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Tissue engineering lung tissue and construction method thereof

A tissue engineering and construction method technology, applied in artificial cell constructs, animal cells, vertebrate cells, etc., to achieve the effects of strong practicability, promotion of development, and simple operation

Active Publication Date: 2012-05-30
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of microcapsules in lung tissue engineering, especially sodium alginate-polylysine-sodium alginate (APA) microcapsules / barium alginate-polylysine-sodium alginate (BPA) microcapsules Capsules as Scaffolds for Tissue-Engineered Lungs Have Not Been Reported

Method used

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  • Tissue engineering lung tissue and construction method thereof
  • Tissue engineering lung tissue and construction method thereof
  • Tissue engineering lung tissue and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Construction of tissue engineered lung tissue using mouse fetal lung cells as seed cells

[0050] (1) Isolation, culture and identification of mouse fetal lung cells

[0051] Fetal lungs were derived from fetal mice of Kunming mice with an embryonic stage of 18 days. The fetal lung was separated according to the conventional method, then washed in 1×PBS, cut into pieces and digested in 0.25% trypsin-EDTA digestion solution for 20-25 minutes at room temperature. Then add 2 volumes of H-DMEM medium containing 10% FBS to stop the enzyme activity, and then use a pipette to blow and grind. The tissue homogenate was filtered with a 70 μm nylon filter and centrifuged at 800 rpm for 5 minutes. The cell pellet was resuspended in 900 μl of distilled water for 30-45 seconds, the red blood cells were removed by hypotonicity, and then 100 μl of 10×PBS was added. The cells were pelleted by centrifugation again, resuspended in a certain volume of H-DMEM medium containing 10% F...

Embodiment 2

[0061] Example 2 Construction of tissue engineered lung tissue using lung progenitor cells obtained by inducing mouse embryonic stem cells as seed cells

[0062] (1) Isolation, culture and differentiation of mouse embryonic stem cells into lung progenitor cells

[0063] (a) Isolation and culture of trophoblast cells, that is, primary mouse embryonic fibroblasts (MEF): Kunming pregnant mice were sacrificed, soaked in 75% alcohol for 30 seconds, and the uterus was taken out and placed in 100-mm PBS Remove the embryos from the tissue culture dish, remove the extra-embryonic tissues, and rinse with PBS to remove blood. Use sterile forceps to remove the embryo head and internal organs, transfer the embryo to a 60-mm tissue culture dish, and cut it into 1mm 3 The left and right tissue blocks are then transferred to the culture flask and washed with PBS. Pour out PBS, add appropriate amount of trypsin solution for digestion, after repeated pipetting and shaking, let it stand for a while,...

Embodiment 3

[0070] Example 3 Histological and immunohistochemical detection of tissue engineered lung tissue constructed in vitro

[0071] HE staining: fix the cell-scaffold complex cultured in vitro for 7 days using the method of Example 1 with 4% paraformaldehyde solution, dehydrate with alcohol with concentration gradient, embed in paraffin, and prepare slices with a thickness of 4 μm. Conventional HE staining, light microscope observation, from Figure 4 It can be seen that the cell activity is good and can form an alveolar-like tissue structure.

[0072] Immunohistochemical staining: The cell-scaffold complexes cultured in vitro for 7 days using the method of Example 1 were paraffin-embedded, sliced ​​with a thickness of 4 μm, and deparaffinized. After microwave repair, the slices were rinsed with PBS for 5min×3 times, in H 2 O 2 Incubate for 10min, rinse with PBS for 5min×3 times, and act on goat blocking serum for 20min. Vimentin antibody (1:1000) and SpC (1:1200) were used as the prim...

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Abstract

The invention discloses a tissue engineering lung tissue and a construction method thereof, belonging to the field of tissue engineering and regenerative medicine. The tissue engineering lung tissue is obtained with the following construction method: (1) preparing lung cell suspension; (2) preparing sodium alginate-polylysine-alginate microcapsules; (3) compounding cells with holder material, andcultivating cell and holder composite. The invention adopts sodium alginate-polylysine-alginate microcapsules and combines liquid state I type collagen with Matrigel matrigel to serve as the holder of tissue engineering lungs; the holder system can effectively accelerate the formation of the alveolus pulmonis type structure to realize the exchange of air and nutritive materials, so that the invention has important significance for clinically applying tissue engineering lung tissues to treat patients with lung diseases in the future. The construction method of the invention has simple technology and moderate application condition.

Description

Technical field [0001] The invention belongs to the field of tissue engineering and regenerative medicine, and particularly relates to a tissue engineered lung tissue and a construction method thereof. Background technique [0002] Tissue engineering is an emerging cross-discipline developed in the late 1980s. In recent years, with the development of life sciences, material sciences, and engineering sciences, researchers have successively focused on bone, cartilage, skin, breast, and nerves. Breakthroughs have been made in the research of tissue and organ reconstruction in vitro, among which some tissue engineering products such as cartilage and skin have been commercialized. Due to the complex tissue structure of the lung itself, coupled with the late start of research on tissue engineered lungs, the current tissue engineered lung tissue constructed in vitro is far from being commercialized, but the construction of tissue engineered lung tissue in vitro is a study The basis of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
Inventor 王常勇张文君邱丽媛王海滨段翠密周瑾
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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