CN104738028B - A kind of stem cell quick-freezing method and the freezing liquid of use thereof - Google Patents
A kind of stem cell quick-freezing method and the freezing liquid of use thereof Download PDFInfo
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Abstract
The invention provides a kind of stem cell quick-freezing method and the freezing liquid of use thereof, described stem cell quick-freezing method comprises the following steps: balanced in cryoprotective extender by stem cell；Go to stem cell No. 1 freezing liquid balances；After going to stem cell No. 2 freezing liquids balance, stem cell is transferred in support tube；Support tube keeps level near liquid nitrogen vapor layer, and carries out rotating to being vertically to liquid nitrogen liquid level to liquid nitrogen liquid level with support tube either end for the center of circle, make support tube is loaded with being partially submerged in liquid nitrogen of cell.Technical scheme can preferably protect stem cell when carrying out rapid glass freezing; it is effectively improved stem cell recovery survival rate after glass freezing, and the totipotency such as Growth and Differentiation and ability to express of keeping stem cell after Cryopreservation is unaffected；Especially for the stem cell that some are very sensitive, recovery survival rate can bring up to more than 95%.
Description
Technical field
The present invention relates to biological technical field, particularly relate to method and the freezing of use thereof of a kind of stem cell quick freezing
Liquid.
Background technology
Existing stem cell freezing method is typically with slow freezing method, namely relies on program mode cooling instrument to carry out journey
Sequence is lowered the temperature, and slow freezing, this frozen process can significantly change the thermodynamics of cell, chemically and physically environment, and accompany
With causing biological injury, even if adopting stem cell successful resuscitation in this way, still its totipotency also can be undermined.
In recent years along with the development of science and technology, glass freezing is subject to because of being formed without ice crystal in cooling refrigerating process
Concern to everybody.Vitrification refers to that liquid is changed into the solidification process of amorphous state (glassy state).Glassy solids molecule
Between relation and liquid without being clearly distinguished from, in cooling refrigerating process, be formed without ice crystal, cellularity will not be by broken
Bad thus cell is survived, and is a kind of preferably freezen protective approach.
At present, in the glass freezing field of stem cell, a lot of enterprises and research staff are primarily upon freezing liquid, and right
Quick-freezing method in stem cell is paid close attention to seldom, it practice, the survival rate that stem cell recovers after glass freezing
In addition to having the biggest relation with freezing liquid, it also has the biggest with the method for whole stem cell glass freezing and recovery
Relation；Compared with the method that the method for the stem cell glass freezing that existing the art uses is freezing with programmed cooling,
After recovery, the survival rate of stem cell improves a lot, but for the few stem cell of some quantity, recovery survival
Rate is not enough so that it is when the follow-up stem cell used after recovering because quantity is little, does not also have well effect.
Summary of the invention
For above technical problem, the invention provides a kind of stem cell quick-freezing method and the freezing liquid of use thereof,
Further increase stem cell recovery survival rate after glass freezing.
Contrast, a kind of method that the invention provides stem cell quick freezing, mainly comprise the steps that
Step A: stem cell is balanced 1～5 minute in the cryoprotective extender of 36～39 DEG C of preheatings；Allow stem cell with freezing
Basal liquid is fully contacted.
Step B: go to stem cell balance 2～5 minutes in No. 1 freezing liquid of 36～39 DEG C of preheatings；
Step C: after going to stem cell balance 20～60s in No. 2 freezing liquids of 36～39 DEG C of preheatings, extracts containing dry
The mixed liquor of cell is transferred in support tube；
Step D: the support tube being loaded with stem cell is carried out glass freezing: the support tube being loaded with stem cell protected
Water holding level state is near liquid nitrogen vapor layer extremely distance liquid nitrogen liquid level 5～10mm, by support tube with support tube either end as circle
The heart carries out rotating to being vertically to liquid nitrogen liquid level to liquid nitrogen liquid level, and make support tube is loaded with cell be partially submerged to liquid
In nitrogen, rotational time is not more than 5s；
Wherein, described No. 1 freezing liquid and No. 2 freezing liquids are the freezing liquid comprising identical cryoprotective extender, and described 1
In number freezing liquid, the concentration of cryoprotective extender is more than the concentration of cryoprotective extender in described No. 2 freezing liquids, the most described No. 1
In freezing liquid, the concentration of other materials is less than the concentration of other materials in described No. 2 freezing liquids, and so, stem cell is 1
In number freezing liquid after balance, transfer to No. 2 freezing liquids that concentration is higher balance, progressively adapt to and balance, this mistake
Journey is more conducive to make the hydrone in stem cell dissociate and out enters in freezing liquid, at the glass freezing of follow-up stem cell
Time, reduce the infringement to stem cell, preferably improve stem cell recovery survival rate after glass freezing.
Described cryoprotective extender can use pre-freeze liquid well known in the prior art, described No. 1 freezing liquid and No. 2 freezings
Liquid can use freezing liquid well known in the prior art, meets the concentration of cryoprotective extender in No. 1 freezing liquid and is more than No. 2
The concentration of cryoprotective extender in freezing liquid；Can be that the component in No. 1 freezing liquid and No. 2 freezing liquids is identical,
Simply proportioning is different, and in No. 1 freezing liquid, the concentration of cryoprotective extender is more than the concentration of cryoprotective extender in No. 2 freezing liquids,
In No. 1 freezing liquid, other material concentrations are less than the concentration of other materials in No. 2 freezing liquids.
In described step A, the consumption of cryoprotective extender is that each population of stem cells is not less than 5 μ L；In described step B,
The consumption of No. 1 freezing liquid is that each population of stem cells is not less than 5 μ L；In described step C, the consumption of No. 2 freezing liquids is
Each population of stem cells is not less than 10 μ L.Preferably, the consumption of described cryoprotective extender is each population of stem cells 5～25 μ L,
The consumption of described No. 1 freezing liquid is each population of stem cells 10 μ L～50 μ L；The consumption of described No. 2 freezing liquids is each
Population of stem cells 10 μ L～100 μ L.
In described step D by support tube with support tube either end for the center of circle when liquid nitrogen liquid level rotates, preferably with carry
Bigger one end, body mouth of pipe footpath is that the center of circle rotates to liquid nitrogen liquid level；Optimizing further, support tube is OPS, with greatly
Bore end is that the center of circle rotates to liquid nitrogen liquid level.
Described support tube is more excellent is the straw of capillary apertures for one end, is so easy to stem cell and is quickly loaded into carrier
Guan Zhong.Optimizing further, described support tube is OPS.
Above-mentioned steps, more excellent operation under ambient temperature is 25～27 degree.
Use technique scheme, stem cell is carried out substep put down at cryoprotective extender, No. 1 freezing liquid and No. 2 freezing liquids
Weighing apparatus, makes the most slowly to balance between stem cell and freezing liquid, reduces concentration difference between the two, can preferably protect dry
Cell so that it is reduce infringement as far as possible；The method using step D allows the support tube being loaded with stem cell rotate, and makes
Its first Fluid Contacting nitrogen vapor, contact liquid nitrogen liquid level the most gradually, until being totally immersed in liquid nitrogen, allow stem cell
Gradually adapt to the liquid nitrogen environment of low temperature, reduce the infringement to stem cell, be conducive to improving stem cell through glass freezing
After recovery survival rate.
As a further improvement on the present invention, in described step D, by support tube with support tube either end for the center of circle to liquid
When nitrogen liquid level rotates, first rotating to support tube and horizontal plane angle is 30～45 degree, stops 0.5～2s, then revolves
Change into and be perpendicular to liquid nitrogen liquid level.Use this technical scheme, first the support tube being loaded with stem cell is rotated to closer to liquid
The state of nitrogen liquid level, stops 0.5～2s in the liquid nitrogen vapor that temperature is lower, progressively adapts to low temperature environment, revolve
Turn until be immersed in liquid nitrogen, then adapt to the liquid nitrogen of lower temperature, use and this stepped gradually adapt to liquid nitrogen, subtract
Few infringement to stem cell, preferably improves stem cell recovery survival rate after glass freezing.
As a further improvement on the present invention, in described step D, by support tube with support tube either end for the center of circle to liquid
When nitrogen liquid level rotates, first rotating to support tube and horizontal plane angle is 30～45 degree, stops 0.5～1.5s, then continues
Continuous rotate to horizontal plane angle 60～70 degree, stop 0.5～1.5s, be finally rotated into and be perpendicular to liquid nitrogen liquid level.Use more
Further staged rotates, and gradually adapts to liquid nitrogen, decreases the infringement to stem cell, preferably improves stem cell warp
Cross the recovery survival rate after glass freezing.
As a further improvement on the present invention, described step D, be loaded with cell in support tube is partially submerged to liquid nitrogen
After in, support tube is kept in liquid nitrogen 2～5s, then sink to support tube liquid nitrogen preserves.Preferably, will carry
Body pipe keeps 2～3s in liquid nitrogen.Use this technical scheme, can further adapt to liquid nitrogen environment, improve cooling
Speed, the time balanced with liquid nitrogen temperature, reaches the purpose of rapid glass, preferably protects after reducing contact liquid nitrogen
Stem cell, improves stem cell recovery survival rate after glass freezing, and after making recovery, stem cell totipotency is not by shadow
Ring.
As a further improvement on the present invention, described step D, be loaded with cell in support tube is partially submerged to liquid nitrogen
After in, by support tube paddling 2～5s in liquid nitrogen, then sink to support tube liquid nitrogen preserves.Preferably, will carry
Body pipe is paddling 2～3s in liquid nitrogen.Use this technical scheme, can further adapt to liquid nitrogen environment, improve cooling
Speed, the time balanced with liquid nitrogen temperature, reaches the purpose of rapid glass, preferably protects after reducing contact liquid nitrogen
Stem cell, improves stem cell recovery survival rate after glass freezing, and after making recovery, stem cell totipotency is not by shadow
Ring.
As a further improvement on the present invention, also include step E: support tube sealed, then sink to liquid nitrogen is protected
Deposit.Using this technical scheme, stem cell can be avoided to be contaminated when freezen protective, more preferable freezen protective is dry thin
Born of the same parents；Preferably, support tube can be encased in protection pipe and seal, then sink to liquid nitrogen preserves.
As a further improvement on the present invention, in described step B, when stem cell is gone to No. 1 freezing liquid balance, will
Stem cell is placed on liquid level or the drop top of No. 1 freezing liquid, allows stem cell naturally sink.Use this technical scheme, allow
Stem cell sinks naturally, contact refrigeration liquid, the minimizing stimulation to stem cell at leisure, is more beneficial for protecting stem cell,
Improving stem cell recovery survival rate after glass freezing, after making recovery, stem cell totipotency is unaffected.
As a further improvement on the present invention, in described step C, go to stem cell No. 2 freezing liquids carry out substep
Balance, described No. 2 freezing liquids are divided into the one No. 2 freezing liquid and the 2nd No. 2 freezing liquid, first stem cell are gone to the 1st
Mixing in number freezing liquid, the first incorporation time is 10～60s；Then go to stem cell the 2nd No. 2 freezing liquid mixes,
Second incorporation time is 10～60s.Use this technical scheme, make stem cell progressively balance in freezing liquid, make stem cell
Hydrone free further out enter in freezing liquid so that in the rapid glass freezing of follow-up stem cell,
It is not easily formed ice crystal, preferably protects stem cell, improve stem cell recovery survival rate after glass freezing, and
After making recovery, stem cell totipotency is unaffected.Wherein, described one No. 2 freezing liquid and the 2nd No. 2 freezing liquid are phase
Same freezing liquid is individually positioned in two different culture apparatuses, or identical freezing liquid is at same culture apparatus
Two diverse locations, one of them named one No. 2 freezing liquid, named 2nd No. 2 freezing liquid of another one,
The title that may be used without other is named, just to making a distinction in subsequent operation.
As a further improvement on the present invention, described first incorporation time is 15～25s, and described second incorporation time is
15～30s.Use this technical scheme, protect stem cell further so that be freezing in the rapid glassization of follow-up stem cell
In, it is not easily formed ice crystal, preferably protects stem cell.
Present invention also offers the freezing liquid used in a kind of stem cell quick-freezing method as above, described freezing
Liquid includes cryoprotective extender, No. 1 freezing liquid and No. 2 freezing liquids；Component and volume that described cryoprotective extender is comprised contain
Amount is: blood serum substituting replenisher 15-25%, Hepes-TCM199 buffer 75-85%.
Component and volume content thereof that described No. 1 freezing liquid comprises be: ethylene glycol 5-15%, dimethyl sulfoxide (DMSO)
5-15% and cryoprotective extender 75-85%；
Component and volume content thereof that described No. 2 freezing liquids comprise be: ethylene glycol 15-25%, dimethyl sulfoxide (DMSO)
15-25% and the cryoprotective extender 55-65% containing 0.5mol/L sucrose.
Preferably, described Hepes-TCM199 buffer is containing 0.22g/L Sodium Pyruvate (Sodium pyruvate)
Hepes-TCM199 buffer with 0.0461g/L l-GLUTAMINE (L-Glutamin).
Use the freezing liquid of this technical scheme, can be that stem cell provides comprehensive nutrition, and don't cause to stem cell
Pollute, preferably protect stem cell, make the stem cell recovery survival rate through glass freezing be improved, after making recovery
Stem cell totipotency is unaffected.
The preparation method of above-mentioned freezing liquid is: at room temperature, respectively by above-mentioned cryoprotective extender, No. 1 freezing liquid and No. 2
The component mixing that freezing liquid comprises, and stir.
The present invention another aspect also relates to a kind of for stem cell freezing test kit, and it comprises the above-mentioned freezing of the present invention
Liquid, preferably, also includes cryoprotective extender.
Method for resuscitation about stem cell can use cell recovery liquid and the method for resuscitation of routine, it is possible to according to the present invention
Resuscitation fluid as provided below and method for resuscitation, the present invention provide resuscitation fluid include recovery basal liquid, No. 1 recovery reagent and
No. 2 recovery reagent；Component and volume content thereof that described basal liquid comprises be: blood serum substituting replenisher 15-25%,
75-85%TCM199 buffer；
Component and volume content thereof that described No. 1 recovery reagent comprises be: the recovery basal liquid of 0.2mol/L sucrose；
Component and volume content thereof that described No. 2 recovery reagent comprise be: the recovery basal liquid of 0.1mol/L sucrose；
Preferably, described TCM199 buffer be containing 0.22g/L Sodium Pyruvate (Sodium pyruvate) and
The Hepes-TCM199 buffer 75-85% of 0.0461g/L l-GLUTAMINE (L-Glutamin)；
Use this technical scheme, can be that stem cell provides comprehensive nutrition, and don't can pollute to stem cell,
Preferably protect stem cell.
Resuscitation process comprises the following steps:
Step A): pre-thermal agent, No. 2 recovery reagent preheatings are reached 25-27 DEG C, is injected in culture apparatus A
To for No. 2 recovery reagent A；The recovery basal liquid that then preheating reaches 25-27 DEG C is injected into culture apparatus B and cultivation
In device C, obtain recovery basal liquid B and recovery basal liquid C.
Wherein, described culture apparatus A, culture apparatus B and culture apparatus C can be different culture apparatuses, in order to
Distinguish, named culture apparatus A, culture apparatus B and culture apparatus C；Described culture apparatus A, culture apparatus B
Can also be the different cavity volume positions of mutually same culture apparatus, in order to distinguish, named culture apparatus with culture apparatus C
A, culture apparatus B and culture apparatus C；Name herein, just to the effect distinguished, can use other name.
Described culture apparatus is preferably four orifice plates, and described culture apparatus A, culture apparatus B and culture apparatus C are respectively
Wherein three hole in four orifice plates.
Step B): keeping sealing to preheat No. 1 recovery reagent, and preheated by culture apparatus, preheating temperature is
36～39 degree, preheating time is more than 30min；Preheated reagent is injected in preheated culture apparatus, and will dress
The culture apparatus of good reagent is put under the microscope；Use micrurgy can reduce mistake.
Step C): take out support tube, carry out rewarming；
Support tube is taken out from liquid nitrogen, if support tube is to seal, then opens sealing device；If support tube is
Put into what protection pipe sealed again, then preservation pipe is opened from sealing state, take out support tube.
Then one end support tube being contained cell is placed in above No. 1 recovery reagent 2～5s, is then immersed in No. 1 recovery
In reagent, and the one end making support tube contain cell is totally immersed in recovery reagent, and the immersion angle of support tube is 20 °
To 30 ° (with respect to the horizontal plane), whole immersion operation operates under the microscope.The preferred stereoscopic microscope of described microscope
Or anatomical lens.
Step D): take out the cell in support tube；
Being OPS for support tube, blocked by the other end of support tube, the solution in support tube just flows from support tube
Go out in the culture dish equipped with No. 1 recovery reagent；Or use liquid-transfering device to add air row from the other end of support tube
Except the solution containing stem cell.
Step E): carry out cellular-restoring；
Stem cell is gone in No. 2 recovery reagent A, stop 1 minute；
Then go to stem cell, in No. 2 recovery reagent B, stop 5 minutes；
Again stem cell is gone in No. 2 recovery reagent C, stop 5 minutes；
Finally stem cell is put in fresh culture medium.Described culture medium is to be conventionally used for the culture medium that cell is cultivated.
In the present invention, without specified otherwise, term " content " is volume content.
Compared with prior art, the invention have the benefit that
Technical scheme can preferably protect stem cell when carrying out rapid glass freezing, is effectively improved
Stem cell recovery survival rate after glass freezing, and keep Growth and Differentiation and the table of stem cell after Cryopreservation
The totipotencies such as Danone power are unaffected；Especially for the stem cell that some are very sensitive, recovery survival rate can bring up to
More than 95%.
Accompanying drawing explanation
Fig. 1 is that the cryovial being loaded with stem cell in the embodiment of the present invention 2 rotates the schematic diagram immersing liquid nitrogen.
Detailed description of the invention
Below in conjunction with the accompanying drawings, the preferably embodiment of the present invention is described in further detail.
Embodiment 1
Prepared by the freezing liquid for stem cell quick freezing:
Described freezing liquid includes cryoprotective extender, No. 1 freezing liquid and No. 2 freezing liquids；
Component and volume content that described cryoprotective extender is comprised be: blood serum substituting replenisher 15%, containing 0.22g/L
Sodium Pyruvate (Sodium pyruvate) and the Hepes-TCM199 of 0.0461g/L l-GLUTAMINE (L-Glutamin)
Buffer 85%；
Component and volume content thereof that described No. 1 freezing liquid comprises be: ethylene glycol 5%, dimethyl sulfoxide (DMSO)
15%, above-mentioned cryoprotective extender 80%；
Component and volume content thereof that described No. 2 freezing liquids comprise be: ethylene glycol 15%, dimethyl sulfoxide (DMSO)
25%, the cryoprotective extender 60% containing 0.5mol/L sucrose.
At room temperature, the component mixing respectively above-mentioned cryoprotective extender, No. 1 freezing liquid and No. 2 freezing liquids comprised, and
Stir, be preheating to 36～39 DEG C, stand-by.
Embodiment 2
Hereinafter operation all operates under the ambient temperature of 25～27 degree.
(1) stem cell glass freezing:
Support tube uses OPS straw, and described OPS straw includes cryovial and protection pipe, and described cryovial one end is big
Bore end, other end is small-caliber end；Freezing liquid uses the freezing liquid that above-described embodiment 1 prepares, stem cell
Glass freezing employing following steps:
Step A: stem cell is balanced 5 minutes in cryoprotective extender；
Step B: stem cell is gone to No. 1 freezing liquid balance, uses the glob top that stem cell is placed on No. 1 freezing liquid
Portion, allows stem cell naturally sink, and waits 5 minutes.
Step C: stem cell goes to carry out in No. 2 freezing liquids substep and balances, on culture dish separated two part 2
Number freezing liquid, respectively obtains the one No. 2 freezing liquid and the 2nd No. 2 freezing liquid, first stem cell is gone to the one No. 2 cold
Freezing in liquid and mix, incorporation time is 15～25s；Then go to stem cell the 2nd No. 2 freezing liquid mixes, during mixing
Between be 15～30s；Extract the mixed liquor containing stem cell to transfer in cryovial；
Above-mentioned steps A, step B and step C are all carried out under the room temperature environment of 25～27 degree.
Step D: by be loaded with stem cell cryovial keep level near liquid nitrogen vapor layer, to distance liquid nitrogen liquid
Face 5～10mm, rotates cryovial for the center of circle to liquid nitrogen liquid level with the heavy caliber end of cryovial, as it is shown in figure 1,
First rotating to cryovial and horizontal plane angle is at 30～45 degree, stops 0.5～1s；Then proceed to rotate to and horizontal plane
Angle 60～70 degree, stop 0.5～1s, be finally rotated into and be perpendicular to liquid nitrogen liquid level, and make to be loaded with the cryovial of cell
Small-caliber end is immersed in liquid nitrogen, by cryovial paddling 2～3s in liquid nitrogen；
Step E: cryovial is encased in protection pipe, carries out adding heat seal, so by the mouth of pipe sealing pincers of protection pipe
After sink in liquid nitrogen preserve.
(2) stem cell resuscitation process:
Resuscitation fluid prepares:
In this example, resuscitation fluid includes recovery basal liquid, No. 1 recovery reagent and No. 2 recovery reagent；
Component and volume content thereof that described recovery basal liquid comprises be: blood serum substituting replenisher 15%, containing 0.22g/L
Sodium Pyruvate (Sodium pyruvate) and the Hepes-TCM199 of 0.0461g/L l-GLUTAMINE (L-Glutamin)
Buffer 85%；
Component and volume content thereof that No. 1 recovery reagent comprises be: the basal liquid of 0.2mol/L sucrose；
Component and volume content thereof that No. 2 recovery reagent comprises be: the basal liquid of 0.1mol/L sucrose；
Stem cell resuscitation comprises the following steps:
Step A): pre-thermal agent, No. 2 recovery reagent preheatings are reached 25-27 DEG C, is injected in the hole 2 of four orifice plates,
Obtain No. 2 recovery reagent A；Then preheating is reached the recovery basal liquid of 25-27 DEG C and is injected into hole 3 and the hole of four orifice plates
In 4, obtaining recovery basal liquid B and recovery basal liquid C, the hole 1 of four orifice plates is vacant；
Step B): keeping sealing to preheat No. 1 recovery reagent, and preheated by culture apparatus, preheating temperature is
36～39 degree, preheating time 60min；Preheated reagent is injected in preheated culture apparatus, and examination will be installed
The culture apparatus of agent is put under the microscope；
Step C): take out support tube, carry out rewarming；
Protection pipe is taken out from liquid nitrogen, cuts off sealed end, cryovial is taken out；Then cryovial is contained cell
Small-caliber end be placed in 2s above No. 1 recovery reagent, be then immersed in 1 with the angle of with respect to the horizontal plane 20 ° to 30 °
In number recovery reagent, and the part containing cell is made in cryovial to be totally submerged in No. 1 recovery reagent；Above-mentioned whole
Immerse operation to operate under stereoscopic microscope；
Step D): take out the cell in support tube；
After step C completes, after the vitrified matter melts in cryovial, the reagent in culture dish flows backwards and enters freezing
Guan Zhong, now, blocks the heavy caliber end of cryovial, and the solution in cryovial is just flowed into equipped with 1 from cryovial
In the culture dish of number recovery reagent；Or use liquid-transfering device to squeeze into air from the heavy caliber end of cryovial to get rid of containing dry
The solution of cell；
Step E): carry out cellular-restoring；
Go to stem cell, in No. 2 recovery reagent A of four orifice plates, stop 1 minute；
Then stem cell is gone in recovery basal liquid B, stop 5 minutes；
Again stem cell is gone in recovery basal liquid C, stop 5 minutes；
Finally stem cell is put in fresh culture medium.Described culture medium is to be conventionally used for the culture medium that cell is cultivated.
According to the method described above stem cell good for 16 batch state is carried out respectively freezing, each batch arrange 4 parallel
Test；And according to above-mentioned method for resuscitation, the freezing stem cell of each batch is recovered, then check depositing of stem cell
Data are added up, are referred to table 1 by the situation of living.
Meanwhile, being provided with matched group, matched group uses the freezing liquid of prior art routine and freezing method to carry out freezing,
And the stem cell survival situation after recovery is added up, refer to table 1.
Survival condition after the chilled rear recovery of stem cell of table 1 embodiment 2 and matched group
Remarks: result after statistics: Mean ± SE:95% ± 0.0625%
Matched group uses the method for prior art, particularly as follows:
The DMSO that freezing liquid is 10% volume content used in matched group and the hyclone of 90% volume content；
The freezing method used in matched group is first cell to be put in freezing liquid, frozen in the refrigerator-freezer of-80 DEG C, then 24
After hour, take out, be put in liquid nitrogen.
The method for resuscitation of matched group is to thaw, after defrosting, at 500rpm in elder generation is put into the water bath of 37 DEG C cell
Centrifugation 5 minutes, add culture fluid and wash 3 times, be then placed on the cell culture fluid of the hyclone of also 10%
Cultivate.
From the data of above-mentioned table 1, use the freezing method of technical scheme, the stem cell after recovery
Survival rate can reach 98%, and average viability reaches 95%, compared with matched group, uses the dry of the technical program
Cell recovery survival rate is higher.
Above content is to combine concrete preferred implementation further description made for the present invention, it is impossible to assert
Being embodied as of the present invention is confined to these explanations.For general technical staff of the technical field of the invention,
Without departing from the inventive concept of the premise, it is also possible to make some simple deduction or replace, should be all considered as belonging to this
The protection domain of invention.
Claims (9)
1. a stem cell quick-freezing method, it is characterised in that comprise the following steps:
Step A: stem cell is balanced 1 ~ 5 minute in the cryoprotective extender of 36 ~ 39 DEG C of preheatings；
Step B: go to stem cell balance 2 ~ 5 minutes in No. 1 freezing liquid of 36 ~ 39 DEG C of preheatings；
Step C: after going to stem cell balance 20 ~ 60s in No. 2 freezing liquids of 36 ~ 39 DEG C of preheatings, extracts the mixed liquor containing stem cell and transfers in support tube；
Step D: keep level near liquid nitrogen vapor layer the support tube being loaded with stem cell, to distance liquid nitrogen liquid level 5 ~ 10mm, carry out rotating to being vertically to liquid nitrogen liquid level to liquid nitrogen liquid level with support tube either end for the center of circle by support tube, and making support tube is loaded with being partially submerged in liquid nitrogen of cell, rotational time is not more than 5s；
Wherein, the concentration of cryoprotective extender during the concentration of cryoprotective extender is more than described No. 2 freezing liquids in described No. 1 freezing liquid；In described step D, by support tube with support tube either end for the center of circle when liquid nitrogen liquid level rotates, first rotate to support tube and horizontal plane angle be 30 ~ 45 degree, stop 0.5 ~ 2s, be then rotated into and be perpendicular to liquid nitrogen liquid level.
Stem cell quick-freezing method the most according to claim 1, it is characterized in that: in described step D, by support tube with support tube either end for the center of circle when liquid nitrogen liquid level rotates, first rotate to support tube and horizontal plane angle be 30 ~ 45 degree, stop 0.5 ~ 1.5s, then proceed to rotate to horizontal plane angle 60 ~ 70 degree, stop 0.5 ~ 1.5s, be finally rotated into and be perpendicular to liquid nitrogen liquid level.
3. according to the stem cell quick-freezing method described in claim 1 to 2 any one, it is characterized in that: after the support tube of described step D is loaded with being partially submerged in liquid nitrogen of cell, support tube is kept in liquid nitrogen 2 ~ 5s, then sinks to support tube liquid nitrogen preserves.
Stem cell quick-freezing method the most according to claim 3, it is characterised in that: after the support tube of described step D is loaded with being partially submerged in liquid nitrogen of cell, by support tube paddling 2 ~ 5s in liquid nitrogen, then sink to support tube liquid nitrogen preserves.
5., according to the stem cell quick-freezing method described in claim 1 to 2 any one, it is characterised in that also include step E: support tube sealed, then sink to liquid nitrogen preserves.
Stem cell quick-freezing method the most according to claim 1, it is characterised in that: in described step B, when stem cell is gone to No. 1 freezing liquid balance, stem cell is placed on liquid level or the drop top of No. 1 freezing liquid, allows stem cell naturally sink.
7. according to the stem cell quick-freezing method described in claim 1 to 2 any one, it is characterized in that: in described step C, stem cell goes to carry out substep in No. 2 freezing liquids balance, described No. 2 freezing liquids are divided into the one No. 2 freezing liquid and the 2nd No. 2 freezing liquid, first going to stem cell mix in the one No. 2 freezing liquid, the first incorporation time is 10 ~ 60s；Then going to stem cell mix in the 2nd No. 2 freezing liquid, the second incorporation time is 10 ~ 60s.
Stem cell quick-freezing method the most according to claim 7, it is characterised in that: described first incorporation time is 15 ~ 25s, and described second incorporation time is 15 ~ 30s.
Stem cell quick-freezing method the most according to claim 1, it is characterised in that: component and volume content thereof that described cryoprotective extender comprises be: blood serum substituting replenisher 15-25%, Hepes-TCM199 buffer 75-85%；
Component and volume content thereof that described No. 1 freezing liquid comprises be: ethylene glycol 5-15%, dimethyl sulfoxide 5-15% and cryoprotective extender 75-85%；
Component and volume content thereof that described No. 2 freezing liquids comprise be: ethylene glycol 15-25%, dimethyl sulfoxide 15-25% and the cryoprotective extender 55-65% containing 0.5mol/L sucrose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510112445.3A CN104738028B (en) | 2015-03-13 | 2015-03-13 | A kind of stem cell quick-freezing method and the freezing liquid of use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510112445.3A CN104738028B (en) | 2015-03-13 | 2015-03-13 | A kind of stem cell quick-freezing method and the freezing liquid of use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104738028A CN104738028A (en) | 2015-07-01 |
| CN104738028B true CN104738028B (en) | 2017-01-04 |
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