Umbilical cord mesenchymal stem cell serum-free cultivation method and application

A technology of mesenchymal stem cells and a culture method, which is applied to the culture method and application field of serum-free mesenchymal stem cells, and can solve the problem that the passage number of serum-free medium cannot exceed the number of passages.

Inactive Publication Date: 2016-05-25
SHANGHAI JIQUAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to overcome the shortcoming that the passage number of mesenchymal stem cells cannot exceed 8-10 passages in the serum-free medium, the purpose of the present invention is to provide a kind of fibronectin combined with recombinant human epidermal growth factor (rhEGF) and recombinant human adult Fiber growth factor (rhbFGF) is coated on plastic culture bottles, so that fibronectin and recombinant human epidermal growth factor, recombinant human fiber growth factor and plastic culture bottle materials are fully combined. Plastic culture bottles are generally made of polystyrene materials, which are harmful to proteins. With a certain...

Method used

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  • Umbilical cord mesenchymal stem cell serum-free cultivation method and application
  • Umbilical cord mesenchymal stem cell serum-free cultivation method and application
  • Umbilical cord mesenchymal stem cell serum-free cultivation method and application

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Experimental program
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Effect test

Embodiment 1

[0032] (1) Prepare a coating solution with a final concentration of 5ug / mL fibronectin, 5ng / mL recombinant human epidermal growth factor and 5ng / mL recombinant human fibroblast growth factor, take 2mL of the coating solution, add it to a T25 plastic culture bottle, Incubate in a 37°C incubator for 2 hours, and discard the coating solution.

[0033] (2) Take the discarded umbilical cord tissue of the full-term fetus, remove the umbilical vein and umbilical artery, remove the capsule of the umbilical cord tissue, take Walton's jelly, put it in DMEM and wash it.

[0034] (3) step (2) obtains Walton's jelly and shreds, the volume is 1mm 3 Size, take 1.5 grams, add in the T25 plastic culture bottle that step (1) gains.

[0035](4) As obtained in step (3), 15 mL of serum-free medium TheraPEAK Chemical Defined Mesenchymal Stem Cell Growth Medium (Lonza, USA) was added.

[0036] (5) In step (4), a plastic culture bottle containing Walton gum and serum-free medium is obtained, and cu...

Embodiment 2

[0048] (1) Prepare a coating solution with a final concentration of 5ug / mL fibronectin, 5ng / mL recombinant human epidermal growth factor and 5ng / mL recombinant human fibroblast growth factor, take 2mL of the coating solution and add it to a T25 plastic culture bottle, After incubating in a 37°C incubator for 2 hours, the coating solution was discarded.

[0049] (2) As obtained in step (1), 125,000-150,000 primary umbilical cord mesenchymal stem cells were added.

[0050] (3) As obtained in step (2), 5-10 mL of serum-free medium TheraPEAK Chemical Defined Mesenchymal Stem Cell Growth Medium (Lonza, USA) was added, and the cells began to adhere to the wall within 2 hours.

[0051] (4) The obtained product in step (3) was placed in a 37°C, 5% carbon dioxide incubator, and the medium was changed every 2 to 3 days. Non-adherent cells were discarded, and 5 to 10 mL of serum-free medium TheraPEAK Chemical Defined Mesenchymal Stem Cell Growth Medium (Lonza, USA) was added. Wait until...

Embodiment 3

[0060] (1) Prepare a coating solution with a final concentration of 5ug / mL fibronectin, 5ng / mL recombinant human epidermal growth factor and 5ng / mL recombinant human fibroblast growth factor, take 2mL of the coating solution and add it to a T25 plastic culture bottle, After incubating in a 37°C incubator for 2 hours, the coating solution was discarded.

[0061] (2) Take the mesenchymal stem cells stored in liquid nitrogen, put them in a water bath at 37°C, resuscitate, count the cells, and take 125,000-150,000 mesenchymal stem cells to inoculate the obtained in step (1).

[0062] (3) As obtained in step (2), 5-10 mL of serum-free medium TheraPEAK Chemical Defined Mesenchymal Stem Cell Growth Medium (Lonza, USA) was added, and the cells began to adhere to the wall within 2 hours.

[0063] (4) Gained in step (3), cultured in a 37° C., 5% carbon dioxide incubator, changing the medium every 2 to 3 days, discarding non-adherent cells, and adding 5 to 10 mL of serum-free medium Ther...

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Abstract

The invention discloses an umbilical cord mesenchymal stem cell serum-free cultivation method, comprising the following steps: (1) preparing coating buffer for fibronectin, a recombinant human epidermal growth factor and a recombinant human fibroblast growth factor, and coating a plastic culture bottle; (2) taking human umbilical cord tissue Whartonps jelly, cutting into pieces, and adding into the culture bottle in the step (1); (3) for a product obtained in the step (2), adding into a serum-free culture medium; (4) putting into a 5-percent carbon dioxide incubator at 37 DEG C for culture, wherein mesenchymal stem cells overgrow in 10-12 days; (5) washing by a phosphate buffer solution, and performing Triple enzymic digestion; (6) adding the phosphate buffer solution, and stopping the digestion; (7) collecting mesenchymal stem cell supernate; (8) centrifuging; (9) collecting sediments to obtain the needed mesenchymal stem cells. By adopting the method, the mesenchymal stem cells can go down to the 20th generation, the characteristics of the mesenchymal stem cells are kept, diseases can be treated, and application prospect is achieved.

Description

technical field [0001] The invention relates to a culture method and application of umbilical cord tissue mesenchymal stem cells, in particular to a serum-free mesenchymal stem cell culture method and application. technical background [0002] Mesenchymal stem cells come from the mesoderm and have multidirectional differentiation. They can differentiate into bone cells, chondrocytes, fat cells, muscle cells, nerve cells, etc., and are used in the treatment of ischemic heart disease, osteoarthritis, and senile dementia. and other diseases have a good outlook. [0003] Mesenchymal stem cells usually come from bone marrow, but as the bone marrow donor ages, the proliferative ability of bone marrow-derived mesenchymal stem cells gradually decreases, and the application is limited. Therefore, it is necessary to find other sources of mesenchymal stem cells. [0004] In 2003, American scientist Kathy published in stemcells journal (stemcells 2003; 21:50-60) that mesenchymal stem c...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
CPCC12N5/0668C12N2501/119C12N2501/11C12N5/0037C12N2533/52
Inventor 仇志根
Owner SHANGHAI JIQUAN BIOTECH CO LTD
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