CN111449054A - Novel cell preservation solution and preparation method thereof - Google Patents
Novel cell preservation solution and preparation method thereof Download PDFInfo
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- CN111449054A CN111449054A CN202010438194.9A CN202010438194A CN111449054A CN 111449054 A CN111449054 A CN 111449054A CN 202010438194 A CN202010438194 A CN 202010438194A CN 111449054 A CN111449054 A CN 111449054A
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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
- A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
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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
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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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Abstract
The invention relates to a novel cell preservation solution and a preparation method thereof, which relate to the field of preservation solutions and aim to solve the problem of low survival rate of oral cells in the existing cell preservation solution, and the novel cell preservation solution comprises the following components of 0.45-1.6wt% of sodium chloride, 0.05-1.1wt% of sodium dihydrogen phosphate, 0.02-1.5wt% of disodium hydrogen phosphate, 15-48wt% of absolute ethyl alcohol, 0.03-1.4wt% of sodium citrate, 0.03-1.8wt% of trypsin, 0.02-0.99wt% of N-acetyl-L-cysteine, 0.01-0.86wt% of phosphatidylcholine, 0.2-0.68wt% of bactericide, 0.03-1.11wt% of stabilizer and the balance of purified water.
Description
Technical Field
The invention relates to the technical field of cell preservation solution, in particular to novel cell preservation solution and a preparation method thereof.
Background
In human structures, the oral cavity and the pharynx are important portals for microorganisms to enter respiratory tract, digestive tract and blood, and the close and complex interaction between oral microbial communities and between microorganisms and hosts maintains the health of the hosts under normal conditions, so that the breaking of the ecological balance can cause diseases, and the detection of oral cells is very important. In the cell detection, the cell preservation solution is a reagent that plays a role of holding and fixing the original morphological structure of the cells, and can prevent the morphological structure of the cells from changing and reduce errors of diagnosis results.
The existing Chinese patent with the application number of CN201710732482.3 discloses a sample preservation solution which is characterized in that: is prepared from alcohol (40-50%), formaldehyde (10-15%), alkali substance (0.1%), humectant (0.5%), potassium chloride solution (10-20%) and water (rest).
However, the oral cavity and the throat can secrete a large amount of mucus, so that cells cannot be well dispersed in a cell preservation solution, the abundance and integrity of the cells are damaged, and the survival rate of the cells is low, so that subsequent experiments and detection are influenced.
Disclosure of Invention
In view of the defects of the prior art, one of the purposes of the invention is to provide a novel cell preservation solution and a preparation method thereof, the cell preservation solution prepared by the method effectively decomposes mucus in oral cells, improves the cell preservation rate, and has a simple preparation method.
The above object of the present invention is achieved by the following technical solutions:
a novel cell preservation solution comprises 0.45-1.6wt% of sodium chloride, 0.05-1.1wt% of sodium dihydrogen phosphate, 0.02-1.5wt% of disodium hydrogen phosphate, 15-48wt% of absolute ethyl alcohol, 0.03-1.4wt% of sodium citrate, 0.03-1.8wt% of trypsin, 0.02-0.99wt% of N-acetyl-L-cysteine, 0.01-0.86wt% of phosphatidylcholine, 0.2-0.68wt% of bactericide, 0.03-1.11wt% of stabilizer and the balance of purified water.
By adopting the technical scheme, the added purified water is used as a matrix of the cell preservation solution, the added sodium chloride is used as an osmotic pressure regulator of the cell preservation solution, the added sodium dihydrogen phosphate and the added disodium hydrogen phosphate are used as a buffer agent of the cell preservation solution, the added absolute ethyl alcohol is used as a fixing agent of the cell preservation solution, and the sodium citrate is used as an anticoagulant of the cell preservation solution, so that basic survival conditions are provided for oral cells in the cell preservation solution;
the oral mucus preservative solution is characterized in that the oral mucus is diluted by N-acetyl-L-cysteine, the N-acetyl-L-cysteine is soluble in water and can promote mucus secretion to change from sticky to thin so as to play a strong mucus dissolving role, and the N-acetyl-L-cysteine has a protection and stabilization effect on the trypsin, and the two are matched with each other, so that the survival rate of cells can be further improved, and meanwhile, the N-acetyl-L-cysteine has a certain anti-oxidation effect and can prolong the storage time of the cell preservative solution.
The phosphatidylcholine, which is a kind of lecithin, is dispersed in the cell preservation solution because the head is hydrophilic and the tail is hydrophobic, and can supply certain nutrition to the oral cavity in the cell preservation solution, thereby further improving the survival rate of the cells. The bactericide and the stabilizer are added to be matched with other components in the cell preservation solution, so that the oral cells are well dispersed in the cell preservation solution, the oral cells keep good abundance and completeness for a long time, and the survival rate of the cells is further improved.
The invention further provides a cell preservation solution which comprises the following components of 1.2-1.36wt% of sodium chloride, 0.68-0.94wt% of sodium dihydrogen phosphate, 0.12-0.34wt% of disodium hydrogen phosphate, 30-38wt% of absolute ethyl alcohol, 0.23-0.36wt% of sodium citrate, 0.25-0.46wt% of trypsin, 0.66-0.86wt% of N-acetyl-L-cysteine, 0.39-0.45wt% of phosphatidylcholine, 0.33-0.46wt% of bactericide, 0.46-0.65wt% of stabilizer and the balance of purified water.
By adopting the technical scheme, the content range of each component in the cell preservation solution is further optimized, so that each component in the cell preservation solution is better compatible with each other, and the survival rate of oral cells in the cell preservation solution is further improved.
The invention further provides a cell preservation solution which comprises 1.21wt% of sodium chloride, 0.77wt% of sodium dihydrogen phosphate, 0.23wt% of disodium hydrogen phosphate, 35wt% of absolute ethyl alcohol, 0.31wt% of sodium citrate, 0.0.34wt% of trypsin, 0.71wt% of N-acetyl-L-cysteine, 0.69wt% of phosphatidylcholine, 0.41wt% of bactericide, 0.581wt% of stabilizer and the balance of purified water.
By adopting the technical scheme, the content of each component in the cell preservation solution is further optimized, so that each component in the cell preservation solution is optimally compatible, and the survival rate of oral cells in the cell preservation solution is improved to the maximum extent.
The invention is further configured that the stabilizer is one of butanediol, sodium gluconate and ethylenediaminetetraacetic acid.
By adopting the technical scheme, the butanediol has good adsorption performance on water, so that super-strong moisturizing performance can be achieved, and the concentration of the cell preservation solution is kept stable; the sodium gluconate serving as a retarder can effectively improve the stability of the cell preservation solution; the ethylene diamine tetraacetic acid is used as an anticoagulant, and can effectively inhibit coagulation of the cell preservation solution.
The invention is further configured that the bactericide is one of polyhexamethylene biguanide, imidazolidinyl urea and isothiazolinone.
By adopting the technical scheme, the polyhexamethylene biguanide serving as an environment-friendly high-molecular polymer sterilization disinfectant is dissolved in water, has stable property, has no side effect, is added into the cell preservation solution, can well sterilize the cell preservation solution, and reduces the deterioration of the cell preservation solution; the imidazolidinyl urea is very soluble in water, can be well dispersed in the cell preservation solution, and can effectively reduce the putrefaction and deterioration of the cell preservation solution; isothiazolinone as a preservative has strong inhibiting and killing effects on common bacteria, fungi, algae and the like, and reduces deterioration of cell preservation solution.
The invention is further configured to further comprise the following components: 0.005-0.058wt% of sugar alcohol calcium zinc liquid.
By adopting the technical scheme, the sugar alcohol calcium zinc solution provides proper amount of calcium element and zinc element for oral cells, and can also improve the delivery efficiency of phosphatidylcholine; meanwhile, calcium ions provided in the sugar alcohol calcium zinc solution are matched with the complexation of sodium gluconate and ethylene diamine tetraacetic acid, so that the concentration of the calcium ions is moderate, the activity of trypsin is improved, and the cell survival rate is further improved.
The invention further provides a cell preservation solution which comprises 1.32wt% of sodium chloride, 0.91wt% of sodium dihydrogen phosphate, 0.21wt% of disodium hydrogen phosphate, 36wt% of absolute ethyl alcohol, 0.32wt% of sodium citrate, 0.39wt% of trypsin, 0.78wt% of N-acetyl-L-cysteine, 0.42wt% of phosphatidylcholine, 0.40wt% of bactericide, 0.52wt% of stabilizer, 0.036wt% of sugar alcohol calcium zinc solution and the balance of purified water;
wherein the stabilizer is ethylenediamine tetraacetic acid, and the bactericide is polyhexamethylene biguanide.
By adopting the technical scheme, after the sugar alcohol calcium zinc solution is added, the content of each component in the cell preservation solution is optimized, so that each component can be fully matched, and the survival rate of oral cells is further improved.
Another object of the present invention is to provide a method for preparing a novel cell preservation solution, comprising the steps of:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 37-38 ℃, preserving heat for 1-2 minutes, adding trypsin and N-acetyl-L-cysteine, stirring at the rotating speed of 35 rpm for 1-2 minutes, adding phosphatidylcholine and a stabilizer, stirring for 1-2 minutes, adding a bactericide, stirring for 1-2 minutes, and sealing for storage.
By adopting the technical scheme, the cell preservation solution with good performance can be obtained by mixing purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate in advance, heating the mixed solution to a proper temperature, adding trypsin and N-acetyl-L-cysteine, improving the activity of the trypsin by utilizing the temperature, and finally adding phosphatidylcholine, a stabilizer and a bactericide.
The invention is further configured that in the step (2), the phosphatidylcholine and the stabilizing agent are added and then stirred for 1-2 minutes, then the sugar alcohol calcium zinc solution is added and stirred for 1-2 minutes, and then the bactericide is added.
By adopting the technical scheme, because the sodium gluconate in the stabilizer and the ethylene diamine tetraacetic acid have a complexing effect, the stabilizer is added firstly, and then the sugar alcohol calcium zinc liquid is added, so that the influence of overhigh calcium ion concentration on the activity of the trypsin can be effectively prevented.
In summary, the invention includes at least one of the following beneficial technical effects:
1. the oral mucus preservative solution is added with the trypsin with the specified content, can selectively hydrolyze a peptide chain consisting of the carboxyl of lysine or arginine in protein, and effectively dilute oral mucus, so that cells can be fully released into the cell preservative solution, and meanwhile, the oral mucus preservative solution is also added with the N-acetyl-L-cysteine, the N-acetyl-L-cysteine is soluble in water, so that mucus secretion can be promoted to be changed from sticky to thin to play a strong mucus dissolving effect, and the N-acetyl-L-cysteine has a protection and stabilization effect on the trypsin, and the two are matched with each other, so that the survival rate of the cells can be further improved, and meanwhile, the N-acetyl-L-cysteine has a certain anti-oxidation effect and can improve the storage time of the cell preservative solution;
2. the invention can supply certain nutrition to the oral cavity in the cell preservation solution by adding the phosphatidylcholine, thereby further improving the survival rate of the cells. The bactericide and the stabilizer are added to be matched with other components in the cell preservation solution, so that the oral cells are well dispersed in the cell preservation solution, the oral cells keep good abundance and completeness for a long time, and the survival rate of the cells is further improved;
3. the sugar alcohol calcium zinc solution is added, and provides proper amount of calcium element and zinc element for oral cells, and can also improve the conveying efficiency of phosphatidylcholine; meanwhile, calcium ions provided in the sugar alcohol calcium zinc solution are matched with the complexation of sodium gluconate and ethylene diamine tetraacetic acid, so that the concentration of the calcium ions is moderate, the activity of trypsin is improved, and the cell survival rate is further improved.
Detailed Description
The present invention will be described in further detail with reference to examples.
The first embodiment is as follows:
a novel cell preservation solution comprises 0.45wt% of sodium chloride, 0.05 wt% of sodium dihydrogen phosphate, 0.02 wt% of disodium hydrogen phosphate, 15 wt% of absolute ethyl alcohol, 0.03 wt% of sodium citrate, 0.03 wt% of trypsin, 0.02 wt% of N-acetyl-L-cysteine, 0.01 wt% of phosphatidylcholine, 0.2 wt% of bactericide (polyhexamethylene biguanide), 0.03 wt% of stabilizer (butanediol), and the balance of purified water.
The preparation method of the novel cell preservation solution comprises the following steps:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 38 ℃, preserving heat for 2 minutes, adding trypsin and N-acetyl-L-cysteine at the rotating speed of 35 rpm, stirring for 2 minutes, adding phosphatidylcholine and a stabilizer, stirring for 1 minute, adding a bactericide, stirring for 2 minutes, and sealing for storage.
Example two:
a novel cell preservation solution comprises the following components of 1.2 wt% of sodium chloride, 0.68wt% of sodium dihydrogen phosphate, 0.12 wt% of disodium hydrogen phosphate, 30 wt% of absolute ethyl alcohol, 0.23wt% of sodium citrate, 0.25 wt% of trypsin, 0.66 wt% of N-acetyl-L-cysteine, 0.39wt% of phosphatidylcholine, 0.33 wt% of bactericide (imidazolidinyl urea), 0.46wt% of stabilizer (sodium gluconate) and the balance of purified water.
The preparation method of the novel cell preservation solution comprises the following steps:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 37 ℃, preserving heat for 2 minutes, adding trypsin and N-acetyl-L-cysteine at the rotating speed of 35 rpm, stirring for 1 minute, adding phosphatidylcholine and a stabilizer, stirring for 2 minutes, adding a bactericide, stirring for 1 minute, and sealing for storage.
Example three:
a novel cell preservation solution is characterized by comprising the following components of 1.21wt% of sodium chloride, 0.77wt% of sodium dihydrogen phosphate, 0.23wt% of disodium hydrogen phosphate, 35wt% of absolute ethyl alcohol, 0.31wt% of sodium citrate, 0.0.34wt% of trypsin, 0.71wt% of N-acetyl-L-cysteine, 0.69wt% of phosphatidylcholine, 0.41wt% of bactericide (polyhexamethylene biguanide), 0.581wt% of stabilizer (ethylene diamine tetraacetic acid), and the balance of purified water.
Example four:
a novel cell preservation solution comprises the following components of 1.36wt% of sodium chloride, 0.94wt% of sodium dihydrogen phosphate, 0.34wt% of disodium hydrogen phosphate, 38wt% of absolute ethyl alcohol, 0.36wt% of sodium citrate, 0.46wt% of trypsin, 0.86wt% of N-acetyl-L-cysteine, 0.45wt% of phosphatidylcholine, 0.46wt% of bactericide (isothiazolinone), 0.65wt% of stabilizer (ethylene diamine tetraacetic acid) and the balance of purified water.
The preparation method of the novel cell preservation solution comprises the following steps:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 37 ℃, preserving heat for 2 minutes, adding trypsin and N-acetyl-L-cysteine at the rotating speed of 35 rpm, stirring for 1 minute, adding phosphatidylcholine and a stabilizer, stirring for 1 minute, adding a bactericide, stirring for 1 minute, and sealing for storage.
Example five:
a novel cell preservation solution comprises 0.45-1.6wt% of sodium chloride, 1.1wt% of sodium dihydrogen phosphate, 1.5wt% of disodium hydrogen phosphate, 48wt% of absolute ethyl alcohol, 1.4wt% of sodium citrate, 1.8wt% of trypsin, 0.99wt% of N-acetyl-L-cysteine, 0.86wt% of phosphatidylcholine, 0.68wt% of bactericide (polyhexamethylene biguanide), 0.03 wt% of stabilizer (sodium gluconate) and the balance of purified water.
The preparation method of the novel cell preservation solution comprises the following steps:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 38 ℃, preserving heat for 2 minutes, adding trypsin and N-acetyl-L-cysteine at the rotating speed of 35 rpm, stirring for 2 minutes, adding phosphatidylcholine and a stabilizer, stirring for 2 minutes, adding a bactericide, stirring for 2 minutes, and sealing for storage.
Example six:
a novel cell preservation solution comprises 1.32wt% of sodium chloride, 0.91wt% of sodium dihydrogen phosphate, 0.21wt% of disodium hydrogen phosphate, 36wt% of absolute ethyl alcohol, 0.32wt% of sodium citrate, 0.39wt% of trypsin, 0.78wt% of N-acetyl-L-cysteine, 0.42wt% of phosphatidylcholine, 0.40wt% of bactericide (polyhexamethylene biguanide), 0.52wt% of stabilizer (ethylene diamine tetraacetic acid), 0.005 wt% of sugar alcohol calcium zinc solution and the balance of purified water.
The preparation method of the novel cell preservation solution comprises the following steps:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 38 ℃, preserving heat for 2 minutes, adding trypsin and N-acetyl-L-cysteine at the rotating speed of 35 revolutions per minute, stirring for 2 minutes, adding phosphatidylcholine and a stabilizer, stirring for 2 minutes, adding sugar alcohol calcium zinc solution, stirring for 2 minutes, adding a bactericide, stirring for 2 minutes, and sealing for storage.
Example seven:
a novel cell preservation solution comprises 1.32wt% of sodium chloride, 0.91wt% of sodium dihydrogen phosphate, 0.21wt% of disodium hydrogen phosphate, 36wt% of absolute ethyl alcohol, 0.32wt% of sodium citrate, 0.39wt% of trypsin, 0.78wt% of N-acetyl-L-cysteine, 0.42wt% of phosphatidylcholine, 0.40wt% of bactericide (polyhexamethylene biguanide), 0.52wt% of stabilizer (ethylene diamine tetraacetic acid), 0.036wt% of sugar alcohol calcium zinc solution and the balance of purified water.
The preparation method of the novel cell preservation solution comprises the following steps:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 38 ℃, preserving heat for 2 minutes, adding trypsin and N-acetyl-L-cysteine at the rotating speed of 35 revolutions per minute, stirring for 2 minutes, adding phosphatidylcholine and a stabilizer, stirring for 2 minutes, adding sugar alcohol calcium zinc solution, stirring for 2 minutes, adding a bactericide, stirring for 2 minutes, and sealing for storage.
Example eight:
a novel cell preservation solution comprises 1.32wt% of sodium chloride, 0.91wt% of sodium dihydrogen phosphate, 0.21wt% of disodium hydrogen phosphate, 36wt% of absolute ethyl alcohol, 0.32wt% of sodium citrate, 0.39wt% of trypsin, 0.78wt% of N-acetyl-L-cysteine, 0.42wt% of phosphatidylcholine, 0.40wt% of bactericide (polyhexamethylene biguanide), 0.52wt% of stabilizer (ethylene diamine tetraacetic acid), 0.058wt% of sugar alcohol calcium zinc solution, and the balance of purified water.
The preparation method of the novel cell preservation solution comprises the following steps:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 38 ℃, preserving heat for 2 minutes, adding trypsin and N-acetyl-L-cysteine at the rotating speed of 35 revolutions per minute, stirring for 2 minutes, adding phosphatidylcholine and a stabilizer, stirring for 2 minutes, adding sugar alcohol calcium zinc solution, stirring for 2 minutes, adding a bactericide, stirring for 2 minutes, and sealing for storage.
Comparative example one:
in contrast to example one, no trypsin was added.
Comparative example two:
in contrast to example one, N-acetyl-L-cysteine was not added.
Comparative example three:
in contrast to example one, no phosphatidylcholine was added.
Comparative example four:
in contrast to example one, trypsin and N-acetyl-L-cysteine were not added.
Comparative example five:
compared with the first embodiment, the preparation method of the novel cell preservation solution comprises the following steps:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) adding trypsin and N-acetyl-L-cysteine into the mixed solution obtained in the step (1), stirring for 1-2 minutes, adding phosphatidylcholine and a stabilizer, stirring for 1-2 minutes, adding a bactericide, stirring for 1-2 minutes, and sealing for storage.
Comparative example six:
compared with the first embodiment, the preparation method of the novel cell preservation solution comprises the steps of mixing all the raw materials at normal temperature once, wherein the rotating speed is 35 revolutions per minute, and the time is 10 minutes.
Comparative example seven:
compared with the sixth example, the sugar alcohol calcium-zinc liquid is replaced by the same amount of calcium polyhydroxitol.
Comparative example eight:
compared with the sixth example, the sugar alcohol calcium zinc liquid content is 0.003 wt%.
Comparative example nine:
compared with the sixth example, the sugar alcohol calcium-zinc liquid content is 0.061 wt%.
And (3) performance detection:
the cell preservation solutions prepared in examples one to eight and comparative examples one to nine were collected, and oral cells were collected using the same sampling swab at a cell concentration of 3 × 10-6-10-7Standing the sampling swab in a sealing and preserving range within the range of one cell/m, and preserving the cell preservation solution in a sealing way at the temperature of 4 ℃; after 24 hours, 48 hours, 72 hours, and 96 hours of storage, the cells were taken out, stained with trypan blue, and the number of viable cells was counted under a microscope.
TABLE 1
TABLE 2
From the test results of the first to fifth examples, it can be seen that the cell preservation solution prepared by the present application can effectively decompose mucus in oral cells to improve the cell preservation rate, and from the first and second comparative examples, the addition of trypsin and N-acetyl-L-cysteine can effectively decompose mucus to improve the cell survival rate, and from the fourth comparative example, trypsin and N-acetyl-L-cysteine can produce a synergistic effect to further improve the cell survival rate, and from the third comparative example, phosphatidylcholine can provide nutrients to cells to further improve the cell survival rate.
Meanwhile, as can be seen from the fifth comparative example and the sixth comparative example, the preparation method provided by the invention can effectively improve the survival rate of cells; furthermore, as can be seen from the seventh to ninth comparative examples, the sugar alcohol calcium zinc solution with the specified content is added to provide the proper amount of calcium element and zinc element for oral cells, so that the delivery efficiency of phosphatidylcholine can be improved, and the cell survival rate can be further improved.
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.
Claims (9)
1. A novel cell preservation solution is characterized by comprising 0.45-1.6wt% of sodium chloride, 0.05-1.1wt% of sodium dihydrogen phosphate, 0.02-1.5wt% of disodium hydrogen phosphate, 15-48wt% of absolute ethyl alcohol, 0.03-1.4wt% of sodium citrate, 0.03-1.8wt% of trypsin, 0.02-0.99wt% of N-acetyl-L-cysteine, 0.01-0.86wt% of phosphatidylcholine, 0.2-0.68wt% of a bactericide, 0.03-1.11wt% of a stabilizer and the balance of purified water.
2. The novel cell preservation solution according to claim 1, comprising 1.2 to 1.36wt% of sodium chloride, 0.68 to 0.94wt% of sodium dihydrogen phosphate, 0.12 to 0.34wt% of disodium hydrogen phosphate, 30 to 38wt% of absolute ethanol, 0.23 to 0.36wt% of sodium citrate, 0.25 to 0.46wt% of trypsin, 0.66 to 0.86wt% of N-acetyl-L-cysteine, 0.39 to 0.45wt% of phosphatidylcholine, 0.33 to 0.46wt% of a bactericide, 0.46 to 0.65wt% of a stabilizer, and the balance of purified water.
3. The novel cell preservation solution according to claim 2, comprising 1.21wt% of sodium chloride, 0.77wt% of sodium dihydrogen phosphate, 0.23wt% of disodium hydrogen phosphate, 35wt% of absolute ethanol, 0.31wt% of sodium citrate, 0.0.34wt% of trypsin, 0.71wt% of N-acetyl-L-cysteine, 0.69wt% of phosphatidylcholine, 0.41wt% of a bactericide, 0.581wt% of a stabilizer, and the balance of purified water.
4. The novel cell preservation solution according to claim 1, wherein: the stabilizer is one of butanediol, sodium gluconate and ethylenediamine tetraacetic acid.
5. The novel cell preservation solution according to claim 4, wherein: the bactericide is one of polyhexamethylene biguanide, imidazolidinyl urea and isothiazolinone.
6. The novel cell preservation solution according to claim 5, wherein: also comprises the following components: 0.005-0.058wt% of sugar alcohol calcium zinc liquid.
7. The novel cell preservation solution according to claim 6, characterized by comprising 1.32wt% of sodium chloride, 0.91wt% of sodium dihydrogen phosphate, 0.21wt% of disodium hydrogen phosphate, 36wt% of absolute ethyl alcohol, 0.32wt% of sodium citrate, 0.39wt% of trypsin, 0.78wt% of N-acetyl-L-cysteine, 0.42wt% of phosphatidylcholine, 0.40wt% of bactericide, 0.52wt% of stabilizer, 0.036wt% of sugar alcohol calcium zinc solution, and the balance of purified water;
wherein the stabilizer is ethylenediamine tetraacetic acid, and the bactericide is polyhexamethylene biguanide.
8. The method for producing a novel cell preservation solution according to any one of claims 1 to 7, characterized by comprising the steps of:
(1) putting purified water, sodium chloride, sodium dihydrogen phosphate, disodium hydrogen phosphate, absolute ethyl alcohol and sodium citrate into a mixer and uniformly mixing;
(2) heating the mixed solution in the step (1) to 37-38 ℃, preserving heat for 1-2 minutes, adding trypsin and N-acetyl-L-cysteine, stirring at the rotating speed of 35 rpm for 1-2 minutes, adding phosphatidylcholine and a stabilizer, stirring for 1-2 minutes, adding a bactericide, stirring for 1-2 minutes, and sealing for storage.
9. The method for producing a novel cell preservation solution according to claim 8, wherein: and (3) in the step (2), adding phosphatidylcholine and a stabilizer, stirring for 1-2 minutes, then adding sugar alcohol calcium zinc solution, stirring for 1-2 minutes, and adding a bactericide.
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| CN (1) | CN111449054A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111869658A (en) * | 2020-08-22 | 2020-11-03 | 南京健邦锦源医疗科技有限公司 | Inactivated virus preservation solution and preparation method thereof |
| CN111961664A (en) * | 2020-09-08 | 2020-11-20 | 武汉中投汉嘉科技有限公司 | Nucleic acid extracting solution |
| CN113383769A (en) * | 2021-06-29 | 2021-09-14 | 广州朗坤生物科技有限公司 | Cell preservation solution and preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108770836A (en) * | 2018-08-21 | 2018-11-09 | 生工生物工程（上海）股份有限公司 | Liquid based cell preservative fluid and its preparation method and application |
-
2020
- 2020-05-21 CN CN202010438194.9A patent/CN111449054A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108770836A (en) * | 2018-08-21 | 2018-11-09 | 生工生物工程（上海）股份有限公司 | Liquid based cell preservative fluid and its preparation method and application |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111869658A (en) * | 2020-08-22 | 2020-11-03 | 南京健邦锦源医疗科技有限公司 | Inactivated virus preservation solution and preparation method thereof |
| CN111961664A (en) * | 2020-09-08 | 2020-11-20 | 武汉中投汉嘉科技有限公司 | Nucleic acid extracting solution |
| CN113383769A (en) * | 2021-06-29 | 2021-09-14 | 广州朗坤生物科技有限公司 | Cell preservation solution and preparation method and application thereof |
| CN113383769B (en) * | 2021-06-29 | 2022-04-19 | 广州朗坤生物科技有限公司 | Cell preservation solution and preparation method and application thereof |
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