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Construction method for tissue engineering liver unit and tissue engineering liver unit

A technology of tissue engineering and construction method, which is applied in the field of biomedicine, and can solve the problems that large-scale vascularized tissues cannot be produced, further deepening is needed, and in vitro culture is easy to lose activity and function.

Active Publication Date: 2008-03-26
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main problems it faces are: the lack of regenerative functional seed cells that are immune compatible with patients; the lack of ideal biological scaffolds in terms of mechanics, biology and chemistry; vascularized tissue, etc.
[0004] Mature hepatocytes have many deficiencies and cannot be a satisfactory source of cells, such as the difficulty of obtaining hepatocytes from liver failure, and the possibility of immune rejection of allogeneic hepatocytes , In vitro culture is easy to lose activity and function, etc.
At present, the research on the source of seed cells for liver tissue engineering mainly focuses on embryonic stem cells (ESC) and adult stem cells (ASC). Although both have their own advantages, the application of ESC still has ethical and moral , legal disputes; the current culture system for inducing differentiation of ESCs into hepatic cell lines is still immature, and its basic research and application research need to be further deepened

Method used

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  • Construction method for tissue engineering liver unit and tissue engineering liver unit
  • Construction method for tissue engineering liver unit and tissue engineering liver unit
  • Construction method for tissue engineering liver unit and tissue engineering liver unit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Isolation, culture and identification of bone marrow-derived mesenchymal stem cells (BMSCs)

[0046] 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.

[0047] 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;

[0048] 3) Equilibrium density centrifugation, discard the supernatant; resuspend the cells with DMEM medium to make a bone marrow cell suspension;

[0049] 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

[0069] Example 2: Inducing BMSCs to differentiate and identify vascular endothelial cells (EC) in vitro

[0070] 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.;

[0071] 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 of conditioned medium to each well, and change the medium every 3 days;

[0072] 3) Observation by optical microscope: dynamic observation of cell morphological changes on 1d, 3d, 5d, 7d, 14d, and 21d after induction of BMSCs. See Figure 1...

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, 3d after induction, the elongated spindle cells gradually become shorter and thicker, and become polygonal or hepatocyte-like round cells. 5-7 days after inducti...

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Abstract

The present invention discloses one kind of tissue engineering liver unit and its construction process. The tissue engineering liver unit containing vascular endothelial cell and liver cell is prepared through in vitro pre-introducing liver cell and vascular endothelial cell with mesenchyme stem cell, separate combining with albumen glue containing induction factor and filling to the inner wall and outer wall of one tubular rack, coating with one outer enclosure to form a cell complex, and further culturing the cell complex in culture liquid. The present invention obtains great amount of seed cells for liver tissue engineering with mesenchyme stem cell and combines the seed cells with proper bioactive material to obtain auxiliary liver function structure unit. The tissue engineering liver unit may be transplanted to body of hepatic failure patient to compensate and replace liver function. The present invention provides one new treating way.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular to a method for inducing differentiation of stem cells to hepatic parenchymal cells and vascular endothelial cells in vitro, and combining the differentiated cells with biological materials to jointly construct a tissue-engineered liver unit. The method can not only obtain a large number of seed cells for liver tissue engineering to prepare auxiliary functional structural units for hepatic insufficiency, but also can be used as a basic structure to further construct transplantable bioartificial liver tissues with specific structures and functions. Background technique [0002] The liver is the largest internal organ in the human body. It is responsible for important and complex physiological functions, such as the center of various substance metabolism, secretion of bile, synthesis of proteins and blood coagulation factors, and detoxification, etc. It plays an important role in mai...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N11/00C12N5/071C12N5/0775
Inventor 裴雪涛王韫芳粱峰张磊林峰岳慧敏南雪颜永年管利东
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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