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
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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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