CN110724663A - Isolated culture method of liver stem cells - Google Patents
Isolated culture method of liver stem cells Download PDFInfo
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- CN110724663A CN110724663A CN201910947258.5A CN201910947258A CN110724663A CN 110724663 A CN110724663 A CN 110724663A CN 201910947258 A CN201910947258 A CN 201910947258A CN 110724663 A CN110724663 A CN 110724663A
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- C12N5/0672—Stem cells; Progenitor cells; Precursor cells; Oval cells
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Abstract
The invention discloses a separation culture method of liver stem cells, which comprises the following steps: A. injecting a preserving fluid with the temperature of 3-5 ℃ into the isolated liver to reduce the temperature of the liver to 15 ℃ within 30 min; B. washing the liver for 20-30 min by using a buffer solution; C. opening the liver and injecting a perfusion liquid into the liver, wherein the temperature of the perfusion liquid is 37 ℃, the injection flow rate is 200-300 mL/min, and the duration is 2-3 min; D. soaking and digesting the liver for 45-60 min by using a collagenase solution; E. cleaning the liver by using a cleaning solution to obtain a liver stem cell suspension, and centrifuging to obtain liver stem cells; F. and inoculating the obtained liver stem cells into a culture solution for culturing at 37 ℃ for 24-36 h. The invention can improve the defects of the prior art and improve the success rate of the liver stem cell separation culture.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for separating and culturing liver stem cells.
Background
Stem cells are a class of primitive undifferentiated cells that have self-replicating capacity and multipotentiality, are in an undifferentiated state, and have proliferative capacity. Under certain conditions, stem cells can be differentiated into different types of mature cells with characteristic morphology, specific molecular markers and specific functions. The liver stem cells can be converted into new liver cells, and have good treatment prospect for serious liver diseases. Because the content of the liver stem cells is very low, the success rate of the isolation and culture of the liver cells in the prior art is low, and the liver stem cells can be obtained by multiple isolation and culture in a laboratory.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for separating and culturing liver stem cells, which can solve the defects of the prior art and improve the success rate of separating and culturing the liver stem cells.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows.
A method for separating and culturing liver stem cells comprises the following steps:
A. injecting a preserving fluid with the temperature of 3-5 ℃ into the isolated liver to reduce the temperature of the liver to 15 ℃ within 30 min;
B. washing the liver for 20-30 min by using a buffer solution;
C. opening the liver and injecting a perfusion liquid into the liver, wherein the temperature of the perfusion liquid is 37 ℃, the injection flow rate is 200-300 mL/min, and the duration is 2-3 min;
D. soaking and digesting the liver for 45-60 min by using a collagenase solution;
E. cleaning the liver by using a cleaning solution to obtain a liver stem cell suspension, and centrifuging to obtain liver stem cells;
F. and inoculating the obtained liver stem cells into a culture solution for culturing at 37 ℃ for 24-36 h.
Preferably, in step a, UW solution is used as the preservation solution.
Preferably, in step B, the buffer comprises the following components,
0.35wt% of KCl and 0.1wt% of CaCl20.55wt% NaHCO3, 0.15wt% Na2HPO4·2H2O and the balance of water.
Preferably, in step C, the perfusion fluid comprises the following components,
0.03wt% of KCl, 0.01wt% of gentamicin, 0.02wt% of penicillin, 0.05wt% of HEPES, 0.75wt% of NaCl and the balance of water.
Preferably, in step D, the collagenase solution comprises the following components,
0.3wt% collagenase type II, 0.1wt% KH2PO40.05wt% of MgSO4·7H2O, 1.5wt% of glucose, and the balance of water;
after the preparation is finished, citric acid is added to enable the pH value of the collagenase solution to reach 6.5-6.8.
Preferably, in step E, the cleaning solution comprises the following components,
0.55wt% KH2PO40.15wt% of MgCl22.5wt% of double-antibody, 0.05wt% of HEPES and the balance of water.
Preferably, in step F, the culture solution comprises the following components,
0.1wt% of dexamethasone, 0.05wt% of nicotinamide, 0.85wt% of D galactose, 0.03wt% of double antibody, 0.25wt% of insulin and the balance of Ultroser G culture solution.
Adopt the beneficial effect that above-mentioned technical scheme brought to lie in: the invention can effectively improve the activity of liver cells by improving the perfusion operation process and adopting a perfusion method of low concentration and large flow, and then can effectively separate liver stem cells by digestion treatment of an acid collagenase solution so as to achieve the aim of improving the success rate of stem cell culture.
Drawings
FIG. 1 is a photograph showing the state of cells under a microscope after culturing the liver stem cells in example 1.
FIG. 2 is a photograph showing the state of cells under a microscope after culturing the liver stem cells in example 2.
FIG. 3 is a photograph showing the state of cells under a microscope after culturing the liver stem cells in example 3.
Detailed Description
Example 1
A method for separating and culturing liver stem cells comprises the following steps:
A. injecting a preservation solution with the temperature of 5 ℃ into the isolated liver to reduce the temperature of the liver to 15 ℃ within 30 min;
B. washing liver with buffer solution for 20 min;
C. opening to inject perfusion liquid into the liver, wherein the temperature of the perfusion liquid is 37 ℃, the injection flow rate is 270mL/min, and the duration is 3 min;
D. soaking and digesting the liver for 50min by using collagenase solution;
E. cleaning the liver by using a cleaning solution to obtain a liver stem cell suspension, and centrifuging to obtain liver stem cells;
F. inoculating the obtained liver stem cells into a culture solution for culturing at 37 ℃ for 30 h.
In the step A, UW liquid is adopted as the preservation liquid.
In the step B, the buffer solution comprises the following components,
0.35wt% of KCl and 0.1wt% of CaCl20.55wt% NaHCO3, 0.15wt% Na2HPO4·2H2O and the balance of water.
In step C, the perfusion fluid comprises the following components,
0.03wt% of KCl, 0.01wt% of gentamicin, 0.02wt% of penicillin, 0.05wt% of HEPES, 0.75wt% of NaCl and the balance of water.
In step D, the collagenase solution comprises the following components,
0.3wt% collagenase type II, 0.1wt% KH2PO40.05wt% of MgSO4·7H2O, 1.5wt% of glucose, and the balance of water;
after the preparation was complete, the collagenase solution was brought to a pH of 6.7 by the addition of citric acid.
In the step E, the cleaning solution comprises the following components,
0.55wt% KH2PO40.15wt% of MgCl22.5wt% of double-antibody, 0.05wt% of HEPES and the balance of water.
In step F, the culture solution comprises the following components,
0.1wt% of dexamethasone, 0.05wt% of nicotinamide, 0.85wt% of D galactose, 0.03wt% of double antibody, 0.25wt% of insulin and the balance of Ultroser G culture solution.
Example 2
This example is an improvement over example 1, in which 0.01wt% of butanediamine and 0.03wt% of magnesium isoglycyrrhizinate were added to the perfusate. By improving the formula of the perfusion liquid, the proliferation of the liver stem cells can be effectively improved.
Example 3
This example is an improvement over example 2, in which 0.1wt% glutathione and 0.03wt% daidzein are added to a collagenase solution. By improving the formulation of the collagenase solution, the loss of stem cells during the digestion process can be reduced.
The isolation and culture experiment of stem cells was performed according to the above three examples using the liver of a mouse, and the purity of the obtained liver stem cells was as shown in the following table:
| group of | Purity (%) |
| Example 1 | 84.1 |
| Example 2 | 89.6 |
| Example 3 | 95.9 |
FIGS. 1 to 3 are photographs under a microscope of the liver stem cells cultured in examples 1 to 3, respectively, and it can be seen that the liver stem cells isolated and cultured by the present invention have clear and complete structure and normal morphology.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A method for separating and culturing liver stem cells is characterized by comprising the following steps:
A. injecting a preserving fluid with the temperature of 3-5 ℃ into the isolated liver to reduce the temperature of the liver to 15 ℃ within 30 min;
B. washing the liver for 20-30 min by using a buffer solution;
C. opening the liver and injecting a perfusion liquid into the liver, wherein the temperature of the perfusion liquid is 37 ℃, the injection flow rate is 200-300 mL/min, and the duration is 2-3 min;
D. soaking and digesting the liver for 45-60 min by using a collagenase solution;
E. cleaning the liver by using a cleaning solution to obtain a liver stem cell suspension, and centrifuging to obtain liver stem cells;
F. and inoculating the obtained liver stem cells into a culture solution for culturing at 37 ℃ for 24-36 h.
2. The isolated culture method of liver stem cells according to claim 1, wherein: in the step A, UW liquid is adopted as the preservation liquid.
3. The isolated culture method of liver stem cells according to claim 1, wherein: in the step B, the buffer solution comprises the following components,
0.35wt% of KCl and 0.1wt% of CaCl20.55wt% NaHCO3, 0.15wt% Na2HPO4·2H2O and the balance of water.
4. The isolated culture method of liver stem cells according to claim 1, wherein: in step C, the perfusion fluid comprises the following components,
0.03wt% of KCl, 0.01wt% of gentamicin, 0.02wt% of penicillin, 0.05wt% of HEPES, 0.75wt% of NaCl and the balance of water.
5. The isolated culture method of liver stem cells according to claim 1, wherein: in step D, the collagenase solution comprises the following components,
0.3wt% collagenase type II, 0.1wt% KH2PO40.05wt% of MgSO4·7H2O, 1.5wt% of glucose, and the balance of water;
after the preparation is finished, citric acid is added to enable the pH value of the collagenase solution to reach 6.5-6.8.
6. The isolated culture method of liver stem cells according to claim 1, wherein: in the step E, the cleaning solution comprises the following components,
0.55wt% KH2PO40.15wt% of MgCl22.5wt% of double antibody, 0.05wt% of HEPES and the balance of water.
7. The isolated culture method of liver stem cells according to claim 1, wherein: in step F, the culture solution comprises the following components,
0.1wt% of dexamethasone, 0.05wt% of nicotinamide, 0.85wt% of D galactose, 0.03wt% of double antibody, 0.25wt% of insulin and the balance of Ultroser G culture solution.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1414944A2 (en) * | 2001-07-10 | 2004-05-06 | Massachusetts Institute Of Technology | Methods for ex vivo propagation of somatic stem cells |
| CN102634480A (en) * | 2012-03-27 | 2012-08-15 | 中国农业大学 | Method for isolating and culturing liver primary cells |
| CN104818242A (en) * | 2015-05-28 | 2015-08-05 | 四川大学华西医院 | Separation and preparation method of primary hepatocytes |
| CN106978388A (en) * | 2017-04-10 | 2017-07-25 | 安徽农业大学 | A kind of separation of dog liver cell and cultural method |
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- 2019-10-08 CN CN201910947258.5A patent/CN110724663A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1414944A2 (en) * | 2001-07-10 | 2004-05-06 | Massachusetts Institute Of Technology | Methods for ex vivo propagation of somatic stem cells |
| CN102634480A (en) * | 2012-03-27 | 2012-08-15 | 中国农业大学 | Method for isolating and culturing liver primary cells |
| CN104818242A (en) * | 2015-05-28 | 2015-08-05 | 四川大学华西医院 | Separation and preparation method of primary hepatocytes |
| CN106978388A (en) * | 2017-04-10 | 2017-07-25 | 安徽农业大学 | A kind of separation of dog liver cell and cultural method |
Non-Patent Citations (7)
| Title |
|---|
| 余跃等: "《干细胞基础与临床》", 29 February 2008, 中国科学技术大学出版社 * |
| 卢春凤等: "《实用医学研究基本技术与方法》", 31 August 2018, 北京知识产权出版社 * |
| 姚崇舜等: "《葛根的药理作用与临床应用》", 30 April 2014, 人民军医出版社 * |
| 巫协宁等: "《临床肝胆系病学 新版》", 30 September 2002, 上海科学技术文献出版社 * |
| 张在其等: "《急危重病临床救治》", 30 September 2010, 湖北科学技术出版社 * |
| 郭静等: "一种分离正常成年大鼠肝脏祖细胞新方法的研究", 《第三届江浙沪中西医结合高峰论坛论文汇编》 * |
| 陈孝治等: "《药物处方手册 第2版》", 30 June 2012, 湖南科学技术出版社 * |
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Application publication date: 20200124 |