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Culture method and application of primary amniotic stem cells

A culture method and stem cell technology, which is applied to animal cells, embryonic cells, vertebrate cells, etc., can solve the problems of low cell purity, difficulty in cell attachment, and complicated removal, so as to accelerate cell attachment, shorten culture time, and improve cell The effect of improving the purity

Pending Publication Date: 2022-07-01
SHENZHEN WINGOR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the existing amniotic membrane stem cell culture method is difficult to adhere to the wall due to the thin amniotic membrane tissue, and the slow digestion process of the tissue makes it more complicated to remove. Finally, the culture time of amniotic membrane stem cells is about 20 days, and during the cell culture process Even if it is cultured in a sterile environment, a small amount of bacterial contamination cannot be avoided, resulting in low purity of cultured cells

Method used

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  • Culture method and application of primary amniotic stem cells
  • Culture method and application of primary amniotic stem cells
  • Culture method and application of primary amniotic stem cells

Examples

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Embodiment 1

[0026] Example 1 A method for culturing primary amniotic stem cells

[0027] The culture method of the primary amniotic stem cells, the preparation process is as follows:

[0028] S1. Under sterile conditions, the amniotic membrane was peeled off from the placenta, washed three times with normal saline to remove blood capillaries and viscous substances on the amniotic membrane, and then cut into 6cm×6cm tissue pieces in a petri dish. Place the smooth side down to obtain a preliminarily processed amniotic tissue block;

[0029] S2. Place the preliminarily processed amniotic tissue pieces obtained in step S1 in a 90mm petri dish, place a small piece of tissue in each petri dish, and then mix trypsin, chymosin and xylanase in a weight ratio of 8: Mix well in a ratio of 2:1 to prepare digestive enzymes, add the digestive enzymes to each petri dish, place them at 23 °C for 28 min at an added amount of 2.5 g / L, and then wash with normal saline twice to prepare Digested amniotic ti...

Embodiment 2

[0032] Example 2 A method for culturing primary amniotic stem cells

[0033] The culture method of the primary amniotic stem cells, the preparation process is as follows:

[0034] S1. Under sterile conditions, the amniotic membrane was peeled off from the placenta, washed 5 times with normal saline to remove blood capillaries and viscous substances on the amniotic membrane, and then cut into 6cm×6cm tissue pieces in a petri dish. Place the smooth side down to obtain a preliminarily processed amniotic tissue block;

[0035] S2. Place the preliminarily processed amniotic tissue pieces obtained in step S1 in a 90mm petri dish, place a small piece of tissue in each petri dish, and then mix trypsin, chymosin and xylanase in a weight ratio of 15: The digestive enzymes were mixed in a ratio of 4:3 to make digestive enzymes. The digestive enzymes were added to each petri dish, placed at 27 °C for 32 min at 2.5 g / L, and then washed 4 times with normal saline to prepare the digested en...

Embodiment 3

[0038] Example 3 A method for culturing primary amniotic stem cells

[0039] The culture method of the primary amniotic stem cells, the preparation process is as follows:

[0040] S1. Under sterile conditions, the amniotic membrane was peeled off from the placenta, washed 4 times with normal saline to remove blood capillaries and viscous substances on the amniotic membrane, and then cut into 6cm×6cm tissue pieces in a petri dish. Place the smooth side down to obtain a preliminarily processed amniotic tissue block;

[0041] S2. Place the preliminarily processed amniotic tissue pieces obtained in step S1 in a 90 mm petri dish, place a small piece of tissue in each petri dish, and then mix trypsin, chymosin and xylanase in a weight ratio of 12: The digestive enzymes were mixed in a ratio of 3:2 to make digestive enzymes. The digestive enzymes were added to each petri dish, placed at 25 °C for 30 min at 2.5 g / L, and then washed with physiological saline for 3 times. the amniotic...

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Abstract

The invention belongs to the technical field of stem cell culture, and particularly relates to a culture method and application of primary amniotic stem cells. The culture method of the primary amniotic stem cells mainly comprises the following steps: stripping a placenta under a sterile condition, washing with normal saline, washing with a mixed solution containing a bactericide, treating with digestive enzyme, culturing with a culture medium and the like. The mixed solution containing the bactericide is prepared from the following components in percentage by weight: 1 to 2 percent of herba taraxaci extract, 0.5 to 0.8 percent of rosemary hydrolat, 0.3 to 0.8 percent of vitamin C and 96.4 to 98.2 percent of purified water; the culture medium is composed of an LB liquid culture medium, tween-20, type IV collagen and BI recombinant vitreous adhesion protein. By adopting the culture method provided by the invention, cell adherence can be accelerated, tissues in the cells can be cleaned up, and the cell purity is improved on the premise of shortening the culture time of the amniotic stem cells.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a culture method of primary amniotic stem cells and application thereof. Background technique [0002] Human amniotic membrane-derived stem cells include human amniotic epithelial cells (humanamniotic epithelial cells, hAEC) and human amniotic mesenchymal stem cells (human amnioticmes-enchymalstromal cells, hAMSC). Among them, human amniotic epithelial cells have the characteristics of expressing a variety of embryonic stem cell markers and have a more comprehensive multi-directional differentiation potential. hMSCs have stronger proliferation ability and stem cell characteristics than bone marrow mesenchymal stem cells. Therefore, due to its totipotency, amniotic stem cells are often used in various cytology and molecular biology research processes. [0003] However, the existing amniotic membrane stem cell culture method is difficult to adhere to due to t...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/071C12N5/073
CPCC12N5/0668C12N5/0605C12N5/0625C12N2533/54C12N2533/52C12N2509/00C12N2509/10
Inventor 谢亮姜舒张芸纪惜銮
Owner SHENZHEN WINGOR BIO TECH
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