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Method for preparing and preserving umbilical arterial and vein vascular peripheral stem cells

A technology of venous blood vessels and umbilical cord blood vessels, applied in the field of bioengineering, can solve the problems of increasing the difficulty of umbilical cord processing, damage to cells, long cycle, etc., to avoid allergic reactions and cross-infection, strong osteogenic differentiation ability, and reduce cell damage. Effect

Active Publication Date: 2016-06-22
THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0007] (1) After the umbilical cord is collected, the umbilical cord and arterial blood coagulate, which increases the difficulty of umbilical cord treatment, and at the same time, too many blood cells increase the chance of stem cell contamination;
[0008] (2) The tissue attachment method is to cut the umbilical cord into small tissue pieces and directly attach them to the culture medium. Generally, primary cells can be obtained in about 15 days; the disadvantage of this method is that the cycle is long; It loses the ability to grow cells, reduces the number of cells, and the cells are not pure enough;
[0009] (3) Enzyme digestion mostly uses collagenase and trypsin together. Although the cycle is short, the cost is high and the conditions are not easy to grasp. Long digestion time at room temperature may damage the cells. cells, and increases the safety risk of allergic reactions and cross-infection caused by animal-derived proteins during clinical application

Method used

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  • Method for preparing and preserving umbilical arterial and vein vascular peripheral stem cells
  • Method for preparing and preserving umbilical arterial and vein vascular peripheral stem cells
  • Method for preparing and preserving umbilical arterial and vein vascular peripheral stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0028] (1) Under sterile conditions, take the umbilical cord of a newborn baby immediately after vaginal delivery from a healthy puerpera, about 30cm long, squeeze out the blood in the umbilical cord vessel, and put it into a container containing 5% blue chain double antibody and 1% heparin Store in a wide-mouthed bottle of PBS at 4°C, and isolate and culture umbilical cord mesenchymal stem cells within 4 hours;

[0029] (2) Take out the umbilical cord of the newborn, cut off the part with clamp marks and hematoma, squeeze out the blood in the umbilical cord, and trim the two sections with scissors. Use ophthalmic scissors to cut the umbilical cord longitudinally, use forceps to bluntly separate the venous blood vessels and arterial blood vessels, and pay attention to remove the tissue around the adventitia of the blood vessels that is close to the blood vessels;

[0030] (3) The umbilical artery and umbilical vein were washed with preheated PBS to completely remove the blood ...

Embodiment 2

[0038] (1) Under aseptic conditions, take the umbilical cord of a newborn baby immediately after the cesarean section of a healthy puerpera, about 40cm long, squeeze out the blood in the umbilical cord vessel, and put it into a container containing 5% blue chain double antibody and 1% heparin in advance Store in a wide-mouthed bottle of PBS at 4°C, and isolate and culture umbilical cord mesenchymal stem cells within 4 hours;

[0039] (2) Take out the umbilical cord of the newborn, cut off the part with clamp marks and hematoma, squeeze out the blood in the umbilical cord, and trim the two sections with scissors. Use ophthalmic scissors to cut the umbilical cord longitudinally, use forceps to bluntly separate the venous blood vessels and arterial blood vessels, and pay attention to remove the tissue around the adventitia of the blood vessels that is close to the blood vessels;

[0040] (3) The umbilical artery and umbilical vein were washed with preheated PBS to completely remo...

Embodiment 3

[0048] (1) Under aseptic conditions, take the umbilical cord of a newborn baby immediately after delivery from a healthy puerpera. Store in a jar of salt-buffered saline PBS at 4°C, and isolate and culture umbilical cord mesenchymal stem cells within 4 hours;

[0049] (2) Take out the umbilical cord of the newborn, cut off the part with clamp marks and hematoma, squeeze out the blood in the umbilical cord, and trim the two sections with scissors. Use ophthalmic scissors to cut the umbilical cord longitudinally, use forceps to bluntly separate the venous blood vessels and arterial blood vessels, and pay attention to remove the tissue around the adventitia of the blood vessels that is close to the blood vessels;

[0050] (3) The umbilical artery and umbilical vein were washed with preheated PBS to completely remove the blood stains. Use scissors to cut the umbilical vein and umbilical artery into small sections of about 0.5 cm, arrange them neatly in a 100 mm culture dish, and ...

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Abstract

The invention discloses a method for preparing and preserving umbilical arterial and vein vascular peripheral stem cells. The umbilical arterial and vein vascular peripheral stem cells are cultured by a cell culture fluid (DMEM low glucose, 10% of fetal calf serum and 1% of penicillin-streptomycin double anti-body). The operation is carried out under strict aseptic conditions; and after umbilical cord collection, the inconvenience caused by blood coagulation is effectively avoided by use of an anticoagulant. Compared with a conventional coating method, the method disclosed by the invention has the advantages that umbilical perivascular sourced mesenchymal stem cells with higher purity are obtained; allergic reaction and cross infection caused by animal-based protein can be avoided since digestive enzymes are not adopted; and the umbilical perivascular stem cells separated by a non-zymogen digestion method have higher positive rate than that of the Wharton's jelly sourced mesenchymal stem cells.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for preparing and preserving mesenchymal stem cells in umbilical cord blood vessel walls, and isolating mesenchymal stem cells from perivascular tissues. Background technique [0002] In recent years, it has been recognized at home and abroad that adult stem cells can be used as seed cells for tissue engineering, cell therapy or gene therapy, but there are still major obstacles to the application of adult stem cells such as immune rejection of allogeneic transplantation, difficult sources, and difficult control of in vitro expansion. The key to application is to isolate and expand adult stem cells to provide sufficient transplantable cells. [0003] Mesenchymal stem cells are a kind of adult stem cells, derived from the mesoderm in early development, and have attracted much attention because of their high self-renewal, immune regulation and multi-linea...

Claims

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Application Information

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IPC IPC(8): C12N5/0775A01N1/02
CPCA01N1/0221C12N5/0665
Inventor 丁利军孙海翔徐璐
Owner THE AFFILIATED DRUM TOWER HOSPITAL MEDICAL SCHOOL OF NANJING UNIV
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