A kind of culture method of human umbilical cord mesenchymal stem cells

A culture method and stem cell technology, applied in the field of stem cells, can solve the problems of easy aging of cells, pollution, insufficient cell purity, etc., and achieve the effect of strong cell viability and proliferation ability, short primary generation time, and maintaining shape.

Active Publication Date: 2017-12-26
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Collagenase and other sources used in the enzymatic hydrolysis method may involve animal sources, which are expensive and cause great damage to cells
In the tissue block culture method, it takes a long time for the cells to climb out. The long primary culture time makes the cells easy to grow old, and the purity of the cells is not enough, so they are easily polluted by other cells in the umbilical cord.

Method used

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  • A kind of culture method of human umbilical cord mesenchymal stem cells
  • A kind of culture method of human umbilical cord mesenchymal stem cells
  • A kind of culture method of human umbilical cord mesenchymal stem cells

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Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1, the cultivation of human umbilical cord mesenchymal stem cells

[0038] Under sterile conditions, the umbilical cord tissue discarded after delivery was transported back to the laboratory in PBS containing double antibody. Use 100mL of sterile PBS to clean the blood on the surface of the umbilical cord, disinfect the surface of the umbilical cord with 100mL of 75% ethanol, and then wash it several times with 100mL of PBS to remove the blood as much as possible. Arteries, cut the umbilical cord to a size of 1mm 3 to 3mm 3 Distribute the tissue pieces in the petri dish at an interval of 0.5cm-1cm, let it air dry for 40min, add 0.08mL / cm 2 complete medium at 37°C, 5% CO 2 After static culture in the incubator for 3 days, add 0.08mL / cm 2 After most of the tissue blocks have cells crawling out, remove the tissue blocks, wash several times with PBS, add 0.18mL / cm 2 complete medium. After most of the cell colonies grow into sheets, they can be passaged.

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Embodiment 2

[0041] Embodiment 2, the cultivation of human umbilical cord mesenchymal stem cells

[0042] Under sterile conditions, the umbilical cord tissue discarded after delivery was transported back to the laboratory in PBS containing double antibody. Use 50mL of sterile PBS to clean the blood on the surface of the umbilical cord, disinfect the surface of the umbilical cord with 150mL of 75% ethanol, and then wash it several times with 150mL of PBS to remove the blood as much as possible. After cutting the umbilical cord into small pieces about 1-3cm long, remove the outermost membrane of the umbilical cord and the veins and arteries , cut the umbilical cord to a size of 1mm 3 to 3mm 3 Distribute the tissue pieces in the petri dish at an interval of 0.5cm-1cm, let it air dry for 20min, add 0.07mL / cm 2 complete medium at 37°C, 5% CO 2 After static culture in the incubator for 3 days, add 0.07mL / cm 2 After most of the tissue blocks have cells crawling out, remove the tissue blocks, ...

Embodiment 3

[0045] Embodiment 3, the cultivation of human umbilical cord mesenchymal stem cells

[0046] Under sterile conditions, the umbilical cord tissue discarded after delivery was transported back to the laboratory in PBS containing double antibodies. Use 150mL of sterile PBS to clean the blood on the surface of the umbilical cord, disinfect the surface of the umbilical cord with 50mL of 75% ethanol, and then wash it several times with 50mL of PBS to remove the blood as much as possible. After cutting the umbilical cord into small pieces about 1-3cm long, remove the outermost membrane of the umbilical cord and the veins and arteries , cut the umbilical cord to a size of 1mm 3 to 3mm 3 Distribute the tissue pieces in the petri dish at an interval of 0.5cm-1cm, let it air dry for 60min, add 0.1mL / cm 2 complete medium at 37°C, 5% CO 2 After static culture in the incubator for 3 days, add 0.1mL / cm 2 After most of the tissue blocks had cells crawling out, remove the tissue blocks, wa...

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Abstract

The invention relates to the field of stem cells and discloses a culture method for a human mesenchymal stem cell. The culture method for the human mesenchymal stem cell comprises the steps of taking an umbilical cord, and removing a membrane at the outermost layer, veins and arteries of the umbilical cord; adding the umbilical cord into a complete medium after shearing; carrying out static culture at the temperature of 37 DEG C and in the existence of 5% of CO2, and meanwhile replenishing the complete medium; removing a tissue block after a cell creeps out of the tissue block; and adding the cell into the complete medium after washing; and carrying out subculture after a cell colony is formed. The culture method disclosed by the invention is simple in operation, safe and effective. Proved by experiments, compared with the existing method, the culture method for the human mesenchymal stem cell has the advantages that the primary culture time is short, more cells are obtained after multiplication, the cell activity and multiplication capacity are strong, the cell still can keep favorable multiplication capacity after being cultured to the P15th generation, and the form and favorable stem cell characteristic of the mesenchymal stem cell can be favorably kept, so that the culture method is suitable for culturing the mesenchymal stem cell on a large scale.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a method for culturing human umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are derived from the mesoderm and ectoderm in the early stages of development. They have the characteristics of self-renewal, multi-directional differentiation, multi-directional differentiation potential, hematopoietic support, and immune regulation. They produce and release nutrients. Sexual substances promote cell repair and angiogenesis through the paracrine pathway, and play an important role in the field of regenerative medicine. [0003] At present, mesenchymal stem cells are mainly extracted from fat, bone marrow, umbilical cord, and placenta tissue. Mesenchymal stem cells still have multi-directional differentiation potential after continuous subculture and cryopreservation, which provides a new way for the clinical treatment of various diseases, such...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
Inventor 王一飞陈海佳葛啸虎麦锦连马岩岩王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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