CN114617121A - Freezing optimization system of non-human embryonic cells and application - Google Patents

Freezing optimization system of non-human embryonic cells and application Download PDF

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CN114617121A
CN114617121A CN202210368421.4A CN202210368421A CN114617121A CN 114617121 A CN114617121 A CN 114617121A CN 202210368421 A CN202210368421 A CN 202210368421A CN 114617121 A CN114617121 A CN 114617121A
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embryonic cells
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CN114617121B (en
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张静
常在
刘世景
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Tsinghua University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
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    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/16—Magnesium; Mg chelators
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • C—CHEMISTRY; METALLURGY
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    • C12N2500/00—Specific components of cell culture medium
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Abstract

The invention discloses a freezing optimization system of non-human embryonic cells and application thereof. The system adopts the combination of the balance liquid, the freezing liquid and the thawing liquid, so that the three liquids form a cryopreservation and recovery system of the embryonic cells, and the embryonic cells are favorably kept in a stable environment. By utilizing the system of the invention, the development rate of the frozen embryo cells after recovery is improved.

Description

Freezing optimization system of non-human embryonic cells and application
Technical Field
The invention belongs to the technical field of biological seed preservation, and particularly relates to a freezing system of non-human embryonic cells and application thereof.
Background
With the rapid development of various gene editing technologies, particularly various precious gene modification strains appear in the experimental animal industry, biological seed preservation technologies for preserving the strains are also widely applied, and the seed preservation technologies with higher efficiency and success rate are sought and deserve deeper research.
The freezing technology mainly comprises a slow freezing technology and a vitrification freezing technology, and the low survival rate of the slow freezing technology limits the application of the method in the biological seed preservation technology. In 1985, after vitrification freezing techniques were proposed by Rall and Fahy, the vitrification freezing technique has been widely used in embryo freezing of various experimental animals. The Vitrification freezing technology (vitrifying) is that the embryo is placed in a high-concentration cryoprotectant at room temperature and is quickly transferred into liquid nitrogen, and the cryoprotectant forms a non-structural state similar to glass from a liquid state, so that the damage to the frozen embryo is reduced by keeping normal ion distribution.
High concentration of cryoprotectants and rapid freezing rates are two key steps in the vitrification freezing technique. The cryoprotectant with high concentration has certain toxicity and may cause certain damage to embryos, and the search for the cryoprotectant with low toxicity is one of the methods for improving the freezing effect. The frozen embryos are finally placed in liquid nitrogen at-196 ℃ with a large temperature span, and the formation of cryogenically damaged ice crystals is caused in the process, generally at-5 ℃ to-80 ℃. The very high freezing rates require a simpler and easier to handle freezing regime.
Disclosure of Invention
In order to solve the technical problems, the survival rate and the development rate of the non-human embryo cells which are frozen and preserved are improved, the freezing and preserving cost of the embryo cells is reduced, and the seed preserving process of the embryo cells is simplified. The invention provides a freezing optimization system of non-human embryonic cells and application thereof, and specifically comprises the following steps:
in a first aspect of the invention, a freezing optimization system for non-human embryonic cells is provided, and the optimization system comprises a carrier for freezing embryonic cells, an embryonic cell balancing solution, an embryonic cell freezing solution and an embryonic cell thawing solution.
Preferably, the vector is selected from any one of straw (CS), straw, OPS tube, CPS tube.
More preferably, the carrier is a half straw, one end of the half straw is provided with a groove which is inclined upwards from the tail end to the center and can be used for bearing embryonic cells, and preferably, the length of the groove is 1.0-1.5cm, and the width of the groove is 0.5-0.6 mm.
Preferably, the equilibration and freezing fluids comprise receptor fluid (HTF), propylene glycol/DMSO;
more preferably, the equilibration fluid comprises serum, receptor fluid, propylene glycol/DMSO, and ethylene glycol;
further preferably, the balancing solution comprises 15-25% of serum, 60-70% of fertilization solution, 6.0-8.5% of propylene glycol/DMSO, and 6.0-8.5% of ethylene glycol;
the above components may be in any range or any value within the above ranges, for example:
serum: 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, etc.
Receiving semen: 60%, 61%, 62%, 63%, 64%, 65%, 67%, 68%, 69%, 70%, etc.
Propylene glycol or DMSO (propylene glycol/DMSO): 6.0%, 6.1%, 6.3%, 6.5%, 6.8%, 7.0%, 7.5%, 7.8%, 8.0%, 8.2%, 8.5%, etc.
Ethylene glycol: 6.0%, 6.1%, 6.3%, 6.5%, 6.8%, 7.0%, 7.5%, 7.8%, 8.0%, 8.2%, 8.5%, etc.
Preferably, the freezing fluid comprises serum, receptor fluid (HTF), propylene glycol/DMSO, ethylene glycol, and sucrose.
More preferably, the freezing fluid comprises 15-25% of serum, 30-35% of fertilization fluid, 12-17% of propylene glycol/DMSO, 12-17% of ethylene glycol and 0.15-0.25g/ml of sucrose.
The above components may be in any range or any value within the above ranges, for example:
serum: 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, etc.
Receiving semen: 30%, 31%, 32%, 33%, 34%, 35%, etc.
Propylene glycol or DMSO (propylene glycol/DMSO): 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15%, 15.5%, 16.0%, 16.5%, 17%, etc.
Ethylene glycol: 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15%, 15.5%, 16.0%, 16.5%, 17%, etc.
Sucrose: 0.15g/ml, 0.16g/ml, 0.18g/ml, 0.20g/ml, 0.22g/ml, 0.25g/ml, and the like.
Preferably, the concentration of the fertilization fluid in the freezing fluid is 1/2 of the concentration of the fertilization fluid in the equilibrium fluid; the concentration of the propylene glycol, DMSO or ethylene glycol in the refrigerating fluid is 2 times that of the propylene glycol, DMSO or ethylene glycol in the equilibrium liquid.
Preferably, the thawing solution comprises a first thawing solution, a second thawing solution and a third thawing solution, the first thawing solution and the second thawing solution comprise serum, semen receiving solution and sucrose, and the third thawing solution comprises serum and semen receiving solution.
More preferably, in the first, second, and third thawing solutions, the concentration of the fertilization solution gradually increases and the concentration of sucrose gradually decreases, and still more preferably, the third thawing solution does not include sucrose.
More preferably, the first thawing solution comprises 10-30% of serum, 50-70% of receptor fluid and 0.25-0.50g/ml of sucrose.
The above components may be in any range or any value within the above ranges, for example:
serum: 10%, 12%, 15%, 18%, 19%, 20%, 22%, 25%, 28%, 30%, etc.;
receiving semen: 50%, 52%, 55%, 58%, 60%, 61%, 62%, 63%, 64%, 65%, 67%, 68%, 69%, 70%;
sucrose: 0.25g/ml, 0.28g/ml, 0.30g/ml, 0.35g/ml, 0.40g/ml, 0.45g/ml, 0.50g/ml, and the like.
More preferably, the second thawing solution comprises 10-30% of serum, 60-80% of fertilization solution and 0.15-0.20g/ml of sucrose;
the above components may be in any range or any value within the above ranges, for example:
serum: 10%, 12%, 15%, 18%, 19%, 20%, 22%, 25%, 28%, 30%, etc.;
receiving semen: 60%, 62%, 65%, 68%, 70%, 72%, 75%, 78%, 80%, etc.;
sucrose: 0.15g/ml, 0.16g/ml, 0.17g/ml, 0.18g/ml, 0.19g/ml, 0.20g/ml, and the like.
More preferably, the third thawing solution comprises 10-30% of serum and 70-90% of receptor fluid.
The above components may be in any range or any value within the above ranges, for example:
serum: 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, etc.;
receiving semen: 70%, 72%, 75%, 78%, 80%, 82%, 85%, 88%, 90%, etc.;
preferably, the percentage of the liquid not particularly specified is a volume percentage.
Preferably, the serum is bovine serum or horse serum, wherein the bovine serum can be fetal bovine serum or newborn calf serum, and is further preferably fetal bovine serum.
Preferably, the receptive material (HTF) comprises electrolytes, carbon and nitrogen sources. Further preferred include sodium, potassium, magnesium, calcium, glucose and serum albumin.
More preferably, the receptor fluid (HTF) contains 5.7-6.1mg of sodium chloride per ml; 0.30-0.40mg of potassium chloride; magnesium sulfate heptahydrate 0.04-0.06 mg; potassium dihydrogen phosphate 0.045-0.065 mg; 0.45-0.75mg of calcium chloride; 30-35mg of sodium bicarbonate; 0.4-0.6mg of glucose; 0.25-0.45ml of sodium lactate; 0.025-0.050mg of sodium pyruvate; 0.5-1.5% double antibody; bovine serum albumin 3.0-6.0 mg.
In a particular embodiment, said receptive semen (HTF) contains 5.938mg of sodium chloride per ml of said receptive semen (HTF); potassium chloride 0.350 mg; magnesium sulfate heptahydrate 0.049 mg; potassium dihydrogen phosphate 0.054 mg; 0.570mg of calcium chloride; sodium bicarbonate 32.1 mg; glucose 0.500 mg; sodium lactate 0.34 ml; 0.037mg of sodium pyruvate; 1% double antibody; bovine serum albumin 4.0 mg.
In a second aspect of the present invention, there is provided a method for optimizing freezing of non-human embryonic cells using the above freezing optimization system, the method comprising:
step 1, placing the embryonic cells in a balancing solution for pretreatment, so that the embryonic cells recover normal forms;
step 2, placing the embryonic cells treated in the step 1 in refrigerating fluid for washing, then using a carrier to carry the embryonic cells, and placing the embryonic cells in liquid nitrogen for preservation;
and 3, unfreezing the embryonic cells preserved in the step 2 in unfreezing liquid to recover the embryonic cells.
Preferably, the embryonic cells in step 1 are selected from mammalian embryos of cattle, sheep, rabbits, pigs, mice, rats, etc.
Preferably, the step 1 comprises: and (3) placing the embryonic cells in a balanced solution at room temperature, and pretreating for 2-8 min until the embryonic cells recover to be in a normal form. More preferably, the pretreatment time is 2-5 min, such as 2min, 3min, 4min, 5 min.
Preferably, in the step 2, the embryonic cells pretreated by the balancing solution are placed in a freezing solution at room temperature, and are washed for 2-5 times, wherein the total washing time is not more than 1 min.
Preferably, the thawing step of the embryonic cells in step 3 comprises subjecting the cryopreserved embryonic cells to a first thawing solution, a second thawing solution and a third thawing solution in this order.
When the frozen cells are thawed in the step 3, the first thawing solution is preheated to 36-38 ℃, preferably to 37 ℃, and the treatment time of the first thawing solution is 30-90 min, more preferably 60min.
Preferably, the second thawing solution is kept at room temperature, and the second thawing solution is processed for 5min or less to restore the processed form of the embryonic cells to a normal state.
Preferably, the third thawing solution is kept at room temperature, and the treatment time of the third thawing solution is 10min or less.
In a third aspect of the invention, there is provided a use of the above-described freeze optimization system or freeze optimization method for non-human embryo conservation.
Preferably, the non-human embryo is a mammalian embryo, such as bovine, ovine, porcine, rabbit, horse, donkey, mouse, rat, and the like.
More preferably, the embryo is a 2-cell stage embryo.
The invention has the beneficial effects that:
1. the invention designs the combination of the formula of the balance liquid, the freezing liquid and the thawing liquid, so that the three liquids form a cryopreservation and resuscitation system of the embryonic cells, and the cryopreservation and resuscitation system is favorable for keeping the embryonic cells in a stable environment. Meanwhile, the development rate of the frozen embryonic cells after recovery is improved by controlling the processing time of the embryonic cells in the three liquids.
2. In the equilibrium solution and the refrigerating fluid, the invention can preferably adopt low-toxicity and cheap propylene glycol to replace dimethyl sulfoxide (DMSO), thereby avoiding the side effect of high toxicity of DMSO on embryonic cells and reducing the cost.
3. In the balancing solution, the freezing solution and the thawing solution, the components and the content of the receiving liquid (HTF) are adjusted, the cost is reduced, and the development rate of the frozen embryo cells after recovery is improved.
Drawings
FIG. 1 shows the recovery rate of B6 mouse embryonic cells under different cryopreservation recovery systems;
FIG. 2 shows the survival rate of B6 mouse embryonic cells under different cryopreservation recovery systems;
FIG. 3 shows the development rate of B6 mouse embryonic cells under different cryopreservation recovery systems.
Detailed Description
The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
It should be noted that the terms "first", "second" and "third" in the claims and the description of the present invention are not specifically described, but are merely used for distinguishing similar objects, and are not necessarily construed as a specific order or sequence. In addition, the experimental methods used in the examples are all conventional in the art unless otherwise specified. The materials and reagents used in the examples are those commercially available unless otherwise specified.
The main reagents of the invention comprise: in vitro working solution M2(M-7167) was obtained from sigma; m199(GIBCO) cat # 12340-; hormones (PMSG and HCG) were purchased from ningbo san corporation. C57BL/6 background genetically modified mice (hereinafter referred to as B6 mice) were from inbred mice of JAX, USA.
Example 1 the specific experimental process of the influence of the freezing optimization method of the invention on the cryopreservation recovery of the embryonic cells of the B6 mouse comprises the following steps:
1.1, preparing a half straw. Taking a common straw, dividing the common straw into two parts from the middle, and then shearing a groove for bearing embryonic cells at the front end by using scissors. The length of the groove is 1-1.5 cm, and the width is 0.5-0.6 mm.
1.2, preparing a balance liquid, a refrigerating liquid and a thawing liquid. Each of the embryonic cell treatment solutions was prepared according to the formulation shown in Table 1.
TABLE 1
Figure BDA0003581487120000061
Note: 1. the total volume of each treatment liquid in the table was 10mL, and the sum of the above components was less than 10mL and filled with water.
2. The formula of the HTF is as follows: 5.938mg of sodium chloride per ml of receptor fluid (HTF); potassium chloride 0.350 mg; magnesium sulfate heptahydrate 0.049 mg; potassium dihydrogen phosphate 0.054 mg; 0.570mg of calcium chloride; sodium bicarbonate 32.1 mg; 0.500mg of glucose; sodium lactate 0.34 ml; sodium pyruvate 0.037 mg; 1% double antibody; bovine serum albumin 4.0 mg.
1.3, cryopreservation of B6 mouse embryonic cells.
1.3.1, placing the prepared B6 mouse 2 organelle embryonic cell into a balance liquid, and enabling the embryonic cell to shrink till the embryonic cell is restored to the initial state.
1.3.2, transferring the embryonic cells treated by the 1.3.1 treatment into a refrigerating fluid to be washed for 3 times, and then transferring the embryonic cells onto a carrier half wheat pipe, wherein the process is controlled to be completed within 1 min. Then, the half straws were placed in liquid nitrogen, and finally transferred to a 5mL centrifuge tube for cryopreservation.
And 1.4, recovering the cryopreserved cells.
Taking out the half straws needing to be thawed from the liquid nitrogen, and then putting the half straws into a first thawing solution preheated to 37 ℃ to ensure that the front ends of the straws in which the embryonic cells are placed are soaked by the first thawing solution. And observing the embryo cells falling off from the front end of the half wheat pipe, transferring the embryo into a second thawing solution, soaking for 5min, then transferring into a third thawing solution E, soaking for 10min, then transferring into M2 operating solution, and waiting for a receptor to perform embryo transplantation or in-vitro culture other related experiments.
Detecting the recovery rate, survival rate and development rate of the frozen and recovered B6 mouse 2 cell-stage embryonic cells:
hormone super-excretion B6 mice, 3-4 weeks old B6 female mice were selected, and 10 IU/mouse dose of PMSG (Pregnant Mare Serum Gonadroporphin) was intraperitoneally injected, followed by 10 IU/mouse dose of HCG (human Choronic Gonadropin). Wherein the HCG injection and the PMSG injection are separated by at least 47 hours; the mice were housed in cage B6 overnight and were checked for emboli the following day.
Collecting 2-cell embryo, euthanizing female mouse, collecting small section of oviduct and uterus, placing in EP tube filled with DPBS, cleaning with DPBS solution for 2 times, and flushing embryo cells from the umbrella part of fallopian tube with insulin needle. Normal 2-cell stage embryos were cryopreserved and revived.
And (4) carrying out statistics on experimental data (recovery rate, survival rate and development rate) of the frozen and recovered embryonic cells of the mouse 2 cell stage B6. By using the freezing optimization system, the recovery rate, the survival rate and the development rate of the B6 mouse embryonic cells in the 2 cell stage after freezing recovery can respectively reach 99.71 percent, 98.92 percent and 97.38 percent.
Example 2 Effect of dimethyl sulfoxide on cryopreservation Resuscitation of B6 mouse embryonic cells
The procedure is as in example 1, the only difference being the replacement of propylene glycol by dimethyl sulphoxide (DMSO). And (4) counting the experimental data of B6 mouse 2 cell stage embryonic cells after freeze recovery.
The results are shown in Table 2:
table 2: effect of dimethyl sulfoxide or propylene glycol on recovery of cryopreserved cells
Figure BDA0003581487120000071
As shown in table 2, DMSO is a commonly used cryoprotectant, and the recovery rate, survival rate and development rate effects of the two are basically the same after the embryonic cells in the cell stage 2 of the B6 mouse are frozen and recovered after DMSO or propylene glycol is used as the cryoprotectant in the invention, and the propylene glycol is more preferably used as the cryoprotectant in consideration of the low toxicity of propylene glycol.
Example 3 Effect of different cryopreservation systems on the recovery of B6 mouse embryonic cells from cryopreservation
In the freezing system commonly used for large animals, M199 is used as a base, and the embryo cell freezing system with different compositions of M199 and HTF of the invention is also used in the invention, wherein the difference is that the balance liquid, the freezing liquid and the HTF in the thawing liquid are replaced by M199.
Wherein M199 comprises: balanced salt solution, L-glutamine, bicarbonate and Hepes.
The specific experimental procedure was the same as in example 1. After the experiment is finished, experimental data of the frozen and recovered embryonic cells of the B6 mouse in the 2 cell stage are counted. Analysis of data differences between different groups one-way ANOVA method was used, and Bonferroni's post hoc was used to test whether differences between groups were significant, indicating that P < 0.05.
The results are shown in Table 3 and FIGS. 1-3.
Table 3: effect of different freezing systems on recovery of cryopreserved cells
Figure BDA0003581487120000081
As can be seen from the results in table 3 and fig. 1-3, the recovery rates of the cells treated by the different embryo cell freezing systems did not differ much, but the survival rate of the HTF freezing system comprising the present invention was 6.88% higher than the M199 freezing system and the development rate was 8.4% higher than the M199 freezing system, showing significant differences.
The above results show that the embryonic cell freezing system and method of the present invention can improve the survival rate of embryonic cells and significantly improve the development rate of embryonic cells.
The above description is only an embodiment of the present invention, and is not intended to limit the scope of the present invention, and all equivalent structural changes made by using the content of the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (10)

1. A system for cryo-optimizing a non-human embryonic cell, comprising: the freezing optimization system comprises a carrier for freezing the embryonic cells, an embryonic cell balancing solution, an embryonic cell freezing solution and an embryonic cell thawing solution.
2. The refrigeration optimization system of claim 1, wherein: the carrier is selected from any one of straw (CS), half straw, OPS tube and CPS tube, preferably, the carrier is selected from half straw, one end of the half straw is provided with a groove which is inclined upwards from the end to the center, and the groove can be used for bearing embryo cells.
3. A freeze optimization system according to any one of claims 1-2, wherein: the balanced solution comprises 15-25% of serum, 60-70% of fertilization solution, 6.0-8.5% of propylene glycol/DMSO and 6.0-8.5% of ethylene glycol.
4. A freeze optimization system according to any one of claims 1 to 3 wherein: the refrigerating fluid comprises 15-25% of serum, 30-35% of fertilization fluid, 12-17% of propylene glycol/DMSO, 12-17% of ethylene glycol and 0.15-0.25g/ml of sucrose.
5. A freeze optimization system according to any one of claims 1 to 4, wherein: the thawing solution comprises a first thawing solution, a second thawing solution and a third thawing solution, the first thawing solution and the second thawing solution comprise serum, semen and sucrose, the third thawing solution comprises serum and semen, and is preferably in the first thawing solution, the second thawing solution and the third thawing solution, the concentration of the semen gradually increases, the concentration of the sucrose gradually decreases, and is further preferably not in the third thawing solution.
6. A freeze optimization system according to any one of claims 1 to 5, wherein: the fertilization fluid comprises sodium salt, potassium salt, magnesium salt, calcium salt, glucose and serum albumin.
7. A method for cryo-optimizing a non-human embryonic cell using the cryo-optimization system of any one of claims 1 to 6, wherein: the freezing optimization method comprises the following steps:
step 1, placing the embryonic cells in a balancing solution for pretreatment, so that the embryonic cells recover normal forms;
step 2, placing the embryonic cells processed in the step 1 in a refrigerating fluid for washing, then using a carrier to carry the embryonic cells, and placing the embryonic cells in liquid nitrogen for preservation;
and 3, unfreezing the embryonic cells preserved in the step 2 in unfreezing liquid to recover the embryonic cells.
8. The freeze optimization method of claim 7, wherein: the washing time in the step 2 is less than or equal to 1 minute.
9. A freeze optimization method according to any one of claims 7 to 8, wherein: step 3, processing the cryopreserved embryonic cells by a first thawing solution, a second thawing solution and a third thawing solution in sequence; preferably, the time of the embryonic cells in the second thawing solution is not more than 5 minutes, and the time of the embryonic cells in the third thawing solution is not more than 10 minutes.
10. Use of a cryo-optimized system according to any of claims 1-6 or a cryo-optimized method according to any of claims 7-9 for non-human embryo conservation.
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