CN113455496A - Egg white cell cryopreservation liquid, preparation method and application thereof - Google Patents
Egg white cell cryopreservation liquid, preparation method and application thereof Download PDFInfo
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- CN113455496A CN113455496A CN202110739822.1A CN202110739822A CN113455496A CN 113455496 A CN113455496 A CN 113455496A CN 202110739822 A CN202110739822 A CN 202110739822A CN 113455496 A CN113455496 A CN 113455496A
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- egg white
- cell
- cryopreservation
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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 relates to an egg white cell cryopreservation solution, a preparation method and application thereof. The egg white cell freezing medium comprises 5-40% of homogeneous egg white, 25-80% of serum-free cell culture medium and 5-10% of dimethyl sulfoxide according to volume percentage, and is a cell freezing medium which uses the egg white to replace animal serum. The invention overcomes the defect that harmful factors in animal serum influence cells, and greatly reduces the cost of the cell cryopreservation agent. The egg white of goose eggs, duck eggs and the like including egg white can be used for replacing animal serum in the traditional cell frozen stock solution after being homogenized and sterilized. The serum-free egg white cryopreservation liquid provided by the invention can be used for preserving various types of cells for a long time, has the same effect with a fetal bovine serum cryopreservation agent after recovery, has the advantages of high cell survival rate, good adherent growth, strong differentiation capability and the like, and is suitable for the long-term cryopreservation of cells, tissues, embryos and organs of animals.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a serum-free freezing medium for long-term freezing of cells, tissues, embryos and organs, and a preparation method and an application method thereof.
Background
Cell freezing and recovery are one of the important techniques in cell culture technology, and cell freezing is one of the important techniques and methods for cell culture. Generally, the basic principle of cell cryopreservation and recovery is slow freezing and fast thawing, so that damage to the cell cryopreservation can be reduced to the maximum extent, and the cell viability can be preserved. The cell freezing solution is used for suspending cells to be frozen in the freezing solution, supplying nutrient substances necessary for the vital metabolism of the cells and preventing or reducing the damage of frozen ice crystals to the cells.
Fetal Bovine Serum (FBS) contains various plasma proteins, polypeptides, fats, carbohydrates, growth factors, hormones, protease inhibitors, minerals, and protects cells from damage during cryopreservation and resuscitation. Currently, in addition to the safety risks and high costs of heterologous animal sera, fetal bovine serum remains the first choice for most researchers to cryopreserve cells, tissues, embryos, organs, and the like. However, the animal-derived serum has a risk of contamination due to different indexes such as individual animal, serum production place, lot number, etc., and, for example, mycoplasma and viruses are introduced to potentially adversely affect cells, which may result in experimental failure or unreliable experimental results.
The egg white protein has high nutritive value and contains all 8 essential amino acids essential for human body. The egg white protein has important functional properties such as gelation, emulsibility, foamability and the like, and is widely applied to processing of nutritional foods and functional foods. The egg white liquid contains dozens of functional protein components, such as ovalbumin, ovotransferrin, ovomucin, ovomucoid, lysozyme, avidin, ovomacroglobulin, an egg inhibitor, a cysteine protease inhibitor, and the like. The abundant proteins, enzymes, inhibitors, growth factors and the like provide reliable guarantee for the protection and development of chicken embryos. Meanwhile, the blastoderm and the yolk are suspended in the middle of the egg by the egg white through the egg belts at the two ends, so that a good and comfortable environment is provided for the development of the embryo. Egg white (egg white), also known as "albumen", accounts for approximately 60% of the total weight of the egg. The egg white mainly comprises water (85%), protein (10%) and a mixture (5%) of carbohydrate, lipid, inorganic components, vitamins and the like, is similar to a colloidal aqueous solution and is used as a second protective layer of the embryo to prevent bacteria from permeating into the egg yolk and protect the smooth development of the chicken embryo. Egg white is also a rich, inexpensive and natural source of important proteins such as ovalbumin and lysozyme. Due to their biological activity, ease of handling, antibacterial activity and biodegradability, egg white has been used for centuries as an excipient for plasters for the treatment of various diseases. In fact, egg white and some of its by-products have been shown to improve tissue implantation and stimulate angiogenesis, making it particularly attractive in wound healing and tissue engineering applications. However, no report has been made on a technical scheme for using it in a cell cryopreservation solution to replace animal-derived serum.
In order to reduce the cost of culture and cryopreservation of cells, animal tissues and organs, a cell cryopreservation solution which is free of animal serum, can improve the cell survival rate and has low pollution risk needs to be provided.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the serum-free cell cryopreservation solution which is convenient to use, high in survival rate after cell recovery, low in pollution risk and low in cost, and the preparation method and the application thereof.
The technical scheme for realizing the aim of the invention is to provide an egg white cell cryopreservation solution which comprises 5-40% of homogenized egg white, 25-80% of serum-free cell culture medium and 5-10% of dimethyl sulfoxide according to volume percentage.
The egg white cell freezing medium provided by the invention is a serum-free cell culture medium comprising RPMI1640, DMEM, alpha-MEM and DMEM/F12.
The technical scheme of the invention also comprises a preparation method of the egg white cell frozen stock solution, which comprises the following steps:
(1) separating egg white from fresh poultry eggs of animal origin, and shearing for 1-10 min under the condition of 500-5000 rpm to obtain egg white homogeneous liquid; centrifuging at 5000-15000 rpm for 10-30 min, and collecting supernatant as homogeneous egg white;
(2) mixing 5-40% of homogenized egg white, 25-80% of serum-free culture medium and 5-10% of dimethyl sulfoxide according to volume percentage to obtain an egg white cell freezing solution.
In the preparation method of the egg white cell freezing solution, homogenized egg white is irradiated by ultraviolet UVC with the wave band of 200-280 nm for 10-60 min; the animal source poultry eggs comprise ostrich eggs, goose eggs, duck eggs, chicken eggs and quail eggs; the serum-free cell culture medium comprises RPMI1640, DMEM, alpha-MEM and DMEM/F12.
The invention provides application of an egg white cell cryopreservation liquid, which is used for long-term cryopreservation of cells, tissues, embryos and organs of animals.
The method for applying the egg white cell cryopreservation liquid to animal cells comprises the following steps:
(1) suspending cells in a logarithmic phase after being washed by PBS buffer solution in egg white cell frozen stock solution for cell counting, and then diluting with the egg white cell frozen stock solution to obtain mixed solution, wherein the cell concentration in the logarithmic phase in the mixed solution is 0.5-5 multiplied by 106Per milliliter;
(2) and (2) subpackaging the mixed solution obtained in the step (1) into 1mL of sterile freezing tubes, standing for 0.5 h at the temperature of 4 ℃, freezing for 1 h at the temperature of-20 ℃, standing for 12 h-24 h at the temperature of-80 ℃, and then transferring the sterile freezing tubes into liquid nitrogen at the temperature of-196 ℃ for preservation.
By adopting the technical scheme provided by the invention, the produced cell frozen stock solution does not contain animal-derived serum, so that potential adverse effects on cells caused by introduction of mycoplasma, viruses and the like can be avoided, and the pollution risk is reduced; meanwhile, the method can also effectively avoid experiment failure or unreliability of experiment results caused by different indexes such as animal individuals, serum production areas, batch numbers and the like. Although egg white also belongs to egg white protein of poultry of animal origin, in particular eggs, the egg white is fundamentally different from serum of animal origin. Poultry such as chicken and the like are suitable for large-scale artificial sterile culture, the quality of eggs is controllable, the processing of egg white is simple, the cost is lower by tens of times compared with animal-derived fetal calf serum, and the cost of serum-free cell freezing medium is greatly reduced.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention fully reserves and utilizes the functional components of egg white, replaces animal-derived serum with scarce resources, high cost and safety risk, and forms a serum-free cell cryopreservation solution together with a basal culture medium and DMSO to protect the cryopreservation of cells, tissues, embryos and organs and the subsequent cell resuscitation.
2. The homogenized and sterilized egg white provided by the invention basically retains the original active ingredients of the egg white, and almost all the active ingredients of the egg white are retained except that high molecular weight ovomucin (ovomucin) is required to be foamed and denatured and removed due to high-speed shearing homogenization during homogenization. Therefore, in the cell cryopreservation protective agent, egg white is the best choice for replacing animal-derived serum.
Drawings
FIG. 1 is a flow chart of the operation of the egg white cell cryopreservation solution for cell cryopreservation, provided by the invention;
FIGS. 2, 3 and 4 are graphs showing the growth of mouse L929 fibroblasts revived at days 1 to 6 after 1 week, 1 month and 3 months of cryopreservation in a serum cryopreservation solution and an egg white cell cryopreservation solution provided by the invention, respectively;
FIG. 5 is a comparison of growth states of mouse L929 fibroblasts revived on day 3 after 3 months of cryopreservation in a serum cryopreservation solution (30% fetal calf serum) and the egg white cell cryopreservation solution provided by the present invention.
Detailed Description
The technical scheme of the invention is further explained by combining the drawings and the embodiment.
Example 1
The embodiment provides an animal serum-free egg white cell cryopreservation liquid which comprises 5-40% of homogeneous egg white, 25-80% of serum-free cell culture medium and 5-10% of dimethyl sulfoxide in percentage by volume. The basic culture medium and the egg white provide necessary nutrient substances for cell life metabolism, and the dimethyl sulfoxide is mainly used as an antifreeze to improve the permeability of cell membranes to water, facilitate the water in cells to seep out of the cells, reduce the formation of ice crystals in the cells and further reduce the cell damage caused by the formation of the ice crystals.
The preparation method of the egg white cell cryopreservation solution provided by the embodiment comprises the following steps:
1. homogenized egg white preparation and sterilization
After the egg white and the yolk of a certain fresh brand of eggs are separated by an egg white separator, because concentrated egg white liquid and dilute liquid are difficult to be fully mixed uniformly, the eggs are placed in a high-speed homogenizer for high-speed shearing at 500-5000 rpm for 5-10 min, then the eggs are transferred to a centrifugal tube for centrifugal separation at 5000-15000 rpm, trace precipitates and a small amount of white floating matters are removed, and the obtained supernatant is homogenized egg white. Then, irradiating for 10-60 min by using an ultraviolet UVC lamp with a wave band of 200-280 nm; finally, the sterilized homogenized egg white is placed at 4 ℃ for standby.
2. Preparation of serum-free egg white frozen stock solution
The animal serum-free cell frozen stock solution is prepared from the following components in percentage by volume: 200 mL of sterilized homogeneous egg white with the solid content of about 12 percent is mixed with 750 mL of DEME serum-free cell basic culture medium and 50 mL of dimethyl sulfoxide and is placed at 4 ℃ for standby.
In this example, a serum cryopreservation solution was used as a control cryopreservation group, and the serum cryopreservation solution consisted of 600 mL of DEME serum-free cell basal medium, 100 mL of dimethyl sulfoxide and 300 mL of fetal bovine serum.
The egg white cell cryopreservation solution without animal serum prepared in the embodiment is used for cell culture and cryopreservation, and cell viability detection is carried out.
Cell culture
When the mouse L929 cells are cultured to a logarithmic phase, sucking off cell culture solution in a culture dish by using a pipette, adding 2 mL of PBS buffer solution into the culture dish, slightly shaking and washing, and sucking off the buffer solution which is just washed; adding about 1mL of 0.25% trypsin solution uniformly on the surface of the cells in the culture dish, digesting the cells in an incubator at 37 ℃ for 1-2 min, adding 3mL of whole cell culture solution into the culture dish to stop digestion of trypsin, blowing the L929 cells off the culture dish by using a pipette, and suspending the L929 cells in the whole cell culture solution; the whole cell culture solution comprises 9:1 (V/V) cell culture medium DMEM (high glucose) and fetal bovine serum.
Cell cryopreservation in egg white cell cryopreservation liquid
Referring to the attached figure 1, it is a flow chart of the operation of the egg white cell cryopreservation liquid for cell cryopreservation provided by the invention; the concentration is 2' 106Centrifuging (800 r/min) L929 cells at logarithmic phase for 5 min, discarding supernatant, mixing with egg white frozen stock solution, and packaging the frozen stock solution into 1mL sterile frozen tubes; and then placing the sterile freezing tubes in a program cooling box, placing for 0.5 h at 4 ℃, then placing for 1 h at-20 ℃, then placing for 12-24 h at-80 ℃, and finally transferring a plurality of sterile freezing tubes to liquid nitrogen for storage.
Cell recovery and culture
And (3) when the egg white cell freezing solution is respectively frozen in liquid nitrogen for 1 week, 1, 3, 6 or 12 months, recovering the cells. Taking out 1 cell cryopreservation tube from liquid nitrogen, immediately placing the sterile cryopreservation tube into a water bath at 37 ℃, and shaking to melt the cryopreserved cells within 2 min; centrifuging at 800 rpm for 5 min, discarding the supernatant and retaining the precipitate; and adding 1mL of the whole cell culture solution to resuspend cells, adding 10 mL of the whole cell culture solution into the suspension, standing and culturing at 37 ℃ for 20 h, and determining the survival rate of the revived cells, wherein the survival rate is 98-100%.
CCK8 cell viability detection method
Murine L929 cell suspension (300. mu.l/well) was inoculated into 48-well plates and the plates were pre-incubated in an incubator at 37 ℃ for a period of time (24 h); add 20 mL of CCK-8 solution to each well (care not to generate bubbles, which would affect the OD reading); placing the culture plate in an incubator for incubation for 1-4 h; the absorbance at 450 nm was measured with an MD-5 microplate reader. For each sample, 5 wells were repeated and the mean and error values (. + -. SD) were calculated.
Referring to the attached fig. 2, 3 and 4, there are shown growth curves of mouse L929 fibroblasts revived at days 1-6 after 1 week, 1 month and 3 months of cryopreservation in the serum cryopreservation solution (control cryopreservation group) and the egg white cell cryopreservation solution provided in this example. FIGS. 2, 3 and 4 show growth curves of mouse L929 cells in logarithmic growth phase frozen in serum-free egg white frozen stock solution and common serum frozen stock solution (30% fetal bovine serum) for 1 week, 1 month and 3 months respectively according to the experimental procedures of FIG. 1, taken out and recovered, and then cultured in normal cell culture solution for 1-6 days. In the experiment, 5 egg white concentrations of 5%, 10%, 20% and 30% h 40% are set in the egg white freezing group. FIG. 2 shows that the growth trend of cells in 6 days of the frozen stock solution of 5% low-concentration egg white or 40% high-concentration egg white is almost similar to that of the frozen stock solution of fetal bovine serum in a control group, the cells enter a logarithmic growth phase in day 3, the cell proliferation reaches a peak value in day 5, and the proliferation is obviously slowed down in day 6; the concentration of the egg white has no obvious influence on the freezing storage of the cells. FIG. 3 shows that after 1 month of cryopreservation, the first 3 egg white concentrations were observed to have a dose effect, and the cell growth rate of the cryopreservation solution with the egg white concentration from 5% to 20% increased to 20% with a peak. The growth speed of the culture medium is obviously higher than that of a serum frozen stock solution. The cell proliferation speed is obviously reduced along with the increase of the concentration of the egg white, but the level of the frozen stock solution of the serum is still achieved. FIG. 4 shows that after 3 months of cryopreservation, the growth state of the recovered cells almost mimics that of 1 month of cryopreservation. Therefore, the cell proliferation speed and the growth condition of the recovered cells are equal to or better than those of fetal bovine serum frozen stock solution after the cells are frozen in the serum-free frozen stock solution added with 5-20% of egg white for 3 months.
FIG. 5 shows the comparison of the growth status of mouse L929 fibroblasts in 3 rd day that survived after 3 months of cryopreservation in the serum cryopreservation solution and the egg white cell cryopreservation solution provided in this example; wherein, the picture (a) is the cells frozen and recovered in a 30% FBS serum frozen stock solution; (b) the figure shows cells cryopreserved and resuscitated in a 20% EW egg white cell cryopreservation solution.
Claims (8)
1. An egg white cell cryopreservation liquid is characterized in that: according to volume percentage, the egg white preservative comprises 5-40% of homogenized egg white, 25-80% of serum-free cell culture medium and 5-10% of dimethyl sulfoxide.
2. The egg white cell cryopreservation solution of claim 1, wherein: the serum-free cell culture medium comprises RPMI1640, DMEM, alpha-MEM and DMEM/F12.
3. A preparation method of an egg white cell frozen stock solution is characterized by comprising the following steps:
(1) separating egg white from fresh poultry eggs of animal origin, and shearing for 1-10 min under the condition of 500-5000 rpm to obtain egg white homogeneous liquid; centrifuging at 5000-15000 rpm for 10-30 min, and collecting supernatant as homogeneous egg white;
(2) mixing 5-40% of homogenized egg white, 25-80% of serum-free culture medium and 5-10% of dimethyl sulfoxide according to volume percentage to obtain an egg white cell freezing solution.
4. The method for preparing an egg white cell cryopreservation solution according to claim 3, wherein the method comprises the following steps: and carrying out ultraviolet UVC irradiation treatment on the homogenized egg white in a wave band of 200-280 nm for 10-60 min.
5. The method for preparing an egg white cell cryopreservation solution according to claim 3, wherein the method comprises the following steps: the animal source fowl egg includes ostrich egg, goose egg, duck egg, chicken egg, and quail egg.
6. The method for preparing an egg white cell cryopreservation solution according to claim 3, wherein the method comprises the following steps: the serum-free cell culture medium comprises RPMI1640, DMEM, alpha-MEM and DMEM/F12.
7. The application of the egg white cell cryopreservation liquid is characterized in that: it can be used for long-term cryopreservation of animal cells, tissues, embryos and organs.
8. The use of the egg white cell cryopreservation solution according to claim 7, wherein: the cryopreservation method of the animal cells comprises the following steps:
(1) cells in log phase after washing with PBS bufferSuspending the egg white cells in the frozen stock solution for cell counting, and diluting with the frozen stock solution to obtain a mixed solution, wherein the cell concentration of the mixed solution in the logarithmic phase is 0.5-5 multiplied by 106Per milliliter;
(2) and (2) subpackaging the mixed solution obtained in the step (1) into 1mL of sterile freezing tubes, standing for 0.5 h at the temperature of 4 ℃, freezing for 1 h at the temperature of-20 ℃, standing for 12 h-24 h at the temperature of-80 ℃, and then transferring the sterile freezing tubes into liquid nitrogen at the temperature of-196 ℃ for preservation.
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| CN202110739822.1A CN113455496A (en) | 2021-06-30 | 2021-06-30 | Egg white cell cryopreservation liquid, preparation method and application thereof |
| PCT/CN2021/126223 WO2023273046A1 (en) | 2021-06-30 | 2021-10-25 | Cell cryopreservation solution with egg white, and preparation method therefor and use thereof |
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| CN202110739822.1A CN113455496A (en) | 2021-06-30 | 2021-06-30 | Egg white cell cryopreservation liquid, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2023273046A1 (en) * | 2021-06-30 | 2023-01-05 | 苏州大学 | Cell cryopreservation solution with egg white, and preparation method therefor and use thereof |
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| US20130189669A1 (en) * | 2006-06-12 | 2013-07-25 | The Jackson Laboratory | Sperm cryoprotective media |
| CN105685015A (en) * | 2016-03-10 | 2016-06-22 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryopreservation solution |
| CN112401157A (en) * | 2020-10-27 | 2021-02-26 | 苏州大学 | Fresh-keeping egg white crystal powder, preparation and fresh-keeping storage method |
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| CN103891709A (en) * | 2012-12-24 | 2014-07-02 | 深圳先进技术研究院 | Cell cryopreservation liquid and cell cryopreservation method |
| CN103404509B (en) * | 2013-08-07 | 2015-04-08 | 彭乐 | Cell preserving fluid as well as preparation method and application of cell preserving fluid |
| CN103783034B (en) * | 2014-01-20 | 2015-10-21 | 广西大学 | One boar stem spermatogonium cryopreserving liquid and using method thereof |
| CN105900973B (en) * | 2016-05-20 | 2018-11-30 | 广州赛莱拉干细胞科技股份有限公司 | Macrophage cryopreservation liquid and macrophage cryopreservation method |
| CN108552158A (en) * | 2018-04-09 | 2018-09-21 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | A kind of attached cell frozen stock solution, cryopreservation methods and defreezing method |
| CN113455496A (en) * | 2021-06-30 | 2021-10-01 | 苏州大学 | Egg white cell cryopreservation liquid, preparation method and application thereof |
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- 2021-10-25 WO PCT/CN2021/126223 patent/WO2023273046A1/en unknown
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| US20130189669A1 (en) * | 2006-06-12 | 2013-07-25 | The Jackson Laboratory | Sperm cryoprotective media |
| CN105685015A (en) * | 2016-03-10 | 2016-06-22 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryopreservation solution |
| CN112401157A (en) * | 2020-10-27 | 2021-02-26 | 苏州大学 | Fresh-keeping egg white crystal powder, preparation and fresh-keeping storage method |
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| WO2023273046A1 (en) * | 2021-06-30 | 2023-01-05 | 苏州大学 | Cell cryopreservation solution with egg white, and preparation method therefor and use thereof |
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