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Multi-enzyme system for separating different tissues-derived primary culture cells of normal human and mammal, application and related kit thereof

A mammalian and primary cell technology, applied in the field of multi-enzyme system, can solve the problems of not correctly reflecting the biological characteristics and physiological functions of body tissues, and achieve the effect of easy cryopreservation and recovery, reasonable design, stable and reliable quality

Inactive Publication Date: 2010-12-15
刘东旭
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, in the research and development of drugs in the biomedical industry in the domestic and foreign markets, most of the research and development at the cell level uses cell lines. The biggest disadvantage of this type of cells is that they are significantly different from primary cells and cannot correctly reflect the biological characteristics of body tissues. Physical features and physiological functions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The multi-enzyme system of primary cell separation kit I: 0.0125-0.125% (weight) trypsin, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U / ml papain, 0.015-0.15% (weight) Pronase, 5-50 U / ml elastase and 0.01-0.1% by weight isolate enzyme.

[0034] Experimental method of isolated normal human umbilical vein endothelial cells:

[0035] 1. Fresh normal human baby umbilical cord;

[0036] 2. At the openings at both ends of the umbilical vein, tie them tightly with silk thread and fix them on a 50ml syringe and a glass cannula with an infusion hose, and inject D-Hanks solution to flush the umbilical vein until there is no blood;

[0037] 3. Clamp the infusion hose on the glass cannula with a hemostat, and slowly inject the multi-enzyme system solution of our primary cell separation kit I into the umbilical vein from the syringe at the other end until the blood vessel is filled. Note that both ends are closed to prevent liquid backflow. Immediately put into sterile D-Hank...

Embodiment 2

[0051] The multi-enzyme system of primary cell isolation kit II: 0.02-0.2% (weight) collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1-10U / ml papain, 0.015-0.15% (weight) pronase, 5-50 U / ml elastase and 0.01-0.1% (weight) isolate enzyme.

[0052] Isolated normal human lung epithelial cells Experimental method:

[0053] 1. Fresh normal human lung tissue, and lavage with alveolar lavage fluid;

[0054] 2. The multi-enzyme system digestion of primary cell isolation kit II perfused in the lung;

[0055] 3. Remove the surrounding connective tissue;

[0056] 4. Add cell dispersion to terminate digestion;

[0057] 5. The suspension is filtered through a stainless steel mesh screen;

[0058] 6. Collect the filtrate and centrifuge;

[0059] 7. Discard the supernatant and suspend the cells with culture medium;

[0060] 8. Add the cell suspension to the Petri dish, at 37°C, 5% CO 2 Incubated in an incubator;

[0061] 9. Purification and detection of isolated normal h...

Embodiment 3

[0070] The multi-enzyme system of primary cell isolation kit III: 0.0125-0.125% (weight) trypsin, 0.02-0.2% (weight) collagenase I-IV, 0.0015-0.015% (weight) deoxyribonuclease I, 1- 10U / ml papain, 0.015-0.15% (weight) pronase, 5-50U / ml elastase and 0.01-0.1% (weight) separase. Experimental method of isolated normal human epidermal keratinocytes:

[0071] 1. Fresh normal human fetal skin;

[0072] 2. Material pretreatment: soak the skin in alcohol for disinfection; cleaning;

[0073] 4. Normal human skin is placed in the multi-enzyme system digestion solution of the primary cell separation kit III;

[0074] 5. Add to stop digestion: stop the enzyme reaction, pass the suspension through a mesh sieve;

[0075] 6. Collect resuspended cells: collect the cell suspension and centrifuge;

[0076] 7. Culture: place at 37°C, 5% CO 2 in the incubator;

[0077] 8. Purification and detection of isolated normal human epidermal keratinocytes, detection of culture survival rate and reco...

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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 [0001] The present invention relates to the technical field of primary cell separation, in particular to the technical field of primary cell separation from different tissues, and specifically refers to a multi-enzyme system for the separation of primary cells from different tissues of normal humans and mammals, its use and related Reagent test kit. Background technique [0002] In the biomedical industry, especially the application industry of primary cell culture in drug research and development, the profit margin is large, the energy consumption is small, and the technical level is high. It is a policy support industry determined by the National Reform and Development Commission of China. Development Committee Guidance List of China's key development industries before 2010. Provincial governments and other local governments will support such projects as key technologies and major projects, and enjoy special preferential treatment in terms of manpower and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N9/76C12N9/50C12N9/26C12N9/22C12N9/52C12N9/66C12N5/071C12N5/077C12N5/079
Inventor 刘东旭
Owner 刘东旭
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