Method for culturing human amniotic mesenchymal stem cells by enzyme digestion and EGF (epidermal growth factor)

A technique of stromal stem cells and enzymatic digestion is applied in the field of culturing human amniotic mesenchymal stem cells, and the enzymatic digestion method combined with epidermal growth factor is used to cultivate human amniotic mesenchymal stem cells. Long time and other problems, to achieve the effect of simple, fast, and effective separation and purification methods

Inactive Publication Date: 2013-04-03
陆华
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Problems solved by technology

The primary culture of hAMSCs is divided into tissue block culture and enzyme digestion method. As not every tissue block can be successfully cultured in tissue block culture, and it is easy to be mixed with fibroblast contamination, it takes a long time to form a monolayer in culture, which is not conducive to rapid A large number of cells are obtained, which is difficult to meet the needs of stem cell research; in view of the compact structure of the amniotic membrane tissue and the structure of the enzyme digestion method, too long digestion time with enzymes will cause great damage to the cells, and the number of cells obtained if the time is too short is too small

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  • Method for culturing human amniotic mesenchymal stem cells by enzyme digestion and EGF (epidermal growth factor)

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Embodiment Construction

[0014] like figure 1 As shown, the method for culturing human amniotic mesenchymal stem cells by enzymatic digestion combined with epidermal growth factor (EGF) in an embodiment of the present invention comprises the following steps:

[0015] S1. Isolation of human amniotic mesenchymal stem cells (hAMSCs):

[0016] The fresh placenta discarded after delivery was taken under aseptic conditions (with the consent of the family members), the amniotic membrane was peeled off from the placental tissue by mechanical method, washed several times with D-Hank's solution to remove residual blood, and the rinsed amniotic membrane was cut with ophthalmic scissors. About 1mm3 fragments, add 2.5g / L trypsin to digest at 370C for 10min; add DMEM containing 5% calf serum to stop the digestion, and gently pipette and mix well, then filter through a 200-mesh cell sieve; add 1.0g / L trypsin to the filtered amnion tissue L type II collagenase digestion solution, digest at 370C for 0.5h; add DMEM co...

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Abstract

The invention provides a method for culturing human amniotic mesenchymal stem cells (hAMSCs) by enzyme digestion and EGF (epidermal growth factor). The method comprises the following steps of hAMSCs separation, hAMSCs primary culture, hAMSCs sub-culture and amplification, hAMSCs cryopreservation and recovery, and detection of hAMSCs cytometric immunophenotyping. According to the method, amniotic extracells are collected by digesting trypsin and collagenase for multiple times step by step, and adherent culture and digestion time control are combined by addition of 4ng / ml EGF, so that human amniotic mesenchymal stem cells (hAMSCs) with high purity and high activity can be obtained. The enzyme digestion and EGF adopted in the method have the advantages of simpleness in operation, quickness and practicality and the like, so that the method is capable of effectively separating and purifying hAMSCs.

Description

technical field [0001] The invention relates to a method for cultivating human amniotic mesenchymal stem cells (hAMSCs) in the field of biotechnology, in particular to a method for cultivating human amniotic mesenchymal stem cells by using an enzyme digestion method combined with epidermal growth factor (EGF). Background technique [0002] The tissue structure of normal human amniotic membrane is divided into epithelial cell layer, basement membrane, compact layer, fibroblast layer and spongy layer, among which human amnion mesenchymal stem cells (human amnion membrane mesenchymal stem cells, hAMSCs) are located in the innermost layer of amniotic membrane. Developed from the ectodermal leaves on the 8th day after, it is possible to maintain the plasticity of embryonic stem cells in the early stage of gastrulation and have greater multi-directional differentiation potential. hAMSCs can theoretically differentiate into various tissue cells. It is convenient and avoids ethical ...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 陆华
Owner 陆华
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