CN109221081A - A kind of multiple groups knit vitrifying in-stiu encapsulation device and method - Google Patents
A kind of multiple groups knit vitrifying in-stiu encapsulation device and method Download PDFInfo
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- CN109221081A CN109221081A CN201811041589.4A CN201811041589A CN109221081A CN 109221081 A CN109221081 A CN 109221081A CN 201811041589 A CN201811041589 A CN 201811041589A CN 109221081 A CN109221081 A CN 109221081A
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- Prior art keywords
- carrier
- tissue
- conserving case
- protective agent
- syringe
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0236—Mechanical aspects
- A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation
- A01N1/0268—Carriers for immersion in cryogenic fluid, both for slow-freezing and vitrification, e.g. open or closed "straws" for embryos, oocytes or semen
Abstract
The invention discloses a kind of multiple groups to knit vitrifying in-stiu encapsulation device and method, including conserving case, and cover board is equipped with above conserving case, is equipped with water bath below conserving case；Fixed structure is equipped in the conserving case, input structure and export structure above conserving case, the input structure is the first injection syringe and the second injection syringe, mixing chamber is equipped between first injection syringe and the second injection syringe and conserving case, the fixed structure is carrier bracket, is equipped with tissue carrier above carrier bracket.By the way that carrier bracket is arranged, it is matched, and distance between the two can be finely tuned by rotary carrier, to change the tension of tissue, guarantee that tissue surrounding medium even concentration is consistent in perfusing course.Research of the various concentration protective agent to tissue glassy state storage influential effect is carried out using the application, it is advantageous that multiple groups sample can be saved simultaneously, and can fully ensure that the consistency of remaining experiment condition, so that experimental result is more accurate and reliable.
Description
Technical field
It is exactly that a kind of multiple groups knit vitrifying in-stiu encapsulation device and side the present invention relates to biological tissue's preservation field
Method.
Background technique
Human body maintains health status to need the various normal co-ordinations of tissue in vivo, once there is lesion and will affect in a certain tissue
The normal life of people even threat to life.The pipeline that blood vessel is transported as blood of human body, once impaired will seriously jeopardize trouble
The life security of person.The transplanting of blood vessel is to repair the effective means of serious defect blood vessel, but vessel origin is limited and artificial blood vessel
Development it is still immature, this supply and demand greatly limits the clinical application of vascular transplant.Tracheae is as connection larynx and branch gas
Pipeline between pipe is not only the channel of air, and has defence, removing foreign matter and other effects.For suffering from severe broncho disease
Patient, need row resection of trachea, if excision length be more than that 6cm needs promoting the circulation of qi pipe transplantation, the wherein preservation of tracheal transplantation is
One main problem of graft of trachea.The movement of skeleton will rely on the draw of tendon, and the damage of tendon is by serious shadow
The proper motion of limbs is rung, Tendon allograft is to repair tendon injury, and especially a plurality of Tendon Defection is ideal
Method, the critical issue that allografting need to consider are how effectively to save transplanting tendon.Saving above-mentioned group
When knitting, the mechanical characteristics such as its viscoplasticity are considered, realize the in-stiu encapsulation of these tissues.
Slow freezing is most common biological tissue's store method, is still kept after group capable of being made to be woven in long-term preservation living
Property.But this store method is also there are many short slab, and slow freezing can cause the formation and growth of ice crystal in solution, this will cause carefully
Cellular damage causes tissue devitalization dead.
When the living tissues such as Vitrification freezen protective blood vessel, the liquid of intraor extracellular, which not will form, destroys the big of organelle
Ice crystal, will not damaging cells structure, therefore, in organizational project mostly use vitrified method to save biological tissue greatly.But glass
Change to save and need to add the protective agent of high concentration, damages tissue due to around protective agent excessive concentration by solution, therefore
Substep is needed to add protective agent, but substep adding procedure is complicated, cumbersome, protectant removal process is also in this way, to experimenter
Bring inconvenience.
In addition, the existing mode for saving the such large scale tissue of such as blood vessel is will to organize to be put into low-temperature frozen after processing
Deposit pipe or freeze in bag, tissue be unable to maintain that pretightning force, bending can be rolled up after the freezing, this will cause institutional framework, mechanics and
The appearance for the problems such as biological characteristics are impaired.And existing Techniques of preserving uses and gradually adds protectant method, temperature-fall period
For program mode cooling, last it is very long, cumbersome, these all seriously affected tissue preservation effect.
Summary of the invention
It is by group the technical problem to be solved by the present invention is to the existing mode for saving the such large scale tissue of such as blood vessel
It knits and is put into cryogenic vial after processing or freezes in bag, tissue is unable to maintain that pretightning force, can roll up bending after the freezing, this meeting
The appearance for the problems such as causing institutional framework, mechanics and biological characteristics impaired.And existing Techniques of preserving is protected using gradually addition
Protect agent method, temperature-fall period be program mode cool down, last it is very long, cumbersome, these all seriously affected organize preservation
Effect.
In order to solve the above technical problems, the present invention uses following technological means:
A kind of multiple groups knit vitrifying in-stiu encapsulation device and method, including conserving case, are equipped with cover board above conserving case, under conserving case
Side is equipped with water bath；Fixed structure is equipped in the conserving case, input structure and export structure above conserving case, described is defeated
Entering structure is the first injection syringe and the second injection syringe, and the first injection syringe and second injects syringe and conserving case
Between be equipped with mixing chamber, the fixed structure is carrier bracket, is equipped with tissue carrier above carrier bracket.By the way that carrier is arranged
Bracket matches it, and can pass through setting by the distance that rotary carrier is finely tuned between the two to change the tension of tissue
Effect is pre-mixed to solution by mixing chamber, so that into the solution concentration uniformity in conserving case, to guarantee to fill
Tissue surrounding medium even concentration is consistent during stream.
Preferably, the further technical solution of the present invention is:
The tissue carrier side is equipped with rotation screw thread, and rotation screw thread is equipped with the matched rotary nut of size therewith.Rotation
Screw thread its cooperate with corresponding nut being fully sealed for device can be achieved.
The tissue carrier is middle flow-through carrier, in lead to carrier be equipped in lead to outer ring and in lead to inner ring, in lead to inner ring
Middle cavity body is equipped in annulus.Middle flow-through tissue carrier can be such that the inside of blood vessel and tracheae also fills for carrying blood vessel and tracheae
Enter protective agent.
The tissue carrier is clamp type carrier, and clamp type carrier is equipped with inlay.Clamp type tissue carrier is then used to press from both sides
Hold tendon.
Sealing structure is equipped between the cover board and conserving case, the sealing structure is cover board equipped with tongue, and
Cover board is equipped with the groove of the matched conserving case surrounding of tongue.
The input structure is that protective agent entrance square on the cover board is arranged, and protective agent entrance passes through hose and connects mixing
The other end of chamber one end, mixing chamber passes through the first injection syringe of hose connection and the second injection syringe, the first injection injection
Device and the second injection syringe are respectively equipped with syringe pump.First injection syringe and the second injection syringe add protective agent respectively
And culture medium, and injection rate is set separately, certain density solution can be obtained, change the available various concentration of injection rate
Solution.
The export structure is the protective agent outlet above cover board, and protective agent outlet is connected by hose extracts injection
Device.
Meander shape water-bath pipeline is distributed in the water-bath trench bottom, and meander shape water-bath pipeline one end is equipped with water-bath pipeline and enters
Mouthful, the other end of meander shape water-bath pipeline is equipped with water-bath tube outlet.Realize circulating for liquid in entire water-bath pipeline, with
Cooled down in advance to conserving case and maintains optimal temperature in protectant adding procedure
The conserving case, water bath, cover board material be silica glass or dimethyl silicone polymer or polyimides or poly- methyl
The material of methacrylate or Parylene or polytetrafluoroethylene (PTFE), the tissue carrier is polytetrafluoroethylene (PTFE), is had good
Biocompatibility.
A kind of multiple groups knit the application method of vitrifying in-stiu encapsulation device and method, by tissue preserration method and group
Removing method composition is knitted, composition store method is selection tissue carrier, conserving case cooling, adjustment carrier distance, injection and extracts
Protective agent and culture medium, conserving case sealing, organizing removing method is that conserving case thaws, extracts protective agent and take out preservation tissue,
It is characterized in that specific step is as follows:
Step 1: selection tissue carrier is blood vessel or tracheae or tendon according to the tissue saved, selects flow-through in corresponding
Carrier or clamp type carrier are mounted on the carrier bracket in conserving case；
Step 2: conserving case cooling, the liquid for being passed through certain temperature enter the water-bath line entry of meander shape water-bath pipeline, are returning
It is extracted at the water-bath tube outlet of line shape water-bath pipeline with identical speed, to be cooled down in advance to conserving case；
Step 3: adjustment carrier distance, rotational tissue carrier adjust the distance between carrier, which is removed with tissue to be saved
Preceding equal length realizes the in-stiu encapsulation of tissue so that group maintains pretightning force when being woven in glassy state storage；
Step 4: injection and extraction protective agent and culture medium, the first injection syringe, the second injection syringe and extraction syringe
Respectively by different syringe pump control injection rate and extraction speed, the first injection syringe, second are injected in syringe respectively
Protective agent and culture medium are added, starting syringe pump carries out protectant continuous pouring, the first injection syringe, the second injection injection
Device first injects mixing chamber, and mixing chamber is pre-mixed effect to solution, so that into the solution concentration uniformity in conserving case,
To guarantee to organize surrounding medium even concentration consistent in perfusing course；
Step 5: conserving case sealing after protective agent adds, is sealed the protective agent entrance on the cover board with nut, and
Conserving case is put into freezen protective in liquid nitrogen；
Step 6: conserving case thaws, and conserving case is removed from liquid nitrogen and is put into the water bath, meander shape water-bath pipeline one end
Equipped with water-bath line entry, water-bath line entry is passed through the liquid of certain temperature, and the other end of meander shape water-bath pipeline is equipped with water
Bathe tube outlet, water-bath tube outlet its extracted with identical speed；
Step 7: extract protective agent, remove the nut on protective agent entrance, the first injection syringe, the second injection syringe and
It extracts syringe to be connected with protective agent entrance and exit respectively, and sets syringe pump parameter and make injection rate and extract speed
It is identical；
Step 8: taking out and save tissue, unload the cover board, will be removed from tissue carrier in case using by the tissue saved.
During the tissue vitrifying in-stiu encapsulation of the application, tissue carrier keeps former during group capable of being made to be woven in vitrifying
Some geometric shapes and certain tension, avoid the damage of tissue by mechanical property and functionally；The embodiment of the present application uses
The method of continuous pouring is added and removal protective agent, uniform lift and the protectant concentration of reduction, to avoid tissue because of surrounding
Solution concentration variation is too fast and generates solution damage；The water-bath pipeline designed in the embodiment of the present application is distributed in water-bath in meander shape
In slot, it can fully ensure that constant and conserving case the temperature of temperature in protective agent adding procedure is uniform.
Further, the tissue carrier in the embodiment of the present application is divided into two types, i.e., middle flow-through and clamp type can be distinguished
For carrying blood vessel, tracheae and tendon, and protective agent can be perfused to blood vessel or pipe in middle flow-through carrier；Two kinds of carriers it is another
One end can adjust the distance between carrier as needed by screw thread to change the tension of tissue.
Detailed description of the invention
Fig. 1 overall system structure schematic diagram provided in an embodiment of the present invention；
Fig. 2 whole multiple groups provided in an embodiment of the present invention knit vitrifying in-stiu encapsulation apparatus structure schematic diagram；
Fig. 3 covering plate structure schematic diagram provided in an embodiment of the present invention；
Fig. 4 conserving case structural schematic diagram provided in an embodiment of the present invention；
Fig. 5 middle flow-through tissue carrier structure schematic diagram provided in an embodiment of the present invention；
Fig. 6 clamp type tissue carrier structure schematic diagram provided in an embodiment of the present invention；
Fig. 7 water-bath slot structure schematic diagram provided in an embodiment of the present invention；
The structural block diagram of Fig. 8 application method of the present invention.
Description of symbols: 1- cover board；2- conserving case；3- water bath；Flow-through carrier in 4-；5- clamp type carrier；
6- first injects syringe；7- second injects syringe；8- extracts syringe；9- mixing chamber；10- protective agent entrance；
The outlet of 11- protective agent；12- syringe pump；13- tongue；14- groove；15- meander shape water-bath pipeline；16- water-bath pipeline enters
Mouthful；17- water-bath tube outlet；18- carrier bracket；Cavity body in 19-；20- rotates screw thread；Lead to outer ring in 21-；22- is embedding
Body.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated.
Specific embodiment 1:
Referring to Fig. 1, Fig. 3, Fig. 7 it is found that a kind of multiple groups of the present invention knit vitrifying in-stiu encapsulation device and method, including conserving case 2,
It is equipped with cover board 1 above conserving case 2, is equipped with water bath 3 below conserving case 2；It is equipped with fixed structure in the conserving case 2, saves
2 top input structure of box and export structure, the input structure is the first injection syringe 6 and second injects syringe 7, the
Mixing chamber 9 is equipped between one injection syringe 6 and the second injection syringe 7 and conserving case 2, the fixed structure is carrier branch
Frame 18 is equipped with tissue carrier above carrier bracket 18, and sealing structure, the sealing structure are equipped between cover board 1 and conserving case 2
Tongue 13 is equipped with for cover board 1 and cover board 1 is equipped with the groove 14 of matched 2 surrounding of conserving case of tongue 13, and input structure is to set
Set the protective agent entrance 10 above cover board 1, protective agent entrance 10 connects 9 one end of mixing chamber by hose, mixing chamber 9 it is another
By the first injection syringe 6 of hose connection and the second injection syringe 7, first injects syringe 6 and the second injection injection at end
Device 7 is respectively equipped with syringe pump 12, and export structure is the protective agent outlet 11 of 1 top of cover board, and protective agent outlet 11 is connected by hose
It connects and extracts syringe 8, meander shape water-bath pipeline 15 is distributed in 3 bottom of water bath, and 15 one end of meander shape water-bath pipeline is equipped with water-bath
Line entry 16, the other end of meander shape water-bath pipeline 15 be equipped with water-bath tube outlet 17, conserving case 2, water bath 3, cover board 1
Material is silica glass or dimethyl silicone polymer or polyimides or polymethyl methacrylate or Parylene or polytetrafluoro
The material of ethylene, the tissue carrier is polytetrafluoroethylene (PTFE), has good biocompatibility.
Referring to fig. 2, Fig. 4, Fig. 5 are it is found that a kind of multiple groups of the present invention knit vitrifying in-stiu encapsulation device and method, tissue carrier
Side is equipped with rotation screw thread 20, and rotation screw thread 20 is equipped with the matched rotary nut of size therewith, organizes carrier for middle flow-through load
Tool 4, in lead to carrier be equipped in lead to outer ring 21 and in lead to inner ring, in lead in the annulus of inner ring and be equipped with middle cavity body 19.
Referring to fig. 2, Fig. 4, Fig. 6 are it is found that a kind of multiple groups of the present invention knit vitrifying in-stiu encapsulation device and method, tissue carrier
Side is equipped with rotation screw thread 20, and rotation screw thread 20 is equipped with the matched rotary nut of size therewith, and the tissue carrier is folder
Clamp carrier 5, clamp type carrier 5 are equipped with inlay 22.
Specific embodiment 2:
Referring to Fig. 8 it is found that a kind of multiple groups of the present invention knit the application method of vitrifying in-stiu encapsulation device and method, by tissue preserration
Method and tissue removing method composition, composition store method are selection tissue carrier, the cooling of conserving case 2, adjustment carrier distance, note
Enter and extract protective agent and culture medium, conserving case 2 seal, tissue removing method is that conserving case 2 thaws, extracts protective agent and taking-up
Save tissue, it is characterised in that specific step is as follows:
Step 1: selection tissue carrier is blood vessel or tracheae or tendon according to the tissue saved, selects flow-through in corresponding
Carrier 4 or clamp type carrier 5 are mounted on the carrier bracket 18 in conserving case 2；
Step 2: conserving case 2 cools down, and the liquid for being passed through certain temperature enters the water-bath line entry 16 of meander shape water-bath pipeline 15,
It is extracted at the water-bath tube outlet 17 of meander shape water-bath pipeline 15 with identical speed, to be dropped in advance to conserving case 2
Temperature；
Step 3: adjustment carrier distance, rotational tissue carrier adjust the distance between carrier, which is removed with tissue to be saved
Preceding equal length realizes the in-stiu encapsulation of tissue so that group maintains pretightning force when being woven in glassy state storage；
Step 4: injection and extraction protective agent and culture medium, the first injection syringe 6, second inject syringe 7 and extract injection
Device 8 controls injection rate by different syringe pumps 12 respectively and extracts speed, and the first injection syringe 6, second injects syringe 7
Middle to add protective agent and culture medium respectively, starting syringe pump 12 carries out protectant continuous pouring, the first injection syringe 6, the
Two injection syringes 7 first inject mixing chamber 9, and mixing chamber 9 is pre-mixed effect to solution, so that into molten in conserving case 2
Liquid even concentration is consistent, to guarantee to organize surrounding medium even concentration consistent in perfusing course；
Step 5: conserving case 2 seals, and after protective agent adds, is sealed the protective agent entrance on the cover board 1 with nut,
And conserving case 2 is put into freezen protective in liquid nitrogen；
Step 6: conserving case 2 thaws, and conserving case 2 is removed from liquid nitrogen and is put into the water bath 3, meander shape water-bath pipeline 15
One end is equipped with water-bath line entry 16, and water-bath line entry 16 is passed through the liquid of certain temperature, meander shape water-bath pipeline 15 it is another
One end be equipped with water-bath tube outlet 17, water-bath tube outlet 17 its extracted with identical speed；
Step 7: extracting protective agent, remove the nut on protective agent entrance, the first injection syringe 6, second injects syringe 7
With extract syringe 8 be connected respectively with protective agent entrance 10 and outlet, and set 12 parameter of syringe pump make injection rate and
It is identical to extract speed；
Step 8: taking out and save tissue, unload the cover board 1, will be removed from tissue carrier in case making by the tissue saved
With.
Specific embodiment 3:
Fig. 1 is that multiple groups provided in an embodiment of the present invention knit vitrifying in-stiu encapsulation system structure diagram.As shown in Figure 1, this hair
Bright embodiment discloses a kind of multiple groups and knits vitrifying in-stiu encapsulation device and method, including cover board 1, conserving case 2, water bath 3, group
It knits carrier, the injection syringe that two speed control respectively, extract syringe 8 and mixing chamber 9；It is in parallel to inject syringe 6 and 7
Afterwards, it is connect with protective agent entrance 10；Syringe 8 is extracted to connect with protective agent outlet 11；In injection syringe and protective agent entrance
Mixing chamber 9 is connected between 10.
Tissue to be saved is tendon.
The embodiment of the present application is configured to vitrification preserving tendons by existing routine experiment method in the prior art first
Culture medium and protection agent solution.
At different temperature due to cell, cell membrane is different to protectant permeability, protectant in order to guarantee
Effect is best, and certain temperature need to be maintained in protective agent adding procedure.The culture medium prepared and protection agent solution are placed in 4
Cooled down in advance in DEG C refrigerator, 4 DEG C of water is passed through by the entrance of the water-bath pipeline, outlet is extracted with phase same rate.
Tendon under ex vivo situation can shrinkage crimping, tendon its histology, biomethanics after freezen protective in this case
Etc. will be damaged.And tendon is banded structure.So in the present embodiment, we select clamp type tissue carrier to use
The in-stiu encapsulation of tendon is realized in carrying tendon to maintain the pretightning force of tendon.
In protectant adding procedure, cell is damaged due to solution concentration variation is too fast by solution in order to prevent, is needed
Step up protectant concentration.The injection rate for controlling protective agent and culture medium respectively using programmable infusion pump 12 can be realized
The accurate control of protective agent concentration.In addition, culture medium and protective agent are injected into mixing chamber 9, solution can be mixed in advance
It closes, guarantees peri-musculotendinous solution concentration uniformity in perfusing course.It is to cover the cover board 1 specific in the present embodiment,
By two speed are controlled by syringe pump 12 respectively injection syringe it is in parallel after be connected with mixing chamber 9, in two injection syringes point
It is not pre-loaded with above-mentioned configured culture medium and protective agent, the protective agent entrance in the other end connecting cover plate 1 of mixing chamber 9
10, syringe 8 will be extracted and be connected with protective agent outlet 11.Wherein, syringe pump 12 can be by soft to the speed control of syringe
Part programming is realized, had both been accurately controlled two respective injection rates of injection syringe in this way, and had been realized that the difference of solution concentration was matched
Than also accurately controlling total injection rate and extracting that speed is always consistent, to guarantee the flat of solution pressure in conserving case 2
Weighing apparatus.
It organizes to infect because contacting with external environment in order to prevent, and guarantees that protective agent will not during preservation
Leakage, so being put into liquid after the protective agent entrance screw cap closures, then by conserving case 2 after to be protected dose of addition
It is saved in nitrogen.
It saves 24 hours or even after the longer time, the embodiment of the present application carries out rewarming and protective agent removal process:
Successful vitrifying cryo-conservation should also be during rewarming in addition to avoid crystallisation solidification in cool down
Devitrification is avoided, therefore is avoided devitrification with the sufficiently fast rate of heat addition during heating.In the application
In, the water-bath pipeline is meander shape, can guarantee that 2 thermally equivalent of conserving case, temperature quickly go up.Specifically, by the preservation
Box 2 is removed from liquid nitrogen and is put into water bath 3, is passed through 37 DEG C of water by the entrance of the water-bath pipeline and carries out rewarming to device.
Protectant concentration around tissue is stepped up, when adding protective agent to prevent tissue because of surrounding medium concentration
Change too fast and damaged by solution, similarly, also to reduce protectant concentration step by step when removing protective agent.Therefore to
After rewarming, the nut on protective agent entrance is backed out, both ends are separately connected the injection injection that speed is controlled by syringe pump 12
Device and extraction syringe 8, wherein injection syringe is provided with culture medium, and setting 12 parameter of syringe pump makes injection rate and pumping
Take speed equal.
After protective agent removes, the cover board 1 is opened, will be removed from tissue carrier by the tendon of glassy state storage
In case using.
Specific embodiment 4:
Multiple groups knit the structure of vitrifying in-stiu encapsulation device referring to Fig. 1-7.The present embodiment content is similar to Example 1, different
It is that the tissue that the present embodiment is saved is blood vessel.
The embodiment of the present application carries out refrigerating process first:
The culture medium and protection agent solution of glassy state storage blood vessel are configured to by existing routine experiment method in the prior art.
The culture medium prepared and protective agent are put into -4 DEG C of refrigerators and cooled down in advance.Led to by the entrance of the water-bath pipeline
Enter -4 DEG C of ethyl alcohol, outlet is extracted with phase same rate.
Due to the blood vessel under ex vivo situation can shrinkage crimping, blood vessel mechanics such as its viscoplasticity after freezen protective in this case
Performance will be damaged.And vessel retraction, bonding in order to prevent, it need to remain intravascular certain to intra-arterial infusion protective agent
Normal pressure.Therefore in the present embodiment, flow-through tissue carrier is for carrying blood vessel in selection, so that internal blood vessel can also pour into
Protective agent, and the pretightning force of blood vessel can be maintained, realize the in-stiu encapsulation of blood vessel.
Cover the cover board 1, after the injection syringe that two speed are controlled by syringe pump 12 respectively is in parallel with mixing chamber 9
It is connected, is pre-loaded with above-mentioned configured culture medium and protective agent, the other end connection of mixing chamber 9 in two injection syringes respectively
Protective agent entrance 10 on cover board 1 will extract syringe 8 and be connected with protective agent outlet 11.12 parameter of syringe pump is set, with reality
The different ratio of existing solution concentration, and control total injection rate and to extract speed always consistent, to guarantee in conserving case 2
The balance of solution pressure.
After to be protected dose of addition, liquid nitrogen is put into after the protective agent entrance screw cap closures, then by conserving case 2
Middle preservation.
It saves 24 hours or even after the longer time, the embodiment of the present application carries out rewarming and protective agent removal process:
The conserving case 2 is removed from liquid nitrogen and is put into water bath 3,37 DEG C of water pair are passed through by the entrance of the water-bath pipeline
Device carries out rewarming.
Back out the nut on protective agent entrance, both ends be separately connected by syringe pump 12 control speed injection syringe and
Extract syringe 8, wherein injection syringe is provided with culture medium, and setting 12 parameter of syringe pump makes injection rate and extracts speed
It spends equal.
After protective agent removes, the cover board 1 is opened, will be removed from tissue carrier by the blood vessel of glassy state storage
In case using.
Specific embodiment 5:
The embodiment of the present application is for studying influence of the different protective agent concentration to preservation effect.
Specifically, it using blood vessel as research object, as shown in Figure 1, 2, by three conserving cases 2 and drains into water bath 3.
- 4 DEG C of ethyl alcohol are passed through by water-bath line entry 16 to cool down to conserving case 2 in advance, and flow-through tissue carrier in three groups is installed
In three conserving cases 2, three groups of blood vessels are fixed.
The cover board 1 is covered, the protective agent entrance 10 on three cover boards 1 is connected with three groups of injection syringes respectively, phase
Accordingly, three protective agent outlets 11 are connected with three extraction syringes 8 respectively, each to inject syringe and extract syringe 8
Its injection is controlled by syringe pump 12 and extracts speed, every group of injection rate and extraction speed is consistent；Every group of injection injection
Protectant concentration is different in device, to study influence of the different protective agent concentration to preservation effect.
After protective agent adds, three groups of conserving cases 2 are put into liquid nitrogen by the protective agent entrance screw cap closures simultaneously
Middle preservation.
Three groups of conserving cases 2, which are taken out, after saving 24 hours and are put into water bath 3 carries out rewarming.
After to rewarming, the nut on protective agent entrance is backed out, the protective agent entrance on every group of cover board 1 connects respectively
It connecing and the injection syringe of speed is controlled by syringe pump 12 and extracts syringe 8, wherein injection syringe is provided with culture medium, if
Determining 12 parameter of syringe pump makes injection rate equal with speed is extracted.
After protective agent removes, the cover board 1 is opened, will be taken from tissue carrier by the blood vessel of glassy state storage
Under.The detection for carrying out the performances such as cell activity, viscoplasticity to every group of blood vessel respectively, to obtain the best of blood vessel glassy state storage
Protective agent concentration.
Research of the various concentration protective agent to tissue glassy state storage influential effect is carried out using the application, it is advantageous that
Multiple groups sample can be saved simultaneously, and can fully ensure that the consistency of remaining experiment condition, so that experimental result is more accurate and reliable.
Since the foregoing is merely a specific embodiment of the invention, but protection of the invention is without being limited thereto, any skill
The technical staff in art field it is contemplated that the equivalent variation or substitution of the technical program technical characteristic, all covers of the invention
Within protection scope.
Claims (10)
1. a kind of multiple groups knit vitrifying in-stiu encapsulation device and method, including conserving case, cover board, conserving case are equipped with above conserving case
Lower section is equipped with water bath；It is characterized by: fixed structure is equipped in the conserving case, input structure and output above conserving case
Structure, the input structure are the first injection syringe and the second injection syringe, and the first injection syringe and second injects
Mixing chamber is equipped between syringe and conserving case, the fixed structure is carrier bracket, is equipped with tissue above carrier bracket and carries
Tool.
2. a kind of multiple groups according to claim 1 knit vitrifying in-stiu encapsulation device and method, it is characterised in that: described
Carrier side is organized to be equipped with rotation screw thread, rotation screw thread is equipped with the matched rotary nut of size therewith.
3. a kind of multiple groups according to claim 1 knit vitrifying in-stiu encapsulation device and method, it is characterised in that: described
Tissue carrier be middle flow-through carrier, in lead to carrier be equipped in lead to outer ring and in lead to inner ring, in lead to inner ring annulus in be equipped in lead to
Cavity.
4. a kind of multiple groups according to claim 1 knit vitrifying in-stiu encapsulation device and method, it is characterised in that: described
Tissue carrier is clamp type carrier, and clamp type carrier is equipped with inlay.
5. a kind of multiple groups according to claim 1 knit vitrifying in-stiu encapsulation device and method, it is characterised in that: described
Sealing structure is equipped between cover board and conserving case, the sealing structure is that cover board is equipped with tongue and cover board is equipped with tongue
The groove of matched conserving case surrounding.
6. a kind of multiple groups according to claim 1 knit vitrifying in-stiu encapsulation device and method, it is characterised in that: described
Input structure is that protective agent entrance square on the cover board is arranged, and protective agent entrance passes through hose and connects mixing chamber one end, mixing chamber
The other end by hose connection first injection syringe and second injection syringe, first injection syringe and second injection note
Emitter is respectively equipped with syringe pump.
7. a kind of multiple groups according to claim 1 knit vitrifying in-stiu encapsulation device and method, it is characterised in that: described
Export structure is the protective agent outlet above cover board, and protective agent outlet is connected by hose extracts syringe.
8. a kind of multiple groups according to claim 1 knit vitrifying in-stiu encapsulation device and method, it is characterised in that: described
Meander shape water-bath pipeline is distributed in water-bath trench bottom, and meander shape water-bath pipeline one end is equipped with water-bath line entry, meander shape water-bath
The other end of pipeline is equipped with water-bath tube outlet.
9. a kind of multiple groups according to claim 1 knit vitrifying in-stiu encapsulation device and method, it is characterised in that: described
Conserving case, water bath, cover board material be silica glass or dimethyl silicone polymer or polyimides or polymethyl methacrylate
Or Parylene or polytetrafluoroethylene (PTFE), the material for organizing carrier are polytetrafluoroethylene (PTFE), have good bio-compatible
Property.
10. a kind of multiple groups according to claim 1 knit the application method of vitrifying in-stiu encapsulation device and method, by organizing
Store method and tissue removing method composition, composition store method be selection tissue carrier, conserving case cooling, adjustment carrier away from
From, injection and extract protective agent and culture medium, conserving case sealing, tissue removing method be conserving case thaw, extract protective agent and
It takes out and saves tissue, it is characterised in that specific step is as follows:
Step 1: selection tissue carrier is blood vessel or tracheae or tendon according to the tissue saved, selects flow-through in corresponding
Carrier or clamp type carrier are mounted on the carrier bracket in conserving case；
Step 2: conserving case cooling, the liquid for being passed through certain temperature enter the water-bath line entry of meander shape water-bath pipeline, are returning
It is extracted at the water-bath tube outlet of line shape water-bath pipeline with identical speed, to be cooled down in advance to conserving case；
Step 3: adjustment carrier distance, rotational tissue carrier adjust the distance between carrier, which is removed with tissue to be saved
Preceding equal length realizes the in-stiu encapsulation of tissue so that group maintains pretightning force when being woven in glassy state storage；
Step 4: injection and extraction protective agent and culture medium, the first injection syringe, the second injection syringe and extraction syringe
Respectively by different syringe pump control injection rate and extraction speed, the first injection syringe, second are injected in syringe respectively
Protective agent and culture medium are added, starting syringe pump carries out protectant continuous pouring, the first injection syringe, the second injection injection
Device first injects mixing chamber, and mixing chamber is pre-mixed effect to solution, so that into the solution concentration uniformity in conserving case,
To guarantee to organize surrounding medium even concentration consistent in perfusing course；
Step 5: conserving case sealing after protective agent adds, is sealed the protective agent entrance on the cover board with nut, and
Conserving case is put into freezen protective in liquid nitrogen；
Step 6: conserving case thaws, and conserving case is removed from liquid nitrogen and is put into the water bath, meander shape water-bath pipeline one end
Equipped with water-bath line entry, water-bath line entry is passed through the liquid of certain temperature, and the other end of meander shape water-bath pipeline is equipped with water
Bathe tube outlet, water-bath tube outlet its extracted with identical speed；
Step 7: extract protective agent, remove the nut on protective agent entrance, the first injection syringe, the second injection syringe and
It extracts syringe to be connected with protective agent entrance and exit respectively, and sets syringe pump parameter and make injection rate and extract speed
It is identical；
Step 8: taking out and save tissue, unload the cover board, will be removed from tissue carrier in case using by the tissue saved.
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| CN201811041589.4A CN109221081B (en) | 2018-09-07 | 2018-09-07 | Multi-tissue vitrification in-situ storage device |
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