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Human umbilical cord mesenchymal stem cell extract freeze-dried powder and preparation method

A technology of stem cells and extracts, applied in the field of stem cells, can solve the problems of loss of cell differentiation ability, complex microenvironment composition, low abundance of MSCs, etc. Effect

Active Publication Date: 2016-11-16
广东省华桑丽皙生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, the abundance of MSCs in tissues is extremely low. Therefore, in vitro culture is a necessary step to realize the application value of MSCs. Studies have shown that, unlike freshly isolated MSCs, the biological characteristics of cells after in vitro culture and expansion will change significantly. Large changes, and with the prolongation of culture time, the ability of cell differentiation is gradually lost, which is related to the environment of cells in vivo and in vitro. These in vitro factors mimic the

Method used

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  • Human umbilical cord mesenchymal stem cell extract freeze-dried powder and preparation method
  • Human umbilical cord mesenchymal stem cell extract freeze-dried powder and preparation method
  • Human umbilical cord mesenchymal stem cell extract freeze-dried powder and preparation method

Examples

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Effect test

Embodiment 1

[0024] Example 1 Isolation, purification, cultivation and identification of human umbilical cord mesenchymal stem cells

[0025] (1) Isolation, purification and culture of human umbilical cord mesenchymal stem cells

[0026] Under sterile conditions, use balanced salt buffer solution containing 100U / ml penicillin and 100g / ml streptomycin to rinse the freshly collected umbilical cord three times repeatedly. , the jelly between the blood vessel and the adventitia (umbilical cord Wharton's glue) and cut into 1mm 3 Tissue pieces of different sizes were added with digestive solution at a volume ratio of 1:3, and digested at 37°C for 3 hours, wherein the digestive solution was a phosphate buffer solution containing collagenase II, hyaluronidase and EDTA, and collagenase II, hyaluronic acid The mass volume percent final concentration of acidase and EDTA in the phosphate buffer solution is 0.12%, 0.06%, 0.02%, respectively. Then use a 150um cell strainer to filter, collect the filtr...

Embodiment 2

[0032] Example 2 Preparation of freeze-dried powder of human umbilical cord mesenchymal stem cell extract

[0033] (1) Obtaining the extract of human umbilical cord mesenchymal stem cells: when the umbilical cord mesenchymal stem cells are nearly 90% confluent, remove the culture medium, wash with phosphate buffer solution for 3 times, and add trypsin digestion solution with a mass fraction of 0.25% , digest at 37°C for 10 minutes, add an equal volume of serum-free medium without antibiotics to terminate the digestion, centrifuge at 1200r / min for 5 minutes, discard the supernatant to obtain cells, and resuspend the cells in serum-free medium according to 1: 3 ratio for subculture inoculation, placed at 37 ° C, 5% CO 2 Culture in an incubator, and replace the culture medium every 2 days. When the umbilical cord mesenchymal stem cells are close to 80% confluent, discard the culture medium, replace with fresh serum-free medium and continue for 72 hours, collect the supernatant, a...

Embodiment 3

[0035] Example 3 Comparison of lyophilized powders of human umbilical cord mesenchymal stem cell extracts prepared by using different lyoprotectants

[0036] Respectively replace trehalose in the present invention with mannitol, anhydrous lactose, and dextran as the freeze-drying protection agent, and make the human umbilical cord mesenchymal stem cell extract into freeze-dried powder according to the process of Example 2, and freeze-dried the obtained Powder and the lyophilized powder that embodiment 2 makes compare table, and the results are shown in Table 2.

[0037] Table 2 Comparison of physical properties of human umbilical cord mesenchymal stem cell extract lyophilized powder prepared by different lyoprotectants

[0038] group

[0039] It can be seen from the above table that the freeze-dried powder of human umbilical cord mesenchymal stem cell extract prepared by using trehalose as a freeze-drying protective agent has good appearance properties; the freeze-dr...

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Abstract

The invention relates to human umbilical cord mesenchymal stem cell extract freeze-dried powder and a preparation method. The preparation method of the human umbilical cord mesenchymal stem cell extract freeze-dried powder comprises the following steps: obtaining human umbilical cord mesenchymal primary stem cells, which are relatively high in purity, by virtue of density gradient centrifugation, conducting subculture, collecting a culture supernatant of the 3rd-18th generations of cells, and mixing the supernatant with trehalose, so that the freeze-dried powder is prepared. The human umbilical cord mesenchymal stem cell extract freeze-dried powder prepared by the invention has good appearance characteristics; the freeze-dried powder, when redissolved, is relatively rapid to dissolve and is completely dissolved; and animal experiments prove that the freeze-dried powder is low in irritation, high in safety and reliable to use. In addition, the freeze-dried powder disclosed by the invention can effectively preserve various bioactive cell factors in the human umbilical cord mesenchymal stem cell culture supernatant, protect skin from ultraviolet damage and promote the synthesis of skin collagen, and the freeze-dried powder has functions of whitening the skin and delaying aging.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to freeze-dried powder of human umbilical cord mesenchymal stem cell extract and a preparation method. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells with self-renewal, proliferation and multi-directional differentiation potential, which can stimulate tissue growth and repair, enhance tissue regeneration ability, and affect immune regulation. Very broad application prospects. MSCs can be obtained from bone marrow, umbilical cord blood, fat, synovium, placenta, connective tissue and other parts. At present, studies have shown that MSCs derived from umbilical cords have many advantages compared with MSCs from other sources, such as wide source, convenient collection, strong proliferation and differentiation ability, weak immunogenicity, and no ethical issues. focus on. [0003] However, the abundance of MSCs in tissues is extremely low. Therefor...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61K47/26A61K9/19A61P39/06A61P17/00
CPCA61K9/19A61K35/28A61K47/26
Inventor 陈松彬易萍倪彦艳张爱萍刘杰森
Owner 广东省华桑丽皙生物技术有限公司
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