Six baffle support pieces 3 are fixed on the filter membrane fixing piece 4 in an equidistant surrounding way.
The liquid inlet and outlet comprises a liquid inlet 8, a cell liquid outlet 9 and a waste liquid outlet 10. The liquid inlet 8 is the liquid inlet of the microcarrier cell mixed liquid and the enzyme liquid to be separated. The cell fluid outlet 9 is a fluid outlet of the target cell. The waste liquid outlet 10 is a liquid outlet of the waste liquid in the tank body 1. The liquid inlet 8 is positioned on the end cover 11 and is used for communicating the outside of the tank body 1 and the outside part of the filter 2 inside the tank body 1. The cell fluid outlet 9 passes through a second through hole 112 in the end cap 11 for communicating the outside of the tank 1 with the inside of the filter 2. The waste liquid outlet 10 is located below the side wall of the tank 1 for communicating the outside of the tank 1 with the outside portion of the filter 2 inside the tank 1. The liquid inlet 8, the cell liquid outlet 9 and the waste liquid outlet 10 are respectively and hermetically fixed with a liquid inlet pipeline, a cell liquid pipeline and a waste liquid pipeline, and each pipeline is provided with a switch for controlling the pipeline to be unblocked and closed.
Example 2
This example describes the procedure for microcarrier and cell separation using the microcarrier separation apparatus of example 1, with reference to the accompanying drawings.
When the separating device works, firstly, the microcarrier cell mixed solution to be separated and the separated enzyme solution are added through the liquid inlet 8, and are fully mixed. The filter 2 is fixed to the end cap 11 by placing a filter membrane (pore diameter 50 to 70 μm) in both the filter membrane holder 4 and the stopper support 3 of the filter 2. And an end cover 11 is covered, a stirring system is started, and a motor 5 drives a stirring paddle 7 to rotate rapidly through a stirring shaft 6, so that a swirling flow field is formed in liquid. At this time, both the microcarriers and cells outside the filter 2 flow toward the center of the vortex, and when flowing to the first filter membrane on the filter membrane holder 4 of the filter 2, the cells can normally pass through and enter the inside of the filter 2 due to the difference in particle size, while the microcarriers are blocked outside.
Meanwhile, the second filter membrane arranged on the baffle support piece 3 on the filter membrane fixing piece 4 intercepts microcarriers in the liquid vortex preferentially, so that the liquid vortex is prevented from blocking caused by too fast passing through the first filter membrane on the filter membrane fixing piece 4, and more cells are ensured to smoothly enter the filter 2 through the first filter membrane.
The baffle support 3 is arranged to incline towards the rotation direction of the stirring shaft 6, so that the baffle support 3 not only can intercept microcarriers in the vortex field, but also can ensure that the intercepted microcarriers have certain fluidity, and the loss of cells is avoided after the microcarriers directly block a second filter membrane on the baffle support 3.
After the stirring system rotates for a period of time, the stable stirring speed is maintained, the target cells are mostly positioned at the center of the vortex field, and at the moment, a pipeline switch of the cell liquid outlet 9 and a driving mechanism for driving liquid in a pipeline to flow are opened, so that the collection of the target cell liquid at the center of the vortex field can be completed. In certain embodiments, some buffer may be added from inlet 8 as appropriate during collection to clean the membrane on filter 2 and increase cell recovery. After the separation is completed, the stirring system stops rotating, and the end cover 11 is opened to collect a small amount of cell liquid at the bottom of the filter 2. Opening the waste outlet 10 allows recovery of microcarriers trapped outside the filter 2.
Claims (5)
1. A cell microcarrier separation apparatus, characterized in that the cell microcarrier separation apparatus comprises:
a tank body;
a filter disposed inside the tank; and
the stirring device is arranged in the stirring system inside the filter;
the filter comprises a container frame structure and one or more baffle support pieces, wherein the container frame structure comprises a structure for forming an upper opening of a container, filter membrane fixing pieces which are connected with the structure for forming the upper opening of the container and are formed around the container and used for fixing filter membranes, and a bottom container connected with the filter membrane fixing pieces; the baffle support piece is arranged outside the container frame structure and fixed on the filter membrane fixing piece, and an included angle of 10-30 degrees is formed between the baffle support piece and the normal line at the connecting point of the baffle support piece and the filter membrane fixing piece; the filter membrane fixing piece is provided with a hollowed-out structure and is used for fixing the filter membrane; the baffle support piece is provided with a hollowed-out structure and is used for fixing the filter membrane;
the filter further comprises a first filter membrane and a second filter membrane, wherein the first filter membrane is fixed on the filter membrane fixing piece, and the second filter membrane is fixed on the baffle support piece; the mesh size of the first filter membrane and the second filter membrane is between the size of a microcarrier used for cell culture and the cell size, so as to entrap the microcarrier;
the filter further comprises an end cover, wherein the end cover is fixed on the structure forming the upper opening of the container and seals the upper opening, and two through holes are formed in the end cover.
2. The cell microcarrier separation apparatus of claim 1, further comprising a liquid inlet disposed on a top or upper sidewall of the tank and a waste outlet disposed on a lower sidewall or bottom of the tank.
3. The cell microcarrier separation apparatus of claim 1, wherein the end cap is configured to seal the tank to form a closed space within the tank, one of the two through holes in the end cap is configured to mount the agitation system and the other through hole is configured to drain cells.
4. The cell microcarrier separation apparatus of claim 1, wherein the stirring system comprises a motor, a stirring shaft, and a stirring paddle, wherein the stirring shaft connects the stirring paddle to a shaft of the motor and extends into the filter.
5. A cell microcarrier separation apparatus, characterized in that the cell microcarrier separation apparatus comprises:
a tank body;
an end cap sealably connected to the can body;
a filter disposed inside the tank; and
the stirring device is arranged in the stirring system inside the filter;
wherein, a liquid inlet is arranged on the top or the upper side wall of the tank body, and a waste liquid outlet is arranged on the lower side wall or the bottom of the tank body;
the end cover is provided with a first through hole and a second through hole which are communicated with the outside of the tank body and the inside of the filter, the first through hole is used for installing the stirring system, and the second through hole is used for discharging cells;
the filter includes a container frame structure and one or more stopper supports; the filter membrane fixing piece is connected with the structure forming the upper opening of the container and is formed around the container; the baffle support piece is arranged outside the container frame structure and fixed on the filter membrane fixing piece, and an included angle of 10-30 degrees is formed between the baffle support piece and the normal line at the connecting point of the baffle support piece and the filter membrane fixing piece; the filter membrane fixing piece is provided with a hollowed-out structure and is used for fixing the filter membrane; the baffle support piece is provided with a hollowed-out structure and is used for fixing the filter membrane; the filter further comprises a first filter membrane and a second filter membrane, wherein the first filter membrane is fixed on the filter membrane fixing piece, and the second filter membrane is fixed on the baffle support piece; the mesh size of the first filter membrane and the second filter membrane is between the size of the microcarrier used for cell culture and the cell size, and is used for intercepting the microcarrier.
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| CN201911221057.3A CN110734847B (en) | 2019-12-03 | 2019-12-03 | Cell microcarrier separation device |
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| CN201911221057.3A CN110734847B (en) | 2019-12-03 | 2019-12-03 | Cell microcarrier separation device |
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| CN1517141A (en) * | 2003-01-16 | 2004-08-04 | ������������ʽ���� | Filter device and filter method using the filter device |
| KR20090063370A (en) * | 2007-12-14 | 2009-06-18 | 한국동서발전(주) | The inner flow cut off system for bucket type strainer |
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| CN108408834A (en) * | 2018-03-25 | 2018-08-17 | 苏州鑫活力环保科技有限公司 | A kind of packaged type cartridge filter for sewage disposal |
| CN108970200A (en) * | 2018-07-23 | 2018-12-11 | 浙江倍世环境科技有限公司 | Filter with self-cleaning strainer function |
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|---|---|---|---|---|
| CN1517141A (en) * | 2003-01-16 | 2004-08-04 | ������������ʽ���� | Filter device and filter method using the filter device |
| KR20090063370A (en) * | 2007-12-14 | 2009-06-18 | 한국동서발전(주) | The inner flow cut off system for bucket type strainer |
| CN205746870U (en) * | 2016-05-13 | 2016-11-30 | 武汉绿色动力再生能源有限公司 | A kind of incinerator for garbage power |
| CN108408834A (en) * | 2018-03-25 | 2018-08-17 | 苏州鑫活力环保科技有限公司 | A kind of packaged type cartridge filter for sewage disposal |
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