CN113999813A - Primary culture method of umbilical cord mesenchymal stem cells - Google Patents
Primary culture method of umbilical cord mesenchymal stem cells Download PDFInfo
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- CN113999813A CN113999813A CN202111187717.8A CN202111187717A CN113999813A CN 113999813 A CN113999813 A CN 113999813A CN 202111187717 A CN202111187717 A CN 202111187717A CN 113999813 A CN113999813 A CN 113999813A
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- 210000003954 umbilical cord Anatomy 0.000 title claims abstract description 35
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 32
- 238000012136 culture method Methods 0.000 title claims abstract description 27
- 210000004027 cell Anatomy 0.000 claims abstract description 27
- 230000007246 mechanism Effects 0.000 claims abstract description 27
- 239000006143 cell culture medium Substances 0.000 claims abstract description 11
- 238000010009 beating Methods 0.000 claims abstract description 8
- 238000007664 blowing Methods 0.000 claims abstract description 8
- 230000001079 digestive effect Effects 0.000 claims abstract description 8
- 239000001963 growth medium Substances 0.000 claims abstract description 8
- GYDJEQRTZSCIOI-LJGSYFOKSA-N tranexamic acid Chemical compound NC[C@H]1CC[C@H](C(O)=O)CC1 GYDJEQRTZSCIOI-LJGSYFOKSA-N 0.000 claims abstract description 8
- 229960000401 tranexamic acid Drugs 0.000 claims abstract description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 230000007910 cell fusion Effects 0.000 claims abstract description 4
- 238000004140 cleaning Methods 0.000 claims abstract description 4
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims abstract description 3
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims abstract description 3
- 238000004891 communication Methods 0.000 claims description 21
- 239000011148 porous material Substances 0.000 claims description 18
- 239000000243 solution Substances 0.000 claims description 11
- 238000002347 injection Methods 0.000 claims description 10
- 239000007924 injection Substances 0.000 claims description 10
- 235000015110 jellies Nutrition 0.000 claims description 10
- 239000008274 jelly Substances 0.000 claims description 10
- 239000002609 medium Substances 0.000 claims description 6
- 238000010008 shearing Methods 0.000 claims description 6
- 230000001502 supplementing effect Effects 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 210000001367 artery Anatomy 0.000 claims description 3
- 210000002808 connective tissue Anatomy 0.000 claims description 3
- 210000003462 vein Anatomy 0.000 claims description 3
- 210000000130 stem cell Anatomy 0.000 abstract description 15
- 238000001816 cooling Methods 0.000 abstract description 5
- 239000000203 mixture Substances 0.000 abstract 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 12
- 238000001125 extrusion Methods 0.000 description 8
- 239000002245 particle Substances 0.000 description 8
- 235000019270 ammonium chloride Nutrition 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 4
- RQPZNWPYLFFXCP-UHFFFAOYSA-L barium dihydroxide Chemical compound [OH-].[OH-].[Ba+2] RQPZNWPYLFFXCP-UHFFFAOYSA-L 0.000 description 3
- 229910001863 barium hydroxide Inorganic materials 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 238000009413 insulation Methods 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/38—Caps; Covers; Plugs; Pouring means
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
- C12M41/20—Heat exchange systems, e.g. heat jackets or outer envelopes the heat transfer medium being a gas
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- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
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- C12M43/00—Combinations of bioreactors or fermenters with other apparatus
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses a primary culture method of umbilical cord mesenchymal stem cells, which is characterized by comprising the following steps: observing cells under a microscope, when the cell fusion degree reaches 85%, absorbing the original culture medium, adding a PBS solution for cleaning once, adding Xeno-Free cell digestive liquid to completely cover the bottom of a bottle, incubating at room temperature for 5-6 minutes or incubating at 37.3 ℃ for 3-4 minutes, adding an umbilical cord mesenchymal stem cell culture medium with the volume of 1.8 times that of the digestive liquid, lightly blowing and beating the cells which are not completely separated on the wall of the bottle by using a pipettor, uniformly blowing and beating to completely disperse the cells, adding the umbilical cord mesenchymal stem cell culture medium, tranexamic acid, G-CSF, EGF, DMEM/F12 culture medium: complementing, and placing the mixture in a box with 37.3 ℃ and 5% CO2 for constant temperature culture in transportation, relating to the technical field of stem cells. Through setting up the automatic invariable mechanism of temperature, in the long-distance transport of high temperature weather, to the inside temperature variation of box, the automatic inside cooling to the box, guaranteed the primary culture efficiency of culture solution in the long-distance transport of high temperature area.
Description
Technical Field
The invention relates to the technical field of stem cells, in particular to a primary culture method of umbilical cord mesenchymal stem cells.
Background
Briefly, stem cells are a class of cells with unlimited or immortal self-renewal capacity, capable of producing at least one type of highly differentiated progeny cells, whose definition has been continuously revised over the years and defined at different levels, and most biologists and physicians now consider stem cells to be a class of cells from embryonic, fetal or adult bodies with unlimited self-renewal and proliferative differentiation capacity under certain conditions, capable of producing daughter cells with the same phenotype as genotype and self-expression, capable of producing specialized cells that make up body tissues, organs, and capable of differentiating into progenitor cells. At present, after stem cells are cultured and placed in a culture solution, the stem cells are often transported to different research institutes for observation and recording of experimental research result reports, but at present, in long-distance transportation and transportation of the stem cells, the time of primary culture of the cells is long, and because part of the stem cell culture solution needs to be placed in a certain low-temperature environment for storage, the temperature in a storage box is gradually influenced by hot weather outside in the transportation process, so that the internal culture cannot be normally carried out, and the original research value of the stem cell culture solution is damaged.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a primary culture method of umbilical cord mesenchymal stem cells, which solves the problems that a stem cell culture solution cannot be stored in a low-temperature environment for a long time in transportation and the primary culture time is long.
In order to achieve the purpose, the invention is realized by the following technical scheme: observing cells under a microscope, sucking off an original culture medium when the cell fusion degree reaches 85%, adding a PBS (phosphate buffer solution) solution for cleaning once, adding Xeno-Free cell digestive juice to completely cover the bottom of a bottle, incubating at room temperature for 5-6 minutes or incubating at 37.3 ℃ for 3-4 minutes, adding an umbilical cord mesenchymal stem cell culture medium with the volume of 1.8 times that of the digestive juice, slightly blowing and beating cells which are not completely separated on the wall of the bottle by using a pipettor, slightly blowing and beating the cells uniformly to completely disperse the cells, and adding the umbilical cord mesenchymal stem cell culture medium:
tranexamic acid: 9600 mg/L;
I-CSF：22ng/L；
EGF：18ng/mL；
DMEM/F12 medium: supplementing fully;
or, umbilical cord mesenchymal stem cell culture medium:
tranexamic acid: 9700 mg/L;
J-CSF：24ng/L；
EGF：12ng/mL；
DMEM/F12 medium: supplement the essence.
Then, peeling off the umbilical cord adventitia, veins and arteries to obtain the Wharton's jelly, fully shearing the Wharton's jelly into pieces, and shearing the Wharton's jelly into pieces with the diameter of 1.3-2.6 mm3Culturing at constant temperature in a box body with the temperature of 37.3 ℃ and the content of 5% CO2, loading the box body into a transport vehicle, transporting to a specified place, and continuously culturing in the transportation process;
the two sides of the box body are in threaded connection with top covers, and an automatic temperature constant mechanism is arranged in the box body;
the automatic invariable mechanism of temperature includes heat conduction piece, intercommunicating pore A, intercommunicating pore B, slide, dead slot, box inner wall bottom is seted up flutedly, heat conduction piece outer wall and recess inner wall fixed connection, the inside and the inside intercommunication of recess of intercommunicating pore A, the inside and the inside intercommunication of intercommunicating pore B of intercommunicating pore A, intercommunicating pore B is seted up inside the box, the spout has been seted up to the box inside, slide outer wall and spout inner wall sliding connection, the dead slot is seted up inside the box, inside opening and shutting of being provided with of box shakes off mechanism and alarm mechanism.
Preferably, the opening and closing shaking-off mechanism comprises an arc-shaped plate, a weight, a push plate, an elastic plate, a small air groove, an air injection groove, a small elastic plate, a lifting block and a opening and closing plate, the arc-shaped plate is arranged inside the box body, the outer wall of the arc-shaped plate is connected with the inner wall of the arc-shaped groove in a sliding manner, the bottom of the arc-shaped plate is fixedly connected with the top of the weight, the outer wall of the push plate is connected with the inner wall of the arc-shaped groove in a sliding manner, the outer wall of the push plate is fixedly connected with one end of the elastic plate, the other end of the elastic plate is fixedly connected with one side of the arc-shaped groove, the small air groove is arranged inside the box body and is communicated with the inside of the arc-shaped groove, the air injection groove is arranged inside the box body and is communicated with the inside of the arc-shaped groove, the lifting groove is arranged inside the box body, one end of the small elastic plate is fixedly connected with one side of the inner wall of the lifting groove, the other end of the small elastic plate is fixedly connected with the bottom of the lifting block, the lifting block outer wall is connected with the lifting groove inner wall in a sliding mode, the top of the lifting block is in contact with the bottom of the opening plate, and one end of the opening plate is hinged to the interior of the box body.
Preferably, the alarm mechanism comprises a communication hole C, a communication hole D, a fixed plate, a rotating rod, a rotating plate, a free plate, a connecting rod, a rotating shaft, an extrusion plate and a thin air outlet groove, wherein the inside of the communication hole C is communicated with the inside of the communication hole D, a circular cavity is formed in the box body, the inside of the communication hole D is communicated with the inside of the circular cavity, one end of the fixed plate is fixedly connected with the outer wall of the rotating rod, the other end of the fixed plate is contacted and sealed with the bottom of the inner wall of the circular cavity, two ends of the rotating rod are rotatably connected with the front surface of the inner wall of the circular cavity and the back surface of the inner wall through bearings, one end of the rotating plate is fixedly connected with the outer wall of the rotating rod, the outer wall of the rotating rod is slidably connected with one end of the free plate, one end of the connecting rod is fixedly connected with the outer wall of the rotating shaft, the outer wall of the rotating shaft is slidably connected with the inner wall of the circular cavity, the outer wall is fixedly connected with one end of the extrusion plate, and a cavity is formed in the box body, the other end of the extrusion plate is connected with the inner wall of the cavity in a sliding mode and is sealed, and the thin air outlet groove is formed in the cavity.
Preferably, the inside check valve that is provided with of jet-propelled groove, dead slot one side intercommunication has the air outlet groove, and air outlet groove inside and box outside intercommunication are provided with the pressure valve to air outlet groove inside to receive certain pressure and just can open its pressure valve and ventilate.
Preferably, the inside intercommunication air duct of seting up of box, inside and the inside intercommunication of cavity of intercommunication air duct to make its inside air can communicate.
Preferably, the inside of the fine air outlet groove is provided with an alarm whistle, so that the alarm whistle can make a sound when being ventilated.
Preferably, free plate bottom fixedly connected with balancing weight, the inside circular port of having seted up of slide to make to increase its weight, communicate through the circular port simultaneously.
Preferably, the Wharton's jelly is sufficiently sheared into 1.5-2.3mm3。
The invention provides a primary culture method of umbilical cord mesenchymal stem cells. The method has the following beneficial effects:
(1) according to the umbilical cord mesenchymal stem cell primary culture method, the automatic constant temperature mechanism is arranged, so that when the temperature inside the box body is continuously raised by the external temperature and is driven to rise in long-distance transportation, the temperature inside the box body is automatically cooled, the temperature is kept within a certain range, and the storage time of the long-distance transportation of the culture solution is prolonged.
(2) This primary culture method of umbilical cord mesenchymal stem cell is through setting up the automatic invariable mechanism of temperature to make through cooling down to the air of giving vent to anger inslot, avoided then appearing the condition of the various adverse reactions that lead to the rapid change of stem cell culture fluid temperature that leads to the inside cooling of box fast, slow its inside temperature of maintenance, thereby promoted stem cell culture's stability.
(3) This primary culture method of umbilical cord mesenchymal stem cell is through setting up the mechanism that shakes off that opens and shuts to make and open and shut along with the rotation of open-close board and shake the fixed granule of ammonium chloride of dead slot inside, thereby promote its whereabouts efficiency, avoided the jam to pile up the condition appearance that can't fall in the dead slot inside, further promoted the speed of both mutual contacts, and fast to the inside real-time measure of cooling down of box, promote cooling efficiency.
(4) This primary culture method of umbilical cord mesenchymal stem cell, through setting up alarm mechanism, when the temperature lasts or risees suddenly, automatic production high decibel's alarm whistle carries out real-time alarm to the guard personnel in the transport vechicle and reminds, has avoided in the transportation box to collide with the damage because jolting and lead to the heat preservation effect loss, and inside receives a large amount of steam invasion and the whole chemical reaction of ammonium chloride solid particle and barium hydroxide solid particle to have ended the condition that the temperature still rises and can't solve and appears.
(5) According to the umbilical cord mesenchymal stem cell primary culture method, the umbilical cord mesenchymal stem cell is cultured in real time during transportation, and simultaneously added into a basic culture medium through G-CSF, tranexamic acid and EGF with corresponding doses, so that the culture time required by primary culture can be shortened, the effect is better than that of a conventional culture medium and a culture medium with a single factor, and the primary culture efficiency is improved.
Drawings
FIG. 1 is a schematic structural view of the present invention;
FIG. 2 is a front cross-sectional view of the present invention;
FIG. 3 is a front view of the automatic temperature constancy mechanism of the present invention;
FIG. 4 is an enlarged view taken at A of FIG. 3 according to the present invention;
FIG. 5 is an enlarged view of FIG. 4 at B according to the present invention;
FIG. 6 is an enlarged view of FIG. 4 at C.
In the figure: 1 top cover, 2 box body, 3 automatic temperature constant mechanism, 301 heat conducting block, 302 communication hole A, 303 communication hole B, 304 sliding plate, 305 empty slot, 4 open-close shaking mechanism, 401 arc plate, 402 weight, 403 push plate, 404 elastic plate, 405 small air slot, 406 air jet slot, 407 small elastic plate, 408 lifting block, 409 open-close plate, 5 alarm mechanism, 501 communication hole C, 502 communication hole D, 503 fixed plate, 504 rotating rod, 505 rotating plate, 506 free plate, 507 connecting rod, 508 rotating shaft, 509 extrusion plate, 510 fine air slot.
Detailed Description
Referring to fig. 1-6, the present invention provides a technical solution: a primary culture method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps: observing cells under a microscope, when the cell fusion degree reaches 85%, absorbing the original culture medium, adding a PBS solution for cleaning once, adding Xeno-Free cell digestive liquid to completely cover the bottom of a bottle, incubating at room temperature for 5-6 minutes or incubating at 37.3 ℃ for 3-4 minutes, adding an umbilical cord mesenchymal stem cell culture medium with the volume of 1.8 times that of the digestive liquid, slightly blowing and beating the cells which are not completely separated from the wall of the bottle by a pipettor, slightly blowing and beating the cells uniformly to completely disperse the cells, and adding the umbilical cord mesenchymal stem cell culture medium:
tranexamic acid: 9600 mg/L;
K-CSF：22ng/L；
EGF：18ng/mL；
DMEM/F12 medium: supplementing fully;
or, umbilical cord mesenchymal stem cell culture medium:
tranexamic acid: 9700 mg/L;
L-CSF：24ng/L；
EGF：12ng/mL；
DMEM/F12 medium: supplementing fully;
then, peeling off the umbilical cord adventitia, veins and arteries to obtain the Wharton's jelly, fully shearing the Wharton's jelly into pieces, and shearing the Wharton's jelly into pieces with the diameter of 1.3-2.6 mm3The cells were incubated in a 5% CO2 tank 2 at 37.3 ℃ and the tank 2 was loaded into a transport vehicle for transportation to a designated place and the cells were continuously incubated during transportation.
The two sides of the box body 2 are in threaded connection with a top cover 1, and an automatic temperature constant mechanism 3 is arranged in the box body 2;
automatic invariable mechanism 3 of temperature includes heat conduction piece 301, intercommunicating pore A302, intercommunicating pore B303, slide 304, dead slot 305, the recess has been seted up to 2 inner wall bottoms of box, heat conduction piece 301 outer wall and recess inner wall fixed connection, the inside and the inside intercommunication of recess of intercommunicating pore A302, the inside and the inside intercommunication of intercommunicating pore B303 of intercommunicating pore A302, intercommunicating pore B303 is seted up inside box 2, the inside spout of having seted up of box 2, slide 304 outer wall and spout inner wall sliding connection, dead slot 305 is seted up inside box 2, the inside mechanism 4 and the alarm mechanism 5 that shakes off that opens and shuts that is provided with of box 2.
The opening and closing shaking-off mechanism 4 comprises an arc-shaped plate 401, a heavy block 402, a push plate 403, an elastic plate 404, a small air groove 405 and an air injection groove 406, wherein a one-way valve is arranged inside the air injection groove 406, one side of a hollow groove 305 is communicated with an air outlet groove, the inside of the air outlet groove is communicated with the outside of the box body 2, a pressure valve is arranged inside the air outlet groove, the small elastic plate 407, a lifting block 408 and a split plate 409 are arranged inside the box body 2, the arc-shaped groove is arranged inside the box body 2, the outer wall of the arc-shaped plate 401 is fixedly connected with the top of the heavy block 402, the outer wall of the push plate 403 is slidably connected with the inner wall of the arc-shaped groove, the outer wall of the push plate 403 is fixedly connected with one end of the elastic plate 404, the other end of the elastic plate 404 is fixedly connected with one side of the arc-shaped groove, the small air groove 405 is arranged inside the box body 2, the inside of the small air groove 405 is communicated with the inside of the arc-shaped groove, the air injection groove 406 is arranged inside the box body 2, and the air injection groove 406 is communicated with the inside of the arc-shaped groove 406, the inside lift groove of having seted up of box 2, little elastic plate 407 one end and lift inslot wall one side fixed connection, little elastic plate 407 other end and 408 bottom fixed connection of elevator, elevator 408 outer wall and lift inslot wall sliding connection, elevator 408 top and the contact of division board 409 bottom, division board 409 one end and the inside articulated of box 2.
The alarm mechanism 5 comprises a communication hole C501, a communication hole D502, a fixed plate 503, a rotating rod 504, a rotating plate 505 and a free plate 506, wherein the bottom of the free plate 506 is fixedly connected with a balancing weight, a circular hole is formed in a sliding plate 304, a connecting rod 507, a rotating shaft 508, a squeezing plate 509 and a thin air outlet groove 510 are formed in the free plate 510, an alarm whistle is arranged in the thin air outlet groove 510, the interior of the communication hole C501 is communicated with the interior of the communication hole D502, a circular cavity is formed in a box body 2, the interior of the circular cavity is communicated with the interior of the circular cavity, one end of the fixed plate 503 is fixedly connected with the outer wall of the rotating rod 504, the other end of the fixed plate 503 is contacted and sealed with the bottom of the inner wall of the circular cavity, two ends of the rotating rod 504 are rotatably connected with the front surface of the inner wall of the circular cavity and the back surface of the inner wall through a bearing, one end of the rotating plate 505 is fixedly connected with the outer wall of the rotating rod 504, the outer wall of the rotating rod is slidably connected with one end of the free plate 506, one end of the connecting rod 507 is fixedly connected with the outer wall of the rotating shaft 508, and the outer wall of the rotating shaft 508 is slidably connected with the inner wall of the circular cavity, the outer wall of the rotating shaft 508 is fixedly connected with one end of the extrusion plate 509, a cavity is formed in the box body 2, the other end of the extrusion plate 509 is connected with the inner wall of the cavity in a sliding mode and is sealed, and the thin air outlet groove 510 is formed in the cavity.
When in use, cultured stem cells are placed in the box body 2, then the box body 2 is placed in a transport vehicle, when the vehicle starts to transport, because the external temperature is higher than the internal temperature of the box body 2, the internal temperature of the box body 2 is influenced by the external temperature along with the passage of long-distance transport time, the temperature begins to rise slowly, the temperature enters the groove at the bottom through the heat conduction block 301, so that the air temperature in the groove continuously rises, the volume expands to enter the communication hole B303 through the communication hole A302, then enters the sliding groove through the communication hole B303 and pushes the sliding plate 304 to descend, so that ammonium chloride solid particles contained in one side of the internal part of the hollow groove 305 enter the other side of the hollow groove 305 through the circular hole in the sliding plate 304 and are combined with barium hydroxide solid particles, so as to generate chemical reaction, a large amount of cold air is generated and enters the air outlet groove in the internal part of the hollow groove 305, hot air originally having the heat insulation effect in the hollow groove 305 is extruded and discharged through a pressure valve at one side of the air outlet groove, cold air enters the air outlet groove, so that the air originally having the heat insulation effect in the air outlet groove in the box body 2 is cooled, and is slowly conducted to the inside of the box body 2 to cool the stem cell culture solution, so that the temperature of the air is kept in a constant state, when the temperature in the box body 2 is gradually reduced to a proper temperature, the air in the groove is conducted by the temperature of the heat conduction block 301, so that the air is synchronously cooled to retract, the sliding plate 304 returns to the original position, the circular hole is not communicated with the inside of the hollow groove 305, channels at two sides of the hollow groove 305 are closed, meanwhile, bumping occurs in the running process of a vehicle, the arc-shaped plate 401 in the arc-shaped groove slides left and right in the arc-shaped groove by the inertia of the weight 402, so as to reciprocally push the pushing push plate 403, the push plate 403 pushes the air inside the arc-shaped groove to enter the lifting groove through the air injection groove 406, and pushes the lifting block 408 to push the opening-closing plate 409 upwards, the opening-closing plate 409 is rotated and opened and closed under the condition that one end is hinged after being pushed upwards, then the reciprocating extrusion air of the push plate 403 enters the air injection groove 406 along with the continuous left and right impact of the arc-shaped plate 401, then the lifting block 408 and the opening-closing plate 409 are pushed, and simultaneously, because the rebound force of the elastic plate 404 is greater than the gravity of the lifting block 408 and the opening-closing plate 409, when the push plate 403 pushes the air to extrude, the push plate 403 on the other side cannot be pushed to slide, and only the lifting block 408 and the opening-closing plate 409 can be pushed to be opened and closed, so that the ammonium chloride fixed particles inside the hollow groove 305 can be vibrated along with the rotation and opening-closing of the opening plate 409, thereby improving the falling efficiency, avoiding the occurrence of the situation that the ammonium chloride fixed particles cannot fall and accumulate inside the hollow groove 305, meanwhile, when an accident happens, when the sliding plate 304 is continuously pushed, the temperature of the air in the groove is continuously raised, when the sliding plate 304 is continuously pushed, the air can enter the inside of the communicating hole D502 through the communicating hole C501, then enter the circular cavity, and push the rotating plate 505 to rotate on the outer wall of the rotating rod 504, the rotating plate 505 can push the free plate 506, after the free plate 506 is pushed to be in a vertical state, the free plate 506 is continuously pushed, the free plate 506 is rapidly inverted to the other side and smashes towards the connecting rod 507 due to the heavy weight of the balancing weight, the connecting rod 507 rapidly drives the rotating shaft 508 to rotate, and drives the extrusion plate 509 to extrude the air in the cavity into the thin air outlet groove 510, and then the air passes through the alarm whistle in the thin air outlet groove 510, so that high-decibel alarm whistle is generated, real-time alarm reminding is carried out on guard personnel in the transport vehicle, and the loss of the heat preservation effect caused by bumping and collision damage of the box body 2 in the transport process is avoided, the inner part is invaded by a large amount of hot gas, and the situation that the temperature is still raised after the chemical reaction of the ammonium chloride solid particles and the barium hydroxide solid particles is finished can not be solved.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.
Claims (8)
1. A primary culture method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps: observing cells under a microscope, when the cell fusion degree reaches 85%, absorbing the original culture medium, adding a PBS solution for cleaning once, adding Xeno-Free cell digestive liquid to completely cover the bottom of a bottle, incubating at room temperature for 5-6 minutes or incubating at 37.3 ℃ for 3-4 minutes, adding an umbilical cord mesenchymal stem cell culture medium with the volume of 1.8 times that of the digestive liquid, slightly blowing and beating the cells which are not completely separated from the wall of the bottle by a pipettor, slightly blowing and beating the cells uniformly to completely disperse the cells, and adding the umbilical cord mesenchymal stem cell culture medium:
tranexamic acid: 9600 mg/L;
G-CSF：22ng/L；
EGF：18ng/mL；
DMEM/F12 medium: supplementing fully;
or, umbilical cord mesenchymal stem cell culture medium:
tranexamic acid: 9700 mg/L;
H-CSF：24ng/L；
EGF：12ng/mL；
DMEM/F12 medium: supplementing fully;
then, peeling off the umbilical cord adventitia, veins and arteries to obtain the Wharton's jelly, fully shearing the Wharton's jelly into pieces, and shearing the Wharton's jelly into pieces with the diameter of 1.3-2.6 mm3Culturing at constant temperature in a box (2) of 5% CO2 at 37.3 deg.C, transporting the box (2) to a designated place in a transport vehicle, and continuously culturing during transportation.
2. The umbilical cord mesenchymal stem cell primary culture method of claim 1, wherein the primary culture method comprises: the two sides of the box body (2) are in threaded connection with a top cover (1), and an automatic temperature constant mechanism (3) is arranged in the box body (2);
automatic invariable mechanism of temperature (3) is including heat conduction piece (301), intercommunicating pore A (302), intercommunicating pore B (303), slide (304), dead slot (305), the recess is seted up to box (2) inner wall bottom, heat conduction piece (301) outer wall and recess inner wall fixed connection, intercommunicating pore A (302) inside and recess inside intercommunication, intercommunicating pore A (302) inside and intercommunicating pore B (303) inside intercommunication, intercommunicating pore B (303) are seted up inside box (2), the inside spout of having seted up of box (2), slide (304) outer wall and spout inner wall sliding connection, dead slot (305) are seted up inside box (2), box (2) inside is provided with to open and shut and shake off mechanism (4) and alarm mechanism (5).
3. The umbilical cord mesenchymal stem cell primary culture method of claim 2, wherein the primary culture method comprises: the opening and closing shaking-off mechanism (4) comprises an arc-shaped plate (401), a weight (402), a push plate (403), an elastic plate (404), a small air groove (405), an air injection groove (406), a small elastic plate (407), a lifting block (408) and an opening plate (409), wherein the arc-shaped groove is formed in the box body (2), the outer wall of the arc-shaped plate (401) is in sliding connection with the inner wall of the arc-shaped groove, the bottom of the arc-shaped plate (401) is fixedly connected with the top of the weight (402), the outer wall of the push plate (403) is in sliding connection with the inner wall of the arc-shaped groove, the outer wall of the push plate (403) is fixedly connected with one end of the elastic plate (404), the other end of the elastic plate (404) is fixedly connected with one side of the arc-shaped groove, the small air groove (405) is formed in the box body (2), the inside of the small air groove (405) is communicated with the inside of the arc-shaped groove, and the air injection groove (406) is formed in the box body (2), jet-propelled groove (406) inside and the inside intercommunication of arc wall, the lift groove has been seted up to box (2) inside, little elastic plate (407) one end and lift inslot wall one side fixed connection, little elastic plate (407) other end and elevator (408) bottom fixed connection, elevator (408) outer wall and lift inslot wall sliding connection, elevator (408) top and open-close board (409) bottom contact, open-close board (409) one end and box (2) inside are articulated.
4. The umbilical cord mesenchymal stem cell primary culture method of claim 3, wherein the primary culture method comprises: the alarm mechanism (5) comprises a communication hole C (501), a communication hole D (502), a fixing plate (503), a rotating rod (504), a rotating plate (505), a free plate (506), a connecting rod (507), a rotating shaft (508), a squeezing plate (509) and a fine air outlet groove (510), wherein the inside of the communication hole C (501) is communicated with the inside of the communication hole D (502), a circular cavity is formed in the box body (2), the inside of the communication hole D (502) is communicated with the inside of the circular cavity, one end of the fixing plate (503) is fixedly connected with the outer wall of the rotating rod (504), the other end of the fixing plate (503) is contacted and sealed with the bottom of the inner wall of the circular cavity, two ends of the rotating rod (504) are rotatably connected with the front surface and the back surface of the inner wall of the circular cavity through bearings, one end of the rotating plate (505) is fixedly connected with the outer wall of the rotating rod (504), and the outer wall of the rotating rod (504) is slidably connected with one end of the free plate (506), connecting rod (507) one end and pivot (508) outer wall fixed connection, pivot (508) outer wall and circular cavity inner wall sliding connection, pivot (508) outer wall and stripper plate (509) one end fixed connection, the cavity has been seted up to box (2) inside, the stripper plate (509) other end and cavity inner wall sliding connection and sealed, thin gas groove (510) are seted up inside the cavity.
5. The umbilical cord mesenchymal stem cell primary culture method of claim 4, wherein the primary culture method comprises: the inside check valve that is provided with of jet-propelled groove (406), dead slot (305) one side intercommunication has the groove of giving vent to anger, gives vent to anger inside of groove and box (2) outside intercommunication, gives vent to anger the inside pressure valve that is provided with of groove.
6. The umbilical cord mesenchymal stem cell primary culture method of claim 5, wherein the primary culture method comprises: a communicating air groove is formed in the box body (2), and the inside of the communicating air groove is communicated with the inside of the cavity.
7. The umbilical cord mesenchymal stem cell primary culture method of claim 6, wherein the primary culture method comprises: an alarm whistle is arranged in the thin air outlet groove (510).
8. The umbilical cord mesenchymal stem cell primary culture method of claim 7, wherein the primary culture method comprises: the bottom of the free plate (506) is fixedly connected with a balancing weight, and a circular hole is formed in the sliding plate (304).
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110032694A (en) * | 2009-09-22 | 2011-03-30 | 일렉트론 바이오 (주) | Microorganism analysis apparatus and its analysis method |
| US20160089673A1 (en) * | 2014-09-25 | 2016-03-31 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Hybrid linear actuator controlled hydraulic cell stretching |
| CN108823160A (en) * | 2018-07-25 | 2018-11-16 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells primary culture medium and its primary culture method |
| CN109055214A (en) * | 2018-08-20 | 2018-12-21 | 唐燕 | A kind of cell incubator of the transfer easy to carry to genetic engineering |
| CN211079133U (en) * | 2019-11-12 | 2020-07-24 | 湖北富之源生物科技有限公司 | Biological incubator |
| CN111979107A (en) * | 2020-09-02 | 2020-11-24 | 佳木斯大学 | Bacteria culture experimental device of aseptic constant temperature and humidity |
| CN112266873A (en) * | 2020-10-19 | 2021-01-26 | 广州丽都生物科技有限公司 | Cell culture box for dynamically adjusting carbon dioxide amount by using acid-base change |
| CN112280658A (en) * | 2020-10-28 | 2021-01-29 | 张金兰 | Multifunctional box for large-scale culture of microorganisms |
| CN112980685A (en) * | 2021-04-20 | 2021-06-18 | 广州赛太特生物医学科技有限公司 | A biological cell breeding device for cultivateing, survey |
| CN112980687A (en) * | 2021-05-19 | 2021-06-18 | 广州维柏生物科技有限公司 | Constant temperature and humidity cell culture box |
| CN113136335A (en) * | 2021-04-27 | 2021-07-20 | 湖北明德健康科技有限公司 | Stem cell culture device and culture method thereof |
-
2021
- 2021-10-12 CN CN202111187717.8A patent/CN113999813A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110032694A (en) * | 2009-09-22 | 2011-03-30 | 일렉트론 바이오 (주) | Microorganism analysis apparatus and its analysis method |
| US20160089673A1 (en) * | 2014-09-25 | 2016-03-31 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Hybrid linear actuator controlled hydraulic cell stretching |
| CN108823160A (en) * | 2018-07-25 | 2018-11-16 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells primary culture medium and its primary culture method |
| CN109055214A (en) * | 2018-08-20 | 2018-12-21 | 唐燕 | A kind of cell incubator of the transfer easy to carry to genetic engineering |
| CN211079133U (en) * | 2019-11-12 | 2020-07-24 | 湖北富之源生物科技有限公司 | Biological incubator |
| CN111979107A (en) * | 2020-09-02 | 2020-11-24 | 佳木斯大学 | Bacteria culture experimental device of aseptic constant temperature and humidity |
| CN112266873A (en) * | 2020-10-19 | 2021-01-26 | 广州丽都生物科技有限公司 | Cell culture box for dynamically adjusting carbon dioxide amount by using acid-base change |
| CN112280658A (en) * | 2020-10-28 | 2021-01-29 | 张金兰 | Multifunctional box for large-scale culture of microorganisms |
| CN112980685A (en) * | 2021-04-20 | 2021-06-18 | 广州赛太特生物医学科技有限公司 | A biological cell breeding device for cultivateing, survey |
| CN113136335A (en) * | 2021-04-27 | 2021-07-20 | 湖北明德健康科技有限公司 | Stem cell culture device and culture method thereof |
| CN112980687A (en) * | 2021-05-19 | 2021-06-18 | 广州维柏生物科技有限公司 | Constant temperature and humidity cell culture box |
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