Complete medium and human amnion-derived mesenchymal stem cell culture method

A technology of complete medium and culture method, applied in the field of culture of complete medium and human amniotic mesenchymal stem cells

Active Publication Date: 2011-09-21
AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the use of autologous hCBS for the cultivation of hAMSCs at home and abroad.

Method used

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  • Complete medium and human amnion-derived mesenchymal stem cell culture method
  • Complete medium and human amnion-derived mesenchymal stem cell culture method
  • Complete medium and human amnion-derived mesenchymal stem cell culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 of the present invention: the preparation of complete culture medium:

[0083] (1) Prepare hCBS first: extract 50-70ml of umbilical cord blood under aseptic conditions, pour it into a sterile glass bottle, and place it in a 37°C incubator for 2 hours. Aspirate the precipitated serum, transfer it into a new sterile centrifuge tube, centrifuge at 1000g at 4°C for 20min, collect 15-20ml of the supernatant, inactivate in a water bath at 56°C for 30min, sterilize with a 0.2μm filter, and distribute the serum into 5ml vials at -20 Store at ℃ for later use; culture and test bacteria at the same time, no bacterial growth is enough.

[0084] (2) Prepare LG-DMEM solution again: take 10g of LG-DMEM (commercially available from Gibco, 10g / bag) and dissolve it in 1000ml of ultrapure water, adjust the pH value to 7.2-7.4, filter and sterilize, subpackage, and store at 4°C Save it for later use.

[0085] (3) Take the LG-DMEM solution prepared in step (2) and the hCBS pr...

Embodiment 2

[0086] Embodiment 2 of the present invention: the culture method of hAMSCs, comprises the following steps:

[0087](1) Separation: Wash the amnion tissue repeatedly with D-Hank's solution to remove the remaining blood, scrape off the blood vessels and mucus on the surface of the amnion, cut the amnion into centrifuge tubes, add 2.5-3 times the volume of 0.05% pancreatic Protease-0.02% EDTA-2Na digestion solution, digest at 37°C for 10 minutes, discard the digestion solution, then rotate at 37°C and 200rpm at a low speed, continue to digest for 20 minutes, filter through a 300-mesh stainless steel mesh, collect tissue fragments in a centrifuge tube; continue to use the same Methods Digestion and filtration were repeated twice; after digestion, the remaining amnion tissue was washed with D-Hank's solution, and 0.75mg / ml type II collagenase-0.075mg / ml Dnase I digestion solution was added, 37°C, 200rpm, rotary digestion for 2h, until the tissue After complete digestion, filter thr...

Embodiment 3

[0090] Embodiment 3 of the present invention: the culture method of hAMSCs, comprises the following steps:

[0091] (1) Separation: Rinse the amnion tissue with D-Hank's solution, cut it into pieces, and put it in a centrifuge tube, add 0.05% trypsin-0.02% EDTA-2Na digestion solution, digest at 37°C for 10 minutes, discard the digestion solution, and then Rotate at 37°C and 200rpm at a low speed, continue to digest for 20 minutes, filter through a 300-mesh stainless steel mesh, collect tissue fragments in a centrifuge tube; continue to repeat digestion and filtration twice in the same way; rinse the remaining amnion tissue with D-Hank's solution after digestion, Add 0.75mg / ml type II collagenase-0.075mg / ml DNase I digestion solution, rotate and digest at 37°C and 200rpm for 2 hours until the tissue is completely digested, filter through a 300-mesh stainless steel mesh, collect the cell filtrate in a centrifuge tube, centrifuge, and discard supernatant.

[0092] (2) Primary cu...

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Abstract

The invention discloses a complete medium and a human amnion-derived mesenchymal stem cell (hAMSCs) culture method. The complete medium is prepared by adding 3 to 10 percent of autologous umbilical cord blood serum into low-sugar Dulbecco minimum essential medium solution according to a volume ratio. The culture method comprises: (1) separation; (2) primary culture; and (3) subculture. The methodusing the complete medium in the hAMSCs culture has the advantages that: the risk of using fetal calf serum is avoided; although the need of adding L-glutamine, non-essential amino acid, 2-mercapitoethanol, pyruvic acid and the like is obviated, the high proliferation properties and phenotypic characteristics of the hAMSCs and expression of multilineage differentiation marker genes sand proteins of some stem cells can still be retained; and in subculture, the wall adherence fastness of the hAMSCs is much lower than that in fetal bovine serum (FBS) culture, the digestion time is reduced obviously, and the damage of trypsinization to cells and loss of cells are reduced.

Description

technical field [0001] The invention relates to a complete culture medium and a culture method of human amniotic mesenchymal stem cells (human amniotic mesenchymalstem cells, hAMSCs), belonging to the technical fields of cell engineering and biomedicine. Background technique [0002] Human amnion is a layer of translucent film attached to the surface of the placental chorion, which is a part of the fetal membrane tissue and evolved from the cytotrophoblast during embryonic development. In addition to human amniotic epithelial cells, hAMSCs are one of the main cell types constituting human amniotic membrane tissue, express embryonic stem cells and mesenchymal stem cells (mesenchymal stem cells, MSCs) markers, and have multi-lineage differentiation potential. MSCs. Under specific induction conditions in vitro, hAMSCs can differentiate into all cells from the three germ layers, including nerve cells, cardiomyocytes, pancreatic cells, and liver cells. Due to its multi-directio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 余丽梅
Owner AFFILIATED HOSPITAL OF ZUNYI MEDICAL COLLEGE
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