WO2023056726A1 - 一种液基细胞保存液及其配制方法 - Google Patents
一种液基细胞保存液及其配制方法 Download PDFInfo
- Publication number
- WO2023056726A1 WO2023056726A1 PCT/CN2022/077616 CN2022077616W WO2023056726A1 WO 2023056726 A1 WO2023056726 A1 WO 2023056726A1 CN 2022077616 W CN2022077616 W CN 2022077616W WO 2023056726 A1 WO2023056726 A1 WO 2023056726A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- liquid
- preservation solution
- mixture
- cell preservation
- based cell
- Prior art date
Links
- 239000007788 liquid Substances 0.000 title claims abstract description 74
- 239000003761 preservation solution Substances 0.000 title claims abstract description 69
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 57
- 239000008213 purified water Substances 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 32
- 210000003097 mucus Anatomy 0.000 claims abstract description 18
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 17
- 239000003146 anticoagulant agent Substances 0.000 claims abstract description 15
- 229940127219 anticoagulant drug Drugs 0.000 claims abstract description 15
- 239000003755 preservative agent Substances 0.000 claims abstract description 14
- 239000000872 buffer Substances 0.000 claims abstract description 11
- 230000002335 preservative effect Effects 0.000 claims abstract description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 72
- 239000000203 mixture Substances 0.000 claims description 59
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 51
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 46
- 238000003756 stirring Methods 0.000 claims description 37
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 36
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 36
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 28
- 239000002243 precursor Substances 0.000 claims description 28
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 24
- 239000011780 sodium chloride Substances 0.000 claims description 23
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical group CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 claims description 20
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 18
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 18
- 239000001632 sodium acetate Substances 0.000 claims description 18
- 235000017281 sodium acetate Nutrition 0.000 claims description 18
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 claims description 16
- 235000011285 magnesium acetate Nutrition 0.000 claims description 16
- 239000011654 magnesium acetate Substances 0.000 claims description 16
- 229940069446 magnesium acetate Drugs 0.000 claims description 16
- 229960001484 edetic acid Drugs 0.000 claims description 15
- 239000001639 calcium acetate Substances 0.000 claims description 14
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 claims description 14
- 229960005147 calcium acetate Drugs 0.000 claims description 14
- 235000011092 calcium acetate Nutrition 0.000 claims description 14
- 239000001103 potassium chloride Substances 0.000 claims description 14
- 235000011164 potassium chloride Nutrition 0.000 claims description 14
- 235000002639 sodium chloride Nutrition 0.000 claims description 14
- 150000001298 alcohols Chemical class 0.000 claims description 13
- 229940009662 edetate Drugs 0.000 claims description 13
- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 claims description 12
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 12
- QCDWFXQBSFUVSP-UHFFFAOYSA-N 2-phenoxyethanol Chemical compound OCCOC1=CC=CC=C1 QCDWFXQBSFUVSP-UHFFFAOYSA-N 0.000 claims description 11
- 229960005323 phenoxyethanol Drugs 0.000 claims description 11
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 8
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 8
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 8
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical group [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 8
- GFIHKCBJSYGAPP-UHFFFAOYSA-N [Cl-].C(=O)=CC[PH+](CC=C=O)CC=C=O Chemical compound [Cl-].C(=O)=CC[PH+](CC=C=O)CC=C=O GFIHKCBJSYGAPP-UHFFFAOYSA-N 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 6
- 235000011009 potassium phosphates Nutrition 0.000 claims description 6
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 claims description 6
- 239000004299 sodium benzoate Substances 0.000 claims description 6
- 235000010234 sodium benzoate Nutrition 0.000 claims description 6
- 239000001488 sodium phosphate Substances 0.000 claims description 6
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 6
- 235000011008 sodium phosphates Nutrition 0.000 claims description 6
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- 229960002668 sodium chloride Drugs 0.000 claims description 4
- -1 (2-carbonylethyl) phosphorus hydrochloride Chemical compound 0.000 claims description 3
- MGDKBCNOUDORNI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;potassium Chemical compound [K].[K].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O MGDKBCNOUDORNI-UHFFFAOYSA-N 0.000 claims description 3
- CAAULPUQFIIOTL-UHFFFAOYSA-N methyl dihydrogen phosphate Chemical compound COP(O)(O)=O CAAULPUQFIIOTL-UHFFFAOYSA-N 0.000 claims description 3
- 230000000510 mucolytic effect Effects 0.000 claims description 3
- 229960002816 potassium chloride Drugs 0.000 claims description 3
- 229960004249 sodium acetate Drugs 0.000 claims description 3
- 239000007983 Tris buffer Substances 0.000 claims description 2
- XCIZXBXDZASUDQ-UHFFFAOYSA-N C(=O)=CCP(CC=C=O)CC=C=O Chemical class C(=O)=CCP(CC=C=O)CC=C=O XCIZXBXDZASUDQ-UHFFFAOYSA-N 0.000 claims 1
- 239000002253 acid Substances 0.000 claims 1
- LFQRKUIOSYPVFY-UHFFFAOYSA-L dipotassium diacetate Chemical compound [K+].[K+].CC([O-])=O.CC([O-])=O LFQRKUIOSYPVFY-UHFFFAOYSA-L 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 238000004321 preservation Methods 0.000 abstract description 7
- 241000894006 Bacteria Species 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 238000009825 accumulation Methods 0.000 abstract description 2
- 244000052769 pathogen Species 0.000 abstract description 2
- 230000035755 proliferation Effects 0.000 abstract description 2
- 238000010494 dissociation reaction Methods 0.000 abstract 2
- 230000005593 dissociations Effects 0.000 abstract 2
- 210000004027 cell Anatomy 0.000 description 65
- ZRSCWSKNSQLFFI-UHFFFAOYSA-N Cl.C(=O)=CCOP(OCC=C=O)(OCC=C=O)=O Chemical compound Cl.C(=O)=CCOP(OCC=C=O)(OCC=C=O)=O ZRSCWSKNSQLFFI-UHFFFAOYSA-N 0.000 description 8
- 206010008342 Cervix carcinoma Diseases 0.000 description 7
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 7
- 201000010881 cervical cancer Diseases 0.000 description 7
- QLBHNVFOQLIYTH-UHFFFAOYSA-L dipotassium;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O QLBHNVFOQLIYTH-UHFFFAOYSA-L 0.000 description 5
- 229940058180 edetate dipotassium anhydrous Drugs 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 5
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 230000002380 cytological effect Effects 0.000 description 2
- 210000000805 cytoplasm Anatomy 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000003205 genotyping method Methods 0.000 description 2
- AEELXMHQIJJMKP-QWWZWVQMSA-N (2r,3r)-3-sulfanylbutane-1,2,4-triol Chemical compound OC[C@@H](O)[C@H](S)CO AEELXMHQIJJMKP-QWWZWVQMSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- KVJXEJFFQNSORF-UHFFFAOYSA-L disodium acetic acid diacetate Chemical compound [Na+].[Na+].CC(O)=O.CC(O)=O.CC([O-])=O.CC([O-])=O KVJXEJFFQNSORF-UHFFFAOYSA-L 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 229940124274 edetate disodium Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003172 expectorant agent Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 229940066491 mucolytics Drugs 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- REJGOFYVRVIODZ-UHFFFAOYSA-N phosphanium;chloride Chemical compound P.Cl REJGOFYVRVIODZ-UHFFFAOYSA-N 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229960003339 sodium phosphate Drugs 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- 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
Definitions
- the present application relates to the field of biotechnology, in particular to a liquid-based cell preservation solution and a preparation method thereof.
- cervical cancer is one of the most common malignant tumors among female cancers. Early diagnosis of cervical cancer and precancerous lesions is the key to improving the cure rate of cervical cancer and the survival rate of patients.
- HPV genotyping examination is expensive and requires high inspection environment and equipment, so it is not suitable for conventional large-scale screening methods.
- Thin-layer liquid-based cytology examination has low cost and low requirements for the examination environment and equipment. It is currently the most widely used clinical method for screening cervical cancer. It is a discipline that diagnoses clinical diseases by observing the changes of cell structure and morphology. For decades, cytological examination has become the main method for large-scale cervical cancer screening and follow-up observation of high-risk groups, which has greatly reduced the incidence of advanced cervical cancer.
- thin-layer liquid-based cytology also has deficiencies. Since the cells are separated from their original living environment, the morphology and structure of the cells will change. Only through the correct method of cell preservation can a correct diagnosis be ensured.
- Existing cell preservation solutions often use more than 40% alcohol or cryopreservation of cell samples, and these cells are stored for a short time and are harmful to cells, which will lead to changes in cell morphology and structure, thereby affecting the pathological changes. judge.
- the purpose of this application is to provide a liquid-based cell preservation solution and its preparation method, which is suitable for thin-layer liquid-based cytological examination.
- the purpose of this application is to provide a liquid-based cell preservation solution and its preparation method to overcome the short storage time of cells in the cell solution in the prior art, and at the same time maintain the cell shape well.
- the first technical solution of the present application is to provide a liquid-based cell preservation solution, specifically as follows:
- a liquid-based cell preservation solution comprising 15-20% alcohols, 0.4-0.7% buffer components, 0.2-0.5% ionic strength regulator, 0.05-2% anticoagulant, and 0.05-0.2% mucolytic solution according to mass percentage Release agent, 0.5-0.9% preservative, and the balance is purified water.
- the second technical solution of the present application is to provide a preparation method for a liquid-based cell preservation solution, specifically as follows:
- a preparation method of the above-mentioned liquid-based cell preservation solution comprising the steps of:
- the pH of the precursor liquid is adjusted to 6.50-7.00, and purified water is supplemented to prepare the liquid-based cell preservation solution.
- composition of the cell preservation solution is set reasonably, and the cells can be preserved for a long time at room temperature and the cell shape can be maintained;
- Adding a mucus dissociating agent can effectively soften the cell mucus and separate the cells adhered to the mucus in the sample to avoid cell accumulation;
- Fig. 1 is the morphological figure before cell preservation in embodiment 1;
- Fig. 2 is a diagram of cell morphology after one year of preservation using the preservation solution prepared in Example 1.
- the liquid-based cell preservation solution provided by this application is composed of purified water, alcohols, buffer components, ionic strength regulators, anticoagulants, mucus dissociating agents, preservatives and other components. According to mass percentage, each component ratio is as follows:
- the alcohol component in the liquid-based cell preservation solution, can be selected from one or any two of methanol, ethanol and isopropanol; for example, the alcohol component can be selected from methanol, ethanol or isopropanol alone; Or the alcohol components are selected from the mixed alcohol of methanol and ethanol, the mixed alcohol of ethanol and isopropanol, and the mixed alcohol of methanol and isopropanol, wherein the mass ratio of methanol to ethanol is 10:1 ⁇ 1:1, ethanol The mass ratio of methanol to isopropanol is 5:1 ⁇ 1:1, and the mass ratio of methanol to isopropanol is 10:1 ⁇ 2:1.
- the alcohol component is preferably methanol. If alcohols are controlled below 20%, it is beneficial to avoid changes in cell morphology and structure.
- the buffer component can be any one of sodium dihydrogen phosphate, methyl dihydrogen phosphate, sodium phosphate or potassium phosphate.
- the component of the ionic strength regulator can be one or any two of sodium acetate, magnesium acetate, calcium acetate, sodium chloride and potassium chloride; for example, the ionic strength regulator The component is selected from sodium acetate, magnesium acetate, calcium acetate, sodium chloride or potassium chloride alone; or the ionic strength modifier component is selected from a mixture of sodium acetate and magnesium acetate, a mixture of magnesium acetate and calcium acetate, calcium acetate and chloride Mixtures of sodium, mixtures of sodium chloride and potassium chloride, mixtures of sodium acetate and calcium acetate, mixtures of sodium acetate and sodium chloride, mixtures of sodium acetate and potassium chloride, mixtures of magnesium acetate and sodium chloride, acetic acid A mixture of a mixture of magnesium and potassium chloride, a mixture of calcium acetate and potassium chloride; when the ionic strength regulator is a mixture, the molar ratio of the corresponding components is 5
- the anticoagulant is selected from one or any two of ethylenediaminetetraacetic acid, disodium edetate and dipotassium edetate; for example, anticoagulant
- the agent is selected from EDTA, disodium EDTA or dipotassium EDTA alone; or the anticoagulant is selected from a mixture of EDTA and disodium EDTA, ethylenediamine A mixture of disodium tetraacetate and dipotassium edetate, a mixture of ethylenediaminetetraacetic acid and dipotassium edetate; when the anticoagulant is a mixture, the moles of the corresponding components The ratio is 3:1 ⁇ 1:1.
- the mucus dissociating agent is selected from one of N-acetyl-L-cysteine, dithiothreitol and tris(2-carbonylethyl) phosphate hydrochloride.
- a mucolytic agent independently selected from N-acetyl-L-cysteine, dithiothreitol, or tris(2-carbonylethyl) phosphate hydrochloride; or mucolytic The agent is selected from a mixture of N-acetyl-L-cysteine and dithiothreitol, a mixture of dithiothreitol and tris (2-carbonylethyl) phosphate hydrochloride, N-acetyl-L -A mixture of cysteine and tris(2-carbonylethyl)phosphonium hydrochloride; when the mucus dissociating agent is a mixture, the molar ratio of the corresponding components is 5:1 ⁇ 1: 1.
- the preservative is selected from one of phenoxyethanol, sodium benzoate, and phenol.
- the function of the preservative is to prevent the growth of bacteria and improve the preservation time of cells.
- the liquid-based cell preservation solution contains 15-20% alcohol according to the mass percentage 0.4-0.7% buffer component, 0.2-0.5% ionic strength regulator, 0.05-2% anticoagulant, 0.05-0.2% mucus dissociative agent, 0.5-0.9% preservative, and the balance is purified water.
- step S3 the adjustment of the pH of the precursor liquid is realized by adding hydrochloric acid or sodium hydroxide.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- Figure 1 is a morphological state diagram of cells before storage
- Figure 2 is a morphological diagram of cells after 1 year of storage using the preservation solution prepared in this example. It can be seen from Figures 1 and 2 that the cells were in a dispersed state after one year, and the cell morphology was complete, and the background of the smear was clear and transparent, and the monolayer of cells was evenly distributed, which was conducive to diagnosis and follow-up observation of pathological conditions.
- the liquid-based cell preservation solution in this example is mainly used to preserve cervical epithelial cells. And use cell preservation solution to preserve the exfoliated epithelial cells at room temperature, and observe the changes in the morphology and structure of the cells. The results showed that exfoliated epithelial cells had a high preservation rate, uniform cell distribution, complete cell shape, clear boundary between cytoplasm and nucleus, no mucus and clear layers, and very good transparency of cytoplasm and nucleus. Epithelial cells can be stored at room temperature for 1 to 2 years under this liquid-based cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
- hydrochloric acid or sodium hydroxide to adjust the pH of the precursor liquid to 6.50-7.00, and finally add purified water to the 1000mL mark, stir well, and make a cell preservation solution.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本申请公开了一种液基细胞保存液及其配制方法；按照质量百分比，该保存液包括15～20％醇类、0.4～0.7％缓冲成分、0.2～0.5％离子强度调节剂、0.05～2％抗凝剂、0.05～0.2％粘液解离剂、0.5～0.9％防腐剂、余量为纯化水。该细胞保存液中成分设置合理，能够常温长时间地保存细胞，维持细胞形态；且粘液解离剂的加入，能有有效软化细胞粘液，可使样本中被粘液粘附的细胞分离出来，避免细胞堆积；而防腐剂的加入，能有效抑制霉菌、细菌或其他病原体增生，有利于细胞保存。
Description
本申请要求于2021年10月8日提交中国专利局、申请号为202111172785.7，发明名称为“一种液基细胞保存液及其配制方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
本申请涉及生物技术领域，尤其涉及一种液基细胞保存液及其配制方法。
目前，宫颈癌是目前女性癌症中最常见的恶性肿瘤之一。早期诊断宫颈癌及癌前病变是提高宫颈癌治愈率及患者生存率的关键，宫颈癌筛查方法有HPV基因分型检查和薄层液基细胞学检查两种方式。HPV基因分型检查费用昂贵，对检查的环境和设备要求较高，不宜适用常规的大规模筛查手段。薄层液基细胞学检查有着费用低，检查的环境和设备要求不高，是目前临床应用最广泛的筛查子宫颈癌的主要方法。它是以观察细胞结构和形态变化来诊断临床病症的一门学科。几十年来细胞学检查已成为宫颈癌的大规模普查和高危人群随访观察的主要方法，使中晚期宫颈癌发生率大幅度下降。
但是，薄层液基细胞学检查也存在不足。由于细胞脱离原来的生存环境，细胞的形态结构都将会发生改变，只有通过正确的细胞保存方法才能确保得到正确的诊断。现有的细胞保存液常用大于40％的醇类或对细胞标本进行冷冻保存，而这些的细胞保存时间很短并对细胞有损伤的危害，会导致细胞形态结构发生改变，从而影响到对病变判断。
本申请的目的是提供一种液基细胞保存液极其制备方法，适合用于薄层液基细胞学检查。
发明内容
基于上述问题，本申请的目的在于提供一种液基细胞保存液及其配制方法，以克服现有技术中细胞液存在的细胞保存时间短，同时很好地维持细胞形态。
本申请的技术方案一是提供液基细胞保存液，具体如下：
一种液基细胞保存液，按照质量百分比，包括15～20％醇类、0.4～0.7％缓冲成分、0.2～0.5％离子强度调节剂、0.05～2％抗凝剂、0.05～0.2％粘液解离剂、0.5～0.9％防腐剂、余量为纯化水。
本申请的技术方案二是提供液基细胞保存液的配制方法，具体如下：
一种上述液基细胞保存液的配制方法，包括如下步骤：
将缓冲成分、离子强度调节剂、抗凝剂、粘液解离剂及防腐剂加入容量器中，获得混合液；
往容量器中加入纯化水，搅拌溶解所述混合液；随后再加入醇类，继续搅拌混合均匀，制得前驱液体；
调节所述前驱液体pH至6.50～7.00，并补充纯化水，制得所述液基细胞保存液。
与现有的常用技术相比，本申请的优点在于：
(1)细胞保存液中成分设置合理，能够常温长时间地保存细胞，维持细胞形态；
(2)加入粘液解离剂，能有有效软化细胞粘液，可使样本中被粘液粘附的细胞分离出来，避免细胞堆积；
(3)加入防腐剂，能有效抑制霉菌、细菌或其他病原体增生，有利于细胞保存。
图1为实施例1中细胞保存前的形态图；
图2为采用实施例1配制的保存液保存1年以后的细胞形态图。
下面结合附图，对本申请的较佳实施例作进一步详细说明。
本申请提供的液基细胞保存液，它由纯化水、醇类、缓冲成分、离子强度调节剂、抗凝剂、粘液解离剂、防腐剂等组分组成。按照质量百分比，各组分比例如下：
15～20％醇类，0.4～0.7％缓冲成分，0.2～0.5％离子强度调节剂，0.05～2％抗凝剂，0.05～0.2％粘液解离剂，0.5～0.9％防腐剂，余量为纯化水。
较好地，液基细胞保存液中，醇类组分可以选自甲醇、乙醇及异丙醇中的一种或任意两种；如，醇类组分单独选用甲醇、乙醇或异丙醇；或者醇类组分选自甲醇和乙醇的混合醇、乙醇与异丙醇的混合醇、甲醇与异丙醇的混合醇,其中，甲醇与乙醇的质量比为10:1～1:1，乙醇与异丙醇的质量比为5:1～1:1，甲醇与异丙醇的质量比为10:1～2:1。在液基细胞保存液中，醇类组分优选为甲醇。如果醇类物质控制在20％以下，有利于避免细胞形态结构改变。
较好地，液基细胞保存液中，缓冲成分可为磷酸二氢钠、磷酸二氢甲、磷酸钠或磷酸钾中的任意一种。
较好地，液基细胞保存液中，离子强度调节剂组分可为乙酸钠、乙酸镁、乙酸钙、氯化钠及氯化钾中的一种或任意两种；如，离子强度调节剂组分单独选用乙酸钠、乙酸镁、乙酸钙、氯化钠或氯化钾；或者离子强度调节剂组分选自乙酸钠和乙酸镁混合物、乙酸镁与乙酸钙的混合物、乙酸钙与氯化钠的混合物、氯化钠及氯化钾的混合物、乙酸钠与乙酸钙的混合物、乙酸钠与氯化钠的混合物、乙酸钠与氯化钾的混合物、乙酸镁与氯化钠的混合物、乙酸镁与氯化钾的混合物、乙酸钙与氯化钾的混合物中的一种混合物；所述离子强度调节剂为混合物时，相应组分的摩尔比为5:1～1:5。
较好地，液基细胞保存液中，抗凝剂选自乙二胺四乙酸、乙二胺四乙酸二钠及乙二胺四乙酸二钾中的一种或任意两种；如，抗凝剂单独选自二胺四乙酸、乙二胺四乙酸二钠或乙二胺四乙酸二钾；或者抗凝剂选自乙二胺四乙酸与乙二 胺四乙酸二钠的混合物、乙二胺四乙酸二钠及乙二胺四乙酸二钾的混合物、乙二胺四乙酸与乙二胺四乙酸二钾的混合物中的一种混合物；所述抗凝剂为混合物时，相应组分的摩尔比为3:1～1:1。
较好地，液基细胞保存液中，粘液解离剂选自N-乙酰基-L-半胱氨酸、二硫苏糖醇及三(2-羰基乙基)磷盐酸盐中的一种或任意两种；如，粘液解离剂单独选自N-乙酰基-L-半胱氨酸、二硫苏糖醇或三(2-羰基乙基)磷盐酸盐；或粘液解离剂选自N-乙酰基-L-半胱氨酸与二硫苏糖醇的混合物、二硫苏糖醇与三(2-羰基乙基)磷盐酸盐的混合物、N-乙酰基-L-半胱氨酸与三(2-羰基乙基)磷盐酸盐的混合物中的一种混合物；所述粘液解离剂为混合物时，相应各组分的摩尔比为5:1～1:1。
较好地，液基细胞保存液中，防腐剂选自苯氧乙醇、苯甲酸钠、苯酚等中的一种。该防腐剂的作用是防止细菌滋生，提高细胞保存时间。
上述提供的液基细胞保存液，其配制工艺步骤如下：
S1、以配置1000mL保存液为例，将缓冲成分、离子强度调节剂、抗凝剂、粘液解离剂及防腐剂等组分物质加入容量器中，得到混合液；
S2、先用500mL纯化水加入容量器中，搅拌溶解混合液中的各组分物质；随后加入醇类，继续搅拌充分混合，制得前驱体液；
S3、调节前驱液体pH至6.50～7.00，最后再补充纯化水至1000mL刻度出，搅拌均匀；制得液基细胞保存液；该液基细胞保存液中，按照质量百分比，包括15～20％醇类，0.4～0.7％缓冲成分，0.2～0.5％离子强度调节剂，0.05～2％抗凝剂，0.05～0.2％粘液解离剂，0.5～0.9％防腐剂，余量为纯化水。
上述步骤S3中，前驱液体pH的调节是加入盐酸或氢氧化钠来实现的。
下面举一些具体实施例来进一步详细说明液基细胞保存液的配制
实施例1
以配置1000mL保存液为例，取6g磷酸二氢钠、1.2g乙酸镁、2g氯化钠、1g乙二胺四乙酸二钠、1.5g N-乙酰基-L-半胱氨酸及6g苯氧乙醇加入容量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸二氢钠、乙酸镁、氯化钠、乙二胺四乙酸二钠、N-乙酰基-L-半胱氨酸、苯氧乙醇；随后再加入180g甲醇，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
如图1和2所示。图1为细胞保存前的形态状态图；图2为采用本实施例配制的保存液保存1年以后的细胞形态图。从图1和2中可以看出，1年后细胞呈分散状态，细胞形态完整，而且涂片背景清晰透明、细胞单层均匀分布，有利于诊断及病态跟踪观察。
本实施例中的液基细胞保存液，主要用于保存宫颈上皮细胞。及采用细胞保存液对脱落的上皮细胞在常温下进行保存，并察细胞的形态结构变化。结果显示脱落的上皮细胞的保存率高、细胞分布均匀，细胞形态完整，胞浆与细胞核分界清楚，无粘液层次分明，胞浆与细胞核透亮性非常好。上皮细胞在这种液基细胞保存液下，常温能够保存1～2年。
实施例2
以配置1000mL保存液为例，取5g磷酸钠、2g乙酸钙、1.5g氯化钠、1.2g乙二胺四乙酸、1g N-乙酰基-L-半胱氨酸及7g苯酚加入容量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸钠、乙酸钙、氯化钠、乙二胺四乙酸、N-乙酰基-L-半胱氨酸、苯酚；随后再加入170g甲醇，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
实施例3
以配置1000mL保存液为例，取6g磷酸二氢钾、1.5g乙酸钠、3g氯化钠、1.2g乙二胺四乙酸二钾、1gL三(2-羰基乙基)磷盐酸盐及7g苯氧乙醇加入容量 器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸二氢钾、乙酸钠、氯化钠、乙二胺四乙酸二钾、三(2-羰基乙基)磷盐酸盐、苯氧乙醇；随后再加入180g异丙醇，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
实施例4
以配置1000mL保存液为例，取5g磷酸钾、2g乙酸钠、1.6g乙二胺四乙酸与乙二胺四乙酸二钠的混合物(其中，乙二胺四乙酸与乙二胺四乙酸二钠的摩尔比为1:1，即0.7g乙二胺四乙酸,0.9g乙二胺四乙酸二钠)、0.9g二硫苏糖醇及6g苯甲酸钠加入容量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸钾、乙酸钠、乙二胺四乙二胺四乙酸、乙二胺四乙酸二钠、二硫苏糖醇、苯甲酸钠；随后再加入180g乙醇与异丙醇的混合醇(其中，乙醇与异丙醇质量比为2:1，乙醇120g,异丙醇60g)，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
实施例5
以配置1000mL保存液为例，取6g磷酸二氢甲、4g氯化钠、2.15g乙二胺四乙酸二钠及乙二胺四乙酸二钾的混合物(其中，乙二胺四乙酸二钠及乙二胺四乙酸二钾的摩尔比为2:1,即1.39g乙二胺四乙酸二钠，0.76g乙二胺四乙酸二钾)、1.27g N-乙酰基-L-半胱氨酸与二硫苏糖醇的混合物(其中，N-乙酰基-L-半胱氨酸与二硫苏糖醇的摩尔比为1:1，即0.65g N-乙酰基-L-半胱氨酸,0.62g二硫苏糖醇)及6g苯氧乙醇加入容量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸二氢钾、氯化钠、乙二胺四乙酸二钠、乙二胺四乙酸二钾、N-乙酰基-L-半胱氨酸、二硫苏糖醇、苯氧乙醇；随后再加入160g甲醇与异丙醇的混合醇(其中，甲醇与异丙醇质量比为3:1，甲醇120g,异丙醇40g)，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
实施例6
以配置1000mL保存液为例，取7g磷酸二氢钠、2.5g氯化钾、1.92g乙二胺四乙酸与乙二胺四乙酸二钾的混合物(其中，乙二胺四乙酸(292)及乙二胺四乙酸二钾的摩尔比为3:1，即1.31g乙二胺四乙酸,0.61g乙二胺四乙酸二钾)、1.5g二硫苏糖醇与三(2-羰基乙基)磷盐酸盐的混合物(其中，二硫苏糖醇与三(2-羰基乙基)磷盐酸盐的摩尔比为3:1，即0.93g二硫苏糖醇、0.57g三(2-羰基乙基)磷盐酸盐)及6.5g苯甲酸钠加入容量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸二氢钠、氯化钾、乙二胺四乙酸、乙二胺四乙酸二钾、二硫苏糖醇、三(2-羰基乙基)磷盐酸盐、苯甲酸钠；随后再加入175g甲醇和乙醇的混合醇(其中，甲醇与乙醇质量比为4:1，甲醇140g,乙醇35g)，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
实施例7
以配置1000mL保存液为例，取4.5g磷酸钠、2.4g乙酸镁、1.8g乙二胺四乙酸二钾、1.88g N-乙酰基-L-半胱氨酸与三(2-羰基乙基)磷盐酸盐的混合物(其中，N-乙酰基-L-半胱氨酸与三(2-羰基乙基)磷盐酸盐的摩尔比为4:1，即1.31g N-乙酰基-L-半胱氨酸，0.57g三(2-羰基乙基)磷盐酸盐)及6g苯氧乙醇加入容 量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸钠、乙酸镁、乙二胺四乙酸二钾、N-乙酰基-L-半胱氨酸、三(2-羰基乙基)磷盐酸盐、苯氧乙醇；随后再加入170g甲醇，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
实施例8
以配置1000mL保存液为例，取6.5g磷酸钾、1.2g乙酸镁、3.5g氯化钠、1.6g乙二胺四乙酸、1.3g N-乙酰基-L-半胱氨酸及7g苯酚加入容量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸钾、乙酸镁、氯化钠、乙二胺四乙酸二钾、N-乙酰基-L-半胱氨酸、苯酚；随后再加入180g乙醇，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
实施例9
以配置1000mL保存液为例，取6g磷酸二氢钠、1.8g乙酸钠、2g氯化钾、1g乙二胺四乙酸二钠、0.8g三(2-羰基乙基)磷盐酸盐及6.5g苯酚加入容量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸二氢钠、乙酸钠、氯化钠、乙二胺四乙酸二钾、三(2-羰基乙基)磷盐酸盐、苯酚；随后再加入180g甲醇，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
实施例10
以配置1000mL保存液为例，取5g磷酸二氢钾、1.2g乙酸钙、0.9g氯化钠、2g乙二胺四乙酸二钠、1.6g二硫苏糖醇及5g苯氧乙醇加入容量器中。
用500mL纯化水加入容量器中，搅拌充分溶解磷酸二氢钾、乙酸钠、氯化钠、乙二胺四乙酸二钠、二硫苏糖醇、苯氧乙醇；随后再加入160g乙醇，继续搅拌充分混合；制得前驱液体。
使用盐酸或氢氧化钠调节前驱液体的pH至6.50～7.00，最后补充纯化水至1000mL刻度处，搅拌均匀，制成细胞保存液。
应当理解的是，上述针对本申请较佳实施例的表述较为详细，并不能因此而认为是对本申请专利保护范围的限制，本申请的专利保护范围应以所附权利要求为准。
Claims (16)
- 一种液基细胞保存液，按照质量百分比，包括15～20％醇类、0.4～0.7％缓冲成分、0.2～0.5％离子强度调节剂、0.05～2％抗凝剂、0.05～0.2％粘液解离剂、0.5～0.9％防腐剂、余量为纯化水。
- 根据权利要求1所述的液基细胞保存液，其中，所述醇类选自甲醇、乙醇及异丙醇中的一种或任意两种。
- 根据权利要求1所述的液基细胞保存液，其中，所述醇类为甲醇。
- 根据权利要求1所述的液基细胞保存液，其中，所述缓冲成分为磷酸二氢钠、磷酸二氢甲、磷酸钠或磷酸钾。
- 根据权利要求1所述的液基细胞保存液，其中，所述离子强度调节剂为乙酸钠、乙酸镁、乙酸钙、氯化钠及氯化钾中的一种或任意两种。
- 根据权利要求1所述的液基细胞保存液，其中，所述抗凝剂选自乙二胺四乙酸、乙二胺四乙酸二钠及乙二胺四乙酸二钾中的一种或任意两种。
- 根据权利要求1所述的液基细胞保存液，其中，所述粘液解离剂为N-乙酰基-L-半胱氨酸、二硫苏糖醇及三(2-羰基乙基)磷盐酸盐中的一种或任意两种。
- 根据权利要求1所述的液基细胞保存液，其中，所述防腐剂为苯氧乙醇、苯甲酸钠及苯酚中的一种。
- 根据权利要求2所述的液基细胞保存液，其中，所述醇类为甲醇和乙醇的混合醇，所述甲醇与所述乙醇的质量比为10:1～1:1。
- 根据权利要求2所述的液基细胞保存液，其中，所述醇类为乙醇与异丙醇的混合醇，所述乙醇与所述异丙醇的质量比为5:1～1:1。
- 根据权利要求2所述的液基细胞保存液，其中，所述醇类为甲醇与异丙醇的混合醇，所述甲醇与所述异丙醇的质量比为10:1～2:1。
- 根据权利要求5所述的液基细胞保存液，其中，所述离子强度调节剂为乙酸钠和乙酸镁混合物、乙酸镁与乙酸钙的混合物、乙酸钙与氯化 钠的混合物、氯化钠及氯化钾的混合物、乙酸钠与乙酸钙的混合物、乙酸钠与氯化钠的混合物、乙酸钠与氯化钾的混合物、乙酸镁与氯化钠的混合物、乙酸镁与氯化钾的混合物、乙酸钙与氯化钾的混合物中的一种混合物，其中，所述混合物相应组分的摩尔比为5:1～1:5。
- 根据权利要求6所述的液基细胞保存液，其中，所述抗凝剂选自乙二胺四乙酸与乙二胺四乙酸二钠的混合物、乙二胺四乙酸二钠及乙二胺四乙酸二钾的混合物、乙二胺四乙酸与乙二胺四乙酸二钾的混合物中的一种混合物，其中，所述混合物相应组分的摩尔比为3:1～1:1。
- 根据权利要求7所述的液基细胞保存液，其中，所述粘液解离剂选自N-乙酰基-L-半胱氨酸与二硫苏糖醇的混合物、二硫苏糖醇与三(2-羰基乙基)磷盐酸盐的混合物、N-乙酰基-L-半胱氨酸与三(2-羰基乙基)磷盐酸盐的混合物中的一种混合物，其中，所述混合物相应各组分的摩尔比为5:1～1:1。
- 一种权利要求1至14任一所述液基细胞保存液的配制方法，包括如下步骤：将缓冲成分、离子强度调节剂、抗凝剂、粘液解离剂及防腐剂加入容量器中，获得混合液；往容量器中加入纯化水，搅拌溶解所述混合液；随后再加入醇类，继续搅拌混合均匀，制得前驱液体；调节所述前驱液体pH至6.50～7.00，并补充纯化水，制得所述液基细胞保存液。
- 根据权利要求15所述的配制方法，其中，所述前驱液体pH的调节是加入盐酸或氢氧化钠来实现的。
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP22717336.6A EP4413857A1 (en) | 2021-10-08 | 2022-02-24 | Liquid-based cell preservation solution and preparation method therefor |
| US17/726,498 US20230116675A1 (en) | 2021-10-08 | 2022-04-21 | Liquid-based cell preservation solution and method for preparing same |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111172785.7 | 2021-10-08 | ||
| CN202111172785.7A CN113907064A (zh) | 2021-10-08 | 2021-10-08 | 一种液基细胞保存液及其配制方法 |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US17/726,498 Continuation US20230116675A1 (en) | 2021-10-08 | 2022-04-21 | Liquid-based cell preservation solution and method for preparing same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2023056726A1 true WO2023056726A1 (zh) | 2023-04-13 |
Family
ID=79238385
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2022/077616 WO2023056726A1 (zh) | 2021-10-08 | 2022-02-24 | 一种液基细胞保存液及其配制方法 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN113907064A (zh) |
| WO (1) | WO2023056726A1 (zh) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101363011A (zh) * | 2008-09-17 | 2009-02-11 | 上海艾迪康临床检验中心有限公司 | 一种宫颈脱落细胞保存液 |
| CN101636649A (zh) * | 2007-02-27 | 2010-01-27 | 恰根有限公司 | 生物材料的固定 |
| CN101999343A (zh) * | 2010-10-26 | 2011-04-06 | 深圳华大基因科技有限公司 | 一种细胞保存液、其制备方法及用途 |
| CN102625831A (zh) * | 2009-07-20 | 2012-08-01 | 通用医院公司以麻省总医院名义经营 | 提高低温保存细胞生活力的方法和组合物 |
| CN103271023A (zh) * | 2013-05-11 | 2013-09-04 | 港龙生物技术（深圳）有限公司 | 一种具有液化功能的宫颈脱落细胞样本细胞保存液 |
| CN105409925A (zh) * | 2014-09-19 | 2016-03-23 | 孝感宏翔生物医械技术有限公司 | 一种液基细胞保存液 |
| CN105432598A (zh) * | 2015-11-09 | 2016-03-30 | 湖南品信生物工程有限公司 | 一种液基细胞保存液及其制备方法 |
| CN108402033A (zh) * | 2018-04-10 | 2018-08-17 | 南京福怡科技发展股份有限公司 | 一种具有预处理功能宫颈脱落细胞保存液及应用 |
| CN113729009A (zh) * | 2021-10-14 | 2021-12-03 | 山东高创医疗器械国家研究院有限公司 | 一种宫颈细胞保存液及其制备方法和保存方法 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101395081B1 (ko) * | 2013-01-10 | 2014-05-16 | 전북대학교산학협력단 | 부인과용 세포 보존액 |
| CN107494519A (zh) * | 2017-09-06 | 2017-12-22 | 岳阳市阿罗格医药技术有限公司 | 一种环保型液基细胞保存液及其制备方法以及应用 |
| CN108770836A (zh) * | 2018-08-21 | 2018-11-09 | 生工生物工程（上海）股份有限公司 | 液基细胞保存液及其制备方法和应用 |
| CN110283726A (zh) * | 2019-07-03 | 2019-09-27 | 孝感宏翔生物医械技术有限公司 | 一种液基细胞和微生物处理保存试剂及其制备方法 |
-
2021
- 2021-10-08 CN CN202111172785.7A patent/CN113907064A/zh active Pending
-
2022
- 2022-02-24 WO PCT/CN2022/077616 patent/WO2023056726A1/zh active Application Filing
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101636649A (zh) * | 2007-02-27 | 2010-01-27 | 恰根有限公司 | 生物材料的固定 |
| CN101363011A (zh) * | 2008-09-17 | 2009-02-11 | 上海艾迪康临床检验中心有限公司 | 一种宫颈脱落细胞保存液 |
| CN102625831A (zh) * | 2009-07-20 | 2012-08-01 | 通用医院公司以麻省总医院名义经营 | 提高低温保存细胞生活力的方法和组合物 |
| CN101999343A (zh) * | 2010-10-26 | 2011-04-06 | 深圳华大基因科技有限公司 | 一种细胞保存液、其制备方法及用途 |
| CN103271023A (zh) * | 2013-05-11 | 2013-09-04 | 港龙生物技术（深圳）有限公司 | 一种具有液化功能的宫颈脱落细胞样本细胞保存液 |
| CN105409925A (zh) * | 2014-09-19 | 2016-03-23 | 孝感宏翔生物医械技术有限公司 | 一种液基细胞保存液 |
| CN105432598A (zh) * | 2015-11-09 | 2016-03-30 | 湖南品信生物工程有限公司 | 一种液基细胞保存液及其制备方法 |
| CN108402033A (zh) * | 2018-04-10 | 2018-08-17 | 南京福怡科技发展股份有限公司 | 一种具有预处理功能宫颈脱落细胞保存液及应用 |
| CN113729009A (zh) * | 2021-10-14 | 2021-12-03 | 山东高创医疗器械国家研究院有限公司 | 一种宫颈细胞保存液及其制备方法和保存方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113907064A (zh) | 2022-01-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102258003A (zh) | 一种液基细胞保存液 | |
| ITMI20120913A1 (it) | Uso della n-acetil-5-metossitriptamina o suoi analoghi per favorire il meccanismo di impianto dell' embrione, e relative composizioni e mezzi di coltura | |
| CN105409925A (zh) | 一种液基细胞保存液 | |
| CN110037013A (zh) | 一种液基薄层细胞保存液及其制备方法 | |
| WO2023109877A1 (zh) | 一种玻色因及其生产方法和用途 | |
| CN101988875A (zh) | 一种痰液液基处理液 | |
| CN101664028B (zh) | 一种液基细胞保存液 | |
| WO2023056726A1 (zh) | 一种液基细胞保存液及其配制方法 | |
| Wei et al. | Orange-emissive carbon quantum dots for ligand-directed Golgi apparatus-targeting and in vivo imaging | |
| CN105432598A (zh) | 一种液基细胞保存液及其制备方法 | |
| Yi et al. | Cardiac electrophysiological changes and downregulated connexin 43 Prompts reperfusion arrhythmias induced by hypothermic ischemia–reperfusion injury in isolated rat hearts | |
| Whistler et al. | The production of l-erythrulose by the action of Acetobacter suboxydans upon erythritol | |
| US20230116675A1 (en) | Liquid-based cell preservation solution and method for preparing same | |
| Roseman | The Reduction of 1, 2-Isopropylidene-D-glucuronolactone with Lithium Aluminum Hydride1 | |
| CN101347412B (zh) | 三水合氨磷汀结晶冻干制剂及其制备方法 | |
| CN114344331B (zh) | 小分子化合物在制备激活原始卵泡的药物中的应用 | |
| TWI746069B (zh) | 3d細胞培養膠體的套組及其用於3d細胞培養的方法 | |
| CN114907332A (zh) | 一种新型高稳定性线粒体荧光探针及其制备方法与应用 | |
| CN107574144A (zh) | 一种添加鼠尾草酸的猪卵母细胞体外成熟培养液及其应用 | |
| CN104164465A (zh) | 一种表达重组蛋白的发酵工艺 | |
| Burton | The histological demonstration of alkaline phosphatase activity in human endometrium | |
| CN108300687A (zh) | 一种基于肾小管上皮细胞培养造影剂损伤模型的方法 | |
| Grossfeld | Production of hyaluronic acid in tissue culture by fibroblasts growing from intestinal, stomach, liver and kidney explants. | |
| Sparrow | Relative X-ray sensitivity of metaphase and interphase chromosomes | |
| CN107058455B (zh) | 猪2细胞胚胎体积的测量方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 22717336 Country of ref document: EP Kind code of ref document: A1 |
|
| WWE | Wipo information: entry into national phase |
Ref document number: 2022717336 Country of ref document: EP |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| ENP | Entry into the national phase |
Ref document number: 2022717336 Country of ref document: EP Effective date: 20240508 |