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Method of mass producing growth factor using adipose derived adult stem cells

a technology of adult stem cells and growth factors, which is applied in the direction of genetically modified cells, skeletal/connective tissue cells, peptide/protein ingredients, etc., can solve the problems of difficult to obtain in large quantities, low differentiation potential, and high risk of infection, and achieve excellent safety and activity, the effect of producing human growth factors

Inactive Publication Date: 2012-03-15
PROSTEMICS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0021]It is an object of the present invention to produce large amounts of human growth factors from adipose-derived adult stem cells, the produced human growth factors having excellent in vivo activity compared to growth factors synthesized by recombinant or chemical methods.
[0022]Another object of the present invention is to provide safe and effective drugs or cosmetics containing either human growth factors produced in large amounts from adipose-derived stem cells, or culture media of the growth factors.
[0026]According to the present invention, it is possible to produce human growth factors in large amounts from adipose-derived adult stem cells. It was found that the growth factors produced using the inventive production method had excellent safety and activity compared to those of growth factors produced according to existing production methods. Furthermore, the growth factors produced using the inventive production method could act in the same fashion as the existing growth factors of the human body. Also, it is expected that a culture medium of adipose-derived stem cells and growth factors isolated from the culture medium can be advantageously applied in drugs, quasi drugs and cosmetics for anti-wrinkle, wound healing, and scar removing.

Problems solved by technology

The embryonic stem cells have advantages of very good differentiation potential and long telomeres, but have ethical problems and in the disadvantage that they are difficult to obtain in large amounts.
In comparison, the adult stem cells can be obtained in large amounts, but have the disadvantage that, when these cells are transplanted into other persons, they carry the risk of infection and have a relatively low differentiation potential.
Despite this progress, many studies have, in fact, not been conducted on the use of human adipose tissue.
Specifically, studies on the structure and synthesis of the signaling substances (growth factors) have been conducted, but the most of the growth factors have protein structures, which are three-dimensionally complex, thus posing various problems in chemical synthesis and significantly increasing costs for the synthesis.
However, studies on adipose-derived adult stem cells mainly relate to the use or differentiation of the cells themselves, and methods of synthesizing growth factors from these cells have not been studied.
Korean Patent Laid-Open Publication No. 2004-94910, entitled “Improved fat cell-differentiated, adipose-derived adult stem cells and the use thereof” discloses a method capable of increasing the in vivo survival rate of adipose-derived adult stem cells, but does not disclose the meaningful synthesis of growth factors.
The reason that studies on the synthesis of growth factors using adipose-derived adult stem cells are insufficient is that the methods of using adult stem cells are focused on their use through differentiation into other cells and that studies on the cytological differences between stem cells are insignificant.

Method used

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  • Method of mass producing growth factor using adipose derived adult stem cells
  • Method of mass producing growth factor using adipose derived adult stem cells
  • Method of mass producing growth factor using adipose derived adult stem cells

Examples

Experimental program
Comparison scheme
Effect test

example 1

(1-1) Isolation of Adipose-Derived Stem Cells

[0121]Human liposuction material collected from Leaders Clinic, Seoul, Korea was washed with an equal volume of phosphate buffer saline, and only adipose tissue was separated from the liposuction material.

[0122]The extracellular matrix of the adipose tissue was enzymatically treated with 0.075% collagenase in a 5% CO2 incubator at 37° C. for 45 minutes, and the enzymatically treated adipose tissue was centrifuged at 1200 g for 5 minutes to collect a stromal vascular fraction containing a high density of stem cells. The pellets were washed with phosphate buffer saline and passed through a 70-μm nylon cell filter to remove other tissues, and only monocyte cells and cell fragments including red blood cells were separated from the remaining material using Histopaque-1077 (SIGMA).

[0123]The separated monocytes were cultured in a non-inducing medium containing Dulbecco's Modified Eagle's Medium (DMEM), 10% fetal bovine serum (FBS), 1% penicillin...

example 2

Identification of Activated Growth Factors of Adipose-Derived Stem Cells

[0132]In order to examine whether adipose-derived stem cells synthesize growth factors, adipose-derived stem cells were cultured in the same culture medium and culture conditions as in Example (1-2), and then RNA in the stem cells was analyzed through a reverse transcription-polymerase chain reaction.

[0133]Specifically, the total RNA of the adipose-derived stem cells was extracted with a RNeasy Plus Mini kit (QIAGEN Corp., Valencia, Calif.), and the RNA was amplified by PCR using a MMLV-reverse transferase (Promega Corp., U.S.A) at 37° C. for 45. Then, the MMLV-reverse transferase was inactivated at 65° C. for 45 minutes. The PCR reaction solution had a total volume of 50 μl and contained 1.5 mM MgCl2, 0.25 mM dNTP, and 2.5 unit Taq polymerase (QIAGEN). The PCR reaction was performed in TGRADIENT (BIOMETRA) in the following conditions: a cDNA denaturation of 3 min at 94° C., and then 30 cycles each consisting of...

example 3

Activity of Adipose-Derived Stem Cells on Collagen Production of Fibroblasts

[0139]One of the causes of formation of skin wrinkles is a lack of collagen. Collagen is a major protein of skin dermis and serves to maintain the skin structure and firmness. It is known that the production of collagen decreases as age increases, and the degradation thereof also increases to induce the collapse of the dermal layer, thus producing skin wrinkles. Thus, the effect of a substance for anti-wrinkle activity can be proven by testing the production and degradation of collagen.

[0140](3-1) Coculture of Adipose-Derived Stem Cells and Fibroblasts

[0141]The effect of adipose-derived stem cells on the collagen production of fibroblasts was estimated through the coculture of adipose-derived stem cells and fibroblasts. In the test, an increase in the production of collagen in fibroblasts, when adipose-derived stem cells were cocultured with fibroblasts in a transwell insert (Costar, Corning), was compared w...

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Abstract

The present invention relates to a method for producing large amounts of human growth factors from human adipose-derived stem cells. More specifically, the invention provides a method capable of synthesizing human growth factors in significantly large amounts by culturing adipose-derived stem cells extracted from human adipose cells in suitable media and conditions. Also, stem cell culture media produced according to the method of the invention, and human growth factors isolated from the culture media, can be advantageously used as raw materials for drugs and cosmetics.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a divisional application of application Ser. No. 12 / 161,899 filed on Jul. 23, 2008, which is a U.S. national phase application, pursuant to 35 U.S.C. §371, of PCT / KR2006 / 004111 filed on Oct. 12, 2006, which claims priority to Korean application 10-2006-0008874, filed Jan. 27, 2006. The entire contents of the aforementioned patent applications are incorporated herein by this reference.TECHNICAL FIELD[0002]The present invention relates to a method for producing large amounts of growth factors from human adipose-derived stem cells. More specifically, the present invention provides a method which comprises culturing adipose-derived stem cells extracted from human fat cells in suitable media and conditions, such that human growth factors, for example, an acidic fibroblast growth factor (acidic FGF), a basic fibroblast growth factor (basic FGF), an insulin-like growth factor-1 (IGF-1), an insulin-like growth factor-2 (IGF-2),...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K8/64A61Q19/08A61Q19/00C12N5/0775
CPCC07K14/495C07K14/503C07K14/52C12N5/0667C12N2510/02C12N2500/38C12N2500/60C12N2500/99C12N2500/02A61P17/00C12N2500/90C12N2502/1382C12N5/00C12N5/06C12N5/0602
Inventor PARK, BYUNGSOONCHOI, BONGGEUNPARK, CHULHONG
Owner PROSTEMICS
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