Construction method for tissue engineering liver unit
A tissue engineering and construction method technology, applied in the field of biomedicine
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Embodiment 1
[0046] 1. Isolation, culture and identification of bone marrow-derived mesenchymal stem cells (BMSCs)
[0047] 1) Bone marrow source: it can be bone marrow in a bone marrow bank, bone marrow squeezed out from discarded bone during surgery, or obtained through bone marrow aspiration. Bone marrow aspiration is a mature and safe method for obtaining bone marrow tissue widely used clinically, so bone marrow aspiration can be used as a special source of the present invention.
[0048] 2) Add an appropriate amount of DMEM culture solution to the obtained bone marrow fluid, mix well, filter with a 200-mesh sieve, and take the under-sieve component;
[0049] 3) Equilibrium density centrifugation, discard the supernatant; resuspend the cells with DMEM medium to make a bone marrow cell suspension;
[0050] 4) Add the cell suspension dropwise on the liquid surface of Percoll separation solution with a specific gravity of 1.073 to carry out density gradient centrifugation, take the monon...
Embodiment 2
[0070] Example 2: Inducing BMSCs to differentiate and identify vascular endothelial cells (EC) in vitro
[0071] 1) Preparation of conditioned medium: 10 ng / ml VEGF (R&D system), 2 ng / ml bFGF (R&D system), 1×ITS (Sigma), nicotinamide 0.61 g / L, dexamethasone were added to low-sugar DMEM containing 2% fetal bovine serum. Methone 0.1μmoL / L, Bovine Serum Albumin (BSA) 2g / L, Ornithine 0.1g / L, Proline 0.03g / L, Glutamine 0.73g / L, Glucose 1g / L, Galactose 2g / L L and appropriate amount of trace elements (zinc chloride, zinc sulfate and manganese chloride), etc.;
[0072] 2)P 2 -P 9 Substituted BMSCs were resuspended in conditioned medium, at 2×10 4 / cm 2 Inoculate in a 96-well plate pre-coated with Matrigel (diluted at a ratio of 1:3), add 200ul conditioned medium to each well, and change the medium every 3 days; 3) Optical microscope observation: dynamic observation of BMSC induction 1d, 3d, 5d , 7d, 14d, 21d cell morphology changes. See results figure 1 , wherein, a) MSC100×; ...
Embodiment 3
[0090] Example 3: Induction and identification of bone marrow mesenchymal stem cells to hepatocyte-like cells
[0091] The reagents and antibodies used in the experiment are: mouse anti-human AFP monoclonal antibody, rabbit anti-human Alb antibody (Santa Cruz); fluorescently labeled goat anti-rabbit IgG, anti-mouse IgG (Beijing Zhongshan Jinqiao Company); , ICG) (Liaoyang Third Pharmaceutical Factory); the rest are the same as before.
[0092] 1. To induce BMSCs to differentiate into hepatocytes in vitro: P 2 -P 9 BMSCs were resuspended in conditioned medium, at 5×10 3 / cm 2 Inoculate in a 96-well plate pre-coated with Matrigel (diluted at a ratio of 1:3), and change the medium every 3 days for induction culture. Dynamically observe the morphological changes of BMSCs 3d, 5d, and 7d after induction, see Figure 4 , 3 days after induction, the elongated spindle cells gradually became shorter and thicker, and became polygonal or hepatocyte-like round cells. 5-7 days after i...
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