CN108823146A - A kind of preparation of liver stem cells and extracting method - Google Patents
A kind of preparation of liver stem cells and extracting method Download PDFInfo
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- CN108823146A CN108823146A CN201810650659.XA CN201810650659A CN108823146A CN 108823146 A CN108823146 A CN 108823146A CN 201810650659 A CN201810650659 A CN 201810650659A CN 108823146 A CN108823146 A CN 108823146A
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- stem cells
- liver
- extracting method
- enzymolysis liquid
- liver stem
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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
- C12N5/067—Hepatocytes
- C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
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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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a kind of preparation of liver stem cells and extracting methods, include the following steps, cleaning and sterilizing, surface layer are rinsed in liver organization water purification, and wipe using alcohol swab, are then immersed in physiological saline, soaking time 3min；Enzymatic hydrolysis, take out liver organization after rinsed well using PBS buffer solution, liver organization is cut into small pieces and is put into protein enzyme solution, dissolution time be 6~for 24 hours, with filter screen filtration enzymolysis liquid；Separation；Enzymolysis liquid after separation is inoculated into basal medium until there is cell cluster by screening, and observation cell appearance determines liver stem cells, and filters out liver stem cells；It saves, the present invention relates to technical field of bioengineering.Liver stem cells preparation and extracting method, keep the screening process of stem cell simpler, reduce the screening cost of stem cell, while effectively eliminating the impurity being likely to occur in stem cell tissue liquid, improve the research and use value of the stem cell after extracting.
Description
Technical field
The present invention relates to technical field of bioengineering, specially a kind of liver stem cells preparation and extracting method.
Background technique
Stem cell is a kind of multipotential cell with the of self-replication capacity, and under certain condition, it can be divided into more
Kind functioning cell, the stage of development according to locating for stem cell is divided into embryonic stem cell and adult stem cell, according to the hair of stem cell
Potential is educated to be divided into three classes:Myeloid-lymphoid stem cell, multipotential stem cell and unipotent stem cell (specially energy stem cell), stem cell are that one kind is not filled
Differentiation, still immature cell have the potential function for regenerating various histoorgans and human body, and referred to as " general-purpose is thin for medical field
Born of the same parents ".But existing stem cell extracting method screening stem cell process complexity, screening cost is higher, in stem cell extraction process
It is easy residual impurity, influences the research and service performance of stem cell.
Summary of the invention
（One）The technical issues of solution
In view of the deficiencies of the prior art, it the present invention provides a kind of preparation of liver stem cells and extracting method, solves existing
Stem cell extracting method screens stem cell process complexity, and screening cost is higher, and residual impurity, shadow are easy in stem cell extraction process
The problem of ringing the research and service performance of stem cell.
（Two）Technical solution
In order to achieve the above object, the present invention is achieved by the following technical programs：A kind of preparation of liver stem cells and extraction side
Method includes the following steps,
（1）Liver organization water purification is rinsed surface layer, and is wiped using alcohol swab by cleaning and sterilizing, is then immersed in physiological saline
In, soaking time 3min；
（2）Enzymatic hydrolysis is rinsed well using PBS buffer solution after taking out liver organization, liver organization is cut into small pieces and is put into protease
In solution, dissolution time be 6~for 24 hours, with filter screen filtration enzymolysis liquid；
（3）Separation pours into 5 times of distilled water dilution enzymolysis liquids, is uniformly mixed with clean glass bar, pours into differential centrifugation machine
In；
（4）Enzymolysis liquid after separation is inoculated into basal medium until there is cell cluster by screening, and observation cell appearance determines
Liver stem cells, and filter out liver stem cells；
（5）It saves, liver stem cells is stored in low temperature chamber, temperature should be 4 DEG C.
Preferably, the protein enzyme solution is trypsase and ETDA, and the concentration of the trypsase and ETDA are respectively
1g/L and 0.5g/L.
Preferably, the volume ratio of the trypsase and ETDA are 1:3.
Preferably, the basal medium is stem cell serum-free basal medium.
Preferably, shading environment should be kept in the liver organization enzymolysis liquid incubation.
Preferably, stem cell serum-free nutritional supplements should be added in the stem cell serum-free basal medium, and
The volume ratio of stem cell serum-free basal medium and stem cell serum-free nutritional supplements is 10:1.
Preferably, the liver organization enzymolysis liquid culture should be inoculated into new basal medium afterwards for a period of time continues to train
It supports.
Preferably, incubation time of the liver organization enzymolysis liquid in basal medium should be 24~48h.
Preferably, centrifugation time of the enzymolysis liquid in differential centrifugation machine should be 10min, and centrifugal force should be 200g.
Preferably, supernatant should be removed after the enzymolysis liquid centrifugal treating.
（Three）Beneficial effect
The present invention provides a kind of preparation of liver stem cells and extracting methods.Has following beneficial effect：
（1）, liver stem cells preparation and extracting method, dilute enzymolysis liquids by pouring into 5 times of distilled water, stirred with clean glass bar
It mixes uniformly mixed, pours into differential centrifugation machine, the enzymolysis liquid after separation is inoculated into basal medium until there is cell cluster,
Observation cell appearance determines liver stem cells, and filters out liver stem cells, is more convenient after simply cultivating and to form cell cluster
The sShape features of cell are observed, convenient directly observation distinguishes stem cell and mature cell, keeps the screening process of stem cell simpler
It is single, reduce the screening cost of stem cell.
（2）, the liver stem cells preparation and extracting method, by by liver organization with water purification rinse surface layer, and use wine
Smart cotton rub is wiped, and is then immersed in physiological saline, soaking time 3min, is rinsed after taking out liver organization using PBS buffer solution
Completely, liver organization being cut into small pieces and is put into protein enzyme solution, dissolution time is 6~for 24 hours, with filter screen filtration enzymolysis liquid,
The impurity being likely to occur in stem cell tissue liquid effectively is eliminated, improve the research of the stem cell after extracting and uses valence
Value.
Specific embodiment
In order to be easy to understand the technical means, the creative features, the aims and the efficiencies achieved by the present invention, tie below
Specific embodiment is closed, the present invention is further explained.
Embodiment one
A kind of preparation of liver stem cells and extracting method, include the following steps,
（1）Liver organization water purification is rinsed surface layer, and is wiped using alcohol swab by cleaning and sterilizing, is then immersed in physiological saline
In, soaking time 3min；
（2）Enzymatic hydrolysis is rinsed well using PBS buffer solution after taking out liver organization, liver organization is cut into small pieces and is put into protease
In solution, dissolution time 6h, with filter screen filtration enzymolysis liquid；
（3）Separation pours into 5 times of distilled water dilution enzymolysis liquids, is uniformly mixed with clean glass bar, pours into differential centrifugation machine
In；
（4）Enzymolysis liquid after separation is inoculated into basal medium until there is cell cluster by screening, and observation cell appearance determines
Liver stem cells, and filter out liver stem cells；
（5）It saves, liver stem cells is stored in low temperature chamber, temperature should be 4 DEG C.
Protein enzyme solution is trypsase and ETDA, and the concentration of the trypsase and ETDA are respectively 1g/L and 0.5g/
L。
The volume ratio of trypsase and ETDA are 1:3.
Basal medium is stem cell serum-free basal medium.
Shading environment should be kept in liver organization enzymolysis liquid incubation.
Stem cell serum-free nutritional supplements, and stem cell serum-free should be added in stem cell serum-free basal medium
The volume ratio of basal medium and stem cell serum-free nutritional supplements is 10:1.
The culture of liver organization enzymolysis liquid should be inoculated into new basal medium afterwards for a period of time to be continued to cultivate.
Incubation time of the liver organization enzymolysis liquid in basal medium should be for 24 hours.
Centrifugation time of the enzymolysis liquid in differential centrifugation machine should be 10min, and centrifugal force should be 200g.
Supernatant should be removed after enzymolysis liquid centrifugal treating.
Embodiment two
A kind of preparation of liver stem cells and extracting method, include the following steps,
（1）Liver organization water purification is rinsed surface layer, and is wiped using alcohol swab by cleaning and sterilizing, is then immersed in physiological saline
In, soaking time 3min；
（2）Enzymatic hydrolysis is rinsed well using PBS buffer solution after taking out liver organization, liver organization is cut into small pieces and is put into protease
In solution, dissolution time 15h, with filter screen filtration enzymolysis liquid；
（3）Separation pours into 5 times of distilled water dilution enzymolysis liquids, is uniformly mixed with clean glass bar, pours into differential centrifugation machine
In；
（4）Enzymolysis liquid after separation is inoculated into basal medium until there is cell cluster by screening, and observation cell appearance determines
Liver stem cells, and filter out liver stem cells；
（5）It saves, liver stem cells is stored in low temperature chamber, temperature should be 4 DEG C.
Protein enzyme solution is trypsase and ETDA, and the concentration of the trypsase and ETDA are respectively 1g/L and 0.5g/
L。
The volume ratio of trypsase and ETDA are 1:3.
Basal medium is stem cell serum-free basal medium.
Shading environment should be kept in liver organization enzymolysis liquid incubation.
The culture of liver organization enzymolysis liquid should be inoculated into new basal medium afterwards for a period of time to be continued to cultivate.
Incubation time of the liver organization enzymolysis liquid in basal medium should be 36h.
Centrifugation time of the enzymolysis liquid in differential centrifugation machine should be 10min, and centrifugal force should be 200g.
Supernatant should be removed after enzymolysis liquid centrifugal treating.
Embodiment three
A kind of preparation of liver stem cells and extracting method, include the following steps,
（1）Liver organization water purification is rinsed surface layer, and is wiped using alcohol swab by cleaning and sterilizing, is then immersed in physiological saline
In, soaking time 3min；
（2）Enzymatic hydrolysis is rinsed well using PBS buffer solution after taking out liver organization, liver organization is cut into small pieces and is put into protease
In solution, dissolution time is for 24 hours, with filter screen filtration enzymolysis liquid；
（3）Separation pours into 5 times of distilled water dilution enzymolysis liquids, is uniformly mixed with clean glass bar, pours into differential centrifugation machine
In；
（4）Enzymolysis liquid after separation is inoculated into basal medium until there is cell cluster by screening, and observation cell appearance determines
Liver stem cells, and filter out liver stem cells；
（5）It saves, liver stem cells is stored in low temperature chamber, temperature should be 4 DEG C.
Protein enzyme solution is trypsase and ETDA, and the concentration of the trypsase and ETDA are respectively 1g/L and 0.5g/
L。
The volume ratio of trypsase and ETDA are 1:3.
Basal medium is stem cell serum-free basal medium.
Shading environment should be kept in liver organization enzymolysis liquid incubation.
Stem cell serum-free nutritional supplements, and stem cell serum-free should be added in stem cell serum-free basal medium
The volume ratio of basal medium and stem cell serum-free nutritional supplements is 5:1.
The culture of liver organization enzymolysis liquid should be inoculated into new basal medium afterwards for a period of time to be continued to cultivate.
Incubation time of the liver organization enzymolysis liquid in basal medium should be 48h.
Centrifugation time of the enzymolysis liquid in differential centrifugation machine should be 10min, and centrifugal force should be 200g.
Supernatant should be removed after enzymolysis liquid centrifugal treating.
It should be noted that, in this document, relational terms such as first and second and the like are used merely to a reality
Body or operation are distinguished with another entity or operation, are deposited without necessarily requiring or implying between these entities or operation
In any actual relationship or order or sequence.Moreover, the terms "include", "comprise" or its any other variant are intended to
Non-exclusive inclusion, so that the process, method, article or equipment including a series of elements is not only wanted including those
Element, but also including other elements that are not explicitly listed, or further include for this process, method, article or equipment
Intrinsic element.In the absence of more restrictions.By sentence " element limited including one ..., it is not excluded that
There is also other identical elements in the process, method, article or apparatus that includes the element ".
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (10)
1. a kind of liver stem cells preparation and extracting method, it is characterised in that：Include the following steps,
（1）Liver organization water purification is rinsed surface layer, and is wiped using alcohol swab by cleaning and sterilizing, is then immersed in physiological saline
In, soaking time 3min；
（2）Enzymatic hydrolysis is rinsed well using PBS buffer solution after taking out liver organization, liver organization is cut into small pieces and is put into protease
In solution, dissolution time be 6~for 24 hours, with filter screen filtration enzymolysis liquid；
（3）Separation pours into 5 times of distilled water dilution enzymolysis liquids, is uniformly mixed with clean glass bar, pours into differential centrifugation machine
In；
（4）Enzymolysis liquid after separation is inoculated into basal medium until there is cell cluster by screening, and observation cell appearance determines
Liver stem cells, and filter out liver stem cells；
（5）It saves, liver stem cells is stored in low temperature chamber, temperature should be 4 DEG C.
2. a kind of liver stem cells preparation according to claim 1 and extracting method, it is characterised in that：The protease is molten
Liquid is trypsase and ETDA, and the concentration of the trypsase and ETDA are respectively 1g/L and 0.5g/L.
3. a kind of liver stem cells preparation according to claim 2 and extracting method, it is characterised in that：The trypsase
Volume ratio with ETDA is 1:3.
4. a kind of liver stem cells preparation according to claim 1 and extracting method, it is characterised in that：The basis culture
Base is stem cell serum-free basal medium.
5. a kind of liver stem cells preparation according to claim 1 and extracting method, it is characterised in that：The liver organization
Shading environment should be kept in enzymolysis liquid incubation.
6. a kind of liver stem cells preparation according to claim 4 and extracting method, it is characterised in that：The stem cell without
Stem cell serum-free nutritional supplements should be added in serum basal medium, and stem cell serum-free basal medium and dry thin
The volume ratio of born of the same parents' serum-free nutrient additive is 10:1.
7. a kind of liver stem cells preparation according to claim 1 and extracting method, it is characterised in that：The liver organization
Enzymolysis liquid culture should be inoculated into new basal medium afterwards for a period of time to be continued to cultivate.
8. a kind of liver stem cells preparation according to claim 1 and extracting method, it is characterised in that：The liver organization
Incubation time of the enzymolysis liquid in basal medium should be 24~48h.
9. a kind of liver stem cells preparation according to claim 1 and extracting method, it is characterised in that：The enzymolysis liquid exists
Centrifugation time in differential centrifugation machine should be 10min, and centrifugal force should be 200g.
10. a kind of liver stem cells preparation according to claim 1 and extracting method, it is characterised in that：The enzymolysis liquid
Supernatant should be removed after centrifugal treating.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080026463A1 (en) * | 2006-06-28 | 2008-01-31 | Vesta Therapeutics, Inc. | Matrix and method for isolation of hepatic progenitor cells |
| US20080311094A1 (en) * | 2005-12-21 | 2008-12-18 | Universite Catholique De Louvain | Isolated Liver Stem Cells |
| CN102399744A (en) * | 2011-11-24 | 2012-04-04 | 吉林省拓华生物科技有限公司 | Method for culturing liver stem cells |
| CN107151651A (en) * | 2017-06-30 | 2017-09-12 | 云南农业大学 | A kind of primary culture method of porcine hepatocyte |
-
2018
- 2018-06-22 CN CN201810650659.XA patent/CN108823146A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080311094A1 (en) * | 2005-12-21 | 2008-12-18 | Universite Catholique De Louvain | Isolated Liver Stem Cells |
| US20080026463A1 (en) * | 2006-06-28 | 2008-01-31 | Vesta Therapeutics, Inc. | Matrix and method for isolation of hepatic progenitor cells |
| CN102399744A (en) * | 2011-11-24 | 2012-04-04 | 吉林省拓华生物科技有限公司 | Method for culturing liver stem cells |
| CN107151651A (en) * | 2017-06-30 | 2017-09-12 | 云南农业大学 | A kind of primary culture method of porcine hepatocyte |
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