CN109479870A - A kind of method for glass frozen preservation of biomaterial - Google Patents
A kind of method for glass frozen preservation of biomaterial Download PDFInfo
- Publication number
- CN109479870A CN109479870A CN201710811425.4A CN201710811425A CN109479870A CN 109479870 A CN109479870 A CN 109479870A CN 201710811425 A CN201710811425 A CN 201710811425A CN 109479870 A CN109479870 A CN 109479870A
- Authority
- CN
- China
- Prior art keywords
- liquid
- biomaterial
- buffer
- fibre web
- sucrose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of method for glass frozen preservation of biomaterial, comprising the following steps: 1) prepares buffer；2) biomaterial to be saved is put into buffer and stands 10-60 seconds, be then transferred into EFS20 and stand 10-60 seconds, be then transferred in EFS40 and stand 10-60 seconds；3) biomaterial in EFS40 is transferred to the nylon fibre web on piece that aperture is 30-50 μm；4) container will be frozen to immerse in liquid nitrogen；5) the nylon fibre web piece of step 3) is quickly transferred to the freezing in container of step 4), then will freezes container and be completely immersed in liquid nitrogen, and close the lid, and will be put into liquid nitrogen and saves for a long time；6) when in use, biomaterial is thawed in thawing solution.The present invention also provides a kind of carrier of glass frozen preservation for biomaterial, the carrier includes nylon fibre web piece and freezes container.Method of the invention is easy to operate, quick, and does not allow to easily cause egg mother cell/embryo loss, the recycling after being conducive to embryo thawing.
Description
Technical field
The invention belongs to field of biotechnology, are related to a kind of method for glass frozen preservation of biomaterial, the invention further relates to
Frozen stock solution used in the cryopreservation methods and freeze carrier.
Background technique
Method for glass frozen preservation is the new cryopreservation methods of one kind occurred in recent years, easy to operate, without using valuableness
Precision instrument carry out temperature control.The techniqueflow of glass frozen preservation is that embryo samples or egg mother cell are immersed high concentration first to freeze
Make embryo's fast dewatering in liquid, then the embryo containing frozen stock solution is put on the small freezing carrying tool of volume, will finally contain
The carrying tool of embryo carries out fast cooling (> 2000 (TC/min)), and the high concentration frozen stock solution containing embryo will be by liquid at this time
Quickly become the solid-state of vitrifying sample.Since embryo is quickly turned to solid-state by liquid in the frozen stock solution of high concentration, avoid thin
The formation of intracellular ice crystal reduces ice crystal to cellular damage possibility, plays the role of cryoprotection, thus for embryo after defrosting
Survival and subsequent development play crucial effect.Since glass frozen preservation technology has simple, time saving, high performance-price ratio etc.
Advantage, the technology are widely used to animal reproduction in agriculture field, breeding and save excellent poultry kind, also have been reported adopt at present
With successful case of the glass frozen preservation technology in terms of the freezing of egg mother cell, body early embryo to the mankind, therefore it is auxiliary in the mankind
Also there is huge application value in terms of helping sexual reproduction field, be the research field for having very big application prospect.
The glass frozen preservation success or not of biomaterial needs to consider there are three key factor: the first, guaranteeing to allow biological material
Material being capable of fast dewatering before entering liquid nitrogen；The second, guarantee to make biomaterial directly contact liquid nitrogen, fast prompt drop in refrigerating process
Temperature reaches glassy state；Third, guarantee will to freeze in resuscitation process biomaterial quickly, be adequately exposed to thaw
Liquid makes embryo be detached from high concentration frozen stock solution environment in time.On the basis of guaranteeing glass freezing principle, researcher is being frozen
The improvement of liquid, the exploitation for freezing carrier, the optimization of embryo thawing liquid etc. are made that very big contribution.Currently, egg mother cell/
The freezing procedure and whole process of embryo often only needs a few minutes that can complete.
In the prior art, at present mainly using Electronic Speculum grid, freezing ring and open drawing in the selection of freezing carrier
Long plastic straw etc., they allow embryo cryopreservation liquid directly to contact with liquid nitrogen, to improve freezing efficiency.However, firstly, Electronic Speculum
In gridding method and open elongation plastic straw method, the embryo samples number that each carrier freezes is limited, as electron microscopy should be limited in
10-15 pieces/every lattice, open elongation plastic straw is 4-6 pieces/pipe (freezing around-France limiting the number to have not been reported).Therefore,
The above method is not appropriate for the experiment frozen for quick, large-scale biomaterial and application.Secondly, above-mentioned carrier
Manufacture craft has certain technical difficulty, improves the expense of cost.In conclusion currently to simple, inexpensive and applicable
Biomaterial frozen stock solution and freeze carrier and can efficiently, simply glass frozen preservation biomaterial method exist need
It asks.
Summary of the invention
Therefore, the purpose of the present invention is to provide a kind of simple, low cost and biomaterial frozen stock solutions easy to use and jelly
Deposit carrier, the present invention also provides a kind of easy to operate, inexpensive, can it is extensive, efficiently, rapidly carry out biomaterial
The method of glass frozen preservation.
On the one hand, the present invention provides a kind of method for glass frozen preservation of biomaterial, the method for glass frozen preservation packets
Include following steps:
1) buffer, frozen stock solution of the preparation for the glass frozen preservation of biomaterial；
Wherein, the buffer is PB1, the frozen stock solution is EFS20 and EFS40；
2) biomaterial to be saved is put into buffer and is stood, be then transferred into EFS20 and stand 10-60 seconds, then
It is transferred in EFS40 and stands 10-60 seconds；
Preferably, the time stood in the buffer is 10-60 seconds；
Preferably, the time stood in EFS20 is 20-60 seconds；
Preferably, the time stood in EFS40 is 10-30 seconds；
3) biomaterial in EFS40 is transferred to aperture is 30-50 μm, preferably 30-39 μm, more preferably 34-36 μ
The nylon fibre web on piece of m；
Preferably, the size of the nylon fibre web piece is 0.3cm2~0.8cm2；
Preferably, the protrusion that the nylon fibre web piece has convenient for clamping；
Preferably, every nylon fibre web piece moves into 1-100 pieces of biomaterial；It is highly preferred that every nylon fibre web piece moves into
30-50 pieces of biomaterial；
4) container will be frozen to immerse in liquid nitrogen；
5) the nylon fibre web piece of step 3) is quickly transferred to the freezing in container of step 4), it is complete then will to freeze container
It immerses in liquid nitrogen, and closes the lid, be put into liquid nitrogen and save for a long time；
6) when in use, biomaterial is thawed in thawing solution.
Preferably, in step 1), the PB1 buffer is prepared by method comprising the following steps:
A. A liquid, B liquid and C liquid are prepared respectively
By 4.00 grams of sodium chloride, 0.1 gram of potassium chloride, 0.1 gram of potassium dihydrogen phosphate, 0.575 gram of disodium hydrogen phosphate, 0.018 gram
Sodium Pyruvate, 0.5 gram of glucose, 1.5 grams of bovine serum albumin(BSA)s and 0.025 gram of penicillin, which are dissolved in the steaming ultrapure water of 200ml tri-, to be stirred
It mixes uniformly, as A liquid；
0.0657 gram of calcium chloride dihydrate is dissolved in the steaming ultrapure water of 50ml tri- and is stirred evenly, as B liquid；
0.05 gram of magnesium chloride hexahydrate is dissolved in the steaming ultrapure water of 50ml tri- and is stirred evenly, as C liquid；
B. A liquid, B liquid and C liquid are placed in 4~10 DEG C 0.5-2h to being completely dissolved；
C. A liquid, B liquid, C liquid after being completely dissolved mix, and are settled to 500 milliliters, as PB1 buffer.
Preferably, in step 1), the frozen stock solution EFS20 includes following components:
PB1 buffer, the ethylene glycol of 20% (V/V), the ficoll 70 (Ficoll-70) of 18% (V/V), 0.3M sugarcane
Sugar.
Preferably, in step 1), the frozen stock solution EFS40 includes following components:
PB1 buffer, the ethylene glycol of 40% (V/V), the ficoll 70 (Ficoll-70) of 18% (V/V), 0.3M sugarcane
Sugar.
Preferably, in step 1), the frozen stock solution EFS20 and EFS40 is prepared by method comprising the following steps:
It is basic liquid with PB1 buffer, ethylene glycol, which is added, makes its volume ratio 20%, and Ficoll-70, which is added, makes its volume
Than being 18%, and sucrose is added, make its final concentration of 0.3M, mix well, dissolution overnight, then uses 0.22 μm of filter mistake
Bacterium is filtered out, that is, is configured to EFS20 frozen stock solution；
It is basic liquid with PB1 buffer, ethylene glycol, which is added, makes its volume ratio 40%, and Ficoll-70, which is added, makes its volume
Than being 18%, and sucrose is added, make its final concentration of 0.3M, mix well, dissolution overnight, then uses 0.22 μm of filter mistake
Bacterium is filtered out, that is, is configured to EFS40 frozen stock solution；
Preferably, the thawing solution is prepared by method comprising the following steps:
It is basic liquid with PB1 buffer, the sucrose solution that sucrose is made into 0.5M, 0.25M and 0.125M respectively is added, to sugarcane
Sugar after completely dissolution, with 0.22 μm of filter filtration sterilization, that is, is configured to thawing solution in PB1 buffer.
Preferably, it is described thaw the following steps are included:
The sucrose that nylon fibre web piece quickly immerses 0.5M is scrubbed 3-5 times first, equilibrium at room temperature 10-30min；Then will
Biomaterial in the sucrose thawing solution of 0.5M moves into the sucrose thawing solution of 0.25M, moves into again after balancing in 10-30min
It in the sucrose thawing solution of 0.125M, after balancing 10-30min, finally moves into PB1 buffer, i.e. completion embryo thawing.
Preferably, the biomaterial is animal embryo or egg mother cell；It is highly preferred that the biomaterial is Development of Mouse Embryos
Tire；It is further preferred that the biomaterial is mouse 2- cell stage.
On the other hand, the present invention provides a kind of carrier of glass frozen preservation for biomaterial, the carrier includes
Nylon fibre web piece and freeze container；
Wherein, the aperture of the nylon fibre web piece is 30-50 μm, preferably 30-39 μm, more preferably 34-36 μm；It is preferred that
Ground, the size of the nylon fibre web piece are 0.3cm2~0.8cm2；
Preferably, the protrusion that the nylon fibre web piece has convenient for clamping；
Preferably, the container that freezes is made by polypropylene；It is highly preferred that the container that freezes is cell cryopreservation tube.
In another aspect, the present invention also provides method for glass frozen preservation of the invention and the purposes for freezing carrier.
Compared with prior art, the invention has the following advantages that
1) the present invention provides the freezing of biomaterial, the full techniqueflow scheme of defrosting whole process, the flow operations
Simply, quickly, work can be frozen in a large amount of embryos of 1h to 2h or so completion；
2) present invention selected low-cost nylon fibre web piece piece and ordinary cells freeze container as freezing carrier,
Only need for the frozen stock solution containing biomaterial to be placed in nylon fibre web on piece when freezing, then by the nylon fibre web containing biomaterial
Piece quickly immerses in the cryopreservation tube containing liquid nitrogen, and the process is easy to operate, can extensive, efficiently, rapidly be given birth to
The glass frozen preservation of object material；
3) it is cold not need complicated Electronic Speculum grid, freezing ring and open elongation plastic straw etc. for method provided by the invention
Freeze carrier, and does not allow to easily cause egg mother cell/embryo loss, the recycling after being conducive to embryo thawing.
4) traditional slow freezing or the freezing of vitrifying tubule method are time-consuming too long in the prior art, and experimental procedure is cumbersome, and adds
The big unstability and insecurity of experiment, therefore be not suitable for the freezing for carrying out body early embryo on a large scale.And the present invention mentions
The freezing scheme of confession is more simpler, and freezing carrier is low in cost, and more applicable mammal embryo is efficient, large-scale cold
Freeze.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 is the picture for freezing carrier of the invention, and wherein Figure 1A is that the nylon with the protrusion convenient for tweezers clamping is fine
The picture of mesh sheet；Figure 1B is the picture of cell cryopreservation tube and nylon fibre web piece.
Fig. 2 shows freezen protective step of the invention, wherein Fig. 2A be will before embryo cryopreservation immerse PB1 buffer, E20 and
The picture of E40 droplet；Fig. 2 B is to prepare the picture for being put into cryopreservation tube after the E40 droplet containing embryo moves to nylon fibre web piece；Fig. 2 C
It is the picture that the nylon fibre web piece containing embryo is put into completely in cryopreservation tube.
Form after the mouse 2- cell stage defrosting recovery that Fig. 3 display uses method of the invention to freeze for a long time.
Fig. 4 shows the form after carrying out in vitro culture 72 hours to the mouse 2- cell after recovery.As the result is shown using this
The 2- cell stage of the method freezing of invention can develop into blastaea after thawing in vitro.
Fig. 5 A shows the transplanting culture photo that internal fallopian tubal is carried out to the mouse 2- cell after recovery；B is 19- after transplanting
20 days newborn mouses generated.Being transplanted to parent after thawing as the result is shown using the 2- cell stage of the present invention program freezing can be in body
Blastaea is developed into outside.
Specific embodiment
Now in conjunction with embodiment, the present invention is further elaborated, but implementation of the invention is not limited to that.Except non-specifically
Illustrate, 2 cell stage of mouse that the present invention uses is derived from second day female rat fallopian tubal of after fertilization.
Unless specifically stated otherwise, reagent used in following embodiment is analytical grade reagent, and can be from regular distributor
Purchase obtains.
Embodiment 1: the preparation of vitrification frozen stock solution of the invention
The preparation of 1.1PB1 buffer
The application need to be divided into A liquid, B liquid and C liquid when preparing PB1 buffer and prepare respectively.
Wherein, each component of A is purchased from respectively:
Sodium chloride (NaCL) is purchased from Sigma company, article No. S5886；
Potassium chloride (KCL) is purchased from Sigma company, article No. P5405；
Potassium dihydrogen phosphate (KH2PO4) it is purchased from Sigma company, article No. P5655；
Disodium hydrogen phosphate (Na2HPO4) it is purchased from Sigma company, article No. S5136；
Sodium Pyruvate (C3H3NaO3) it is purchased from Sigma company, article No. P4562；
Glucose (Glucose) is purchased from Sigma company, article No. G6152；
Bovine serum albumin(BSA) (BSA) is purchased from Sigma company, article No. A3311；
Penicillin (Penicillin) is purchased from Sigma company, article No. P7794.
The component of B liquid is purchased from:
Calcium chloride dihydrate (CaCL2﹒ 2H2O Sigma company, article No. C7902) are purchased from.
The component of C liquid is purchased from:
Magnesium chloride hexahydrate (MgCL2﹒ 6H2O Sigma company, article No. M2393) are purchased from.
It prepares PB1 buffer of the present invention to need first to dissolve the component in A liquid, B liquid and C liquid respectively, remix.
Specifically, the PB1 buffer is prepared by the following method (for preparing 500 milliliters):
3 clean glass beakers are taken first, put on A, B, C printed words, then prepare A liquid, B liquid and C liquid respectively；
A. by 4.00 grams of sodium chloride, 0.1 gram of potassium chloride, 0.1 gram of potassium dihydrogen phosphate, 0.575 gram of disodium hydrogen phosphate, 0.018
Gram Sodium Pyruvate, 0.5 gram of glucose, 1.5 grams of bovine serum albumin(BSA)s and 0.025 gram of penicillin are dissolved in 200ml tri- and steam in ultrapure water
It stirs evenly, as A liquid；
0.0657 gram of calcium chloride dihydrate is dissolved in the steaming ultrapure water of 50ml tri- and is stirred evenly, as B liquid；
0.05 gram of magnesium chloride hexahydrate is dissolved in the steaming ultrapure water of 50ml tri- and is stirred evenly, as C liquid；
B. A liquid, B liquid and C liquid are placed in 4~10 DEG C 0.5-2h to being completely dissolved；
C. A liquid, B liquid, C liquid after being completely dissolved mix, and are settled to 500 milliliters, as PB1 buffer.
Other than distinguishing A, B, C liquid, the addition of various chemical reagent is prepared without stringent sequence in normal-temperature operation
Process need to be placed under 4~10 DEG C of low temperature environments for a period of time without heating, but before the mixing of A, B, C liquid.
The frozen stock solution EFS20 and EFS40 is prepared by method comprising the following steps:
It is basic liquid with PB1 buffer, ethylene glycol, which is added, makes its volume ratio 20%, and Ficoll-70, which is added, makes its volume
Than being 18%, and sucrose is added, make its final concentration of 0.3M, mix well, dissolution overnight, then uses 0.22 μm of filter mistake
Bacterium is filtered out, that is, is configured to EFS20 frozen stock solution；
It is basic liquid with PB1 buffer, ethylene glycol, which is added, makes its volume ratio 40%, and Ficoll-70, which is added, makes its volume
Than being 18%, and sucrose is added, make its final concentration of 0.3M, mix well, dissolution overnight, then uses 0.22 μm of filter mistake
Bacterium is filtered out, that is, is configured to EFS20 frozen stock solution；
The preparation of the vitrifying thawing solution of the invention of embodiment 2
Prepare the sucrose solution that thawing solution of the present invention is 0.5M, 0.25M and 0.125M.
Specifically, the thawing solution is prepared by the following method (for preparing 100 milliliters):
First by the 100 milliliters of PB1 solution prepared in embodiment 1 be added 17.1 grams of sucrose (be purchased from Sigma, article No.:
S4097), stir evenly, be sterile filtered until completely dissolved using 0.22 micron of filter to liquid, that is, be made into 0.5M's
Sucrose solution.
Then it takes the sucrose solution of the 0.5M of 50 milliliters of above-mentioned preparations that 50 milliliters of PB1 solution are added, dilutes 1 times, that is, be made into
The sucrose solution of 0.25M.
It takes the sucrose solution of the 0.25M of 50 milliliters of above-mentioned preparations that 50 milliliters of PB1 solution are added, dilutes 1 times, that is, be made into
The sucrose solution of 0.125M.
The production of 3 mouse embryo cryopreservation carrier of embodiment
The embryo cryopreservation carrier that the present invention uses is nylon fibre web piece and cell cryopreservation tube.
Specifically: the nylon fibre web piece that aperture is 30-50 μm is chosen, if the nylon fibre web piece by one piece big is cut into dry plate ruler
Very little is 0.3cm2~0.8cm2Small pieces of cloth (size is advisable with can easily be put into cell cryopreservation tube).By the small cloth of the nylon wire sheared
Piece with needlework carry out the company of wearing be made section be fabricated to embryo cryopreservation carrier, the result is shown in Figure 1 A.
The glass freezing of 4 mouse 2- cell stage of embodiment
Tetra- kinds of cryoprotectors of bibliography Lei Xiaohua, Cao Yujing, Duan Enkui, Ma Baohua seep mouse 2- cell stage
The comparison animal journal .2008,54 (4): method described in 725-732 of permeability and toxic effect to the female mice of 6-8 week old into
The super row of row PMSG and hCG sees that fastening mouse collects fallopian tubal 2- cell stage after injection hCG 44 hours with male mouse post-coitum, will
The embryo of collection is washed 1-2 times with PB1 buffer, to be frozen.
Taking a diameter is 10 centimetres of plastic culture dish, drips upper 40-50 microlitres of PB1, E20, E40 liquid respectively in ware lid
Body, as a result as shown in Figure 2.2 cell mouse embryos of above-mentioned collection are successively moved into PB1, are balanced in EFS20 and EFS40 droplet,
Wherein immerse time for being balanced into EFS20 be 20-60 second preferably, time 10-30 second that immersion is balanced into EFS40 is preferably
(be no more than 1min), embryo, which is immersed after EFS40, quickly to be moved it into nylon fibre web on piece (every nylon wire puts 30-50 pieces of embryo
Preferably), then fibre web piece is moved rapidly into the cell cryopreservation tube being previously positioned in liquid nitrogen, covers cryopreservation tube lid, then will be thin
It is frozen for a long time in born of the same parents' cryopreservation tube investment liquid nitrogen container.
Embodiment 5 freezes the defrosting of rear mouse 2- cell stage, recovery
When embryo thawing, cell cryopreservation tube in example 4 is removed from liquid nitrogen, cryopreservation tube lid is opened, will be contained with tweezers
The nylon fibre web piece of embryo presss from both sides out from cell cryopreservation tube, rapidly immerses pieces of cloth in the sucrose thawing solution of 0.5M and is incited somebody to action with tweezers
Pieces of cloth repeatedly rinse so that embryo is in the sucrose thawing solution that nylon fibre web on piece is disengaged to 0.5M, and equilibrium at room temperature 10-15 minutes,
Embryo is moved into later in the sucrose thawing solution of 0.25M again and balanced 10-15 minutes, then the sucrose that embryo moves into 0.125M is thawed
It is balanced 15-20 minutes in liquid, finally embryo is moved in PB1 solution and washs the 1-2 solution for completing mouse 2- cell stage
Freeze, resuscitation process.2- cell stage form after defrosting is as shown in Figure 3.
The in vitro culture of mouse 2- cell stage after embodiment 6 is recovered
Mouse 2- cell stage progress in vitro culture after recovering in collection embodiment 5, reference literature Lei Xiaohua, Cao Yujing,
Comparison animal journal .2008 of the two kinds of cryoprotectors of Ning Lina, Ma Baohua to mouse 2- blast refrigerating effect, 54 (6):
Method described in 1098-1105.By the mouse 2- cell stage after thawing in embodiment 5 in experiment, select body complete
Embryo moves into the droplet of KSOM-AA culture solution, then puts it into CO2 incubator and cultivate 72 hours, observes the development of embryo
Situation simultaneously counts blastocyst rate.The result shows that embryo's in vitro culture as fresh embryo can develop blastaea after Cryopreservation
Stage (result is shown in Fig. 4), and blastocyst rate and fresh group of embryo are without statistical discrepancy.It the results are shown in Table 1.
2- cell cryopreservation embryo and fresh embryo ectogenesis situation after table 1 is recovered
The common oviduct transplantation of mouse 2- cell stage after embodiment 7 is recovered
The internal transplantation experiments of mouse 2- cell stage progress after recovering in collection embodiment 5, method reference Ning L,
Lei X,Cao Y,Zhang Y,Cao Z,Chen Q,Duan E.Effect of Short-Term Hypergravity
Treatment on Mouse 2-Cell Embryo Development.Microgravity Sci.Technol.2015；
Method described in 27:465-471 is improved.By the mouse 2- cell stage after thawing in embodiment 5 in experiment, shape is selected
The complete embryo of body moves into the fallopian tubal of false pregnancy female rat, as a result sees Fig. 5 A.The female rat of becoming pregnant of transplanting embryo produces after 19-20 days
Newborn mouse, postnatal newborn mouse result are shown in Fig. 5 B.It is transplanted in vivo after the defrosting recovery of statistics 2- cell cryopreservation embryo after newborn mouse birth
As a result.
The result shows that the mouse 2- cell stage after cryopreservation resuscitation is transplanted to as fresh mouse 2- cell stage
Can bear individual after parent fallopian tubal, and pregnancy receptor embryo at young rate and fresh group of embryo without statistical discrepancy.It the results are shown in Table
2。
The result transplanted in vivo after the defrosting recovery of table 2.2- cell cryopreservation embryo
After having read above content of the invention, those skilled in the art can make various changes or be repaired to the present invention
Change, these equivalent forms also fall within the scope of the appended claims of the present application.
Claims (8)
1. a kind of method for glass frozen preservation of biomaterial, the method for glass frozen preservation the following steps are included:
1) buffer, frozen stock solution of the preparation for the glass frozen preservation of biomaterial；
Wherein, the buffer is PB1, the frozen stock solution is EFS20 and EFS40；
2) biomaterial to be saved is put into buffer and is stood, be then transferred into EFS20 and stand 10-60 seconds, retransfer
10-60 seconds are stood into EFS40；
Preferably, the time stood in the buffer is 10-60 seconds；
Preferably, the time stood in EFS20 is 20-60 seconds；
Preferably, the time stood in EFS40 is 10-30 seconds；
3) biomaterial in EFS40 is transferred to aperture is 30-50 μm, preferably 30-39 μm, more preferably 34-36 μm
Nylon fibre web on piece；
Preferably, the size of the nylon fibre web piece is 0.3cm2~0.8cm2；
Preferably, the protrusion that the nylon fibre web piece has convenient for clamping；
Preferably, every nylon fibre web piece moves into 1-100 pieces of biomaterial；It is highly preferred that every nylon fibre web piece moves into 30-50
Piece biomaterial；
4) container will be frozen to immerse in liquid nitrogen；
5) the nylon fibre web piece of step 3) is quickly transferred to the freezing in container of step 4), then will freezes container and is completely immersed in
It in liquid nitrogen, and closes the lid, is put into liquid nitrogen and saves for a long time；
6) when in use, biomaterial is thawed in thawing solution.
2. the method according to claim 1, wherein the PB1 buffer is by including following in step 1)
It is prepared by the method for step:
A. A liquid, B liquid and C liquid are prepared respectively
By 4.00 grams of sodium chloride, 0.1 gram of potassium chloride, 0.1 gram of potassium dihydrogen phosphate, 0.575 gram of disodium hydrogen phosphate, 0.018 gram of acetone
It is equal that sour sodium, 0.5 gram of glucose, 1.5 grams of bovine serum albumin(BSA)s and 0.025 gram of penicillin are dissolved in stirring in the steaming ultrapure water of 200ml tri-
It is even, as A liquid；
0.0657 gram of calcium chloride dihydrate is dissolved in the steaming ultrapure water of 50ml tri- and is stirred evenly, as B liquid；
0.05 gram of magnesium chloride hexahydrate is dissolved in the steaming ultrapure water of 50ml tri- and is stirred evenly, as C liquid；
B. A liquid, B liquid and C liquid are placed in 4~10 DEG C 0.5-2h to being completely dissolved；
C. A liquid, B liquid, C liquid after being completely dissolved mix, and are settled to 500 milliliters, as PB1 buffer.
3. method according to claim 1 or 2, which is characterized in that in step 1), the frozen stock solution EFS20 include with
Lower component:
PB1 buffer, the ethylene glycol of 20% (V/V), the ficoll 70 (Ficoll-70) of 18% (V/V), 0.3M sucrose；
Preferably, in step 1), the frozen stock solution EFS40 includes following components:
PB1 buffer, the ethylene glycol of 40% (V/V), the ficoll 70 (Ficoll-70) of 18% (V/V), 0.3M sucrose.
4. method according to any one of claim 1-3, which is characterized in that in step 1), the frozen stock solution EFS20
It is prepared with EFS40 by method comprising the following steps:
It is basic liquid with PB1 buffer, ethylene glycol, which is added, makes its volume ratio 20%, and Ficoll-70, which is added, makes its volume ratio
18%, and sucrose is added, make its final concentration of 0.3M, mix well, dissolution overnight, is then crossed using 0.22 μm of filter and filtered out
Bacterium is configured to EFS20 frozen stock solution；
It is basic liquid with PB1 buffer, ethylene glycol, which is added, makes its volume ratio 40%, and Ficoll-70, which is added, makes its volume ratio
18%, and sucrose is added, make its final concentration of 0.3M, mix well, dissolution overnight, is then crossed using 0.22 μm of filter and filtered out
Bacterium is configured to EFS40 frozen stock solution.
5. method according to any of claims 1-4, which is characterized in that the thawing solution is by including the following steps
Method preparation:
It is basic liquid with PB1 buffer, the sucrose solution that sucrose is made into 0.5M, 0.25M and 0.125M respectively is added, exists to sucrose
In PB1 buffer after completely dissolution, with 0.22 μm of filter filtration sterilization, that is, it is configured to thawing solution.
6. method according to any one of claims 1-5, which is characterized in that it is described thaw the following steps are included:
The sucrose that nylon fibre web piece quickly immerses 0.5M is scrubbed 3-5 times first, equilibrium at room temperature 10-30min；Then by 0.5M's
Biomaterial in sucrose thawing solution moves into the sucrose thawing solution of 0.25M, moves into 0.125M's again after balancing in 10-30min
It in sucrose thawing solution, after balancing 10-30min, finally moves into PB1 buffer, i.e. completion embryo thawing.
7. method according to claim 1 to 6, which is characterized in that the biomaterial is animal embryo or ovum
Mother cell；Preferably, the biomaterial is mice embryonic；It is highly preferred that the biomaterial is mouse 2- cell stage.
8. a kind of carrier of the glass frozen preservation for biomaterial, the carrier includes nylon fibre web piece and freezes container；
Wherein, the aperture of the nylon fibre web piece is 30-50 μm, preferably 30-39 μm, more preferably 34-36 μm；
Preferably, the size of the nylon fibre web piece is 0.3cm2~0.8cm2；
Preferably, the protrusion that the nylon fibre web piece has convenient for clamping；
Preferably, the container that freezes is made by polypropylene；It is highly preferred that the container that freezes is cell cryopreservation tube.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710811425.4A CN109479870A (en) | 2017-09-11 | 2017-09-11 | A kind of method for glass frozen preservation of biomaterial |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710811425.4A CN109479870A (en) | 2017-09-11 | 2017-09-11 | A kind of method for glass frozen preservation of biomaterial |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109479870A true CN109479870A (en) | 2019-03-19 |
Family
ID=65687518
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710811425.4A Pending CN109479870A (en) | 2017-09-11 | 2017-09-11 | A kind of method for glass frozen preservation of biomaterial |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109479870A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110024775A (en) * | 2019-05-10 | 2019-07-19 | 友康恒业生物科技（北京）有限公司 | The clinical cytology preparation formula and purposes for saving liquid |
| WO2020227843A1 (en) * | 2019-05-10 | 2020-11-19 | 友康恒业生物科技（北京）有限公司 | Formulation and use of preservation solution for clinical cell preparations |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302590A (en) * | 1999-10-22 | 2001-07-11 | 朱士恩 | Simple and efficient freeze-thaw method for vitrification of embryo |
| CN101779623A (en) * | 2010-03-11 | 2010-07-21 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| CN104488853A (en) * | 2015-01-12 | 2015-04-08 | 中国农业大学 | Bovine embryo vitrification freezing tube swinging thawing and direct transplanting method |
| CN205196832U (en) * | 2015-11-09 | 2016-05-04 | 中国农业大学 | Freezing carrier of oocyte embryo glass ization |
-
2017
- 2017-09-11 CN CN201710811425.4A patent/CN109479870A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1302590A (en) * | 1999-10-22 | 2001-07-11 | 朱士恩 | Simple and efficient freeze-thaw method for vitrification of embryo |
| CN101779623A (en) * | 2010-03-11 | 2010-07-21 | 中国农业大学 | Cryopreservation method for oocyte / embryo and frozen carrier thereof |
| CN104488853A (en) * | 2015-01-12 | 2015-04-08 | 中国农业大学 | Bovine embryo vitrification freezing tube swinging thawing and direct transplanting method |
| CN205196832U (en) * | 2015-11-09 | 2016-05-04 | 中国农业大学 | Freezing carrier of oocyte embryo glass ization |
Non-Patent Citations (1)
| Title |
|---|
| 梁洋等: ""小鼠早期胚胎的EFS20／40、EFS40、DAP213和GP25玻璃化法冷冻保存"", 《江西农业大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110024775A (en) * | 2019-05-10 | 2019-07-19 | 友康恒业生物科技（北京）有限公司 | The clinical cytology preparation formula and purposes for saving liquid |
| WO2020227843A1 (en) * | 2019-05-10 | 2020-11-19 | 友康恒业生物科技（北京）有限公司 | Formulation and use of preservation solution for clinical cell preparations |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Antinori et al. | Cryotop vitrification of human oocytes results in high survival rate and healthy deliveries | |
| Vajta et al. | Are programmable freezers still needed in the embryo laboratory? Review on vitrification | |
| EP2605644B1 (en) | Method of forming vitrification droplets | |
| JP2003517276A (en) | Systems and continuous cultures for in vitro fertilization | |
| CN102093978A (en) | Prophase culture solution and anaphase culture solution for porcine early embryonic development | |
| CN102899286A (en) | Application of C-type natriuretic peptide to promotion on in vitro maturation of bovine oocyte | |
| CN100334205C (en) | Method for producing sex controllable in vitro embryo of buffalo | |
| CN107365738B (en) | Method for preparing cow and cattle xenogenesis in-vitro fertilization embryo | |
| CN109479870A (en) | A kind of method for glass frozen preservation of biomaterial | |
| CN107099553A (en) | A kind of Mouse Somatic Cells method of nuclear transfer | |
| CN103074294A (en) | Pig embryo culture solution capable of improving ectogenetic efficiency of pig embryo and preparation method thereof | |
| CN110628709A (en) | Culture solution and culture method for improving in-vitro maturation quality of porcine oocytes | |
| CN101560494B (en) | Mouse denuded oocyte in vitro maturation technology | |
| CN100404675C (en) | Production process of somatic cell clone pig | |
| Sotomaru et al. | Production of monozygotic twins after freezing and thawing of bisected mouse embryos | |
| CN105349485B (en) | Improved method for cutting buffalo blastocyst by bare hand and cutting fluid | |
| CN1226378A (en) | Technology for crossbreeding sheep in 'tubes' | |
| CN110684720B (en) | Method for obtaining male pronucleus by taking oocyte among heterogeneous animals as medium | |
| CN102174467A (en) | Extraction method and application of mouse ovarian tissue fluid | |
| Kim et al. | The effect of artificial shrinkage and assisted hatching on the development of mouse blastocysts and cell number after vitrification | |
| CN100494358C (en) | Vitrifiation freezing method for chicken embryo primordial germ cell | |
| CN111117950A (en) | Composition for promoting fertilization of frozen semen of mouse | |
| CN105861424B (en) | A kind of rats in vitro fertilization nutrient solution and its application | |
| CN111304158B (en) | Application of caprylylated Ghrelin in promoting secretion of estradiol and progesterone of oocyte cumulus cell complex of cow | |
| Libermannn et al. | Vitrification of human blastocysts: clinical realities and neonatal outcomes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190319 |
|
| RJ01 | Rejection of invention patent application after publication |