CN115786120A - Disposable fixed bed for adherent cell culture - Google Patents
Disposable fixed bed for adherent cell culture Download PDFInfo
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- CN115786120A CN115786120A CN202211355215.6A CN202211355215A CN115786120A CN 115786120 A CN115786120 A CN 115786120A CN 202211355215 A CN202211355215 A CN 202211355215A CN 115786120 A CN115786120 A CN 115786120A
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- 238000004115 adherent culture Methods 0.000 title claims abstract description 43
- 238000007789 sealing Methods 0.000 claims abstract description 51
- 230000007246 mechanism Effects 0.000 claims abstract description 36
- 238000003756 stirring Methods 0.000 claims abstract description 29
- 239000012530 fluid Substances 0.000 claims abstract description 25
- 238000003760 magnetic stirring Methods 0.000 claims abstract description 17
- 238000003825 pressing Methods 0.000 claims description 23
- 239000007788 liquid Substances 0.000 claims description 17
- 238000004321 preservation Methods 0.000 claims description 17
- 238000005452 bending Methods 0.000 claims description 11
- 230000006835 compression Effects 0.000 claims description 5
- 238000007906 compression Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000010977 jade Substances 0.000 claims description 4
- 230000006978 adaptation Effects 0.000 claims description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical group [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 8
- 238000004113 cell culture Methods 0.000 abstract description 8
- 239000001301 oxygen Substances 0.000 abstract description 8
- 229910052760 oxygen Inorganic materials 0.000 abstract description 8
- 230000010261 cell growth Effects 0.000 abstract description 5
- 238000010008 shearing Methods 0.000 abstract description 5
- 238000011109 contamination Methods 0.000 abstract 1
- 230000007547 defect Effects 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 14
- 238000009413 insulation Methods 0.000 description 12
- 238000000034 method Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 238000003306 harvesting Methods 0.000 description 6
- 230000010412 perfusion Effects 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 3
- 238000009825 accumulation Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000002657 fibrous material Substances 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002861 polymer material Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 239000000565 sealant Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000003466 welding 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
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/02—Apparatus for enzymology or microbiology with agitation means; with heat exchange means
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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
- C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
- C12M3/04—Tissue, human, animal or plant cell, or virus culture apparatus with means providing thin layers
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- Organic Chemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
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Abstract
The invention discloses a disposable fixed bed for adherent cell culture, which comprises a pump shell, an upper cover plate, an annular pressure plate, a carrier, an annular bottom plate, a first supporting seat, a second supporting seat, a sealing connection mechanism and a magnetic stirring mechanism, wherein the annular bottom plate, the first supporting seat and the second supporting seat are sequentially arranged on the inner bottom surface of the pump shell from bottom to top, the upper cover plate is arranged at the opening end of the pump shell and is in sealing connection with the pump shell through the sealing connection mechanism, a stirring blade of the magnetic stirring mechanism is sleeved on a connecting column at the bottom of the pump shell, the annular pressure plate and a groove structure of the second supporting seat form an accommodating space together, and the carrier is arranged in the accommodating space. In the disposable fixed bed for adherent cell culture provided by the invention, the curtain-distributed oxygen exchange is realized through the specific fluid channel, and the stirring blade is arranged outside the cell growth space, so that the defects of large occupied area, over-intensive labor, easy contamination, large shearing force and low dissolved oxygen of the conventional cell culture equipment are overcome.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a disposable fixed bed for adherent cell culture.
Background
When the anchorage-dependent cells are cultured, the cells are attached to the wall of a culture (bottle) vessel, spread rapidly once attached to the wall, then start mitosis and enter the logarithmic growth phase quickly. The culture surface is generally confluent after several days and a dense cell monolayer is formed. The traditional disposable adherent cell culture equipment has some disadvantages at present: the occupied area is large; the labor required is too intensive; most of the devices are non-sealed devices which are easy to be infected with bacteria; the shearing force of the stirring damages cells; the oxygen exchange efficiency is low. Therefore, a more efficient and more suitable device for adherent cell culture is needed.
Disclosure of Invention
The invention aims to provide a disposable fixed bed for adherent cell culture, which solves the problems that the existing disposable adherent cell culture equipment has large floor area, needs intensive labor, is easy to contaminate bacteria, and has damage to cells and low oxygen exchange efficiency due to the shearing force of stirring.
In order to solve the technical problems, the invention provides a disposable fixed bed for adherent cell culture, which comprises: the device comprises a pump shell, an upper cover plate, an annular pressure plate, a carrier, an annular bottom plate, a first supporting seat, a second supporting seat, a first sealing connecting mechanism and a magnetic stirring mechanism;
the annular bottom plate, the first supporting seat and the second supporting seat are sequentially arranged on the bottom surface of the inner side of the pump shell from bottom to top, the upper cover plate is arranged at the opening end of the pump shell and is in sealing connection with the pump shell through the first sealing connection mechanism, and a first closed space is formed between the upper cover plate and the pump shell; the magnetic stirring mechanism comprises a driving part and a stirring blade which rotates under the driving of the driving part, a connecting column is arranged on the bottom surface of the inner side of the pump shell, the stirring blade is sleeved on the connecting column, and the stirring blade and the connecting column are both positioned in a central through hole of the annular bottom plate;
the first supporting seat and the second supporting seat are respectively provided with a first side wall, a bottom wall, a second side wall and a top wall which are sequentially connected, the first side wall of the first supporting seat is tightly attached to the first side wall of the second supporting seat, a first preset gap is formed between the second side wall of the first supporting seat and the second side wall of the second supporting seat so as to form a first fluid channel, the top wall of the second supporting seat is higher than the top wall of the first supporting seat, the bottom wall of the first supporting seat is arranged in a downward inclined mode, and the distance between the bottom wall of the first supporting seat and the bottom wall of the second supporting seat gradually increases along the inclined direction;
the bottom of the first supporting seat abuts against the outer edge of the annular bottom plate, the first supporting seat and the annular bottom plate are enclosed to form a first cavity, the first supporting seat and the second supporting seat are enclosed to form a second cavity, and the second supporting seat and the upper cover plate are enclosed to form a third cavity;
the first cavity is communicated with the bottom of the pump shell through a central through hole of the annular bottom plate, a fluid through hole is formed in the top wall of the first supporting seat, the first cavity is communicated with the second cavity through the fluid through hole, the bottom wall of the second supporting seat is of a grid structure, the second cavity is communicated with the third cavity through grid holes in the bottom wall of the second supporting seat, a second preset gap is formed between the first side wall of the first supporting seat and the side wall of the pump shell to form a second fluid channel, and the third cavity is communicated with the bottom of the pump shell through the second fluid channel;
the annular pressing plate is of a grid structure, the annular pressing plate is fixedly arranged on the second supporting seat, an accommodating space is formed by the annular pressing plate, the first side wall, the bottom wall and the second side wall of the second supporting seat, and the carrier is arranged in the accommodating space.
Optionally, in the disposable fixed bed for adherent cell culture, the center of the bottom surface of the pump housing is an upward convex structure, the upward convex structure is internally provided with accommodating spaces, the accommodating spaces are used for arranging driving parts of the magnetic stirring mechanism, and the connecting column is located above the upward convex structure;
at least two positioning columns are arranged on the bottom surface of the inner side of the pump shell, at least two clamping holes are correspondingly formed in the annular bottom plate, and the top ends of the positioning columns are inserted into the clamping holes of the annular bottom plate;
in the first supporting seat, the bottom wall is obliquely arranged, a chamfer structure is arranged at the joint of the bottom wall and the second side wall, an oblique pressing surface is arranged on the outer side edge of the annular bottom plate, and the oblique pressing surface is matched with the chamfer structure.
Optionally, the disposable fixed bed for adherent cell culture further comprises a support column, and the two opposite ends of the support column are provided with thread structures;
the pump casing is characterized in that a first mounting hole is formed in a connecting column of the pump casing, one end of the supporting column is inserted into the first mounting hole and is fixedly connected with the pump casing in a threaded connection mode, a second mounting hole is formed in the top wall of the first supporting seat, and the other end of the supporting column is inserted into the second mounting hole and is fixedly connected with the first supporting seat in a threaded connection mode.
Optionally, in the disposable fixed bed for adherent cell culture, an open end of the pump casing extends radially outward to form a first annular flange, an open end of the upper cover plate extends radially outward to form a second annular flange, and the first annular flange and the second annular flange are arranged opposite to each other and are provided with bolt holes;
the first sealing connection mechanism includes: the first flexible sealing ring, the two flexible compression rings and the first bolt and nut assembly are arranged on the first flexible sealing ring; first flexible sealing washer set up in first annular flange with between the second annular flange, two flexible clamping rings set up respectively in the dorsad of first annular flange the terminal surface of second annular flange and the dorsad of second annular flange on the terminal surface of first annular flange, the pump case two flexible clamping rings with the upper cover plate passes through first bolt and nut subassembly fixed connection.
Optionally, in the disposable fixed bed for adherent cell culture, the disposable fixed bed further comprises: the heat preservation base and the second sealing connection mechanism;
the pump shell is arranged on the heat-insulating base and is in sealing connection with the upper end edge of the heat-insulating base through the second sealing connection mechanism, a gap is reserved between the heat-insulating base and the pump shell to form a second closed space, and the second closed space is a water bath temperature control cavity.
Optionally, in the disposable fixed bed for adherent cell culture, an outward flange is annularly arranged at the top of the heat preservation base, a plurality of connecting through holes are arranged on the outward flange, a bent part corresponding to the outward flange is arranged on the side wall of the pump shell, a plurality of screw sleeves are arranged on the bent part, the bent part of the pump shell abuts against the outward flange of the heat preservation base, and the inner side surface of the bent part is an inclined plane;
the second sealing connection mechanism further comprises: the second flexible sealing ring, the flexible gasket and the second bolt-nut assembly; the second flexible sealing ring is arranged between the bent part of the pump shell and the outward turning edge of the heat preservation base, the flexible gasket is arranged on one side, back to the bent part, of the outward turning edge, and the heat preservation base, the flexible gasket and the pump shell are fixedly connected through the second bolt and nut assembly.
Optionally, be used for adherent cell culture's disposable fixed bed in, be provided with a plurality of recessed structure on the bottom surface of heat preservation base, the height of a plurality of recessed structure equals and uses the center pin of spliced pole is the center and is the symmetric distribution.
Optionally, the disposable fixed bed for adherent cell culture further comprises; the electrode holder comprises a liquid inlet, a liquid outlet, a first electrode holder, a second electrode holder and a third electrode holder;
the liquid inlet, the liquid outlet, the first electrode holder, the second electrode holder and the third electrode holder are respectively arranged on the upper cover plate.
Optionally, a disposable fixed bed for adherence cell culture in, the top jade wall of second supporting seat with the last electrode through-hole of having all seted up of annular clamp plate, the bottom of first electrode holder and third electrode holder insert respectively in the electrode through-hole that annular clamp plate corresponds, and with annular clamp plate's lower terminal surface flushes, the bottom of second electrode holder pass a spacing ring insert in the electrode through-hole of second supporting seat, the up end of spacing ring support by in on the step face of second electrode holder, the lower terminal surface of spacing ring support by in on the top jade wall of second supporting seat.
Optionally, in the disposable fixed bed for adherent cell culture, a descending step is arranged at the edge of the top wall of the second support seat, and the edge of the annular pressing plate is arranged on the step surface of the descending step and is fixedly connected with the step surface of the descending step through a connecting piece.
The invention has the beneficial effects that:
1. the culture medium is operated in a perfusion mode, all cells do not need centrifugal force action in the harvesting period, the cells are thawed to be harvested to the final product, the device only needs one step, and the process of early culture can be obviously simplified.
2. Magnetic stirring mechanism is built-in to the bottom, magnetic stirring mechanism's stirring paddle sets up outside the cell growth space, does not contact with the cell of cultivateing, shearing force greatly reduced that the cell received.
3. The curtain-distributed oxygen exchange can be realized through a specific structure (a second fluid channel), so that the volume dissolved oxygen coefficient is improved, the mixing uniformity of materials is improved, and the temperature control stability is improved.
4. The culture area of 500 square meters can be expressed by 0.64 square meters in floor area, which is equivalent to 3000 cell culture rotating bottles (1700 square centimeters per rotating bottle), the inoculation density can be reduced, and the culture cost of target cells can be reduced.
Drawings
FIG. 1 is a quarter sectional view of a disposable fixed bed for adherent cell culture according to an embodiment of the invention;
FIG. 2 is an exploded view of a disposable fixed bed for adherent cell culture according to an embodiment of the present invention;
FIG. 3 is a schematic flow diagram of the medium inside a disposable fixed bed for adherent cell culture according to an embodiment of the present invention in an operating state;
FIG. 4 is a cross-sectional view of a disposable fixed bed for adherent cell culture according to another embodiment of the invention;
FIG. 5 is a sectional view of a disposable fixed bed for adherent cell culture according to still another embodiment of the present invention.
In the figure: 1. a heat-insulating base; 2. a flexible gasket; 3. a pump housing; 4. a flexible compression ring; 5. an upper cover plate; 6. a first electrode holder; 7. a second electrode holder; 8. a third electrode holder; 9. an annular pressure plate; 10. a carrier; 11. a support column; 12. a stirring paddle; 13. an annular base plate; 14. a first support base; 15. a second support seat; 16. a seal ring; 17. a liquid inlet; 18. a liquid outlet; 19. a limiting ring; 1a, a concave structure; 3a, a positioning column; 3b, connecting columns.
Detailed Description
The technical scheme of the invention is clearly and completely described in the following with reference to the accompanying drawings. In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of description and simplicity of description, but do not indicate or imply that the device or element being referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention. Moreover, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance.
The invention will be further explained with reference to the drawings.
Please refer to fig. 1 to fig. 3, which are schematic structural diagrams of a disposable fixed bed for adherent cell culture according to an embodiment of the present invention. As shown in fig. 1-3, the disposable fixed bed for adherent cell culture comprises: the device comprises a pump shell 3, an upper cover plate 5, an annular pressure plate 9, a carrier 10, an annular bottom plate 13, a first supporting seat 14, a second supporting seat 15, a sealing connection mechanism and a magnetic stirring mechanism; the annular bottom plate 13, the first supporting seat 14 and the second supporting seat 15 are sequentially mounted on the inner bottom surface of the pump shell 3 from bottom to top, the upper cover plate 5 is arranged at the opening end of the pump shell 3 and is in sealing connection with the pump shell 3 through the sealing connection mechanism, and a first closed space is formed between the upper cover plate 5 and the pump shell 3; the magnetic stirring mechanism comprises a driving part (not shown in the figure) and a stirring blade 12 which rotates under the driving of the driving part, the bottom surface of the inner side of the pump shell 3 is provided with a connecting column 3b, the stirring blade 12 is sleeved on the connecting column 3b, and the stirring blade 12 and the connecting column 3b are both positioned in a central through hole of the annular bottom plate 13; the first support seat 14 and the second support seat 15 both have a first side wall, a bottom wall, a second side wall and a top wall (reference numerals in the drawing are not shown) which are connected in sequence, the first side wall of the first support seat 14 is tightly attached to the first side wall of the second support seat 15, a first preset gap is formed between the second side wall of the first support seat 14 and the second side wall of the second support seat 15 so as to form a first fluid channel, the top wall of the second support seat 15 is higher than the top wall of the first support seat 14, the bottom wall of the first support seat 14 is arranged in a downward inclination manner, and the distance between the bottom wall of the first support seat 14 and the bottom wall of the second support seat 15 gradually increases along an inclination direction; the bottom of the first supporting seat 14 abuts against the outer edge of the annular bottom plate 13, the first supporting seat 14 and the annular bottom plate 13 enclose to form a first cavity, the first supporting seat 14 and the second supporting seat 15 enclose to form a second cavity, and the second supporting seat 15 and the upper cover plate 5 enclose to form a third cavity; the first cavity is communicated with the bottom of the pump shell 3 through a central through hole of the annular bottom plate 13, a top wall of the first support seat 14 is provided with a fluid through hole, the first cavity is communicated with the second cavity through the fluid through hole, a bottom wall of the second support seat 15 is of a grid structure, the second cavity is communicated with the third cavity through grid holes in the bottom wall of the second support seat 15, a second preset gap is formed between the first side wall of the first support seat 14 and the side wall of the pump shell 3 to form a second fluid channel, and the third cavity is communicated with the bottom of the pump shell 3 through the second fluid channel; the annular pressing plate 9 is of a grid structure, the annular pressing plate 9 is fixedly arranged on the second supporting seat 15, an accommodating space is formed by the annular pressing plate 9 and the first side wall, the bottom wall and the second side wall of the second supporting seat 15, and the carrier 10 is arranged in the accommodating space.
Specifically, a central through hole (the reference number in the figure is not shown) and at least two clamping holes (not shown) are formed in the annular bottom plate 13, the diameter of the central through hole is required to be larger than that of the stirring paddle blade 12, at least two positioning columns 3a are arranged on the bottom surface of the inner side of the pump casing 3, the at least two positioning columns 3a and the clamping holes of the annular bottom plate 13 are arranged in a one-to-one correspondence manner, and the top ends of the positioning columns 3a are inserted into the clamping holes of the annular bottom plate 13, so that the annular bottom plate 13 and the bottom of the pump casing 3 are clamped and fixed. Preferably, the at least two clamping holes are uniformly distributed along the circumferential direction of the annular bottom plate 13, and the positioning columns 3a and the pump shell 3 are of an integrally formed structure.
The first supporting seat 14 and the second supporting seat 15 are basically similar in structure and are provided with a first side wall, a bottom wall, a second side wall and a top wall which are connected in sequence, and the first side wall, the bottom wall and the second side wall form a groove structure. The groove structure of the first support seat 14 and the groove structure of the second support seat 15 are not completely overlapped. In first supporting seat 14, the department of meeting of end wall and second lateral wall is provided with chamfer structure (reference numeral not shown in the figure), the outside edge of annular bottom plate 13 is provided with the slant and presses the face (reference numeral not shown in the figure) correspondingly, the slant press the face with chamfer structure looks adaptation, the bottom of first supporting seat 14 support lean on in annular bottom plate 13's slant presses the face. Second supporting seat 15 is placed on first supporting seat 14, just the first side wall of second supporting seat 15 with the first side wall of second supporting seat 15 closely laminates, under the stress, first supporting seat 14 with second supporting seat 15 is difficult to separate under the exogenic action.
Wherein, the bottom wall of the first supporting seat 14 is obliquely arranged, the bottom wall of the second supporting seat 15 is horizontally arranged or obliquely arranged, one end of the first supporting seat 14 is contacted with the bottom wall of the second supporting seat 15, and the other end (i.e. harvesting point) of the first supporting seat 14 is kept at a certain distance from the bottom wall of the second supporting seat 15. The bottom wall of the first supporting seat 14 is obliquely arranged, so that cell harvesting work can be conveniently carried out from a harvesting point, and complete harvesting is ensured without residues.
Preferably, the inner side edge of the annular bottom plate 13 is set as an inclined surface, and the inclined surface forms an included angle with the upper end surface of the annular bottom plate 13, so that fluid can smoothly flow into a second cavity surrounded by the first supporting seat 14 and the second supporting seat 15 from a first cavity surrounded by the annular bottom plate 13 and the first supporting seat 14.
The annular pressing plate 9 is of a grid structure and is provided with a plurality of grid holes, and the grid holes penetrate through the annular pressing plate 9 along the thickness direction of the annular pressing plate 9. The edge of the top wall of the second support seat 15 is provided with a descending step, the edge of the annular pressing plate 9 is placed on the step surface of the descending step, the step surface of the descending step and the edge of the annular pressing plate 9 are provided with through holes for inserting connecting pieces, and the edge of the annular pressing plate 9 is fixedly connected with the step surface of the second support seat 15 through the connecting pieces (such as screw and nut assemblies). As shown in fig. 3, the annular pressing plate 9 is fixedly disposed on the second supporting seat 15, and forms an accommodating space together with the first side wall, the bottom wall, and the second side wall of the second supporting seat 15, and a fluid (e.g., a culture medium) in the accommodating space can smoothly flow out through the plurality of mesh holes on the annular pressing plate 9. The carrier 10 is disposed in the accommodating space, the carrier 10 is made of a medical polymer material or a fiber material, and the medical polymer material or the fiber material is of a porous structure.
With continued reference to fig. 1, the pump casing 3 and the upper cover plate 5 are both of a disk-shaped structure as a whole, the height of the pump casing 3 is greater than the height of the upper cover plate 5, the upper cover plate 5 is covered on the pump casing 3, the open end of the pump casing 3 extends radially outward to form a first annular flange, the open end of the upper cover plate 5 extends radially outward to form a second annular flange, the first annular flange and the second annular flange are disposed opposite to each other and are respectively provided with a bolt hole, the first sealing and connecting mechanism includes a first flexible sealing ring (no reference numeral shown in the figure), two flexible pressing rings 4 and a first bolt and nut assembly (no reference numeral shown in the figure), the first flexible sealing ring is disposed as a sealing element between the first annular flange and the second annular flange, respectively with first annular flange with the medial surface contact of second annular flange, two flexible clamping rings 4 set up respectively in first annular flange with on the outside end face of second annular flange, one of them flexible clamping ring 4 set up promptly in on the first annular flange of pump case 3, and be located dorsad one side of upper cover plate 5, another flexible clamping ring 4 set up in on the second annular flange of upper cover plate 5, and be located dorsad one side of pump case 3, all be provided with a plurality of connecting holes on two flexible clamping rings 4, the bolt in the first bolt nut subassembly passes the bolt hole on pump case 3, the upper cover plate 5 and connecting hole and nut on two flexible clamping rings 4 are connected. After being screwed, the bolt and nut assembly applies pressure to the pump shell 3 and the upper cover plate 5 to be close to each other, and the first flexible sealing ring is pressed and fixed between the pump shell 3 and the upper cover plate 5, so that a first closed space is formed between the upper cover plate 5 and the pump shell 3. In the fixing process, the two flexible compression rings 4 can protect the edge structures of the pump shell 3 and the upper cover plate 5.
In this embodiment, the pump housing 3 and the upper cover plate 5 are hermetically connected through a bolt and nut assembly and a flexible sealing ring. In other embodiments, the joint between the pump housing 3 and the upper cover plate 5 may adopt other structural forms, and other sealing connection manners may also be adopted between the two, as long as the pump housing 3 and the upper cover plate 5 can be connected in a sealing manner to form the first closed space. For example, the edge of the upper cover plate 5 is an annular flange structure, the annular flange structure is fixed with the annular flange structure of the pump shell 3 in a snap-fit manner, and a sealing element can be arranged in the annular flange structure or the annular groove of the annular flange structure.
As shown in fig. 1-3, a disposable fixed bed for adherent cell culture still includes a support column 11, the relative both ends of support column 11 all are provided with helicitic texture, the bottom of pump case 3 is provided with spliced pole 3b, spliced pole 3b is vertical setting, first mounting hole (reference numeral does not show in the picture) has been seted up at spliced pole 3 b's top, wherein one end of support column 11 insert in the first mounting hole to through threaded connection mode with pump case 3 fixed connection, the second mounting hole (reference numeral does not show in the picture) has been seted up on the top wall of first support seat 14, the other end of support column 11 insert in the second mounting hole to through threaded connection mode with the top wall fixed connection of first support seat 14.
In this embodiment, the supporting column 11 is connected between the first supporting seat 14 and the pump case 3, and not only can fix the first supporting seat 14 on the pump case 3 to prevent the first supporting seat 14 from moving up and down, but also the lower step surface of the supporting column 11 abuts against the stirring blade 12, so that the height of the connecting column 3b above the stirring blade 12 can be limited, and the stirring blade 12 cannot move up and down.
It should be noted that the arrangement and fixing manner of the supporting column 11 are only examples, and are not limited. In other embodiments, the opposite ends of the supporting column 11 may not be provided with screw structures, but may be fixed between the first supporting seat 14 and the pump housing 3 by other means. The disposable fixed bed for adherent cell culture may not be provided with the support column 11, or the upper end of the support column 11 may not be fixed. In another embodiment, as shown in fig. 4, the lower end of the supporting pillar 11 is fixedly connected to the pump housing 3 by a screw connection, and the upper end of the supporting pillar 11 is not fixedly connected to the first supporting seat 14, but abuts against the top wall of the first supporting seat 14. In a further embodiment, as shown in fig. 5, the stirring blade 12 is directly fixed by a compression nut (not shown), so that it cannot move up and down on the connecting column 3 b.
With reference to fig. 1, the disposable fixed bed for adherent cell culture includes a heat preservation base 1 and a second sealing connection mechanism, an outward flange is annularly disposed at an upper end edge of the heat preservation base 1, a plurality of connection through holes are disposed on the outward flange, a bending portion corresponding to the outward flange is disposed on a side wall of the pump housing 3, a plurality of screw sleeves are disposed on the bending portion, the plurality of screw sleeves and the plurality of connection through holes are disposed in a one-to-one correspondence manner, the pump housing 3 is mounted on the heat preservation base 1, the bending portion thereof is pressed against the outward flange of the heat preservation base 1, and the second sealing connection mechanism includes a second flexible sealing ring (no reference numeral in the figure), a flexible gasket 2 and a second bolt and nut assembly (no reference numeral in the figure); the second flexible sealing ring is arranged between the bent part of the pump shell 3 and the outward turning edge of the heat insulation base 1 as a sealing piece, the flexible gasket 2 is arranged on one side, back to the bent part, of the outward turning edge and used for protecting a structure of a joint when the heat insulation base 1 is fixedly connected with the pump shell 3, and the heat insulation base 1, the flexible gasket 2 and the pump shell 3 are fixedly connected through the second bolt and nut assembly.
A gap is reserved between the heat preservation base 1 and the pump shell 3 to form a second closed space, and the second closed space is a water bath temperature control cavity and is used for introducing pure water. The temperature of the medium in the first closed space can be controlled by controlling the temperature of the water in the second closed space.
In this embodiment, the outer flange of the heat insulation base 1 is horizontally arranged, correspondingly, the outer side of the bending portion of the pump case 3 (i.e., the side abutted against the outer flange) is a horizontal plane, and the pump case 3 and the heat insulation base 1 are in sealing connection through a bolt and nut component and a flexible sealing ring.
In other embodiments, the joint between the pump casing 3 and the heat-insulating base 1 may adopt other structural forms, and other sealing connection manners may also be adopted between the pump casing 3 and the heat-insulating base 1 as long as the pump casing 3 and the heat-insulating base 1 can be connected in a sealing manner to form a second closed space. For example, the outer flanging of the heat insulation base 1 and the outer side of the bending part of the pump shell 3 are obliquely arranged, and the pump shell 3 and the heat insulation base 1 are sealed and fixed by adopting sealant, welding or other modes.
Preferably, the inner side of the bent part of the pump housing 3 is an inclined surface to ensure that no liquid accumulation occurs at the bent part.
With reference to fig. 1 and fig. 2, the pump case 3 and the thermal insulation base 1 are respectively provided with an upward protruding structure (no reference numeral is shown in the figures) at a central position, the upward protruding structure of the thermal insulation base 1 corresponds to the upward protruding structure of the pump case 3, a sealing ring 16 is arranged between the upward protruding structure and the upward protruding structure, the upward protruding structures of the thermal insulation base 1 and the pump case 3 are both hollow so as to be convenient for arranging a driving part of the magnetic stirring mechanism, the connecting column 3b is located on the upward protruding structure of the pump case 3, the stirring blade 12 of the magnetic stirring mechanism is sleeved on the connecting column 3b, and a certain distance is maintained between the upward protruding structure of the pump case 3 and the annular bottom plate 13, so that a fluid (such as a culture medium) can pass through smoothly.
With reference to fig. 3, a plurality of concave structures 1a are disposed on the bottom surface of the heat-insulating base 1, and the plurality of concave structures 1a have the same height and are symmetrically distributed around the central axis of the connecting column 3 b. The plurality of concave structures 1a are used for limiting the positions of the heat preservation base 1 and the pump shell 3 so as to facilitate the alignment and installation of the stirring paddle 12.
With continuing reference to fig. 1 and 3, the disposable fixed bed for adherent cell culture further includes a first electrode holder 6, a second electrode holder 7, a third electrode holder 8, a liquid inlet 17 and a liquid outlet 18, wherein the liquid inlet 17 and the liquid outlet 18 are both disposed on the upper cover plate 5, a threaded hole is respectively disposed at the center and both sides of the upper end surface of the upper cover plate 5, threads are respectively disposed on the outer sidewalls of the first electrode holder 6, the second electrode holder 7 and the third electrode holder 8, and the first electrode holder 6, the second electrode holder 7 and the third electrode holder 8 are respectively inserted into the corresponding threaded holes and fixed on the upper cover plate 5 in a threaded connection manner.
In this embodiment, the first electrode holder 6 is used for detecting PH and D0, the second electrode holder 7 is used for detecting temperature and D0, and the third electrode holder 8 is used for detecting biomass (i.e., cell density).
In this embodiment, the annular pressing plate 9 and the top wall of the second supporting seat 15 are both provided with electrode through holes, the bottoms of the first electrode holder 6 and the third electrode holder 8 are respectively inserted into the electrode through holes of the annular pressing plate 9, and the bottom of the second electrode holder 7 is inserted into the electrode through holes of the second supporting seat 15.
Preferably, the bottoms of the first electrode holder 6 and the third electrode holder 8 are both flush with the lower end surface of the annular pressure plate 9.
Preferably, the bottom of the second electrode holder 7 passes through a limiting ring 19 and is inserted into the electrode through hole of the second support seat 15, the lower end of the second electrode holder 7 has a step structure, the upper end face of the limiting ring 19 abuts against the step face of the second electrode holder 7, and the lower end face of the limiting ring 19 abuts against the top wall of the second support seat 15. As shown in fig. 3 and 5, the limiting ring 19 is clamped between the step surface of the second electrode holder 7 and the top wall of the second support seat 15, so as to limit the upward movement of the second support seat 15.
In this embodiment, the heat-insulating base 1, the pump case 3, the upper cover plate 5, the annular pressure plate 9, the support columns 11, the annular bottom plate 13, the first support seat 14, and the second support seat 15 are all made of a rigid material, the pump case 3 and the upper cover plate 5 are hermetically connected to form a first closed space, a driving component of the magnetic stirring mechanism is located outside the first closed space, the stirring blades 12 of the magnetic stirring mechanism are located inside the first closed space, the carrier 10 is located in a containing space defined by the groove structure (formed by the first side wall, the bottom wall, and the second side wall) of the second support seat 15 and the annular pressure plate 9 together, the containing space is a cell growth space, and the stirring blades 12 are located outside the cell growth space.
Preferably, the heat insulation base 1, the pump case 3, the upper cover plate 5, the annular pressure plate 9, the carrier 10, the annular bottom plate 13, the first support seat 14 and the second support seat 15 are integrally of a central symmetrical structure, and the central shafts of the heat insulation base 1, the upper convex structure of the pump case 3 and the connecting column 3b are located on the central shaft of the symmetrical structure.
When the device is used, a culture medium is filled into the pump housing 3 through the liquid inlet 17 on the upper cover plate 5, the magnetic stirring mechanism is started, the stirring paddle 12 is driven by the driving part to rotate, so that the culture medium flows into a first cavity enclosed by the annular bottom plate 13 and the first supporting seat 14 from the bottom of the pump housing 3 through the central through hole of the annular bottom plate 13, the stirring paddle 12 continuously runs, so that the culture medium can fill the supporting seat of the first cavity, further flows into a second cavity enclosed by the first supporting seat 14 and the second supporting seat 15 through the through hole on the top wall of the first supporting seat 14, flows into a space between the bottom wall of the first supporting seat 14 and the bottom wall of the second supporting seat 15 through a first fluid channel between the first supporting seat 14 and the second supporting seat 15, and enters the accommodating space where the carrier 10 is located through the grid holes on the bottom wall of the second supporting seat 15. When the accommodating space is filled with the culture medium, the culture medium flows into the third cavity between the second support seat 15 and the upper cover plate 5 through the mesh holes of the annular pressure plate 9, and returns to the bottom of the pump housing 3 through the second fluid channel between the pump housing 3 and the first support seat 14. The perfusion culture is realized by the circulation.
The disposable fixed bed for adherent cell culture provided by the embodiment adopts a perfusion mode to realize cell culture, but not a perfusion mode, can discharge waste liquid in the culture process, prevents the accumulation of harmful metabolites, is not only beneficial to reaching higher cell culture density, but also can reduce subsequent treatment steps, only needs one step from unfreezing to the harvest of final products, can recover finished cells without operations such as centrifugation, greatly simplifies the operation process, and is easy for industrialization.
Moreover, the disposable fixed bed for adherent cell culture adopts a sealing structure, so that a totally-enclosed integrated process from cell activation, infection and amplification to finished product recovery can be realized, and bacteria are not easily infected in the culture process. More importantly, because the stirring paddle 12 is arranged outside the cell growth space, when the stirring paddle stirs the culture medium in the reactor to ensure that the culture medium flows at a constant speed, the shearing force applied to the cells is very small, so that the cells can be effectively protected, and the cell yield is improved.
In addition, in the perfusion culture process, the culture medium needs to flow through the first fluid channel between the second side wall of the first support seat 14 and the second side wall of the second support seat 15 and the second fluid channel between the first side wall of the first support seat 14 and the side wall of the pump housing 3, so that the culture medium can realize curtain-distributed oxygen exchange when passing through the two fluid channels, the contact area between the gas and the culture medium is effectively increased, the volume dissolved oxygen coefficient is improved, high-density culture is realized, the temperature uniformity and the mixing uniformity of the culture medium can be improved, and the culture environment is more stable.
In the embodiment, the disposable fixed bed for adherent cell culture is flat as a whole, the floor area is only 0.64 square meter, and the surface area of a carrier 10 can reach 500 square meters. That is, the floor area is 0.64 square meter, which can express the culture area of 500 square meters, equivalent to 3000 cell culture rotary bottles (1700 square centimeters per rotary bottle). Compared with the existing cell culture equipment, the disposable fixed bed for adherent cell culture provided by the embodiment can reduce the inoculation density, improve the culture efficiency and further effectively reduce the culture cost of target cells.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A disposable fixed bed for adherent cell culture comprising: the device comprises a pump shell (3), an upper cover plate (5), an annular pressure plate (9), a carrier (10), an annular bottom plate (13), a first supporting seat (14), a second supporting seat (15), a first sealing connecting mechanism and a magnetic stirring mechanism;
the annular bottom plate (13), the first supporting seat (14) and the second supporting seat (15) are sequentially arranged on the bottom surface of the inner side of the pump shell (3) from bottom to top, the upper cover plate (5) is arranged at the opening end of the pump shell (3) and is in sealing connection with the pump shell (3) through the first sealing connection mechanism, and a first closed space is formed between the upper cover plate (5) and the pump shell (3); the magnetic stirring mechanism comprises a driving part and a stirring blade (12) which rotates under the driving of the driving part, a connecting column (3 b) is arranged on the bottom surface of the inner side of the pump shell (3), the stirring blade (12) is sleeved on the connecting column (3 b), and the stirring blade (12) and the connecting column (3 b) are both positioned in a central through hole of the annular bottom plate (13);
the first supporting seat (14) and the second supporting seat (15) are provided with a first side wall, a bottom wall, a second side wall and a top wall which are connected in sequence, the first side wall of the first supporting seat (14) is tightly attached to the first side wall of the second supporting seat (15), a first preset gap is formed between the second side wall of the first supporting seat (14) and the second side wall of the second supporting seat (15) to form a first fluid channel, the top wall of the second supporting seat (15) is higher than the top wall of the first supporting seat (14), the bottom wall of the first supporting seat (14) is arranged in a downward inclined mode, and the distance between the bottom wall of the first supporting seat (14) and the bottom wall of the second supporting seat (15) is gradually increased along the inclined direction;
the bottom of the first supporting seat (14) abuts against the outer edge of the annular bottom plate (13), the first supporting seat (14) and the annular bottom plate (13) are enclosed to form a first cavity, the first supporting seat (14) and the second supporting seat (15) are enclosed to form a second cavity, and the second supporting seat (15) and the upper cover plate (5) are enclosed to form a third cavity;
the first cavity is communicated with the bottom of the pump shell (3) through a central through hole of the annular bottom plate (13), a fluid through hole is formed in the top wall of the first supporting seat (14), the first cavity is communicated with the second cavity through the fluid through hole, the bottom wall of the second supporting seat (15) is of a grid structure, the second cavity is communicated with the third cavity through grid holes in the bottom wall of the second supporting seat (15), a second preset gap is formed between the first side wall of the first supporting seat (14) and the side wall of the pump shell (3) to form a second fluid channel, and the third cavity is communicated with the bottom of the pump shell (3) through the second fluid channel;
the annular pressing plate (9) is of a grid structure, the annular pressing plate (9) is fixedly arranged on the second supporting seat (15), an accommodating space is formed by the annular pressing plate and the first side wall, the bottom wall and the second side wall of the second supporting seat (15), and the carrier (10) is arranged in the accommodating space.
2. The disposable fixed bed for adherent cell culture according to claim 1, wherein the center of the bottom surface of the pump housing (3) is an upward convex structure, the interior of the upward convex structure is provided with a containing space for arranging a driving part of the magnetic stirring mechanism, and the connecting column (3 b) is positioned above the upward convex structure;
at least two positioning columns (3 a) are arranged on the bottom surface of the inner side of the pump shell (3), at least two clamping holes are correspondingly arranged on the annular bottom plate (13), and the top ends of the positioning columns (3 a) are inserted into the clamping holes of the annular bottom plate (13);
in first supporting seat (14), end the jade, for the slant setting, and the department of meeting of end jade and second lateral wall is provided with the chamfer structure, the outside edge of annular bottom plate (13) is provided with the slant and presses the face, the slant press the face with chamfer structure looks adaptation.
3. The disposable fixed bed for adherent cell culture according to claim 1, further comprising a support column (11), wherein opposite ends of the support column (11) are provided with a screw structure;
the pump casing is characterized in that a first mounting hole is formed in a connecting column (3 b) of the pump casing (3), one end of a supporting column (11) is inserted into the first mounting hole and is fixedly connected with the pump casing (3) in a threaded connection mode, a second mounting hole is formed in the top wall of a first supporting seat (14), and the other end of the supporting column (11) is inserted into the second mounting hole and is fixedly connected with the first supporting seat (14) in a threaded connection mode.
4. The disposable fixed bed for adherent cell culture according to claim 1, wherein the open end of the pump housing (3) extends radially outwards to form a first annular flange, the open end of the upper cover plate (5) extends radially outwards to form a second annular flange, and the first annular flange and the second annular flange are arranged opposite to each other and are provided with bolt holes;
the first sealing connection mechanism includes: the first flexible sealing ring, the two flexible compression rings (4) and the first bolt and nut assembly; first flexible sealing washer set up in first annular flange with between the second annular flange, two flexible clamping rings (4) set up respectively in the dorsad of first annular flange the terminal surface of second annular flange and the dorsad of second annular flange on the terminal surface of first annular flange, pump case (3) two flexible clamping rings (4) with upper cover plate (5) pass through first bolt and nut subassembly fixed connection.
5. The disposable fixed bed for adherent cell culture of claim 1, further comprising: the heat preservation base (1) and the second sealing connection mechanism;
the pump shell (3) is arranged on the heat-insulating base (1) and is in sealing connection with the upper end edge of the heat-insulating base (1) through the second sealing connection mechanism, a gap is reserved between the heat-insulating base (1) and the pump shell (3) to form a second closed space, and the second closed space is a water bath temperature control cavity.
6. The disposable fixed bed for adherent cell culture according to claim 5, wherein the top of the heat preservation base (1) is annularly provided with an outward flange, the outward flange is provided with a plurality of connecting through holes, the side wall of the pump shell (3) is provided with a bending part corresponding to the outward flange, the bending part is provided with a plurality of screw sleeves, the bending part of the pump shell (3) is abutted against the outward flange of the heat preservation base (1), and the inner side surface of the bending part is an inclined surface;
the second sealing connection mechanism further comprises: the second flexible sealing ring, the flexible gasket (2) and the second bolt-nut assembly; the second flexible sealing ring is arranged between the bending part of the pump shell (3) and the outward turning edge of the heat preservation base (1), the flexible gasket (2) is arranged on one side of the outward turning edge, back to the bending part, and the heat preservation base (1) is fixedly connected with the pump shell (3) through the second bolt and nut component.
7. The disposable fixed bed for adherent cell culture according to claim 5, wherein a plurality of concave structures (1 a) are arranged on the bottom surface of the heat-insulating base (1), and the plurality of concave structures (1 a) have the same height and are symmetrically distributed with the central axis of the connecting column (3 b) as the center.
8. The disposable fixed bed for adherent cell culture of claim 1, further comprising; a liquid inlet (17), a liquid outlet (18), a first electrode seat (6), a second electrode seat (7) and a third electrode seat (8);
the liquid inlet (17), the liquid outlet (18), the first electrode holder (6), the second electrode holder (7) and the third electrode holder (8) are respectively arranged on the upper cover plate (5).
9. The disposable fixed bed for adherent cell culture according to claim 8, wherein the top wall of the second support seat (15) and the annular pressure plate (9) are provided with electrode through holes, the bottoms of the first electrode holder (6) and the third electrode holder (8) are respectively inserted into the electrode through holes corresponding to the annular pressure plate (9) and flush with the lower end surface of the annular pressure plate (9), the bottom of the second electrode holder (7) is inserted into the electrode through holes of the second support seat (15) through a limiting ring (19), the upper end surface of the limiting ring (19) abuts against the step surface of the second electrode holder (7), and the lower end surface of the limiting ring (19) abuts against the top wall of the second support seat (15).
10. The disposable fixed bed for adherent cell culture according to claim 1, wherein the edge of the top wall of the second support seat (15) is provided with a descending step, and the edge of the annular pressure plate (9) is arranged on the step surface of the descending step and is fixedly connected with the step surface of the descending step through a connecting piece.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211355215.6A CN115786120A (en) | 2022-11-01 | 2022-11-01 | Disposable fixed bed for adherent cell culture |
| PCT/CN2023/126806 WO2024093788A1 (en) | 2022-11-01 | 2023-10-26 | Disposable fixed bed for adherent cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211355215.6A CN115786120A (en) | 2022-11-01 | 2022-11-01 | Disposable fixed bed for adherent cell culture |
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| CN115786120A true CN115786120A (en) | 2023-03-14 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN202211355215.6A Pending CN115786120A (en) | 2022-11-01 | 2022-11-01 | Disposable fixed bed for adherent cell culture |
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| WO (1) | WO2024093788A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024093788A1 (en) * | 2022-11-01 | 2024-05-10 | 东富龙生命科技有限公司 | Disposable fixed bed for adherent cell culture |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0820779D0 (en) * | 2008-11-13 | 2008-12-17 | Artelis S A | Cell culture device and method of culturing cells |
| JP2016504024A (en) * | 2012-12-11 | 2016-02-12 | ポール テクノロジー ユーケイ リミテッドＰａｌｌ Ｔｅｃｈｎｏｌｏｇｙ Ｕｋ Ｌｉｍｉｔｅｄ | Receptors for cell culture |
| CN106047700B (en) * | 2016-07-28 | 2018-09-14 | 江南大学 | A kind of fixed bed device and its application for cell culture |
| CN112708562A (en) * | 2021-01-11 | 2021-04-27 | 昆明柏特生物科技有限公司 | Fixed bed type bioreactor |
| CN113637585A (en) * | 2021-08-11 | 2021-11-12 | 四川大学 | High-efficient cell culture reactor and mixing system thereof |
| CN114736797A (en) * | 2022-03-30 | 2022-07-12 | 昆明柏特生物科技有限公司 | Cell culture reactor |
| CN115786120A (en) * | 2022-11-01 | 2023-03-14 | 上海东富龙医疗装备有限公司 | Disposable fixed bed for adherent cell culture |
-
2022
- 2022-11-01 CN CN202211355215.6A patent/CN115786120A/en active Pending
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2023
- 2023-10-26 WO PCT/CN2023/126806 patent/WO2024093788A1/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024093788A1 (en) * | 2022-11-01 | 2024-05-10 | 东富龙生命科技有限公司 | Disposable fixed bed for adherent cell culture |
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