Method for extracting subtotipotent stem cells from placental fetal face chorion

Abstract

The invention relates to a method for extracting sub-totipotent stem cells from a chorion of a fetal surface of a placenta. The method comprises the steps of removing an amnion and stagnated blood from the placenta, and then repetitively washing the surface of the placenta to perform sterilization; shearing the chorion of the fetal surface in a glass utensil, removing placenta lobule tissues remained on the surface as much as possible, and shearing the chorion into small blocks; washing small tissue blocks by using a screen and a great amount of normal saline, and removing residual blood cells; performing tissue digestion: performing oscillatory digestion in a constant temperature shaker by using mixed enzymes; adding a proper amount of FBS for termination after digestion, performing filtration through a filter screen, and adding a great amount of normal saline to wash filter residues to obtain cells as many as possible; performing centrifugation, abandoning supernatant, adding saline water for washing and performing centrifugation to obtain mononuclear cells; performing magnetic bead sorting (OCT-4 positive, Nanog positive and STRO-1 negative) to obtain target cells. The invention further relates to the sub-totipotent stem cells obtained by adopting the method and pharmaceutical use thereof. The sub-totipotent stem cells have excellent characteristics.

Description

technical field

[0001] The present invention relates to the field of stem cells, relates to a method for subtotipotent stem cells derived from placenta fetal chorion, and also relates to a method for extracting subtotipotent stem cells from placenta fetal chorion, and the present invention further relates to the production of the subtotipotent stem cells use. The resulting sub-totipotent stem cells can be further expanded. Background technique

[0002] Mammalian organisms are made up of trillions of cells. Cells are the basic structural and functional units of the human body, which are differentiated from stem cells, so stem cells are the origin cells of various tissues and organs in the human body. Stem cells are characterized by self-replication and multilineage differentiation. During normal reproductive physiology, stem cells are differentiated from fertilized eggs. The fertilized egg initially develops into primitive embryonic stem cells, which have the full potenti...

Examples

Embodiment 1

[0086] Example 1. Subpotent stem cells were extracted from placental fetal face chorion, and detected and amplified

[0087] (1) Take out the human placenta from the collection box and place it in a white porcelain plate, after removing the amniotic membrane and blood stasis, rinse the surface of the placenta repeatedly with physiological saline (sterile, the same below) for disinfection;

[0088] (2) Transfer to a new white porcelain plate, cut the fetal chorion and put it in a 100mm glass dish, try to remove the remaining placental lobular tissue on the surface, and cut it to 0.5-1.5mm 3 Left and right (1mm in this example 3 left and right) small pieces;

[0089] (3) Using a 300-mesh sieve, rinse the small tissue pieces with a large amount of normal saline to remove residual blood cells;

[0090] (4) Tissue digestion: Use 1 to 2 times (1.5 times in this case) tissue volume of mixed enzymes (0.1mg / ml type I collagenase, 0.1mg / ml type II collagenase, 0.1mg / ml hyaluronic ac...

Embodiment 2

[0098] Example 2. Subpotent stem cells were extracted from the placental fetal face chorion, and detected and amplified

[0099] (1) Take out the human placenta from the collection box and place it in a white porcelain plate, after removing the amniotic membrane and blood stasis, rinse the surface of the placenta repeatedly with physiological saline (sterile, the same below) for disinfection;

[0100] (2) Transfer to a new white porcelain plate, cut the fetal chorion and put it in a 100mm glass dish, try to remove the remaining placental lobular tissue on the surface, and cut it to 0.5mm 3 left and right small pieces;

[0101] (3) Using a 300-mesh sieve, rinse the small tissue pieces with a large amount of normal saline to remove residual blood cells;

[0102] (4) Tissue digestion: Use a mixed enzyme (0.1 mg / ml type I collagenase, 0.1 mg / ml type II collagenase, 0.1 mg / ml hyaluronidase and 0.05 mg / ml neutral enzyme) of 1 tissue volume Protease, which also added 0.01mol / L ci...

Embodiment 3

[0109] Example 3. Subpotent stem cells were extracted from placental fetal face chorion, and detected and amplified

[0110] (1) Take out the human placenta from the collection box and place it in a white porcelain plate, after removing the amniotic membrane and blood stasis, rinse the surface of the placenta repeatedly with physiological saline (sterile, the same below) for disinfection;

[0111] (2) Transfer to a new white porcelain plate, cut the fetal chorion and put it in a 100mm glass dish, try to remove the residual placental lobular tissue on the surface, and cut it to 1.5mm 3 left and right small pieces;

[0112] (3) Using a 300-mesh sieve, rinse the small tissue pieces with a large amount of normal saline to remove residual blood cells;

[0113] (4) Tissue digestion: use 2 times the tissue volume of mixed enzymes (0.1mg / ml type I collagenase, 0.1mg / ml type II collagenase, 0.1mg / ml hyaluronidase and 0.05mg / ml neutral Protease, which also added 0.03mol / L citric aci...

technical field

[0001] The present invention relates to the field of stem cells, relates to a method for subtotipotent stem cells derived from placenta fetal chorion, and also relates to a method for extracting subtotipotent stem cells from placenta fetal chorion, and the present invention further relates to the production of the subtotipotent stem cells use. The resulting sub-totipotent stem cells can be further expanded. Background technique

[0002] Mammalian organisms are made up of trillions of cells. Cells are the basic structural and functional units of the human body, which are differentiated from stem cells, so stem cells are the origin cells of various tissues and organs in the human body. Stem cells are characterized by self-replication and multilineage differentiation. During normal reproductive physiology, stem cells are differentiated from fertilized eggs. The fertilized egg initially develops into primitive embryonic stem cells, which have the full potenti...

Examples

Embodiment 1

[0086] Example 1. Subpotent stem cells were extracted from placental fetal face chorion, and detected and amplified

[0087] (1) Take out the human placenta from the collection box and place it in a white porcelain plate, after removing the amniotic membrane and blood stasis, rinse the surface of the placenta repeatedly with physiological saline (sterile, the same below) for disinfection;

[0088] (2) Transfer to a new white porcelain plate, cut the fetal chorion and put it in a 100mm glass dish, try to remove the remaining placental lobular tissue on the surface, and cut it to 0.5-1.5mm 3 Left and right (1mm in this example 3 left and right) small pieces;

[0089] (3) Using a 300-mesh sieve, rinse the small tissue pieces with a large amount of normal saline to remove residual blood cells;

[0090] (4) Tissue digestion: Use 1 to 2 times (1.5 times in this case) tissue volume of mixed enzymes (0.1mg / ml type I collagenase, 0.1mg / ml type II collagenase, 0.1mg / ml hyaluronic ac...

Embodiment 2

[0098] Example 2. Subpotent stem cells were extracted from the placental fetal face chorion, and detected and amplified

[0099] (1) Take out the human placenta from the collection box and place it in a white porcelain plate, after removing the amniotic membrane and blood stasis, rinse the surface of the placenta repeatedly with physiological saline (sterile, the same below) for disinfection;

[0100] (2) Transfer to a new white porcelain plate, cut the fetal chorion and put it in a 100mm glass dish, try to remove the remaining placental lobular tissue on the surface, and cut it to 0.5mm 3 left and right small pieces;

[0101] (3) Using a 300-mesh sieve, rinse the small tissue pieces with a large amount of normal saline to remove residual blood cells;

[0102] (4) Tissue digestion: Use a mixed enzyme (0.1 mg / ml type I collagenase, 0.1 mg / ml type II collagenase, 0.1 mg / ml hyaluronidase and 0.05 mg / ml neutral enzyme) of 1 tissue volume Protease, which also added 0.01mol / L ci...

Embodiment 3

[0109] Example 3. Subpotent stem cells were extracted from placental fetal face chorion, and detected and amplified

[0110] (1) Take out the human placenta from the collection box and place it in a white porcelain plate, after removing the amniotic membrane and blood stasis, rinse the surface of the placenta repeatedly with physiological saline (sterile, the same below) for disinfection;

[0111] (2) Transfer to a new white porcelain plate, cut the fetal chorion and put it in a 100mm glass dish, try to remove the residual placental lobular tissue on the surface, and cut it to 1.5mm 3 left and right small pieces;

[0112] (3) Using a 300-mesh sieve, rinse the small tissue pieces with a large amount of normal saline to remove residual blood cells;

[0113] (4) Tissue digestion: use 2 times the tissue volume of mixed enzymes (0.1mg / ml type I collagenase, 0.1mg / ml type II collagenase, 0.1mg / ml hyaluronidase and 0.05mg / ml neutral Protease, which also added 0.03mol / L citric aci...

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