CN114794081B - Embryo stem cell preservation method, adopted frozen stock solution, preparation method and application thereof - Google Patents
Embryo stem cell preservation method, adopted frozen stock solution, preparation method and application thereof Download PDFInfo
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- CN114794081B CN114794081B CN202210309332.2A CN202210309332A CN114794081B CN 114794081 B CN114794081 B CN 114794081B CN 202210309332 A CN202210309332 A CN 202210309332A CN 114794081 B CN114794081 B CN 114794081B
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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
- 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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- 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
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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
- 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
Abstract
The invention discloses an embryo stem cell cryopreservation liquid, which belongs to the technical field of cell biology and comprises the following components: platelet lysate, polyvinyl alcohol and polyvinylpyrrolidone can obtain the preservation effect equivalent to that of serum+DMSO by combining PVA and PVP with only low concentration of platelet lysate under the condition of no serum and DMSO, and even under certain content, the preservation effect is better than that of 90% serum+10% DMSO combination, but obviously the freezing solution has lower cytotoxicity and wider application.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to an embryonic stem cell preservation method, a freezing solution adopted by the method, a preparation method and application of the freezing solution.
Background
Embryonic stem cells (embryonic stem cells, ESCs) are a stem cell with unlimited proliferation and omni-differentiation capacity isolated and cloned from early embryonic inner cell mass or embryonic primordial germ cells. Under specific conditions, ESCs can differentiate into neural cells, bone cells, islet cells, cardiomyocytes, etc., known as "universal cells". The main biological properties of ESCs are: the cell surface antigen and enzyme can maintain specific cell morphology and growth characteristics under proper culture conditions, has permanent self-renewal capacity, expresses specific cell surface antigens and enzymes, and has a normal diploid karyotype; (II) has the potential of multi-differentiation, and can differentiate differentiated cells belonging to three germ layers in vitro under proper induction conditions; (III) easy to genetically modify, in vitro can introduce heterologous genes, reporter genes or marker genes, and in theory ESCs can still maintain the totipotency of amplification and differentiation after genetic modification.
Since ESCs may have abnormal karyotype, a corresponding decrease in the number of clones of undifferentiated cells, etc. after long-term culture, they need to be stored at low temperature. In low-temperature environment, the cells are easy to be damaged, and the cryopreservation protective solution is needed to be added for protecting the cells. The cell cryopreservation protective solution commonly used at present is 10% DMSO+90% Fetal Bovine Serum (FBS), which has good cytoprotective effect and high universality, but contains FBS of animal origin and DMSO which is toxic to cells, and the existence of the factors limits the application of the cell cryopreservation protective solution. Therefore, development of a serum-free cryopreservation solution without toxicity is particularly necessary.
At present, park et al use human albumin to replace FBS (FBS), namely physiological saline, 10% DMSO and 9% human albumin as cryoprotectants, and the cryopreservation effect is not obviously different from that of serum [1] But the cryoprotectant still needsDMSO toxic to cells was added. Furthermore, lu Ruizhou et al frozen umbilical cord hematopoietic stem cells using a 5% DMSO+hydroxyethyl starch (HES) combination, which have no significant difference in cell viability compared to 10% DMSO [2] The cryoprotectant, although free of serum and having reduced DMSO concentration, reduces damage to cells, is still not free of both serum and DMSO. It is therefore difficult to develop a cryoprotectant that does not contain serum, does not contain DMSO, and does not sacrifice the cryopreservation effect.
Platelet lysate has been reported for cell cryopreservation, such as CN107027743a, the inventors used 5% platelet lysate in combination with bFGF, L-glutamine, trehalose, hydroxyethyl starch, sodium carboxymethyl cellulose, and glycerol to cryopreserve adipose stem cells, wherein the platelet lysate can reduce the concentration of electrolyte in solution, reducing the number of cations entering the cells. After the freezing solution is adopted to freeze and store the adipose-derived stem cells for 12 months, the cell survival rate can reach 93 percent, and the differentiation capacity of the adipose-derived stem cells is not affected. In CN108617638B, the inventor adopts 5-20% of platelet lysate combined with DMSO, HSA, PVP, D-trehalose and serum to freeze skin tissues or cells, and compared with a freezing protection liquid without the platelet lysate, the protection liquid can better protect the frozen cells or tissues, and the activity rate of the frozen cells are obviously improved. It can be seen that the addition of platelet lysate to the cryopreservation solution may still require the addition of DMSO and/or serum to achieve good cryopreservation effects.
[1]Park S,Lee DR,Nam JS,et al.Fetal bovine serum-free cryopreservation methods for clinical banking of human adipose-derived stem cells[J].Cryobiolog y,2018,81:65-73.
[2] Lu Ruizhou, he Xueping, wang Dandan, etc. DMSO in combination with HES cryoprotectant for freezing impact on umbilical cord hematopoietic stem cells [ J ]. Modern diagnosis and treatment 2012, 23 (12): 2098-2099.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide the embryo stem cell cryopreservation solution which does not depend on serum and DMSO, and the cell resuscitating survival rate after the cryopreservation solution is adopted for cryopreservation has no obvious difference compared with that of the serum and DMSO, but the cell resuscitating survival rate is less damaged by the serum and DMSO.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides an embryo stem cell freezing solution, which comprises the following components: platelet lysate, polyvinyl alcohol and polyvinylpyrrolidone.
Polyvinyl alcohol and polyvinylpyrrolidone belong to impermeable cryoprotectants, and the polyvinyl alcohol has the effect of inhibiting ice crystal formation in the cooling process; polyvinylpyrrolidone can provide a certain protection to the cell membrane. Platelet lysate, polyvinyl alcohol and polyvinylpyrrolidone are not currently seen as cryoprotectants, independent of serum and DMSO.
Experiments have shown that after addition of the platelet lysate, the cell viability is higher and more cells remain in an undifferentiated state than in a frozen stock without the platelet lysate formulation.
In addition, the invention also provides an embryo stem cell cryopreservation solution, wherein the volume fraction of the platelet lysate in the cryopreservation solution is less than 1.5 percent but more than 0.2 percent.
In addition, the invention also provides an embryo stem cell cryopreservation solution, wherein the volume fraction of the platelet lysate in the cryopreservation solution is less than 1 percent but more than 0.2 percent.
In addition, the invention also provides an embryo stem cell cryopreservation solution, wherein the content of platelet lysate is 0.3-1.5%, the content of polyvinyl alcohol is 1-3% and the content of polyvinylpyrrolidone is 1-5% in terms of volume fraction.
In addition, the invention also provides an embryo stem cell cryopreservation solution, wherein the content of platelet lysate is 0.3-1%, the content of polyvinyl alcohol is 1-3% and the content of polyvinylpyrrolidone is 1-5% in terms of volume fraction.
In addition, the invention also provides an embryo stem cell cryopreservation solution, wherein the content of platelet lysate is 0.3-0.8%, the content of polyvinyl alcohol is 1-3% and the content of polyvinylpyrrolidone is 1-5% in terms of volume fraction.
The present invention is further limited in the amount of platelet lysate, since the inventors have unexpectedly found that at low levels of platelet lysate (< 1.5%), in combination with polyvinyl alcohol and polyvinylpyrrolidone, a more desirable cryopreservation effect is exhibited, which is regular when the amount of platelet lysate is maintained between 0.3 and 1.5%, but not as low as better, and significantly reduced when < 0.3%. This is contrary to prior studies, because in prior studies, the skilled person often used platelet lysate in an amount of more than 5%, e.g. the two prior art documents in the background art used 5% and 5-15% values, respectively, which showed good cryopreservation effects, and it is generally believed that platelet lysate with a high content is more favorable for cell preservation, as it is generally believed that platelet lysate with a higher content is more favorable for cell growth, as opposed to the present invention. This may be related to the other two components of polyvinyl alcohol and polyvinylpyrrolidone present in the frozen stock solution, and there is some effect between the low concentration of platelet lysate and polyvinyl alcohol and polyvinylpyrrolidone, which appears to facilitate cell protection.
In some embodiments, the polyvinyl alcohol may generally be present in an amount of anywhere between 1 and 3% and the polyvinylpyrrolidone may be present in an amount of anywhere between 1 and 5%, in which case the platelet lysate is preferably present in an amount of anywhere between 0.3% and 1.5%, more preferably between 0.3 and 1%, even more preferably between 0.3 and 0.8%, and preferably between 0.3%, 0.4%, 0.5%, 0.6%, 0.7% and 0.8%. Among these contents, the platelet lysate contents of 0.4%, 0.5% and 0.7% are the best in combination with 1 to 3% of polyvinyl alcohol and 1 to 5% of polyvinylpyrrolidone, and the contents of the latter two may be any value within the interval.
In addition, the invention also provides an embryo stem cell cryopreservation solution, and the cryopreservation solution further comprises a buffer solution.
In addition, the invention also provides embryo stem cell cryopreservation liquid, wherein the buffer solution is PBS or Hepes buffer solution.
In addition, the invention also provides embryo stem cell cryopreservation liquid, wherein the buffer solution is PBS buffer solution.
In addition, the invention also provides an embryo stem cell cryopreservation solution, wherein the content of serum and DMSO in the cryopreservation solution is 0.
Compared with the prior art, the embryo stem cell cryopreservation solution provided by the invention has the obvious advantages that the equivalent cryopreservation effect can be obtained without depending on serum and DMSO, and the equivalent excellent cryopreservation effect can be obtained by combining the platelet lysate with PVA and PVP with low concentration.
In addition, the invention also provides a preparation method of the embryo stem cell cryopreservation liquid, which is obtained by mixing the components at normal temperature.
In addition, the invention also provides a preservation method of the embryonic stem cells, which comprises the step of freezing the embryonic stem cells in the frozen stock solution.
The frozen stock solution can be used in combination with a conventional freezing preservation method, such as a conventional program type cooling method, a vitrification freezing method and the like, and in order to achieve a better freezing effect, a three-section program type cooling method is preferably adopted. The specific operation is as follows:
digesting the human embryo stem cells by collagenase, blowing a plurality of lumps by a suction tube, centrifuging to remove the supernatant, taking 80 lumps, transferring into the frozen stock solution to prepare cell suspension, loading into a wheat tube, placing the wheat tube into a freezer, adopting a three-stage program cooling method (cooling from 22 ℃ to-7 ℃ at the speed of 2.5 ℃/min, standing for 5min after nuclear placement, cooling from-7 ℃ to-30 ℃ at the speed of 0.3 ℃/min, cooling from-30 ℃ to-150 ℃ at the speed of 10 ℃/min), immediately taking out the wheat tube when cooling to-150 ℃ and placing the wheat tube into a liquid nitrogen tank for preservation.
In addition, the embryo stem cell freezing solution can be prepared into a kit for use.
The invention has the beneficial effects that:
(1) The invention provides an embryo stem cell cryopreservation solution, which can obtain a preservation effect equivalent to that of serum+DMSO by combining PVA and PVP with platelet lysate with low concentration under the condition of no serum and DMSO, and is better than 90% serum+10% DMSO combination even under certain content, but obviously, the cryopreservation solution has lower cytotoxicity and wider application.
(2) The embryo stem cell freezing solution of the invention can be applied to freezing of embryo stem cells, especially preservation of human embryo stem cells, and the freezing effect of other types of cells needs to be further studied.
Detailed Description
For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples.
Example one Effect of different levels of platelet lysate on human embryonic Stem cell cryopreservation
1.1 cryopreservation solution formulation used in the test:
(1) 0.1% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
(2) 0.2% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
(3) 0.3% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
(4) 0.4% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
(5) 0.5% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
(6) 0.6% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
(7) 0.7% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
(8) 0.8% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
(9) 0.9% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
1.0% platelet lysate+1.5 v/v% pva+3v/v% pvp+balance PBS buffer;
control group: 10% DMSO+90% fetal bovine serum.
1.2 human embryonic stem cell freezing procedure: digesting the human embryo stem cells by collagenase, blowing a plurality of lumps by a suction tube, centrifuging to remove the supernatant, respectively transferring 80 lumps into the frozen stock solution to prepare cell suspension, loading the cell suspension into a wheat tube, placing the wheat tube into a freezer, adopting a three-stage program cooling method (cooling from 22 ℃ to-7 ℃ at the speed of 2.5 ℃/min, standing for 5min after nuclear placement, cooling from-7 ℃ to-30 ℃ at the speed of 0.3 ℃/min, cooling from-30 ℃ to-150 ℃ at the speed of 10 ℃/min), immediately taking out the wheat tube when cooling to-150 ℃ and placing the wheat tube into a liquid nitrogen tank for preserving for 1 month.
1.3 resuscitation procedure of human embryonic stem cells: taking out the wheat tube from the liquid nitrogen tank, staying in the air for 30s, placing the wheat tube in a constant-temperature water bath box at 37 ℃ until the liquid in the wheat tube is liquefied, transferring the wheat tube into a human embryonic stem cell culture solution, centrifugally cleaning, adding the human embryonic stem cell culture solution again, lightly blowing and uniformly mixing the wheat tube with a suction tube, recovering the human embryonic stem cell mass which keeps the complete form and has no scattered cells after recovery, and calculating the recovery rate.
Recovery (%) =number of pellet recovery/total number of frozen pellets x 100%.
2. Growth and differentiation conditions after cell resuscitation: resuscitated human embryonic stem cell pellet was planted on MEF feeder layer (murine fibroblast feeder layer, prepared according to conventional techniques, methods of preservation of human embryonic stem cells can be referred to as Wang Jing, li Yali, etc.), and its growth status and differentiation were observed under a microscope and compared with that of the unfrozen embryonic stem cells cultured in the same period.
3. Test results
TABLE 1 Effect of varying levels of platelet lysate on human embryonic Stem cell resuscitation and differentiation (n=80)
Note that: in comparison with the control group, * p is less than 0.05; in contrast to the group (5), # p is less than 0.05; undifferentiated means differentiated cells < 30%; the partial differentiation means that differentiated cells account for 30-70%; fully differentiated cells means that the differentiated cells are > 70%.
The analysis results show that the cryopreservation effect of the platelet lysate and PVA and PVP combined with 10% DMSO+90% fetal bovine serum is obtained in the specific content range (0.3% -1.5%), and the two groups of cells have no obvious difference in terms of cell survival rate and undifferentiated rate.
The analysis of the whole test results shows that in the freezing system containing PVA and PVP, the freezing system keeps a lower amount of platelet lysate, which is more beneficial to the freezing of stem cells.
Finally, it should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, and that those skilled in the art will understand that the technical scheme of the invention may be modified or equally substituted without departing from the spirit and scope of the technical scheme of the invention.
Claims (8)
1. The embryo stem cell cryopreservation liquid is characterized in that the content of platelet lysate is 0.3-1.5%, the content of polyvinyl alcohol is 1-3%, the content of polyvinylpyrrolidone is 1-5% and the balance is PBS buffer solution in the cryopreservation liquid according to volume fraction.
2. The frozen stock solution of claim 1, wherein the frozen stock solution comprises, in volume fraction, 0.3-1% of platelet lysate, 1-3% of polyvinyl alcohol, 1-5% of polyvinylpyrrolidone, and the balance of PBS buffer.
3. The frozen stock solution of claim 2, wherein the frozen stock solution comprises, in volume fraction, 0.3-0.8% of platelet lysate, 1-3% of polyvinyl alcohol, 1-5% of polyvinylpyrrolidone, and the balance of PBS buffer.
4. A frozen stock solution as claimed in claim 3, characterized in that in the frozen stock solution, the platelet lysate content is 0.5%, the polyvinyl alcohol content is 1.5%, the polyvinylpyrrolidone content is 3% and the balance is PBS buffer solution, in terms of volume fraction.
5. A frozen stock solution as claimed in claim 3, characterized in that in the frozen stock solution, the platelet lysate content is 0.6%, the polyvinyl alcohol content is 1.5%, the polyvinylpyrrolidone content is 3% and the balance is PBS buffer solution, in terms of volume fraction.
6. The frozen stock solution of claim 2, wherein the frozen stock solution has a platelet lysate content of 0.9%, a polyvinyl alcohol content of 1.5%, a polyvinylpyrrolidone content of 3%, and the balance PBS buffer, by volume fraction.
7. The frozen stock solution of claim 1, wherein the frozen stock solution has a platelet lysate content of 1.5%, a polyvinyl alcohol content of 1.5%, a polyvinylpyrrolidone content of 3%, and the balance PBS buffer, by volume fraction.
8. A method for preserving embryonic stem cells, comprising the step of freezing the embryonic stem cells in the frozen stock solution according to any one of claims 1 to 7.
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| CN102349500B (en) * | 2011-11-10 | 2013-04-03 | 成都清科生物科技有限公司 | Mesenchymal stem cell self-preserving liquid |
| CN108617638B (en) * | 2017-03-22 | 2020-01-24 | 拜西欧斯（北京）生物技术有限公司 | Tissue and/or cell cryopreservation protective solution and preparation and application thereof |
| CN107183008A (en) * | 2017-05-27 | 2017-09-22 | 魏方萌 | A kind of placenta mesenchyma stem cell frozen stock solution and its cryopreservation methods |
| CN107027743B (en) * | 2017-06-14 | 2020-12-29 | 沃昕生物科技(深圳)有限公司 | Cell cryopreservation solution and cell cryopreservation method |
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