Human amniotic epithelial cell as well as isolated culture method and application thereof

An epithelial cell, separation and culture technology, applied in cell dissociation methods, embryonic cells, animal cells, etc., can solve the problem of inability to industrially prepare high-purity amniotic epithelial cells, achieve stable properties, improve enzymatic hydrolysis treatment efficiency, and purity. high effect

Pending Publication Date: 2016-12-21
吉林省中科生物工程股份有限公司
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The first object of the present invention is to provide a method for isolating and culturing human amniotic membrane epithelial cells. In the method, the amniotic membrane epithelial cells are extracted, separated and purified from the placenta, and then expanded and cultur

Method used

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  • Human amniotic epithelial cell as well as isolated culture method and application thereof
  • Human amniotic epithelial cell as well as isolated culture method and application thereof
  • Human amniotic epithelial cell as well as isolated culture method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0062] (1) Under sterile conditions, the amniotic membrane was peeled off from the placental tissue by mechanical means, and then washed several times with HBSS buffer solution containing 3% double antibody to remove residual blood;

[0063] (2) Shred the washed amnion tissue, add trypsin, and digest in a 37°C water bath for 3 times, each time for 30 minutes;

[0064] (3) Pass the digested liquid obtained after digestion through a 300-mesh sieve to remove undigested tissue, and collect the filtered cell filtrate; then centrifuge the cell filtrate at 1500 rpm for 10 min;

[0065] (4) Resuspend the pelleted cells obtained after centrifugation with low-sugar DMEM medium, and 6 The cell density was seeded in a 10cm culture dish; and the culture medium was placed in 37°C CO 2 In the incubator, after culturing for 48 hours, the figure 1 The medium of the indicated cell morphology, and replaced with a new low-glucose DMEM medium;

[0066] (5) if figure 2 As shown, when the confl...

experiment example 1

[0068] Flow cytometry detection

[0069] Collect the human amniotic membrane epithelial cells obtained by filtration and centrifugation after digestion in step (3) of Example 1, and collect about 10 6 cells;

[0070] The collected cells were washed twice with PBS buffer solution, and diluted appropriately; then loaded on the flow cytometer for detection;

[0071] After the detection, software was used to analyze the expression of five cell surface molecules including CD73, CD90, CD44, CD45, and CD105 on human amniotic epithelial cells, and the analysis results were as follows: Figure 5 shown;

[0072] further based on Figure 5 The analysis results shown were integrated and calculated to obtain the expression of the five cell surface molecules in each generation. The results are as follows:

[0073] CD73 expression:

[0074] population

[0075] CD90 expression:

[0076] population

[0077] CD105 expression:

[0078] population

[0079] CD4...

experiment example 2

[0087] Experimental Study on Adipogenic and Osteogenic Differentiation

[0088] Stem cells have the potential for self-replication and multiple differentiations. So far, people have successively isolated mesenchymal stem cells from umbilical cord, placenta, bone marrow, fat, skin, hair follicle and other tissues. The separation methods mainly include planting method and enzyme digestion method. Different methods and experimental procedures have great differences in the quality and purity of the extracted stem cells. Therefore, the stem cell differentiation experiment is the universally recognized stem cell identification standard in the world.

[0089] The specific detection method is as follows:

[0090] a, the P3 generation amniotic epithelial cells in the embodiment 1 step (5) were mixed with 1×10 5 ml -1 Inoculate in a 12-well plate at a density of 70% to 80%, add adipogenic induction solution, and have control wells. Change the medium twice a week, and after 14-18 d...

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Abstract

The invention provides a human amniotic epithelial cell, as well as an isolated culture method and application thereof. In the method, the amniotic epithelial cell can be simply and conveniently obtained on a large scale by extracting and isolating the amniotic epithelial cell from a placenta, purifying the amniotic epithelial cell and then carrying out enlarged multiplication culture in vitro. Meanwhile, the human amniotic epithelial cell obtained through culture has high purity, can be used in treatment of diseases, such as diabetes mellitus, and can be used for effectively controlling the blood glucose levels of patients.

Description

technical field [0001] The invention relates to the field of cell isolation and culture, in particular to a human amniotic epithelial cell, a method for isolation and culture, and an application thereof. Background technique [0002] Diabetes is a common chronic lifelong disease caused by metabolic disorders of the endocrine system. Long-term illness can lead to chronic damage to the nervous system, cardiovascular system, urinary system and other systems. [0003] With the development of my country's economy and the improvement of people's living standards, the prevalence of diabetes has increased year by year, seriously endangering people's health and bringing a heavy economic burden to families and society. The latest large-sample epidemiological survey shows that the prevalence of diabetes in Chinese adults is 11.6%, among which the prevalence rate of men is 12.1%, that of women is 11%, that of urban residents is 14.3%, and that of rural residents is 10.3%; the survey res...

Claims

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Application Information

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IPC IPC(8): C12N5/073A61K35/50A61P3/10
CPCA61K35/50C12N5/0605C12N2509/00
Inventor 张璐邢邵良王红艳贾琳
Owner 吉林省中科生物工程股份有限公司
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