CN113621568B - Umbilical cord mesenchymal stem cell serum-free culture equipment and culture method thereof - Google Patents
Umbilical cord mesenchymal stem cell serum-free culture equipment and culture method thereof Download PDFInfo
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- CN113621568B CN113621568B CN202110830785.5A CN202110830785A CN113621568B CN 113621568 B CN113621568 B CN 113621568B CN 202110830785 A CN202110830785 A CN 202110830785A CN 113621568 B CN113621568 B CN 113621568B
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- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 27
- 238000012136 culture method Methods 0.000 title abstract description 12
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- 239000011521 glass Substances 0.000 claims abstract description 19
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- 241001330002 Bambuseae Species 0.000 description 6
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- 239000011425 bamboo Substances 0.000 description 6
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M23/08—Flask, bottle or test tube
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- C12M39/00—Means for cleaning the apparatus or avoiding unwanted deposits of microorganisms
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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Abstract
The invention discloses serum-free culture equipment and a culture method for umbilical cord mesenchymal stem cells, and relates to the field of cell culture. The outer surface fixed mounting of aseptic incubator has the glass board, fixedly connected with latex gloves on the lateral wall of glass board, and the top fixed mounting of aseptic incubator has the microscope, and the inner bottom fixed mounting of aseptic incubator has the centrifuge, and the inside of aseptic incubator is equipped with the bracing piece, and the lower surface activity joint of bracing piece has the fixed disk, and the inside activity grafting of fixed disk has the culture tube. The serum-free culture equipment and the culture method for the umbilical cord mesenchymal stem cells are convenient for separating, treating and culturing the umbilical cord mesenchymal stem cells in a sterile environment, and reduce external pollution.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to serum-free culture equipment and a culture method for umbilical cord mesenchymal stem cells.
Background
The umbilical cord mesenchymal stem cells refer to multifunctional stem cells existing in umbilical cord tissues of newborns, can be differentiated into a plurality of tissue cells, have higher differentiation potential, can differentiate towards a plurality of directions, have wide clinical application prospects in tissue engineering aspects such as bones, cartilages, muscles, tendons, ligaments, nerves, livers, endothelium and cardiac muscles, and the like, have reported that the stem cells are separated from human umbilical cords, have cell content and proliferation capacity superior to those of bone marrow stem cells, have immunogenicity lower than those of bone marrow stem cells, have the advantages of convenience in material acquisition, no ethical disputes and the like, and are therefore becoming more and more popular with researchers.
At present, in the process of culturing umbilical cord mesenchymal stem cells, the isolated culture is usually carried out by a collagenase and pancreatin digestion method, and the isolated culture is required to be placed in a culture medium for about 15-30 days during the culture, in the process, the growth condition of the cells needs to be closely concerned, and meanwhile, the culture medium needs to be prevented from being influenced by external environment and bacterial infection, so that the isolated culture is usually carried out in an ultra clean bench and then placed into a sterile culture bin for culturing, but in the process of culturing, numerous operations such as centrifugation, separation and culture medium replacement are required to be carried out, and therefore, the movement is required, so that the risk of pollution caused by the contact of the culture medium with the outside is increased, and therefore, the serum-free culture equipment and the culture method for umbilical cord mesenchymal stem cells are required to be provided for solving the problems.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention discloses umbilical cord mesenchymal stem cell serum-free culture equipment and a culture method thereof, which are used for solving the problems in the background art.
(II) technical scheme
In order to achieve the above purpose, the invention is realized by the following technical scheme: an umbilical cord mesenchymal stem cell serum-free culture device and a culture method thereof, comprising;
the sterile incubator comprises a sterile incubator body, wherein a glass plate is fixedly arranged on the outer surface of the sterile incubator body, latex gloves are fixedly connected to the side wall of the glass plate, a side-opening incubator door is movably hinged to the side surface of the sterile incubator body, a storage box is fixedly arranged on the inner side wall of the side-opening incubator door, a microscope is fixedly arranged at the top of the sterile incubator body, a centrifugal machine is fixedly arranged at the inner bottom of the sterile incubator body, a supporting rod is arranged in the sterile incubator body, a fixing disc is movably clamped on the lower surface of the supporting rod, and a culture test tube is movably inserted in the fixing disc;
the suction pump, the inside movable mounting that one end of suction pump extends to aseptic incubator has the suction tube, the other end of suction pump is equipped with a sewage section of thick bamboo, movable mounting has the pull shower nozzle on the inside wall of aseptic incubator, movable joint has the cleaning disc on the inside wall of aseptic incubator.
Preferably, a drawing hose is fixedly connected between the suction pipe and the suction pump, a water inlet pipe is fixedly connected to the back of the sterile incubator, the tail end of the drawing spray head is connected with the water inlet pipe through the drawing hose, a clamping groove is formed in the back of the sterile incubator, a clamping block is fixedly connected to the outer surface of the sewage cylinder, and the clamping block is movably clamped in the clamping groove.
Preferably, the inside wall of aseptic incubator is last to have seted up first spout, the top side fixedly connected with spacing axle of washing dish, spacing axle sliding connection is in the inside of first spout, the inside joint subassembly that is equipped with in bottom of washing dish, joint subassembly and the inside wall activity joint of aseptic incubator.
Preferably, the clamping assembly comprises a rotating head, a gear, a rack, a first spring, a fixed shaft and a clamping strip, wherein the rotating head is rotationally connected to the outer surface of the cleaning disc, the gear is fixedly connected to the end part of the rotating head, the rack is slidably connected to the inside of the cleaning disc, the gear is meshed with the rack, the first spring is movably clamped inside one end of the rack, the clamping strip is fixedly connected to the other end of the rack, one end of the fixed shaft is fixedly connected to the inside of the cleaning disc, and the other end of the fixed shaft abuts against the first spring.
Preferably, the second spout has been seted up on the lateral wall of aseptic incubator, the both sides of side opening the chamber door all swing joint has the connecting strip, the other end sliding connection of connecting strip is in the inside of second spout, fixedly connected with sealing rubber board on the inside wall of side opening the chamber door, storing box fixed connection is at sealing rubber board's surface, the top of side opening the chamber door rotates and is connected with fixing bolt, fixing bolt's tip threaded connection is at aseptic incubator's surface.
Preferably, the top activity of centrifuge articulates there is the rotation apron, the lower surface of rotation apron is equipped with circular separation blade, square groove has been seted up to the upper surface of centrifuge, the lower surface fixedly connected with of fixed disk and square strip of square groove looks adaptation.
Preferably, the upper surface of rotation apron has seted up the movable groove, the inside sliding connection in movable groove has the bolt, the bolt is pegged graft with the centrifuge activity, the lower surface fixedly connected with fixed sleeve of rotation apron, fixed sleeve's inside activity joint has the second spring, the top rotation of circular separation blade is connected with the inserted bar, the top activity of inserted bar is pegged graft in fixed sleeve's interior bottom.
Preferably, fixedly connected with fixing base on the inside wall of aseptic incubator, the one end activity of bracing piece articulates the lower surface at the fixing base, the observation hole has been seted up to the surface of bracing piece, the observation hole is just to the bottom of microscope, the other end rotation of bracing piece is connected with the cassette, the fixed slot has been seted up at the upper surface middle part of fixed disk, the slot has been seted up all around to the surface of fixed disk, the inside at the fixed slot is articulated to the bottom activity joint of cassette, cultivate the inside of test tube activity grafting at the slot.
Preferably, the upper surface activity grafting of cassette has the movable rod, the surface activity of movable rod has cup jointed the third spring, the bottom fixedly connected with fixed plate of movable rod, the bottom fixedly connected with triangle ejector pad of fixed plate, the bottom fixedly connected with fixed block of cassette, the equal movable joint in inside both sides of fixed block has spacing connecting block, two sets of the relative one side movable joint of spacing connecting block has the fourth spring, the bottom of triangle ejector pad offsets with the one side surface that two sets of spacing connecting blocks are on the back mutually.
Preferably, firstly, taking a fresh umbilical cord, fully flushing the fresh umbilical cord, removing residual blood in the umbilical cord, then cutting the umbilical cord, adding the cut umbilical cord into a culture tube, then adding collagenase IV with one thousandth concentration into the culture tube, standing for thirty minutes, waiting for digestion of cell tissues, then adding FBS with one tenth concentration into the culture tube to terminate digestion, filtering the mixture, and centrifuging the obtained cell filtrate;
adding LG-DMEM-containing culture solution into a culture tube, culturing for three and four days, replacing the culture solution, adding pancreatin with a final concentration of one thousandth into the culture tube, stirring for thirty minutes, waiting for further digestion of tissues, adding PBS with a concentration of one tenth, stopping digestion, filtering again, centrifuging the filtrate again, adding LG-DMEM-containing culture solution into the centrifuged cell filtrate, culturing for three and four days, filtering, replacing the culture solution, removing cells which are not adhered to the culture solution, filtering and replacing the culture solution every three days until the cells are fully fused and passaged;
finally, after the cells are fused and grown to seventy-eight, EDTA with the concentration of two thousandths is added into the culture tube again for digestion, at the moment, the culture tube is gently shaken to enable the digestion liquid to cover the whole cell surface, then three to five milliliters of digestion liquid is added after the cell culture liquid is filtered, and at the moment, the growth condition of umbilical cord mesenchymal stem cells is observed under an optical microscope.
The invention discloses umbilical cord mesenchymal stem cell serum-free culture equipment and a culture method thereof, which have the following beneficial effects:
1. according to the serum-free umbilical cord mesenchymal stem cell culture equipment and the culture method thereof, the glass plate is fixedly arranged on the outer surface of the sterile incubator, the latex glove is fixedly connected to the side wall of the glass plate, the microscope is fixedly arranged on the top of the sterile incubator, the centrifuge is fixedly arranged at the inner bottom of the sterile incubator, the support rod is arranged in the sterile incubator, the fixing disc is movably clamped on the lower surface of the support rod, the culture test tube is movably inserted in the fixing disc, and all operation steps in the umbilical cord mesenchymal stem cell treatment and culture process are conveniently carried out in the sterile incubator, so that the condition of contact pollution with the outside is reduced.
2. According to the serum-free umbilical cord mesenchymal stem cell culture equipment and the culture method thereof, the drawing spray head and the suction pipe are movably mounted on the inner side wall of the sterile incubator, meanwhile, the cleaning disc is movably clamped on the inner side wall of the sterile incubator through the clamping assembly, when the umbilical cord mesenchymal stem cell serum-free culture equipment is used, the cleaning disc is placed into the cleaning disc to be washed through the drawing spray head, sewage is pumped out through the suction pump and the suction pipe after washing, then the cleaning disc is restored and leaned against the inner side wall of the sterile incubator, the cleaning disc is conveniently washed, and pollution to the inside caused by washing is reduced.
3. This umbilical cord mesenchymal stem cell does not have serum culture apparatus and cultivates method thereof, through pressing down movable rod when using for two sets of triangle ejector pad will two sets of limit connecting blocks to middle part extrusion, take off the fixed disk from the surface of bracing piece and carry out the centrifugation this moment, then with fixed disk activity joint to the lower surface of bracing piece through rotating the fixed disk for fixed disk and cassette rotate at the tip of bracing piece, conveniently observe each group culture tube, be convenient for remove holistic culture tube, convenient operation.
Drawings
FIG. 1 is a schematic diagram of the overall structure of the present invention;
FIG. 2 is a schematic view of the back structure of the sterile incubator of the present invention;
FIG. 3 is a schematic view of the outer surface structure of a glass sheet according to the present invention;
FIG. 4 is a schematic view of the internal structure of the sterile incubator of the present invention;
FIG. 5 is an exploded view of the internal structure of the sterile incubator of the present invention;
FIG. 6 is an exploded view showing the internal structure of the cleaning disc of the present invention;
FIG. 7 is an enlarged view of the portion A of FIG. 6 in accordance with the present invention;
FIG. 8 is an exploded view of the outer surface structure of the rotary cover plate of the present invention;
FIG. 9 is an exploded view of the structure of the outer surface of the culture tube according to the present invention;
FIG. 10 is a schematic view of the outer surface structure of the holding pan of the present invention;
FIG. 11 is an exploded view of the internal structure of the cartridge of the present invention.
In the figure: 1. a sterile incubator; 2. a glass plate; 3. a side opening box door; 4. a microscope; 5. a suction pump; 6. a sewage drum; 7. a water inlet pipe; 8. a clamping groove; 9. a clamping block; 10. latex gloves; 11. cleaning a tray; 12. a second chute; 13. a connecting strip; 14. a first chute; 15. culturing a test tube; 16. a centrifuge; 17. a square groove; 18. drawing the spray head; 19. a suction tube; 20. sealing the rubber plate; 21. a storage box; 22. a fixing bolt; 23. a clamping assembly; 231. a rotating head; 232. a gear; 233. a rack; 234. a first spring; 235. a fixed shaft; 236. clamping strips; 24. rotating the cover plate; 25. a circular baffle; 26. a movable groove; 27. a plug pin; 28. a fixed sleeve; 29. a second spring; 30. a support rod; 31. a fixed plate; 32. square strips; 33. a fixing seat; 34. an observation hole; 35. a clamping seat; 36. a fixing groove; 37. a slot; 38. a fixed block; 39. a movable rod; 40. a third spring; 41. a fixing plate; 42. triangular pushing blocks; 43. a limit connecting block; 44. and a fourth spring.
Detailed Description
The embodiment of the invention discloses serum-free culture equipment and a culture method of umbilical cord mesenchymal stem cells, which are shown in figures 1-7 and comprise the following steps of;
the sterile incubator 1, the outer surface of the sterile incubator 1 is fixedly provided with a glass plate 2, the side wall of the glass plate 2 is fixedly connected with latex gloves 10, the side face of the sterile incubator 1 is movably hinged with a side-opening incubator door 3, the inner side wall of the side-opening incubator door 3 is fixedly provided with a storage box 21, the top of the sterile incubator 1 is fixedly provided with a microscope 4, the inner bottom of the sterile incubator 1 is fixedly provided with a centrifuge 16, the inside of the sterile incubator 1 is provided with a supporting rod 30, the lower surface of the supporting rod 30 is movably clamped with a fixed disc 31, the inside of the fixed disc 31 is movably inserted with a culture tube 15, and the inside of the top end of the culture tube 15 is provided with a sealing plug;
the suction pump 5, the inside movable mounting that one end of suction pump 5 extends to aseptic incubator 1 has suction tube 19, and the other end of suction pump 5 is equipped with sewage section of thick bamboo 6, and movable mounting has pull shower nozzle 18 on aseptic incubator 1's the inside wall, and movable joint has cleaning disc 11 on aseptic incubator 1's the inside wall.
Referring to fig. 2 and 5, fixedly connected with pull hose between suction tube 19 and the suction pump 5, the back fixedly connected with inlet tube 7 of aseptic incubator 1, draw-in groove 8 has been seted up to the end of pull shower nozzle 18 through pull hose with inlet tube 7, the surface fixedly connected with fixture block 9 of sewage section of thick bamboo 6, the inside at fixture block 8 of fixture block 9 activity joint, make pull shower nozzle 18 and suction tube 19 can remove in the inside of aseptic incubator 1 through setting up the pull hose, conveniently wash the umbilical cord, simultaneously after washing the end with the inside that sewage was taken out into sewage section of thick bamboo 6 through suction tube 19, carry out centralized processing.
Referring to fig. 4, 6 and 7, the first chute 14 is provided on the inner sidewall of the aseptic incubator 1, the top side surface of the cleaning disc 11 is fixedly connected with a limiting shaft, the limiting shaft is slidably connected in the first chute 14, the bottom end of the cleaning disc 11 is internally provided with a clamping assembly 23, the clamping assembly 23 is movably clamped with the inner sidewall of the aseptic incubator 1, the clamping assembly 23 comprises a rotating head 231, a gear 232, a rack 233, a first spring 234, a fixed shaft 235 and a clamping strip 236, the rotating head 231 is rotatably connected to the outer surface of the cleaning disc 11, the gear 232 is fixedly connected to the end of the rotating head 231, the rack 233 is slidably connected in the cleaning disc 11, the gear 232 is meshed with the rack 233, the first spring 234 is movably clamped in one end of the rack 233, the clamping strip 236 is fixedly connected to the other end of the rack 233, one end of the fixed shaft 235 is fixedly connected in the cleaning disc 11, the other end of the fixed shaft 235 is abutted against the first spring 234, and the gear 232 is rotated to rotate the rotating head 231, so that the gear 232 drives the two groups of racks 233 to move, and the two groups of clamping strips 236 are moved out of the inner sidewall of the aseptic incubator 1, and then the bottom end 11 is moved to the top side of the cleaning disc 11, which is close to the top surface of the centrifuge disc 11, and the top surface is pulled upwards by the centrifuge disc 11.
Referring to fig. 4-5, a second chute 12 is formed in the side wall of the sterile incubator 1, two sides of the side-opening box door 3 are movably hinged with a connecting strip 13, the other end of the connecting strip 13 is slidably connected in the second chute 12, a sealing rubber plate 20 is fixedly connected to the inner side wall of the side-opening box door 3, a storage box 21 is fixedly connected to the outer surface of the sealing rubber plate 20, a fixing bolt 22 is rotatably connected to the top of the side-opening box door 3, the end of the fixing bolt 22 is in threaded connection with the outer surface of the sterile incubator 1, the sealing performance of the sterile incubator 1 is improved by arranging the sealing rubber plate 20, and meanwhile, equipment required to be used in the processes of cell tissue treatment and culture is conveniently stored by arranging the storage box 21.
Referring to fig. 8, the top of the centrifuge 16 is movably hinged with a rotary cover plate 24, the lower surface of the rotary cover plate 24 is provided with a circular baffle 25, the upper surface of the centrifuge 16 is provided with a square groove 17, the lower surface of a fixed disc 31 is fixedly connected with a square strip 32 matched with the square groove 17, the upper surface of the rotary cover plate 24 is provided with a movable groove 26, the inside of the movable groove 26 is slidably connected with a plug 27, the plug 27 is movably inserted with the centrifuge 16, the lower surface of the rotary cover plate 24 is fixedly connected with a fixed sleeve 28, the inside of the fixed sleeve 28 is movably clamped with a second spring 29, the top end of the circular baffle 25 is rotatably connected with a plug rod, the top end of the plug rod is movably inserted at the inner bottom of the fixed sleeve 28, the fixed disc 31 and the culture tube 15 are synchronously placed into the centrifuge 16, then the rotary cover plate 24 is closed, and at this time, the circular baffle 25 abuts against the upper surface of the culture tube 15 under the action of the second spring 29, and the sealing plug at the top of the culture tube 15 is prevented from falling.
Referring to fig. 9-11, a fixing base 33 is fixedly connected to the inner side wall of the sterile incubator 1, one end of a supporting rod 30 is movably hinged to the lower surface of the fixing base 33, an observation hole 34 is formed in the outer surface of the supporting rod 30, the observation hole 34 is opposite to the bottom end of a microscope 4, the other end of the supporting rod 30 is rotatably connected with a clamping seat 35, a fixing groove 36 is formed in the middle of the upper surface of the fixing plate 31, slots 37 are formed around the outer surface of the fixing plate 31, the bottom end of the clamping seat 35 is movably clamped in the fixing groove 36, a culture tube 15 is movably inserted in the slots 37, a movable rod 39 is movably inserted in the upper surface of the clamping seat 35, a third spring 40 is movably sleeved on the outer surface of the movable rod 39, a fixing plate 41 is fixedly connected to the bottom end of the movable rod 39, a triangular push block 42 is fixedly connected to the bottom end of the fixing plate 41, the bottom fixedly connected with fixed block 38 of cassette 35, the inside both sides of fixed block 38 all activity joint has spacing connecting block 43, the relative one side activity joint of two sets of spacing connecting block 43 has fourth spring 44, the bottom of triangle ejector pad 42 offsets with the one side surface that two sets of spacing connecting blocks 43 are opposite, through pressing down movable rod 39 for third spring 40 is compressed, make the bottom of movable rod 39 drive triangle ejector pad 42 simultaneously and remove downwards, two sets of triangle ejector pads 42 extrude two sets of spacing connecting blocks 43 towards the middle part this moment, make fourth spring 44 compressed, make the bottom of spacing connecting block 43 remove to the inside of fixed block 38 simultaneously, take off fixed disk 31 from the surface of bracing piece 30 this moment.
Referring to fig. 1 to 11, a fresh cord is first taken and fully washed to remove residual blood in the cord, then the cord is sheared and added into a culture tube 15, collagenase IV with one thousandth concentration is then added into the cord, the cord is then left to stand for thirty minutes to wait for digestion of cell tissues, FBS with one tenth concentration is then added into the cord to terminate digestion, and then the cord is filtered, and the obtained cell filtrate is centrifuged;
adding LG-DMEM culture solution into a culture tube 15, culturing for three and four days, replacing the culture solution, adding pancreatin with a final concentration of one thousandth into the culture tube 15, stirring for thirty minutes, waiting for further digestion of tissues, adding PBS with a concentration of one tenth, stopping digestion, filtering again, centrifuging the filtrate again, adding LG-DMEM culture solution into the centrifuged cell filtrate, culturing for three and four days, filtering, replacing the culture solution, removing cells which are not adhered to the culture solution, filtering and replacing the culture solution every three days until the cells are fully fused and passaged;
finally, after the cells are grown to seventy-eight, EDTA with the concentration of two thousandths is added into the culture tube 15 again for digestion, at the moment, the culture tube 15 is gently shaken to enable the digestion liquid to cover the whole cell surface, then three to five milliliters of digestion liquid is added after the cell culture liquid is filtered, and at the moment, the growth condition of umbilical mesenchymal stem cells is observed under an optical microscope.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below by way of examples with reference to the accompanying drawings.
Embodiment 1,
The umbilical cord mesenchymal stem cell serum-free culture equipment comprises an aseptic culture box 1, wherein a glass plate 2 is fixedly arranged on the outer surface of the aseptic culture box 1, latex gloves 10 are fixedly connected to the side wall of the glass plate 2, a side opening box door 3 is movably hinged to the side surface of the aseptic culture box 1, a storage box 21 is fixedly arranged on the inner side wall of the side opening box door 3, a microscope 4 is fixedly arranged at the top of the aseptic culture box 1, a centrifugal machine 16 is fixedly arranged at the inner bottom of the aseptic culture box 1, a supporting rod 30 is arranged in the aseptic culture box 1, a fixed disc 31 is movably clamped on the lower surface of the supporting rod 30, and a culture tube 15 is movably inserted in the fixed disc 31;
still include suction pump 5, suction pipe 19 is movably installed in the inside movable mounting that one end of suction pump 5 extends to aseptic incubator 1, the other end of suction pump 5 is equipped with sewage section of thick bamboo 6, movable mounting pull shower nozzle 18 is gone up to aseptic incubator 1's inside wall, movable joint has cleaning disc 11 on aseptic incubator 1's the inside wall, fixedly connected with pull hose between suction pipe 19 and suction pump 5, the back fixedly connected with inlet tube 7 of aseptic incubator 1, the terminal of pull shower nozzle 18 is connected with inlet tube 7 through the pull hose, draw-in groove 8 has been seted up at aseptic incubator 1's back, the surface fixedly connected with fixture block 9 of sewage section of thick bamboo 6, fixture block 9 movable joint is in draw-in groove 8's inside, first spout 14 has been seted up on aseptic incubator 1's the inside wall, the top side fixedly connected with spacing axle of cleaning disc 11, the limiting shaft is slidably connected in the first chute 14, the clamping assembly 23 is arranged in the bottom end of the cleaning disc 11 and movably clamped with the inner side wall of the sterile incubator 1, the clamping assembly 23 comprises a rotating head 231, a gear 232, a rack 233, a first spring 234, a fixed shaft 235 and a clamping strip 236, the rotating head 231 is rotationally connected to the outer surface of the cleaning disc 11, the gear 232 is fixedly connected to the end of the rotating head 231, the rack 233 is slidably connected in the cleaning disc 11, the gear 232 is meshed with the rack 233, the first spring 234 is movably clamped in one end of the rack 233, the clamping strip 236 is fixedly connected to the other end of the rack 233, one end of the fixed shaft 235 is fixedly connected in the cleaning disc 11, and the other end of the fixed shaft 235 abuts against the first spring 234;
in the initial state, the cleaning disc 11 is movably clamped on the inner side wall of the sterile incubator 1, at this time, an operator inserts two hands into the latex glove 10 from the glass plate 2, at this time, the hands wearing the latex glove 10 operate the inside of the sterile incubator 1, the gears 232 drive the two sets of racks 233 to move by rotating the rotating heads 231, the two sets of clamping strips 236 are removed from the inner side wall of the sterile incubator 1, the first springs 234 are compressed, then the bottom end of the cleaning disc 11 is pulled towards the side close to the side opening door 3, the top end of the cleaning disc 11 slides upwards, finally the cleaning disc 11 is positioned on the upper surface of the centrifuge 16, as shown in fig. 4, the prepared umbilical cord is placed on the upper surface of the cleaning disc 11 through the side opening door 3, then placing the cutting and stirring equipment required for subsequent culture and the bottle required for the subsequent culture and containing collagenase solution and pancreatin solution into the storage box 21, closing the side opening box door 3, locking and fixing by the fixing bolt 22, flushing the umbilical cord by taking the drawing nozzle 18 off the inner side wall of the sterile incubator 1, removing residual blood in the umbilical cord, reserving the waste water generated by flushing in the interior of the cleaning disc 11, shearing the umbilical cord, taking the culture test tube 15 off the outer surface of the fixing disc 31, adding a small part of the umbilical cord tissue into all the culture test tubes 15, adding collagenase IV with a concentration of one thousandth into the interior, standing for thirty minutes, waiting for digestion of the cell tissue, withdrawing the waste water generated by cleaning the umbilical cord on the cleaning disc 11 outwards by the suction tube 19, rotating the cleaning disc 11 upwards, so that the cleaning disc 11 is movably clamped on the inner side wall of the sterile incubator 1 again through the clamping strip 236, and the preliminary treatment of stem cells is completed at this time.
Embodiment II,
The umbilical cord mesenchymal stem cell serum-free culture equipment comprises an aseptic culture box 1, wherein a glass plate 2 is fixedly arranged on the outer surface of the aseptic culture box 1, latex gloves 10 are fixedly connected to the side wall of the glass plate 2, a side opening box door 3 is movably hinged to the side surface of the aseptic culture box 1, a storage box 21 is fixedly arranged on the inner side wall of the side opening box door 3, a microscope 4 is fixedly arranged at the top of the aseptic culture box 1, a centrifugal machine 16 is fixedly arranged at the inner bottom of the aseptic culture box 1, a supporting rod 30 is arranged in the aseptic culture box 1, a fixed disc 31 is movably clamped on the lower surface of the supporting rod 30, and a culture tube 15 is movably inserted in the fixed disc 31;
the inner side wall of the sterile incubator 1 is fixedly connected with a fixed seat 33, one end of a supporting rod 30 is movably hinged to the lower surface of the fixed seat 33, an observation hole 34 is formed in the outer surface of the supporting rod 30, the observation hole 34 is opposite to the bottom end of a microscope 4, the other end of the supporting rod 30 is rotatably connected with a clamping seat 35, a fixed groove 36 is formed in the middle of the upper surface of the fixed disc 31, slots 37 are formed in the periphery of the outer surface of the fixed disc 31, the bottom end of the clamping seat 35 is movably clamped in the fixed groove 36, a culture tube 15 is movably inserted in the slots 37, a movable rod 39 is movably inserted in the upper surface of the clamping seat 35, a third spring 40 is movably sleeved on the outer surface of the movable rod 39, a fixed plate 41 is fixedly connected to the bottom end of the movable rod 39, a triangular push block 42 is fixedly connected to the bottom end of the fixed plate 41, a fixed block 38 is movably clamped on two sides of the inner part of the fixed block 38, one side opposite to the two groups of the limiting connecting blocks 43 is movably clamped with a fourth spring 44, and the bottom end of the triangular push block 42 is abutted to the outer surface of one side opposite to the two groups of the limiting connecting blocks 43;
after the operation of the first embodiment is completed, the digestion is terminated by adding the FBS solution into the culture tube 15, then sliding the latch 27 to one side inside the movable groove 26, then rotating the rotary cover plate 24 to open, then pressing the movable rod 39 downward so that the bottom ends of the movable rod 39 drive the triangular push blocks 42 to move downward, at this time, the two sets of triangular push blocks 42 press the two sets of stopper connecting blocks 43 toward the middle, so that the fourth springs 44 are compressed, and at the same time, the bottom ends of the stopper connecting blocks 43 move toward the inside of the fixed block 38, at this time, the fixed disk 31 is removed from the outer surface of the support rod 30.
Third embodiment,
The umbilical cord mesenchymal stem cell serum-free culture equipment comprises an aseptic culture box 1, wherein a glass plate 2 is fixedly arranged on the outer surface of the aseptic culture box 1, latex gloves 10 are fixedly connected to the side wall of the glass plate 2, a side opening box door 3 is movably hinged to the side surface of the aseptic culture box 1, a storage box 21 is fixedly arranged on the inner side wall of the side opening box door 3, a microscope 4 is fixedly arranged at the top of the aseptic culture box 1, a centrifugal machine 16 is fixedly arranged at the inner bottom of the aseptic culture box 1, a supporting rod 30 is arranged in the aseptic culture box 1, a fixed disc 31 is movably clamped on the lower surface of the supporting rod 30, and a culture tube 15 is movably inserted in the fixed disc 31;
the side wall of the sterile incubator 1 is provided with a second chute 12, both sides of the side opening box door 3 are movably hinged with a connecting strip 13, the other end of the connecting strip 13 is slidably connected in the second chute 12, the inner side wall of the side opening box door 3 is fixedly connected with a sealing rubber plate 20, a storage box 21 is fixedly connected to the outer surface of the sealing rubber plate 20, the top of the side opening box door 3 is rotatably connected with a fixing bolt 22, the end part of the fixing bolt 22 is in threaded connection with the outer surface of the sterile incubator 1, the top of the centrifuge 16 is movably hinged with a rotary cover plate 24, the lower surface of the rotary cover plate 24 is provided with a circular baffle 25, the upper surface of the centrifuge 16 is provided with a square groove 17, the lower surface of a fixed disc 31 is fixedly connected with a square strip 32 matched with the square groove 17, the upper surface of the rotary cover plate 24 is provided with a movable groove 26, the inside of the movable groove 26 is slidably connected with a plug 27, the plug 27 is movably spliced with the centrifuge 16, the lower surface of the rotary cover plate 24 is fixedly connected with a fixing sleeve 28, the inside of the fixing sleeve 28 is movably spliced with a second spring 29, the top of the circular baffle 25 is rotatably connected with a plug rod, and the top of the plug rod is movably spliced at the inner bottom of the fixing sleeve 28;
then the fixed disk 31 and the culture tube 15 are synchronously placed into the centrifuge 16, then the rotary cover plate 24 is closed, at the moment, the round baffle 25 is propped against the upper surface of the culture tube 15 under the action of the second spring 29, at the moment, the centrifuge 16 is started to centrifuge, after the centrifugation is finished, the culture tube 15 and the fixed disk 31 are taken out from the inside of the centrifuge 16, then the culture solution is movably clamped to the lower surface of the support rod 30 to culture for three and four days, then the culture solution is replaced, the obtained cell filtrate is centrifuged, the culture solution containing LG-DMEM is added into the culture tube 15 after three and four days, the culture solution is replaced, then the pancreatin with the final concentration of one thousandth is added into the culture tube 15, stirring is carried out for thirty minutes, the tissue is further digested, then the PBS with the concentration of one tenth is added to stop digestion, then the filtration is carried out again, the centrifuged filtrate is added into the cell filtrate after centrifugation, the culture solution is filtered after three and the culture solution is replaced, cells which are not adhered to the culture solution are removed, and the filtration and the replacement solution is carried out once every three days until the cells are fully passaged.
Finally, after the cells are fused and grown to seventy-eight, EDTA with the concentration of two thousandths is added into the culture tube 15 again for digestion, at this time, the culture tube 15 is gently shaken to enable the digestion liquid to cover the whole cell surface, then three to five milliliters of digestion liquid are added after the cell culture liquid is filtered, at this time, the growth condition of umbilical cord mesenchymal stem cells is observed under an optical microscope, the growth condition of the stem cells in the culture tube 15 is observed through the observation holes 34 by the microscope 4, and the fixing disc 31 and the clamping seat 35 can be rotated at the end part of the supporting rod 30 by rotating the fixing disc 31, so that the observation of each group of culture tubes 15 is facilitated.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (3)
1. A serum-free culture apparatus for umbilical cord mesenchymal stem cells, comprising;
the sterile incubator comprises a sterile incubator body (1), wherein a glass plate (2) is fixedly arranged on the outer surface of the sterile incubator body (1), latex gloves (10) are fixedly connected to the side wall of the glass plate (2), a side-opening incubator door (3) is movably hinged to the side surface of the sterile incubator body (1), a storage box (21) is fixedly arranged on the inner side wall of the side-opening incubator door (3), a microscope (4) is fixedly arranged at the top of the sterile incubator body (1), a centrifugal machine (16) is fixedly arranged at the inner bottom of the sterile incubator body (1), a supporting rod (30) is arranged in the sterile incubator body (1), a fixing disc (31) is movably clamped on the lower surface of the supporting rod (30), and a culture test tube (15) is movably inserted in the fixing disc (31).
The device comprises a suction pump (5), wherein one end of the suction pump (5) extends to the inside of the sterile incubator (1) and is movably provided with a suction pipe (19), the other end of the suction pump (5) is provided with a sewage cylinder (6), the inner side wall of the sterile incubator (1) is movably provided with a drawing spray head (18), and the inner side wall of the sterile incubator (1) is movably clamped with a cleaning disc (11); a drawing hose is fixedly connected between the suction pipe (19) and the suction pump (5), a water inlet pipe (7) is fixedly connected to the back of the sterile incubator (1), the tail end of the drawing spray head (18) is connected with the water inlet pipe (7) through the drawing hose, a clamping groove (8) is formed in the back of the sterile incubator (1), a clamping block (9) is fixedly connected to the outer surface of the sewage cylinder (6), and the clamping block (9) is movably clamped in the clamping groove (8); the inner side wall of the sterile incubator (1) is provided with a first chute (14), the side surface of the top end of the cleaning disc (11) is fixedly connected with a limiting shaft, the limiting shaft is slidably connected inside the first chute (14), the bottom end of the cleaning disc (11) is internally provided with a clamping assembly (23), and the clamping assembly (23) is movably clamped with the inner side wall of the sterile incubator (1); the clamping assembly (23) comprises a rotating head (231), a gear (232), a rack (233), a first spring (234), a fixed shaft (235) and a clamping strip (236), wherein the rotating head (231) is rotationally connected to the outer surface of the cleaning disc (11), the gear (232) is fixedly connected to the end part of the rotating head (231), the rack (233) is slidingly connected to the inside of the cleaning disc (11), the gear (232) is meshed with the rack (233), the first spring (234) is movably clamped inside one end of the rack (233), the clamping strip (236) is fixedly connected to the other end of the rack (233), one end of the fixed shaft (235) is fixedly connected to the inside of the cleaning disc (11), and the other end of the fixed shaft (235) is abutted to the first spring (234); the side wall of the sterile incubator (1) is provided with a second chute (12), two sides of the side-opening incubator door (3) are movably hinged with connecting strips (13), the other ends of the connecting strips (13) are slidably connected in the second chute (12), the inner side wall of the side-opening incubator door (3) is fixedly connected with a sealing rubber plate (20), the storage box (21) is fixedly connected to the outer surface of the sealing rubber plate (20), the top of the side-opening incubator door (3) is rotatably connected with a fixing bolt (22), and the end part of the fixing bolt (22) is in threaded connection with the outer surface of the sterile incubator (1); the top of the centrifugal machine (16) is movably hinged with a rotary cover plate (24), the lower surface of the rotary cover plate (24) is provided with a circular baffle (25), the upper surface of the centrifugal machine (16) is provided with a square groove (17), and the lower surface of the fixed disc (31) is fixedly connected with a square strip (32) matched with the square groove (17);
fixedly connected with fixing base (33) on the inside wall of aseptic incubator (1), the one end activity of bracing piece (30) articulates the lower surface at fixing base (33), observation hole (34) have been seted up to the surface of bracing piece (30), observation hole (34) are just right the bottom of microscope (4), the other end rotation of bracing piece (30) is connected with cassette (35), fixed slot (36) have been seted up at the upper surface middle part of fixed disk (31), slot (37) have been seted up all around to the surface of fixed disk (31), the inside at fixed slot (36) of the bottom activity joint of cassette (35), cultivate inside of test tube (15) activity grafting at slot (37).
2. The umbilical cord mesenchymal stem cell serum-free culture apparatus of claim 1, wherein: the upper surface of rotation apron (24) has seted up movable groove (26), the inside sliding connection of movable groove (26) has bolt (27), bolt (27) are pegged graft with centrifuge (16) activity, the lower fixed surface of rotation apron (24) is connected with fixed sleeve (28), the inside movable joint of fixed sleeve (28) has second spring (29), the top rotation of circular separation blade (25) is connected with the inserted bar, the top activity of inserted bar is pegged graft in the interior bottom of fixed sleeve (28).
3. The umbilical cord mesenchymal stem cell serum-free culture apparatus of claim 1, wherein: the upper surface activity of cassette (35) is pegged graft and is had movable rod (39), the surface activity of movable rod (39) has been cup jointed third spring (40), the bottom fixedly connected with fixed plate (41) of movable rod (39), the bottom fixedly connected with triangle ejector pad (42) of fixed plate (41), the bottom fixedly connected with fixed block (38) of cassette (35), the equal movable joint in inside both sides of fixed block (38) has spacing connecting block (43), two sets of one side activity joint that spacing connecting block (43) are relative has fourth spring (44), the bottom of triangle ejector pad (42) offsets with one side surface that two sets of spacing connecting blocks (43) are opposite.
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