CN108812643A - Human placenia membrane tissue prepares cryopreservation methods and application - Google Patents
Human placenia membrane tissue prepares cryopreservation methods and application Download PDFInfo
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- CN108812643A CN108812643A CN201810787958.8A CN201810787958A CN108812643A CN 108812643 A CN108812643 A CN 108812643A CN 201810787958 A CN201810787958 A CN 201810787958A CN 108812643 A CN108812643 A CN 108812643A
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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
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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
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
Abstract
The invention discloses a kind of cryopreservation methods that prepare of human placenia membrane tissue, including：(1) placenta amnion, decidua separation and freeze；(2) it the separation of Chorionic villi of placenta Xia great vascular tissue and freezes；(3) it the separation of placental villi membrane tissue and freezes；(4) it the processing of placental villus tissues and freezes.The present invention gives the method recovered after freezen protective.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, organize inner cell overall survival rate up to 90% or more, the tissue saved can be used not only for separation stem cell, 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 placenia film group are prepared the present invention relates to human placenia membrane tissue
The freezing and storing method and method for resuscitation knitted.
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, cryopreservation methods and application, tool are prepared the present invention provides human placenia membrane tissue
Body is related to the freezing and storing method and method for resuscitation of human placenia membrane tissue.
The present invention is achieved by the following technical solutions：
A kind of human placenia membrane tissue prepares cryopreservation methods, includes 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：Choose 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 together, with physiological saline or PBS
Buffer solution for cleaning, and rinse blood vessel to remove intravascular delay clot, it will be under the big scissors for vessels that connect with chorionic plate.
(3) being removed in step (2) from placental fetal surface, eliminate amnion and big blood vessel after remaining white tissues be
Placental villi membrane tissue (because of the larger also known as chorionic plate of ulking thickness), is longitudinally cut into strip piece, width, thickness for chorionic plate
It is respectively 0.5cm~2cm, length 3cm~15cm；
The strip piece being cut into is put into and freezes bag or cryopreservation tube, frozen stock solution is added in the following manner：Firstly, under the conditions of 4 DEG C,
Frozen stock solution 1 is added, balances 5min；Secondly, frozen stock solution 2 is added under the conditions of 0 DEG C, 15min is balanced；Finally, frozen stock solution 3 is added；Three
The volume ratio of the frozen stock solution of secondary addition is 3:1:1；Then, it is transferred to programmed cooling instrument, is cooled to -80 according to the cooling process of setting
DEG C~-90 DEG C, it is transferred to liquid nitrogen frozen preservation；
The cooling process is：4 DEG C are cooled to, 2~5min is kept；0 DEG C is reduced to 1 DEG C/min rate, keep 5~
10min；It is cooled to -10 DEG C in 5~10min, keeps 5~10min；- 40 DEG C are cooled in 40~60min, then in 1~3min
- 80 DEG C~-90 DEG C are inside down to, 5min is kept.
The frozen stock solution 1, is made of, wherein dimethyl sulfoxide accounts for 5~10wt% MEM culture medium and dimethyl sulfoxide.
The frozen stock solution 2, is made of, wherein dimethyl sulfoxide accounts for 5 MEM culture medium, dimethyl sulfoxide and Dextran 40
~10wt%, Dextran 40 account for 50~70wt%.
The frozen stock solution 3 (with the frozen stock solution in step 1), human serum albumin solution, diformazan by mass concentration for 20%
Base sulfoxide, propylene glycol, ethoxy urea and trehalose composition, wherein each component proportion is：5~10wt% of dimethyl sulfoxide,
5~10wt% of propylene glycol, ethoxy 5~10wt% of urea, 5~10wt% of trehalose, surplus is people's albumin solution.
Big blood vessel under above-mentioned amnion, decidua, chorion, villus membrane tissue according to sequencing successively from placenta removing,
It is processed into suitable size, be reloaded into cryopreservation tube or is frozen in the special containers such as bag, saves that be composed of organization name on container special
Number, sample place into liquid nitrogen container specific position long term storage after being iced to specific temperature, and the specificity number on container is used
Sample is searched when recovery.
After carrying out above-mentioned freezen protective, it can recover, include the following steps whenever necessary：
The placental villi membrane tissue of freezen protective is removed from liquid nitrogen, will be frozen rapidly after placing gas phase 10min balance
Bag or cryopreservation tube are placed in 37 DEG C~42 DEG C water-baths, or use number of patent application for 2017107960072 (publication numbers
Device disclosed in patent application 107365700A) is recovered；Bag will be frozen rapidly after dissolution or cryopreservation tube is transferred to peace
In full cabinet or super-clean bench, opening freezes bag or cryopreservation tube, gently takes out Chorionic villi of placenta with tweezers, is put into 4 DEG C of three times benchmark
It in concentration resuscitation fluid (being cooled to 4 DEG C in advance in advance), balances 1 minute, takes out, place into 4 DEG C of two times of benchmark concentration resuscitation fluids (in advance
It is cooled to 4 DEG C in advance) in, it balances 1~3 minute, takes out, place into 4 DEG C of one times of benchmark concentration resuscitation fluid (being cooled to 4 DEG C in advance in advance)
In, it balances 3 minutes；It is rinsed 2~5 times with 4 DEG C of physiological saline or PBS solution (being cooled to 4 DEG C in advance in advance), is put into 4 DEG C of physiology
In salt water or PBS solution (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 or MEM culture medium when using；
The concentration of each component is in one times of benchmark concentration resuscitation fluid：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 is in two times of benchmark concentration resuscitation fluids：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 is in three times benchmark concentration resuscitation fluid：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.
Chorionic villi of placenta after recovery can be used for separating Chorionic villi of placenta mescenchymal stem cell, placenta source candidate stem cell
Deng.The compositions such as antibody, hormone can also be extracted from Chorionic villi of placenta.
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 membrane tissue of the invention prepares cryopreservation methods and method for resuscitation, has the advantages that：
1) blood vessel, villus membrane tissue under placenta amnion, decidua, chorion have not been carried out respectively to separation, saved, has been placenta
The stem-cell research in 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 villus membrane tissue HE colored graph.
Fig. 2：Freeze rear placental villi membrane tissue recovery HE colored graph.
Fig. 3：Mescenchymal stem cell figure (upper left side), the inducible skeletonization obtained from the placental villi membrane tissue frozen is thin
Born of the same parents scheme (lower left side) and streaming phenotypic map (right side).
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 membrane tissue of embodiment
Placenta acquisition：Free from infection, the healthy placenta without obstetric complication are chosen, 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 removed
Amnion.
(2) the above-mentioned placental fetal surface chorion for having removed amnion and big blood vessel are separated together, are cleaned with physiological saline,
And rinse blood vessel to remove intravascular delay clot, it will be under the big scissors for vessels that connect with chorionic plate.
(3) being removed in step (2) from placental fetal surface, eliminate amnion and big blood vessel after remaining white tissues be
Placental villi membrane tissue (because of the larger also known as chorionic plate of ulking thickness), is longitudinally cut into strip piece for cbor onic plate, width, thickness are each
For 0.5cm~2cm, length 3cm~15cm；
The strip piece being cut into is put into and freezes bag, frozen stock solution is added in the following manner：Firstly, addition freezes under the conditions of 4 DEG C
Liquid 1 balances 5min；Secondly, frozen stock solution 2 is added under the conditions of 0 DEG C, 15min is balanced；Finally, frozen stock solution 3 is added；It is added three times
The volume ratio of frozen stock solution is 3:1:1；Then, it is transferred to programmed cooling instrument, is cooled to -90 DEG C according to the cooling process of setting, transfer
It is saved to liquid nitrogen frozen；
The cooling process is：4 DEG C are cooled to, 2min is kept；It is reduced to 0 DEG C with 1 DEG C/min rate, keeps 10min；
It is cooled to -10 DEG C in 10min, keeps 10min；It is cooled to -40 DEG C in 45min, -90 DEG C are then down in 2min, keeps
5min。
The frozen stock solution 1, is made of, wherein dimethyl sulfoxide accounts for 8wt% MEM culture medium and dimethyl sulfoxide.
The frozen stock solution 2, is made of, wherein dimethyl sulfoxide is accounted for MEM culture medium, dimethyl sulfoxide and Dextran 40
8wt%, Dextran 40 account for 60wt%.
The frozen stock solution 3, human serum albumin solution, dimethyl sulfoxide, propylene glycol, ethoxy by mass concentration for 20%
Urea and trehalose composition, wherein each component proportion is：Dimethyl sulfoxide 8wt%, propylene glycol 6wt%, ethoxy urea
6wt%, trehalose 6wt%, surplus are people's albumin 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 placental villi membrane tissue of freezen protective is removed from liquid nitrogen, place gas phase 10min balance after will freeze rapidly bag or
Cryopreservation tube is 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, uses tweezer
Son gently takes out Chorionic villi of placenta, is put into 4 DEG C of three times benchmark concentration resuscitation fluid (being cooled to 4 DEG C in advance in advance), balances 1 minute,
It takes out, places 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, take out, place into 4
DEG C one times of benchmark concentration resuscitation fluid (being cooled to 4 DEG C in advance in advance) in, balance 3 minutes；Physiological saline with 4 DEG C (is cooled to 4 in advance in advance
DEG C) rinse 2~5 times, it is put into 4 DEG C of physiological saline (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；
The concentration of each component is in one times of benchmark concentration resuscitation fluid：Trehalose accounts for 5wt%, and Dextran 40 accounts for 6wt%, and third
Propylhomoserin accounts for 3wt%, and glycine accounts for 3wt%, and surplus is that Hydroxyethyl starch sodium chloride injection or MEM cache solution；
The concentration of each component is in two times of benchmark concentration resuscitation fluids：Trehalose accounts for 10wt%, and Dextran 40 accounts for 12wt%,
Alanine 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 is in three times benchmark concentration resuscitation fluid：Trehalose accounts for 15wt%, and Dextran 40 accounts for 18wt%,
Alanine accounts for 9wt%, and glycine accounts for 9wt%, and surplus is that Hydroxyethyl starch sodium chloride injection or MEM cache solution.
HE dyeing：Fresh placental villi membrane tissue samples before freezing carries out HE dyeing；Chorionic villi of placenta after recovery
Tissue, sampling carry out HE dyeing；As a result as shown in Figure 1 and Figure 2, it can be seen that：Chorionic villi of placenta institutional framework after recovery is still
It is maintained, it is complete that the placental villi membrane tissue after illustrating cryopreservation resuscitation maintains institutional framework.Compare fresh human placenta chorion
HE coloration result is organized, rear placental villi membrane tissue recovery HE is frozen and dyes, nothing obvious great difference close with flesh tissue,
Illustrate that institutional framework is suitable with flesh tissue after freezing the recovery of placental villi membrane tissue.
Detect the motility rate of recovery villus membrane tissue inner cell：0.25% pancreatin is added after the chorion that recovery obtains is shredded
Digestive juice, 37 DEG C digest 1 hour, add the clostridiopetidase A IV of 2g/L, 37 DEG C digest two hours.With life after 1500 turns/min centrifugation
It manages salt water to be resuspended, crosses cell sieve, sampling, Trypan Blue detects Cell viability.
Using 1 disclosure of embodiment in Chinese invention patent CN201210288706 (Authorization Notice No. CN 102763642B)
Method freeze villus membrane tissue and recover, using same method detect motility rate, the results are shown in Table 1, examines through Cell viability
Display is surveyed, the cell survival rate in amnion tissue is much higher than the prior art up to 95% or more.
1 cryopreservation resuscitation chorion histocyte motility rate situation of table
Chorionic villi of placenta is separable to obtain placenta mesenchyma stem cell (method uses existing public technology), as a result such as Fig. 3
It is shown, as seen from the figure, it can have successfully been isolated to fill between obtaining after the placental villi membrane tissue recovery frozen using method of the invention
Matter stem cell.
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 membrane tissue prepares cryopreservation methods, it is characterised in that：Include the following steps：
(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
It, will be under the big scissors for vessels that connect with chorionic plate except intravascular delay clot；
(3) it the separation of placental villi membrane tissue and freezes：Chorionic plate is longitudinally cut into strip piece；
The strip piece being cut into is put into and freezes bag or cryopreservation tube, frozen stock solution is added in the following manner：Firstly, being added under the conditions of 4 DEG C
Frozen stock solution 1 balances 5min；Secondly, frozen stock solution 2 is added under the conditions of 0 DEG C, 15min is balanced；Finally, frozen stock solution 3 is added；Three times plus
The volume ratio of the frozen stock solution entered is 3:1:1；Then, it is transferred to programmed cooling instrument, is cooled to -80 DEG C according to the cooling process of setting
~-90 DEG C, it is transferred to liquid nitrogen frozen preservation；
The cooling process is：4 DEG C are cooled to, 2~5min is kept；It is reduced to 0 DEG C with 1 DEG C/min rate, keeps 5~10min；
It is cooled to -10 DEG C in 5~10min, keeps 5~10min；It is cooled to -40 DEG C in 40~60min, is then dropped in 1~3min
To -80 DEG C~-90 DEG C, 5min is kept；
The frozen stock solution 1 caches solution by MEM and dimethyl sulfoxide forms, wherein dimethyl sulfoxide accounts for 5~10wt%；
The frozen stock solution 2 is made of MEM caching solution, dimethyl sulfoxide and Dextran 40, wherein and dimethyl sulfoxide accounts for 5~
10wt%, Dextran 40 account for 50~70wt%；
The frozen stock solution 3, by mass concentration be 20% human serum albumin solution, dimethyl sulfoxide, propylene glycol, ethoxy urea and
Trehalose composition, wherein each component proportion is：5~10wt% of dimethyl sulfoxide, 5~10wt% of propylene glycol, ethoxy urea
5~10wt%, 5~10wt% of trehalose, surplus are people's albumin solution.
2. human placenia membrane tissue according to claim 1 prepares cryopreservation methods, it is characterised in that：The step
(2) in, when cleaning, is cleaned with physiological saline or PBS buffer solution.
3. human placenia membrane tissue according to claim 1 prepares cryopreservation methods, it is characterised in that：The step
(3) in, width, the thickness of strip piece are respectively 0.5cm~2cm, and length is 3cm~15cm.
4. human placenia membrane tissue according to claim 1 prepares cryopreservation methods, it is characterised in that：The step
(3) in, the cooling process is：4 DEG C are cooled to, 2min is kept；It is reduced to 0 DEG C with 1 DEG C/min rate, keeps 10min；
It is cooled to -10 DEG C in 10min, keeps 10min；It is cooled to -40 DEG C in 45min, -90 DEG C are then down in 2min, keeps
5min。
5. human placenia membrane tissue according to claim 1 prepares cryopreservation methods, it is characterised in that：The step
(3) in, in the frozen stock solution 1, dimethyl sulfoxide accounts for 8wt%.
6. human placenia membrane tissue according to claim 1 prepares cryopreservation methods, it is characterised in that：The step
(3) in, in the frozen stock solution 2, dimethyl sulfoxide accounts for 8wt%, and Dextran 40 accounts for 60wt%.
7. human placenia membrane tissue according to claim 1 prepares cryopreservation methods, it is characterised in that：The step
(3) in, each component proportion is in the frozen stock solution 3：Dimethyl sulfoxide 8wt%, propylene glycol 6wt%, ethoxy urea
6wt%, trehalose 6wt%, surplus are people's albumin solution.
8. a kind of method for resuscitation of human placenia membrane tissue, it is characterised in that：By the placental villi membrane tissue of freezen protective from
It is taken out in liquid nitrogen, bag will be frozen rapidly after placing gas phase 10min balance or cryopreservation tube is placed in 37 DEG C~42 DEG C water-baths, or
Number of patent application is used to recover for device disclosed in 2017107960072 patent application；It will be frozen rapidly after dissolution
Bag or cryopreservation tube are transferred in safety cabinet or super-clean bench, and opening freezes bag or cryopreservation tube, gently take out Chorionic villi of placenta with tweezers,
It is put into 4 DEG C of three times benchmark concentration resuscitation fluid, balances 1 minute, take out, place into 4 DEG C of two times of benchmark concentration resuscitation fluids,
Balance 1~3 minute is taken out, is placed into 4 DEG C of one times of benchmark concentration resuscitation fluid, balances 3 minutes；It rinses；
The resuscitation fluid is made of trehalose, Dextran 40, alanine, glycine and Hydroxyethyl starch sodium chloride injection,
Concentration is adjusted as solvent by Hydroxyethyl starch sodium chloride injection or MEM culture medium when using；
The concentration of each component is in one times of benchmark concentration resuscitation fluid：Trehalose accounts for 5wt%, and Dextran 40 accounts for 6wt%, alanine
3wt% is accounted for, glycine accounts for 3wt%, and surplus is Hydroxyethyl starch sodium chloride injection or MEM culture medium；
The concentration of each component is in two times of benchmark concentration resuscitation fluids：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 Hydroxyethyl starch sodium chloride injection or MEM culture medium；
The concentration of each component is in three times benchmark concentration resuscitation fluid：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 Hydroxyethyl starch sodium chloride injection or MEM culture medium.
9. the method for resuscitation of human placenia membrane tissue according to claim 8, it is characterised in that：After " balance 3 minutes ",
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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810787958.8A CN108812643B (en) | 2018-07-18 | 2018-07-18 | Preparation and cryopreservation method and application of human placental chorionic villus tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810787958.8A CN108812643B (en) | 2018-07-18 | 2018-07-18 | Preparation and cryopreservation method and application of human placental chorionic villus tissue |
Publications (2)
| Publication Number | Publication Date |
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| CN108812643A true CN108812643A (en) | 2018-11-16 |
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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 |
| CN112569407A (en) * | 2019-12-25 | 2021-03-30 | 北京光捷扬基健康科技有限公司 | Preparation method of placenta tissue engineering immunogen-removing skin scaffold |
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