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Production and freeze-drying method and application for supernate of culture solution of mesenchymal stem cell

A quality stem cell and production method technology, applied in the field of cells, can solve the problem of limited source of self-sera, and achieve the effects of shortening culture time, reducing cost and convenient operation

Inactive Publication Date: 2015-09-30
MILTENYI TIANJIN BIOLOGICAL SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the source of autoserum is extremely limited, and when the culture supernatant of human mesenchymal stem cells is prepared for external use, it is not necessary to use autoserum

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] A method for producing human mesenchymal stem cell culture supernatant, the steps are as follows:

[0061] ⑴ Umbilical cord sampling:

[0062] ① High-temperature disinfection of surgical instruments and large glass dishes, while ultraviolet disinfection of ultra-clean workbench;

[0063] ② Strict screening of umbilical cord donors, blood tests to exclude common infectious diseases including AIDS, hepatitis A, B, and C;

[0064] ③Transport the umbilical cord in a low-temperature ice box, put the umbilical cord in a large glass dish, cut off 3 sections, 5 cm long, and then cut it longitudinally, wash the umbilical cord twice with phosphate buffered saline (PBS) + penicillin, and wash away the blood ;

[0065] ④ Cut the umbilical cord tissue into small fragments of about 2-3 mm to obtain the umbilical cord fragments;

[0066] ⑤ Add 5 ml of umbilical cord fragments and 20 ml of 2 mg / ml collagenase IV to two 50 ml centrifuge tubes, and digest on a shaker at 37°C for 1.5-2...

Embodiment 2

[0082] A method for producing human mesenchymal stem cell culture supernatant, the steps are as follows:

[0083] ⑴ Umbilical cord sampling:

[0084] ① High-temperature disinfection of surgical instruments and large glass dishes, while ultraviolet disinfection of ultra-clean workbench;

[0085] ② Strict screening of umbilical cord donors, blood tests to exclude common infectious diseases including AIDS, hepatitis A, B, and C;

[0086] ③Transport the umbilical cord in a low-temperature ice box, put the umbilical cord in a large glass dish, cut off 3 sections, 5 cm long, and then cut it longitudinally, wash the umbilical cord twice with phosphate buffered saline (PBS) + penicillin, and wash away the blood ;

[0087] ④ Cut the umbilical cord tissue into small fragments of about 2-3 mm to obtain the umbilical cord fragments;

[0088] ⑤ Add 10 ml of umbilical cord fragments and 20 ml of 2 mg / ml collagenase IV to two 50 ml centrifuge tubes, and digest on a shaker at 37°C for 3-3....

Embodiment 3

[0104] A method for producing human mesenchymal stem cell culture supernatant, the steps are as follows:

[0105] Step (1) and (2) are with embodiment 2;

[0106] (3) Culture human mesenchymal stem cells in Nunc cell factory and collect culture supernatant:

[0107] ① Trypsinization and passaging to Nunc cell factory: wash the amplified cells cultured in T75 twice with PBS, digest with 0.25% trypsin-EDTA (5-10 ml per T175), 37°C, 10 minutes. After the cells were suspended, 5 ml of 5% FBS medium was added to each T175. Centrifuge at 1000 rpm for 10 minutes. Resuspend in a total of 1300 ml of 5% FBS medium. Subculture into two four-layer cell factories (650 ml medium each). Compared with the 10-layer cell factory, the four-layer cell factory can be observed under a microscope, and because of its relatively light weight, it is easier to handle (especially to collect the supernatant).

[0108] ②The concentration of FBS was reduced to 3% until the cells covered 60% of the area...

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Abstract

The invention relates to a production and freeze-drying method and application for supernate of a culture solution of a mesenchymal stem cell. a culture solution supernate freeze drying agent can be used with skin care products simultaneously after being dissolved again, the function of improving a skin is achieved, the production method adopts umbilical cord tissues of a healthy person as raw materials, the umbilical cord tissues are digested through collagenase and cultured outside the body, amplification and trypsin digestive passage are conducted, an NuncTM cell factory system with ten layers or four layers is utilized to replace a traditional cell culture bottle finally, the cost is greatly reduced, needed space is reduced, a large number of mesenchymal stem cells are obtained in shorter culture time, low concentration fetal bovine serum (3%FBS) is reserved in cell culture media to make the condition of the cells healthier compared with that of the cells without serum, the quality of culture supernatant fluid is guaranteed, the culture supernatant fluid can be stored under room temperature after vacuum freeze drying is conducted on the culture supernatant fluid directly, and activity of various growth factors of the culture supernatant fluid is maintained.

Description

technical field [0001] The invention belongs to the field of cell technology, and relates to a stem cell derivative biological product and a preparation method thereof, in particular to a low-cost mass production of human mesenchymal stem cell culture supernatant and its freeze-drying method and application. Background technique [0002] Human mesenchymal stem cells (MSC) secrete basic fibroblast growth factor (bFGF), human epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF) in culture -1) and hepatocyte growth factor (HGF), a variety of growth factors that promote skin cell regeneration. MSCs also secrete large amounts of extracellular matrix that can increase the health and appearance of the skin. The culture fluid of MSC is collected and freeze-dried, which is easy to store and can be used together with skin care products to achieve the effect of beauty and skin care. [0003] The transplantation of human mesenchy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K8/98A61K8/97A61Q19/08
Inventor 任峰张称闫军
Owner MILTENYI TIANJIN BIOLOGICAL SCI & TECH
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