CN116656468A - Efficient exosome preparation system and method - Google Patents

Efficient exosome preparation system and method Download PDF

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Publication number
CN116656468A
CN116656468A CN202310456990.9A CN202310456990A CN116656468A CN 116656468 A CN116656468 A CN 116656468A CN 202310456990 A CN202310456990 A CN 202310456990A CN 116656468 A CN116656468 A CN 116656468A
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plate
reagent bottle
driving motor
plugging
supernatant
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梁高峰
杨如月
毛龙飞
何向锋
王伟
杨家蘅
张楠
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Henan Academy of Sciences
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Henan Academy of Sciences
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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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/50—Means for positioning or orientating the apparatus
    • 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
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/08—Flask, bottle or test tube
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/10—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by centrifugation ; Cyclones
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • 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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
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  • General Health & Medical Sciences (AREA)
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Abstract

The invention discloses an efficient exosome preparation system and method, comprising an incubator and three reagent bottles arranged in the incubator, wherein an electric telescopic rod is fixedly arranged at the top of the incubator. According to the high-efficiency preparation system and method for the exosome, circumferential limitation of the reagent bottle is achieved by utilizing the cooperation of the iron square plate and the square groove, under the arrangement of the electric telescopic rod and the hanging plate, the arrangement of the cover cylinder and the transparent square ring is matched, limitation of the vertical direction of the reagent bottle is achieved, when the driving motor is guaranteed to rotate, the reagent bottle can be driven to rotate effectively, further guarantee is provided for centrifugation of liquid in the reagent bottle, and under the arrangement of the liquid level adjusting assembly, the blocking plate and the liquid outlet pipe, after centrifugation is completed, the reagent bottle is directly placed, convenient separation of supernatant can be achieved, and the preparation efficiency and the preparation quality of the exosome are improved while the quality of the supernatant is guaranteed.

Description

Efficient exosome preparation system and method
Technical Field
The invention relates to the technical field of exosome preparation, in particular to a high-efficiency exosome preparation system and method.
Background
Exosomes refer to small vesicles containing complex RNAs and proteins, which nowadays refer to disc vesicles with diameters of 40-100nm, and various cells can secrete exosomes under normal and pathological conditions, and mainly originate from multivesicles formed by the invagination of intracellular lysosome particles, and are released into extracellular matrix after fusion of the outer membrane of the multivesicles with cell membranes.
When exosome extraction is performed by centrifugation, frequent supernatant extraction is required, supernatant is directly extracted, precipitate is easily raised to affect the purity of extracted supernatant, and in order to solve the above problems, a protein precipitation supernatant separation device as described in application number 202122575363.6, performs supernatant separation by adjusting the height of the liquid level after standing.
Based on the retrieval of the above data, it can be seen that the separation mode of the supernatant needs to adopt a special standing device, so that centrifugation and standing cannot be performed in the same container, the centrifugal effect is affected, and the purity of the supernatant is easily affected by adopting a conventional supernatant separation mode, and the preparation quality of exosomes is seriously affected.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention provides a high-efficiency exosome preparation system and method, which solve the problems that in the exosome preparation process, the supernatant is troublesome to separate and the exosome preparation quality is affected.
(II) technical scheme
In order to achieve the above purpose, the present invention provides the following technical solutions: the utility model provides an high-efficient preparation system of exosome, includes the thermostated container and sets up three reagent bottle in the thermostated container, the top fixed mounting of thermostated container has electric telescopic handle, electric telescopic handle's flexible end runs through the thermostated container and through link fixedly connected with hanger plate, the periphery of hanger plate and the inner wall sliding contact of thermostated container, between the inner wall of thermostated container and be located the below fixed mounting of hanger plate have the assembly plate, the top fixed mounting of assembly plate has two division boards, two the division board is divided into three working chamber with the inside of thermostated container;
the bottom fixed mounting of working chamber inner chamber has three driving motor, driving motor's output passes through shaft coupling fixedly connected with iron square board, and the square groove with iron square board looks adaptation is seted up to the bottom of reagent bottle, the bottom rotation of hanger plate is installed and is covered a section of thick bamboo, the bottom fixed mounting of cover a section of thick bamboo has transparent square ring, and the ring channel of reagent bottle looks adaptation has been seted up to the bottom of transparent square ring, the shutoff groove has been seted up to one side of transparent square ring and the top that is located the ring channel, the inside slidable mounting in shutoff groove has the shutoff board, through bolt fixed connection between shutoff board and the transparent square ring, the opposite side of transparent square ring is provided with the drain pipe with shutoff groove intercommunication, the inside of reagent bottle is provided with liquid level control subassembly for adjust the inside culture solution of reagent bottle.
Through adopting above-mentioned technical scheme, utilize the cooperation of iron square board and square groove, realize spacing to the circumference of reagent bottle, under the setting of electric telescopic handle and hanger plate, the cooperation covers the setting of a section of thick bamboo and transparent square ring, realize spacing to the vertical direction of reagent bottle, when guaranteeing driving motor and rotate, can drive the reagent bottle and carry out effectual rotation, and then provide the guarantee for the centrifugation of liquid in the reagent bottle, and under the setting of liquid level regulation subassembly, shutoff board and drain pipe, after accomplishing centrifugation, directly stew in the reagent bottle, can realize the convenient separation of supernatant fluid, when guaranteeing the supernatant fluid quality, the preparation efficiency and the preparation quality of exosome have been improved.
In order to realize effective rising of the standing liquid in the reagent bottle, the invention is further provided with: the liquid level adjusting component comprises a piston plate, the piston plate is matched with the inner surface of the reagent bottle, a sealing gasket is arranged on the periphery of the piston plate and used for preventing liquid from flowing to the lower portion of the piston plate, extension rods are fixedly connected to the left side and the right side of the bottom of the piston plate, the bottom ends of the extension rods penetrate through the reagent bottle and extend to the lower portion of the reagent bottle, balancing weights are fixedly connected to the bottom ends of the extension rods, and a magnetic block is fixedly inlaid at the bottom of the piston plate and is matched with the iron square plate.
Through adopting above-mentioned technical scheme, utilize the magnetic force piece to adsorb indisputable square board, when guaranteeing contact stability between reagent bottle and the indisputable square board, guarantee the stability of piston board position, guarantee the inside effective centrifugation process of liquid of reagent bottle.
In order to facilitate the ascending adjustment of the piston plate, the invention is further provided with: the bottom of assembly plate is connected with the regulation threaded rod through the bearing rotation, the periphery cover of regulation threaded rod is established and threaded connection has the regulating plate, the equal fixedly connected with ejector pin in the left and right sides at regulating plate top, the top of ejector pin runs through the assembly plate setting, and two the common fixedly connected with top ring in top of ejector pin, the top ring sets up in driving motor's periphery.
Through adopting above-mentioned technical scheme, utilize the cooperation of adjusting threaded rod and regulating plate, under the setting of ejector pin and backing ring, can realize the altitude mixture control of regulating plate through rotating adjusting threaded rod, and then for extension rod altitude mixture control is convenient, realizes the altitude mixture control of piston board, for the altitude mixture control of the inside liquid of reagent bottle is convenient.
The invention is further provided with: the top fixed mounting of covering a section of thick bamboo has first revolving plate, first revolving plate fixed mounting is at the top of covering a section of thick bamboo, the top fixed mounting of first revolving plate has the pivot, the top of pivot runs through the hanger plate and fixedly connected with second revolving plate, the ball is all inlayed and is rotated to the opposite one side of first revolving plate and second revolving plate, and the surface of ball contacts with the surface of assembly plate.
The invention is further provided with: the periphery interval even fixed mounting of reagent bottle has a plurality of first gag lever post, the bottom interval even fixed mounting of transparent square ring has a plurality of and the second gag lever post of first gag lever post looks adaptation.
The invention is further provided with: the left side and the right side of the inner cavity of the working cavity are fixedly provided with silencing plates, and an operation port is formed in the front side of the incubator and below the assembly plate.
The invention is further provided with: the surface of drain pipe is provided with the control valve of control drain pipe circulation, the front of thermostated container is provided with the sealing door with working chamber looks adaptation, and the surface of sealing door is provided with the observation window.
The invention also discloses a high-efficiency preparation method of the exosome, which comprises the following steps:
step one, assembling: collecting culture solution of mesenchymal stem cells to P5 through subculture, adding into the reagent bottle, opening sealing door, with square groove card on the reagent bottle on the iron square plate, back control electric telescopic handle extends, and electric telescopic handle drives the hanger plate and moves down, and hanger plate extrusion first rotating plate makes covering section of thick bamboo move down, and covering section of thick bamboo drives transparent square ring and moves down, until the ring channel cover on the transparent square ring is in the periphery of reagent bottle, stops electric telescopic handle extension, accomplishes the material loading assembly:
step two, low-speed centrifugation is carried out: controlling a driving motor to rotate, driving an iron square plate by the driving motor to enable a reagent bottle to rotate, driving a transparent square ring by the reagent bottle to enable a covering cylinder to rotate, controlling the rotating speed of the driving motor in the process, centrifuging at 300g and keeping for 10min, closing the driving motor, standing, and turning to the step six to obtain supernatant;
step three, low-speed centrifugation is carried out: placing the supernatant taken out in the second step into a new reagent bottle, repeating the operation of the first step, controlling the rotating speed of a driving motor after finishing feeding and assembling, centrifuging at 2000g and keeping for 10min, closing the driving motor, standing, turning to the sixth step, and taking the supernatant;
step four, high-speed centrifugation is carried out: placing the supernatant taken out in the third step into a new reagent bottle, repeating the operation of the first step, controlling the rotating speed of a driving motor after finishing feeding and assembling, centrifuging at 10000g and keeping for 70min, closing the driving motor, standing, turning to the sixth step, and taking the supernatant;
step five, high-speed centrifugation is carried out: placing the supernatant taken out in the fourth step into a new reagent bottle, repeating the operation of the first step, controlling the rotating speed of a driving motor after finishing feeding and assembling, centrifuging at 100000g and keeping for 70min, closing the driving motor, standing, turning to the seventh step, and taking sediment;
step six, liquid taking: after standing, rotating the bolts to separate the bolts from the transparent square rings, pulling out parts of the plugging grooves of the plugging plates, then enabling the bolts to pass through the operation openings by hands, rotating the adjusting threaded rods, driving the adjusting plates to ascend by the adjusting plates, driving the ejector rods to ascend by the adjusting plates, enabling the ejector rods to extrude the balancing weights to ascend by the extending rods, enabling the extending rods to extrude the pistons to ascend by the extending rods until the contact surface of the supernatant and the lower liquid is positioned below the plugging plates, pushing the plugging plates to slide in the plugging grooves, screwing the bolts, opening the control valves after limiting of the plugging plates is completed, taking new reagent bottles to be placed below the liquid outlet pipes, and collecting the supernatant;
step seven: separating: when the sediment is required to be collected, repeating the operation of the step six, enabling the stationary culture solution to rise until the liquid and the sediment contact surface are positioned below the plugging plate, pushing the plugging plate to slide in the plugging groove, screwing the bolt after limiting the plugging plate, opening the control valve, taking the garbage can to be placed below the liquid outlet pipe, after the liquid is collected, controlling the electric telescopic rod to shrink, enabling the electric telescopic rod to shrink to drive the hanging plate to move upwards, enabling the hanging plate to drive the covering barrel to move upwards, enabling the transparent square ring to be separated from the reagent bottle, taking out the reagent bottle, and pouring out the precipitated exosome.
(III) beneficial effects
The invention provides an efficient exosome preparation system and method. The beneficial effects are as follows:
(1) According to the invention, circumferential limitation of the reagent bottle is realized by utilizing the cooperation of the iron square plate and the square groove, under the arrangement of the electric telescopic rod and the hanging plate, the limitation of the reagent bottle in the vertical direction is realized by cooperation of the covering cylinder and the transparent square ring, when the driving motor rotates, the reagent bottle can be driven to effectively rotate, further, the guarantee is provided for the centrifugation of liquid in the reagent bottle, and under the arrangement of the liquid level adjusting assembly, the blocking plate and the liquid outlet pipe, after the centrifugation is completed, the reagent bottle is directly placed in the reagent bottle, so that the supernatant can be conveniently separated, the quality of the supernatant is ensured, and meanwhile, the preparation efficiency and the preparation quality of exosome are improved.
(2) The invention ensures the contact stability between the reagent bottle and the iron square plate and the stability of the position of the piston plate and the effective centrifuging process of the liquid in the reagent bottle by utilizing the magnetic block to adsorb the iron square plate.
(3) According to the invention, through the cooperation of the adjusting threaded rod and the adjusting plate, under the arrangement of the ejector rod and the backing ring, the height adjustment of the adjusting plate can be realized by rotating the adjusting threaded rod, so that the convenience is provided for the height adjustment of the extension rod, the height adjustment of the piston plate is realized, and the convenience is provided for the height adjustment of liquid in the reagent bottle.
(4) According to the invention, through the cooperation of the first limiting rod and the second limiting rod, the reagent bottle can effectively drive the transparent square ring to rotate the covering cylinder in the rotating process, and the friction force between the reagent bottle and the hanging plate in the rotating process is effectively reduced under the cooperation of the first rotating plate, the second rotating plate, the rotating shaft and the balls, so that the device is ensured to be used stably for a long time.
Drawings
FIG. 1 is a schematic view of the external structure of the present invention;
FIG. 2 is a schematic view of the internal structure of the present invention;
FIG. 3 is a schematic view of the structure of the plugging groove and the plugging plate of the present invention;
FIG. 4 is a schematic view of the internal structure of the transparent square ring of the present invention;
FIG. 5 is a schematic illustration of the connection of the liquid level adjustment assembly and the reagent bottle structure of the present invention;
FIG. 6 is an enlarged schematic view of the structure of FIG. 2A in accordance with the present invention;
in the figure, 1, a constant temperature box; 2. a reagent bottle; 3. an electric telescopic rod; 4. a hanger plate; 5. an assembly plate; 6. a partition plate; 7. a working chamber; 8. a driving motor; 9. an iron square plate; 10. a square groove; 11. a cover cylinder; 12. a transparent square ring; 13. an annular groove; 14. plugging the groove; 15. a plugging plate; 16. a liquid outlet pipe; 17. a liquid level adjustment assembly; 18. a piston plate; 19. an extension rod; 20. balancing weight; 21. a magnetic block; 22. adjusting a threaded rod; 23. an adjusting plate; 24. a push rod; 25. a top ring; 26. a first rotating plate; 27. a rotating shaft; 28. a second rotating plate; 29. a ball; 30. a first stop lever; 31. a second limit rod; 32. a muffler plate; 33. an operation port; 34. a control valve; 35. sealing the door; 36. and an observation window.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
Referring to fig. 1-6, the embodiment of the present invention provides a technical solution: the utility model provides an high-efficient preparation system of exocrine, including incubator 1 and three reagent bottle 2 of setting in incubator 1, built-in temperature regulation spare of incubator 1, as conventional subassembly, hereinafter unnecessary, a constant temperature 4 ℃ is kept to the inside temperature of for adjust incubator 1, in order to guarantee that reagent bottle 2 can carry out effectual supernatant separation, the inside of reagent bottle 2 is provided with liquid level control assembly 17, a height for adjust the inside culture solution of reagent bottle 2, specifically, liquid level control assembly 17 includes piston plate 18, the equal fixedly connected with extension pole 19 in left and right sides of piston plate 18 bottom, reagent bottle 2 is run through to the bottom of reagent bottle 2 is stretched to the bottom of extension pole 19, and the bottom fixedly connected with balancing weight 20 of extension pole 19.
As the preferred scheme, electric telescopic rod 3 is fixed mounting in the top of thermostated container 1, electric telescopic rod 3 and external power supply electric connection, control through control switch, electric telescopic rod 3's flexible end runs through thermostated container 1 and through link fixedly connected with hanger plate 4, the periphery of hanger plate 4 and the inner wall sliding contact of thermostated container 1, just be located the below fixed mounting of hanger plate 4 between the inner wall of thermostated container 1 has assembly plate 5, the top fixed mounting of assembly plate 5 has two division boards 6, the internal partition of two division boards 6 with thermostated container 1 is three working chamber 7, the bottom fixed mounting of working chamber 7 inner chamber has three driving motor 8, driving motor 8 adopts servo motor, control through control switch, the output of driving motor 8 passes through shaft coupling fixedly connected with iron square plate 9, and the square groove 10 with iron square plate 9 looks adaptation is seted up to the bottom of reagent bottle 2, in order to guarantee the stability of piston plate 18 structure, the bottom of piston plate 18 is inlayed and is fixed with magnetic force piece 21, magnetic force piece 21 and iron square plate 9 are used in coordination.
As the preferred scheme, the bottom rotation of hanger plate 4 is installed and is covered a section of thick bamboo 11, and the bottom fixed mounting that covers a section of thick bamboo 11 has transparent square ring 12, and the annular channel 13 of reagent bottle 2 looks adaptation has been seted up to the bottom of transparent square ring 12, and shutoff groove 14 has been seted up to one side of transparent square ring 12 and the top that is located annular channel 13, and the inside slidable mounting of shutoff groove 14 has shutoff board 15, through bolt fixed connection between shutoff board 15 and the transparent square ring 12, the opposite side of transparent square ring 12 is provided with drain pipe 16 with shutoff groove 14 intercommunication.
To describe in detail, the surface of the liquid outlet pipe 16 is provided with a control valve 34 for controlling the liquid outlet pipe 16 to circulate, the front surface of the incubator 1 is provided with a sealing door 35 matched with the working chamber 7, and the surface of the sealing door 35 is provided with an observation window 36.
As the preferred scheme, for the convenience carries out high regulation to the piston plate 18, the bottom of assembly plate 5 is connected with adjusting threaded rod 22 through the bearing rotation, for the rotation of convenient adjusting threaded rod 22, the bottom fixedly connected with knob at adjusting threaded rod 22, the periphery cover of adjusting threaded rod 22 is established and threaded connection has regulating plate 23, the equal fixedly connected with ejector pin 24 in left and right sides at regulating plate 23 top, the top of ejector pin 24 runs through assembly plate 5 setting, and the common fixedly connected with top ring 25 in top of two ejector pins 24, top ring 25 sets up in driving motor 8's periphery, and operation mouth 33 has been seted up to the front of thermostated container 1 and the below that is located assembly plate 5.
As a preferred scheme, in order to ensure that the reagent bottle 2 rotates, the covering cylinder 11 is driven to effectively rotate, a plurality of first limit rods 30 are fixedly mounted on the periphery of the reagent bottle 2 at uniform intervals, and a plurality of second limit rods 31 matched with the first limit rods 30 are fixedly mounted on the bottom of the transparent square ring 12 at uniform intervals.
As a preferred scheme, in order to ensure low friction force in the rotation process of the covering cylinder 11, a first rotating plate 26 is fixedly arranged at the top of the covering cylinder 11, a rotating shaft 27 is fixedly arranged at the top of the first rotating plate 26, a second rotating plate 28 is fixedly connected with the top end of the rotating shaft 27, balls 29 are embedded and rotatably arranged on the opposite sides of the first rotating plate 26 and the second rotating plate 28, and the outer surface of the balls 29 is in contact with the surface of the assembly plate 5.
Preferably, in order to reduce noise during centrifugal operation, muffler plates 32 are fixedly installed on both left and right sides of the inner cavity of the working chamber 7.
The high-efficiency exosome preparation method specifically comprises the following steps:
step one, assembling: collecting culture solution of mesenchymal stem cells after subculturing to P5, adding into reagent bottle 2, opening sealing door 35, clamping square groove 10 on reagent bottle 2 on iron square plate 9, controlling electric telescopic rod 3 to extend afterwards, electric telescopic rod 3 driving hanger plate 4 to move downwards, hanger plate 4 extruding first rotating plate 26 to make covering cylinder 11 move downwards, covering cylinder 11 driving transparent square ring 12 to move downwards until annular groove 13 on transparent square ring 12 is sleeved on the periphery of reagent bottle 2, stopping electric telescopic rod 3 to extend, finishing feeding assembly:
step two, low-speed centrifugation is carried out: controlling the driving motor 8 to rotate, driving the iron square plate 9 to drive the reagent bottle 2 to rotate by the driving motor 8, driving the transparent square ring 12 to drive the covering cylinder 11 to rotate by the reagent bottle 2, controlling the rotating speed of the driving motor 8 in the process, centrifuging at 300g and keeping for 10min, closing the driving motor 8, standing, turning to the step six, and taking the supernatant for removing cell components;
step three, low-speed centrifugation is carried out: placing the supernatant taken out in the second step into a new reagent bottle 2, repeating the operation of the first step, controlling the rotating speed of a driving motor 8 after finishing feeding and assembling, centrifuging at 2000g and keeping for 10min, closing the driving motor 8, standing, turning to the sixth step, and taking the supernatant for removing dead cells;
step four, high-speed centrifugation is carried out: placing the supernatant taken out in the third step into a new reagent bottle 2, repeating the operation of the first step, controlling the rotating speed of a driving motor 8 after finishing loading and assembling, centrifuging at 10000g and keeping for 70min, closing the driving motor 8, standing, turning to the sixth step, and taking the supernatant for removing cell fragments;
step five, high-speed centrifugation is carried out: placing the supernatant taken out in the fourth step in a new reagent bottle 2, repeating the operation of the first step, controlling the rotating speed of a driving motor 8 after finishing feeding and assembling, centrifuging with 100000g and keeping for 70min, closing the driving motor 8, standing, turning to the seventh step, and taking the precipitate as an exosome at the moment;
step six, liquid taking: after standing, rotating the bolts to separate the bolts from the transparent square rings 12, plugging the plugging plate 15 into the extraction part in the slot 14, then passing through the operation port 33 by hand, rotating the adjusting threaded rod 22, driving the adjusting plate 23 to ascend by the adjusting threaded rod 22, driving the ejector rod 24 to ascend by the adjusting plate 23, enabling the ejector ring 25 to ascend by the ejector rod 24, extruding the balancing weight 20 by the ejector ring 25 to ascend by the extension rod 19, extruding the piston by the extension rod 19 to ascend by the standing culture solution until the contact surface of the supernatant and the lower liquid is positioned below the plugging plate 15, pushing the plugging plate 15 to slide in the plugging slot 14, screwing the bolts, opening the control valve 34 after limiting of the plugging plate 15 is completed, taking a new reagent bottle 2 to be placed below the liquid outlet pipe 16, and collecting the supernatant;
step seven: separating: when the sediment needs to be collected, repeating the operation of the step six, lifting the rest culture solution until the position below the plugging plate 15 at the contact surface of the liquid and the sediment pushes the plugging plate 15 to slide in the plugging groove 14, screwing the bolt after the limit of the plugging plate 15 is completed, opening the control valve 34, taking the garbage can to be placed below the liquid outlet pipe 16, controlling the electric telescopic rod 3 to shrink after the liquid is collected, enabling the electric telescopic rod 3 to shrink to drive the hanging plate 4 to move upwards, enabling the hanging plate 4 to drive the covering can 11 to move upwards, enabling the transparent square ring 12 to be separated from the reagent bottle 2, taking out the reagent bottle 2, and pouring out the precipitated exosome.

Claims (8)

1. The utility model provides an high-efficient preparation system of exosome, includes incubator (1) and sets up three reagent bottle (2) in incubator (1), its characterized in that: the automatic constant temperature box is characterized in that an electric telescopic rod (3) is fixedly arranged at the top of the constant temperature box (1), the telescopic end of the electric telescopic rod (3) penetrates through the constant temperature box (1) and is fixedly connected with a hanging plate (4) through a connecting frame, the periphery of the hanging plate (4) is in sliding contact with the inner wall of the constant temperature box (1), an assembly plate (5) is fixedly arranged between the inner walls of the constant temperature box (1) and below the hanging plate (4), two isolation plates (6) are fixedly arranged at the top of the assembly plate (5), and the interior of the constant temperature box (1) is divided into three working cavities (7) by the two isolation plates (6);
three driving motor (8) are fixedly installed at the bottom of the inner cavity of the working cavity (7), iron square plates (9) are fixedly connected to the output end of the driving motor (8) through a coupler, square grooves (10) matched with the iron square plates (9) are formed in the bottom of the reagent bottles (2), a covering cylinder (11) is installed at the bottom of the hanging plate (4) in a rotating mode, a transparent square ring (12) is fixedly installed at the bottom of the covering cylinder (11), annular grooves (13) matched with the reagent bottles (2) are formed in the bottom of the transparent square ring (12), a plugging groove (14) is formed in one side of the transparent square ring (12) and above the annular grooves (13), a plugging plate (15) is installed in the interior of the plugging groove (14) in a sliding mode, a liquid outlet pipe (16) communicated with the plugging groove (14) is arranged on the other side of the transparent square ring (12), and a liquid level adjusting component (17) of the reagent bottles (2) is used for adjusting the height of the reagent bottles (2).
2. The efficient exosome preparation system according to claim 1, wherein: the liquid level adjusting assembly (17) comprises a piston plate (18), extension rods (19) are fixedly connected to the left side and the right side of the bottom of the piston plate (18), the bottom ends of the extension rods (19) penetrate through the reagent bottle (2) and extend to the lower side of the reagent bottle (2), balancing weights (20) are fixedly connected to the bottom ends of the extension rods (19), magnetic blocks (21) are inlaid and fixed at the bottom of the piston plate (18), and the magnetic blocks (21) are matched with the square iron plate (9).
3. The efficient exosome preparation system according to claim 2, wherein: the bottom of assembly plate (5) is connected with adjusting threaded rod (22) through the bearing rotation, the periphery cover of adjusting threaded rod (22) is established and threaded connection has regulating plate (23), the equal fixedly connected with ejector pin (24) in the left and right sides at regulating plate (23) top, the top of ejector pin (24) runs through assembly plate (5) setting, and two common fixedly connected with top ring (25) in top of ejector pin (24), top ring (25) set up in driving motor (8) periphery.
4. The efficient exosome preparation system according to claim 1, wherein: the top fixed mounting of covering section of thick bamboo (11) has first rotating plate (26), the top fixed mounting of first rotating plate (26) has pivot (27), the top of pivot (27) runs through hanger plate (4) and fixedly connected with second rotating plate (28), ball (29) are all inlayed and are rotated to one side that first rotating plate (26) and second rotating plate (28) are relative, and the surface of ball (29) contacts with the surface of assembly board (5).
5. The efficient exosome preparation system according to claim 1, wherein: the reagent bottle (2) periphery interval even fixed mounting has a plurality of first gag lever post (30), the bottom interval even fixed mounting of transparent square ring (12) has a plurality of second gag lever post (31) with first gag lever post (30) looks adaptation.
6. The efficient exosome preparation system according to claim 1, wherein: the left side and the right side of the inner cavity of the working cavity (7) are fixedly provided with silencing plates (32), and an operation opening (33) is formed in the front face of the incubator (1) and below the assembly plate (5).
7. The efficient exosome preparation system according to claim 1, wherein: the surface of drain pipe (16) is provided with control valve (34) that control drain pipe (16) circulate, the front of thermostated container (1) is provided with sealing door (35) with working chamber (7) looks adaptation, and the surface of sealing door (35) is provided with observation window (36).
8. An efficient preparation method of exosomes is characterized by comprising the following steps: the method specifically comprises the following steps:
step one, assembling: collecting culture solution of mesenchymal stem cells to P5 through subculture, add in reagent bottle (2), open sealing door (35), block square groove (10) on reagent bottle (2) on indisputable square board (9), afterwards control electric telescopic handle (3) extension, electric telescopic handle (3) drive hanger plate (4) move down, hanger plate (4) extrusion first rotating plate (26) make covering cylinder (11) move down, covering cylinder (11) drive transparent square ring (12) move down, until ring channel (13) cover on transparent square ring (12) are in the periphery of reagent bottle (2), stop electric telescopic handle (3) extension, accomplish the material loading assembly:
step two, low-speed centrifugation is carried out: controlling a driving motor (8) to rotate, driving the iron square plate (9) to rotate the reagent bottle (2) by the driving motor (8), driving the transparent square ring (12) to rotate the covering cylinder (11) by the reagent bottle (2), controlling the rotating speed of the driving motor (8) in the process, centrifuging at 300g and keeping for 10min, closing the driving motor (8), standing, and turning to the step six to obtain supernatant;
step three, low-speed centrifugation is carried out: placing the supernatant taken out in the second step into a new reagent bottle (2), repeating the operation of the first step, controlling the rotating speed of a driving motor (8) after finishing feeding and assembling, centrifuging at 2000g and keeping for 10min, closing the driving motor (8), standing, turning to the sixth step, and taking the supernatant;
step four, high-speed centrifugation is carried out: placing the supernatant taken out in the third step in a new reagent bottle (2), repeating the operation of the first step, controlling the rotating speed of a driving motor (8) after finishing feeding and assembling, centrifuging at 10000g and keeping for 70min, closing the driving motor (8), standing, turning to the sixth step, and taking the supernatant;
step five, high-speed centrifugation is carried out: placing the supernatant taken out in the fourth step in a new reagent bottle (2), repeating the operation of the first step, controlling the rotating speed of a driving motor (8) after finishing feeding and assembling, centrifuging with 100000g and keeping for 70min, closing the driving motor (8), standing, turning to the seventh step, and taking the precipitated exosomes;
step six, liquid taking: after standing, rotating the bolts to separate the bolts from the transparent square rings (12), pulling out parts of the plugging grooves (14) of the plugging plates (15), then enabling the bolts to pass through the operation openings (33) by hand, rotating the adjusting threaded rods (22), driving the adjusting plates (23) to ascend by the adjusting threaded rods (22), driving the ejector rods (24) to ascend by the adjusting plates (23), enabling the ejector rods (24) to ascend by the ejector rods (25), enabling the extension rods (19) to ascend by the extrusion weights (20), enabling the stationary culture solution to ascend by the extrusion pistons () of the extension rods (19), until the contact surface of the supernatant and the lower-layer liquid is located below the plugging plates (15), pushing the plugging plates (15) to slide in the plugging grooves (14), screwing the bolts after limiting of the plugging plates (15) is completed, opening the control valves (34), taking a new reagent bottle (2) to be placed below the liquid outlet pipe (16), and collecting the supernatant;
step seven: separating: when the sediment needs to be collected, the operation of the step six is repeated, the stationary culture solution rises until the liquid and the sediment contact surface position plugging plate (15) are below, the plugging plate (15) is pushed to slide in the plugging groove (14), then the bolt is screwed, after the limit of the plugging plate (15) is completed, the control valve (34) is opened, the garbage can is placed below the liquid outlet pipe (16), after the liquid is collected, the electric telescopic rod (3) is controlled to shrink, the electric telescopic rod (3) shrinks to drive the lifting plate (4) to move upwards, the lifting plate (4) drives the covering can (11) to move upwards, the transparent square ring (12) is separated from the reagent bottle (2), then the reagent bottle (2) is taken out, and the precipitated exosome is poured out.
CN202310456990.9A 2023-04-25 2023-04-25 Efficient exosome preparation system and method Pending CN116656468A (en)

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CN202310456990.9A CN116656468A (en) 2023-04-25 2023-04-25 Efficient exosome preparation system and method

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CN202310456990.9A CN116656468A (en) 2023-04-25 2023-04-25 Efficient exosome preparation system and method

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CN116656468A true CN116656468A (en) 2023-08-29

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