CN101914504A - Multi-enzyme system for separating different tissues-derived primary culture cells of normal human and mammal, application and related kit thereof - Google Patents
Multi-enzyme system for separating different tissues-derived primary culture cells of normal human and mammal, application and related kit thereof Download PDFInfo
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Abstract
The invention relates to a multi-enzyme system for separating different tissues-derived primary culture cells of a normal human and a mammal. The system comprises a plurality of 0.0125 to 0.125 weight percent of trypsin, 0.02 to 0.2 weight percent of collagenase I-IV, 0.0015 to 0.015 weight percent of hyaluronidase, 0.02 to 0.2 weight percent of neutral protease, 0.0015 to 0.015 weight percent of deoxyribonuclease I, 1 to 10 U/ml papain, 0.015 to 0.15 weight percent of pronase, 5 to 50 U/ml elastase and 0.01 to 0.1 weight percent of separase. The invention also relates to the application of the multi-enzyme system and a kit formed by the multi-enzyme system. The multi-enzyme system of the invention has the characteristics of reasonable design and capacity of separating the different tissues-derived primary culture cells of the normal human and the mammal highly specifically and highly sensitively; and the separated primary culture cells have the characteristics of high survival rate, high purity, easy cryopreservation and recovery, can be used for medicament experimental evaluation, medicament toxicity test and medicament therapeutic judgment, and are suitable for large-scale popularization and application.
Description
Technical field
The present invention relates to the primary cell separation technology field, particularly the different tissues-derived primary culture cells separation technology field specifically is meant a kind of normal people and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells, its purposes and related kit.
Background technology
In the biological medicine industry, especially primary cell culture is in the application industry of medicament research and development, profit margin is big, energy consumption is little, the state of the art height, be the support on policy industry that the China national reform and development council determines, listed before the national reform and development council 2010 China in and given priority to industry and instruct register.Local governments such as each provincial government are to this intermediate item, all list gordian technique in, major project is helped, and enjoys special Preferential Treatment on manpower, financial resources.
Primary cell refers to from body tissue (as normal people, patient, Mammals etc.) after the special technique method is handled, and obtains individual cells and carry out in vitro culture, right-on analog machine body tissue physiologic function, and this cell is called primary cell.Cultivate 1-10 for being referred to as primary cell culture with interior cell.
The basic biological property of primary cell:
1, just the same with the biological property of body cell growth.
2, can right-on reflection body tissue biological property.
3, the right-on reflection body tissue of energy is to the result of test experience.
4, the right-on reflection body tissue of energy is to the result of drug study.
At present, on the domestic and international market in the medicament research and development of biological medicine industry, the research and development overwhelming majority is to use clone on the cell levels, and this class cell disadvantage is with primary cell otherness significantly to be arranged, and can not correctly react body tissue biological property and physiologic function.The separation of primary cell and cultivation are difficult points always, its reason is that cell dissociation enzyme application problem makes the primary cell after the separation be difficult for external surviving, because normal people and Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) different tissues-derived primary culture cells are variant to the reaction of different digestive ferments, influencing chorista on the one hand is one primary cell, and influence separates the one-tenth activity of primary cell on the other hand.Thereby be biological medicine industrial hot spot and difficult point with primary cell assessment medicament research and development always.
Therefore, a kind of normal people and the isolating reagent of Mammals different tissues-derived primary culture cells need be provided, it can separate normal people and Mammals different tissues-derived primary culture cells by height specifically, in high sensitivity, and isolating primary cell can be used for to the drug study assessment, to the drug toxicity test, therapeutic drug is judged.
Summary of the invention
The objective of the invention is to have overcome above-mentioned shortcoming of the prior art, a kind of normal people and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells, its purposes and related kit are provided, this multi-enzyme system is reasonable in design, can separate normal people and Mammals different tissues-derived primary culture cells specifically, in high sensitivity by height, isolating primary cell surviving rate height, purity height, be easy to frozen and the recovery, can be used for to the drug study assessment, to the drug toxicity test, therapeutic drug is judged, be suitable for large-scale promotion application.
To achieve these goals, in a first aspect of the present invention, a kind of normal people and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells are provided, be characterized in, comprise 0.0125-0.125% (weight) trypsin Trypsin), 0.02-0.2% (weight) Collagenase I-IV (CollagenaseI-IV), 0.0015-0.015% (weight) Unidasa (Hyaluronidase), 0.02-0.2% (weight) neutral protease (Neutral Protease), 0.0015-0.015% (weight) deoxyribonuclease I (Deoxyribonuclease I), 1-10U/ml papoid (Papain), 0.015-0.15% (weight) PRONASE A (Pronase), 5-50U/ml elastoser (Elastase) is separated several in the enzyme (Dispase) with 0.01-0.1% (weight).
For example, every milliliter of above-mentioned normal people and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells comprise 0.0125-0.125 gram trypsin Trypsin), 0.02-0.2 gram Collagenase I-IV (CollagenaseI-IV), 0.0015-0.015 gram Unidasa (Hyaluronidase), 0.02-0.2 gram neutral protease (Neutral Protease), 0.0015-0.015 gram deoxyribonuclease I (Deoxyribonuclease I), 1-10U papoid (Papain), 0.015-0.15 gram PRONASE A (Pronase), 5-50U elastoser (Elastase) is separated enzyme (Dispase) with the 0.01-0.1 gram.
Above-mentioned multi-enzyme system can be used for separating normal people and Mammals different tissues-derived primary culture cells.Wherein, multi-enzyme system comprise at least two kinds of above-mentioned enzyme and more than.Mammals comprises mouse, rat, cavy, rabbit, dog, pig, ox, monkey etc.Can be wherein a kind of among the Collagenase I-IV, also can be wherein multiple and even whole.
Preferably, multi-enzyme system comprises that 0.0125-0.125% (weight) trypsinase, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser separate enzyme with 0.01-0.1% (weight).
Above-mentioned multi-enzyme system can be used for separating of normal people and the different tissues-derived endotheliocyte of Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey), epithelial cell, liver cell, nephrocyte, corneal epithelial cell.
Preferably, multi-enzyme system comprises that 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser separate enzyme with 0.01-0.1% (weight).
Above-mentioned multi-enzyme system can be used for the normal people and separates with Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) lung tissue cell, gastrointestinal tissue's cell, the parotid gland, Tiroidina, prostate gland, mammary gland, suprarenal gland, hypophysis, pancreas, amnion cell.
Preferably, multi-enzyme system comprises that 0.0125-0.125% (weight) trypsinase, 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser separate enzyme with 0.01-0.1% (weight).
Above-mentioned multi-enzyme system can be used for separating of normal people and the different tissues-derived skin cells of Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey), osteocyte, cartilaginous tissue cell, Skeletal Muscle Cell, smooth muscle cell, myocardial cell, brain and neurocyte.
In a second aspect of the present invention, provide above-mentioned normal people and the application of the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells in separating normal people and Mammals different tissues-derived primary culture cells.
In a third aspect of the present invention, provide above-mentioned normal people and the application of the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells in separating normal people and Mammals endotheliocyte, epithelial cell, liver cell, nephrocyte or corneal epithelial cell.
In a fourth aspect of the present invention, provide above-mentioned normal people and the application of the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells in separating normal people and Mammals lung tissue cell, gastrointestinal tissue's cell, the parotid gland, Tiroidina, prostate gland, mammary gland, suprarenal gland, hypophysis, pancreas, amnion cell.
In a fifth aspect of the present invention, provide above-mentioned normal people and the application of the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells in separating normal people and mammal skin cell, osteocyte, cartilaginous tissue cell, Skeletal Muscle Cell, smooth muscle cell, myocardial cell, brain and neurocyte.
In a sixth aspect of the present invention, a kind of normal people and Mammals different tissues-derived primary culture cells separating kit are provided, be characterized in, comprise the reagent container that above-mentioned normal people and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells are housed.
Beneficial effect of the present invention is:
1, the invention provides a kind of normal people and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells, several in comprising trypsinase, Collagenase I-IV, Unidasa, neutral protease, deoxyribonuclease I, papoid, PRONASE A, elastoser and separating enzyme are characterized in: (1) separates pointed and specificity to different tissues-derived primary culture cells; (2) primary cell separates back cultivation surviving rate height; (3) primary cell separates back cultivation purity height; (4) primary cell is easy to frozen and recovery.Be suitable for large-scale promotion application.
2, the great advantage of normal people provided by the invention and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells is: (1) tissue is made up of the broad variety cell, uses plurality of enzymes digestion tissue, can make cellular segregation more thorough; (2) consumption of every kind of enzyme is few, and is little to the primary cell damage of separate tissue; (3) the primary cell vitro culture after the separate tissue is easy to survival; (4) isolating primary cell keeps source sex organization biological characteristics.The isolating multi-enzyme system of this tissues-derived primary culture cells is reasonable in design, steady quality is reliable.
3, the invention provides the test kit that comprises normal people and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells, make the user simple, fast and height separate normal people and Mammals different tissues-derived primary culture cells specifically, in high sensitivity, isolating primary cell surviving rate height, purity height, be easy to frozen and the recovery, can be used for to the drug study assessment, to the drug toxicity test, therapeutic drug is judged, be suitable for large-scale promotion application.
Embodiment
The inventor is through extensive and deep research, obtain the isolating multi-enzyme system of a kind of normal people and Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) different tissues-derived primary culture cells, this multi-enzyme system is Collagenase I, II, III, IV, trypsinase, deoxyribonuclease I, Unidasa, neutral protease, papoid, PRONASE A, elastoser and separates the multiple of enzyme.3 kinds of primary cells of main formation separate the multi-enzyme system test kits: primary cell separates multi-enzyme system test kit I, primary cell separation multi-enzyme system test kit II, primary cell separates multi-enzyme system test kit III.Separate normal people and Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) different tissues-derived primary culture cells respectively.Finished the present invention on this basis.
In order more to be expressly understood technology contents of the present invention, describe in detail especially exemplified by following examples.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
The multi-enzyme system of primary cell separating kit I: 0.0125-0.125% (weight) trypsinase, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).
Isolating normal human umbilical vein endothelial cell experimental technique:
1. fresh normal people's infant umbilical cord;
2. at umbilical vein both ends open place, tighten and be fixed on silk thread respectively on the glass intubate of 50ml syringe and band transfusion sebific duct, inject D-Hanks liquid flushing umbilical vein, to there not being bloodstain;
3. clamp transfusion sebific duct on the glass intubate with mosquito forceps, inject the multi-enzyme system solution of our primary cell separating kit I from the syringe of the other end slowly to umbilical vein, to angioplerosis.Note closed at both ends, in case liquid backflows.Put into 37 ℃ aseptic D-Hanks liquid immediately;
4. draw by syringe and collect enzyme liquid, inject the nutrient solution flushing vein that contains calf serum simultaneously, be incorporated in the same centrifuge tube, centrifugal;
5. discard the supernatant liquor after the centrifugation.The nutrient solution that adds calf serum, suspension cell again.And be adjusted to cell density, be inoculated in culturing bottle or the culture dish.Put 37 ℃, the 5%CO2 incubator is cultivated;
6. abandoned nutrient solution in second day, wash gently 1 time, to remove not adherent cell or dead cell with D-Hanks liquid.Change to the fresh nutrient solution that contains calf serum, 100IU/ml penicillin and 100 μ g/ml Streptomycin sulphates.Continue to put 37 ℃, the 5%CO2 incubator is cultivated;
7. surviving rate is cultivated in isolating normal human umbilical vein endothelial cell purifying and detection and the surviving rate detection is cultivated in the recovery of frozen back.
The basic characteristic of isolating normal human umbilical vein endothelial cell biology:
(1) vascular endothelial cell is identified: vWF, and Factor VIII, CD31 (P-CAM), positive.
(2) vascular endothelial cell keeps the blood vessel barrier function.
(3) vascular endothelial cell eight factor related antigen immunofluorescence dyeings are verified as the positive.
(4) vascular endothelial cell does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.
Isolating normal people's vascular endothelial cell:
Surviving rate 70% is cultivated in purity 95%, cultivation surviving rate 90%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been respectively applied for the separation of the different tissues-derived epithelial cell of normal people, liver cell, nephrocyte, corneal epithelial cell.The experimental technique tissue digestion is with the multi-enzyme system of above-mentioned primary cell separating kit I (detailed process slightly, can referring to above-mentioned experimentation).
Embodiment 2
The multi-enzyme system of primary cell separating kit II: 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).
Isolating normal people's pulmonary epithelial cells experimental technique:
1. fresh normal people's lung tissue, and use the bronchoalveolar lavage fluid lavation;
2. the multi-enzyme system digestion of perfusion primary cell separating kit II in the lung;
3. the reticular tissue around removing;
4. add the cell dispersion liquid, to stop digestion;
5. suspension filters through the stainless steel mesh screen;
6. it is centrifugal to collect filtrate;
7. abandon supernatant liquor, use the nutrient solution suspension cell;
8. cell suspension is added on the culture dish, at 37 ℃, 5%CO
2Incubator in hatch;
9. surviving rate is cultivated in isolating normal people's pulmonary epithelial cells purifying and detection and the surviving rate detection is cultivated in the recovery of frozen back.The basic characteristic of isolating normal people's pulmonary epithelial cells biology:
(1) normal people's pulmonary epithelial cells is identified: Cytokeratin-18, and-19 and the Vimentin immunofluorescence dyeing, positive.
(2) normal people's pulmonary epithelial cells keeps the blood vessel barrier function.
(3) normal people's pulmonary epithelial cells produces, secretes and utilize pulmonary surfactant.
(4) normal people's pulmonary epithelial cells does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.
Isolating normal people's pulmonary epithelial cells:
Surviving rate 68% is cultivated in purity 96%, cultivation surviving rate 92%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been respectively applied for the separation of the different tissues-derived lung tissue cell of normal people, gastrointestinal tissue's cell, the parotid gland, Tiroidina, prostate gland, mammary gland, suprarenal gland, hypophysis, pancreas, amnion cell.The experimental technique tissue digestion is with the multi-enzyme system of above-mentioned primary cell separating kit II (detailed process slightly, can referring to top experimentation).
Embodiment 3
The multi-enzyme system of primary cell separating kit III: 0.0125-0.125% (weight) trypsinase, 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).Isolating normal people's keratinization of epidermis cell experiment method:
1, fresh normal people's fetal skin;
2, material pre-treatment: skin graft is immersed in the alcohol sterilizes; Clean;
4, normal human skin places the multi-enzyme system Digestive system of primary cell separating kit III;
5, add termination digestion: end enzyme reaction, suspension is crossed mesh screen;
6, collect re-suspended cell: the collecting cell suspension, centrifugal;
7, cultivate: be positioned over 37 ℃, 5%CO
2In the incubator;
8, surviving rate is cultivated in isolating normal people's keratinization of epidermis cell purification and detection and the surviving rate detection is cultivated in the recovery of frozen back.
Isolating normal people's keratinization of epidermis cytobiology fundamental characteristics:
(1) normal people's keratinization of epidermis cell derives from normal people's fetal skin tissue.
(2) normal people's keratinization of epidermis cell is identified: Cytokeratine-8 ,-18and-19 immunofluorescence dyeing.
(3) normal people's keratinization of epidermis cell primary cell expression of adhesion molecules and cytokine participate in the natural immunity.
(4) normal people's keratinization of epidermis cell does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.Isolating normal people's keratinization of epidermis cell:
Surviving rate 72% is cultivated in purity 98%, cultivation surviving rate 94%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been respectively applied for separating of the different tissues-derived skin cells of normal people, osteocyte, cartilaginous tissue cell, Skeletal Muscle Cell, smooth muscle cell, myocardial cell, brain and neurocyte.The experimental technique tissue digestion is with the multi-enzyme system of primary cell separating kit III (detailed process slightly, can referring to above-mentioned experimentation).
Embodiment 4
The multi-enzyme system of primary cell separating kit I: 0.0125-0.125% (weight) trypsinase, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).
Isolating normal rat vascular endothelial cell experimental technique:
1. fresh normal rat blood vessel;
2. at normal rat blood vessel both ends open place, tighten and be fixed on silk thread respectively on the glass intubate of 50ml syringe and band transfusion sebific duct, inject D-Hanks liquid flushing umbilical vein, to there not being bloodstain;
3. clamp transfusion sebific duct on the glass intubate with mosquito forceps, inject the multi-enzyme system solution of our primary cell separating kit I from the syringe of the other end slowly to umbilical vein, to angioplerosis.Note closed at both ends, in case liquid backflows.Put into 37 ℃ aseptic D-Hanks liquid immediately;
4. draw by syringe and collect enzyme liquid, inject the nutrient solution flushing vein that contains calf serum simultaneously, be incorporated in the same centrifuge tube, centrifugal;
5. discard the supernatant liquor after the centrifugation.The nutrient solution that adds calf serum, suspension cell again.And be adjusted to cell density, be inoculated in culturing bottle or the culture dish.Put 37 ℃, 5%CO
2Incubator is cultivated;
6. abandoned nutrient solution in second day, wash gently 1 time, to remove not adherent cell or dead cell with D-Hanks liquid.Change to the fresh nutrient solution that contains calf serum, 100IU/ml penicillin and 100 μ g/ml Streptomycin sulphates.Continue to put 37 ℃, 5%CO
2Incubator is cultivated;
7. surviving rate is cultivated in isolating normal rat vascular endothelial cell purifying and detection and the surviving rate detection is cultivated in the recovery of frozen back.
The basic characteristic of isolating normal rat vascular endothelial cell biology:
(1) the normal rat vascular endothelial cell is identified: vWF, and Factor VIII, CD31 (P-CAM), positive.
(2) the normal rat vascular endothelial cell keeps the blood vessel barrier function.
(3) normal rat vascular endothelial cell eight factor related antigen immunofluorescence dyeings are verified as the positive.
(4) the normal rat vascular endothelial cell does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.
Isolating rat aorta endotheliocyte:
Surviving rate 69% is cultivated in purity 96%, cultivation surviving rate 92%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been respectively applied for the separation of the different tissues-derived epithelial cell of normal rat, liver cell, nephrocyte, corneal epithelial cell.The experimental technique tissue digestion is with the multi-enzyme system of primary cell separating kit I (detailed process slightly, can referring to above-mentioned experimentation).
Embodiment 5
The multi-enzyme system of primary cell separating kit II: 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).
Isolating normal rat pulmonary epithelial cells experimental technique:
1. fresh normal rat pulmonary epithelial cells lung tissue; And use the bronchoalveolar lavage fluid lavation;
2. the multi-enzyme system digestion of perfusion primary cell separating kit II in the lung;
3. the reticular tissue around removing;
4. add the cell dispersion liquid, to stop digestion;
5. suspension filters through the stainless steel mesh screen;
6. it is centrifugal to collect filtrate;
7. abandon supernatant liquor, use the nutrient solution suspension cell;
8. cell suspension is added on the culture dish, at 37 ℃, 5%CO
2Incubator in hatch;
9. surviving rate is cultivated in isolating normal rat vascular endothelial cell purifying and detection and the surviving rate detection is cultivated in the recovery of frozen back.
The basic characteristic of isolating normal rat pulmonary epithelial cells biology:
(1) the normal rat pulmonary epithelial cells is identified: Cytokeratin-18, and-19 and the Vimentin immunofluorescence dyeing, positive.
(2) the normal rat pulmonary epithelial cells keeps the blood vessel barrier function.
(3) the normal rat pulmonary epithelial cells produces, secretes and utilize pulmonary surfactant.
(4) the normal rat pulmonary epithelial cells does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.
Isolating induced lung epithelial cell:
Surviving rate 66% is cultivated in purity 95%, cultivation surviving rate 90%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been respectively applied for the separation of the different tissues-derived lung tissue cell of normal rat, gastrointestinal tissue's cell, the parotid gland, Tiroidina, prostate gland, mammary gland, suprarenal gland, hypophysis, pancreas, amnion cell.The experimental technique tissue digestion is with the multi-enzyme system of primary cell separating kit II (detailed process slightly, can referring to above-mentioned experimentation).
Embodiment 6
The multi-enzyme system of primary cell separating kit III: 0.0125-0.125% (weight) trypsinase, 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).Isolating normal rat keratinization of epidermis cell experiment method:
1, fresh normal rat skin;
2, material pre-treatment: skin graft is immersed in the alcohol sterilizes; Clean;
4, the normal people rat places the multi-enzyme system Digestive system of primary cell separating kit III;
5, add termination digestion: end enzyme reaction, suspension is crossed mesh screen;
6, collect re-suspended cell: the collecting cell suspension, centrifugal;
7, cultivate: be positioned over 37 ℃, 5%CO
2In the incubator;
8, surviving rate is cultivated in isolating normal rat keratinization of epidermis cell purification and detection and the surviving rate detection is cultivated in the recovery of frozen back.
Isolating normal rat keratinization of epidermis cytobiology fundamental characteristics:
(1) normal rat keratinization of epidermis cell derives from the normal rat skin histology.
(2) normal rat keratinization of epidermis cell is identified: Cytokeratine-8 ,-18and-19 immunofluorescence dyeing.
(3) normal rat keratinization of epidermis cell primary cell expression of adhesion molecules and cytokine participate in the natural immunity.
(4) normal rat keratinization of epidermis cell does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.
Isolating rat keratinization of epidermis cell:
Surviving rate 70% is cultivated in purity 97%, cultivation surviving rate 93%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been respectively applied for separating of the different tissues-derived skin cells of normal rat, osteocyte, cartilaginous tissue cell, Skeletal Muscle Cell, smooth muscle cell, myocardial cell, brain and neurocyte.The experimental technique tissue digestion is with the multi-enzyme system of primary cell separating kit III (detailed process slightly, can referring to above-mentioned experimentation).
Embodiment 7
The multi-enzyme system of primary cell separating kit I: 0.0125-0.125% (weight) trypsinase, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).
Isolating normal big rabbit vascular endothelial cell experimental technique:
1. fresh normal big rabbit vascular endothelial cell;
2. at normal big rabbit blood vessel both ends open place, tighten and be fixed on silk thread respectively on the glass intubate of 50ml syringe and band transfusion sebific duct, inject D-Hanks liquid flushing umbilical vein, to there not being bloodstain;
3. clamp transfusion sebific duct on the glass intubate with mosquito forceps, inject the multi-enzyme system solution of our primary cell separating kit I from the syringe of the other end slowly to umbilical vein, to angioplerosis.Note closed at both ends, in case liquid backflows.Put into 37 ℃ aseptic D-Hanks liquid immediately;
4. draw by syringe and collect enzyme liquid, inject the nutrient solution flushing vein that contains calf serum simultaneously, be incorporated in the same centrifuge tube, centrifugal;
5. discard the supernatant liquor after the centrifugation.The nutrient solution that adds calf serum, suspension cell again.And be adjusted to cell density, be inoculated in culturing bottle or the culture dish.Put 37 ℃, 5%CO
2Incubator is cultivated;
6. abandoned nutrient solution in second day, wash gently 1 time, to remove not adherent cell or dead cell with D-Hanks liquid.Change to the fresh nutrient solution that contains calf serum, 100IU/ml penicillin and 100 μ g/ml Streptomycin sulphates.Continue to put 37 ℃, 5%CO
2Incubator is cultivated;
7. isolating normal big rabbit vascular endothelial cell purifying and detection, cultivation surviving rate and the recovery of frozen back are cultivated surviving rate and are detected.
The isolating normal big basic characteristic of rabbit vascular endothelial cell biology:
(1) normal big rabbit vascular endothelial cell is identified: vWF, and Factor VIII, CD31 (P-CAM), positive.
(2) normal big rabbit vascular endothelial cell keeps the blood vessel barrier function.
(3) normal big rabbit vascular endothelial cell eight factor related antigen immunofluorescence dyeings are verified as the positive.
(4) normal big rabbit vascular endothelial cell does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.
Isolating big rabbit vascular endothelial cell:
Surviving rate 74% is cultivated in purity 95%, cultivation surviving rate 90%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been used for the separation of the normal big different tissues-derived epithelial cell of rabbit, liver cell, nephrocyte, corneal epithelial cell respectively.The experimental technique tissue digestion is with the multi-enzyme system of primary cell separating kit I (detailed process slightly, can referring to above-mentioned experimentation).
Embodiment 8
The multi-enzyme system of primary cell separating kit II: 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).
Isolating normal big rabbit pulmonary epithelial cells experimental technique:
1. fresh big rabbit pulmonary epithelial cells lung tissue, and use the bronchoalveolar lavage fluid lavation;
2. the multi-enzyme system digestion of perfusion primary cell separating kit II in the lung;
3. the reticular tissue around removing;
4. add the cell dispersion liquid, to stop digestion;
5. suspension filters through the stainless steel mesh screen;
6. it is centrifugal to collect filtrate;
7. abandon supernatant liquor, use the nutrient solution suspension cell;
8. cell suspension is added on the culture dish, at 37 ℃, 5%CO
2Incubator in hatch;
9. isolating normal big rabbit pulmonary epithelial cells purifying and detection, cultivation surviving rate and the recovery of frozen back are cultivated surviving rate and are detected.The isolating normal big basic characteristic of rabbit pulmonary epithelial cells biology:
(1) normal big rabbit pulmonary epithelial cells is identified: Cytokeratin-18, and-19 and the Vimentin immunofluorescence dyeing, positive.
(2) normal big rabbit pulmonary epithelial cells keeps the blood vessel barrier function.
(3) normal big rabbit pulmonary epithelial cells produces, secretes and utilize pulmonary surfactant.
(4) normal big rabbit pulmonary epithelial cells does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.
Isolating big rabbit pulmonary epithelial cells:
Surviving rate 69% is cultivated in purity 98%, cultivation surviving rate 93%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been respectively applied for the separation of the normal big different tissues-derived lung tissue cell of rabbit pulmonary epithelial cells, gastrointestinal tissue's cell, the parotid gland, Tiroidina, prostate gland, mammary gland, suprarenal gland, hypophysis, pancreas, amnion cell.The experimental technique tissue digestion is with the multi-enzyme system of primary cell separating kit II (detailed process slightly, can referring to above-mentioned experimentation).
Embodiment 9
The multi-enzyme system of primary cell separating kit III: 0.0125-0.125% (weight) trypsinase, 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser are separated enzyme with 0.01-0.1% (weight).
Isolating normal big rabbit keratinization of epidermis cell experiment method:
1, fresh normal big rabbit skin;
2, material pre-treatment: skin graft is immersed in the alcohol sterilizes; Clean;
4, normal big rabbit skin places the multi-enzyme system Digestive system of primary cell separating kit III;
5, add termination digestion: end enzyme reaction, suspension is crossed mesh screen;
6, collect re-suspended cell: the collecting cell suspension, centrifugal;
7, cultivate: be positioned over 37 ℃, 5%CO
2In the incubator;
8, isolating normal big rabbit keratinization of epidermis cell purification and detection, cultivation surviving rate and the recovery of frozen back are cultivated surviving rate and are detected.
Isolating normal big rabbit keratinization of epidermis cytobiology fundamental characteristics:
(1) normal big rabbit keratinization of epidermis cell derives from the normal rabbit skin histology.
(2) normal big rabbit keratinization of epidermis cell is identified: Cytokeratine-8 ,-18and-19 immunofluorescence dyeing.
(3) normal big rabbit keratinization of epidermis cell primary cell expression of adhesion molecules and cytokine participate in the natural immunity.
(4) normal big rabbit keratinization of epidermis cell does not contain HIV-1, HBV, HCV, mycoplasma, bacterium, yeast and fungi.
Isolating big rabbit keratinization of epidermis cell:
Surviving rate 74% is cultivated in purity 96%, cultivation surviving rate 95%, the recovery of frozen back.
Above-mentioned multi-enzyme system also has been respectively applied for separating of the normal big different tissues-derived skin cells of rabbit, osteocyte, cartilaginous tissue cell, Skeletal Muscle Cell, smooth muscle cell, myocardial cell, brain and neurocyte.The experimental technique tissue digestion is with the multi-enzyme system of primary cell separating kit III (detailed process slightly, can referring to above-mentioned experimentation).
The invention provides separation normal people and Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) multi-enzyme system of different tissues-derived primary culture cells, normal people and Mammals are (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) the isolating multi-enzyme system of different tissues-derived primary culture cells is the high specific of the inventor through the years of researches exploitation, highly sensitive primary cell separates multi-enzyme system, invented primary cell multi-enzyme system separating kit of new generation on this basis, be used for normal people and Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) different tissues-derived primary culture cells separates.
Peculiar advantage of the present invention and great advantage are: (1) tissue is made up of the broad variety cell, uses plurality of enzymes digestion tissue, can make cellular segregation more thorough; (2) consumption of every kind of enzyme is few, and is little to the primary cell damage of separate tissue; (3) the primary cell vitro culture after the separate tissue is easy to survival; (4) isolating primary cell keeps source sex organization biological characteristics.The isolating multi-enzyme system of this tissues-derived primary culture cells is reasonable in design, steady quality is reliable.
Because China is to the attention of life science and biological medicine enterprise, the inventor specially to normal people and Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) multi-enzyme system of different tissues-derived primary culture cells separation use is studied, new understanding has been arranged, normal people and Mammals are (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) the different tissues-derived primary culture cells separating kit not only will generally be accepted by life science market, also will be subjected to the research and development of Chinese biological pharmaceutical manufacturer and the common concern of Application Areas deeply, will be used widely.Similar product of the present invention does not occur as yet in market at present.
Adopt the present invention, the isolating specificity of primary cell, susceptibility and accuracy have had huge raising, can separate for 900 kinds with Mammals (as mouse, rat, cavy, rabbit, dog, pig, ox, monkey) different tissues-derived primary culture cells the normal people exactly.Therefore this project has significant social value, economic benefit and technical advance, meets country and local industry support policy.
To sum up, normal people of the present invention and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells are reasonable in design, can separate normal people and Mammals different tissues-derived primary culture cells specifically, in high sensitivity by height, isolating primary cell surviving rate height, purity height, be easy to frozen and the recovery, can be used for to the drug study assessment, to the drug toxicity test, therapeutic drug is judged, be suitable for large-scale promotion application.
In this specification sheets, the present invention is described with reference to its certain embodiments.But, still can make various modifications and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets is regarded in an illustrative, rather than a restrictive.
Claims (9)
1. normal people and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells, it is characterized in that, comprise that 0.0125-0.125% (weight) trypsinase, 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) Unidasa, 0.02-0.2% (weight) neutral protease, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser separate several in the enzyme with 0.01-0.1% (weight).
2. normal people according to claim 1 and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells, it is characterized in that, comprise that 0.0125-0.125% (weight) trypsinase, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser separate enzyme with 0.01-0.1% (weight).
3. normal people according to claim 1 and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells, it is characterized in that, comprise that 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser separate enzyme with 0.01-0.1% (weight).
4. normal people according to claim 1 and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells, it is characterized in that, comprise that 0.0125-0.125% (weight) trypsinase, 0.02-0.2% (weight) Collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U/ml papoid, 0.015-0.15% (weight) PRONASE A, 5-50U/ml elastoser separate enzyme with 0.01-0.1% (weight).
5. normal people according to claim 1 and the application of the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells in separating normal people and Mammals different tissues-derived primary culture cells.
6. normal people according to claim 2 and the application of the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells in separating normal people and Mammals endotheliocyte, epithelial cell, liver cell, nephrocyte or corneal epithelial cell.
7. normal people according to claim 3 and the application of the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells in separating normal people and Mammals lung tissue cell, gastrointestinal tissue's cell, the parotid gland, Tiroidina, prostate gland, mammary gland, suprarenal gland, hypophysis, pancreas, amnion cell.
8. normal people according to claim 4 and the application of the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells in separating normal people and mammal skin cell, osteocyte, cartilaginous tissue cell, Skeletal Muscle Cell, smooth muscle cell, myocardial cell, brain and neurocyte.
9. normal people and Mammals different tissues-derived primary culture cells separating kit is characterized in that, comprise the reagent container that is equipped with according to arbitrary described normal people of claim 1-4 and the isolating multi-enzyme system of Mammals different tissues-derived primary culture cells.
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| CN107267438A (en) * | 2016-04-08 | 2017-10-20 | 江苏齐氏生物科技有限公司 | A kind of separation of rat pituitary cell and cultural method |
| CN113025567A (en) * | 2021-03-31 | 2021-06-25 | 中国人民解放军陆军特色医学中心 | Separation method of intervertebral disc single cells |
| CN113388599A (en) * | 2021-04-22 | 2021-09-14 | 浙江大学 | Enzyme kit for dispersing biological tissue and method for obtaining single cell suspension using the same |
| CN116396921A (en) * | 2023-04-13 | 2023-07-07 | 杭州普罗亭医学检验实验室有限公司 | Kit special for pancreatic tissue digestion and pancreatic tissue digestion method |
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| CN116396921A (en) * | 2023-04-13 | 2023-07-07 | 杭州普罗亭医学检验实验室有限公司 | Kit special for pancreatic tissue digestion and pancreatic tissue digestion method |
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