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Method of preparing hepatic cells by using human minor salivary gland epithelial stem/progenitor cells and application of method

A technology of epithelial cells and minor salivary glands, applied in artificial cell constructs, non-embryonic pluripotent stem cells, artificially induced pluripotent cells, etc. The effect of strong amplification ability, wide distribution and hidden donor area

Inactive Publication Date: 2016-01-06
赵振民
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the shortage of donor sites, difficulties in collection, etc. severely limit the acquisition of cells, and the lack of appropriate cell culture solutions for storage and activity maintenance of donor site cells also constitutes an obvious obstacle to the application of this technology.

Method used

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  • Method of preparing hepatic cells by using human minor salivary gland epithelial stem/progenitor cells and application of method
  • Method of preparing hepatic cells by using human minor salivary gland epithelial stem/progenitor cells and application of method
  • Method of preparing hepatic cells by using human minor salivary gland epithelial stem/progenitor cells and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Materials: Human minor salivary glands are derived from the minor salivary gland tissues of patients who need to be resected during clinical operations. After surgical resection, they are placed in centrifuge tubes pre-added with complete medium and stored at 4°C.

[0044] Pretreatment and cutting of minor salivary gland tissue: transfer the tissue to a petri dish, use small scissors to remove excess connective tissue, separate the complete minor salivary gland, absorb all the liquid in the petri dish, and use ophthalmic scissors to cut it into approximately 1mm 3 of pieces.

[0045] In the process of cutting the minor salivary gland tissue in the pretreatment, after the tissue is transferred to a petri dish, the culture solution of thistle extract can also be added to the petri dish, soaked for 10 minutes, and the excess connective tissue is removed with small scissors, and the obtained Complete minor salivary glands, suck all the liquid in the petri dish, and cut it ...

Embodiment 2

[0072] Example 2: Cell Hepatogenic Induction

[0073] Matrigel preparation: use the night before inoculating cells into the Matrigel3D culture system, dissolve Matrigel at -20°C in a 4°C environment. On the second day, Matrigel was dissolved into a liquid state and operated in an ice box. All experimental equipment and consumables used had to be pre-cooled at 4°C before use. Mix Matrigel and epithelial cell culture medium at a ratio of 2 to 1, add to a 24-well cell culture plate, 350ul per well, and spread out. Place it in a 37°C cell culture incubator. After about 30 minutes, Matrigel solidifies into a gel and can be used for seeding cells.

[0074] Cell preparation: The primary cultured epithelial stem / progenitor cells from small salivary gland tissue were expanded and cultured to P4 for hepatogenic induction experiments. When the P3 cells reached 80-90% confluence, the original medium in the culture flask was aspirated, the cells were washed twice with 1×PBS, and the PBS ...

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Abstract

The invention belongs to the technical field of preparation of tissue engineering products and relates to a method of preparing hepatic cells by using human minor salivary gland epithelial stem / progenitor cells and application of the method. The method includes: subjecting human minor salivary gland tissues to in-vitro separation and cultivation to obtain epithelial stem / progenitor cells, subjecting the cells to further cultivation and amplification in an epithelial cell culture medium, performing induced differentiation through a flat culture or three-dimensional culture system in a hepatic induced culture medium containing growth factors so as to obtain hepatocyte-like cells and cholangiocyte-like cells. The method has the advantages that material sources are wide, taking by incision is easy, wounds are minor, oral donor sites are hidden, the prepared hepatocyte-like cells and cholangiocyte-like cells are applicable to the study on Physiological function or pathological changes of the liver cells, the establishment of a cell model for liver diseases, in-vitro drug testing, in-vitro toxicity testing, the establishment of in-vitro bio-engineered liver-like organs, and the treatment of liver diseases by in-vivo hepatocellular transplantation.

Description

technical field [0001] The invention belongs to the technical field of preparation of tissue engineering products, in particular to a method and application for preparing hepatocyte-like cells and cholangiocyte-like cells by using human minor salivary gland epithelial stem / progenitor cells. Background technique [0002] Liver transplantation is one of the effective methods for the treatment of advanced liver disease, but many problems such as lack of donor sources, high surgical costs, and lifelong use of immunosuppressants after surgery restrict the clinical application of this technology. Therefore, by culturing, inducing, and differentiating stem cells into hepatocyte-like cells for cell transplantation, or as seed cells for bioengineered liver organoids, stem cells provide new treatment methods and technologies for the treatment of liver diseases, as well as for drug metabolism and Toxicity testing provides new methods and techniques. However, the shortage of donor site...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/074
Inventor 赵振民张辰张翔宇吕璘韩婷璐童海州刘磊杜水果
Owner 赵振民
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