CN109042624A - Human placenia Mo Xia great vascular tissue prepares cryopreservation methods and application - Google Patents
Human placenia Mo Xia great vascular tissue prepares cryopreservation methods and application Download PDFInfo
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- CN109042624A CN109042624A CN201810787908.XA CN201810787908A CN109042624A CN 109042624 A CN109042624 A CN 109042624A CN 201810787908 A CN201810787908 A CN 201810787908A CN 109042624 A CN109042624 A CN 109042624A
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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
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
Cryopreservation methods are prepared the invention discloses human placenia Mo Xia great vascular tissue, comprising: (1) are cleaned placenta；The decidua to have fallen off is cut along placental edge, removes amnion；(2) the placental fetal surface chorion for removing amnion and big blood vessel are separated together, is cleaned with physiological saline or PBS buffer solution, and rinse blood vessel to remove intravascular delay clot；Then, the big blood vessel connecting with chorionic plate is cut one by one；(3) blood vessel is transferred to cryopreservation tube or frozen in bag, vitrification frozen stock solution is imported with three-step approach, programmed cooling instrument is transferred to, is cooled to -80 DEG C~-90 DEG C, be transferred to liquid nitrogen frozen preservation.Freezing and storing method of the invention is conducive to improve the activity of cryopreserved tissue and cell, form, function, structure are consistent with flesh tissue after recovery, the tissue saved can be used not only for separation stem cell, and the fields such as epithelial cell can be also used for the fields such as organizational project transplanting.
Description
Technical field
Cryopreservation methods and application more particularly to Human plactnta are prepared the present invention relates to human placenia Mo Xia great vascular tissue
The freezing and storing method and method for resuscitation of chorion Xia great vascular tissue.
Background technique
Placenta is the vitals of mass exchange between fetus and parent, by embryo's embryophoric membrane and parent during being human pregnancy
Endometrium organizes colligator official between combining the mothers and sons grown up to.Recently as going deep into for scientific research, people can be from placenta
In sort out including a plurality of types of cells such as candidate stem cell, mescenchymal stem cell, epithelial cell, so that it is long to change placenta
Phase is with the situation as Biohazard Waste.
Modern scientific research prompt, placenta amnion can be used to expand amnion-derived mescenchymal stem cell, can also be with
In ophthalmologic operation.Chorionic villi of placenta can isolate mescenchymal stem cell, can also isolate candidate stem cell.Placenta
In fluff structures be capillary tissue in fact, can therefrom sort out endothelial stem cell, epithelial stem cell etc..Deciduate placenta
It is the structure that placenta is attached to parent, research also obtains the decidua stem cell of similar mescenchymal stem cell.And in amnion and suede
There is the interstitial tissue rich in mescenchymal stem cell between trichilemma, chorion and decidua.At present to placenta utilization be it is extensive, it is past
Toward taking placenta very small part tissue only to isolate one or several kinds of stem cells of placenta and be stored, the tire of rest part
Disk is still used as MEDICAL WASTE TREATMENT, forms waste.Also, with going deep into for scientific research, placenta has blood forming organ etc.
Function can synthesize a large amount of hormone and the factor, still nourish sertoli cell containing other in addition to candidate stem cell.
It is worth noting that, in the prior art from placenta tissue extract stem cell save method, be only limitted to it is existing
It is carried out under conditions of technological means and cell quality standard, technology and cell quality mark after WeiLai Technology progress may not be met
Alignment request.Therefore, research and develop a kind of method, by the tissue freezen protective of each component part of placenta, decades even long-term preservation its
Activity, when needing in future, according to following technical conditions and cell quality standard resuscitation tissue, separation etc. is obtained more again
Cell or the stem cell for meeting forward requirement, to meet clinical research and application, it appears particularly significant.
Chinese invention patent CN201210288706 (102763642 B of Authorization Notice No. CN) discloses a kind of freezing guarantor
Protect liquid and freezen protective Human plactnta amnion and chorial method.The amnion and chorion size that this method saves are only 1cm2
Left and right, cannot be in the large area freezen protective of complete tissue by amnion, and relatively simple single, the tissue frozen of the mode frozen
Cell survival rate is lower, prepares stem cell after being simply possible to use in amplification, membrane tissue of preservation living cells itself is less, and rich in placenta
Rich vascular tissue is not saved.And studies have shown that the activity that the form of cryopreserved tissue can preferably save cell is to freeze
Depositing harmful ingredient in protective agent not can enter the gap of the membrane structure rich in cell.Therefore, if the tissue volume frozen too
It is small, it can make cell is more to be exposed in the toxic component of freezing protective agent, cell activity will be affected, and lose and freeze
The effect of organization protection's cell.In addition, studies have shown that different tissues, even different cells should be applicable in distinctive frost side
What formula can just have freezes effect, and the various organization of placenta should be distinguished, is independent using different frost modes, can just obtain preferably
Freeze.
Summary of the invention
For the above-mentioned prior art, the present invention provides human placenia Mo Xia great vascular tissue prepare cryopreservation methods and
Using, and in particular to the freezing and storing method and method for resuscitation of human placenia Mo Xia great vascular tissue.
The present invention is achieved by the following technical solutions:
Human placenia Mo Xia great vascular tissue prepares cryopreservation methods, comprising the following steps:
(1) placenta is cleaned into (remove dirt and microbial contamination)；The decidua to have fallen off is cut along placental edge, is removed
Amnion；
The placenta, collects in the following manner: choosing free from infection, the healthy placenta without obstetric complication, warp
Puerpera agrees to and signs informed consent form；Normal acquisition uses number of patent application for 2017106869531 (publication number of CN
Placenta acquisition method described in patent application 107320332A) will be transported to experiment in collected placenta 48 hours
Room, and various necessary detections are carried out, such as the detection of virus infectious disease, germ contamination detection etc..
(2) the above-mentioned placental fetal surface chorion for having removed amnion and big blood vessel are separated into (reservation villus as far as possible together
Big blood vessel is complete under film), it is cleaned with physiological saline or PBS buffer solution, and rinse blood vessel to remove intravascular delay clot；So
Afterwards, the big blood vessel connecting with chorionic plate is cut one by one (by scissors for vessels at the cylindrical type segment of length 2cm~5cm for most
It is good)；
(3) blood vessel is transferred to cryopreservation tube or is frozen in bag (when transfer, can use the finer wire that can be used for ultra-low temperature surroundings
Or the materials such as plastic wire pass through blood vessel, and to facilitate transfer), vitrification frozen stock solution is imported with three-step approach, programmed cooling instrument is transferred to, presses
- 80 DEG C~-90 DEG C are cooled to according to the cooling process of setting, is transferred to liquid nitrogen frozen preservation；The cooling process are as follows: protected at 4 DEG C
Hold 10min；- 20 DEG C are reduced to 1 DEG C/min rate；It is cooled to -80 DEG C~-90 rapidly with the rate of 50 DEG C~60 DEG C/min
DEG C, it keeps for 24 hours, being transferred quickly in liquid nitrogen.
The vitrification frozen stock solution, by mass concentration be 20% human serum albumin solution, dimethyl sulfoxide, propylene glycol,
Ethoxy urea and trehalose composition, wherein each component proportion are as follows: 15~22wt% of dimethyl sulfoxide, propylene glycol 10~
18wt%, ethoxy 10~16wt% of urea, 8~15wt% of trehalose, surplus is people's albumin solution.Preferably, each component
Proportion are as follows: dimethyl sulfoxide 18wt%, propylene glycol 12wt%, ethoxy urea 12wt%, trehalose 12wt%, surplus is
20% human serum albumin solution.
The three-step approach imports the concrete mode of vitrification frozen stock solution are as follows:
The first step imports the dimethyl sulfoxide and groups of people's albumin solution of trehalose, 50%, in 4 DEG C of balance 5min；
Second step imports the dimethyl sulfoxide and groups of people's albumin solution of ethoxy urea, remaining 50%, in 4 DEG C
2min is balanced,
Third step imports propylene glycol and groups of people's albumin solution, is immediately placed in programmed cooling instrument；
The volume ratio of the liquid imported three times is 3:1:1, is adjusted with 20% human serum albumin solution to suitable concentration, entirely
After portion imports, the concentration of each component reaches above-mentioned final concentration requirement in vitrification frozen stock solution.
Big blood vessel is removed from placenta, is processed into suitable size under above-mentioned chorion, be reloaded into cryopreservation tube or freeze bag etc.
It in special container, saves and is composed of the special number of organization name on container, sample places into liquid nitrogen appearance after being iced to specific temperature
Device specific position long term storage, the specificity number on container is for searching sample when recovery.
After carrying out above-mentioned freezen protective, it can recover whenever necessary, comprising the following steps:
The Chorionic villi of placenta Xia great vascular tissue of freezen protective is removed from liquid nitrogen, is placed fast after gas phase 10min is balanced
Speed will freeze bag or cryopreservation tube is placed in 37 DEG C~42 DEG C water-baths, or use number of patent application (public for 2017107960072
The number of opening 107365700A) patent application disclosed in device recover；Bag or cryopreservation tube transfer will be frozen after dissolution rapidly
Into safety cabinet or super-clean bench, opening freezes bag or cryopreservation tube, and Chorionic villi of placenta Xia great vascular tissue is gently taken out with tweezers, is put
Enter in 4 DEG C of three times benchmark concentration resuscitation fluid (being cooled to 4 DEG C in advance in advance), balance 1 minute, takes out, place into 4 DEG C of two times of benchmark
It in concentration resuscitation fluid (being cooled to 4 DEG C in advance in advance), balances 1~3 minute, takes out, place into 4 DEG C of one times of benchmark concentration resuscitation fluid
In (being cooled to 4 DEG C in advance in advance), balance 3 minutes；2~5 are rinsed with 4 DEG C of physiological saline or PBS solution (being cooled to 4 DEG C in advance in advance)
Time, it is put into 4 DEG C of physiological saline or PBS solution (being cooled to 4 DEG C in advance in advance), stands, it is spare；
The resuscitation fluid, by trehalose, Dextran 40, alanine, glycine and Hydroxyethyl starch sodium chloride injection
Composition adjusts concentration as solvent by Hydroxyethyl starch sodium chloride injection or MEM culture medium when using；
The concentration of each component in one times of benchmark concentration resuscitation fluid are as follows: trehalose accounts for 5wt%, and Dextran 40 accounts for 6wt%, and third
Propylhomoserin accounts for 3wt%, and glycine accounts for 3wt%, and surplus is Hydroxyethyl starch sodium chloride injection or MEM culture medium；
The concentration of each component in two times of benchmark concentration resuscitation fluids are as follows: trehalose accounts for 10wt%, and Dextran 40 accounts for 12wt%,
Alanine accounts for 6wt%, and glycine accounts for 6wt%, and surplus is Hydroxyethyl starch sodium chloride injection or MEM culture medium；
The concentration of each component in three times benchmark concentration resuscitation fluid are as follows: trehalose accounts for 15wt%, and Dextran 40 accounts for 18wt%,
Alanine accounts for 9wt%, and glycine accounts for 9wt%, and surplus is Hydroxyethyl starch sodium chloride injection or MEM culture medium.
Vascular tissue can be used for separating blood vessel and form related stem cell under Chorionic villi of placenta after recovery.
Freezing and storing method of the invention can be used for setting up the tissue or cellular resources sample database in placenta source.This hair
Solid foundation and foundation are established in the bright mankind's inheritance resources library to improve placenta source.
Human placenia Mo Xia great vascular tissue of the invention prepares cryopreservation methods and method for resuscitation, has beneficial below
Effect:
1) vascular tissue under Chorionic villi of placenta separation has been subjected to, saved, the stem-cell research for placenta source provides
Important living resources.
2) placental membrane dress tissue is carried out complete, large area to save, it is dry thin that the tissue saved can be used not only for separation
Born of the same parents, the fields such as epithelial cell can be also used for the fields such as organizational project transplanting；Various tissue shears are broken into 1 in the prior art~
3cm3The unified cryopreservation methods of fritter, only can guarantee there is cell survival on a cellular level, can only be used to isolate cell progress
Amplification, it is impossible to be used in the fields such as big block film transplanting of organizational project.
3) frost mode of the invention is conducive to the activity for improving cryopreserved tissue and cell.In the prior art by various groups
It knits and shreds into 1~3cm3The unified cryopreservation methods of fritter do not meet every kind of tissue, cell wants the adaptability of specific frost mode
It asks, it is poor to freeze rear tissue activity, and institutional framework deformation, cell obtains inefficient.
4) placental membrane saved fills tissue, and form, function, structure are consistent with flesh tissue after recovery, and tissue inner cell is total
Body survival rate is up to 90% or more；And the method for cryopreserved tissue in the prior art, generally separation amplify cell activity and reach
So-and-so is worth, and actually survivaling cell is few in resuscitation team, and the cell isolated is that a small number of survivals are thin after cryopreserved tissue is recovered
Born of the same parents obtain after being proliferated.
Detailed description of the invention
Fig. 1: fresh human placenta chorion Xia great vascular tissue's HE colored graph.
Fig. 2: rear Chorionic villi of placenta Xia great vascular tissue recovery HE colored graph is frozen.
Fig. 3: the Umbilical Vein Endothelial Cells aspect graph obtained from the big vascular tissue frozen.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated.
Instrument involved in following embodiments, reagent, material etc. are unless otherwise noted existing in the prior art
Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Experimental method involved in following embodiments, inspection
Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..
The freezing and storing method of 1 human placenia Mo Xia great vascular tissue of embodiment
Placenta acquisition: choosing free from infection, the healthy placenta without obstetric complication, and multipara agrees to and signs informed consent
Book；Normal acquisition will be transported to laboratory in collected placenta 48 hours, and carry out various necessary detections, such as virus
Infectious disease detection, germ contamination detection etc..
The method of freezen protective, steps are as follows:
(1) placenta is cleaned into (remove dirt and microbial contamination)；The decidua to have fallen off is cut along placental edge, is gone out
Amnion.
(2) the above-mentioned placental fetal surface chorion for having removed amnion and big blood vessel are separated into (reservation villus as far as possible together
Big blood vessel is complete under film), it is cleaned with physiological saline, and rinse blood vessel to remove intravascular delay clot；It then, will be with chorion
The big blood vessel of plate connection cuts (the cylindrical type segment by scissors for vessels at length 2cm~5cm) one by one；
(3) blood vessel is transferred in cryopreservation tube (when transfer, it can be used for the finer wire of ultra-low temperature surroundings or pass through blood vessel, with
Facilitate transfer), vitrification frozen stock solution is imported with three-step approach, programmed cooling instrument is transferred to, is cooled to -80 according to the cooling process of setting
DEG C, it is transferred to liquid nitrogen frozen preservation；The cooling process are as follows: in 4 DEG C of holding 10min；- 20 DEG C are reduced to 1 DEG C/min rate；
It is cooled to -80 DEG C rapidly with the rate of 50 DEG C/min, keeps for 24 hours, being transferred quickly in liquid nitrogen.
The vitrification frozen stock solution, by mass concentration be 20% human serum albumin solution, dimethyl sulfoxide, propylene glycol,
Ethoxy urea and trehalose composition, wherein each component proportion are as follows: dimethyl sulfoxide 18wt%, propylene glycol 12wt%, hydroxyl second
Base urea 12wt%, trehalose 12wt%, the human serum albumin solution that surplus is 20%.
The three-step approach imports the concrete mode of vitrification frozen stock solution are as follows:
The first step imports the dimethyl sulfoxide and groups of people's albumin solution of trehalose, 50%, in 4 DEG C of balance 5min；
Second step imports the dimethyl sulfoxide and groups of people's albumin solution of ethoxy urea, remaining 50%, in 4 DEG C
2min is balanced,
Third step imports propylene glycol and groups of people's albumin solution, is immediately placed in programmed cooling instrument；
The volume ratio of the liquid imported three times is 3:1:1, is adjusted with 20% human serum albumin solution to suitable concentration, entirely
After portion imports, the concentration of each component reaches above-mentioned final concentration requirement in vitrification frozen stock solution.
Recovery after 2 freezen protective of embodiment
It according to the method for embodiment 1 after freezen protective 6 months, recovers, and induces separating mesenchymal stem cell, step
It is as follows:
The Chorionic villi of placenta Xia great vascular tissue of freezen protective is removed from liquid nitrogen, is placed fast after gas phase 10min is balanced
Speed will freeze bag and be placed in 37 DEG C~42 DEG C water-baths；Bag will be frozen after dissolution rapidly to be transferred in safety cabinet, opening freezes bag,
Chorionic villi of placenta Xia great vascular tissue is gently taken out with tweezers, is put into 4 DEG C of three times benchmark concentration resuscitation fluid and (is cooled to 4 in advance in advance
DEG C) in, it balances 1 minute, takes out, place into 4 DEG C of two times of benchmark concentration resuscitation fluids (being cooled to 4 DEG C in advance in advance), balance 1~3
Minute, it takes out, places into 4 DEG C of one times of benchmark concentration resuscitation fluid (being cooled to 4 DEG C in advance in advance), balance 3 minutes；With 4 DEG C of life
It manages salt water (being cooled to 4 DEG C in advance in advance) to rinse 2~5 times, be put into 4 DEG C of physiological saline (being cooled to 4 DEG C in advance in advance), stand, it is spare；
The resuscitation fluid, by trehalose, Dextran 40, alanine, glycine and Hydroxyethyl starch sodium chloride injection
Composition adjusts concentration as solvent by Hydroxyethyl starch sodium chloride injection when using；
The concentration of each component in one times of benchmark concentration resuscitation fluid are as follows: trehalose accounts for 5wt%, and Dextran 40 accounts for 6wt%, and third
Propylhomoserin accounts for 3wt%, and glycine accounts for 3wt%, and surplus is Hydroxyethyl starch sodium chloride injection；
The concentration of each component in two times of benchmark concentration resuscitation fluids are as follows: trehalose accounts for 10wt%, and Dextran 40 accounts for 12wt%,
Alanine accounts for 6wt%, and glycine accounts for 6wt%, and surplus is Hydroxyethyl starch sodium chloride injection；
The concentration of each component in three times benchmark concentration resuscitation fluid are as follows: trehalose accounts for 15wt%, and Dextran 40 accounts for 18wt%,
Alanine accounts for 9wt%, and glycine accounts for 9wt%, and surplus is Hydroxyethyl starch sodium chloride injection.
HE dyeing: fresh Chorionic villi of placenta Xia great vascular tissue samples before freezing carries out HE dyeing；Tire after recovery
Disk chorion Xia great vascular tissue, sampling carry out HE dyeing；As a result as shown in Figure 1 and Figure 2, it may be seen that the big blood vessel after recovery
Institutional framework is still maintained, and it is complete that the big vascular tissue after illustrating cryopreservation resuscitation maintains institutional framework.It compares fresh big
Vascular tissue's HE coloration result freezes Hou great vascular tissue recovery HE and dyes, nothing obvious great difference close with flesh tissue,
Illustrate that institutional framework is suitable with flesh tissue after freezing big vascular tissue's recovery.
It can be used for tissue transplantation after the big vascular tissue recovery frozen, it can also be used to isolated Umbilical Vein Endothelial Cells
(separation method is existing mature technology), as shown in figure 3, as seen from the figure, the big vascular tissue frozen using method of the invention
It can have successfully been isolated to obtain Umbilical Vein Endothelial Cells after recovery.
Above-described embodiment is provided to those skilled in the art, how to implement and use to be advocated with full disclosure and description
Embodiment, rather than for limiting range disclosed herein.Obvious modification will to those skilled in the art
Within the scope of the appended claims.The all publications, patents and patent applications of this specification citation are incorporated by reference into this
Text, as these publications, patents and patent applications respectively show particularly and individually to be incorporated herein by reference.
Claims (10)
1. a kind of human placenia Mo Xia great vascular tissue prepares cryopreservation methods, it is characterised in that: the following steps are included:
(1) placenta is cleaned；The decidua to have fallen off is cut along placental edge, removes amnion；
(2) the above-mentioned placental fetal surface chorion for having removed amnion and big blood vessel are separated together, is cleaned, and rinse blood vessel to go
Except intravascular delay clot；Then, the big blood vessel connecting with chorionic plate is cut one by one；
(3) blood vessel is transferred to cryopreservation tube or frozen in bag, vitrification frozen stock solution is imported with three-step approach, is transferred to programmed cooling instrument,
- 80 DEG C~-90 DEG C are cooled to according to the cooling process of setting, is transferred to liquid nitrogen frozen preservation；The cooling process are as follows: at 4 DEG C
Keep 10min；- 20 DEG C are reduced to 1 DEG C/min rate；It is cooled to -80 DEG C~-90 rapidly with the rate of 50 DEG C~60 DEG C/min
DEG C, it keeps for 24 hours, being transferred quickly in liquid nitrogen；
The vitrification frozen stock solution, human serum albumin solution, dimethyl sulfoxide, propylene glycol, hydroxyl second by mass concentration for 20%
Base urea and trehalose composition, wherein each component proportion are as follows: 15~22wt% of dimethyl sulfoxide, 10~18wt% of propylene glycol,
Ethoxy 10~16wt% of urea, 8~15wt% of trehalose, surplus is people's albumin solution.
2. human placenia Mo Xia great according to claim 1 vascular tissue prepares cryopreservation methods, it is characterised in that: institute
It states in step (2), when cleaning is cleaned with physiological saline or PBS buffer solution.
3. human placenia Mo Xia great according to claim 1 vascular tissue prepares cryopreservation methods, it is characterised in that: institute
It states in step (2), by scissors for vessels at the cylindrical type segment of length 2cm~5cm.
4. human placenia Mo Xia great according to claim 1 vascular tissue prepares cryopreservation methods, it is characterised in that: institute
It states in step (3), each component proportion in the vitrification frozen stock solution are as follows: dimethyl sulfoxide 18wt%, propylene glycol 12wt%,
Ethoxy urea 12wt%, trehalose 12wt%, the human serum albumin solution that surplus is 20%.
5. human placenia Mo Xia great according to claim 1 vascular tissue prepares cryopreservation methods, it is characterised in that: institute
It states in step (3), the three-step approach imports the concrete mode of vitrification frozen stock solution are as follows:
The first step imports the dimethyl sulfoxide and groups of people's albumin solution of trehalose, 50%, in 4 DEG C of balance 10min；
Second step imports the dimethyl sulfoxide and groups of people's albumin solution of ethoxy urea, remaining 50%, balances in 4 DEG C
5min,
Third step imports propylene glycol and groups of people's albumin solution, is immediately placed in programmed cooling instrument；
The volume ratio of the liquid imported three times is 3:1:1.
6. human placenia Mo Xia great according to claim 1 vascular tissue prepares cryopreservation methods, it is characterised in that: institute
It states in step (3), the cooling process are as follows: in 4 DEG C of holding 10min；- 20 DEG C are reduced to 1 DEG C/min rate；With 50 DEG C~60
DEG C/rate of min is cooled to rapidly -80 DEG C~-90 DEG C, it keeps for 24 hours, being transferred quickly in liquid nitrogen.
7. human placenia Mo Xia great according to claim 1 vascular tissue prepares cryopreservation methods, it is characterised in that: institute
It states in step (3), the cooling process are as follows: in 4 DEG C of holding 10min；- 20 DEG C are reduced to 1 DEG C/min rate；With 50 DEG C/min
Rate be cooled to -80 DEG C rapidly, keep for 24 hours, being transferred quickly in liquid nitrogen.
8. a kind of method for resuscitation of human placenia Mo Xia great vascular tissue, it is characterised in that: by the placental villi of freezen protective
Mo Xia great vascular tissue is removed from liquid nitrogen, bag will be frozen rapidly after placing gas phase 10min balance or cryopreservation tube be placed in 37 DEG C~
In 42 DEG C of water-baths, or number of patent application is used to recover for device disclosed in 2017107960072 patent application；
Bag will be frozen rapidly after dissolution or cryopreservation tube is transferred in safety cabinet or super-clean bench, and opening freezes bag or cryopreservation tube, light with tweezers
Chorionic villi of placenta Xia great vascular tissue is beaten easily out, is put into 4 DEG C of three times benchmark concentration resuscitation fluid, is balanced 1 minute, is taken out, then
It is put into 4 DEG C of two times of benchmark concentration resuscitation fluids, balances 1~3 minute, take out, place into 4 DEG C of one times of benchmark concentration resuscitation fluid
In, it balances 3 minutes；It rinses；
The resuscitation fluid is made of trehalose, Dextran 40, alanine, glycine and Hydroxyethyl starch sodium chloride injection；
The concentration of each component in one times of benchmark concentration resuscitation fluid are as follows: trehalose accounts for 5wt%, and Dextran 40 accounts for 6wt%, alanine
3wt% is accounted for, glycine accounts for 3wt%, and surplus is that Hydroxyethyl starch sodium chloride injection or MEM cache solution；
The concentration of each component in two times of benchmark concentration resuscitation fluids are as follows: trehalose accounts for 10wt%, and Dextran 40 accounts for 12wt%, the third ammonia
Acid accounts for 6wt%, and glycine accounts for 6wt%, and surplus is that Hydroxyethyl starch sodium chloride injection or MEM cache solution；
The concentration of each component in three times benchmark concentration resuscitation fluid are as follows: trehalose accounts for 15wt%, and Dextran 40 accounts for 18wt%, the third ammonia
Acid accounts for 9wt%, and glycine accounts for 9wt%, and surplus is that Hydroxyethyl starch sodium chloride injection or MEM cache solution.
9. the method for resuscitation of human placenia Mo Xia great according to claim 8 vascular tissue, it is characterised in that: " balance 3
Minute " after, it is rinsed 2~5 times, is put into 4 DEG C of physiological saline or PBS solution with 4 DEG C of physiological saline or PBS solution, stood.
10. the tissue in placenta source or the method for building up of cellular resources sample database, it is characterised in that: using in claim 1~7
Described in any item freezing and storing methods handle Human plactnta and store.
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| CN110892890A (en) * | 2019-12-17 | 2020-03-20 | 银丰低温医学科技有限公司 | Low-temperature preservation method and rewarming method for blood vessels |
| CN111602651A (en) * | 2020-06-20 | 2020-09-01 | 河南和泽干细胞基因工程有限公司 | Placental cell freezing device and freezing method thereof |
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| CN110892890A (en) * | 2019-12-17 | 2020-03-20 | 银丰低温医学科技有限公司 | Low-temperature preservation method and rewarming method for blood vessels |
| CN110892890B (en) * | 2019-12-17 | 2021-09-17 | 银丰低温医学科技有限公司 | Low-temperature preservation method and rewarming method for blood vessels |
| CN111602651A (en) * | 2020-06-20 | 2020-09-01 | 河南和泽干细胞基因工程有限公司 | Placental cell freezing device and freezing method thereof |
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