CN114557339A - Embryonic stem cell serum-free preservation solution and preparation method and application thereof - Google Patents
Embryonic stem cell serum-free preservation solution and preparation method and application thereof Download PDFInfo
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- 239000003761 preservation solution Substances 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 238000007710 freezing Methods 0.000 claims abstract description 36
- 230000008014 freezing Effects 0.000 claims abstract description 36
- 238000005138 cryopreservation Methods 0.000 claims abstract description 26
- 229920002643 polyglutamic acid Polymers 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000004017 vitrification Methods 0.000 claims abstract description 19
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- 230000008569 process Effects 0.000 claims description 5
- DILRJUIACXKSQE-UHFFFAOYSA-N n',n'-dimethylethane-1,2-diamine Chemical compound CN(C)CCN DILRJUIACXKSQE-UHFFFAOYSA-N 0.000 claims description 4
- 239000012980 RPMI-1640 medium Substances 0.000 claims description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 abstract description 65
- 239000000243 solution Substances 0.000 abstract description 17
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- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
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- 238000011084 recovery Methods 0.000 description 8
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- 229910052757 nitrogen Inorganic materials 0.000 description 4
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- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 2
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- 239000011521 glass Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
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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
-
- 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
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- General Health & Medical Sciences (AREA)
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- Environmental Sciences (AREA)
- Biophysics (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of cytobiology, and particularly relates to an embryonic stem cell serum-free preservation solution, and a preparation method and application thereof. The preservation solution comprises the following components in percentage by volume: 10-30% of platelet lysate, 1-5% of low molecular weight gamma-polyglutamic acid and the balance of a basic culture medium. The cryopreservation solution provided by the invention does not contain serum and DMSO, is particularly suitable for cryopreservation of embryonic stem cells, particularly human embryonic stem cells, and multiple batch tests show that the cryopreservation solution provided by the invention is combined with a vitrification freezing method to preserve embryonic stem cells, the cryopreservation effect is equivalent to that of the combination of classical serum and DMSO, but the toxicity is lower, and the cryopreservation solution is remarkably improved compared with the prior art.
Description
Technical Field
The invention belongs to the field of biotechnology. More particularly, relates to an embryonic stem cell serum-free preservation solution, a preparation method and an application thereof.
Background
Human embryonic stem (hES) cells are pluripotent stem cells that are isolated and cultured from the Inner Cell Mass (ICM) of the early embryo, which can maintain the undifferentiated state of the stem cells, normal diploid karyotype and wireless proliferation capacity, have internal differentiation capacity and germ line chimerism capacity, and can be subjected to various genetic manipulations in vitro, thus hES cells have a wide clinical application prospect.
Currently, common cryopreservation methods for hES cells include slow freezing and vitrification freezing, both of which require the use of cryoprotectants in conjunction with the freezing process. Wherein, the recovery rate of the slow freezing method is about 16 percent, and a large number of cells can be differentiated after recovery; internationally reported survival rates of hES cells cryopreserved by vitrification cryopreservation are around 75%.
The existing cryoprotectants are mainly divided into permeable cryoprotectants and impermeable cryoprotectants, wherein the permeable cryoprotectants are mostly small-molecule neutral substances which can weaken the water crystallization process and reduce the damage to the cell structure and function, and are commonly used glycerol, DMSO, propylene glycol, ethylene glycol, acetamide, methanol and the like. Among them, DMSO is a common cryoprotectant in clinical use, and is often used in combination with 90% FBS to cryopreserve stem cells, but a series of studies show that DMSO may change the stability of chromosomes and cause the change of telomere length of cells, thereby causing the formation of tumors, and the DMSO itself has certain toxicity, and the toxicity of DMSO to tissues and cells depends on exposure time, temperature and concentration; in addition, FBS, as a heterologous animal component, presents the risk of introducing viruses, and may be adversely affected by serum production lot, source and composition, and cryopreserved stem cells. Therefore, there is a need for a low-toxicity, serum-free cryopreservation solution.
The inventor provides a cell cryopreservation protective solution which comprises a platelet lysate, a permeability protective agent, a non-permeability protective agent and serum, and proves that compared with the cryopreservation protective solution without the platelet lysate, the preservation solution can better protect cryopreserved cells, the activity and the survival rate of the cryopreserved cells are obviously improved, and the preservation solution has no influence on the cell survival rate after primary culture. But the preservation solution still needs to add 4-70% volume fraction of serum. Wang et al discovered that the survival rate of cells after recovery is higher than 80% by using 97% human platelet lysate + 3% DMSO to freeze and store hASCs, has no obvious difference from the survival rate of 95% FBS + 5% DMSO, can reduce the concentration of DMSO, can release various growth factors to promote the in vitro proliferation of hASCs, and further needs to understand the growth matrix of platelet lysate-promoted cells and the freezing and storing effects of the platelet lysate-promoted cells in other stem cells[1]. However, 3% DMSO was still added to the frozen stock solution.
There are currently no examples of the use of human platelet lysates for culturing hESCs.
[1]Wang C，Xiao R，Cao YL，etal.Evaluation of human plateletlysate an d dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adip ose-derived stem cells[J].Biochem Biophys Res Commun,2017,491(1):198-203.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects of the existing cryopreservation solution and provides the serum-free preservation solution for the embryonic stem cells, which is characterized by containing no serum or DMSO. The freezing preservation solution can improve the freezing survival rate of cells, the recovery rate of the embryonic stem cells can reach more than 95 percent by freezing preservation combined with vitrification freezing, and the cells can maintain an undifferentiated state after recovery.
The invention aims to provide a serum-free preservation solution for embryonic stem cells, which comprises the following components in percentage by volume:
10-30% of platelet lysate
1-5% of low molecular weight gamma-polyglutamic acid
The balance of the buffer solution.
Further, the embryonic stem cell serum-free preservation solution comprises the following components in percentage by volume:
10-25% of platelet lysate
1-3% of low molecular weight gamma-polyglutamic acid
The balance of the buffer solution.
Further, the embryonic stem cell serum-free preservation solution comprises the following components in percentage by volume:
platelet lysate 16%
1% of low molecular weight gamma-polyglutamic acid
The balance of the buffer solution.
In the above-mentioned scheme, the platelet lysate is extracted and separated from human blood, and contains platelet-derived growth factors, transforming growth factors, insulin-like growth factors, and the like, which are advantageous for the survival of cells, and currently, the platelet lysate is applied to cryopreservation of hASCD, mesenchymal stem cells, adipose-derived stem cells, and the like, but the effect of cryopreserving embryonic stem cells is not known.
The gamma-polyglutamic acid belongs to an impermeable cryoprotectant, has strong water molecule complexing ability, can reduce the formation of ice crystals in cells in a freezing process, and the survival rate of stem cells is still up to more than 97 percent after the stem cells are frozen for 12 months by using human serum albumin, stem cell growth factors, gamma-polyglutamic acid and glycerol. However, it is not known how effective the combination of platelet lysate with it for cryopreserving embryonic stem cells.
Generally, gamma-polyglutamic acids of different molecular weights exhibit different physiological activities, and the inventors have unexpectedly found that gamma-polyglutamic acids of different molecular weights exhibit greatly different effects in binding to platelet lysate frozen embryonic stem cells. It was found through a research experiment that gamma-polyglutamic acid having a low molecular weight is more advantageous in combination with platelet lysate for improving the cell survival rate and maintaining the cell undifferentiated state.
Further, the low molecular weight gamma-polyglutamic acid is gamma-polyglutamic acid with the molecular weight of 5-70 kDa; furthermore, the molecular weight of the gamma-polyglutamic acid is preferably between 10-70 kDa, 20-70 kDa, 30-70 kDa, 40-70 kDa, 50-70 kDa and 60-70 kDa; further, the gamma-polyglutamic acid has a molecular weight of 10kDa, 20kDa, 30kDa, 40kDa, 50kDa, 60kDa or 70 kDa; further, the gamma-polyglutamic acid has a molecular weight of 10 kDa; further, the gamma-polyglutamic acid has a molecular weight of 70 kDa.
Those skilled in the art know that low molecular weight gamma-polyglutamic acid can be obtained by fermentation of Bacillus subtilis (refer to Chinese patent application CN 104805143A).
Further, the basic culture medium is one of DMEN-12, low-sugar DMEN, high-sugar DMEN or RPMI-1640.
It is another object of the present invention to provide a method for cryopreservation of embryonic stem cells, wherein the cryopreservation solution of the present invention can be used in combination with conventional cryopreservation methods for embryonic stem cells, including but not limited to: freezing and storing at-80 deg.c, conventional liquid nitrogen freezing and storing in glass. More preferably, the cryopreservation method is a vitrification freezing method.
The "vitrification freezing method" is a conventional vitrification freezing method in the prior art, and the specific steps thereof may include:
firstly, balancing the embryonic stem cells in a vitrification balancing liquid at the temperature of 37-40 ℃ for 5-10 min;
and secondly, transferring the embryonic stem cells into a cryopreservation solution at the temperature of 37-40 ℃, after balancing for 10-30 seconds, transferring the embryonic stem cells onto a vitrification freezing carrier, and quickly immersing the vitrification carrier into liquid nitrogen.
Further, the vitrification balance liquid is a conventional vitrification balance liquid, which can be obtained from commercially available technologies of Wuhan Cornstai technology, Inc., containing DMSO, ethylene glycol and 10mg/ml HSA, and used
w/HEPES buffer.
Furthermore, because the existing cryopreservation solution contains DMSO and has toxicity to cells, the time for transferring the embryonic stem cells onto the vitrified carrier in the step II must be controlled within 1min, while the cryopreservation solution does not contain DMSO, and the operation time is not strictly required in the step.
Further, the carrier used in the vitrification freezing process may be OPS or Cryotop.
Experiments show that the average survival rate of the stem cells is greatly improved by adopting the cryopreservation liquid of the invention to freeze and store the human embryonic stem cells by combining the vitrification freezing method, and the freezing and storing effect is equivalent to the combination of classical serum and DMSO.
As used herein, "embryonic stem cells" include, but are not limited to: human embryonic stem cells, bovine embryonic stem cells, porcine embryonic stem cells, murine embryonic stem cells, or ovine embryonic stem cells, and the like.
The invention also aims to provide an embryonic stem cell freezing kit which comprises the preservation solution.
The invention has the following beneficial effects:
(1) the invention provides a cryopreservation liquid, which is particularly suitable for cryopreservation of embryonic stem cells, particularly human embryonic stem cells.
(2) The cryopreservation liquid provided by the invention does not contain serum and DMSO, has lower toxicity, and is more suitable for clinic.
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
Vitrification equilibrium liquid: comprising DMSO, ethylene glycol and 10mg/ml HSA, using
w/HEPES buffer from Wuhan-Heitai science, Inc
EXAMPLE one preparation of freezing fluid for embryonic Stem cells (volume percent)
The preparation method of each group of refrigerating fluid comprises the following steps: and uniformly mixing the components at room temperature to obtain refrigerating fluid.
Example two comparison of recovery effect of different refrigerating fluid formulations
2.1 test methods:
2.1.1 embryonic stem cell freezing: placing human embryonic stem cells in a vitrification balancing solution at 39 ℃ for balancing for 10 min; respectively transferring the embryonic stem cells into a refrigerating fluid, balancing for 30s, sucking the embryonic stem cells by a suction pipe, transferring the embryonic stem cells to a Cryotop vitrified carrier, quickly immersing the carrier into liquid nitrogen, and respectively freezing and storing for 1 month, 3 months and 6 months.
2.1.2 recovery of embryonic stem cells: taking the carrier out of the liquid nitrogen tank, inserting the carrier into a culture dish filled with vitrified thawing solution (WS1, 0.2 mol/sucrose + FBS with volume fraction of 20%) preheated at 37 ℃, and determining that the embryonic stem cells stay for 1min after falling from the carrier into the culture dish; transferring into preheated vitrification thawing solution (WS2, 0.1 mol/sucrose + 20% FBS) at 37 deg.C with pipette, and standing for 4 min; then transferring the embryonic stem cells into a preheated resuscitation solution 1 (Hepes buffer solution containing 20 v/v% FBS) at 37 ℃ for 5 min; transferring it to a pre-warmed resuscitation solution 2 (Hepes buffer containing 20 v/v% FBS) at 37 deg.C for 5 min; finally, the cells are transferred into a cell culture solution (PZM-3), placed in an incubator to be cultured continuously, observed under a mirror and counted.
Freezing 10 batches of embryonic stem cells with good growth state by adopting freezing liquid 1# -12 # respectively according to the method, replacing the freezing liquid with conventional freezing liquid (prepared by 1mol/LHepes buffer solution and containing 20% of FBS, 10% of DMSO and 10% of ethylene glycol) as a control group, setting 3 parallel tests for each batch, and calculating the average survival rate of the cells after 10 batches of each group are stored for different time.
2.2 test results
TABLE 110 average cell viability after different time of batch storage (20 cells frozen per batch)
Note: compared with the control group, the compound of the formula,*P＜0.05，**P＜0.05。
as can be seen from the above table, the freezing liquid (1# -9 #) of the invention is combined with the vitrification freezing method to freeze the embryonic stem cells for 1 month for recovery, the average survival rate of the cells is more than 85%, the average survival rate of the cells is more than 75% after 3 months of freezing, and the freezing effect is equivalent to that of the combination of the serum of the classical freezing liquid and the DMSO (a control group); meanwhile, the results of other groups are analyzed, and it is known that the freezing effect of the combination of the gamma-polyglutamic acid with higher molecular weight (120kDa, 200kDa and 500kDa) and the platelet lysate is obviously different (P is less than 0.05) compared with the serum + DMSO combination (control group), but the effect changes irregularly among the three groups.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. The serum-free preservation solution for the embryonic stem cells is characterized by comprising the following components in percentage by volume:
10-30% of platelet lysate
1-5% of low molecular weight gamma-polyglutamic acid
The balance of basic culture medium.
2. The serum-free preservation solution for the embryonic stem cells according to claim 1, which comprises the following components in percentage by volume:
10-25% of platelet lysate
1-3% of low molecular weight gamma-polyglutamic acid
The balance of basic culture medium.
3. The serum-free preservation solution for embryonic stem cells according to claim 1, which comprises the following components in percentage by volume:
platelet lysate 16%
2% of low molecular weight gamma-polyglutamic acid
The balance of basic culture medium.
4. The serum-free preservation solution for embryonic stem cells according to any one of claims 1 to 3, wherein the molecular weight of the gamma-polyglutamic acid is 5-70 kDa.
5. The serum-free preservation solution for embryonic stem cells according to claim 4, wherein the molecular weight of the gamma-polyglutamic acid is 10-70 kDa.
6. The serum-free preservation solution for the embryonic stem cells according to any one of claims 1 to 5, wherein the basic culture medium is one of DMEN-12, low-sugar DMEN, high-sugar DMEN or RPMI-1640.
7. A cryopreservation method of embryonic stem cells, which is a vitrification cryopreservation method, and particularly comprises a step of preserving embryonic stem cells by using the preservation solution according to any one of claims 1 to 6 in a freezing process.
8. A freezing kit for embryonic stem cells, which comprises the serum-free preservation solution for embryonic stem cells according to any one of claims 1 to 6.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116138250A (en) * | 2023-04-20 | 2023-05-23 | 深圳市森盈智能科技有限公司 | Puncture cell preservation solution, kit, preservation method and application |
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| CN108617638A (en) * | 2017-03-22 | 2018-10-09 | 拜西欧斯（北京）生物技术有限公司 | Tissue and/or cell cryopreservation protection liquid and its preparation and application |
| CN109511651A (en) * | 2019-01-04 | 2019-03-26 | 青岛麦迪赛斯生物科技有限公司 | A kind of preparation method of human umbilical cord mesenchymal stem cells serum-free frozen stock solution |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102124098A (en) * | 2008-06-27 | 2011-07-13 | 博傲沃德株式会社 | Cryopreservative composition for cell and tissue |
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Application publication date: 20220531 |