Method for inducing adipose tissue-derived stromal cells into liver sample cells

A kind of stem cell and cell induction technology, applied in the field of artificial liver, can solve the problem of long induction time, and achieve the effect of short induction time, shortened induction time and good induction effect

Inactive Publication Date: 2016-12-07
GENERAL HOSPITAL OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of the above-mentioned induction schemes take 2 weeks, and the time to induce hepatoblast-like cells is longer

Method used

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  • Method for inducing adipose tissue-derived stromal cells into liver sample cells

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Experimental program
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Effect test

Embodiment 1

[0048] 1. Isolation, extraction and primary culture of mouse ADSCs:

[0049] (1) 5 BALB / c male 3-day-old suckling mice were killed by cervical dislocation, soaked in 75% alcohol for disinfection for 3 minutes, and then transferred to an ultra-clean table;

[0050] (2) In an ultra-clean bench, use sterilized ophthalmic scissors and ophthalmic tweezers to peel off the mouse skin from below the umbilical cord, expose the inguinal adipose tissue, completely separate the inguinal adipose tissue, add 5v / v% double-antibody PBS and wash for 3 minutes, Transfer to serum-free medium for treatment;

[0051] (3) Remove the subcutaneous and fascial tissue around the mouse adipose tissue, and use ophthalmic scissors to cut it into pieces less than 1cm 3 , transferred into a 15mL centrifuge tube, added three times the volume of PBS containing 1mg / mL collagenase I, and shaken at room temperature for 1h;

[0052] (4) Equal volume of serum-containing medium to stop digestion, incubate at room...

Embodiment 2

[0065] 1. Isolation, extraction and primary culture of mouse ADSCs:

[0066] Concrete steps are with this step in embodiment 1;

[0067] 2. Subculture and cell culture;

[0068] After 72 hours of inoculation of the primary isolated cells, observe the growth of the cells, and when the cell density reaches 91%, passage at a ratio of 1:3;

[0069] Then inoculate after subculture, take the logarithmic growth of the 4th passage cells, and inoculate 1×10 4 / cm 2 After subculture, inoculate and place in 5v / v% CO 2 , Cultivated in a constant temperature incubator at 37°C; the medium is a medium containing a-MEM+20v / v%FBS;

[0070] 3. Induction culture of hepatoblast-like cells;

[0071] After 24 hours after inoculation to observe cell adhesion, the medium was replaced with hepatic induction medium, and the hepatic induction medium was changed every 4 days; the hepatic induction medium contained 50 ng / mL FGF4, 150 ng / mL HGF, 40 ng The DMEM medium containing 10v / v% FBS of / mL OSM,...

Embodiment 3

[0076] 1. Isolation, extraction and primary culture of mouse ADSCs:

[0077] Concrete steps are with this step in embodiment 1;

[0078] 2. Subculture and cell culture;

[0079] After the primary isolated cells were inoculated for 72 hours, observe the growth of the cells. If the cell density does not reach 90%, change the medium at half the volume, and pass the culture at 1:2.5 after extending the culture for one day;

[0080] Then inoculate after subculture, take the logarithmic growth of the 4th passage cells, and inoculate 1×10 4 / cm 2 After subculture, inoculate and place in 5v / v% CO 2 , Cultivated in a constant temperature incubator at 37°C; the medium is a medium containing DMEM+10v / v% FBS;

[0081] 3. Hepatoblast-like cell induction culture;

[0082] After 24 hours after inoculation to observe cell adhesion, the medium was replaced with hepatogenic induction medium, and the hepatic induction medium was changed every 4 days; the hepatic induction medium contained 3...

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Abstract

The invention relates to a method for inducing adipose tissue-derived stromal cells into liver sample cells. The method comprises the following steps: adopting an inducing system containing various cell factors (HGF, FGF4, OSM, EGF, bFGF and aFGF) and growth factors (Dex, Vc, Vpp and ITS) for inducing the adipose-derived stem cells and acquiring a large amount of liver sample cells with liver functions in a short period of time. The adipose-derived stem cells induced by the induction medium provided by the invention have a polygonal form and can express the liver sample cell markers of ALB, AFP, CK18, CK19 and CYP1A1. Compared with the other inducing system, the inducing system has the advantages that the inducing time is shortened and the inducing rate of the liver sample cells is increased; the adipose-derived stem cells are easily acquired, the cells can multiply in quantity in vitro and can be induced into the liver sample cells, so that the adipose tissue-derived stromal cells are ideal seed cells for cell transplantation and bioartificial liver.

Description

technical field [0001] The invention belongs to the technical field of artificial liver and relates to a method for inducing adipose-derived mesenchymal stem cells into liver-like cells. Background technique [0002] At present, researchers have established a variety of systems to induce mesenchymal stem cells into hepatic-like cells, using a variety of growth factors and nutrients in combination, such as fibroblast growth factor (FGF), hepatocyte growth factor (HGF), insulin transduction Iron selenium (ITS), and dexamethasone (Dex) etc. induce BMMSCs to transform into cells with polygonal morphology and stem cell function. In addition, some chemical factors such as insulin-like growth factor 1, nicotinamide, hepatocyte nuclear factor (HNF-4α) and valproic acid can significantly increase the rate of differentiation of human BMMSCs into hepatic-like cells, as well as enhance the liver-specific expression level. Lu et al. induced BMMSCs with HGF and FGF-4 for seven days, and ...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/0775
CPCC12N5/067C12N2500/25C12N2500/38C12N2501/11C12N2501/113C12N2501/115C12N2501/119C12N2501/12C12N2501/237C12N2501/30C12N2506/1353C12N2506/1369C12N2506/1384
Inventor 阎丽徐丽娟王军王淑芳
Owner GENERAL HOSPITAL OF PLA
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