CN101338299A - Vitrification freezing and storing method of a little somatic cells for cloning domestic animal - Google Patents
Vitrification freezing and storing method of a little somatic cells for cloning domestic animal Download PDFInfo
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- 238000004017 vitrification Methods 0.000 title claims abstract description 14
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- 238000010257 thawing Methods 0.000 claims abstract description 32
- 238000004321 preservation Methods 0.000 claims abstract description 17
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Abstract
The invention discloses a vitrification freezing preservation method of a small aqueous cell used for cloning domestic animals and belongs to the field of vitrification freezing preservation. The operation steps of the method are as follows: (I) liquid preparation; (II) vitrification freezing; (III) thawing. Compared with the prior art, the vitrification freezing preservation method of a small aqueous cell used for cloning domestic animals of the invention has the characteristics of low cost expense, simple method, needing no expensive instruments, good vitrification freezing preservation effect and high survival rate after thawing.
Description
Technical field
The present invention relates to a kind of vitrification freezing and storing method, specifically a kind of vitrification freezing and storing method that is used for the little somatic cells of cloning domestic animal.
Background technology
After somatic cell clone sheep " many jasmines " in 1997 birth, multiple somatic cell clone animal is come out one after another.As everyone knows, in the somatic cell clone research, reconstructed embryo is a cytoplasmic-nuclear hybridization body, and nucleus derives from donorcells, and tenuigenin derives from enucleation oocyte.Only carry the donorcells of normal genetic material, incorporate in the non-nucleus egg mother cell after, the spilting of an egg that could start reconstructed embryo for nuclear under the effect of the kytoplasm factor is also and then instruct it to grow normally.If the genetic material of donorcells is unusual, then can causes the reconstructed embryo spilting of an egg unusual after the fusion, and then its growth is blocked.Therefore, provide that form is good, the normal donorcells of caryogram is the prerequisite that the nuclear transplantation experiment is succeedd.
The cultivation because somatocyte can only go down to posterity in certain algebraically scope, passage number too much can cause the cell proterties to change, and cell is stored in the freeze pipe preserves and can address this problem in very low temperature (80 ℃ or-196 ℃) environment.The freezing method that uses mainly contains program cold method, substep cold method and vitrifying freeze process at present.The program cold method is used frigorimeter, and it is freezing to carry out batch under the program that is provided with, and it is less respectively to manage surviving rate difference after thawing, and spends bigger but buy instrument.The substep cold method is that freeze pipe is placed 4 ℃ 10 minutes ,-20 ℃ 30 minutes ,-80 ℃ 16～18 hours (or overnight), puts into the medium-term and long-term storage of liquid nitrogen then.Because this method does not need frigorimeter, only needs general refrigerator, Ultralow Temperature Freezer and liquid nitrogen container just can satisfy the experiment needs, therefore be widely used in each laboratory.The vitrifying freeze process early start is used for the freezing preservation of body early embryo and ovocyte, is meant that the carrier instrument that will embryo/ovocyte be housed directly drops in the liquid nitrogen, the back surviving rate height that thaws, and also lighter to the infringement of later stage developmental potency.But the problem that vitrifying freeze process exists is a high density glass freezing liquid can cause chemical toxicity damage and osmotic pressure damage to ovocyte inevitably.Therefore, the scientific worker updates freezing method, by accelerating freeze-thaw speed, shortens the duration of contact of ovocyte and vitrification solution, to reduce the damage of its chemical toxicity and osmotic pressure, the Ultrastructural damage of the freezing ovocyte that causes is dropped to minimum level.These methods comprise electron microscope copper mesh method (Martino etc., 1996), OPS method (the open stretched thin-tube of minor diameter) (Vajta etc., 1998), SSV method (150 ℃ solid surface) (Dinnyes etc., 2000) and CLV method (the CPA film on the little nylon ring) (Lane etc., 1999), thus its principle all is to improve rate of temperature fall greatly by ovocyte being suspended in the very little drop of volume.But the electron microscope that uses in the electron microscope copper mesh method is valuable, and it is bigger to buy the instrument cost; Because the OPS pipe all is to draw with 0.25 milliliter of plastics tubing is manual,, cause the repeatable relatively poor of refrigerating effect in the OPS method so the caliber size is not very consistent; The refrigerated small-particle is easy to lose in the SSV method; The problem that the CLV method exists is that operation easier is bigger.
Donorcells mainly contains three kinds of sources in the nuclear transplantation: first kind is the cell (Chen Dayuan etc., 1999) that directly digests fresh culture; Second kind is to thaw the cell of freezing preservation, and renewed vaccination is cultivated, and treats that cell 80-100% is adherent to converge back digestion and use (Lan etc., 2006); The third is directly to use the back cell (Liang Suli etc., 2007) that thaws.The cell in these three kinds of sources all has the animal birth, illustrates that the cell in three kinds of sources all has the totipotent ability of recapturing of dedifferenting in the ooecium matter of stoning.But use the cell work amount of fresh culture big, and the cell proterties change easily.Therefore, the researchist generally is inclined to use the cell in second kind and the third source.In the nuclear transplantation experiment, operate ovocyte mostly below 100 at every turn.Therefore, the donorcells that only needs to prepare to carry about 100 normal genetic material just can satisfy the test needs.And every freeze pipe is equipped with about 1 * 10 in the present cell freezing
6-5 * 10
6Individual cell is if directly use the back cell that thaws to cause a large amount of wastes of cell; The back renewed vaccination is cultivated and can be strengthened workload again if thaw.
Summary of the invention
Technical assignment of the present invention provides a kind of vitrification freezing and storing method that is used for the little somatic cells of cloning domestic animal.
Operation steps of the present invention is as follows:
(1), liquid dosage:
Basal liquid: in 100ml cell culture fluid (DMEM liquid), add 20ml-30ml foetal calf serum (V/V) and 0.5ml-1.5ml antibiotic liquid (Antibiotic-Antimycotic);
Glass freezing liquid I: in the 100ml basal liquid, add 2ml-4ml ethylene glycol (V/V) and 2ml-4ml dimethyl sulfoxide (DMSO) (V/V);
Glass freezing liquid II: in the 100ml basal liquid, add 30ml-35ml ethylene glycol (V/V), 30ml-35ml part dimethyl sulfoxide (DMSO) (V/V) and 0.2-0.3 mole trehalose;
Thawing solution: in the 100ml basal liquid, add 0.25-0.35 mole sucrose;
(2), glass freezing:
With 6 well culture plate culturing cells, digestion 1 porocyte earlier when treating that cell converges degree and is 80%-90% with 1.5 milliliters of centrifuge tube collecting cell suspensions, the centrifugal 5-8 of 800g-1000g minute, removes supernatant; Then add 1ml glass freezing liquid I, the centrifugal 5-8 of 800g-1000g minute again, remove supernatant; Add 0.5ml glass freezing liquid II then, mixing; Afterwards, draw the blank space that 2 microlitre cell suspensions are transferred to a plate with pipettor; A micro-Glass tubing inserts wherein, sucks liquid to the mark line place, micro-Glass tubing is dropped in the liquid nitrogen to store immediately; Carry out refrigeration operation so repeatedly, the time that cell enters glass freezing liquid II was controlled in 1 minute, surpassed 1 minute, and remaining cell does not remake freezing treatment; Digest 1 porocyte again, repeat preceding step, until with the freezing preservation of the porose inner cell of institute.
(3), thaw:
At first in a plate, place the 2ml thawing solution, then from liquid nitrogen, take out 1 micro-Glass tubing, in the end insertion thawing solution with micro-Glass tubing mark line, block the other end with forefinger, utilize the temperature of pointing to order about cell and enter into thawing solution, stopped 3-5 minute; Afterwards, with mouthful suction pipe with cell transfer in another plate that the 2ml basal liquid is housed, stopped 3-5 minute; At last cell transfer is got final product in the plate that carries out the nuclear transplantation experiment.
Described its external diameter of micro-Glass tubing is 1 millimeter, and internal diameter is 0.75 millimeter, and length is 10 centimetres.
The vitrification freezing and storing method of the little somatic cells that is used for cloning domestic animal of the present invention compared with prior art, it is low to have a cost cost, method is simple, does not need expensive instrument, and freezing preservation effect is good, the high characteristics of the back surviving rate of thawing.
Embodiment
Embodiment 1:
The glass freezing preservation step of bovine ear fibroblast is as follows:
(1), configuration liquid:
Basal liquid: in 100ml cell culture fluid (DMEM liquid), add 20ml foetal calf serum (V/V) and 0.5ml antibiotic liquid (Antibiotic-Antimycotic);
Glass freezing liquid I: in the 100ml basal liquid, add 2ml ethylene glycol (V/V) and 2ml dimethyl sulfoxide (DMSO) (V/V);
Glass freezing liquid II: in the 100ml basal liquid, add 30ml ethylene glycol (V/V), 30ml part dimethyl sulfoxide (DMSO) (V/V) and 0.2 mole of trehalose;
Thawing solution: in the 100ml basal liquid, add 0.25 mole of sucrose;
(2), freezing: with 6 well culture plate culturing cells, treat that it is 85% o'clock digestion 1 porocyte earlier that cell converges degree, with 1.5 milliliters of centrifuge tube collecting cell suspensions, centrifugal 5 minutes of 1000g removes supernatant.Then add 1 milliliter of glass freezing liquid I, 1000g is centrifugal 5 minutes again, removes supernatant.Add 0.5 milliliter of glass freezing liquid II then, mixing.Afterwards, draw the blank space that 2 microlitre cell suspensions are transferred to a plate with pipettor.Micro-Glass tubing inserts wherein, sucks liquid to the mark line place, micro-Glass tubing is dropped in the liquid nitrogen to store immediately; Carry out refrigeration operation so repeatedly, the time that cell enters glass freezing liquid II was controlled in 1 minute, surpassed 1 minute, and remaining cell does not remake freezing treatment; Digest 1 porocyte again, repeat preceding step, until with the freezing preservation of the porose inner cell of institute;
(3), thaw: at first in a plate, place 2 milliliters of thawing solutions.From liquid nitrogen, take out 1 micro-Glass tubing then, an end of micro-Glass tubing line is inserted in the thawing solution, block the other end, utilize the temperature of finger to order about cell and enter into thawing solution, stopped 5 minutes with forefinger.Afterwards, with mouthful suction pipe with cell transfer in another plate that 2 milliliters of basal liquids are housed, stopped 5 minutes.At last cell transfer is got final product in the plate that carries out the nuclear transplantation experiment.
Freezing bovine ear fibroblast 100 pipes in the test, the back surviving rate of thawing is at 85%-92%, and the karyotype natural rate of interest is 95%.
Embodiment 2:
The glass freezing preservation step of freezing ox uterine tubal epithelium cell is as follows:
(1), configuration liquid:
Basal liquid: in 100ml cell culture fluid (DMEM liquid), add 30ml foetal calf serum (V/V) and 1.5ml antibiotic liquid (Antibiotic-Antimycotic);
Glass freezing liquid I: in the 100ml basal liquid, add 4ml ethylene glycol (V/V) and 4ml dimethyl sulfoxide (DMSO) (V/V);
Glass freezing liquid II: in the 100ml basal liquid, add 35ml ethylene glycol (V/V), 35ml part dimethyl sulfoxide (DMSO) (V/V) and 0.3 mole of trehalose;
Thawing solution: in the 100ml basal liquid, add 0.35 mole of sucrose;
(2), freezing: with 6 well culture plate culturing cells, treat that it is 80% o'clock digestion 1 porocyte earlier that cell converges degree, with 1.5 milliliters of centrifuge tube collecting cell suspensions, centrifugal 5 minutes of 800g removes supernatant.Then add 1 milliliter of glass freezing liquid I, 800g is centrifugal 5 minutes again, removes supernatant.Add 0.5 milliliter of glass freezing liquid II then, mixing.Afterwards, draw the blank space that 2 microlitre cell suspensions are transferred to a plate with pipettor.Micro-Glass tubing inserts wherein, sucks liquid to the mark line place, micro-Glass tubing is dropped in the liquid nitrogen to store immediately; Carry out refrigeration operation so repeatedly, the time that cell enters glass freezing liquid II was controlled in 1 minute, surpassed 1 minute, and remaining cell does not remake freezing treatment; Digest 1 porocyte again, repeat preceding step, until with the freezing preservation of the porose inner cell of institute;
(3), thaw: at first in a plate, place 2 milliliters of thawing solutions.From liquid nitrogen, take out 1 micro-Glass tubing then, an end of micro-Glass tubing line is inserted in the thawing solution, block the other end, utilize the temperature of finger to order about cell and enter into thawing solution, stopped 3 minutes with forefinger.Afterwards, with mouthful suction pipe with cell transfer in another plate that 2 milliliters of basal liquids are housed, stopped 3 minutes.At last cell transfer is got final product in the plate that carries out the nuclear transplantation experiment.
Freezing ox uterine tubal epithelium cell 100 pipes in the test, the back surviving rate of thawing is at 81%-87%, and the karyotype natural rate of interest is 90%.
Embodiment 3:
The glass freezing preservation step of freezing goat uterine tubal epithelium cell is as follows:
(1), configuration liquid:
Basal liquid: in 100ml cell culture fluid (DMEM liquid), add 25ml foetal calf serum (V/V) and 1ml antibiotic liquid (Antibiotic-Antimycotic);
Glass freezing liquid I: in the 100ml basal liquid, add 3ml ethylene glycol (V/V) and 3ml dimethyl sulfoxide (DMSO) (V/V);
Glass freezing liquid II: in the 100ml basal liquid, add 30ml ethylene glycol (V/V), 30ml part dimethyl sulfoxide (DMSO) (V/V) and 0.25 mole of trehalose;
Thawing solution: in the 100ml basal liquid, add 0.3 mole of sucrose;
(2), freezing: with 6 well culture plate culturing cells, treat that it is 80% o'clock digestion 1 porocyte earlier that cell converges degree, with 1.5 milliliters of centrifuge tube collecting cell suspensions, centrifugal 7 minutes of 900g removes supernatant.Then add 1 milliliter of glass freezing liquid I, 900g is centrifugal 7 minutes again, removes supernatant.Add 0.5 milliliter of glass freezing liquid II then, mixing.Afterwards, draw the blank space that 2 microlitre cell suspensions are transferred to a plate with pipettor.Micro-Glass tubing inserts wherein, sucks liquid to the mark line place, micro-Glass tubing is dropped in the liquid nitrogen to store immediately; Carry out refrigeration operation so repeatedly, the time that cell enters glass freezing liquid II was controlled in 1 minute, surpassed 1 minute, and remaining cell does not remake freezing treatment; Digest 1 porocyte again, repeat preceding step, until with the freezing preservation of the porose inner cell of institute;
(3), thaw: at first in a plate, place 2 milliliters of thawing solutions.From liquid nitrogen, take out 1 micro-Glass tubing then, an end of micro-Glass tubing line is inserted in the thawing solution, block the other end, utilize the temperature of finger to order about cell and enter into thawing solution, stopped 4 minutes with forefinger.Afterwards, with mouthful suction pipe with cell transfer in another plate that 2 milliliters of basal liquids are housed, stopped 4 minutes.At last cell transfer is got final product in the plate that carries out the nuclear transplantation experiment.
Freezing goat uterine tubal epithelium cell 120 pipes in the test, the back surviving rate of thawing is at 83%-89%, and the karyotype natural rate of interest is 87%.
Embodiment 4:
The fibroblastic glass freezing preservation of freezing pig ear step is as follows:
(1), configuration liquid:
Basal liquid: in 100ml cell culture fluid (DMEM liquid), add 20ml foetal calf serum (V/V) and 1.5ml antibiotic liquid (Antibiotic-Antimycotic);
Glass freezing liquid I: in the 100ml basal liquid, add 2ml ethylene glycol (V/V) and 4ml dimethyl sulfoxide (DMSO) (V/V);
Glass freezing liquid II: in the 100ml basal liquid, add 30ml ethylene glycol (V/V), 35ml part dimethyl sulfoxide (DMSO) (V/V) and 0.3 mole of trehalose;
Thawing solution: in the 100ml basal liquid, add 0.23 mole of sucrose;
(2), freezing: with 6 well culture plate culturing cells, treat that it is 90% o'clock digestion 1 porocyte earlier that cell converges degree, with 1.5 milliliters of centrifuge tube collecting cell suspensions, centrifugal 5 minutes of 1000g removes supernatant.Then add 1 milliliter of glass freezing liquid I, 1000g is centrifugal 5 minutes again, removes supernatant.Add 0.5 milliliter of glass freezing liquid II then, mixing.Afterwards, draw the blank space that 2 microlitre cell suspensions are transferred to a plate with pipettor.Micro-Glass tubing inserts wherein, sucks liquid to the mark line place, micro-Glass tubing is dropped in the liquid nitrogen to store immediately; Carry out refrigeration operation so repeatedly, the time that cell enters glass freezing liquid II was controlled in 1 minute, surpassed 1 minute, and remaining cell does not remake freezing treatment; Digest 1 porocyte again, repeat preceding step, until with the freezing preservation of the porose inner cell of institute;
(3), thaw: at first in a plate, place 2 milliliters of thawing solutions.From liquid nitrogen, take out 1 micro-Glass tubing then, an end of micro-Glass tubing line is inserted in the thawing solution, block the other end, utilize the temperature of finger to order about cell and enter into thawing solution, stopped 3 minutes with forefinger.Afterwards, with mouthful suction pipe with cell transfer in another plate that 2 milliliters of basal liquids are housed, stopped 5 minutes.At last cell transfer is got final product in the plate that carries out the nuclear transplantation experiment.
Freezing pig ear inoblast 90 pipes in the test, the back surviving rate of thawing is at 68%-75%, and the karyotype natural rate of interest is 85%.
Claims (2)
1, the vitrification freezing and storing method that is used for the little somatic cells of cloning domestic animal is characterized in that the concrete steps of this method are as follows:
(1), liquid dosage:
Basal liquid: in 100ml cell culture fluid (DMEM liquid), add 20ml-30ml foetal calf serum (V/V) and 0.5ml-1.5ml antibiotic liquid (Antibiotic-Antimycotic);
Glass freezing liquid I: in the 100ml basal liquid, add 2ml-4ml ethylene glycol (V/V) and 2ml-4ml dimethyl sulfoxide (DMSO) (V/V);
Glass freezing liquid II: in the 100ml basal liquid, add 30ml-35ml ethylene glycol (V/V), 30ml-35ml part dimethyl sulfoxide (DMSO) (V/V) and 0.2-0.3 mole trehalose;
Thawing solution: in the 100ml basal liquid, add 0.25-0.35 mole sucrose;
(2), glass freezing:
With 6 well culture plate culturing cells, digestion 1 porocyte earlier when treating that cell converges degree and is 80%-90% with 1.5 milliliters of centrifuge tube collecting cell suspensions, the centrifugal 5-8 of 800g-1000g minute, removes supernatant; Then add 1ml glass freezing liquid I, the centrifugal 5-8 of 800g-1000g minute again, remove supernatant; Add 0.5ml glass freezing liquid II then, mixing; Afterwards, draw the blank space that 2 microlitre cell suspensions are transferred to a plate with pipettor; A micro-Glass tubing inserts wherein, sucks liquid to the mark line place, micro-Glass tubing is dropped in the liquid nitrogen to store immediately; Carry out refrigeration operation so repeatedly, the time that cell enters glass freezing liquid II was controlled in 1 minute, surpassed 1 minute, and remaining cell does not remake freezing treatment; Digest 1 porocyte again, repeat preceding step, until with the freezing preservation of the porose inner cell of institute;
(3), thaw:
At first in a plate, place the 2ml thawing solution, then from liquid nitrogen, take out 1 micro-Glass tubing, in the end insertion thawing solution with micro-Glass tubing mark line, block the other end with forefinger, utilize the temperature of pointing to order about cell and enter into thawing solution, stopped 3-5 minute; Afterwards, with mouthful suction pipe with cell transfer in another plate that the 2ml basal liquid is housed, stopped 3-5 minute; At last cell transfer is got final product in the plate that carries out the nuclear transplantation experiment.
2, the vitrification freezing and storing method that is used for the little somatic cells of cloning domestic animal according to claim 1 is characterized in that described its external diameter of micro-Glass tubing is 1 millimeter, and internal diameter is 0.75 millimeter, and length is 10 centimetres.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101983562A (en) * | 2010-05-26 | 2011-03-09 | 赛业（广州）生物科技有限公司 | Cell freezing solution without complicated procedures for freezing |
| CN102428910A (en) * | 2010-11-03 | 2012-05-02 | 江苏省北科生物科技有限公司 | Human umbilical cord Wharton's jelly tissue block cryopreservation protective solution |
| CN108094412A (en) * | 2018-03-01 | 2018-06-01 | 安徽工业大学 | A kind of novel freezing protective agent preserved for cell freezing and its application |
| CN111678282A (en) * | 2020-06-25 | 2020-09-18 | 余召 | Environment-friendly freezing sampling liquid nitrogen quick freezing device |
| CN113729006A (en) * | 2021-09-22 | 2021-12-03 | 华中农业大学 | Method for rapidly preserving pig germplasm resources |
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2008
- 2008-07-29 CN CNA2008101388076A patent/CN101338299A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101983562A (en) * | 2010-05-26 | 2011-03-09 | 赛业（广州）生物科技有限公司 | Cell freezing solution without complicated procedures for freezing |
| CN101983562B (en) * | 2010-05-26 | 2013-04-17 | 赛业（广州）生物科技有限公司 | Cell freezing solution without complicated procedures for freezing |
| CN102428910A (en) * | 2010-11-03 | 2012-05-02 | 江苏省北科生物科技有限公司 | Human umbilical cord Wharton's jelly tissue block cryopreservation protective solution |
| CN108094412A (en) * | 2018-03-01 | 2018-06-01 | 安徽工业大学 | A kind of novel freezing protective agent preserved for cell freezing and its application |
| CN111678282A (en) * | 2020-06-25 | 2020-09-18 | 余召 | Environment-friendly freezing sampling liquid nitrogen quick freezing device |
| CN111678282B (en) * | 2020-06-25 | 2021-01-19 | 厦门市妇幼保健院（厦门市计划生育服务中心） | Environment-friendly freezing sampling liquid nitrogen quick freezing device |
| CN113729006A (en) * | 2021-09-22 | 2021-12-03 | 华中农业大学 | Method for rapidly preserving pig germplasm resources |
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