CN116179342A - Neural stem cell expansion device and method thereof - Google Patents
Neural stem cell expansion device and method thereof Download PDFInfo
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- CN116179342A CN116179342A CN202211425901.6A CN202211425901A CN116179342A CN 116179342 A CN116179342 A CN 116179342A CN 202211425901 A CN202211425901 A CN 202211425901A CN 116179342 A CN116179342 A CN 116179342A
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- 238000000034 method Methods 0.000 title claims abstract description 37
- 210000001178 neural stem cell Anatomy 0.000 title claims abstract description 21
- 230000010261 cell growth Effects 0.000 title claims abstract description 18
- 239000007788 liquid Substances 0.000 claims abstract description 275
- 238000007789 sealing Methods 0.000 claims abstract description 41
- 210000004027 cell Anatomy 0.000 claims abstract description 35
- 239000000872 buffer Substances 0.000 claims abstract description 33
- 239000000243 solution Substances 0.000 claims abstract description 28
- 238000004113 cell culture Methods 0.000 claims abstract description 24
- 238000007599 discharging Methods 0.000 claims abstract description 17
- 239000002699 waste material Substances 0.000 claims abstract description 17
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- 230000001464 adherent effect Effects 0.000 claims description 6
- 210000000130 stem cell Anatomy 0.000 claims description 6
- 210000005036 nerve Anatomy 0.000 claims description 2
- 230000003321 amplification Effects 0.000 abstract description 12
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 12
- 238000011010 flushing procedure Methods 0.000 abstract description 3
- 238000009827 uniform distribution Methods 0.000 abstract description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 2
- 239000012531 culture fluid Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000004248 oligodendroglia Anatomy 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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- 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
- C12M27/10—Rotating vessel
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0623—Stem cells
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
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Abstract
The invention relates to the technical field of biology, in particular to a neural stem cell amplification device which comprises a culture bottle, a liquid inlet component for adding buffer liquid and culture liquid into the culture bottle and a liquid outlet component for discharging waste liquid, wherein the top end of the culture bottle is connected with a bottle cap in a sealing manner, the liquid inlet component is arranged in the center of the bottle cap, the center of the bottom of the culture bottle is provided with a liquid exchange port, and the liquid outlet component is arranged in the liquid exchange port. An expansion method of a neural stem cell expansion device, comprising the following steps: s1, preparing work; s2, adding a culture solution; s3, cleaning by using a buffer solution; s4, cell liquid exchange. According to the invention, the liquid inlet and outlet processes of the culture liquid and the buffer liquid are mutually independent through the double-cavity channel structure of the liquid inlet pipe, so that the uniform distribution of the culture liquid in the cell liquid exchange process and the uniform coverage of the buffer liquid in the liquid exchange flushing process are ensured, meanwhile, a bottle cap is not required to be opened in the cell liquid exchange process, and the pollution to the cell culture and amplification caused by the external environment is ensured.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a neural stem cell expansion device and a neural stem cell expansion method.
Background
Neural stem cells refer to a population of cells that exist in the nervous system, have the potential to differentiate into neurons, astrocytes and oligodendrocytes, thereby being capable of producing a large amount of brain tissue, and are capable of self-renewal and sufficient to provide a large amount of brain tissue cells. Stem cell expansion refers to a process of isolating and purifying autologous, allogeneic or xenogeneic stem cells, and culturing the stem cells under aseptic conditions to increase the number of stem cells.
At present, the culture and amplification process of the neural stem cells in a laboratory is generally carried out through a culture bottle, in the cell culture and amplification process, culture fluid in the culture bottle needs to be replaced at regular time to ensure a proper culture environment in the bottle, in the cell culture and amplification process, operators need to open a bottle cap, waste liquid in the culture bottle is sucked out firstly, then the residual waste liquid in the culture bottle needs to be cleaned by using buffer fluid, then the cell buffer fluid is sucked out, and finally new culture fluid is replenished, in the whole cell culture and amplification process, the bottle cap of the culture bottle is always in an open state, although the bottle cap is subjected to disinfection treatment by an alcohol lamp, the open state still easily causes pollution, the cell culture and amplification is not facilitated, meanwhile, operators need to strictly execute operation specifications in the cell culture and amplification process, such as arms do not need to sweep from the upper part of an open bottle mouth, a liquid gun head cannot touch other places except the culture bottle during liquid transferring operation, and the like, and the cell culture and amplification process may be influenced by slight operation non-specifications.
Disclosure of Invention
The invention aims to provide a neural stem cell amplification device and a neural stem cell amplification method, and aims to solve the technical problems.
The aim of the invention can be achieved by the following technical scheme:
the nerve stem cell expansion device comprises a culture bottle, a liquid inlet component for adding buffer liquid and culture liquid into the culture bottle and a liquid discharge component for discharging waste liquid, wherein the top end of the culture bottle is connected with a bottle cap in a sealing manner, the liquid inlet component is arranged in the center of the bottle cap, the center of the bottom of the culture bottle is provided with a liquid exchange port, and the liquid discharge component is arranged in the liquid exchange port;
the utility model provides a liquid feed assembly, including feed liquor pipe and outer chamber box, bottle lid central authorities are provided with the mounting hole that supplies the feed liquor pipe to pass, be provided with the connecting seat in the mounting hole, the feed liquor pipe adaptation runs through the connecting seat and stretches into in the blake and is connected with the feed liquor box, the feed liquor pipe top is connected with the rotation joint, be provided with the bearing between feed liquor pipe and the connecting seat, the feed liquor pipe passes through bearing and connecting seat normal running fit, the feed liquor pipe is inside to be provided with first chamber way along the axis direction, first chamber way outside is provided with the second chamber way, first chamber way and second chamber way top all are connected with the inlet, first chamber way bottom is provided with first connecting portion, second chamber way bottom is provided with second connecting portion, outer chamber box and second connecting portion sealing connection, outer chamber box both sides intercommunication has the second drain pipe, the inside coaxial inner chamber box that is provided with of outer chamber box, inner chamber box and first connecting portion sealing connection, first chamber both sides intercommunication have first drain pipe and equal interval to distribute, first drain pipe and be close to the equal to the inner wall.
As a further scheme of the invention: the first liquid outlet pipe is provided with a first control valve, and the second liquid outlet pipe is provided with a second control valve.
As a further scheme of the invention: and a sealing ring is arranged between the liquid inlet pipe and the connecting seat, and sealing gaskets are arranged at the first connecting part and the second connecting part.
As a further scheme of the invention: sealing plugs are arranged in the liquid inlets, and cross sockets for inserting the pipette tips are arranged on the end faces of the sealing plugs.
As a further scheme of the invention: the liquid draining assembly comprises a liquid draining joint, a stand column and a liquid draining plate, wherein the liquid draining joint is in sealing connection with the liquid exchanging port, a liquid draining base plate with liquid draining holes is arranged on the outer side of the stand column, the liquid draining base plate is arranged at the upper end of the liquid draining joint, a connecting hole matched with the top end of the stand column is formed in the center of the liquid draining plate, and the outer edge of the liquid draining plate is in sealing connection with the liquid exchanging port.
As a further scheme of the invention: the liquid discharging device comprises a liquid discharging plate, and is characterized in that a plurality of liquid discharging ports are uniformly arranged on the liquid discharging plate, a mounting groove is formed in one side, close to the upright post, of the liquid discharging ports, a flashboard is movably mounted in the mounting groove, movable cavities corresponding to the mounting grooves one by one are formed in the upright post, piston pushing plates are arranged in the movable cavities, one side, close to the flashboard, of each piston pushing plate is connected with the corresponding movable cavity through a reset spring, the piston pushing plates are connected with one end of a push rod, and the other end of the push rod penetrates through the liquid discharging plate and stretches into the mounting groove to be connected with the corresponding flashboard.
As a further scheme of the invention: the piston is characterized in that a piston cavity is arranged in the upright post, each movable cavity is communicated with the piston cavity, a piston pad is arranged in the piston cavity, a push-pull cylinder is arranged below the piston cavity, and an output shaft of the push-pull cylinder extends into the piston cavity to be connected with the piston pad.
As a further scheme of the invention: and a sealing gasket is arranged at the joint of the push rod and the liquid discharge plate.
An expansion method of a neural stem cell expansion device, comprising the following steps:
s1, preparing: sucking cell culture solution through a liquid transfer device, inserting a gun head of the liquid transfer device into a liquid inlet communicated with the first cavity channel, and transferring the culture solution into the inner cavity box;
s2, adding a culture solution: the driving device drives the liquid inlet pipe to rotate through the rotating joint, and the culture solution is ejected out through the first liquid outlet pipe under the action of centrifugal force and uniformly flows along the inner wall of the culture bottle;
s3, cleaning by using a buffer solution: before liquid change culture, the waste liquid in the culture bottle is discharged through a liquid discharge assembly, cell buffer liquid is sucked through a liquid shifter, the gun head of the liquid shifter is inserted into a liquid inlet communicated with a second cavity, the buffer liquid is transferred into the outer cavity box, a liquid inlet pipe is driven to rotate through a rotating joint, the buffer liquid is ejected through the second liquid outlet pipe under the action of centrifugal force and flows uniformly along the inner wall of the culture bottle, and residual waste liquid on the culture bottle is washed;
s4, cell replacement: repeating S2, replacing the cell culture solution, and completing culture expansion of the adherent cells in the culture flask in the cell culture solution.
The invention has the beneficial effects that:
(1) According to the invention, in the process of cell liquid exchange, the liquid inlet pipe is driven to rotate through the rotary joint, the internal culture liquid or buffer liquid can be thrown out under the action of centrifugal force, the liquid inlet and outlet processes of the culture liquid and the buffer liquid which are mutually independent are realized through the double-cavity channel structure of the liquid inlet pipe, meanwhile, the liquid discharge assembly realizes the opening and closing control of the liquid discharge port by utilizing the cylinder piston structure, and the waste liquid can be timely discharged, so that a bottle cap is not required to be opened in the process of cell liquid exchange, the pollution to cell culture and amplification caused by external environment is avoided, meanwhile, the adverse effect possibly brought by manual operation is eliminated by utilizing mechanical automation control, the operation difficulty during cell liquid exchange is reduced, and the cell pollution risk is reduced;
(2) In the liquid inlet and outlet process, the internal culture liquid or buffer liquid is injected onto the inner wall of the culture bottle through the corresponding liquid outlet pipe under the action of centrifugal force, and uniform liquid outlet is realized along with the uniform rotation process of the liquid inlet pipe relative to the culture bottle, so that the culture liquid or buffer liquid can uniformly flow along the inner wall of the culture bottle, and uniform distribution of the culture liquid in the cell liquid exchange process and uniform coverage of the buffer liquid in the liquid exchange flushing process are ensured;
(3) According to the invention, a buffer area is formed by a certain interval between the liquid discharge plate and the liquid discharge base plate, so that waste liquid can flow uniformly, and the influence on the culture process of adherent cells due to turbulence of liquid flow is avoided.
Drawings
The invention is further described below with reference to the accompanying drawings.
FIG. 1 is a schematic view of the overall structure of the present invention;
FIG. 2 is a schematic view showing the internal structure of the culture flask according to the present invention;
FIG. 3 is a schematic view of the structure of the liquid inlet pipe in the invention;
FIG. 4 is a schematic view of the structure of the outer chamber box of the present invention;
FIG. 5 is a schematic view of a drain assembly according to the present invention;
FIG. 6 is a schematic view of the internal structure of the column according to the present invention;
fig. 7 is an enlarged partial schematic view at a in fig. 6.
In the figure: 1. a culture bottle; 11. a bottle cap; 111. a mounting hole; 112. a connecting seat; 113. a bearing; 114. a seal ring; 12. a liquid exchange port; 2. a liquid inlet component; 21. a liquid inlet pipe; 211. a first channel; 212. a second channel; 213. a first connection portion; 214. a second connecting portion; 215. a sealing gasket; 22. a rotary joint; 23. an outer chamber box; 231. an inner chamber box; 24. a first liquid outlet pipe; 241. a first control valve; 25. a second liquid outlet pipe; 251. a second control valve; 26. a liquid inlet; 261. a sealing plug; 3. a liquid discharge assembly; 31. a liquid discharge joint; 32. a column; 321. a movable cavity; 322. a piston push plate; 323. a return spring; 324. a push rod; 325. a piston chamber; 326. a piston pad; 327. a push-pull cylinder; 328. a sealing gasket; 33. a liquid discharge plate; 331. a connection hole; 332. a liquid outlet; 333. a mounting groove; 334. a flashboard; 34. and a liquid draining backing plate.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Referring to fig. 1 and 2, the invention relates to a neural stem cell expansion device, which comprises a culture flask 1, a liquid inlet assembly 2 for adding buffer liquid and culture liquid into the culture flask 1, and a liquid outlet assembly 3 for discharging waste liquid, wherein the top end of the culture flask 1 is connected with a bottle cap 11 in a sealing manner, the liquid inlet assembly 2 is arranged in the center of the bottle cap 11, a liquid exchange port 12 is arranged in the center of the bottom of the culture flask 1, and the liquid outlet assembly 3 is arranged in the liquid exchange port 12.
As shown in fig. 3 and 4, the liquid inlet assembly 2 includes a liquid inlet tube 21 and an outer cavity box 23, a mounting hole 111 through which the liquid inlet tube 21 passes is provided in the center of the bottle cap 11, a connecting seat 112 is provided in the mounting hole 111, the liquid inlet tube 21 is adapted to penetrate through the connecting seat 112 and extend into the culture flask 1 to be connected with the liquid inlet box, a rotary joint 22 is connected to the top of the liquid inlet tube 21, a bearing 113 is provided between the liquid inlet tube 21 and the connecting seat 112, the liquid inlet tube 21 is in rotary fit with the connecting seat 112 through the bearing 113, a first cavity 211 is provided in the liquid inlet tube 21 along the central axis direction, a second cavity 212 is provided outside the first cavity 211, both the first cavity 211 and the second cavity 212 are connected with a liquid inlet 26, a first connecting portion 213 is provided at the bottom of the first cavity 211, a second connecting portion 214 is provided at the bottom of the second cavity 212, the outer cavity box 23 is in sealing connection with the second connecting portion 214, both sides of the outer cavity box 23 are communicated with a second liquid outlet tube 25, an inner cavity box 231 is coaxially provided in the outer cavity box 23, an inner cavity 231 is coaxially arranged in the inner cavity 231 is closely connected with the first connecting portion 213 and both sides of the first connecting portion 24 are communicated with the first inner wall 24 and the inner wall 25 is closely communicated with the first inner wall of the culture flask 1.
Wherein, the liquid inlet pipe 21 adopts the structural design of dual-cavity way, and first cavity way 211 is linked together through the inner space of first connecting portion 213 and inner chamber box 231, and the cell culture liquid that flows in the first cavity way 211 flows out through first drain pipe 24, and simultaneously second cavity way 212 is linked together through the inner space of second connecting portion 214 and outer chamber box 23, and the cell buffer that flows in the second cavity way 212 flows out through second drain pipe 25 to the mutually independent liquid inlet and outlet process of dual-cavity way has been realized.
Specifically, in the process of liquid feeding and liquid discharging, the liquid feeding pipe 21 is connected with the driving device through the rotary joint 22, the liquid feeding pipe 21 rotates in the connecting seat 112 through the bearing 113, in the rotating process, the internal culture solution or buffer solution is injected onto the inner wall of the culture bottle 1 through the corresponding liquid discharging pipe under the action of centrifugal force, and uniform liquid discharging is realized along with the uniform rotation process of the liquid feeding pipe 21 relative to the culture bottle 1, the culture solution or buffer solution can uniformly flow along the inner wall of the culture bottle 1, and uniform distribution of the culture solution during cell culture and uniform coverage of the buffer solution during liquid changing flushing are ensured.
As shown in fig. 4, the first control valve 241 is mounted on the first liquid outlet pipe 24, and the second control valve 251 is mounted on the second liquid outlet pipe 25.
Wherein, in order to avoid the liquid remained in the inner chamber box 231 and the outer chamber box 23 from being thrown out simultaneously when the liquid inlet pipe 21 rotates, the first liquid outlet pipe 24 and the second liquid outlet pipe 25 are respectively controlled to be opened and closed by the first control valve 241 and the second control valve 251, when the culture liquid is replaced, the first control valve 241 is controlled to be closed by the control switch (not shown), and the second control valve 251 is opened, at the moment, the first liquid outlet pipe 24 is closed, the second liquid outlet pipe 25 is closed, namely, when the liquid inlet pipe 21 rotates, only the culture liquid in the inner chamber box 231 is thrown out; similarly, when washing is performed, the first drain pipe 24 is closed, the second drain pipe 25 is connected, and only the cell buffer solution in the outer chamber box 23 is thrown out.
Further, a sealing ring 114 is disposed between the liquid inlet pipe 21 and the connecting seat 112, and sealing gaskets 215 are disposed at the first connecting portion 213 and the second connecting portion 214.
Wherein, the sealing performance between the liquid inlet pipe 21 and the connecting seat 112 is improved by the sealing ring 114, so that the process of culturing and amplifying cells in the culture flask is prevented from being interfered by external environment; the sealing gasket 215 improves the sealing property when the outer chamber box 23 and the inner chamber box 231 are connected, and prevents mutual seepage, thereby keeping the first chamber channel 211 and the second chamber channel 212 mutually independent.
Further, the liquid inlet 26 is internally provided with a sealing plug 261, and the end surface of the sealing plug 261 is provided with a cross socket for inserting a pipette gun head.
Wherein, sealing plug 261 adopts elastic rubber material, and under the normal condition, cross socket can extrude and seal to guaranteed the sealing performance of inlet 26, also can directly insert the pipettor rifle head through cross socket simultaneously, thereby need not to pull out sealing plug 261, avoid causing the pollution to the inside of feed liquor pipe 21.
As shown in fig. 5-7, the liquid draining assembly 3 comprises a liquid draining joint 31, a column 32 and a liquid draining plate 33, wherein the liquid draining joint 31 is in sealing connection with the liquid exchanging port 12, a liquid draining base plate 34 with liquid draining holes is arranged on the outer side of the column 32, the liquid draining base plate 34 is arranged at the upper end of the liquid draining joint 31, a connecting hole 331 matched with the top end of the column 32 is arranged in the center of the liquid draining plate 33, and the outer edge of the liquid draining plate 33 is in sealing connection with the liquid exchanging port 12.
Wherein, there is a certain interval between drain board 33 and the drain backing plate 34 to form the buffer area, make the waste liquid can evenly flow, avoid liquid flow disorder turbulence and cause the influence to the culture process of adherent cells.
Further, a plurality of liquid discharge ports 332 are uniformly arranged on the liquid discharge plate 33, a mounting groove 333 is arranged on one side, close to the upright post 32, of the liquid discharge ports 332, a flashboard 334 is movably arranged in the mounting groove 333, movable cavities 321 in one-to-one correspondence with the mounting groove 333 are arranged in the upright post 32, a piston push plate 322 is arranged in the movable cavities 321, one side, close to the flashboard 334, of the piston push plate 322 is connected with the movable cavities 321 through a reset spring 323, the piston push plate 322 is connected with one end of a push rod 324, and the other end of the push rod 324 penetrates through the liquid discharge plate 33 and stretches into the mounting groove 333 to be connected with the corresponding flashboard 334.
Further, a piston cavity 325 is provided in the upright post 32, each movable cavity 321 is communicated with the piston cavity 325, a piston pad 326 is provided in the piston cavity 325, a push-pull cylinder 327 is provided below the piston cavity 325, and an output shaft of the push-pull cylinder 327 extends into the piston cavity 325 to be connected with the piston pad 326.
In the liquid draining process, the piston pad 326 is pushed by the push-pull air cylinder 327, so that the piston push plate 322 is pushed by air pressure, and the piston push plate 322 moves in a straight line in the movable cavity 321, so that the flashboard 334 is driven to move by the push rod 324, and further, the opening and closing control of the liquid draining port 332 is realized. The return spring 323 can apply a force to the piston push plate 322, so that the piston push plate 322 can drive the flashboard 334 to retract into the mounting groove 333 when not pushed by air pressure.
Further, a sealing gasket 328 is provided at the junction of the push rod 324 and the drain plate 33.
Wherein, sealing performance between the push rod 324 and the drain plate 33 is improved through the sealing gasket 328, and liquid is prevented from penetrating into the upright post 32 from the gap.
An expansion method of a neural stem cell expansion device, comprising the following steps:
s1, preparing: sucking the cell culture solution through the pipette, inserting the pipette tip into the liquid inlet 26 communicated with the first cavity channel 211, and transferring the culture solution into the inner cavity box 231;
s2, adding a culture solution: the driving device drives the liquid inlet pipe 21 to rotate through the rotary joint 22, and the culture solution is ejected out through the first liquid outlet pipe 24 under the action of centrifugal force and uniformly flows along the inner wall of the culture bottle 1;
s3, cleaning by using a buffer solution: before liquid change culture, the waste liquid in the culture flask 1 is discharged through the liquid discharge assembly 3, the cell buffer liquid is sucked through the liquid transfer device, the gun head of the liquid transfer device is inserted into the liquid inlet 26 communicated with the second cavity 212, the buffer liquid is transferred into the outer cavity box 23, the liquid inlet pipe 21 is driven to rotate through the rotary joint 22, the buffer liquid is ejected through the second liquid outlet pipe 25 under the action of centrifugal force and uniformly flows along the inner wall of the culture flask 1, and the residual waste liquid on the culture flask 1 is washed;
s4, cell replacement: repeating S2, replacing the cell culture solution, and completing culture expansion of the adherent cells in the culture bottle 1 in the cell culture solution.
The working principle of the invention is as follows: when in use, the culture bottle 1 is placed on a sterile operation table, cell culture solution is sucked by a liquid transfer device, the gun head of the liquid transfer device is inserted into a liquid inlet 26 communicated with a first cavity channel 211, the cell culture solution enters the inner cavity box 231 along the first cavity channel 211, then a rotary joint 22 is connected with driving equipment, the rotary joint 22 drives a liquid inlet pipe 21 to rotate at a constant speed, the culture solution in the inner cavity box 231 is ejected out through a first liquid outlet pipe 24 under the action of centrifugal force, and simultaneously the cell culture solution can flow uniformly along the inner wall of the culture bottle 1 in cooperation with a constant speed rotation process, so that adherent cells in the culture bottle 1 are cultured in the cell culture solution;
when the culture medium needs to be replaced, the piston pad 326 is pulled back through the push-pull air cylinder 327, so that the air pressure is utilized to apply force to the piston push plate 322, the piston push plate 322 is retracted in the movable cavity 321, the push rod 324 drives the flashboard 334 to move, the liquid drain port 332 is opened, waste liquid in the culture flask 1 sequentially passes through the liquid drain plate 33 and the liquid drain pad 34, the liquid drain process uniformly flows, then the cell buffer is sucked through the liquid transfer device, the gun head of the liquid transfer device is inserted into the liquid inlet 26 communicated with the second cavity 212, the cell buffer enters the outer cavity box 23 through the second cavity 212, the liquid inlet 21 is driven to rotate through the rotary joint 22, meanwhile, the first liquid outlet 24 is closed, the second liquid outlet 25 is communicated, the phenomenon that residual liquid in the inner cavity box 231 and the outer cavity box 23 is simultaneously thrown out is avoided, the cell buffer is ejected out through the second liquid outlet 25 under the centrifugal force effect, the waste liquid remained on the culture flask 1 uniformly flows along the inner wall of the culture flask, after the liquid drain process is washed, the cell buffer is sucked through the liquid transfer device, the piston pad 326 is pushed by the push-pull air cylinder 327 again, so that the air pressure 322 is provided, the piston push rod 322 is driven by the push rod 324 to drive the liquid inlet 21 to rotate, the liquid drain port 332 is sealed again, the cell buffer is sealed in the culture flask 1, and the cell buffer is replaced in the culture flask 1, the cell culture medium is replaced, and the cell buffer is completely, and the cell buffer is amplified, and the cell buffer is replaced in the culture flask 1, and the cell culture flask is cultured, and the cell culture medium is cultured, and the cell culture medium is cultured and then replaced.
The foregoing describes one embodiment of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.
Claims (9)
1. The nerve stem cell expansion device is characterized by comprising a culture bottle (1), a liquid inlet component (2) for adding buffer solution and culture solution into the culture bottle (1) and a liquid discharge component (3) for discharging waste liquid, wherein the top end of the culture bottle (1) is connected with a bottle cap (11) in a sealing manner, the liquid inlet component (2) is arranged in the center of the bottle cap (11), a liquid exchange port (12) is arranged in the center of the bottom of the culture bottle (1), and the liquid discharge component (3) is arranged in the liquid exchange port (12);
the liquid inlet component (2) comprises a liquid inlet pipe (21) and an outer cavity box (23), a mounting hole (111) for the liquid inlet pipe (21) to pass through is formed in the center of the bottle cap (11), a connecting seat (112) is arranged in the mounting hole (111), the liquid inlet pipe (21) is matched with the connecting seat (112) and stretches into the culture bottle (1) to be connected with the liquid inlet box, a rotary joint (22) is connected to the top of the liquid inlet pipe (21), a bearing (113) is arranged between the liquid inlet pipe (21) and the connecting seat (112), the liquid inlet pipe (21) is in rotary fit with the connecting seat (112) through the bearing (113), a first cavity channel (211) is arranged inside the liquid inlet pipe (21) in the central axis direction, a second cavity channel (212) is arranged outside the first cavity channel (211), liquid inlets (26) are respectively connected to the top of the first cavity channel (211), a first connecting cavity (213) is arranged at the bottom of the first cavity channel (211), two sides of the second cavity channel (212) are connected with the outer cavity (23), the outer cavity (23) is connected with the outer cavity box (23), the inner cavity box (231) is in sealing connection with the first connecting part (213), a first liquid outlet pipe (24) is communicated with two sides of the inner cavity box (231), and the first liquid outlet pipe (24) and the second liquid outlet pipe (25) are distributed at equal intervals and are all close to the inner wall of the culture bottle (1).
2. The neural stem cell expansion device according to claim 1, wherein the first liquid outlet pipe (24) is provided with a first control valve (241), and the second liquid outlet pipe (25) is provided with a second control valve (251).
3. The neural stem cell expansion device according to claim 2, wherein a sealing ring (114) is arranged between the liquid inlet tube (21) and the connecting seat (112), and the first connecting portion (213) and the second connecting portion (214) are both provided with sealing gaskets (215).
4. A neural stem cell expansion device according to claim 3, wherein sealing plugs (261) are installed in the liquid inlets (26), and cross sockets for inserting pipette tips are arranged on the end faces of the sealing plugs (261).
5. The neural stem cell expansion device according to claim 1, wherein the liquid draining assembly (3) comprises a liquid draining joint (31), a stand column (32) and a liquid draining plate (33), the liquid draining joint (31) is in sealing connection with the liquid exchanging port (12), a liquid draining base plate (34) with liquid draining holes is arranged on the outer side of the stand column (32), the liquid draining base plate (34) is arranged at the upper end of the liquid draining joint (31), a connecting hole (331) matched with the top end of the stand column (32) is formed in the center of the liquid draining plate (33), and the outer edge of the liquid draining plate (33) is in sealing connection with the liquid exchanging port (12).
6. The neural stem cell expansion device according to claim 5, wherein a plurality of liquid discharging ports (332) are uniformly arranged on the liquid discharging plate (33), one side, close to the upright post (32), of the liquid discharging ports (332) is provided with a mounting groove (333), a flashboard (334) is movably mounted in the mounting groove (333), movable cavities (321) corresponding to the mounting grooves (333) one by one are arranged in the upright post (32), piston pushing plates (322) are arranged in the movable cavities (321), one side, close to the flashboard (334), of each piston pushing plate (322) is connected with the corresponding movable cavity (321) through a reset spring (323), one end of each piston pushing plate (322) is connected with one end of each pushing rod (324), and the other end of each pushing rod (324) penetrates through the liquid discharging plate (33) and stretches into the corresponding flashboard (334) to be connected with the corresponding flashboard (334).
7. The neural stem cell expansion device according to claim 6, wherein a piston cavity (325) is provided in the upright (32), each of the movable cavities (321) is communicated with the piston cavity (325), a piston pad (326) is provided in the piston cavity (325), a push-pull cylinder (327) is provided below the piston cavity (325), and an output shaft of the push-pull cylinder (327) extends into the piston cavity (325) to be connected with the piston pad (326).
8. The neural stem cell expansion device according to claim 7, wherein a sealing gasket (328) is provided at the junction of the pushrod (324) and the drainage plate (33).
9. The method for amplifying a neural stem cell amplifying device according to claim 1, comprising the steps of:
s1, preparing: sucking the cell culture solution through a liquid transfer device, inserting a gun head of the liquid transfer device into a liquid inlet (26) communicated with a first cavity (211), and transferring the culture solution into an inner cavity box (231);
s2, adding a culture solution: the driving device drives the liquid inlet pipe (21) to rotate through the rotary joint (22), and the culture solution is ejected out through the first liquid outlet pipe (24) under the action of centrifugal force and uniformly flows along the inner wall of the culture bottle (1);
s3, cleaning by using a buffer solution: before liquid change culture, the waste liquid in the culture bottle (1) is discharged through a liquid discharge assembly (3), cell buffer liquid is sucked through a liquid shifter, a gun head of the liquid shifter is inserted into a liquid inlet (26) communicated with a second cavity (212), the buffer liquid is transferred into an outer cavity box (23), a liquid inlet pipe (21) is driven to rotate through a rotary joint (22), the buffer liquid is ejected through a second liquid outlet pipe (25) under the action of centrifugal force and uniformly flows along the inner wall of the culture bottle (1), and the residual waste liquid on the culture bottle (1) is washed;
s4, cell replacement: repeating S2, replacing the cell culture solution, and completing culture expansion of the adherent cells in the culture bottle (1) in the cell culture solution.
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