CN117660323A - Stem cell culture solution freeze-drying process - Google Patents
Stem cell culture solution freeze-drying process Download PDFInfo
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- CN117660323A CN117660323A CN202311705542.4A CN202311705542A CN117660323A CN 117660323 A CN117660323 A CN 117660323A CN 202311705542 A CN202311705542 A CN 202311705542A CN 117660323 A CN117660323 A CN 117660323A
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- drying
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- lyophilization
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- 238000004108 freeze drying Methods 0.000 title claims abstract description 165
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000008569 process Effects 0.000 title claims abstract description 26
- 238000004113 cell culture Methods 0.000 title claims description 10
- 239000007788 liquid Substances 0.000 claims abstract description 79
- 239000000843 powder Substances 0.000 claims abstract description 67
- 239000006228 supernatant Substances 0.000 claims abstract description 67
- 238000007790 scraping Methods 0.000 claims abstract description 49
- 230000005484 gravity Effects 0.000 claims abstract description 44
- 238000005507 spraying Methods 0.000 claims abstract description 42
- 238000007710 freezing Methods 0.000 claims abstract description 26
- 230000008014 freezing Effects 0.000 claims abstract description 26
- 210000003954 umbilical cord Anatomy 0.000 claims abstract description 26
- 230000007306 turnover Effects 0.000 claims abstract description 19
- 239000012930 cell culture fluid Substances 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 7
- 239000006143 cell culture medium Substances 0.000 claims abstract description 6
- 238000005119 centrifugation Methods 0.000 claims abstract description 6
- 238000012258 culturing Methods 0.000 claims abstract description 6
- 238000007781 pre-processing Methods 0.000 claims abstract description 4
- 238000010438 heat treatment Methods 0.000 claims description 32
- 238000007789 sealing Methods 0.000 claims description 23
- 238000005192 partition Methods 0.000 claims description 22
- 238000009413 insulation Methods 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 4
- 239000012535 impurity Substances 0.000 claims description 4
- 239000008176 lyophilized powder Substances 0.000 claims description 4
- 230000000149 penetrating effect Effects 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 20
- 239000007787 solid Substances 0.000 description 18
- 238000007599 discharging Methods 0.000 description 12
- 238000003892 spreading Methods 0.000 description 10
- 230000007480 spreading Effects 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 7
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 6
- 238000000859 sublimation Methods 0.000 description 5
- 230000008022 sublimation Effects 0.000 description 5
- 238000005092 sublimation method Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 4
- 238000005057 refrigeration Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000010009 beating Methods 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000005086 pumping Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
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- 239000003102 growth factor Substances 0.000 description 1
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- 238000001802 infusion Methods 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
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Landscapes
- Drying Of Solid Materials (AREA)
Abstract
The invention discloses a freeze-drying process of stem cell culture fluid, which comprises the following steps: step one, preprocessing an umbilical cord; culturing stem cells, namely transferring crushed umbilical cord tissues into a culture flask, adding a stem cell culture medium into the culture flask, and placing the culture flask into an incubator after the addition is completed; step three, centrifuging the culture solution, collecting the culture solution in a culture bottle, centrifuging the culture solution, taking supernatant after centrifugation, and filtering; uniformly spraying the supernatant, and uniformly spraying the supernatant into a linkage turnover type tubular freezing device by using a lifting type liquid spraying device; step five, quick freeze-drying of supernatant; and step six, collecting freeze-dried powder, namely scraping the freeze-dried powder on the inner wall of the linkage overturning type tubular freezing device by using a gravity scraping device and collecting the freeze-dried powder in a concentrated manner so as to obtain finished supernatant freeze-dried powder. The invention relates to a preparation process which is convenient for uniformly distributing and freeze-drying supernatant.
Description
Technical Field
The invention mainly relates to the technical field of cells, in particular to a freeze-drying process of stem cell culture solution.
Background
The supernatant fluid of human stem cell culture refers to liquid obtained by filtering supernatant fluid produced in the process of culturing mesenchymal stem cells from human body to take out the stem cells, the remained liquid consists of three parts of a culture medium, growth factors and exosomes, the liquid is rich in various nutrients, the liquid has nourishing effect on the stem cells, the supernatant fluid can enable the healthy living and proliferation of the stem cells, and the supernatant fluid of human stem cell culture is favorable for storage after freeze-drying.
According to a mesenchymal stem cell culture solution freeze-drying process provided by patent document with the application number of CN202210136504.0, the process comprises the following steps: s1: screening umbilical cord donors, and carrying out pathological detection; culturing umbilical cord mesenchymal stem cells by taking umbilical cord stem cells with good health, and collecting mesenchymal stem cell conditioned medium; s2: centrifuging the mesenchymal stem cell conditioned medium, filtering the suspension, adding a freeze-drying protective agent and glycerol, and uniformly stirring by ultrasonic waves; s3: putting the obtained liquid into a liquid adding port of a mesenchymal stem cell culture solution freeze-drying device; s4: and (5) waiting for the freeze-drying process of the organism to obtain freeze-dried powder. The invention has simple process operation steps and high production quantity, and can be used for beautifying and protecting skin; the device provided by the invention is simple to operate, low in cost and applicable to large-scale production.
The process steps in the patent are simple, the production quantity is high, but the uniform distribution and the freeze-drying of the supernatant and the automatic centralized collection of the freeze-dried materials are inconvenient.
Disclosure of Invention
The invention mainly provides a freeze-drying process of stem cell culture fluid, which is used for solving the technical problems in the background technology.
The technical scheme adopted for solving the technical problems is as follows:
a process for lyophilizing a stem cell culture fluid, comprising the steps of:
step one, preprocessing an umbilical cord, namely taking one umbilical cord, and crushing the umbilical cord after cleaning and impurity removal to obtain an umbilical cord tissue I;
culturing stem cells, namely transferring a unit amount of umbilical cord tissue I into a culture flask, adding a stem cell culture medium into the culture flask, and placing the culture flask into an incubator after the addition is completed;
step three, centrifuging the culture solution, collecting the culture solution in the culture bottle after stem cells in the culture bottle grow on the wall, centrifuging the culture solution, taking supernatant after centrifugation is completed, and filtering to obtain supernatant I;
uniformly spraying the supernatant, and uniformly spraying the supernatant into the linkage turnover type tubular freezing device by using a lifting type liquid spraying device;
step five, quick freeze-drying of the supernatant, wherein the linkage turnover type tubular freezing device quickly freeze-dries the supernatant sprayed on the inner wall;
and step six, collecting freeze-dried powder, namely scraping the freeze-dried powder on the inner wall of the linkage overturning type tubular freezing device by using a gravity scraping device and collecting the freeze-dried powder in a concentrated manner so as to obtain finished supernatant freeze-dried powder.
Preferably, the lifting liquid scattering device comprises a partition plate, a sealing cover arranged on the partition plate, a feeding hole penetrating through the partition plate, an L-shaped sealing plate arranged at the top of the feeding hole, a driving cylinder arranged on the partition plate and with an execution end connected with the outer wall of the L-shaped sealing plate, and uniformly distributed liquid scattering components arranged on the partition plate and positioned on one side of the L-shaped sealing plate away from the driving cylinder. In the preferred embodiment, the supernatant is conveniently uniformly sprayed in the linkage turnover type tubular freezing device through the lifting type liquid spraying device.
Preferably, the uniformly distributed liquid scattering component comprises a linear guide rail arranged on the partition board, a positioning column arranged at the execution end of the linear guide rail, a plurality of positioning frames arranged on one side of the L-shaped sealing plate and close to the positioning column from top to bottom in sequence, two rollers arranged on the positioning frames, a liquid spraying ring arranged below the bottommost positioning frame, a liquid conveying pipe with one end communicated with the liquid spraying ring and the other end passing through gaps between the two rollers on the plurality of positioning frames and extending to the outside of the sealing cover, and a miniature motor arranged on the outer wall of one positioning frame and used for driving one roller to rotate. In the preferred embodiment, lifting adjustment of the liquid scattering position is realized through uniformly distributing the liquid scattering components, and annular spraying of the liquid to be freeze-dried is realized at the same time, so that the liquid to be freeze-dried is uniformly attached to the inner wall of the freeze-drying tube.
Preferably, the linkage overturning type tubular refrigerating device comprises a freeze-drying box arranged at the bottom of the partition plate, a plurality of freeze-drying pipes which are arranged in the freeze-drying box and distributed in a linear array, a shaft rod symmetrically arranged on the outer wall of the freeze-drying pipes, a support plate arranged at the bottom of the inner wall of the freeze-drying box and rotationally connected with the shaft rod, a gear disc arranged at the end part of the shaft rod, a stepping motor arranged at the outer wall of the freeze-drying box and an execution end of the stepping motor extending to the inside of the freeze-drying box, a driving fluted disc arranged at the execution end of the stepping motor and meshed with one of the gear discs, a refrigerating part and a heating part arranged on the outer wall of the freeze-drying box, and gear discs arranged between two adjacent freeze-drying pipes are meshed with each other. In the preferred embodiment, efficient and uniform lyophilization of the supernatant is achieved by a linked flip-type tube freezer.
Preferably, the refrigerating component comprises refrigerating sheets symmetrically arranged on the outer wall of the freeze-drying tube. In the preferred embodiment, the cooling of the freeze-drying tube is facilitated by the refrigeration component to achieve rapid freeze-solidification of the supernatant on the inner wall of the freeze-drying tube.
Preferably, the heating component comprises heat insulation boards symmetrically arranged at the top and the bottom of the outer wall of the freeze-drying tube, heating plates arranged on the outer wall of the heat insulation board, C-shaped heat conduction rings sleeved on the outer wall of the freeze-drying tube and both ends of the C-shaped heat conduction rings are connected with the heating plates, first extension heat conduction plates arranged on the outer wall of the freeze-drying tube and one ends of the C-shaped heat conduction rings are connected with the first extension heat conduction plates, and second extension heat conduction plates arranged on the outer wall of the freeze-drying tube and one ends of the second extension heat conduction plates are connected with the heating plates. In the present preferred embodiment, heat provision during sublimation of solid water is facilitated by a heating element to increase the rate of sublimation.
Preferably, the outer wall of the freeze-drying box is provided with a vacuum suction device, the vacuum suction device comprises a plurality of negative suction pipes, one ends of the negative suction pipes are communicated with the outer wall of the freeze-drying box, the plurality of negative suction pipes are communicated with one another and far away from a communicating pipe at one end of the freeze-drying box, one ends of the negative suction pipes are communicated with a main pipe of the communicating pipe, and an electric control valve is arranged on the main pipe, and one of the negative suction pipes corresponds to one of the freeze-drying pipe positions. In the preferred embodiment, the vacuum suction device is used for facilitating vacuum suction in the freeze-drying box and facilitating suction and discharge of sublimated gaseous water.
Preferably, the gravity scraping device comprises extension pipes symmetrically arranged at the top and the bottom of the freeze-drying pipe, electromagnetic rings arranged in the extension pipes, a gravity scraping ring arranged on the inner wall of one of the electromagnetic rings, and a vibration shaking part arranged on the inner ring of the gravity scraping ring. In the preferred embodiment, the gravity scraping device is used for conveniently scraping the freeze-dried powder attached to the inner wall of the freeze-drying tube after freeze-drying.
Preferably, the vibration shaking component comprises a column box, wherein the outer wall of the column box is connected with the inner ring of the gravity scraping ring through a plurality of connecting rods, magnetic blocks symmetrically arranged at the top and the bottom of the inner wall of the column box, and a vibration beating ball which is positioned in the column box and magnetically attracted with the magnetic blocks. In the preferred embodiment, the vibration shake-out part is used for conveniently vibrating the gravity scraping ring when the gravity scraping ring is suddenly stopped, so that freeze-dried powder attached to the gravity scraping ring falls off.
Preferably, the freeze-drying powder collecting part is arranged at the bottom of the inner wall of the freeze-drying box, the freeze-drying powder collecting part comprises a plurality of powder collecting boxes arranged at the bottom of the inner wall of the freeze-drying box, an electric cylinder arranged at the end part of the powder collecting box and the execution end of the electric cylinder and the scraper blade arranged at the execution end of the electric cylinder, and a discharging box arranged at the bottom of the freeze-drying box and the top of the freeze-drying box and communicated with the powder collecting boxes, wherein one of the powder collecting boxes corresponds to one of the freeze-drying pipes in position. In the present preferred embodiment, concentrated discharge of the lyophilized supernatant powder is facilitated by the lyophilized powder collecting means.
Compared with the prior art, the invention has the beneficial effects that:
the process is convenient for uniformly distributing and freeze-drying the supernatant, and simultaneously is convenient for automatically and intensively collecting freeze-dried materials, so that the freeze-drying quality of the supernatant is improved, and the freeze-drying efficiency of the supernatant is accelerated;
according to the invention, the supernatant is conveniently and uniformly sprayed in the linkage turnover type tubular freezing device through the lifting type liquid spraying device, lifting adjustment of the liquid spraying position is realized through uniformly distributing liquid spraying components in the lifting type liquid spraying device, and meanwhile, annular spraying of the liquid to be freeze-dried is realized, so that the inner wall of the freeze-dried tube is uniformly attached to the liquid to be freeze-dried;
the efficient and uniform freeze-drying of the supernatant is realized through the linkage overturning type tubular freezing device, the temperature of the freeze-drying tube is conveniently reduced through the refrigerating component in the linkage overturning type tubular freezing device, so that the supernatant is rapidly frozen and solidified on the inner wall of the freeze-drying tube, the heat supply is conveniently carried out in the sublimation process of solid water through the heating component, the sublimation rate is increased, and the continuous freeze-drying production is realized through the freeze-drying mode of the overturning type freeze-drying tube;
the vacuum suction device is convenient for carrying out vacuum suction on the freeze-drying box, and is convenient for sucking and discharging the sublimated gaseous water;
the freeze-drying powder attached to the inner wall of the freeze-drying tube after freeze-drying is convenient to scrape through the gravity scraping device, the gravity scraping ring is convenient to shake through the vibration shaking part when the gravity scraping ring is suddenly stopped, so that the freeze-drying powder attached to the gravity scraping ring falls, and the supernatant powder after freeze-drying is convenient to concentrate and discharge through the freeze-drying powder collecting part.
The invention will be explained in detail below with reference to the drawings and specific embodiments.
Drawings
FIG. 1 is an overall process flow diagram of the present invention;
FIG. 2 is an isometric view of the overall structure of the device of the present invention;
FIG. 3 is an exploded view of the overall structure of the device of the present invention;
FIG. 4 is an exploded view of the structure of the lifting type liquid spreading device of the present invention;
FIG. 5 is an exploded view of the structure of the linked and inverted tube refrigeration device of the present invention;
FIG. 6 is a top view of the structure of the linked and inverted tube refrigeration device of the present invention;
FIG. 7 is a cross-sectional view of the overall structure of the device of the present invention;
fig. 8 is an enlarged view of the structure at a of the present invention.
Description of the drawings: 10. a lifting type liquid spreading device; 11. a partition plate; 12. a cover; 13. a feed hole; 14. an L-shaped sealing plate; 15. a drive cylinder; 16. uniformly distributing liquid scattering components; 161. a linear guide rail; 162. positioning columns; 163. a positioning frame; 164. a roller; 165. a liquid spraying ring; 166. an infusion tube; 167. a micro motor; 20. a linkage turnover type tubular refrigerating device; 21. a lyophilization cassette; 22. freeze-drying the tube; 23. a shaft lever; 24. a support plate; 25. a gear plate; 26. a stepping motor; 27. driving the fluted disc; 28. a refrigerating part; 281. a cooling sheet; 29. a heating member; 291. a heat insulating plate; 292. a heating sheet; 293. a C-shaped heat conducting ring; 294. a first extended heat transfer plate; 295. a second extended heat transfer plate; 30. a gravity scraping device; 31. extending the tube; 32. an electromagnetic ring; 33. a gravity scraping ring; 34. a vibrating shaking component; 341. a column box; 342. a magnetic block; 343. a beating ball; 35. a lyophilized powder collection member; 351. a powder collection box; 352. an electric cylinder; 353. a scraper; 354. a discharge box; 40. a vacuum suction device; 41. a negative suction pipe; 42. a communicating pipe; 43. a header pipe; 44. an electrically controlled valve.
Detailed Description
In order that the invention may be more fully understood, a more particular description of the invention will be rendered by reference to the appended drawings, in which several embodiments of the invention are illustrated, but which may be embodied in different forms and are not limited to the embodiments described herein, which are, on the contrary, provided to provide a more thorough and complete disclosure of the invention.
It will be understood that when an element is referred to as being "mounted" on another element, it can be directly on the other element or intervening elements may be present, and when an element is referred to as being "connected" to the other element, it may be directly connected to the other element or intervening elements may also be present, the terms "vertical", "horizontal", "left", "right" and the like are used herein for the purpose of illustration only.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the terms used herein in this description of the invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention, with the term "and/or" as used herein including any and all combinations of one or more of the associated listed items.
Referring to fig. 1, 2, 3, 4 and 7, in a preferred embodiment of the present invention, a process for lyophilizing stem cell culture fluid comprises the following steps: step one, preprocessing an umbilical cord, namely taking one umbilical cord, and crushing the umbilical cord after cleaning and impurity removal to obtain an umbilical cord tissue I; culturing stem cells, namely transferring a unit amount of umbilical cord tissue I into a culture flask, adding a stem cell culture medium into the culture flask, and placing the culture flask into an incubator after the addition is completed; step three, centrifuging the culture solution, collecting the culture solution in the culture bottle after stem cells in the culture bottle grow on the wall, centrifuging the culture solution, taking supernatant after centrifugation is completed, and filtering to obtain supernatant I; step four, uniformly spraying the supernatant, namely uniformly spraying the supernatant into a linkage turnover type tubular freezing device 20 by using a lifting type liquid spraying device 10; the lifting type liquid spreading device 10 comprises a partition plate 11, a sealing cover 12 arranged on the partition plate 11, a feeding hole 13 penetrating through the partition plate 11, an L-shaped sealing plate 14 arranged at the top of the feeding hole 13, a driving cylinder 15 arranged on the partition plate 11 and with an execution end connected with the outer wall of the L-shaped sealing plate 14, a uniformly distributed liquid spreading component 16 arranged on the partition plate 11 and positioned at one side of the L-shaped sealing plate 14 far away from the driving cylinder 15, wherein the uniformly distributed liquid spreading component 16 comprises a linear guide rail 161 arranged on the partition plate 11, a positioning column 162 arranged at the execution end of the linear guide rail 161, a plurality of positioning racks 163 sequentially arranged at the side, close to the L-shaped sealing plate 14, of the positioning column 162 from top to bottom, two rollers 164 arranged on the positioning racks 163, a liquid spraying ring 165 arranged below the bottommost positioning rack, a liquid spraying pipe 166 with one end communicated with the liquid spraying ring 165 and the other end sequentially passing through gaps between the two rollers 164 on the positioning racks 163 and extending to the outer side of the sealing cover 12, and a liquid spraying pipe 166 arranged on the outer wall of the positioning rack 163 and used for driving one of the micro rollers 167.
In this embodiment, when the stem cell supernatant is freeze-dried, umbilical cord is taken and crushed after being cleaned and decontaminated, a unit amount of crushed umbilical cord tissue is taken and transferred into a culture flask, a stem cell culture medium is added into the culture flask, the culture flask is placed into a culture box after the addition is completed, after stem cells in the culture flask grow on the wall, culture solution in the culture flask is collected, the culture solution is centrifuged, supernatant is taken after the centrifugation is completed and filtered, the filtered supernatant can be conveyed into a lifting type liquid scattering device 10 through a pressure conveying system such as a liquid pump, and the supernatant is uniformly sprayed into a linkage turnover type tubular freezing device 20 by the lifting type liquid scattering device 10;
further, when the lifting liquid spreading device 10 works, the driving cylinder 15 drives the L-shaped sealing plate 14 to move, so that the L-shaped sealing plate 14 leaves the feeding hole 13, the feeding hole 13 is in an open state, and at the moment, liquid spreading components 16 are uniformly distributed to spray the freeze-drying liquid on the inner walls of the plurality of freeze-drying pipes 22 one by one;
further, when the uniformly distributed liquid scattering component 16 works, the linear guide rail 161 drives the positioning column 162 to move until the liquid spraying ring 165 corresponds to one of the freeze-drying pipes 22, the micro motor 167 is started, the execution end of the micro motor 167 drives the roller 164 to rotate, the roller 164 drives the liquid conveying pipe 166 to move, the liquid spraying ring 165 is gradually lowered into the corresponding freeze-drying pipe 22 through the liquid conveying pipe 166, at the moment, the pressure conveying system can convey the supernatant from the liquid conveying pipe 166 to the liquid spraying ring 165, the supernatant can be sprayed out through the liquid spraying ring 165 and is attached to the inner wall of one freeze-drying pipe 22, after the liquid spraying on the inner wall of the other freeze-drying pipe 22 is completed, the micro motor 167 drives the liquid spraying ring 165 to reset, and at the moment, the supernatant can be sprayed on the inner wall of the other freeze-drying pipe 22.
Referring to fig. 1, 3, 5 and 6, in another preferred embodiment of the present invention, the step five, quick freeze-drying of the supernatant, the linkage overturning type tube type freezing device 20 quick-freezes the supernatant sprayed on the inner wall; the linkage turnover type tube refrigeration device 20 comprises a freeze-drying box 21 arranged at the bottom of the partition plate 11, a plurality of freeze-drying tubes 22 which are arranged in the freeze-drying box 21 and distributed in a linear array, a shaft rod 23 symmetrically arranged at the outer wall of the freeze-drying tube 22, a supporting plate 24 arranged at the bottom of the inner wall of the freeze-drying box 21 and rotationally connected with the shaft rod 23, a gear disc 25 arranged at the end part of the shaft rod 23, a stepping motor 26 arranged at the outer wall of the freeze-drying box 21 and extending to the inside of the freeze-drying box 21 at the execution end, a driving fluted disc 27 arranged at the execution end of the stepping motor 26 and meshed with one of the gear discs 25, a refrigerating part 28 arranged at the outer wall of the freeze-drying box 21 and a heating part 29, two adjacent refrigerating parts 25 meshed with each other, wherein the refrigerating part 28 comprises refrigerating sheets 281 symmetrically arranged at the outer wall of the freeze-drying tube 22, the heating part 29 comprises heating sheets 292 symmetrically arranged at the top and bottom of the outer wall of the freeze-drying tube 22, heating sheets 292 sleeved at the outer wall of the freeze-drying tube 22 and arranged at the outer wall of the heating sheets 291, heating sheets 293C-shaped heat conducting plates 293 are respectively connected with the heating sheets 21 and the heating sheets 42, the two heat conducting sheets are arranged at the outer wall of the vacuum conducting tube 22 and the vacuum conducting tube 42 are connected with one end of the vacuum conducting tube 42 and the vacuum conducting device, the vacuum conducting tube 42 is communicated with the vacuum conducting tube 42, and the vacuum conducting device is communicated with one end of the vacuum conducting tube 42 and the vacuum conducting device 42, and the vacuum conducting device is connected with the vacuum conducting tube 42, and the vacuum conducting tube and the vacuum conducting device 41, and the vacuum drying tube device 41, and the vacuum drying tube device. One of the negative pipettes 41 corresponds in position to one of the lyophilization tubes 22.
It should be noted that, in this embodiment, when the coordinated turnover type tube-type freezing device 20 works, after the inner wall of each freeze-drying tube 22 finishes spraying the supernatant, the refrigerating component 28 cools the freeze-drying tube 22, the supernatant is quickly frozen and solidified on the inner wall of the freeze-drying tube 22, the vacuum pumping device 40 pumps the inside of the freeze-drying box 21 so as to make the inside of the freeze-drying box 21 in a vacuum state, at this time, the solid water needs to absorb heat to sublimate into a gaseous state, the solid heating component 29 provides heat to increase the sublimation speed of the solid water, when the solid water is completely sublimated, the freeze-drying process is completed on the supernatant, the dry powder of the supernatant adheres to the inner wall of the freeze-drying tube 22, the execution end of the stepper motor 26 drives the plurality of freeze-drying tubes 22 to turn over one hundred eighty degrees simultaneously through the driving fluted disc 27 and the gear disc 25, the gravity scraping device 30 can automatically collect the freeze-dried powder after the turning is completed, and the freeze-drying powder collection operation can be repeatedly scattered;
further, when the refrigerating component 28 works, the refrigerating sheets 281 symmetrically distributed on two sides of the freeze-drying tube 22 directly cool the freeze-drying tube 22, and the heat-fixing conduction efficiency is high because the refrigerating sheets 281 directly contact the freeze-drying tube 22;
further, when the heating component 29 works, the heating plate 292 heats, heat is uniformly transferred to all the outer walls of the freeze-drying tube 22 through the C-shaped heat conducting ring 293, the first extension heat conducting plate 294 and the second extension heat conducting plate 295, and then the heat is transferred to solid water to be sublimated through the freeze-drying tube 22 so as to accelerate the sublimation process of the solid water, the heating plate 292 contacts with the outer walls of the freeze-drying tube 22 through the heat insulating plate 291, so that the heat of the heating plate 292 is prevented from being directly transferred to the freeze-drying tube 22, and the possibility of partial solid water liquefaction on the inner walls of the freeze-drying tube 22 is avoided;
further, when the vacuum suction device 40 works, the main pipe 43 can be connected with a vacuum system, the electric control valve 44 on the main pipe 43 is opened, the negative pressure system can suck the freeze-drying box 21 through the main pipe 43, the communicating pipe 42 and the negative suction pipe 41, and in the freeze-drying solid water sublimation process, the freeze-drying pipe 22 can rotate to be parallel to the negative suction pipe 41, so that the sublimated gaseous water can be directly sucked away, and the freeze-drying efficiency is increased.
Referring to fig. 1, 3, 5, 7 and 8, in another preferred embodiment of the present invention, step six, freeze-dried powder is collected, the gravity scraping device 30 scrapes and collects the freeze-dried powder on the inner wall of the linkage turnover type tube freezing device 20 in a concentrated manner, so as to obtain the finished product supernatant freeze-dried powder, the gravity scraping device 30 includes an extension tube 31 symmetrically disposed at the top and bottom of the freeze-drying tube 22, an electromagnetic ring 32 disposed in the extension tube 31, a gravity scraping ring 33 disposed on the inner wall of one of the electromagnetic rings 32, and a vibration scraping member 34 disposed on the inner wall of the gravity scraping ring 33, the vibration scraping member 34 includes a column box 341 with an outer wall connected to the inner ring of the gravity scraping ring 33 by a plurality of connecting rods, a magnetic block 342 symmetrically disposed at the top and bottom of the inner wall of the column box 341, and a vibration ball 343 magnetically absorbed by the magnetic block 342, a freeze-dried powder collecting member 35 disposed at the bottom of the inner wall of the column box 21, the powder collecting member 35 includes a plurality of vibration balls 351 disposed at the bottom of the inner wall of the column box 21 and a plurality of vibration scraping members 351 disposed at the bottom of the one of the electric cylinder 351 and extending to the one of the collection cylinder 351 disposed at the bottom of the collection cylinder 351, and the one of the collection cylinder 351 is disposed at the end of the collection cylinder 351.
In this embodiment, when the gravity scraping device 30 collects the supernatant freeze-dried powder, the electromagnetic ring 32 located at the top of the freeze-drying tube 22 is powered off, the electromagnetic ring 32 located at the bottom of the freeze-drying tube 22 is powered on, the gravity scraping ring 33 slides from top to bottom due to gravity on the inner wall of the freeze-drying tube 22, and is magnetically and rapidly stopped when reaching the powered electromagnetic ring 32 located at the bottom of the freeze-drying tube 22, the vibration shaking part 34 gives vibrating force to the gravity scraping ring 33 during rapid stopping, so that the freeze-dried powder attached to the gravity scraping ring 33 falls to the freeze-dried powder collecting part 35, the freeze-dried powder on the inner wall of the freeze-drying tube 22 can be scraped in the process that the gravity scraping ring 33 slides down, the freeze-dried powder directly falls to the freeze-dried powder collecting part 35, and the freeze-dried powder collecting part 35 can intensively discharge the freeze-dried powder;
further, when the vibration shaking component 34 works and the gravity scraping ring 33 suddenly stops, the inertia thrust of the vibration ball 343 is larger than the magnetic attraction of the magnetic block 342, namely, the vibration ball 343 falls down in the column box 341 and contacts the bottom of the inner wall of the column box 341, so that a vibration effect can be generated;
further, when the freeze-dried powder collecting part 35 works, the electric cylinder 352 is started, the execution end of the electric cylinder 352 drives the scraper 353 to move, the scraper 353 pushes freeze-dried powder in the powder collecting box 351 into the discharging box 354, the discharging hole of the discharging box 354 is opened, and the freeze-dried powder can be intensively discharged from the discharging hole of the discharging box 354.
The specific flow of the invention is as follows:
the electrical components in the invention are triggered to work by a PLC (programmable logic controller) model of which is '6 ES7315-2EH14-0AB 0'.
When stem cell supernatant is freeze-dried, taking one umbilical cord, carrying out crushing treatment on the umbilical cord after cleaning and impurity removal, taking a unit amount of crushed umbilical cord tissue, transferring the unit amount of crushed umbilical cord tissue into a culture bottle, adding a stem cell culture medium into the culture bottle, placing the culture bottle into an incubator after the addition, collecting culture solution in the culture bottle after the stem cells in the culture bottle grow on the wall, carrying out centrifugal processing on the culture solution, taking supernatant after the centrifugation is completed, carrying out filtering processing, conveying the filtered supernatant into a lifting type liquid scattering device 10 through a pressure conveying system such as a liquid pump, and uniformly spraying the supernatant into a linkage turnover type tubular freezing device 20 by utilizing the lifting type liquid scattering device 10;
when the lifting liquid spreading device 10 works, the driving cylinder 15 drives the L-shaped sealing plate 14 to move so as to enable the L-shaped sealing plate 14 to leave the feeding hole 13, the feeding hole 13 is in an open state, and liquid spreading components 16 are uniformly distributed at the moment so as to spray the uniform freeze-drying liquid on the inner walls of the plurality of freeze-drying pipes 22 one by one;
when the uniformly distributed liquid spreading component 16 works, the linear guide rail 161 drives the positioning column 162 to move until the liquid spraying ring 165 corresponds to the position of one of the freeze-drying pipes 22, the micro motor 167 is started, the execution end of the micro motor 167 drives the roller 164 to rotate, the roller 164 drives the liquid conveying pipe 166 to move, the liquid spraying ring 165 is gradually lowered into the corresponding freeze-drying pipe 22 through the liquid conveying pipe 166, at the moment, the pressure conveying system can convey supernatant from the liquid conveying pipe 166 to the liquid spraying ring 165, the supernatant can be sprayed out through the liquid spraying ring 165 and is attached to the inner wall of the freeze-drying pipe 22, after the liquid spraying on the inner wall of one freeze-drying pipe 22 is completed, the micro motor 167 drives the liquid spraying ring 165 to reset, and at the moment, the supernatant can be sprayed on the inner wall of the other freeze-drying pipe 22;
when the linkage turnover type tubular freezing device 20 works, after the inner wall of each freezing tube 22 is sprayed with the supernatant, the refrigerating component 28 cools the freezing tubes 22, the supernatant is quickly frozen and solidified on the inner wall of each freezing tube 22, the vacuum pumping device 40 pumps the inside of the freezing box 21 so as to enable the inside of the freezing box 21 to be in a vacuum state, at the moment, the solid water needs to absorb heat to sublimate into a gaseous state, the solid heating component 29 carries out heat supply so as to increase the sublimation speed of the solid water, when the solid water is completely sublimated, the supernatant is completely freeze-dried, the supernatant dry powder is adhered on the inner wall of each freezing tube 22, the execution end of the stepping motor 26 drives the plurality of freezing tubes 22 to turn over one hundred eighty degrees simultaneously through the driving fluted disc 27 and the gear disc 25, the gravity scraping device 30 can automatically collect freeze-dried powder after the turning over is completed, and the freeze-drying operation can be repeated after the freeze-drying powder collection is completed;
when the refrigerating component 28 works, the refrigerating sheets 281 symmetrically distributed on two sides of the freeze-drying pipe 22 directly cool the freeze-drying pipe 22, and the heat fixing conduction efficiency is high because the refrigerating sheets 281 directly contact the freeze-drying pipe 22;
when the heating component 29 works, the heating plate 292 heats, heat is uniformly transferred to all parts of the outer wall of the freeze-drying tube 22 through the C-shaped heat conducting ring 293, the first extension heat conducting plate 294 and the second extension heat conducting plate 295, and then the heat is transferred to solid water to be sublimated through the freeze-drying tube 22 so as to accelerate the sublimation process of the solid water, the heating plate 292 contacts with the outer wall of the freeze-drying tube 22 through the heat insulation plate 291, the heat of the heating plate 292 is prevented from being directly transferred to the freeze-drying tube 22, and the possibility of partial solid water liquefaction on the inner wall of the freeze-drying tube 22 is avoided;
when the vacuum suction device 40 works, the main pipe 43 can be connected with a vacuum system, the electric control valve 44 on the main pipe 43 is opened, the negative pressure system can suck the freeze-drying box 21 through the main pipe 43, the communicating pipe 42 and the negative suction pipe 41, and in the freeze-drying solid water sublimation process, the freeze-drying pipe 22 can rotate to be parallel to the negative suction pipe 41, so that sublimated gaseous water can be directly sucked away, and the freeze-drying efficiency is improved;
when the gravity scraping device 30 collects the supernatant freeze-dried powder, the electromagnetic ring 32 positioned at the top of the freeze-drying tube 22 is powered off, the electromagnetic ring 32 positioned at the bottom of the freeze-drying tube 22 is powered on, the gravity scraping ring 33 slides from top to bottom under the action of gravity on the inner wall of the freeze-drying tube 22, and is magnetically stopped when the gravity scraping ring 32 positioned at the bottom of the freeze-drying tube 22 is reached, the vibration shaking part 34 gives vibrating force to the gravity scraping ring 33 when the gravity scraping ring is suddenly stopped, so that the freeze-dried powder attached to the gravity scraping ring 33 falls to the freeze-dried powder collecting part 35, the freeze-dried powder on the inner wall of the freeze-drying tube 22 can be scraped in the process of sliding down the gravity scraping ring 33, the freeze-dried powder directly falls to the freeze-dried powder collecting part 35, and the freeze-dried powder collecting part 35 can intensively discharge the freeze-dried powder;
when the vibration shaking component 34 works and the gravity scraping ring 33 is suddenly stopped, the inertia thrust of the vibration ball 343 is larger than that of the magnetic block 342, namely, the vibration ball 343 falls down in the column box 341 and contacts the bottom of the inner wall of the column box 341, so that a vibration effect can be generated;
when the freeze-dried powder collecting part 35 works, the electric cylinder 352 is started, the execution end of the electric cylinder 352 drives the scraper 353 to move, the scraper 353 pushes freeze-dried powder in the powder collecting box 351 into the discharging box 354, the discharging hole of the discharging box 354 is opened, and the freeze-dried powder can be discharged from the discharging hole of the discharging box 354 in a concentrated mode.
While the invention has been described above with reference to the accompanying drawings, it will be apparent that the invention is not limited to the embodiments described above, but is intended to be within the scope of the invention, as long as such insubstantial modifications are made by the method concepts and technical solutions of the invention, or the concepts and technical solutions of the invention are applied directly to other occasions without any modifications.
Claims (10)
1. A process for lyophilizing a stem cell culture fluid, comprising the steps of:
step one, preprocessing an umbilical cord, namely taking one umbilical cord, and crushing the umbilical cord after cleaning and impurity removal to obtain an umbilical cord tissue I;
culturing stem cells, namely transferring a unit amount of umbilical cord tissue I into a culture flask, adding a stem cell culture medium into the culture flask, and placing the culture flask into an incubator after the addition is completed;
step three, centrifuging the culture solution, collecting the culture solution in the culture bottle after stem cells in the culture bottle grow on the wall, centrifuging the culture solution, taking supernatant after centrifugation is completed, and filtering to obtain supernatant I;
uniformly spraying the supernatant, namely uniformly spraying the supernatant into a linkage turnover type tubular refrigerating device (20) by using a lifting type liquid spraying device (10);
step five, quick freeze-drying of the supernatant, wherein a linkage turnover type tubular freezing device (20) quickly freeze-dries the supernatant sprayed on the inner wall;
and step six, collecting freeze-dried powder, namely scraping the freeze-dried powder on the inner wall of the linkage turnover type tubular freezing device (20) by using a gravity scraping device (30) and collecting the freeze-dried powder in a concentrated manner to obtain finished supernatant freeze-dried powder.
2. The stem cell culture liquid freeze-drying process according to claim 1, wherein the lifting liquid scattering device (10) comprises a partition plate (11), a sealing cover (12) arranged on the partition plate (11), a feeding hole (13) penetrating through the partition plate (11), an L-shaped sealing plate (14) arranged at the top of the feeding hole (13), a driving cylinder (15) arranged on the partition plate (11) and with an execution end connected with the outer wall of the L-shaped sealing plate (14), and uniformly distributed liquid scattering components (16) arranged on the partition plate (11) and positioned on one side of the L-shaped sealing plate (14) away from the driving cylinder (15).
3. The stem cell culture liquid freeze-drying process according to claim 2, wherein the uniformly-distributed liquid scattering component (16) comprises a linear guide rail (161) arranged on the partition board (11), a positioning column (162) arranged at the execution end of the linear guide rail (161), a plurality of positioning frames (163) arranged on one side of the positioning column (162) close to the L-shaped sealing plate (14) from top to bottom in sequence, two rollers (164) arranged on the positioning frames (163), a liquid spraying ring (165) arranged below the bottommost positioning frames (163), a liquid conveying pipe (166) with one end communicated with the liquid spraying ring (165) and the other end sequentially passing through gaps between the two rollers (164) on the positioning frames (163) and extending to the outside of the sealing cover (12), and a micro motor (167) arranged on the outer wall of one of the positioning frames (163) and used for driving one of the rollers (164) to rotate.
4. The process for lyophilizing stem cell culture fluid according to claim 2, wherein the linkage turnover type tube type freezing device (20) comprises a lyophilization box (21) arranged at the bottom of the partition plate (11), a plurality of lyophilization tubes (22) which are arranged in the lyophilization box (21) and distributed in a linear array, a shaft rod (23) symmetrically arranged at the outer wall of the lyophilization tube (22), a support plate (24) arranged at the bottom of the inner wall of the lyophilization box (21) and rotationally connected with the shaft rod (23), a gear disc (25) arranged at the end part of the shaft rod (23), a stepping motor (26) arranged at the outer wall of the lyophilization box (21) and with an execution end extending into the lyophilization box (21), a driving fluted disc (27) arranged at the execution end of the stepping motor (26) and meshed with one of the gear discs (25), and a refrigerating part (28) and a heating part (29) arranged at the outer wall of the lyophilization box (21), wherein the gear discs (25) between two adjacent lyophilization tubes (22) are meshed with each other.
5. The process according to claim 4, wherein the refrigerating unit (28) comprises refrigerating sheets (281) symmetrically arranged on the outer wall of the freeze-drying tube (22).
6. The process according to claim 4, wherein the heating unit (29) comprises a heat insulation plate (291) symmetrically arranged at the top and the bottom of the outer wall of the freeze-drying tube (22), a heating plate (292) arranged on the outer wall of the heat insulation plate (291), a C-shaped heat conduction ring (293) sleeved on the outer wall of the freeze-drying tube (22) and both ends of which are connected with the heating plate (292), a first extension heat conduction plate (294) arranged on the outer wall of the freeze-drying tube (22) and one end of which is connected with the C-shaped heat conduction ring (293), and a second extension heat conduction plate (295) arranged on the outer wall of the freeze-drying tube (22) and one end of which is connected with the heating plate (292).
7. The process for lyophilizing a stem cell culture fluid according to claim 4, wherein a vacuum suction device (40) is disposed on an outer wall of the lyophilization cassette (21), the vacuum suction device (40) comprises a plurality of negative suction pipes (41) with one end communicating with the outer wall of the lyophilization cassette (21), a communicating pipe (42) with one end communicating with the plurality of negative suction pipes (41) and one end far away from the lyophilization cassette (21), a manifold (43) with one end communicating with the communicating pipe (42), and an electric control valve (44) disposed on the manifold (43), wherein one of the negative suction pipes (41) corresponds to one of the lyophilization pipes (22).
8. The process according to claim 4, wherein the gravity scraping device (30) comprises extension pipes (31) symmetrically arranged at the top and the bottom of the freeze-drying pipe (22), electromagnetic rings (32) arranged in the extension pipes (31), gravity scraping rings (33) arranged on the inner wall of one of the electromagnetic rings (32), and vibration shaking parts (34) arranged on the inner ring of the gravity scraping rings (33).
9. The stem cell culture liquid freeze-drying process according to claim 8, wherein the vibration shaking component (34) comprises a column box (341) with an outer wall connected with an inner ring of the gravity scraping ring (33) through a plurality of connecting rods, magnetic blocks (342) symmetrically arranged at the top and the bottom of the inner wall of the column box (341), and a vibration ball (343) which is positioned in the column box (341) and magnetically attracted with the magnetic blocks (342).
10. The process for lyophilizing a stem cell culture fluid according to claim 4, wherein a lyophilized powder collecting member (35) is disposed at the bottom of the inner wall of the lyophilization cassette (21), the lyophilized powder collecting member (35) comprises a plurality of powder collecting cassettes (351) disposed at the bottom of the inner wall of the lyophilization cassette (21), an electric cylinder (352) disposed at the end of the powder collecting cassette (351) and having an execution end extending into the powder collecting cassette (351), a scraper (353) disposed at the execution end of the electric cylinder (352), and a discharge box (354) disposed at the bottom of the lyophilization cassette (21) and having a top portion communicating with the plurality of powder collecting cassettes (351), wherein one of the powder collecting cassettes (351) corresponds to one of the lyophilization tubes (22).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311705542.4A CN117660323A (en) | 2023-12-12 | 2023-12-12 | Stem cell culture solution freeze-drying process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311705542.4A CN117660323A (en) | 2023-12-12 | 2023-12-12 | Stem cell culture solution freeze-drying process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117660323A true CN117660323A (en) | 2024-03-08 |
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ID=90086181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311705542.4A Pending CN117660323A (en) | 2023-12-12 | 2023-12-12 | Stem cell culture solution freeze-drying process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117660323A (en) |
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2023
- 2023-12-12 CN CN202311705542.4A patent/CN117660323A/en active Pending
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