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Separation method for human amniotic mesenchymal stem cells

A technology of stem cells and separation method, applied in the field of separation of human amniotic mesenchymal stem cells and culture of human amniotic mesenchymal stem cells, can solve the problems of small number of cells, difficult to meet stem cell research, long time, etc., and achieve simple and fast operation. , the effect of effective separation and purification methods

Inactive Publication Date: 2014-05-14
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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  • Separation method for human amniotic mesenchymal stem cells
  • Separation method for human amniotic mesenchymal stem cells

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Embodiment Construction

[0015] Such as 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:

[0016] S1. Isolation of human amniotic mesenchymal stem cells (hAMSCs):

[0017] see figure 2 As shown, this step is specifically as follows: take the fresh placenta discarded after delivery under sterile conditions (with the consent of the family members), use mechanical methods to peel off the amniotic membrane from the placenta tissue, rinse with D-Hank's solution several times to remove residual blood, and rinse the placenta. The final amniotic membrane was cut into about 1mm3 fragments with ophthalmic scissors, and 2.5g / L trypsin was added to digest at 370C for 10 minutes; DMEM containing 5% calf serum was added to stop the digestion, and the 200-mesh cell sieve was filtered after being gently blown and mixed; Add 1.0g / L type II colla...

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Abstract

The invention provides a separation method for human amniotic mesenchymal stem cells (hAMSCs) in a process of cultivating the hAMSCs through enzyme digestion and epidermal growth factor (EGF). The cultivating process comprises the steps of separation of hAMSCs, primary culture of hAMSCs, hAMSCs subculturing and amplification, hAMSCs cryopreservation and recovery, and hAMSCs immunophenotyping detection. According to the method disclosed by the invention, step-by-step digestion is implemented for multiple times through trypsin and collagenase to collect amnion cells, and adherent culture and digestion time control are combined through adding of 4ng / ml of EGF (Epidermal Growth Factor), so as to obtain human amniotic mesenchymal stem cells (hAMSCs) which are relatively high in both purity and activity. The method disclosed by the invention, which combines enzyme digestion and EGF, has the advantages of being simple to operate, rapid, practical and the like, being an effective method for 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 using an enzyme digestion method combined with epidermal growth factor (EGF). Isolation method of amniotic mesenchymal stem cells. 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 ...

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 陆华
Owner 陆华
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