CN105483077A - Cell culture method for improving hepatocyte proliferation activity - Google Patents
Cell culture method for improving hepatocyte proliferation activity Download PDFInfo
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- CN105483077A CN105483077A CN201510982296.6A CN201510982296A CN105483077A CN 105483077 A CN105483077 A CN 105483077A CN 201510982296 A CN201510982296 A CN 201510982296A CN 105483077 A CN105483077 A CN 105483077A
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- 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
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
- C12N2502/1352—Mesenchymal stem cells
- C12N2502/1382—Adipose-derived stem cells [ADSC], adipose stromal stem cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/28—Vascular endothelial cells
Abstract
The invention relates to a cell culture method for improving the hepatocyte proliferation activity. The method comprises the following step: co-culturing hepatocyte, vascular endothelial cells and adipose-derived stem cells in a co-culture medium for 3 days or over 3 days according to a ratio of 14: (2 to 4): (2 to 4). The cell culture method disclosed by the invention has the beneficial effect mainly reflected in that a real structure and function of hepatocyte in vivo can be well simulated by adopting the cell culture method, and the activity of the cultured hepatocyte can well maintain the function and activity of the hepatocyte in vitro.
Description
(1) technical field
The present invention relates to a kind of cell culture processes improving hepatocyte proliferation activity.
(2) background technology
Hepatopathy is the common disease of human body, the especially health of the liver failure serious harm mankind, and liver transplantation is treatment is caused liver distribution effective ways by hepatitis, liver cirrhosis etc., but the in short supply of donor liver cannot meet clinical needs.Liver tissue engineering is exactly progressively formed in this context a kind of by the subject built for the purpose of transplantable liver, and it will bring hope for liver regeneration.The core of liver tissue engineering sets up a kind of cell and the biomaterial three-dimensional composite with vitality, the liver organization of disease damage carried out to the reconstruction of form, structure and fuction, and the permanence realizing liver substitutes.
Hepatocellular three-dimensional vitro culture, it is the main method of liver tissue engineering, usually the dimensional culture of liver cell spheroid is carried out using Poly(D,L-lactide-co-glycolide (PLGA), sodium alginate, chitosan etc. as timbering material, though the method can maintain the external longer function of liver cell with active, differ greatly with the real structure of liver in body.Cell in conjunction with liver organization forms, and Cell Co culturing Techenique, by non-liver cell and liver cell Dual culture, liver cell microenvironment in vivo can better be simulated, contribute to improving hepatocellular function with active.
Angiogenesis is the Major Difficulties of liver tissue engineering research.Fat stem cell is the one of mescenchymal stem cell (MSCs), have draw materials easily, source is sufficient, cylinder storage amount is large, without advantages such as immunological rejections.Research shows, after fat stem cell and endotheliocyte Dual culture, pericyte can be divided into, can promote the angiogenesis of endotheliocyte, and this is applied in that animal body is inside and outside to be all confirmed, and illustrates that fat stem cell can be urged to build angiogenesis (MaJ to a certain extent, YangF, BothSK, etal.InvitroandinvivoangiogeniccapacityofBM-MSCs/HUVECsa ndAT-MSCs/HUVECscocultures.Biofabrication.2014,6 (1): 015005; TakebeT, EnomuraM, YoshizawaE, etal.VascularizedandComplexOrganBudsfromDiverseTissuesvi aMesenchymalCell-DrivenCondensation.CellStemCell.2015,16 (5): 556-65.).On the other hand, fat stem cell is also ideal seed cell, because it has very strong self-renewal capacity and multi-lineage potential (as to hepatocyte differentiation), may be used for treating various End-stage liver disease as the (ZhangY such as fatty liver, liver cirrhosis, ChenXM, SunDL.Effectsofcoencapsulationofhepatocyteswithadipose-d erivedstemcellsinthetreatmentofratswithacute-on-chronicl iverfailure.IntJArtifOrgans.2014,37:133-141; SaitoY, ShimadaM, UtsunomiyaT, etal:Theprotectiveeffectofadipose-derivedstemcellsagains tliverinjurybytrophicmolecules.JSurgRes.2013,180:162-168; HarnHJ, LinSZ, HungSH, etal:Adipose-derivedstemcellscanabrogatechemical-induced liverfibrosisandfacilitaterecoveryofliverfunction.CellTr ansplant.2012,21:2753-2764.).Therefore, by fat stem cell, endotheliocyte and liver cell Dual culture, the difficult problem solving liver tissue engineering vascularization further will be contributed to.
In sum, develop a kind of energy enhance hepatocyte function and active cell culture processes, there is great application prospect.
(3) summary of the invention
The object of the invention be to provide a kind of can the cell culture processes of enhance hepatocyte proliferation activity.
The technical solution used in the present invention is:
A kind of cell culture processes improving hepatocyte proliferation activity, described method comprises: by liver cell and vascular endothelial cell, fat stem cell cell according to the ratio (number ratio) of 14:2 ~ 4:2 ~ 4, Dual culture more than 3 days in Dual culture substratum, cultivates the cell obtained and can be further used for treatment or prevention; Described Dual culture substratum is composed as follows: 10% foetal calf serum, 100IU/mL penicillin, 100 μ g/mL Streptomycin sulphates, 50 μMs of dexamethasone, 10ng/mL people source pHGF, and 20ng/mL people source endothelial cell growth factor (ECGF), solvent is 1640 substratum.Cell culture condition is regular growth culture condition, 37 DEG C, 5%CO
2, change liquid every other day.Penicillin and Streptomycin sulphate are generally buy dual anti-mixing solutions, wherein containing penicillin 10000IU/mL and Streptomycin sulphate 10000 μ g/mL, add in substratum during use by 1% volumetric usage.
Preferably, described liver cell is LO2 cell strain (people liver immortalized cell line), described vascular endothelial cell is HUVECs cell strain (human umbilical vein endothelial cell strain), and described fat stem cell cell is human adipose-derived stem cell, and proportions is 14:3:3.
Concrete, described method is as follows:
(1) when liver cell LO2 cell strain adopts perfect medium cultivation to grow to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs, 0.25% trysinization is after 2 minutes, add equal-volume perfect medium to stop, centrifugal, collecting cell, adds Dual culture substratum, and adjustment cell solubility is 2.8 × 10
6individual/mL, obtains hepatocyte cell liquid;
(2) when vascular endothelial cell HUVECs cell strain adopts perfect medium cultivation to grow to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs, 0.25% trysinization is after 2 minutes, add equal-volume perfect medium to stop, centrifugal, collecting cell, adds Dual culture substratum, and adjustment cell solubility is 0.6 × 10
6individual/mL, obtains vascular endothelial cell enchylema;
(3) when human adipose-derived stem cell adopts perfect medium cultivation to grow to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs, 0.25% trysinization is after 2 minutes, add equal-volume perfect medium to stop, centrifugal, collecting cell, adds Dual culture substratum, and adjustment cell solubility is 0.6 × 10
6individual/mL, obtains human adipose-derived stem cell enchylema; In step (1) ~ (3), described perfect medium is composed as follows: 10% foetal calf serum, 100IU/mL penicillin, 100 μ g/mL Streptomycin sulphates, and solvent is 1640 substratum; Described Dual culture substratum is composed as follows: 10% foetal calf serum, 100IU/mL penicillin, 100 μ g/mL Streptomycin sulphates, 50 μMs of dexamethasone, 10ng/mL people source pHGF, 20ng/mL people source endothelial cell growth factor (ECGF), and solvent is 1640 substratum;
(4) by after aforementioned liver cell, vascular endothelial cell, the mixing of fat stem cell enchylema equal-volume, be inoculated in the Dual culture substratum in 6 orifice plates, 37 DEG C, 5%CO
2dual culture 3 ~ 7 days, changes liquid every other day.
Preferably, described step (4) every hole inoculating cell adds up to 0.4 × 10
6individual, namely the cell density of LO2 cell, HUVECs cell, fat stem cell is respectively 2.8 × 10
5individual/hole, 0.6 × 10
5individual/hole, 0.6 × 10
5individual/hole.
In order to microenvironment in better analogue body, the present invention selects the vascular endothelial cell and the liver cell Dual culture that account for larger proportion in vivo in non-liver cell, is more conducive to hepatocellular growth.
In order to improve the angiogenesis function of vascular endothelial cell, the present invention selects fat stem cell and vascular endothelial cell, the liver cell Dual culture that can promote vascular endothelial cell angiogenesis, for vascularization liver tissue engineering provides possibility.
Beneficial effect of the present invention is mainly reflected in: adopt cell culture processes of the present invention, can better simulate liver cell real structure and function in vivo, and the hepatocyte activity of turning out can better maintain liver cell function in vitro with active.
(4) accompanying drawing explanation
Fig. 1 is the activity of cell conditioned medium serum lactic dehydrogenase (LDH) after liver cell and vascular endothelial cell, fat stem cell Dual culture.
Fig. 2 is the content of cell conditioned medium urea (Urea) after liver cell and vascular endothelial cell, fat stem cell Dual culture.
Fig. 3 is the content of cell conditioned medium albumin (ALB) after liver cell and vascular endothelial cell, fat stem cell Dual culture.
Fig. 4 is after liver cell and vascular endothelial cell, fat stem cell Dual culture, the expression of liver cell albumin (ALB).A figure is the immunofluorescence picture that liver cell ALB expresses; B is the picture quantitative result that liver cell ALB expresses.
Fig. 5 is after liver cell and vascular endothelial cell, fat stem cell Dual culture, the expression of hepatocyte cell chromo-oxidase (CYP450) subfamily 3A4 (CYP3A4).A figure is the immunofluorescence picture that liver cell CYP3A4 expresses; B is the picture quantitative result that liver cell CYP3A4 expresses.
Fig. 6 is after liver cell and vascular endothelial cell, fat stem cell Dual culture, the expression of liver cell Hepatocyte nuclear factor 4-alpha (HNF-4a).A figure is the immunofluorescence picture that liver cell HNF-4a expresses; B is the picture quantitative result that liver cell HNF-4a expresses.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Reagent source in embodiment: 1640 substratum (Gibco, LifeTechnologies, USA), foetal calf serum (FBS, Gibco, LifeTechnologies, USA), penicillin/streptomycin dual anti-(Gibco, LifeTechnologies, USA), dexamethasone (Sigma-Aldrich, USA), people source endothelial cell growth factor (ECGF) (BD, USA), people source pHGF (BD, USA).
Substratum forms:
Perfect medium: 10% foetal calf serum, 1% penicillin/streptomycin is dual anti-, and solvent is 1640 substratum;
Dual culture substratum: 10% foetal calf serum, 1% penicillin/streptomycin is dual anti-, 50 μMs of dexamethasone, 10ng/mL people source pHGF, 20ng/mL people source endothelial cell growth factor (ECGF), and solvent is 1640 substratum.
Embodiment 1:
Liver cell (LO2 cell strain, purchased from Shanghai Inst. of Life Science, CAS cellular resources center) adopt perfect medium to cultivate when growing to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs 1 time, 0.25% trysinization 2 minutes; Add equal-volume to stop, after 300g is centrifugal 5 minutes, collecting cell, and add Dual culture substratum, adjustment cell solubility is 2.8 × 10
6individual/mL.
Embodiment 2:
Vascular endothelial cell (HUVECs, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's preclinical medicine cell centre) adopt perfect medium to cultivate when growing to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs 1 time, 0.25% trysinization 2 minutes; Add equal-volume perfect medium to stop, after 300g is centrifugal 5 minutes, collecting cell, and add Dual culture substratum, adjustment cell solubility is 0.6 × 10
6individual/mL.
Embodiment 3:
When human adipose-derived stem cell (the American Type Culture Collection council of Chinese Academy of Sciences Kunming cell bank) adopts perfect medium cultivation to grow to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs 1 time, 0.25% trysinization 2 minutes; Add equal-volume perfect medium to stop, after 300g is centrifugal 5 minutes, collecting cell, and add Dual culture substratum, adjustment cell solubility is 0.6 × 10
6individual/mL.
Embodiment 4:
By aforementioned liver cell, vascular endothelial cell, the mixing of fat stem cell enchylema equal-volume, be inoculated in the 2mL Dual culture substratum in 6 orifice plates, every porocyte adds up to 0.4 × 10
6individual, 37 DEG C, 5%CO
2continuous Dual culture 3 ~ 7 days, changes liquid every other day.
Hepatocellular structure and fuction after the method detection Dual culture such as use ultraviolet-visible spectrophotometer (UV-Vis), laser confocal microscope, concrete test result is as follows:
(1) lactate dehydrogenase activity
Collect the cell conditioned medium of after Dual culture 1 day, 3 days respectively, and adopt serum lactic dehydrogenase (LDH) detection kit (Japanese colleague's chemistry institute (Shanghai)) to detect.
The results are shown in Figure 1, Dual culture after 3 days the LDH content of cell conditioned medium obviously reduce, illustrate that the liver cell of Dual culture has good function of detoxification, it is active better.
(2) urea synthesis
Collect the cell conditioned medium of after Dual culture 1 day, 3 days, 5 days, 7 days respectively, and adopt urea (Urea) detection kit (Biovision, (U.S.)) to detect.
Result is see Fig. 2, and after Dual culture, the urea content of cell conditioned medium is obviously many than independent hepatocellular supernatant, illustrates that Dual culture can promote that liver cell synthesizes more diuresis element.
(3) albumin secretion
Collect the cell conditioned medium of after Dual culture 1 day, 3 days, 5 days, 7 days respectively, and adopt ELISA detection kit (Bioengineering Research Institute is built up in Nanjing, (Nanjing)) to detect.
Result see Fig. 3, Dual culture after 5 days emiocytosis albumin amount obviously many than independent hepatocellular supernatant, illustrate that Dual culture can improve the function of hepatocytes secrete albumin.
(4) liver cell Expression of Albumin situation detects
Adopt the empty plasmid transfection vascular endothelial cell of slow virus (carrying Ds-red), for label vascular endotheliocyte; The empty plasmid transfected hepatocytes of slow virus (carrying EGFP), for marking liver cell.Then after 3 days, the method for immunofluorescence is adopted to detect the expression of liver cell ALB in the ratio Dual culture of 14:3:3 the vascular endothelial cell of the liver cell of transfection, transfection, fat stem cell.
Result is see Fig. 4, and figure A is the immunofluorescence picture that ALB expresses, and after Dual culture, hepatocellular quantity increases, and the expression of its ALB also increases; Figure B is the quantitative result of ALB expression and liver cell quantity.Show that Dual culture can promote the activity of hepatocellular propagation and secretion ALB.
(5) Terminal oxidase expression detects
Adopt the empty plasmid transfection vascular endothelial cell of slow virus (carrying Ds-red), for label vascular endotheliocyte; The empty plasmid transfected hepatocytes of slow virus (carrying EGFP), for marking liver cell.Then after 3 days, the method for immunofluorescence is adopted to detect the expression of liver cell CYP3A4 in the ratio Dual culture of 14:3:3 the vascular endothelial cell of the liver cell of transfection, transfection, fat stem cell.
Result is see Fig. 5, and figure A is the immunofluorescence picture that CYP3A4 expresses, and after Dual culture, hepatocellular quantity increases, and the expression of its CYP3A4 also increases; Figure B is the quantitative result of CYP3A4 expression and liver cell quantity.Show that Dual culture can promote the function of hepatocellular propagation and metabolic detoxification.
(6) Hepatocyte nuclear factor 4-alpha expression detects
Adopt the empty plasmid transfection vascular endothelial cell of slow virus (carrying Ds-red), for label vascular endotheliocyte; The empty plasmid transfected hepatocytes of slow virus (carrying EGFP), for marking liver cell.Then after 3 days, the method for immunofluorescence is adopted to detect the expression of liver cell HNF-4a in the ratio Dual culture of 14:3:3 the vascular endothelial cell of the liver cell of transfection, transfection, fat stem cell.
Result is see Fig. 6, and figure A is the immunofluorescence picture that HNF-4a expresses, and after Dual culture, hepatocellular quantity increases, and the expression of its HNF-4a also increases; Figure B is the quantitative result of HNF-4a expression and liver cell quantity.Show that Dual culture can promote hepatocellular propagation and activity, be further used for treatment for liver cell or prevent to provide the foundation.
Claims (4)
1. improve a cell culture processes for hepatocyte proliferation activity, described method comprises: by liver cell and vascular endothelial cell, fat stem cell cell according to the ratio of 14:2 ~ 4:2 ~ 4, Dual culture more than 3 days in Dual culture substratum; Described Dual culture substratum is composed as follows: 10% foetal calf serum, 100IU/mL penicillin, 100 μ g/mL Streptomycin sulphates, 50 μMs of dexamethasone, 10ng/mL people source pHGF, and 20ng/mL people source endothelial cell growth factor (ECGF), solvent is 1640 substratum.
2. the method for claim 1, it is characterized in that described liver cell is LO2 cell strain, described vascular endothelial cell is HUVECs cell strain, and described fat stem cell cell is human adipose-derived stem cell, and proportions is 14:3:3.
3. the method for claim 1, is characterized in that described method is as follows:
(1) when liver cell LO2 cell strain adopts perfect medium cultivation to grow to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs, 0.25% trysinization is after 2 minutes, add equal-volume perfect medium to stop, centrifugal, collecting cell, adds Dual culture substratum, and adjustment cell solubility is 2.8 × 10
6individual/mL, obtains hepatocyte cell liquid;
(2) when vascular endothelial cell HUVECs cell strain adopts perfect medium cultivation to grow to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs, 0.25% trysinization is after 2 minutes, add equal-volume perfect medium to stop, centrifugal, collecting cell, adds Dual culture substratum, and adjustment cell solubility is 0.6 × 10
6individual/mL, obtains vascular endothelial cell enchylema;
(3) when human adipose-derived stem cell adopts perfect medium cultivation to grow to 80% ~ 90% degrees of fusion, remove perfect medium, 0.01MPBS washs, 0.25% trysinization is after 2 minutes, add equal-volume perfect medium to stop, centrifugal, collecting cell, adds Dual culture substratum, and adjustment cell solubility is 0.6 × 10
6individual/mL, obtains human adipose-derived stem cell enchylema;
In step (1) ~ (3), described perfect medium is composed as follows: 10% foetal calf serum, 100IU/mL penicillin, 100 μ g/mL Streptomycin sulphates, and solvent is 1640 substratum; Described Dual culture substratum is composed as follows: 10% foetal calf serum, 100IU/mL penicillin, 100 μ g/mL Streptomycin sulphates, 50 μMs of dexamethasone, 10ng/mL people source pHGF, 20ng/mL people source endothelial cell growth factor (ECGF), and solvent is 1640 substratum;
(4) by after aforementioned liver cell, vascular endothelial cell, the mixing of fat stem cell enchylema equal-volume, be inoculated in the Dual culture substratum in 6 orifice plates, 37 DEG C, 5%CO
2dual culture 3 ~ 7 days, changes liquid every other day.
4. method as claimed in claim 3, is characterized in that described step (4) every hole inoculating cell adds up to 0.4 × 10
6individual.
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Cited By (3)
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| WO2018036119A1 (en) * | 2015-11-19 | 2018-03-01 | 中国人民解放军第二军医大学 | Method for in-vitro induction of ductal metaplasia of primary hepatocyte and for long-term cultivation, proliferation and differentiation thereof, and application thereof |
| CN109136185A (en) * | 2017-06-28 | 2019-01-04 | 中国科学院生物物理研究所 | The preparation method and application of one type brain organ device |
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