CN117379401A

CN117379401A - Live cell atomizing inhalation device and use method thereof

Info

Publication number: CN117379401A
Application number: CN202311366391.4A
Authority: CN
Inventors: 臧伟仲; 阿卡尔·阿尔普
Original assignee: Ark Qiyuan Beijing Biotechnology Co ltd
Current assignee: Ark Qiyuan Beijing Biotechnology Co ltd
Priority date: 2023-10-20
Filing date: 2023-10-20
Publication date: 2024-01-12

Abstract

The application provides a living cell aerosol inhalation device and a use method thereof, wherein the method comprises the following steps: s1, in vitro culture is carried out on living cells needing to be inhaled by atomization; s2, performing relevant quality inspection on living cells cultured in vitro, and identifying specific living cell markers; s3, removing supernatant from living cells cultured in vitro by centrifugation, and then adding a certain amount of living cell culture solution and lung cell protection solution for resuspension; s4, freezing and preserving living cells; s5, resuscitating living cells; s6, preparing a cell atomizing agent, and continuously atomizing through an atomizing device. The application aims at improving the cell activity problem after cell atomization through in-vitro cell culture aiming at the improvement of the in-vitro cell culture medium; through the aerosol inhalation device, the portable cell inhalation device has the characteristics of portability, and can realize lung inhalation after cell atomization, thereby solving the clinical input pain point of the current clinical cell therapy.

Description

Live cell atomizing inhalation device and use method thereof

Technical Field

The invention relates to the technical field of atomization, in particular to a living cell atomization inhalation device and a using method thereof.

Background

Cell therapy is widely used in the fields of multi-tissue regeneration, organ defects and the like. However, the treatment method is usually carried out by means of surgical treatment, exosome delivery, vascular infusion, in-situ surgical injection and the like, so that the cell treatment technical threshold can only be completed by medical institutions. Therefore, the method and the scheme for cell input into the human body have many technical scheme designs, no good technical research exists on portable and household cell input modes, more cell input modes are performed at present, intravenous injection and in-situ surgical injection of defective organs are adopted, or cells are prepared into powder or derivatives (such as exosomes) and then are input into the human body through atomization or oral administration and other modes, and the defects of over-high technical thresholds exist in cell activity preservation and cell input, so that the development of a set of methods for improving the cell inhalation/input mode is expected to be improved so as to facilitate better promotion of the technology to commercialization and portability, and the establishment of the method is favorable for the development or application of the cell therapy or cell health care industry. For example: the preparation of a stem cell active peptide aerosol inhalation method for treating pulmonary fibrosis and an aerosol disclosed in Chinese patent (application number: CN 201910747093.7) is disclosed in the specification: studies have shown that the active peptides secreted by stem cells are mostly well fused with cell membranes, thereby transporting the internal active substances into the targeted cells for action. At present, mesenchymal stem cells are reported to be applied to lung injury and lung fibrosis repair, but due to the short survival time of active cells, the requirements on preservation and transportation are harsh, and the effect is limited; the above patent can be used to demonstrate the drawbacks of the prior art. Therefore, we have made improvements to this and have proposed a live cell aerosol inhalation device and method of use.

Disclosure of Invention

The invention aims at: aiming at the problems that the existing cell activity preservation and cell input have the defect of over-high technical threshold. In order to achieve the above object, the present invention provides a living cell aerosol inhalation device and a method of using the same, which are capable of improving the above problems, and the present invention is specifically as follows:

a method of using a live cell aerosol inhalation device comprising the steps of: s1, in vitro culture is carried out on living cells needing to be inhaled by atomization; s2, performing relevant quality inspection on living cells cultured in vitro, and identifying specific living cell markers; s3, removing supernatant from living cells cultured in vitro by centrifugation, and then adding a certain amount of living cell culture solution and lung cell protection solution for resuspension; s4, freezing and preserving living cells; s5, resuscitating living cells; s6, preparing a cell atomizing agent, and continuously atomizing through an atomizing device.

As a preferred technical scheme of the application, the live cell cryopreservation comprises the following steps: s1, preparing a frozen stock solution with the concentration of the protective agent being 2 times of the final concentration and half of the final volume, slowly adding the frozen stock solution into a culture medium along the pipe wall, and placing the culture medium in a specific environment for more than 10 minutes; s2, counting 10 mu l of cell suspension to be frozen, calculating cell density and activity rate, and adding a certain amount of preservation solution into a centrifuge tube according to the cell counting result to enable the cell density in the tube to be 2 times of the frozen density; s3, slowly adding a cell freezing solution with the same volume as the preservation solution in the tube along the tube wall into the cell suspension, and lightly blowing and uniformly mixing to ensure that the cell density is consistent with the instruction requirement; s4, sub-packaging the cell suspension in a freezing tube; s5, placing the cells to be frozen in a freezing box filled with isopropanol, standing for a period of time in a specific environment, and transferring to a liquid nitrogen tank.

As a preferred technical scheme of the application, the live cell cryopreservation also comprises a cryopreservation tube marker and sampling detection.

As a preferred technical solution of the present application, the living cell resuscitation comprises the following steps: s1, regulating the temperature of a constant-temperature water bath box to a certain range; s2, taking out the cell freezing tube from the liquid nitrogen tank, immediately placing the cell freezing tube into warm water, and slightly shaking the cell freezing tube until the freezing liquid is completely dissolved.

As a preferable technical scheme of the application, the freezing solution used in the freezing step of the living cells is 10% DMSO, 5% human serum albumin, 1% dextran and compound electrolyte injection.

The utility model provides a living cell atomizing inhalation device, includes the shell, the bottom of shell is equipped with the installation component, one side of shell is equipped with intercommunication portion and transfer portion, and the intercommunication position is located directly over the transfer portion, the top of intercommunication portion is equipped with atomizing portion, the inside of shell is equipped with drive portion, input portion and output portion, the output position is located one side near the transfer portion, drive portion is located between input portion and the output portion.

Compared with the prior art, the invention has the beneficial effects that:

1. in order to solve the problem that the technical threshold is too high in the living cell activity preservation in the prior art, the method can improve the cell activity problem after cell atomization by improving the living cell in-vitro culture medium through the living cell in-vitro culture;

2. in order to solve the problem that the technical threshold is too high in living cell input in the prior art, the atomizing inhalation device has the characteristics of portability, and the pulmonary inhalation after living cell atomization solves the clinical input pain point of the current clinical living cell treatment;

3. the drive part and the transfer part are matched with the communication part, so that the function of quantitative liquid conveying is realized, the waste caused by excessive or insufficient liquid use can be reduced by accurately controlling the liquid conveying amount, and the operation is simple and is suitable for personal operation;

4. the automatic fixing function is realized by the matching of the arranged mounting assembly and the driving part, and the tank body in the transfer part structure can be automatically fixed in the operation process of the driving part, so that the mounting assembly is simple in dismounting operation and beneficial to use;

5. the centrifugal separation function is realized through the combination of the driving part, the input part and the output part, and the centrifugal separation device can be applied to cell centrifugal work and has diversified functions;

6. through the transmission switching assembly that sets up and remove the subassembly and cooperate, realized the speed limit function, drive portion running speed is too fast, can automatic cutout power transmission, improves operating stability.

Drawings

FIG. 1 is a flow chart of a method of using a live cell aerosol inhalation device provided herein;

FIG. 2 is a graph I of the experiment and data of example 2 in the present application;

FIG. 3 is a second experimental and data diagram of example 2 of the present application;

FIG. 4 is a third experimental and data plot of example 2 of the present application;

FIG. 5 is a fourth experimental and data plot of example 2 of the present application;

FIG. 6 is a fifth experimental and data plot of example 2 of the present application;

FIG. 7 is a sixth experimental and data plot of example 2 of the present application;

FIG. 8 is a front view of a living cell aerosol inhalation device provided herein;

FIG. 9 is a schematic diagram of a live cell aerosol inhalation device provided herein;

FIG. 10 is an enlarged view of A in the present application;

FIG. 11 is a schematic diagram of the first and second drive rods of the aerosol inhalation device for living cells according to the present disclosure;

FIG. 12 is a schematic view of the structure of the movable bar and the mounting cylinder in the aerosol inhalation device for living cells provided by the application;

FIG. 13 is a schematic view showing the internal structure of a mounting cylinder in the living cell aerosol inhalation device provided in the present application;

FIG. 14 is a schematic view showing the structure of a moving component in a living cell aerosol inhalation device provided by the present application;

FIG. 15 is a cross-sectional view of the housing of the living cell aerosol inhalation device provided herein;

FIG. 16 is an enlarged view of C in the present application;

FIG. 17 is a schematic view of the mounting assembly of the aerosol inhalation device for living cells provided herein;

fig. 18 is an enlarged view of D in the present application;

FIG. 19 is an enlarged view of E in the present application;

FIG. 20 is an enlarged view of B in the present application;

FIG. 21 is a schematic view of the structure of a pressure switching assembly in a living cell aerosol inhalation device provided herein;

FIG. 22 is a schematic view of the structure of a ring in a device for atomizing and inhaling living cells according to the present application;

FIG. 23 is a schematic view showing the structure of a support bar in a living cell aerosol inhalation device according to the present application;

FIG. 24 is a schematic view showing the structure of a cap in a living cell aerosol inhalation device provided herein;

fig. 25 is a schematic structural view of a support rod and a stabilizer rod in the aerosol inhalation device for living cells provided in the present application.

1. A mounting assembly; 11. a base; 12. a switch assembly; 121. a knob; 122. a threaded rod; 123. a rotating ring; 124. a sealing plug; 125. a first connecting rod; 126. a movable rod; 127. a support ring; 128. a second connecting rod; 13. a negative pressure fixing assembly; 131. a cylinder; 132. a winding wheel; 134. a first spring; 135. a first piston; 136. a pull rope; 137. a locking hook; 138. a thread cylinder; 139. a mounting base; 1310. a clamping rod; 14. an exhaust hole; 15. a first cavity; 16. a second cavity; 17. a first communicating hole; 18. a third cavity; 19. a second communicating hole; 110. a fourth cavity; 111. a third communicating hole; 112. a fifth cavity; 2. a housing; 21. a first partition board; 22. a second separator; 3. a communication section; 31. a communication table; 32. pressing the switch; 33. an output channel; 34. a pressure conversion assembly; 341. a housing; 342. a second spring; 343. an input port; 344. an output port; 345. a slide switch; 346. a limit rod; 35. a third spring; 36. an elastic buckle; 37. a clamping groove; 4. a transfer section; 41. a tank body; 42. a hose; 43. an end cap; 44. a spring IV; 45. an output head; 5. an atomizing unit; 51. an atomizer; 52. a connector; 54. a filter screen; 6. a side cover; 7. a driving section; 71. a second piston; 72. pressing head; 73. a first transmission rod; 74. a second transmission rod; 75. a transmission switching assembly; 751. a push rod; 752. a connecting rod; 753. a first sliding block; 754. a movable bar; 755. a mounting cylinder; 756. a chute; 757. a second slide block; 758. a first ball head buckle; 759. a limiting ring; 7510. a fifth spring; 7511. a connecting rope; 76. a moving assembly; 761. a mobile station; 763. a spring number six; 764. a telescopic block; 765. a first driving block; 766. a second driving block; 8. an input unit; 81. a one-way valve; 82. a knob post; 83. a hollow rod; 84. a support rod; 85. a circular ring; 86. a fourth slider; 87. a fifth slider; 88. a filter screen; 89. an inner ring; 810. a support bar; 811. a second ball head buckle; 812. a spring number seven; 813. a pull rod; 814. an operation block; 815. a top cover; 816. a stabilizer bar; 817. a spring No. eight; 818. a third ball head buckle; 9. an output unit; 91. a driving rod; 92. a rectangular frame; 93. triangle block number one; 94. a drive plate; 95. triangular blocks II; 96. a movable frame; 97. a rack; 98. a strip-shaped through hole; 99. a pressure bearing head; 910. a sleeve; 911. a telescopic rod; 912. and a linkage rod.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.

In order to solve the technical problem, the invention provides a living cell aerosol inhalation device and a use method thereof.

Example 1

Referring to fig. 1, a method for using a living cell aerosol inhalation device specifically includes the following steps: s1, in vitro culture is carried out on living cells needing to be inhaled by atomization; s2, performing relevant quality inspection on living cells cultured in vitro, and identifying specific living cell markers; s3, removing supernatant from living cells cultured in vitro by centrifugation, and then adding a certain amount of living cell culture solution and lung cell protection solution for resuspension; s4, freezing and preserving living cells; s5, resuscitating living cells; s6, preparing a cell atomizing agent, and continuously atomizing through an atomizing device. Living cells include, but are not limited to, mesenchymal stem cells, immune cells, mature tissue cells differentiated from umbilical cord mesenchymal stem cells, tissue cells differentiated from IPS pluripotent stem cells, CART cells, CAR-NK cells.

Further, the freezing and storing of the living cells comprises the following steps: s1, preparing a frozen stock solution with the concentration of the protective agent being 2 times of the final concentration and half of the final volume, slowly adding the frozen stock solution into a culture medium along the pipe wall, and placing the culture medium in an environment of 4 ℃ for more than 10 minutes; s2, counting 10 mu l of cell suspension to be frozen, calculating cell density and activity rate, and adding a certain amount of preservation solution into a centrifuge tube according to the cell counting result to enable the cell density in the tube to be 2 times of the frozen density; s3, slowly adding a cell freezing solution with the same volume as the preservation solution in the tube along the tube wall into the cell suspension, and lightly blowing and uniformly mixing to ensure that the cell density is consistent with the instruction requirement; s4, sub-packaging the cell suspension into frozen storage tubes, wherein each tube is 1ml; s5, placing the cells to be frozen in a freezing box filled with isopropanol, standing for a period of time in an environment of-80 ℃, and transferring to a liquid nitrogen tank.

Furthermore, the live cell cryopreservation also comprises cryopreservation tube labeling and sampling detection.

Further, the live cell resuscitation comprises the following steps: s1, regulating the temperature of a constant-temperature water bath box to 37 ℃;

s2, taking out the cell freezing tube from the liquid nitrogen tank, immediately placing the cell freezing tube into warm water at 37 ℃ and slightly shaking the cell freezing tube until the freezing liquid is completely dissolved.

Further, the frozen stock solution used in the frozen stock step of living cells is 10% DMSO, 5% human serum albumin, 1% dextran and compound electrolyte injection.

Further, the markers in the live cell cryopreservation step include, but are not limited to, numbering information, cell algebra information, cell cryopreservation lot information, cryopreservation date information, and operator information.

Further, sampling assays include, but are not limited to, microbiological assay items, endotoxin assay items.

Further, the cryopreservation density in the step of cryopreserving living cells is in the range of 0.5 to 2X 10 ⁸ /ml。

Further, the cell atomizing agent is prepared by mixing physiological saline, 5% human serum albumin and cells.

The cell activity problem after stem cell atomization can be improved by in vitro cell culture aiming at improvement of in vitro culture medium of living cells.

Example 2

Referring to fig. 2, 3, 4, 5, 6 and 7, a laboratory performs animal experiments on live cell aerosol inhalation;

cell type: the tested cells are umbilical cord mesenchymal stem cells and can be extended to any beneficial living cells;

cell density: 1X 10 ⁸ /ml；

Cell atomizing agent composition: DMEM cell culture broth, 5% human serum albumin and cells;

experimental animals: wild-type mice;

inhalation dose: 3 to 5ml of 1X 10 ⁸ The cell suspension is/ml, and the atomization inhalation is completed within 15 minutes;

cell labeling: green Fluorescent Protein (GFP) markers, animal live imaging shows that there are larger cells residing in the lungs;

number of inhalations, interval time: dosing every other day, 5 times in succession;

results: tissue section identification (secondary staining), 1-2 umbilical cord mesenchymal stem cells were present per 400-fold field.

Example 3

Referring to fig. 8 and 9, an aerosol inhalation device for living cells includes a housing 2, a mounting assembly 1 is disposed at the bottom of the housing 2, a communication portion 3 and a transfer portion 4 are disposed at one side of the housing 2, the communication portion 3 is located right above the transfer portion 4, an aerosol portion 5 is disposed at the top of the communication portion 3, a driving portion 7, an input portion 8 and an output portion 9 are disposed inside the housing 2, the output portion 9 is located at one side close to the transfer portion 4, and the driving portion 7 is located between the input portion 8 and the output portion 9. The atomization inhalation device has the characteristics of portability, and can realize pulmonary inhalation after stem cell atomization, thereby solving the problem of clinical input pain point of the current clinical stem cell treatment.

Example 4

For further optimization of the aerosol inhalation device for living cells provided in embodiment 3, specifically, as shown in fig. 9 and 10, a first partition plate 21 and a second partition plate 22 are fixedly installed inside the housing 2, the first partition plate 21 and the second partition plate 22 divide the inside of the housing 2 into a first area, a second area and a third area, the driving part 7 comprises a second piston 71 slidably arranged inside the housing 2 and a first transmission rod 73 arranged in the second area, a pressing head 72 is arranged above the housing 2, the top end of the first transmission rod 73 penetrates through the housing 2 and is fixedly connected with the pressing head 72, the first transmission rod 73 is in linkage with the pressing head 72, a second transmission rod 74 is arranged at the bottom end of the first transmission rod 73, the first transmission rod 73 is in linkage with the second transmission rod 74, the bottom end of the second transmission rod 74 penetrates through the second area and is fixedly connected with the second piston 71, and the second transmission rod 74 is in linkage with the second piston 71. By controlling the pressing head 72 to perform linear reciprocating motion, the No. two piston 71 can be driven to perform linear displacement motion.

Further, as shown in fig. 8, 9, 10 and 22, an air inlet is formed on one side of the casing 2 far away from the transfer part 4, a side cover 6 is detachably mounted below the air inlet, the input part 8 comprises a one-way valve 81 fixedly mounted in the air inlet and a supporting rod 84 arranged in a first area, the bottom end of the supporting rod 84 penetrates through the first area and is in threaded connection with a second piston 71, the supporting rod 84 is linked with the second piston 71, the characteristics of convenient disassembly are provided, at least four inner rings 89 are detachably mounted on the outer wall of the supporting rod 84, the supporting rod 84 is linked with the inner rings 89, a circular ring 85 is arranged on the outer side of the inner rings 89, supporting rods 810 are arranged between the inner rings 89 and the circular ring 85, the inner rings 89 are linked with the circular ring 85, and a filter screen 88 is fixedly arranged in a circumferential gap between the inner rings 89 and the circular ring 85. The filter screen 88 can filter partial impurity in the input air, and in the linear displacement process of the second piston 71, the circular ring 85 and the inner ring 89 move synchronously, static electricity is generated by friction between the circular ring 85 and the inner wall of the first area, and the impurity of which the fixed part is not treated by the filter screen 88 is adsorbed by the static electricity.

Further, as shown in fig. 9, 10, 15 and 16, the exhaust port is opened at one side of the casing 2 near the transfer portion 4, the output portion 9 includes a driving rod 91, a rectangular frame 92, a driving plate 94, a sleeve 910, a telescopic rod 911, a linkage rod 912 and a generator which are arranged in the third region, the bottom end of the driving rod 91 passes through the third region and is fixedly connected with the second piston 71, the driving rod 91 is linked with the second piston 71, the top end of the driving rod 91 is fixedly connected with the rectangular frame 92, the driving rod 91 is linked with the rectangular frame 92, the bottom end of the telescopic rod 911 is slidably arranged in the sleeve 910, the bottom end of the sleeve 910 is fixedly connected with the rectangular frame 92, the sleeve 910 is linked with the rectangular frame 92, two ends of the linkage rod 912 are respectively connected with the telescopic rod 911 and the driving plate 94 in a rotating way, the connection point of the linkage rod 912 and the driving plate 94 is deviated from the center point of the driving plate 94, thereby the linear motion is converted into rotary motion, and the driving plate 94 is fixedly mounted on the transmission shaft of the generator. By converting the linear motion into the rotary motion, the driving disc 94 is driven to perform the rotary motion, so that the generator generates electric energy, and the electric energy is stored in a battery arranged above the generator to provide electric energy for the electricity-requiring unit.

Further, as shown in fig. 9 and 20, the transfer portion 4 includes a tank body 41, a hose 42 is provided in the tank body 41, an end cover 43 is detachably mounted at the top of the tank body 41, a limiting hole is provided at the top of the end cover 43, an output head 45 is slidably provided in the limiting hole, the output head 45 can passively perform telescopic motion, a fourth spring 44 is provided below the output head 45, the fourth spring 44 is arranged, the output head 45 can automatically reset after the acting force of the control motion disappears, the top end of the hose 42 passes through the end cover 43, the fourth spring 44 and the output head 45 to be slidably connected, a gas pipe is communicated with the outer wall of the tank body 41, and the gas pipe is inserted into the exhaust port. When the second piston 71 performs linear reciprocating motion, the second piston includes upward motion and downward motion, during the downward motion, air enters the first area, the second area and the third area through the check valve 81, during the upward motion, air cannot be discharged at the check valve 81, so that the air enters the tank 41 through the air pipe, the tank 41 is pressurized, and liquid in the tank 41 moves upwards through the hose 42 and the output head 45.

Further, as shown in fig. 8, 9, 20 and 21, the communicating portion 3 includes a communicating table 31 fixedly mounted on one side of the housing 2, an output channel 33 and a limit slide are provided in the communicating table 31, a pressure conversion assembly 34 is provided in the output channel 33, a pressing switch 32 is provided in the limit slide in a sliding manner, the pressing switch 32 is subjected to a pressing force, a third spring 35 is fixedly sleeved on the pressing switch 32, the third spring 35 is arranged to enable the pressing switch 32 to automatically reset after the action force of the control movement disappears, one end of the pressing switch 32 passes through the limit slide and extends into the output channel 33, a clamping groove 37 is provided in the limit slide, an elastic buckle 36 is fixedly provided on the pressing switch 32, the elastic buckle 36 is linked with the pressing switch 32, when the pressing switch 32 is pressed once, the elastic buckle 36 is clamped into the clamping groove 37, the third spring 35 is forced to shrink, when the pressing switch 32 is pressed twice, the elastic buckle 36 exits in the clamping groove 37 and continues to displace forwards, the action force of the pressing switch 32 is controlled to disappear, and after the third spring 35 rebounds rapidly, the reset speed is far greater than the first reset speed of the pressing buckle, and the reset speed is far greater than the first reset speed of the pressing switch 37, thereby realizing the reset speed in the reset process.

The pressure conversion assembly 34 is controlled by pressing the switch 32, so that the function of quantitative liquid delivery is realized, the delivery quantity of liquid is accurately controlled, the waste caused by excessive use or insufficient use of liquid can be reduced, the operation is simple, and the device is suitable for personal operation.

Further, as shown in fig. 20 and 21, the pressure conversion assembly 34 includes a casing 341 fixedly installed inside the output channel 33, an input port 343 is provided at the bottom of the casing 341, an output port 344 is provided at the top of the casing 341, a sliding switch 345 is slidably provided inside the casing 341, liquid enters the output channel 33 through the output head 45 to push the sliding switch 345 to slide and displace, a second spring 342 is fixedly connected to one side of the sliding switch 345 and one side inner wall of the casing 341, the second spring 342 is arranged to control the acting force of the sliding switch 345 to slide and displace automatically after disappearing, a groove is provided at the other side of the sliding switch 345, a through hole is provided at the middle of the sliding switch 345, a limit rod 346 is fixedly connected to the other side inner wall of the casing 341 to limit the moving range of the sliding switch 345, and a perforation opposite to the pressing switch 32 is provided at the middle of the side. When the sliding switch 345 is not subjected to fluid pressure, a small part of the output port 344 is still closed, the caliber of the through hole is smaller than that of the input port 343, liquid enters through the input port 343 and is subjected to certain resistance when passing through the through hole, so that thrust is generated to the sliding switch 345, when the liquid pressure is high, the thrust to the sliding switch 345 is high, when the liquid pressure is low, the thrust to the sliding switch 345 is low, the larger the displacement distance of the sliding switch 345 is, the more the area of the output port 344 is closed, the smaller the displacement distance of the sliding switch 345 is, the less the area of the output port 344 is closed, and therefore the liquid pressure is small under uneven condition, and stable output is maintained.

Further, as shown in fig. 8 and 9, the atomizing unit 5 includes an atomizer 51 fixedly mounted on the top of the communication table 31, a channel communicating with the output channel 33 is provided at the bottom of the atomizer 51, liquid enters the atomizer 51 through the output port 344, a connector 52 is fixedly connected to the outer wall of the atomizer 51, a filter mesh 54 is fixedly provided in the connector 52, an atomizing sheet is provided in the atomizer 51, the atomizing sheet in the atomizer 51 atomizes the liquid, and the filter mesh 54 filters the atomized liquid again. The liquid can be atomized and filtered again, and the connector 52 can be docked with the mask for inhalation. The atomizing caliber of the atomizer (i.e., the individual atomizers Kong Koujing) is: the pore diameter is 5-100um, and the atomization rate is 0.8ml/min-20ml/min; the atomization rate of the individual wells was 0.53 μl/min to 13.4 μl/min calculated as 1500 atomization wells; .

Example 5

In the embodiment 3 or 4, concretely, as shown in fig. 8, 9, 17, 18 and 19, the installation component 1 includes a base 11 fixedly installed at the bottom of the housing 2, a first cavity 15, a second cavity 16, a third cavity 18, a fourth cavity 110 and a fifth cavity 112 are provided in the base 11, the third cavity 18 is communicated with the first cavity 15 and the second cavity 16, a first communication hole 17 communicated with the second cavity 16 is provided at the top of the base 11, a third communication hole 111 is provided between the first cavity 15 and the fourth cavity 110, a negative pressure fixing component 13 is provided in the first cavity 15, the negative pressure fixing component 13 includes a threaded cylinder 138 fixedly installed in the first cavity 15 and a first piston 135 slidingly installed in the third cavity 18, the first piston 135 is capable of sliding in the third cavity 18, a cylinder 131 is installed in the internal thread of the threaded cylinder 138, a rolling wheel 132 is fixedly installed at the top of the cylinder 131, a rolling wheel 132 is fixedly installed at the bottom of the cylinder 131, a third communication hole 111 is provided between the first cavity 15 and the fourth cavity 110, a third communication hole 111 is provided in the first cavity 15 and the fourth cavity 110, a third piston 135 is slidingly installed in the first piston 135 is slidingly installed in the third piston 135, and the rolling wheel is connected with a first piston 135, and a second piston 134 is slidingly installed at the second piston 132 is connected to the first piston 132, and the second piston is fixedly connected with a third piston 132, and the second piston 132 is fixedly connected with a third piston, and the second piston 132, and the third piston is connected with the third piston 132, and the third piston is provided with the piston through the third piston and the piston is further has a third piston through the piston. When the second piston 71 is displaced downwards, a downward force is applied to the winding wheel 132, so that the cylinder 131 rotates and is displaced downwards, during the rotation process of the winding wheel 132, the pull rope 136 is wound, when the pull rope 136 is wound, the first piston 135 is pulled to displace, a negative pressure environment is formed in the second cavity 16, and when the bottom of the tank 41 is closed with the first communication hole 17, the tank 41 is automatically fixed through negative pressure.

Further, as shown in fig. 8, 9, 17, 18 and 19, an exhaust hole 14 communicated with the first cavity 15 is formed in one side of the base 11, when the second piston 71 is displaced downwards, air in the first cavity 15 is exhausted through the exhaust hole 14, a threaded hole communicated with the fifth cavity 112 is formed in the other side of the base 11, a switch assembly 12 is arranged on one side of the base 11, the switch assembly 12 comprises a threaded rod 122 which is installed in the threaded hole in a threaded manner, one end of the threaded rod 122 is fixedly connected with a knob 121, the other end of the threaded rod 122 is fixedly connected with an extension rod, the end part of the extension rod is fixedly connected with a sealing plug 124, and a limiting channel is formed between the fifth cavity 112 and the second cavity 16. The knob 121 controls the threaded rod 122 to perform rotary motion, so that the threaded rod 122 can perform linear motion and drive the sealing plug 124 to perform synchronous motion, the sealing plug 124 exits from the limiting channel, the inside of the second cavity 16 can be communicated with the outside, the negative pressure environment is recovered to be normal, and the disassembly of the tank 41 is completed.

Further, as shown in fig. 9, 17, 18 and 19, a fourth communication hole is formed between the fourth cavity 110 and the fifth cavity 112, a third communication hole 111 is formed between the fourth cavity 110 and the first cavity 15, a movable rod 126 is arranged in the fourth cavity 110, a support ring 127 is fixedly sleeved on the movable rod 126, a rotating ring 123 is movably sleeved on the extending rod, the extending rod is in linkage with the threaded rod 122, the extending rod can move synchronously with the rotating ring 123, a first connecting rod 125 is fixedly connected to the outer wall of the rotating ring 123, the bottom end of the first connecting rod 125 passes through the fourth communication hole and is fixedly connected with one end of the movable rod 126, the first connecting rod 125 is in linkage with the rotating ring 123 and the movable rod 126, a second connecting rod 128 is fixedly connected to the end of the movable rod 126, the movable rod 126 is in linkage with the second connecting rod 128, the mounting seat 139 is positioned in the inside the third communication hole 111, one end of the mounting seat 139 is rotatably connected with a clamping rod 1310, and a first torsion spring is arranged at the connecting position. When the cylinder 131 moves downwards, the locking hook 137 is driven to synchronously move, the locking hook 137 pushes the clamping rod 1310 to rotate anticlockwise, the locking hook 137 continues to move downwards, the first torsion spring enables the clamping rod 1310 to reset automatically, the locking hook 137 and the clamping rod 1310 are clamped, the cylinder 131 keeps the current position, the threaded rod 122 moves linearly, the sealing plug 124 is driven to synchronously move, the sealing plug 124 exits from the limiting channel, and meanwhile, the clamping rod 1310 is driven to move towards the direction far away from the locking hook 137 through the rotating ring 123, the first connecting rod 125, the movable rod 126, the second connecting rod 128 and the mounting seat 139, so that locking is released.

Example 6

For further optimization of the living cell aerosol inhalation device provided in embodiments 3, 4 or 5, specifically, as shown in fig. 9, 10, 11 and 14, a transmission switching component 75 is disposed in the first transmission rod 73, a special-shaped hole is formed in the first partition 21, a moving component 76 is disposed in the special-shaped hole, the moving component 76 includes a moving table 761 slidably disposed in the special-shaped hole, a sixth spring 763 and a telescopic block 764 are disposed in the moving table 761, the telescopic block 764 is located above the sixth spring 763, a through hole is formed in the top of the moving table 761, a first driving block 765 and a second driving block 766 are fixedly disposed on two sides of the moving table 761, edges of the first driving block 765 are double inclined planes, edges of the second driving block 766 are cambered surfaces, tops of the special-shaped hole are inclined planes, and holes are formed on two sides of the special-shaped hole. The part of the structure of the first driving block 765 is located in the first area, and when the second piston 71 performs linear reciprocating motion, the ring 85 is driven to move synchronously, the ring 85 is repeatedly contacted with the inclined surface of the first driving block 765, and pushing is applied to the first driving block 765, so that the moving table 761 and the second driving block 766 are pushed to move towards the direction close to the second area, the faster the moving speed of the second piston 71 is, and the longer the second driving block 766 stays in the second area.

Further, fig. 9, 10, 11, 12 and 13, the transmission switching assembly 75 includes a push rod 751 rotatably mounted inside a first transmission rod 73, a second torsion spring is disposed at a connection point, a first slider 753 is slidably mounted inside the first transmission rod 73, a connecting rod 752 is disposed between the push rod 751 and the first slider 753, two ends of the connecting rod 752 are respectively rotatably connected with the push rod 751 and the first slider 753, a movable strip 754 is fixedly connected to the bottom of the first slider 753, a sliding groove 756 is formed in the rear side of the movable strip 754, a second slider 757 is slidably disposed inside the sliding groove 756, a mounting cylinder 755 is fixedly mounted inside the first transmission rod 73, two limit rings 759 are fixedly disposed inside the mounting cylinder 755, a fifth spring 7510 is fixedly connected between the first ball buckle 758 and the limit rings 759, a connecting rope 7511 is fixedly connected to the bottom of the second slider 757, two ends of the connecting rope 7511 are respectively fixedly connected with the two first ball buckles 757 through two connecting belts, and two holes are formed in the second ball buckle 758.

When the second driving block 766 enters the second area, the first driving rod 73 is in a linear reciprocating displacement state, the first driving rod 73 drives the push rod 751 when being upwards displaced, the push rod 751 is in contact with the second driving block 766 and rotates anticlockwise, the first sliding block 753 slides downwards through the connecting rod 752, the movable bar 754 synchronously moves downwards and is staggered with the mounting cylinder 755, the first driving rod 73 drives the push rod 751 when being downwards displaced, the push rod 751 is in contact with the second driving block 766 and rotates clockwise, the first sliding block 753 slides upwards through the connecting rod 752, the movable bar 754 synchronously moves upwards, the connecting rope 751 is pulled by the second sliding block 757, the first ball buckle 758 is pulled by the connecting rope 751 to move towards the middle of the mounting cylinder 755, the first driving rod 73 and the first driving rod 73 are in a non-linkage state, power transmission can be automatically cut off, and running stability is improved.

Further, as shown in fig. 9, 10, 22, 23 and 24, a mounting groove is formed in the top of the supporting bar 810, a second ball head buckle 811 is slidably arranged in the mounting groove and fixedly provided with a seventh spring 812, a pull rod 813 is further arranged in the mounting groove, one end of the pull rod 813 penetrates through the seventh spring 812 and is fixedly connected with the supporting bar 810, the other end of the pull rod 813 is fixedly connected with an operation block 814, a blind hole is formed in the inner wall of the circular ring 85, a notch is formed in the outer wall of the circular ring 85, a fourth slider 86 is rotatably arranged in the notch, a fifth slider 87 is fixedly arranged in the outer wall of the circular ring 85, a guide groove matched with the fourth slider 86 and the fifth slider 87 is formed in the first region, a mounting hole communicated with the first region is formed in the top of the housing 2, a top cover 815 is fixedly arranged in the mounting hole in a threaded manner, a knob post 82 is fixedly arranged at the top of the top cover 815, an anti-slip tooth socket is formed in the outer wall of the knob post 82, a hollow rod 83 is fixedly arranged at the bottom of the top cover 815, and a round hole 83 is slidably sleeved on the hollow rod 83. The knob post 82 can be inserted into the exhaust port by controlling the knob post 82 to rotate and be disassembled, the second ball head buckle 811 is controlled to slide through the operation block 814 and the pull rod 813, the blind hole is withdrawn, and the inner ring 89 and the circular ring 85 can be disassembled.

Further, as shown in fig. 25, a stabilizer bar 816 is disposed in the support bar 84, a No. eight spring 817 is fixedly disposed in the stabilizer bar 816, a No. three ball head buckle 818 is fixedly disposed at one end of the No. eight spring 817, mutually aligned through holes are formed in the support bar 84 and the stabilizer bar 816, a bayonet is further formed in an inner wall of the inner ring 89, and an end portion of the No. three ball head buckle 818 is mounted in the bayonet through holes in a clamping manner. The side cover 6 is disassembled, the side cover can be disassembled by controlling the supporting rod 84 to rotate, the stabilizer bar 816 is pulled to bear pulling force, the third ball head buckle 818 is withdrawn from the bayonet, the stabilizer bar 816 is disassembled from the supporting rod 84, and meanwhile, the supporting rod 84 is disassembled from the inner ring 89.

Further, as shown in fig. 15 and 16, a movable frame 96 is slidably disposed in the rectangular frame 92, racks 97 are fixedly disposed on inner walls of two sides of the movable frame 96, strip-shaped through holes 98 are formed on two sides of the rectangular frame 92, pressure-bearing heads 99 are fixedly connected to two sides of the movable frame 96, the two pressure-bearing heads 99 respectively pass through the two strip-shaped through holes 98 and extend to the outside of the rectangular frame 92, a first triangular block 93 and a second triangular block 95 are fixedly disposed in a third region, and the first triangular block 93 and the second triangular block 95 are respectively located on two sides of the rectangular frame 92. The tank body 41 is disassembled, the knob post 82 is inserted into the exhaust port, an anti-slip tooth socket formed on the outer wall of the knob post 82 is meshed with the rack 97, the supporting rod 84 and the stabilizing rod 816 are respectively inserted into a first communication hole 17 and the output channel 33, after one group of inner rings 89 and the supporting rod 84 are separated, a fourth sliding block 86 is arranged at the end part of the supporting rod 84 provided with internal threads in a threaded manner, a fifth sliding block 87 is inserted into the end part of the stabilizing rod 816, the end part of the hollow rod 83 is inserted into the inner ring 89, the control lantern ring slides, the part of the lantern ring which is not provided with a round hole is filled into a circumferential gap between the hollow rod 83 and the inner ring 89, the hollow rod 83 is supported and limited, the rest groups of inner rings 89 and the round rings 85 are separated, the fourth sliding blocks 86 on the outer wall of the round rings 85 are arranged in round holes on the lantern ring in a threaded manner, when the second piston 71 moves, the driving rod 91 drives the rectangular frame 92 to linearly reciprocate and drives the movable frame 96 to synchronously move, the mounting positions of the two pressure bearing heads 99 are respectively close to the top and the bottom of the side face of the movable frame 96, the mounting position of the first triangular block 93 is equal to the bottom of the rectangular frame 92, the inclined surface faces upwards, the mounting position of the second triangular block 95 is equal to the top of the rectangular frame 92, the inclined surface faces downwards, and when the movable frame 96 linearly reciprocates, the movable frame 96 is enabled to linearly reciprocate in the vertical direction and linearly reciprocate in the horizontal direction, the knob post 82 and the hollow rod 83 can be driven to perform rotary motion, the whole structure is horizontal, the centrifuge tube is inserted into the circular ring 85, and the centrifugal tube can be subjected to centrifugal steps.

The invention provides a living cell atomizing inhalation device and a using method thereof, wherein the using process is as follows: the method comprises the steps of carrying out in-vitro culture on living cells needing to be atomized and inhaled, carrying out relevant quality inspection on the living cells cultured in vitro, identifying specific living cell markers, carrying out centrifugation on the living cells cultured in vitro, discarding supernatant, then adding a certain amount of living cell culture solution and lung cell protection solution, carrying out resuspension, freezing and preserving the living cells, resuscitating the living cells, preparing a cell atomizing agent, carrying out continuous atomization through an atomization device, wherein in centrifugal operation, a driving part 7, an input part 8 and an output part 9 are matched, realizing centrifugal separation function through structural change assembly, being applicable to cell centrifugation operation, in atomization operation, the driving part 7, the output part 9 and a transfer part 4 are matched with a communication part 3, uniformly inputting normal saline mixed with stem cells into an atomizer 51, enabling a connector 52 to be in butt joint with a mask, and outputting for inhalation through the connector 52 after atomization.

It is apparent that the above-described embodiments are only some embodiments of the present invention, but not all embodiments, and the preferred embodiments of the present invention are shown in the drawings, which do not limit the scope of the patent claims. This invention may be embodied in many different forms, but rather, embodiments are provided in order to provide a thorough and complete understanding of the present disclosure. Although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described in the foregoing description, or equivalents may be substituted for elements thereof.

Claims

1. A method of using a live cell aerosol inhalation device, comprising the steps of:

s1, in vitro culture is carried out on living cells needing to be inhaled by atomization;

s2, performing relevant quality inspection on living cells cultured in vitro, and identifying specific living cell markers;

s3, removing supernatant from living cells cultured in vitro by centrifugation, and then adding a certain amount of living cell culture solution and lung cell protection solution for resuspension;

s4, freezing and preserving living cells;

s5, resuscitating living cells;

s6, preparing a cell atomizing agent, and continuously atomizing through an atomizing device.

2. A method of using a live cell aerosol inhalation device according to claim 1, wherein the live cell cryopreservation comprises the steps of:

s1, preparing a frozen stock solution with the concentration of the protective agent being 2 times of the final concentration and half of the final volume, slowly adding the frozen stock solution into a culture medium along the pipe wall, and placing the culture medium in a specific environment for more than 10 minutes;

s2, counting 10 mu l of cell suspension to be frozen, calculating cell density and activity rate, and adding a certain amount of preservation solution into a centrifuge tube according to the cell counting result to enable the cell density in the tube to be 2 times of the frozen density;

s3, slowly adding a cell freezing solution with the same volume as the preservation solution in the tube along the tube wall into the cell suspension, and lightly blowing and uniformly mixing to ensure that the cell density is consistent with the instruction requirement;

s4, sub-packaging the cell suspension in a freezing tube;

s5, placing the cells to be frozen in a freezing box filled with isopropanol, standing for a period of time in a specific environment, and transferring to a liquid nitrogen tank.

3. The method of claim 2, wherein the freezing of the living cells further comprises a freezing tube label and sampling.

4. A method of using a live cell aerosol inhalation device according to claim 3, wherein the live cell resuscitation comprises the steps of:

s1, regulating the temperature of a constant-temperature water bath box to a certain range;

s2, taking out the cell freezing tube from the liquid nitrogen tank, immediately placing the cell freezing tube into warm water, and slightly shaking the cell freezing tube until the freezing liquid is completely dissolved.

5. The method of claim 4, wherein the frozen stock solution used in the frozen stock step is 10% DMSO, 5% human serum albumin, 1% dextran and compound electrolyte injection.

6. A method of using a live cell aerosol inhalation device according to claim 5, wherein the indicia of the live cell cryopreservation step include, but are not limited to, numbering information, cell algebra information, cell cryopreservation lot information, cryopreservation date information and operator information.

7. A method of using a live cell aerosol inhalation device according to claim 6 wherein the sampling test comprises but is not limited to a microbiological test item, an endotoxin test item.

8. The method of claim 7, wherein the freezing density in the freezing step of living cells is in the range of 0.5 to 2 x 10 ⁸ /ml。

9. The method of claim 8, wherein the cell atomizing agent is prepared by mixing physiological saline, human serum albumin and cells.

10. The living cell atomizing inhalation device and the use method thereof according to claim 9 are characterized by comprising a shell (2), wherein an installation component (1) is arranged at the bottom of the shell (2), one side of the shell (2) is provided with a communication part (3) and a transfer part (4), the communication part (3) is positioned right above the transfer part (4), the top of the communication part (3) is provided with an atomizing part (5), a driving part (7), an input part (8) and an output part (9) are arranged in the shell (2), the output part (9) is positioned at one side close to the transfer part (4), and the driving part (7) is positioned between the input part (8) and the output part (9).