CN110734847A - cell microcarrier separating device - Google Patents
cell microcarrier separating device Download PDFInfo
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- CN110734847A CN110734847A CN201911221057.3A CN201911221057A CN110734847A CN 110734847 A CN110734847 A CN 110734847A CN 201911221057 A CN201911221057 A CN 201911221057A CN 110734847 A CN110734847 A CN 110734847A
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- 239000012528 membrane Substances 0.000 claims abstract description 167
- 239000007788 liquid Substances 0.000 claims abstract description 83
- 238000003756 stirring Methods 0.000 claims abstract description 73
- 238000000926 separation method Methods 0.000 claims abstract description 37
- 239000002699 waste material Substances 0.000 claims description 22
- 238000004113 cell culture Methods 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 230000000903 blocking effect Effects 0.000 claims description 5
- 238000007599 discharging Methods 0.000 claims description 4
- 210000004027 cell Anatomy 0.000 description 95
- 238000000034 method Methods 0.000 description 9
- 238000001914 filtration Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 229910001220 stainless steel Inorganic materials 0.000 description 4
- 239000010935 stainless steel Substances 0.000 description 4
- 230000001464 adherent effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 210000000172 cytosol Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003670 easy-to-clean Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000010419 fine particle Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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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
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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
- 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
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Abstract
The invention provides an 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, the filter can be fixed with a filter membrane, the stirring system is used for forming a vortex flow field with the vortex center positioned in the filter in liquid, after the stirring system is started, microcarriers and cells outside the filter flow to the vortex center in the filter, when the microcarriers flow 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 intercepted, so that the microcarriers and cells in a microcarrier cell mixed liquid are separated.
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 common technologies in the field of cell culture at present, and is cell culture technologies which adopt fine particles as carriers (about 100 microns in size) to be fully mixed with cell (about 10 microns in size) suspensions, and then the cells can be attached to the surfaces of the carriers to be cultured and proliferated.
In microcarrier culture of adherent cells, the harvesting of the cells of interest is the final critical step in the process , where the cells are separated from the microcarriers by adding a lytic enzyme after the culture is complete and collected by centrifugation or filtration.
Cell therapy is used as accurate medical means, exogenous genes are mostly transfected to immune cells by adopting a lentiviral vector at present, adherent cells are used in a virus production process, the adherent cells are far lower than a large-batch kiloliter production-level suspension cell bioreactor used by conventional production enterprises, a multi-layer cell factory or a microcarrier production process is mostly adopted, because the harvested products are different, a filtering and clarifying method in an antibody production process cannot be adopted, a centrifugal method can be adopted when the cells are simply collected, but when the cells and the microcarriers need to be separated after being cultured together, the simple centrifugal method cannot achieve an ideal separation effect, the complexity of the process flow is increased, in addition, the cell density of the microcarrier culture is higher, and the cell density can reach 1 multiplied by 107More than ml, the separation filter membrane is easily blocked when the filtration membrane is penetrated by adopting a filtration method.
Therefore, there is still a need in the art for more efficient cell microcarrier separation devices that are suitable for separating cell microcarrier mixtures of various volume scales.
Disclosure of Invention
The invention provides filters, which comprise a container frame structure and or a plurality of stopper supporting pieces, wherein the container frame structure comprises a structure forming an upper opening of a container, a filter membrane fixing piece which is connected with the structure forming the upper opening of the container and is formed on the periphery of the container and used for fixing a filter membrane, and a bottom container connected with the filter membrane fixing piece, wherein the stopper supporting 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 stopper supporting piece and a normal line at a connecting point of the stopper supporting piece and the filter membrane fixing piece.
In or more embodiments, the filter membrane holder has a recess for holding a filter membrane, and the barrier support has a recess for holding a filter membrane.
In or more embodiments, the filter further comprises a filter membrane and a second filter membrane, the filter membrane is fixed on the filter membrane fixing member, the second filter membrane is fixed on the baffle supporting member, and the mesh size of the 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 trapping the microcarrier.
In 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 present invention also provides kinds of cell microcarrier separation devices, 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 forming an upper opening of the container, a filter membrane fixing piece which is connected with the structure forming the upper opening of the container and is formed on the periphery of the container and used for fixing a filter membrane, and a bottom container connected with the filter membrane fixing piece.
In or more embodiments, the filter is a filter according to any embodiment of the present invention.
In or more embodiments, the separation device further comprises a liquid inlet disposed at the top or upper sidewall of the tank and a waste liquid outlet disposed at the lower sidewall or bottom of the tank.
In or more embodiments, the cell microcarrier separation device further comprises an end cap for sealing the canister to form a closed space inside the canister, the end cap having two through holes communicating the outside of the canister and the inside of the filter, wherein through holes are used for installing the agitation system and another through holes are used for discharging cells.
In or more embodiments, the stirring system comprises a motor, a stirring shaft and a stirring paddle, wherein the stirring shaft is connected with the rotating shaft of the motor and extends into the filter.
The present invention also provides kinds of cell microcarrier separation devices, comprising:
a tank body;
an end cap sealably connected to the tank;
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 upper side wall of the tank body, and a waste liquid outlet is arranged on the lower side wall or bottom of the tank body;
the end cover is provided with an th through hole and a second through hole which are communicated with the outside of the tank body and the inside of the filter, the th through hole is used for installing the stirring system, and the second through hole is used for discharging cells;
the filter comprises a container frame structure and or more stopper supporting pieces, wherein the container frame structure comprises a structure forming an upper opening of the container, a filter membrane fixing piece which is connected with the structure forming the upper opening of the container and is formed on the periphery of the container and used for fixing a filter membrane, and a bottom container connected with the filter membrane fixing piece, the stopper supporting piece is arranged outside the container frame structure and fixed on the filter membrane fixing piece, an included angle of 10-30 degrees is formed between the stopper supporting piece and a normal line of a connecting point of the stopper supporting piece and the filter membrane fixing piece, the filter membrane fixing piece is provided with a hollowed-out structure and used for fixing the filter membrane, the stopper supporting piece is provided with a hollowed-out structure and used for fixing the filter membrane, the filter further comprises a th filter membrane and a second filter membrane, the th filter membrane is fixed on the filter membrane fixing piece, the second filter membrane is fixed on the stopper supporting piece, and the mesh sizes of the th filter membrane and the second filter membrane are between the size of a microcarrier for cell culture and.
Drawings
FIG. 1 is a schematic structural view of a cell microcarrier separation device 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 below:
1. the device comprises a tank body, a filter, a stopper supporting piece, a filter membrane fixing piece, a motor, a stirring shaft, a stirring paddle, a liquid inlet, a cell liquid outlet, a waste liquid outlet, an end cover, a 111 through hole, an through hole and a 112 through hole, wherein the filter membrane fixing piece is arranged on the tank body, the filter is arranged at the position of the stopper supporting piece, the filter membrane fixing piece is arranged at the position of the stopper supporting piece.
Detailed Description
To achieve the objects and advantages of the invention, as will be apparent to those skilled in the art, the specification and claims are to be regarded in an -generic specification and definition.
The cell microcarrier separation device can be suitable for cell sap treatment of various volume scales, and is particularly suitable for cell sap treatment of small and medium-sized volume scales of about 1-50L.
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 a filter membrane can be fixed on the filter; the stirring system is used for forming a vortex flow field with a vortex center positioned inside the filter in the liquid. After the stirring system is started, the microcarrier and the cells outside the filter flow to the vortex center in the filter and flow to the filter membrane on the filter, the cells with the size smaller than the pore size of the filter membrane can normally pass through the filter membrane, and the microcarrier with the size larger than the pore size of the filter membrane is intercepted, so that the microcarrier and the 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 device may also include an end cap sealably connected to the tank, in certain embodiments, the filter is mounted inside the tank and sealably secured to the end cap, the end cap having a th opening therethrough communicating the outside of the tank with the inside of the filter, a stirring system including a motor, a stirring shaft and a stirring paddle, the motor being mounted to the end cap, the stirring shaft connected to a shaft of the motor extending into the filter through a th opening in the end cap, the stirring shaft having the stirring paddle secured thereto, the stirring device of the stirring system including the stirring shaft and the stirring paddle being disposed within the filter.
The cell microcarrier separation device of the invention may further comprise a liquid inlet and outlet. The liquid inlet and outlet comprises a liquid inlet, a cell sap 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 space outside the tank body and the space outside the filter in the tank body. The end cover is provided with a second through hole for communicating the outside of the tank body with the inside of the filter, and a cell sap 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 with the outside of the filter inside the tank body.
The filter of the present invention comprises a container frame structure and optionally a th filter membrane, the container frame structure comprises a structure forming an upper opening of the container, a filter membrane holder connected to the structure forming the upper opening of the container for holding the filter membrane, and a bottom container connected to the filter membrane holder, the container frame structure forms a structure in which the end is open and the end is closed, the open end is referred to herein as the top of the filter and the closed end is referred to herein as the bottom of the filter, the filter membrane holder is preferably cylindrical, the filter membrane holder has a hollowed-out structure, the th filter membrane is detachably held on the filter membrane holder, thus, the th filter membrane can be provided in the form of a consumable, the mesh size (pore size) of the th filter membrane is set to allow cells to pass through while trapping microcarriers, typically not more than 100 μm, for example, may be not more than 70 μm, 50-70 μm, etc., typically, the hollowed-out area of the filter membrane holder is a majority of the total area of the hollowed-out area of the filter membrane holder, to fully perform the filtering function of the filter membrane holder, the filter is designed to fully, the filter membrane holder is designed to hold the filter membrane holder, the filter membrane holder has a top of a shape of a top cover, the filter membrane holder is preferably designed to be fixed to the top cover, the top cover of a conical shape of a structure, the top cover, the filter membrane holder, the cover is designed to be fixed to cover, the cover of the cover, the cover.
In some embodiments, the filter of the present invention further comprises or more stopper supports and optionally a second filter membrane, wherein the stopper supports have a plate-like shape and a perforated structure, the second filter membrane is detachably fixed to the stopper supports, and thus the second filter membrane can be additionally provided in the form of a consumable, the stopper supports and the second filter membrane constitute a stopper, the mesh size (pore size) of the second filter membrane is set to allow cells to pass through the filter membrane while retaining microcarriers, generally not more than 100 μm, for example, not more than 70 μm, 50 to 70 μm, etc. typically, the perforated portion of the stopper supports has an area occupying a large part of the total surface area to fully perform the filtering function of the second filter membrane.
In some embodiments, as shown in FIG. 2, the support of the stopper is disposed to be inclined toward the rotation direction of the stirring shaft, so that the stopper can trap the microcarrier in the swirling flow field while ensuring that the microcarrier has -degree flowability and avoiding blocking the second filter membrane, preferably, the support of the stopper is at an angle of 10-30 degrees (as shown in ∠α in FIG. 2) with respect to the normal line at the connection point A of the stopper and the filter membrane fixing member.
The length of the stopper support (in the axial direction of the filter) is not particularly limited, and may be the same as or smaller than the height of the filter membrane fixing member, and may be, for example, 1/5 to 4/5 or 1/2 to 4/5. The width of the stopper support is also not particularly limited and can be, for example, 1/10 to 1/2 of the filter inner diameter, or 1/5 to 1/3 of the filter inner diameter. It will be appreciated that the width-wise extension of the flight support should not touch the inner wall of the tank. When the length of the stopper supporting member is shorter than the height of the filter membrane fixing member, there is no particular limitation on the placement position of the stopper supporting member in the axial direction of the filter, for example, the stopper supporting member can be placed at the center of the outside of the filter membrane fixing member (i.e., the distance from the upper end of the stopper supporting member to the upper end of the filter membrane fixing member is the same as the distance from the lower end of the stopper supporting member to the lower end of the filter membrane fixing member), or the lower end of the stopper supporting member is flush with the lower end of.
The present invention may employ any of the conventional attachment mechanisms in the art, such as a shaft seal, connecting the shaft of a motor to a stirring shaft, typically the stirring shaft is rotatably sealed through th through hole, for example, the connection of the stirring shaft to th through hole may be provided with a rubber seal, etc. in certain embodiments, the cell microcarrier separation apparatus of the present invention further comprises a controller electrically connected to the motor for controlling the operation of the motor, th through hole is preferably located on the axis of the filter affixed to the end cap, preferably th through hole, the axis of the stirring shaft and the axis of the filter affixed to the end cap are located on the same straight line, the stirring paddle on the stirring shaft is rotated by the motor to agitate the liquid inside the filter, the stirring paddle may be a known stirring paddle capable of generating a swirling flow field, typically having 2 to 5 blades, such as a propeller with 3 blades.
In the invention, the liquid inlet is a liquid inlet of a microcarrier cell mixed liquid and an enzyme liquid to be separated, the cell liquid outlet is a liquid outlet of target cells, and the waste liquid outlet is a liquid outlet of 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 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 close to the bottom of the tank body. The liquid inlet, the cell liquid outlet and/or the waste liquid outlet are respectively and optionally fixed 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 certain embodiments, the cytosol line on the cytosol 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 smoothness and the closing of the corresponding pipelines. The liquid inlet pipeline, the cell liquid pipeline and/or the waste liquid pipeline can be provided with a driving mechanism for driving the liquid in the pipeline to flow, for example, various pumps can be arranged.
The cell microcarrier separation device of the present invention further comprises a controller that is electrically connected to one or more switches and/or drive mechanisms for controlling the operation of the switches and/or drive mechanisms.
The materials of the tank body, the filter membrane fixing piece and the baffle supporting piece are usually stainless steel which is easy to clean and disinfect, and the th filter membrane and the second filter membrane can be polymers, metals, ceramics, composite materials and the like.
When the cell microcarrier separation device is used for separating cells and microcarriers in a microcarrier cell mixed solution, firstly adding a microcarrier cell mixed solution to be separated into a tank body, wherein the cells in the microcarrier cell mixed solution can be attached to the microcarriers or can be detached from the microcarriers, if the cells in the microcarrier cell mixed solution are attached to the microcarriers, then separating enzyme liquid needs to be added and fully mixed, a corresponding filter membrane and an optional second filter membrane are placed in a filter membrane fixing piece and an optional baffle piece supporting piece of a filter, the filter is fixed on an end cover, the end cover is covered, a stirring system is started, a motor drives a stirring paddle to rotate through a stirring shaft, a vortex field is formed in the liquid, at the moment, the microcarriers and the cells outside the filter flow to a vortex center, when the cells flow to a filter membrane fixing piece of the filter, the cells can normally enter the filter through the filter membranes due to different particle sizes, and the microcarriers are blocked outside the filter.
In the embodiment that the filter comprises the stopper support member, the second filter membrane arranged on the stopper support member on the filter membrane fixing member preferentially intercepts the microcarriers in the liquid vortex, so that the liquid vortex is prevented from passing through the th filter membrane on the filter membrane fixing member too fast to cause blockage, and more cells are ensured to smoothly enter the filter through the th filter membrane on the filter membrane fixing member.
After the stirring system rotates for periods, the stable stirring speed is kept, most target cells are positioned in the center of the vortex field, at the moment, a pipeline switch of a cell sap outlet and a driving mechanism for driving liquid in the pipeline to flow are opened, the collection of the target cell sap in the center of the vortex field can be completed, buffer solutions can be added from a liquid inlet as appropriate in the collection process, a filter membrane on a filter is cleaned, the cell recovery rate is increased, after the separation is completed, the stirring system stops rotating, an end cover is opened, a small amount of cell sap at the bottom of the filter is collected, is also collected, and microcarriers intercepted outside the filter can be recovered by opening a waste liquid outlet.
The cell microcarrier separation device is suitable for separating microcarriers and cells with various volume scales, and has the following beneficial effects:
1. the cell microcarrier separation device disclosed by the invention utilizes the vortex flow field and the filter membrane to separate microcarriers and cells, so that the operation time is short and the shunting efficiency is high;
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 stopper which is arranged to be inclined at a fixed angle along the rotation direction of the stirring motor, so that the microcarrier can be intercepted in the vortex field, the microcarrier can be ensured to have fixed fluidity, and the loss of cells caused by directly blocking the filter membrane of the stopper is avoided;
3. the th filter membrane and the second filter membrane on the filter are respectively detachably fixed on the filter membrane fixing piece and the stopper supporting piece, can be conveniently removed from the filter membrane fixing piece and the stopper supporting piece for cleaning or replacement, and can select different filter membranes according to actual requirements, so that the production efficiency is improved, and the equipment configuration and maintenance cost is reduced;
4. after the cell microcarrier separation device is used for separation, target cell sap can be directly taken out from a cell sap outlet, so that the operation is convenient and fast, and the experimental operation is simplified.
The invention will now be further described with reference to specific examples, it being understood that these examples are intended only to illustrate the invention and are not intended to limit the scope of the invention, and that any devices and constructions similar or equivalent to those described herein may be used in the practice of the invention.
Example 1
This example describes microcarrier separation devices according to the invention in conjunction with the accompanying drawings.
As shown in fig. 1, the separation apparatus includes 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 body 1 has an end cap 11 to seal the can body 1. The material of the tank body 1 is stainless steel.
The filter 2 comprises a stopper support 3 and a container frame structure, and optionally further comprises th filter membrane and a second filter membrane, wherein the container frame structure comprises a structure forming an upper opening of the container, a filter membrane fixing member 4 connected with the structure forming the upper opening of the container and formed on the periphery of the container for fixing the filter membrane, and a bottom container connected with the fixing member, the stopper support 3 is made of stainless steel, the stopper support 3 has an elongated double-layer frame structure, the second filter membrane can be placed in the middle, the stopper support 3 and the second filter membrane form a stopper, the filter membrane fixing member 4 is made of stainless steel, the filter membrane fixing member 4 is cylindrical, the bottom container connected with the filter membrane fixing member 4 is in a shallow conical shape protruding downwards, the filter membrane fixing member 4 has a double-layer frame structure, the th filter membrane can be placed in the middle, the th filter membrane is fixed on the filter membrane fixing member 4, the materials of the th filter membrane and the second filter membrane are polymers, the mesh sizes of the th filter membrane and the second filter membrane are set to allow cells to pass through the filter membrane and retain microcarriers, the filter membrane is usually not more than 100 mu m, the length direction of the stopper support 3 is parallel to.
The stirring system comprises a motor 5, a stirring shaft 6 and a stirring paddle 7, wherein the motor 5 is fixed on an end cover 11, the stirring shaft 6 is connected with a rotating shaft of the motor 5, the stirring shaft 6 enters the filter 2 through a th through hole 111 in the end cover 11, the stirring paddle 7 is fixed on the stirring shaft 6, the stirring paddle 7 rotates under the driving of the motor 5, and then liquid in the filter 2 is stirred, and a vortex flow field is formed in the liquid.
As shown in FIG. 2, the stopper support member 3 is disposed to be inclined toward the rotation direction of the stirring shaft 6. the stopper support member 3 is formed at an angle of 10 to 30 degrees (i.e., ∠α in FIG. 2) with respect to the normal line at the connecting point A of the stopper support member 3 and the filter membrane fixing member 4. six stopper support members 3 are circumferentially fixed to the filter membrane fixing member 4 at equal intervals.
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 a liquid inlet for 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 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 with the outside part of the filter 2 inside the tank body 1. The cell sap outlet 9 passes through the second through hole 112 of the end cap 11 for communicating the outside of the tank 1 and 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 of the filter 2 inside the tank 1. A liquid inlet pipeline, a cell liquid pipeline and a waste liquid pipeline are respectively fixed on the liquid inlet 8, the cell liquid outlet 9 and the waste liquid outlet 10 in a sealing way, and each pipeline is provided with a switch for controlling the smoothness and the closing of the pipeline.
Example 2
This example describes the procedure of microcarrier and cell separation using the microcarrier separation device of example 1 with reference to the accompanying drawings.
When the separation device works, firstly, a microcarrier cell mixed solution to be separated and a separated enzyme solution are added from a liquid inlet 8 and are fully mixed, filter membranes (with the aperture of 50-70 mu m) are placed in a filter membrane fixing part 4 and a blocking part supporting part 3 of a filter 2, the filter 2 is fixed on an end cover 11, the end cover 11 is covered, a stirring system is started, a motor 5 drives a stirring paddle 7 to rapidly rotate through a stirring shaft 6, a vortex flow field is formed in liquid, at the moment, microcarriers and cells outside the filter 2 flow to the vortex center, when the microcarriers flow to an th filter membrane on the filter membrane fixing part 4 of the filter 2, the cells can normally pass through and enter the filter 2 due to different particle sizes, and the microcarriers are blocked outside.
Meanwhile, the second filter membrane arranged on the stopper support 3 on the filter membrane fixing part 4 intercepts the microcarriers in the liquid vortex preferentially, so that the liquid vortex is prevented from passing through the th filter membrane on the filter membrane fixing part 4 too fast to cause blockage, and more cells are ensured to enter the filter 2 through the th filter membrane smoothly.
The stopper support 3 is inclined towards the rotation direction of the stirring shaft 6, so that the stopper support 3 can intercept microcarriers in the vortex field, and ensure that the intercepted microcarriers have -determined flowability, thereby avoiding cell loss caused by the microcarriers directly blocking the second filter membrane on the stopper support 3.
When the stirring system rotates time, the stirring speed is kept stable, the target cells are mostly positioned at the center of the vortex field, at this time, the pipeline switch of the cell sap outlet 9 and the driving mechanism for driving the liquid in the pipeline to flow are opened, the collection of the target cell sap at the center of the vortex field can be completed, in some embodiments, buffer solution can be added from the liquid inlet 8 as appropriate during the collection process, the filter membrane on the filter 2 is cleaned, the cell recovery rate is increased, after the separation is completed, the stirring system stops rotating, the end cover 11 is opened, the small amount of cell sap at the bottom of the filter 2 is collected, and is also collected, and the microcarriers trapped outside the filter 2 can be recovered by opening the waste liquid outlet 10.
Claims (10)
- The filter is characterized by comprising a container frame structure and or a plurality of stopper supporting pieces, wherein the container frame structure comprises a structure forming an upper opening of a container, a filter membrane fixing piece which is connected with the structure forming the upper opening of the container and is formed on the periphery of the container and used for fixing a filter membrane, and a bottom container connected with the filter membrane fixing piece, the stopper supporting pieces are 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 stopper supporting pieces and a normal line at a connecting point of the stopper supporting pieces and the filter membrane fixing piece.
- 2. The filter of claim 1, wherein the filter membrane fixing member has a hollowed-out structure for fixing the filter membrane; the blocking piece supporting piece is provided with a hollow structure and used for fixing the filter membrane.
- 3. The filter of claim 1, further comprising a th filter membrane and a second filter membrane, wherein the th filter membrane is fixed to the filter membrane holder, the second filter membrane is fixed to the stopper support, and wherein the th filter membrane and the second filter membrane have a mesh size between a size of the microcarriers used for cell culture and a cell size for trapping the microcarriers.
- 4. The filter of claim 1 further comprising 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.
- 5, cell microcarrier separation device, wherein said cell microcarrier separation device comprises:a tank body;a filter disposed inside the tank; andthe 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 forming an upper opening of the container, a filter membrane fixing piece which is connected with the structure forming the upper opening of the container and is formed on the periphery of the container and used for fixing a filter membrane, and a bottom container connected with the filter membrane fixing piece.
- 6. The cell microcarrier separation device of claim 5, wherein the filter is according to any of claims 1-4 to .
- 7. The cell microcarrier separation device of claim 5, further comprising a liquid inlet disposed on the top or upper sidewall of the tank and a waste liquid outlet disposed on the lower sidewall or bottom of the tank.
- 8. The cell microcarrier separation device of claim 5, further comprising an end cap for sealing the canister to form a closed space inside the canister, wherein the end cap has two through holes for connecting the outside of the canister and the inside of the filter, and wherein through holes are used for installing the stirring system and another through holes are used for discharging cells.
- 9. The cell microcarrier separation device of claim 5, wherein the stirring system comprises a motor, a stirring shaft and a stirring paddle, and the stirring shaft is connected with the rotating shaft of the stirring paddle and the motor and extends into the filter.
- 10, cell microcarrier separation device, wherein said cell microcarrier separation device comprises:a tank body;an end cap sealably connected to the tank;a filter disposed inside the tank; andthe stirring device is arranged in the stirring system inside the filter;wherein, a liquid inlet is arranged on the top or upper side wall of the tank body, and a waste liquid outlet is arranged on the lower side wall or bottom of the tank body;the end cover is provided with an th through hole and a second through hole which are communicated with the outside of the tank body and the inside of the filter, the th through hole is used for installing the stirring system, and the second through hole is used for discharging cells;the filter comprises a container frame structure and or more stopper supporting pieces, wherein the container frame structure comprises a structure forming an upper opening of the container, a filter membrane fixing piece which is connected with the structure forming the upper opening of the container and is formed on the periphery of the container and used for fixing a filter membrane, and a bottom container connected with the filter membrane fixing piece, the stopper supporting piece is arranged outside the container frame structure and fixed on the filter membrane fixing piece, an included angle of 10-30 degrees is formed between the stopper supporting piece and a normal line of a connecting point of the stopper supporting piece and the filter membrane fixing piece, the filter membrane fixing piece is provided with a hollowed-out structure and used for fixing the filter membrane, the stopper supporting piece is provided with a hollowed-out structure and used for fixing the filter membrane, the filter further comprises a th filter membrane and a second filter membrane, the th filter membrane is fixed on the filter membrane fixing piece, the second filter membrane is fixed on the stopper supporting piece, and the mesh sizes of the th filter membrane and the second filter membrane are between the size of a microcarrier for cell culture and.
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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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| CN110734847B CN110734847B (en) | 2024-03-19 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021164509A1 (en) * | 2020-02-19 | 2021-08-26 | 上海艾众生物科技有限公司 | Cell separation apparatus for bioreactor |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
-
2019
- 2019-12-03 CN CN201911221057.3A patent/CN110734847B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021164509A1 (en) * | 2020-02-19 | 2021-08-26 | 上海艾众生物科技有限公司 | Cell separation apparatus for bioreactor |
| EP4095226A4 (en) * | 2020-02-19 | 2023-08-02 | Alit Biotech (Shanghai) Co., Ltd. | Cell separation apparatus for bioreactor |
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| Publication number | Publication date |
|---|---|
| CN110734847B (en) | 2024-03-19 |
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