CN115433704A - Cell digestive juice and method for separating and culturing oral mucosa epithelial cells by using same - Google Patents
Cell digestive juice and method for separating and culturing oral mucosa epithelial cells by using same Download PDFInfo
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- 210000002919 epithelial cell Anatomy 0.000 title claims abstract description 73
- 210000002200 mouth mucosa Anatomy 0.000 title claims abstract description 71
- 230000001079 digestive effect Effects 0.000 title claims abstract description 49
- 235000011389 fruit/vegetable juice Nutrition 0.000 title claims abstract description 43
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000012258 culturing Methods 0.000 title abstract description 15
- 108091005804 Peptidases Proteins 0.000 claims abstract description 33
- 239000004365 Protease Substances 0.000 claims abstract description 33
- 230000029087 digestion Effects 0.000 claims abstract description 32
- 238000000926 separation method Methods 0.000 claims abstract description 31
- 239000003248 enzyme activator Substances 0.000 claims abstract description 30
- TTWYZDPBDWHJOR-IDIVVRGQSA-L adenosine triphosphate disodium Chemical compound [Na+].[Na+].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O TTWYZDPBDWHJOR-IDIVVRGQSA-L 0.000 claims abstract description 17
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract 4
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- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 claims description 7
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- 238000012360 testing method Methods 0.000 description 12
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- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 7
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- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 3
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- YVBGRQLITPHVOP-UHFFFAOYSA-L disodium;[hydroxy-[hydroxy(oxido)phosphoryl]oxyphosphoryl] hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])(=O)OP(O)(=O)OP(O)([O-])=O YVBGRQLITPHVOP-UHFFFAOYSA-L 0.000 description 3
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
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- 238000001356 surgical procedure Methods 0.000 description 2
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- MUTDXQJNNJYAEG-UHFFFAOYSA-N 2-[4-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-3-(dimethylamino)pyrazol-1-yl]-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C=1C(=NN(C=1)CC(=O)N1CC2=C(CC1)NN=N2)N(C)C MUTDXQJNNJYAEG-UHFFFAOYSA-N 0.000 description 1
- APLNAFMUEHKRLM-UHFFFAOYSA-N 2-[5-[2-(2,3-dihydro-1H-inden-2-ylamino)pyrimidin-5-yl]-1,3,4-oxadiazol-2-yl]-1-(3,4,6,7-tetrahydroimidazo[4,5-c]pyridin-5-yl)ethanone Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C1=NN=C(O1)CC(=O)N1CC2=C(CC1)N=CN2 APLNAFMUEHKRLM-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- MKYBYDHXWVHEJW-UHFFFAOYSA-N N-[1-oxo-1-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propan-2-yl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(C(C)NC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 MKYBYDHXWVHEJW-UHFFFAOYSA-N 0.000 description 1
- 208000018359 Systemic autoimmune disease Diseases 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0632—Cells of the oral mucosa
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
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- Microbiology (AREA)
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- Bioinformatics & Cheminformatics (AREA)
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- Analytical Chemistry (AREA)
- Genetics & Genomics (AREA)
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- Medicinal Chemistry (AREA)
- Dermatology (AREA)
- Rheumatology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The application relates to the technical field of cell separation culture, and particularly discloses a cell digestive juice and a method for separating and culturing oral mucosa epithelial cells by using the same. The application discloses a cell digestive juice, which comprises the following components in concentration: 4-8g/L of protease; 0.2-0.6g/L of disodium adenosine triphosphate; the enzyme activator is 0.09-0.15g/L. The application also discloses application of the cell digestive juice in the field of cell separation culture. When the cell digestive juice is used for carrying out separation culture on the epithelial cells of the oral mucosa, the digestion time can be effectively shortened, and cells with large quantity and high survival rate can be obtained.
Description
Technical Field
The application relates to the technical field of cell separation culture, in particular to cell digestive juice and a method for separating and culturing oral mucosa epithelial cells by using the cell digestive juice.
Background
Rheumatoid arthritis is a systemic autoimmune disease mainly manifested by erosive arthritis, and anti-perinuclear factor autoantibodies can appear in the serum of most patients. The oral mucosa epithelial cells can be used as target antigens of the anti-perinuclear factor antibodies and used for detecting the anti-perinuclear factor antibodies, thereby further providing early specific diagnosis indexes for the rheumatoid arthritis and having good prompting function on the judgment and prognosis of the rheumatoid arthritis. Therefore, the isolated culture of the oral mucosa epithelial cells is important for researching the rheumatoid arthritis diseases.
At present, in the method of cell isolation and culture, it is necessary to digest tissues by using pancreatin and collagenase in sequence to obtain a cell suspension, and then culture the cell suspension by using a serum-containing medium. However, the above method makes the separation process of cells more complicated, the digestion time is too long, and the test cost is increased; in addition, in the process of tissue digestion, multiple times of shaking are needed, and various cell treating agents and the like are added, so that the integrity of cells is easily damaged, the survival rate of the cells is low, and the yield of the cells is reduced.
Based on the above, a method for separating and culturing oral mucosa epithelial cells needs to be found, which improves the separation efficiency of the cells, and effectively relieves the damage to the cells in the separation process, so that the oral mucosa epithelial cells with large quantity and high survival rate can be obtained.
Disclosure of Invention
In order to effectively shorten the digestion time and reduce the damage of cells so as to obtain a large number of oral mucosa epithelial cells with high survival rate, the application provides a cell digestive juice and a method for separating and culturing the oral mucosa epithelial cells by using the cell digestive juice.
In a first aspect, the present application provides a cell digest comprising the following concentrations of components: 4-8g/L of protease; 0.2-0.6g/L of disodium adenosine triphosphate; the enzyme activator is 0.09-0.15g/L.
According to the application, the cell digestive juice is prepared by using the protease, the adenosine disodium triphosphate and the enzyme activator with the concentrations, and then the cell digestive juice is used in the separation culture method of the oral mucosa epithelial cells, so that the oral mucosa epithelial cells with a large number and high survival rate can be obtained.
Since a protein with high viscosity is connected between the cell surface and the extracellular fluid, the cells are connected together and are difficult to separate. The protease used in the application can effectively hydrolyze proteins among cells, accelerate the separation speed among the cells and further obtain a large number of cells.
In addition, the application utilizes the disodium adenosine triphosphate as an effective component of the cell digestive juice, can obviously reduce the surface tension of the liquid, improves the solubilizing capability of the solution, and is favorable for promoting the separation speed of cells. The enzyme activator can promote the enzyme activity of the protease and enhance the separation effect of the protease on cells, thereby obtaining a large number of cells. In addition, the effect of the disodium adenosine triphosphate and the enzyme activator substance on cells is mild, and the survival rate of the cells can be effectively improved.
In a specific embodiment, the concentration of the disodium adenosine triphosphate may be 0.2g/L, 0.4g/L, 0.6g/L.
In some specific embodiments, the concentration of the disodium adenosine triphosphate can also be 0.2-0.4g/L, 0.4-0.6g/L.
Test analysis shows that when the concentration of the adenosine disodium triphosphate is controlled within the range, the number and the survival rate of the oral mucosa epithelial cells can be effectively improved. Therefore, the present application controls the concentration of disodium adenosine triphosphate to be within the above range.
In a specific embodiment, the concentration of the enzyme activator may be 0.09g/L, 0.12g/L, 0.15g/L.
In some specific embodiments, the concentration of the enzyme activator may also be 0.09 to 0.12g/L, 0.12 to 0.15g/L.
As proved by experimental analysis, when the concentration of the enzyme activator is controlled within the range, the number and the survival rate of the epithelial cells of the oral mucosa can be effectively improved. Therefore, the present application controls the concentration of the enzyme activator within the above range.
Preferably, the protease is selected from one or more of collagenase type i, pronase and trypsin.
Further, said protease comprises said collagenase type i and said pronase; the weight ratio of the collagenase type I to the pronase protease is 5: (0.8-1.6).
In a particular embodiment, the weight ratio of said collagenase type i and said pronase protease may be 5:0.8, 5:1.2, 5:1.6.
in some specific embodiments, the weight ratio of the collagenase type i to the pronase protease may also be 5: (0.8-1.2), 5: (1.2-1.6).
As proved by experimental analysis, when the weight ratio of the collagenase type I to the pronase is controlled to be within the range, the number and the survival rate of the epithelial cells of the oral mucosa can be further improved. Therefore, the present application controls the weight ratio of collagenase type I and pronase to be within the above range.
Preferably, the enzyme activator is selected from one or more of phosphatidylethanolamine, reduced glutathione and phosphatidylserine.
The application finds that phosphatidylethanolamine, reductive glutathione and phosphatidylserine can be used as enzyme activators of protease, so that enzymatic reaction is accelerated, the digestion time is shortened, and a large number of oral mucosa epithelial cells can be obtained.
Further, the enzyme activator is phosphatidylethanolamine.
According to experimental analysis, compared with the method of selecting reduced glutathione or phosphatidylserine, the method of selecting phosphatidylethanolamine as the enzyme activator can further improve the number and survival rate of the oral mucosa epithelial cells. Thus, the present application chooses to use phosphatidylethanolamine as the enzyme activator.
In a second aspect, the application provides an application of the cell digestive juice in the field of cell separation and culture.
In a third aspect, the application provides oral mucosa epithelial cells obtained by utilizing the above cell digestive juice separation culture.
In a fourth aspect, the application provides the above method for isolating and culturing the epithelial cells of the oral mucosa, which specifically comprises the following steps:
taking the oral mucosa tissue after disinfection, cleaning and shearing, uniformly mixing the oral mucosa tissue with the cell digestive juice, and digesting to obtain separated cell suspension; pipetting the cell suspension into a cell culture medium and placing at 37 ℃ 5% 2 The cell culture box of (1) is kept still for culture.
This application carries out isolated culture to oral mucosa epithelial cell through one-step digestion method, can obtain the many and high cell of survival rate of quantity, compares in traditional two-step digestion method, has simplified the operation flow, and has shortened test time to effectively reduce test cost.
In addition, when the cell digestive juice provided by the application is used for separating the oral mucosa epithelial cells, metal catalytic reaction is not required to be added, so that the digestive reaction is not required to be stopped by using a serum-containing culture solution in the follow-up process, and the safety of the oral mucosa epithelial cells is favorably improved.
Preferably, the cleaning step of the oral mucosa tissue is as follows: transferring the oral mucosa tissue into PBS buffer solution containing 100U/mL penicillin and 100U/mL streptomycin and having pH of 7.2, and washing for 5-6 times; the washed tissue was then placed in PBS buffer containing 50U/mL penicillin and 50U/mL streptomycin and stored at 4 ℃ until use.
Penicillin and streptomycin are dissolved in PBS buffer solution with the pH value of 7.2 to prepare a mixed reagent which is used for cleaning oral mucosa tissues and can effectively remove the pollution of bacteria and mould, thereby being beneficial to improving the quality safety of oral mucosa epithelial cells.
Preferably, the digestion parameter conditions are: the temperature is 22-28 ℃; the time is 15-25min.
When the digestive juice is used for separating different cell lines, the temperature and time in the digestion process can have important influence on the separation effect of the cells. Compared with the traditional digestion method, the cell digestive juice provided by the application can achieve a good separation effect of cells at a lower temperature in a shorter time, and has a mild effect on the cells, so that oral mucosa epithelial cells with a large number and a high survival rate can be obtained.
In a specific embodiment, the digestion temperature may be 22 ℃, 25 ℃, 28 ℃.
In some specific embodiments, the digestion temperature may be 22-25 ℃ and 25-28 ℃.
According to experimental analysis, when the digestion temperature is controlled within the range, the number and the survival rate of the oral mucosa epithelial cells can be further improved. Therefore, the present application controls the digestion temperature within the above range.
In a specific embodiment, the digestion time may be 15min, 20min, 25min.
In some specific embodiments, the digestion time may also be 15-20min, 20-25min.
According to experimental analysis, when the digestion time is controlled within the range, the number and the survival rate of the oral mucosa epithelial cells can be further improved. Therefore, the present application controls the digestion time within the above range.
Further, the cell culture medium is a serum-free medium.
Further, the serum-free medium is DMEM/F12 medium.
According to the method, the cell suspension obtained by separation is cultured by using the DMEM/F12 serum-free culture medium, the introduction of animal-derived components in the serum-containing culture medium is avoided, the risk of virus, fungus and other microbial pollution caused by serum is reduced, the normal biological characteristics and functions of the oral mucosa epithelial cells can be stably maintained by using the serum-free culture system, the quality of the oral mucosa epithelial cells obtained by culture is stable, and the safety of the cells in clinical application is ensured.
In a fifth aspect, the application provides an application of the oral mucosal epithelial cells in the field of detection of anti-perinuclear factor antibodies.
The oral mucosa epithelial cells obtained by using the cell digestive juice and the separation culture method provided by the application can keep complete surface antigens, do not contain any animal or bacteria source components, and can meet the requirements of various experiments such as cell marking, virus analysis, flow cytometry related detection and the like. Therefore, the oral mucosa epithelial cells provided by the application are used as target antigens of the anti-perinuclear factor antibodies, and are favorable for further application in detection of the anti-perinuclear factor antibodies.
To sum up, the technical scheme of this application has following effect:
according to the application, the cell digestive juice is prepared by using protease, disodium adenosine triphosphate and an enzyme activator, and then the cell digestive juice is used in the separation culture method of the oral mucosa epithelial cells, so that the oral mucosa epithelial cells with large quantity and high survival rate can be obtained.
When the cell digestive juice provided by the application is used for carrying out separation culture on oral mucosa epithelial cells, only a one-step digestion method is adopted, so that the operation flow is simplified; and the digestion temperature is lower, the digestion time is shorter, and the test cost is effectively reduced.
According to the method for separating and culturing the oral mucosa epithelial cells, a serum-containing substance is not required to be used for stopping the digestion reaction, and in addition, the serum-free culture medium is adopted for culturing the cells, so that the oral mucosa epithelial cells with stable quality can be obtained, the safety of the cells in clinical application is ensured, and the method is further favorably applied to the field of detection of the anti-perinuclear factor antibodies.
Detailed Description
In a first aspect, the present application provides a cell digest comprising the following concentrations of components: 4-8g/L of protease; 0.2-0.6g/L of disodium adenosine triphosphate; the enzyme activator is 0.09-0.15g/L.
Wherein the protease is selected from one or more of collagenase type I, pronase and trypsin.
Further, the protease includes collagenase type i and pronase; the weight ratio of collagenase type I to pronase is 5: (0.8-1.6).
Meanwhile, the enzyme activator is selected from one or more of phosphatidylethanolamine, reduced glutathione and phosphatidylserine.
Further, the enzyme activator is phosphatidylethanolamine.
In a second aspect, the application provides an application of the cell digestive juice in the field of cell separation and culture.
In a third aspect, the application provides oral mucosa epithelial cells obtained by utilizing the above cell digestive juice separation culture.
In a fourth aspect, the application provides the above method for isolating and culturing the epithelial cells of the oral mucosa, which specifically comprises the following steps:
s1: obtaining oral mucosa tissues: taking redundant mucous membrane tissues discarded in hospital surgeries, immediately storing the redundant mucous membrane tissues in a pre-cooled DMEM (DMEM) culture solution at 4 ℃, conveying the mucous membrane tissues to a sterile operating platform of a cell culture room, disinfecting the mucous membrane tissues for 2min by using 75% alcohol, transferring the mucous membrane tissues into a PBS (phosphate buffer solution) buffer solution containing 100U/mL penicillin and 100U/mL streptomycin and having the pH value of 7.2, and cleaning for 5-6 times; the washed mucosal tissue is placed in PBS buffer containing 50U/mL penicillin and 50U/mL streptomycin and stored at 4 ℃ for later use.
S2: separation of oral mucosa epithelial cells: taking a mucous membrane tissue, shearing a macroscopic submucosal tissue by utilizing an ophthalmic scissors, and shearing the macroscopic submucosal tissue into mucous membrane tissue blocks with the size of 2mm multiplied by 2 mm; taking 1g of trimmed mucous membrane tissues and placing the trimmed mucous membrane tissues in 10-15mL of digestive juice for digestion; gently blowing and beating the tissue digestive juice to form a cell suspension, and filtering the cell suspension by using a 200-mesh sterile filter screen; the filtrate was centrifuged at 1000rpm for 10min, the supernatant was discarded to obtain a cell pellet, and then the cell pellet was resuspended in 10mL of PBS buffer having a pH of 7.2 to obtain a cell suspension.
S3: culturing oral mucosa epithelial cells: 0.1mL of the aspirated cell suspension was added to 10mL of DMEM/F12 serum-free medium, then placed at 37 ℃ and 5% CO 2 The cell culture box is kept still for culture, during the culture period, trypan blue is used for staining live cells every 2 days, the survival rate and the number of the cells are calculated, and then culture is continued by changing culture solution until the cell fusion rate is 80-90%.
Specifically, the digestion parameter conditions were: the temperature is 22-28 ℃; the time is 15-25min.
In a fifth aspect, the application provides an application of the oral mucosa epithelial cells in the field of detecting anti-perinuclear factor antibodies.
The present application is described in further detail below in connection with preparations 1-22, examples 1-24, comparative examples 1-8, and performance testing tests, which are not to be construed as limiting the scope of the invention as claimed.
Preparation example
Preparation examples 1 to 9
Preparation examples 1 to 9 each provide a cell digest.
The difference of the preparation examples is as follows: the addition amount of each component in the cell digestive juice. Specifically, the results are shown in Table 1.
The preparation method of each preparation example comprises the following steps: weighing 6.2g of protease, disodium adenosine triphosphate and enzyme activator phosphatidylethanolamine according to the formula of table 1, fully dissolving in 950mL of phosphate buffer solution, adjusting the pH to 7.2, fixing the volume to 1L, and then filtering and sterilizing by using a 0.2 mu m filter membrane to obtain cell digestive juice; of these, 6.2g of protease were 5g of collagenase type I and 1.2g of pronase.
TABLE 1 addition amount of each component in cell digests in preparation examples 1 to 9
Preparation examples 10 to 15
Preparation examples 10 to 15 each provide a cell digest.
The above-mentioned preparation examples differ from preparation example 3 in that: the weight ratio of collagenase type I to pronase. Specifically, as shown in table 2.
TABLE 2 weight ratio of collagenase type I to pronase in preparation examples 3, 10-15
Preparation example 16
The present preparation example provides a cell digest.
The difference between the preparation example and the preparation example 3 is that: the protease was 5g collagenase type I and 1.2g trypsin.
Preparation example 17
The present preparation provides a cell digest.
The difference between the preparation example and the preparation example 3 is that: the protease was 5g trypsin and 1.2g streptokinase protease.
Preparation example 18
The present preparation provides a cell digest.
The difference between the preparation example and the preparation example 3 is that: the protease was 6.2g trypsin.
Preparation examples 19 to 20
Preparation examples 19 to 20 each provide a cell digest.
The above-mentioned preparation examples differ from preparation example 3 in that: the kind of enzyme activator. The details are shown in Table 3.
TABLE 3 kinds of enzyme activators in preparation examples 3, 19 to 20
| Preparation example | Classes of enzyme activators |
| 3 | Phosphatidylethanolamine |
| 19 | Reduced glutathione |
| 20 | Phosphatidylserine |
Preparation example 21
The present preparation example provides a cell digest.
The preparation method of the preparation example comprises the following steps: weighing 6.2g of protease and 0.4g of adenosine disodium triphosphate, fully dissolving in 950mL of phosphate buffer solution, adjusting the pH to 7.2, fixing the volume to 1L, and then filtering and sterilizing by using a 0.2 mu m filter membrane to obtain cell digestive juice; of these, 6.2g of protease were 5g of collagenase type I and 1.2g of pronase.
Preparation example 22
The present preparation provides a cell digest.
The preparation method of the preparation example comprises the following steps: weighing 6.2g of protease and 0.12g of phosphatidylethanolamine, fully dissolving in 950mL of phosphate buffer solution, adjusting the pH to 7.2, fixing the volume to 1L, and then filtering and sterilizing by using a 0.2 mu m filter membrane to obtain cell digestive juice; of these, 6.2g of protease were 5g of collagenase type I and 1.2g of pronase.
Examples
Examples 1 to 16
Examples 1-16 provide a method for isolating and culturing oral mucosal epithelial cells, respectively.
The above embodiments differ in that: the types of cellular digests vary. Specifically, as shown in table 2.
The isolated culture method of the embodiment specifically comprises the following steps:
s1: obtaining oral mucosa tissues: taking redundant mucous membrane tissues discarded in hospital surgeries, immediately storing the redundant mucous membrane tissues in a pre-cooled DMEM (DMEM) culture solution at 4 ℃, conveying the tissue to a sterile operating platform of a cell culture room, disinfecting the tissue for 2min by using 75% alcohol, transferring the oral mucous membrane tissues into a PBS (phosphate buffer solution) containing 100U/mL penicillin and 100U/mL streptomycin and having a pH value of 7.2, and cleaning for 5-6 times; the oral mucosa tissue obtained by washing is placed in PBS buffer solution containing 50U/mL penicillin and 50U/mL streptomycin, and is preserved at 4 ℃ for standby.
S2: separation of oral mucosa epithelial cells: taking oral mucosa tissue, shearing macroscopic submucosal tissue by utilizing an ophthalmic scissors, and shearing into mucosa tissue blocks with the size of 2mm multiplied by 2 mm; taking 1g of the trimmed mucous membrane tissue, placing the trimmed mucous membrane tissue in 12mL of digestive juice, placing the digestive juice under the condition of 25 ℃, and digesting the mucous membrane tissue for 20min; gently blowing and beating the tissue digestive juice to form a cell suspension, and filtering the cell suspension by using a 200-mesh sterile filter screen; the filtrate was centrifuged at 1000rpm for 10min, the supernatant was discarded to obtain a cell pellet, and then the cell pellet was resuspended in 10mL of PBS buffer having a pH of 7.2 to obtain a cell suspension.
S3: culturing oral mucosa epithelial cells: 0.1mL of the aspirated cell suspension was added to 10mL of DMEM/F12 medium, which was then subjected to 37 ℃ C. And 5% CO 2 The cell culture chamber (2) was left to stand for culture, and during the culture, viable cells were grown using trypan blue at intervals of 1 dayAnd (3) staining cells, calculating the survival rate and the number of the cells, and then changing the culture solution to continue culturing until the cell fusion rate reaches 80-90%.
TABLE 4 types of cell digests in examples 1-16
| Examples | Type of cell digestive fluid | Examples | Type of cell digestive fluid |
| 1 | Preparation example 2 | 9 | Preparation example 13 |
| 2 | Preparation example 3 | 10 | Preparation example 14 |
| 3 | Preparation example 4 | 11 | Preparation example 15 |
| 4 | Preparation example 7 | 12 | Preparation example 16 |
| 5 | Preparation example 8 | 13 | Preparation example 17 |
| 6 | Preparation example 10 | 14 | Preparation example 18 |
| 7 | Preparation example 11 | 15 | Preparation example 19 |
| 8 | Preparation example 12 | 16 | Preparation example 20 |
Examples 17 to 24
Examples 17-24 each provide a method for isolating and culturing oral mucosal epithelial cells.
The above embodiments are different from embodiment 2 in that: digestion temperature and digestion time. Specifically, the results are shown in Table 5.
TABLE 5 digestion time in examples 2, 17-24
Comparative examples 1 to 6
Comparative examples 1-6 provide a method for isolated culture of oral mucosal epithelial cells, respectively.
The above comparative examples differ from example 2 in that: the types of cellular digests vary. Specifically, the results are shown in Table 6. The remaining steps were the same as in example 2.
TABLE 6 types of cell digests in comparative examples 1-6
| Examples | Type of cell digestive fluid | Examples | Type of cell digestive fluid |
| 1 | Preparation example 1 | 4 | Preparation example 9 |
| 2 | Preparation example 5 | 5 | Preparation example 21 |
| 3 | Preparation example 6 | 6 | Preparation example 22 |
Comparative example 7
Comparative example 7 provides a method for isolated culture of oral mucosal epithelial cells.
The comparative example differs from example 2 in that: the cell digest used in step S2 was commercially available pancreatin digest (purchased from Shanghai culture Biotech Co., ltd.). The remaining steps were the same as in example 2.
Comparative example 8
Comparative example 8 provides a method for isolated culture of oral mucosal epithelial cells.
This comparative example differs from example 2 in that: the separation methods of the oral mucosa epithelial cells in the step S2 are different, and in the comparative example, the oral mucosa epithelial cells are separated by using the method provided by the patent CN 108546673B. The remaining steps were the same as in example 2.
Performance test
Taking the separation culture method of the oral mucosa epithelial cells provided by the examples 1-24 and the comparative examples 1-8 as a detection object, counting the cells of the cell suspension obtained in the step S2, and inspecting the separation effect of the oral mucosa epithelial cells; and counting the cells of the cell suspension obtained after the culture for 3 days in the step S3, and inspecting the culture effect of the epithelial cells of the oral mucosa.
The detection method comprises the following steps: 0.5mL of the cell suspension was taken, and diluted to 1X 10 times with PBS buffer solution having pH of 7.2 3 (ii) a Adding 0.5mL of diluent into a test tube, adding 0.5mL of 4% trypan blue staining solution into the test tube, and staining for 2min; then, 0.1mL of the suspension was applied to the slide, a cover slip was added, the number of dead cells and the number of live cells in 0.1mL of the cell suspension were counted under a microscope, and the cell survival rate was calculated.
And (3) detection results: as shown in table 7.
TABLE 7 results of examining the number of cells and the survival rate of cells in examples 1 to 24 and comparative examples 1 to 8
By combining table 5 and comparing the detection results of examples 1-24 with the detection results of comparative examples 1-8, the application prepares cell digestive juice by selecting protease, disodium adenosine triphosphate and enzyme activator, and the cell digestive juice is used in the isolation culture method of oral mucosa epithelial cells, the number of the isolated living cells is higher than 254, and the cell survival rate is higher than 90.4%; after the separated cells are cultured for 3 days, the number of the obtained living cells is higher than 2720, and the cell survival rate is higher than 92.7%. The detection results show that the cell digestive juice and the separation culture method provided by the application can effectively improve the separation culture effect of the oral mucosa epithelial cells, so that the oral mucosa epithelial cells with high number and high survival rate can be obtained.
By comparing the test results of example 2 with comparative examples 7 to 8, when the oral mucosal epithelial cells were separated using commercially available pancreatin digestive juice or the separation method provided in comparative example 6, the number of cells and the survival rate were low; and the selective use of the separation method provided by the application can effectively improve the number and survival rate of the epithelial cells of the oral mucosa.
By comparing the detection results of example 2 and comparative examples 5-6, compared with the selection of using disodium adenosine triphosphate and protease or the selection of using an enzyme activator and protease, the application selects the simultaneous use of disodium adenosine triphosphate, the enzyme activator and the protease as cell digestive juice, so that the number and the survival rate of the oral mucosa epithelial cells can be further improved. Therefore, the application chooses to use disodium adenosine triphosphate, enzyme activator and protease together as the cell digest.
By comparing the test results of examples 1-3 with comparative examples 1-2, the number and survival rate of oral mucosal epithelial cells can be further improved when the concentration of disodium adenosine triphosphate in the cell digestive fluid is controlled within the range of 0.2-0.6 g/L. Therefore, the present application controls the concentration of disodium adenosine triphosphate in the cell digest to be within the above range.
By comparing the detection results of examples 2 and 15-16, compared with the selection of reduced glutathione or phosphatidylserine, the application selects phosphatidylethanolamine as an enzyme activator, and the number and the survival rate of the oral mucosa epithelial cells can be further improved. Thus, the present application chooses to use phosphatidylethanolamine as the enzyme activator.
By comparing the detection results of the examples 2, 4-5 and the comparative examples 3-4, when the concentration of phosphatidylethanolamine in the cell digestive juice is controlled within the range of 0.09-0.15g/L, the number and the survival rate of the oral mucosa epithelial cells can be effectively improved. Therefore, the present application controls the concentration of phosphatidylethanolamine in the cell digest to be within the above range.
According to the detection results of comparative examples 10-11 and 14, compared with the use of trypsin alone, the use of collagenase I alone or streptokinase protease alone as protease for preparing cell digestive juice can further improve the number and survival rate of oral mucosa epithelial cells. Thus, the present application selects the use of type I collagenase alone or the use of the streptokinase protease alone as the protease.
By comparing the detection results of examples 2 and 12-13, compared with the simultaneous use of trypsin and collagenase type I or the simultaneous use of trypsin and streptokinase, collagenase type I and streptokinase are selected to be used together as proteases for preparing cell digestive juice, so that the number and the survival rate of epithelial cells of the oral mucosa can be further improved. Thus, the present application chooses to use both collagenase type i and pronase.
According to the detection results of comparative examples 2, 6-9, when the weight ratio of the collagenase type I to the pronase protease is controlled to be 5: (0.8-1.6), the number and survival rate of oral mucosa epithelial cells can be further improved. Thus, the present application controls the weight ratio of collagenase type I and pronase to be within the above range.
By comparing the test results of examples 2 and 17-20, the number and survival rate of the oral mucosa epithelial cells can be further improved when the digestion temperature is controlled within the range of 22-28 ℃. Therefore, the present application controls the digestion temperature within the above range.
Through the detection results of comparative examples 2 and 21-24, when the digestion time is controlled within the range of 15-25min, the number and survival rate of the oral mucosa epithelial cells can be further improved. Therefore, the present application controls the digestion time within the above range.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. A cell digest comprising the following concentrations of components: 4-8g/L of protease; 0.2-0.6g/L of disodium adenosine triphosphate; the enzyme activator is 0.09-0.15g/L.
2. The cell lysate of claim 1, wherein the protease is selected from one or more of collagenase type i, pronase, and trypsin.
3. The cell lysate of claim 2, wherein the protease comprises the collagenase type i and the pronase protease; the weight ratio of the collagenase type I to the pronase protease is 5: (0.8-1.6).
4. The cellular digest of claim 1, wherein the enzyme activator is selected from one or more of phosphatidylethanolamine, reduced glutathione, and phosphatidylserine.
5. Use of a cell digest according to any one of claims 1 to 4 in the field of cell separation and culture.
6. An oral mucosal epithelial cell obtained by isolated culture of an oral mucosal tissue using the cell digest of any one of claims 1 to 4.
7. The isolated culture method of the oral mucosal epithelial cells according to claim 6, comprising the following steps:
taking the oral mucosa tissue after disinfection, cleaning and shearing, uniformly mixing the oral mucosa tissue with the cell digestive juice, and digesting to obtain separated cell suspension; pipetting the cell suspension into a cell culture medium and placing at 37 ℃ 5% 2 The cell culture box of (1) is kept still for culture.
8. The isolated culture method of oral mucosal epithelial cells according to claim 7, wherein the digestion parameter conditions are: the temperature is 22-28 ℃; the time is 15-25min.
9. The method for isolated culture of oral mucosal epithelial cells according to claim 7, wherein the cell culture medium is a serum-free medium.
10. The use of the oral mucosal epithelial cells according to claim 6 for detecting anti-perinuclear factor antibodies.
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| JP2013249299A (en) * | 2012-05-30 | 2013-12-12 | Kai:Kk | Anti-infective treatment agent using enzyme treatment liquid, and method for using the same |
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| JP2013249299A (en) * | 2012-05-30 | 2013-12-12 | Kai:Kk | Anti-infective treatment agent using enzyme treatment liquid, and method for using the same |
| CN105505860A (en) * | 2016-01-13 | 2016-04-20 | 河南科技大学第一附属医院 | Method for isolated culture of esophagus epithelial stem cells |
| CN106047801A (en) * | 2016-05-30 | 2016-10-26 | 深圳大学 | Nucleus pulposus cell separating and purifying method |
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