CN101864394A - Method for separating and culturing macaque adult hepatic precursor cells - Google Patents
Method for separating and culturing macaque adult hepatic precursor cells Download PDFInfo
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- CN101864394A CN101864394A CN 201010198776 CN201010198776A CN101864394A CN 101864394 A CN101864394 A CN 101864394A CN 201010198776 CN201010198776 CN 201010198776 CN 201010198776 A CN201010198776 A CN 201010198776A CN 101864394 A CN101864394 A CN 101864394A
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Abstract
The invention relates to a method for separating and culturing macaque adult hepatic precursor cells, in particular to a separation and culture solution of the macaque adult hepatic precursor cells, belonging to a separation and culture solution of animal precursor cells. The method comprises the following steps: digesting a small quantity of hepatic tissues obtained by a macaque liver removal surgery or other ways by utilizing IV-type collagenase; collecting and suspending the cells in 10% of a fetal calf serum DMEM for culture; selecting and then inoculating a single cell block in the culture plate of a rat tail collagen bed board, wherein the culture solution is the special one; and finally sorting E-cad+cells from the cultured cells by the flow cytometry to obtain the target cells. The cells separated by the method of the invention can be applied to cell differentiation mechanism study and can become ideal master cells for treating late-period hepatic diseases.
Description
Technical field
The invention belongs to the separation and the nutrient solution of the separation of animal precursor cell and nutrient solution, particularly macaque adult hepatic precursor cells.
Background technology
China has chronic hepatitis patient 3,000 ten thousand now, and wherein a part progressively develops into hepatic fibrosis, if the further deterioration of hepatic fibrosis will cause liver cirrhosis, liver failure, portal hypertension etc.In general, early stage hepatic fibrosis is a reversible, and the hepatic fibrosis in late period is that liver cirrhosis is irreversible, and organ transplantation is to treat the unique feasible program of hepatic diseases in late period at present, yet the donor organ famine has limited its application.
More and more studies show that: do the new hope of cell replacement treatment having become treatment hepatic diseases of (precursor) cell based on liver.Adult hepatic precursor cells (Hepatic Progenitor cells, HPCs) has stronger cell proliferation and be divided into the ability of particular cell types, lower teratoma production rate, and do not have the HPCs of fetal origin and the ethics problem that embryonic stem cell exists, therefore be considered to the ideal kind source cell of cell replacement treatment hepatic diseases in late period.
Be separated to polytype adult HPCs at present.The elliptocyte (oval cells) that derives from people, mouse and the rat pathological liver is identified the cell with HPCs function the earliest, elliptocyte has very strong multiplication capacity in vivo and in vitro, and can be divided into liver cell or bile duct epithelial cell (biliaryepithelial cells, ability BECs).But because cellular segregation is a kind of very complicated technology, accomplish the cell that enrichment to greatest extent is required, reduce the per-cent of other cells simultaneously, each link that requires cellular segregation to cultivate all will be passed through comparative analysis, just can obtain simple, efficient, a stable system.Also from normal adult hepatic, do not obtain the report of the consistent cell of identical phenotype with elliptocyte at present.Mature liver cells is owing to can be divided into bile duct epithelial cell or express the associated protein of bile duct epithelial cell external, and the reinventing of the bile duct that can participate in regenerating, therefore also be considered to a kind of in the adult HPCs cell, regrettably mature liver cells is in external very difficult amplification cultivation.
Except elliptocyte and liver cell, derive from adult liver epithelial cell (the liver epithelial progenitor cells of mouse, rat and pig health, LEPCs) has the characteristic of HPC, these epithelial cells present the distinctive amplification ability of stem cell, also can be divided into the specific cell type of liver, can recover the liver function level of impaired liver after the transplanting, all these characteristics show: liver epithelium precursor cell is a potential cell replacement treatment ideal donor cell.Equally, from rodents, the fetus liver of the cynomolgus monkey of people and SV40T transfection is also separable to similar liver epithelial cell.
Many researchs have carried out separating the trial of HPCs from people's adult hepatic.Utilize specific culture condition can be separated in the patient's that carries out hepatectomy the liver a kind of can the liver stem cells of external long-term propagation (human hepatic stem cells, HHSC).Yet, the expression pattern of this cell mass and differentiation potential all show they be similar to more mescenchymal stem cell (mensenchymal stem cells, MSCs) rather than traditional HPCs.Recently, obtained presenting the liver epithelial cell of liver and gall expression pattern from the liver of the adult of people's health or hepatectomy, pointing out them may be the potential precursor cell.But report does not confirm the characteristic of hepatic precursor cells fully at present, as two-way (liver and courage) differentiation capability of cell.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, a kind of isolation cultivation method that can be applicable to macaque adult hepatic precursor cells is provided, separate the cell that obtains and can be the separation and Culture of the adult hepatic precursor cells of each species of primates and the research of mechanism of cell differentiation, the ideal kind source cell for the treatment of the hepatic diseases in late period especially as an alternative lays the foundation.
The object of the invention realizes by following steps:
(1) gets the macaque adult hepatic tissue that 5-10 restrains, use PBS washing by soaking 3 times, be cut into small pieces, use the DMEM of the IV Collagen Type VI enzyme that contains 5mg/ml to digest tissue block 30～60 minutes;
(2) collect cell in the supernatant liquor, give a baby a bath on the third day after its birth time back with centrifugal 5 minutes of 1200 rev/mins of speed with DMEM;
(3) cell of collecting is placed the sealing centrifuge tube suspension culture of 10% foetal calf serum DMEM, to form cell mass;
(4) choose one cell mass, be inoculated in the culture plate of rat mouse tail collagen bed board;
(5) cell culture fluid contains 10mmol/L nicotinamide, 1x ITS, 100U/mlpenicillin, the DMEM/F12 (3: 1) of 100 μ g/ml streptomycin, add 10%FBS, 50ng/mL Urogastron EGF, 10ng/ml pHGF HGF;
(6) treat cell after covering with on the culture plate, 37 ℃ of digestion 10～30 minutes,, collect macaque adult hepatic precursor cells with flow cytometry sorting E-cad+ cell with trypsin-EDTA.
Described cell culture condition is 37 ℃, 5% CO
2, changed one time nutrient solution in per two days.
Described step (1) uses the DMEM of the IV Collagen Type VI enzyme that contains 5mg/ml to digest tissue block 30 minutes in 37 ℃ shaking table.
Cell suspending liquid suspension culture in 37 ℃ shaking table in the centrifuge tube of sealing in the described step (3),
Described macaque adult hepatic precursor cells is cultivated on the culture plate of rat mouse tail collagen bed board with the nutrient solution in the step (5), can go down to posterity by 1: 2 or more than 1: 3 time.
Positive effect that the present invention has and progress are:
Through comparing repeatedly, we have successfully set up a suitable system in the present invention, being used for separation and Culture comes from hepatic precursor cells (the monkey liver epithelialprogenitor cells of normal adult macaque, mLEPCs), the length that comprises the liver organization fritter digestion time in the sepn process, the concentration of digestive ferment, the rat mouse tail collagen bed board of culture plate, selecting of the preparation of cell mass and individual cells agglomerate, the combination of various compositions in the nutrient solution, and the key links such as flow cytometry sorting of E-cad+ cell.The present invention can provide condition for the separation and Culture of the adult hepatic precursor cells of each species of primates and the research of mechanism of cell differentiation, the more important thing is, because macaque and people's high similarity, utilize same technology, may separate the people's hepatic precursor cells that obtains being similar to macaque, thereby make cell replacement treatment hepatic diseases in late period become possibility.
The inventive method is simple, and required equipment all is the equipment of Cell Culture Lab indispensability, is easy to promote.Experimental studies results shows, method of the present invention can be separated effectively and obtained macaque adult hepatic precursor cells, and this precursor cell can be the various kinds of cell that comprises liver cell and bile duct cell in vitro differentiation.This precursor cell is transplanted to the liver injury mouse, can participate in the hepatic tissue regeneration of liver injury mouse, and can be divided into functional liver cell.
Embodiment
Embodiment 1:
Get the macaque adult hepatic tissues of 5 grams,, be cut into small pieces,, collect the cell in the supernatant liquor, give a baby a bath on the third day after its birth inferior and centrifugal 5 minutes with 1200 rev/mins speed with DMEM with the DMEM digestion tissue block of the IV Collagen Type VI enzyme that contains 5mg/ml 30 minutes with PBS washing by soaking 3 times.The cell suspension collected in the DMEM of 10% foetal calf serum, is placed the centrifuge tube suspension culture of sealing again, to form cell mass.At microscopically, one cell mass is chosen, and be inoculated in the culture plate of rat mouse tail collagen bed board.Treat cell after covering with on the culture plate, 37 ℃ of digestion 10-30 minute, the cell that digestion obtains obtained macaque adult hepatic precursor cells with flow cytometry sorting E-cad+ cell with trypsin-EDTA.
In the aforesaid method, rat mouse tail collagen preparation method sees (Malhi, 2002).
Cell culture fluid contains 10mmol/L nicotinamide, 1x ITS, and 100U/ml penicillin, the DMEM/F12 (3: 1) of 100 μ g/ml streptomycin adds 10%FBS, 50ng/mL Urogastron, 10ng/ml pHGF.
Embodiment 2:
Get the macaque adult hepatic tissue of about 8 grams, use PBS washing by soaking 3 times, be cut into small pieces, in 37 ℃ shaking table, with the DMEM of the IV Collagen Type VI enzyme that contains 5mg/ml digestion tissue block 50 minutes, collect the cell in the supernatant liquor, give a baby a bath on the third day after its birth time and centrifugal 5 minutes with DMEM with 1200 rev/mins speed.In the DMEM of 10% foetal calf serum, the centrifuge tube that places sealing again is 37 ℃ shaking table suspension culture, to form cell mass with the cell suspension collected.At microscopically, one cell mass is chosen, and be inoculated in the culture plate of rat mouse tail collagen bed board.Cell culture condition is 37 ℃, 5% CO
2, changed one time nutrient solution in per two days.Treat cell after covering with on the culture plate, 37 ℃ of digestion 10-30 minute, the cell that digestion obtains obtained macaque adult hepatic precursor cells with flow cytometry sorting E-cad+ cell with trypsin-EDTA.
Rat mouse tail collagen preparation method, cell culture fluid are with embodiment 1.
Embodiment 3:
With separating the macaque adult hepatic precursor cells that the obtains cell culture fluid of embodiment 1, on the culture plate of rat mouse tail collagen bed board, cultivate, can go down to posterity the growth amplification by 1: 2 or more than 1: 3 time.
Claims (10)
1. the separation of a macaque adult hepatic precursor cells and cultural method may further comprise the steps:
(1) gets the macaque adult hepatic tissue that 5-10 restrains, use PBS washing by soaking 3 times, be cut into small pieces, use the DMEM of the IV Collagen Type VI enzyme that contains 5mg/ml to digest tissue block 30～60 minutes;
(2) collect cell in the supernatant liquor, give a baby a bath on the third day after its birth time back with centrifugal 5 minutes of 1200 rev/mins of speed with DMEM;
(3) cell of collecting is placed the sealing centrifuge tube suspension culture of 10% foetal calf serum DMEM, to form cell mass;
(4) choose one cell mass, be inoculated in the culture plate of rat mouse tail collagen bed board;
(5) cell culture fluid contains 10mmol/L nicotinami de, 1x I TS, 100U/mlpenicillin, the DMEM/F12 (3: 1) of 100 μ g/ml streptomycin, add 10%FBS, 50ng/mL Urogastron EGF, 10ng/ml pHGF HGF;
(6) treat cell after covering with on the culture plate, 37 ℃ of digestion 10～30 minutes,, collect macaque adult hepatic precursor cells with flow cytometry sorting E-cad+ cell with t ryps in-EDTA.
2. the separation of macaque adult hepatic precursor cells according to claim 1 and cultural method is characterized in that cell culture condition is 37 ℃, 5% CO
2, changed one time nutrient solution in per two days.
3. the separation of macaque adult hepatic precursor cells according to claim 1 and 2 and cultural method is characterized in that step (1) in 37 ℃ shaking table, use the DMEM of the IV Collagen Type VI enzyme that contains 5mg/ml to digest tissue block 30 minutes.
4. the separation of macaque adult hepatic precursor cells according to claim 1 and 2 and cultural method is characterized in that cell suspending liquid suspension culture in 37 ℃ shaking table in the middle centrifuge tube that seals of step (3).
5. the separation of macaque adult hepatic precursor cells according to claim 3 and cultural method is characterized in that cell suspending liquid suspension culture in 37 ℃ shaking table in the middle centrifuge tube that seals of step (3).
6. according to the separation and the cultural method of claim 1,2 described macaque adult hepatic precursor cells, it is characterized in that the nutrient solution of described macaque adult hepatic precursor cells in the step (5), on the culture plate of rat mouse tail collagen bed board, cultivate, can go down to posterity by 1: 2 or more than 1: 3 time.
7. the separation of macaque adult hepatic precursor cells according to claim 3 and cultural method, it is characterized in that the nutrient solution of described macaque adult hepatic precursor cells in the step (5), on the culture plate of rat mouse tail collagen bed board, cultivate, can go down to posterity by 1: 2 or more than 1: 3 time.
8. the separation of macaque adult hepatic precursor cells according to claim 4 and cultural method, it is characterized in that the nutrient solution of described macaque adult hepatic precursor cells in the step (5), on the culture plate of rat mouse tail collagen bed board, cultivate, can go down to posterity by 1: 2 or more than 1: 3 time.
9. the separation of macaque adult hepatic precursor cells according to claim 5 and cultural method, it is characterized in that the nutrient solution of described macaque adult hepatic precursor cells in the step (5), on the culture plate of rat mouse tail collagen bed board, cultivate, can go down to posterity by 1: 2 or more than 1: 3 time.
10. the separation of macaque adult hepatic precursor cells according to claim 6 and cultural method, it is characterized in that the nutrient solution of described macaque adult hepatic precursor cells in the step (5), on the culture plate of rat mouse tail collagen bed board, cultivate, can go down to posterity by 1: 2 or more than 1: 3 time.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952776A (en) * | 2012-11-05 | 2013-03-06 | 浙江省医学科学院 | Culture method for eriones unguiculatus primary liver cells |
| CN103031270A (en) * | 2013-01-05 | 2013-04-10 | 绍兴文理学院 | Efficient amplifying and culturing method for biliary epithelial cells |
| CN104293731A (en) * | 2014-10-13 | 2015-01-21 | 中国水产科学研究院淡水渔业研究中心 | Separation culture method of primary hepatocyte of jian carp |
-
2010
- 2010-06-12 CN CN 201010198776 patent/CN101864394A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 《Cell Research》 20090120 Ji Weizhi 等 Isolation and characterization of liver epithelial progenitorcells from normal adult rhesus monkeys 第268-270页 1-3 第19卷, 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102952776A (en) * | 2012-11-05 | 2013-03-06 | 浙江省医学科学院 | Culture method for eriones unguiculatus primary liver cells |
| CN102952776B (en) * | 2012-11-05 | 2014-06-18 | 浙江省医学科学院 | Culture method for eriones unguiculatus primary liver cells |
| CN103031270A (en) * | 2013-01-05 | 2013-04-10 | 绍兴文理学院 | Efficient amplifying and culturing method for biliary epithelial cells |
| CN104293731A (en) * | 2014-10-13 | 2015-01-21 | 中国水产科学研究院淡水渔业研究中心 | Separation culture method of primary hepatocyte of jian carp |
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Application publication date: 20101020 |