CN109797147A - A kind of biological sample freeze-thaw method for vitrification of combination laser technology - Google Patents
A kind of biological sample freeze-thaw method for vitrification of combination laser technology Download PDFInfo
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- CN109797147A CN109797147A CN201910096921.5A CN201910096921A CN109797147A CN 109797147 A CN109797147 A CN 109797147A CN 201910096921 A CN201910096921 A CN 201910096921A CN 109797147 A CN109797147 A CN 109797147A
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Abstract
The invention discloses a kind of biological sample freeze-thaw method for vitrification of combination laser technology.It includes S1, leaching is placed in the freezing liquid containing nano-C particles and keeps quiet leaching state at least 1.5min after taking out biological sample in Cell Buffer, then by include cell sample freezing liquid droplet transfer to glass freezing carry bar on, finally directly will glass freezing carry bar immerse liquid nitrogen in；S2, after being removed from liquid nitrogen glass freezing load bar, apply heat to drop using laser pulse；S3, the cell sample after defrosting is sequentially washed in the descending multiple thawing solutions of sucrose concentration content；S4, the cell sample after recovery is transferred in carbon dioxide incubator and is cultivated.The present invention carries out vitrificated cryopreserration processing to biological sample using the freezing liquid of low concentration, handles the quick-thawing of biological sample and combine subsequent recovery processing step using laser realization, can greatly improve survival rate and potentiality of development of the biological sample in the glassy state storage liquid of low concentration.
Description
Technical field
The present invention relates to technical field of bioengineering, especially a kind of biological sample glass freezing of combination laser technology
Defreezing method.
Background technique
Assisted reproductive technology is a kind of to integrate the technologies such as embryo separating, chimeric, transplanting, in vitro fertilization, transgenosis
Integrated technology, the not still important technology revolution in animal reproduction field, and the important technology hand that the treatment mankind are infertile
Section can sufficiently excavate the heredity and fertility, the rear algebra for increasing purebred animal and excellent individual of animal using this technology
Amount, the improvement period for shortening domestic animal and preservation have embryo and the gene of excellent hereditary capacity, in biology and medical domain
Be extensively studied and apply.Wherein, the glass freezing of biological sample (such as ovum, embryo) and defrosting processing are auxiliary
Two the most conventional operational sequences in reproductive technology；Such as: the glass freezing of embryo and defrosting processing can make patient at one
With the chance of multiple embryo transfer in the treatment cycle of IVF-ET (vitro fertilization-embryo implanting, it may be assumed that test-tube baby), greatly
Ground improves the success rate for the treatment of, and embryo vitrifying freeze and the quality of defrosting then can the treatment knot of direct relation to the end
Office.
Currently, the method for most common vitrificated cryopreserration biological sample is that biological sample is placed in high concentration in the industry
Save liquid in carry out short time processing, then again rapidly in liquid nitrogen cooling and the long-term freezen protective in liquid nitrogen.Biological sample
This defreezing method is then to carry out defrosting processing to the biological sample being removed from liquid nitrogen using 37 DEG C of water-bath mode.Wherein,
During vitrificated cryopreserration, due to save biological sample solution (that is: cryopreservation solution) have higher concentration and
Viscosity can inhibit the formation of ice-nucleus, to avoid the formation of intraor extracellular ice crystal when temperature is lower than fusing point；But it is thawing
In the process, if Thawing Rate is not fast enough, when being higher than glass transition temperature, it may result in ice crystal and quickly form and give birth to
It is long, and then fatal damage is caused to biological sample；And traditional water-bath thawing mode be then conduct and convection current by way of pair
Biological sample is heated, and is affected by biological sample size, preservation carrier itself and external environmental condition, defrosting sample
Rate be limited by very large.Therefore, it is influenced by above-mentioned factor, so that biological sample, especially ovum is as human body
Maximum cell has that survival rate and potentiality of development are relatively low after thawing.Research in recent years also indicates that: Thawing Rate
It is the most important factor for influencing cell and obtaining high-survival rate.
In consideration of it, it is necessary to existing biological sample vitrificated cryopreserration and defrosting processing method propose improvement side
Case.
Summary of the invention
In view of the deficiency of the prior art, the purpose of the present invention is to provide a kind of biologies of combination laser technology
Sample glass freeze-thaw method.
To achieve the goals above, the present invention adopts the following technical scheme:
A kind of biological sample freeze-thaw method for vitrification of combination laser technology, it the following steps are included:
S1, cell sample freezing: after taking out cell sample in Cell Buffer, leaching is placed in the freezing containing nano-C particles
In liquid and keep quiet leaching state at least 1.5min, then by include cell sample freezing liquid droplet transfer it is cold to vitrifying
Freeze and carry on bar and by the control of the size of drop within 3 μ L, finally will directly be carried out in glass freezing load bar immersion liquid nitrogen cold
Freeze and saves；
S2, cell sample thaw: after glass freezing load bar is removed from liquid nitrogen, being applied using laser pulse to drop
Heat makes drop take the lead in thawing and transferring heat to the cell sample in drop simultaneously after absorbing laser heat with final real
The defrosting of existing cell sample；
S3, the removal of cryopreservative ingredient: the cell sample after defrosting is sequentially descending in sucrose concentration content
Multiple thawing solutions in washed, to remove the low-temperature protection material composition in cell sample；
S4, cell sample culture: the cell sample after recovery is transferred in carbon dioxide incubator and is cultivated.
Preferably, the freezing liquid is ethylene glycol of the basic liquid by 0.7mol/kg, 0.71mol/ with PBS Cell Buffer
The acetamide of kg, the sucrose of 0.5mol/kg, the ficoll of 0.0062mol/kg and 0.4 μ g/ml nano-C particles mix.
Preferably, in step s3, the cell sample after defrosting the molality in sucrose is first dipped into exist
At least 1min is washed in the thawing solution of 1.0mol/kg, and then the molality that cell sample is immersed in sucrose exists again
At least 5.0min is washed in the thawing solution of 0.5mol/kg, and cell sample is finally immersed in the thawing solution without containing sugar composition
Middle washing at least 5.0min, to remove the low-temperature protection material composition in cell sample.
As the above scheme is adopted, the present invention carries out biological sample by using the freezing liquid of low concentration (nontoxicity)
Vitrificated cryopreserration processing, and by subsequent defrosting recovery processing step, it is big etc. using the had energy density of laser
Feature improves the Thawing Rate of freezing biological sample, realizes that the supper-fast defrosting to biological sample is handled, is solving biological sample
It can avoid causing due to the generation because of deglass phenomenon or recrystallization phenomenon quickly through glass transition temperature during jelly
The formation of cell interior ice crystal, thus greatly improve survival rate of the biological sample in low concentration glassy state storage liquid and
Potentiality of development is conducive to improve the utilization rate for biological samples such as ovum, embryos, especially for important diseases model
The fertility preservation of experimental animal, economic big animal and the mankind has very important significance.
Specific embodiment
The embodiment of the present invention is described in detail below, but what the present invention can be defined by the claims and cover
Multitude of different ways is implemented.
Embodiment one
A kind of biological sample freeze-thaw method for vitrification of combination laser technology, it the following steps are included:
S1, cell sample freezing: the ovum that will acquire, which soaks to be placed in Cell Buffer (that is: PBS solution) first, to carry out in advance
Processing, ovum is then taken out from Cell Buffer, is soaked and is placed in the freezing liquid containing nano-C particles and keeps quiet leaching shape
Then state at least 1.5min will include that droplet transfer to the glass freezing of freezing liquid of ovum carries on bar and by drop again
Size controls within 3 μ L, and glass freezing is directly finally carried bar within the 2min for completing above-mentioned steps and is immersed in liquid nitrogen
Carry out freezen protective；Ovum is completed in freezing liquid after quiet leaching, before droplet transfer to glass freezing is carried bar, it can benefit
Ovum sufficiently wash to clean buffer entrained by ovum table side with freezing liquid；
S2, cell sample thaw: being 1064 μm, power using wavelength after glass freezing load bar is removed from liquid nitrogen
Laser pulse less than 10kw applies heat to drop, and drop is made to take the lead in thawing and simultaneously passing heat after absorbing laser heat
The ovum in drop is passed finally to realize the defrosting of ovum；This process is mainly inhaled using the carbon granules being scattered in freezing liquid
Receive quick-thawing of the laser energy realization to drop and comprising ovum in the inner from outside to inside；
S3, the removal of cryopreservative ingredient: by the ovum after defrosting sequentially in descending more of sucrose concentration content
Washing is balanced in a thawing solution, to remove the low-temperature protection ingredient of ovum table side；
S4, cell sample culture: the ovum after recovery is transferred in carbon dioxide incubator and is cultivated, body is then made
Outer fertilization, the case where observing subsequent egg development.
Embodiment two
A kind of biological sample freeze-thaw method for vitrification of combination laser technology, it the following steps are included:
S1, cell sample freezing: the 2 cell stage embryos that will acquire soak first to be placed in Cell Buffer (that is: PBS solution)
It is pre-processed, 2 cell stage embryos is then taken out from Cell Buffer, is soaked and is placed in the freezing liquid containing nano-C particles
And keep quiet leaching state at least 1.5min, then again by include 2 cell stage embryos freezing liquid droplet transfer to vitrifying
Freezing carries on bar and by the control of the size of drop within 3 μ L, finally directly by glass within the 2min for completing above-mentioned steps
Change in freezing load bar immersion liquid nitrogen and carries out freezen protective；Turn after quiet leaching, by drop in freezing liquid in 2 cell stage embryos of completion
It moves to before glass freezing load bar, sufficiently washing is carried out to clean 2 cell stage embryos to 2 cell stage embryos using freezing liquid
The buffer of table side；
S2, cell sample thaw: being 1064 μm, power using wavelength after glass freezing load bar is removed from liquid nitrogen
Laser pulse less than 10kw applies heat to drop, and drop is made to take the lead in thawing and simultaneously passing heat after absorbing laser heat
2 cell stage embryos in drop are passed finally to realize the defrosting of 2 cell stage embryos；This process, which mainly utilizes, is scattered in freezing
Carbon granules in liquid absorbs and transfer laser energy is realized to drop and comprising from outside to inside fast of 2 cell stage embryos in the inner
Speed is thawed；
S3, cryopreservative ingredient removal: by 2 cell stage embryos after defrosting sequentially sucrose concentration content by greatly to
It is balanced washing in small multiple thawing solutions, to infiltrate into the low-temperature protection material composition of inside embryo before removal freezing；
S4, cell sample culture: 2 cell stage embryo transfers after recovery are cultivated into carbon dioxide incubator.
Preferably, the freezing liquid of the present embodiment one and two with PBS Cell Buffer be basic liquid and mainly by
The ethylene glycol of 0.7mol/kg, the acetamide of 0.71mol/kg, the sucrose of 0.5mol/kg, 0.0062mol/kg ficoll and
The nano-C particles of 0.4 μ g/ml mix.Freezing liquid made of ingredient configuration according to this, plays the low temperature as protection cell
The concentration of the ingredient (such as ethylene glycol, acetamide) of preservative agent is very low, to be equivalent to the toxicity and energy for reducing freezing liquid itself
Enough play the role of protecting cell, creates prerequisite to carry out quick-thawing using laser.
Preferably, in the step S3 of embodiment one and two, by cell sample (that is: the ovum or 2 thin after defrosting
Born of the same parents' phase embryo) molality that is first dipped into sucrose washs at least 1min in the thawing solution of 1.0mol/kg, then again
The molality that cell sample is immersed in sucrose is washed at least 5.0min in the thawing solution of 0.5mol/kg, finally will
Cell sample, which is immersed in the thawing solution without containing sugar composition, washs at least 5.0min, to remove the guarantor of the low temperature in cell sample
Protect material composition.
Based on this, vitrificated cryopreserration processing is carried out to biological sample by using the freezing liquid of low concentration, and lead to
Cross subsequent recovery processing step, it is big using the had energy density of laser the features such as, in a short time promptly improve freezing
Biological sample heating rate, realize and the supper-fast defrosting of biological sample handled, enable biological sample quickly through glass
Glass transition temperature, to greatly improve the survival rate and potentiality of development of biological sample.
Using ovum and 2 cell stage embryos as the object for implementing this method, by using traditional defreezing method and laser solution
Jelly method carries out defrosting processing, can sufficiently verify the implementation and beneficial effect of this method.
It can be seen that treated the biological sample of the method through the present embodiment is compared to using tradition from table one and table two
The biological sample of defreezing method processing, either ovum or 2 cell stage embryos, survival rate and potentiality of development have obtained significantly
Raising, especially the survival rate of ovum and developmental rate are greatly improved in table one.
In conclusion combining laser technology, low concentration vitrificated cryopreserration, defrosting and subsequent provided by the present embodiment
Method for resuscitation, can be using very low concentrations and in the case where almost without the freezing liquid of toxicity, improving biological sample guarantor
The survival rate and potentiality of development deposited are conducive to improve the survival rate and developmental potency for biological samples such as ovum, embryos,
It saves especially for the fertility of the experimental animal of important diseases model, economic big animal and the mankind with very important meaning
Justice.
The above description is only a preferred embodiment of the present invention, is not intended to limit the scope of the invention, all utilizations
Equivalent structure or equivalent flow shift made by present specification is applied directly or indirectly in other relevant technologies
Field is included within the scope of the present invention.
Claims (3)
1. a kind of biological sample freeze-thaw method for vitrification of combination laser technology, it is characterised in that: it the following steps are included:
S1, cell sample freezing: after taking out cell sample in Cell Buffer, leaching is placed in the freezing liquid containing nano-C particles
And keep quiet leaching state at least 1.5min, then by include cell sample freezing liquid droplet transfer to glass freezing carry
On bar and by the control of the size of drop within 3 μ L, finally directly glass freezing is carried in bar immersion liquid nitrogen and carries out freezing guarantor
It deposits；
S2, cell sample thaw: after glass freezing load bar is removed from liquid nitrogen, applying heat to drop using laser pulse
Amount makes drop take the lead in defrosting and the cell sample that transfers heat in drop simultaneously after absorbing laser heat finally to realize
The defrosting of cell sample；
S3, the removal of cryopreservative ingredient: by the cell sample after defrosting sequentially in descending more of sucrose concentration content
It is washed in a thawing solution, to remove the low-temperature protection material composition in cell sample；
S4, cell sample culture: the cell sample after recovery is transferred in carbon dioxide incubator and is cultivated.
2. a kind of biological sample freeze-thaw method for vitrification of combination laser technology as described in claim 1, feature exist
In: the freezing liquid with PBS Cell Buffer be basic liquid by the ethylene glycol of 0.7mol/kg, 0.71mol/kg acetamide,
The nano-C particles of the sucrose of 0.5mol/kg, the ficoll of 0.0062mol/kg and 0.4 μ g/ml mix.
3. a kind of biological sample freeze-thaw method for vitrification of combination laser technology as described in claim 1, feature exist
In: in step s3, the cell sample after defrosting is first dipped into the molality in sucrose in the defrosting of 1.0mol/kg
At least 1min is washed in liquid, then cell sample is immersed in thawing solution of the molality in 0.5mol/kg of sucrose again
Cell sample, is finally immersed in the thawing solution without containing sugar composition and washs at least 5.0min by middle washing at least 5.0min,
To remove the low-temperature protection material composition in cell sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763623A (en) * | 2020-07-21 | 2020-10-13 | 四川省人民医院 | Auxiliary activation device for frozen embryos |
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|---|---|---|---|---|
| CN101779623A (en) * | 2010-03-11 | 2010-07-21 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| CN104745528A (en) * | 2015-03-13 | 2015-07-01 | 深圳普若赛斯生物科技有限公司 | Method for performing cryopreservation resuscitation on oocyte or embryo and resuscitation solution used therein |
| WO2017184721A1 (en) * | 2016-04-19 | 2017-10-26 | Regents Of The University Of Minnesota | Cryopreservation compositions and methods involving nanowarming |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101779623A (en) * | 2010-03-11 | 2010-07-21 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| CN104745528A (en) * | 2015-03-13 | 2015-07-01 | 深圳普若赛斯生物科技有限公司 | Method for performing cryopreservation resuscitation on oocyte or embryo and resuscitation solution used therein |
| WO2017184721A1 (en) * | 2016-04-19 | 2017-10-26 | Regents Of The University Of Minnesota | Cryopreservation compositions and methods involving nanowarming |
Non-Patent Citations (1)
| Title |
|---|
| BO JIN等: "Survivals of mouse oocytes approach 100% after vitrification in", 《CRYOBIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763623A (en) * | 2020-07-21 | 2020-10-13 | 四川省人民医院 | Auxiliary activation device for frozen embryos |
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Application publication date: 20190524 |