KR20120087625A

KR20120087625A - Enriched media of human adipose tissue-derived stem cells enhancing migration, proliferation or keratin synthesis of keratinocyte and uses thereof

Info

Publication number: KR20120087625A
Application number: KR1020110008934A
Authority: KR
Inventors: 김동수; 박예형
Original assignee: (주)프로스테믹스
Priority date: 2011-01-28
Filing date: 2011-01-28
Publication date: 2012-08-07

Abstract

The present invention is the passage of human fat-derived stem cells in serum medium, followed by continuous culture in serum-free medium, followed by migration, proliferation or keratin synthesis of keratinocytes obtained by filtration of stem cell culture medium or its concentrate as an active ingredient. The present invention relates to a method for promoting migration, proliferation or keratin synthesis of keratinocytes, the method comprising promoting the composition, topically applying or intradermal injection to the skin, and a therapeutic agent for intractable skin diseases comprising the composition as an active ingredient. .

Description

Enriched media of human adipose tissue-derived stem cells enhancing migration, proliferation or keratin synthesis of keratinocyte and uses according to the present invention for promoting the migration, proliferation or keratin synthesis of keratinocytes.

The present invention relates to a culture concentrate of human adipose derived stem cells having a migration, proliferation, or keratin synthesis promoting effect of keratinocytes, and more particularly, the present invention relates to a serum-free medium after passage of human adipose derived stem cells in a serum medium. After continuous culture in a medium, a composition for promoting migration, proliferation or keratin synthesis of keratinocytes containing the stem cell culture medium obtained by filtration or the concentrated solution thereof as an active ingredient, topically applying or intradermal injection to the skin The present invention relates to a method for promoting migration, proliferation or keratin synthesis of keratinocytes, and a therapeutic agent for refractory skin diseases comprising the composition as an active ingredient.

The skin consists of the epidermis, the dermis and the subcutaneous tissue. The epidermis, the outer layer of skin, consists of the stratified squamous epithelium and acts as a protective barrier for the body to the outside environment. The epidermis is divided from the outside into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale. The dermis is the layer between the epidermis and the subcutaneous tissue, divided into papillary and reticular dermis, blood vessels, collagen, elastin fibers, pores, hair roots, sebaceous glands, Korean glands, various Sensory nerves, fibroblasts and macrophages are present and occupy the largest part of the skin.

The dermis consists primarily of collagen and elastin fibers, which support the skin. Therefore, when such a problem occurs in the dermis, wrinkles occur and skin elasticity is lost, thereby aging the skin. Collagen is known to play the most important role in skin regeneration, skin moisture content, wound healing and wrinkle improvement, and is produced from fibroblasts. Collagen has a function that can contain a large amount of moisture, which serves to supply moisture to the dermis. When aging, the collagen loses its water-containing function and wrinkles increase. Collagen can also heal wounds by filling fibroblasts with sustained collagen production when the wound is injured.

Keratinocytes make up about 95% of the cells in the epidermis. The main function of keratinocytes is the formation of a keratin layer that protects the skin and its underlying tissues from damage such as heat, ultraviolet rays, and water loss. The keratin layer is formed through keratinization and cornification, and keratinocytes that produce keratin eventually undergo a programmed cell death process.

Stem cells are cells that have been differentiated into specific cells without progress and, if necessary, have the ability to differentiate into all kinds of cells constituting the body such as nerves, blood, and cartilage. There are two ways to obtain such stem cells, firstly from embryos derived from fertilized eggs (embryonic stem cells) and secondly from stem cells (adult stem cells) stored in each part of our adult body. ) Is recovered. Although functionally different, both embryonic and adult stem cells have the ability to differentiate into different cell types. Embryonic stem cells have very good differentiation ability and long telomeres, but they have ethical problems and difficult to obtain a large amount of cells. On the other hand, adult stem cells can obtain a large number of cells, but can be transplanted to others. The risk of infection or differentiation is relatively low.

Adult stem cells are very safe for medical applications. Specifically, cancer does not occur even when transplanted into the body for long-term regeneration, and since the immune rejection reaction does not occur because it is in an adult body, autologous transplantation using its own cells is possible. In addition, there is a site-specific differentiation ability to differentiate itself according to the characteristics of the surrounding tissue, and since injection does not cause cancer even when injected in the undifferentiated state, in addition to producing the cells needed immediately after transplantation The advantage is the ability to self-renewal to regenerate and store the necessary undifferentiated stem cells. Therefore, the importance of adult stem cells has recently been highlighted, and various studies are under way to reveal its usefulness.

Korean Patent Publication No. 2010-0114729 discloses a composition for skin regeneration using umbilical cord blood stem cell-derived angiogenic precursor cells, and Korean Patent Publication No. 2010-0032099 for skin wrinkle improvement using a mammalian stem cell culture solution or Disclosed is a composition for inhibiting skin aging.

The present invention is derived from the above requirements, the present inventors subcultured human adipose-derived stem cells in serum medium and then continuous culture in serum-free medium, the stem cell culture obtained by filtration or the concentrate is keratin The present invention has been found to be effective in promoting cell migration, proliferation, or promoting keratin synthesis.

In order to solve the above problems, the present invention is a keratinocyte containing the stem cell culture medium or its concentrate obtained by filtration after culturing human fat-derived stem cells in serum medium and then continuously cultured in serum-free medium. It provides a composition for promoting migration, proliferation or keratin synthesis.

The present invention also provides a method for promoting migration, proliferation or keratin synthesis of keratinocytes, characterized in that the composition is topically applied or intradermally injected to the skin.

The present invention also provides an intractable skin disease treatment agent comprising the composition as an active ingredient.

The culture solution of human adipose derived stem cells of the present invention or the concentrate thereof promotes migration, proliferation, or keratin synthesis of keratinocytes, and thus is expected to be effectively used for the treatment of refractory skin diseases.

Figure 1 shows the culture of human adipose derived stem cells (ADSC) under normal oxygen (normoxia) and hypoxia (hypoxia).
FIG. 2 shows the migration pattern of keratinocytes after 24 hours of culture when AAPE was added (1.22 μg / mL) in the culture of keratinocytes. The wound line in the middle of each picture was made using a microtip. Medium: control group treated with culture medium, AAPE: experimental group to which 1.22 ㎍ / mL of AAPE was added.
Figure 3 shows the results of analysis of the expression pattern of AAPE 44K-8 molecule group using a DNA chip (Agilent DNA microarray).
4 is a reconstruction of FIG. 3 into a visual pie graph.

In order to achieve the object of the present invention, the present invention comprises a subculture of human adipose derived stem cells in serum medium and then continuous culture in serum-free medium, containing the stem cell culture obtained by filtration or the concentrate thereof as an active ingredient. Provided is a composition for promoting migration, proliferation or keratin synthesis of keratinocytes.

The stem cell culture or its concentrate is preferably

(a) passage of human adipose derived stem cells in serum medium followed by continuous culture in serum-free medium;

(b) applying any one or more physical stimuli selected from low oxygen culture, ultraviolet irradiation, low frequency treatment, nutrient deprivation, and mechanical friction to the cultured stem cells;

(c) culturing by adding at least one vitamin selected from vitamin A, vitamin B, vitamin C and vitamin D to the culture medium; and

(d) may be prepared by filtering the culture solution.

Steps (b) and (c) may be performed in combination under conditions in which maximum production of human growth factors occurs. For the production of stem cell culture concentrate, refer to Korean Patent Publication No. 2008-0109725, the entire contents of which are included in the present invention.

In the composition of the present invention, the serum-free medium may preferably be a mixed medium of DMEM (Dulbecco's Modified Eagle's Medium) and Ham's F-12 nutrient mixture (see Korean Patent Publication No. 2008-0109725), The mixing ratio may preferably be 1: 1 with DMEM and Ham's F-12 nutrient mixture, but is not limited thereto.

The stem cells refer to undifferentiated cells having the ability to differentiate into various types of tissue cells, and are separated from embryonic stem cells and adult tissues separated from the inner cell mass of the blastocyst. It can be broadly classified into adult stem cells. Adult stem cells refer to undifferentiated cells having mutipotency derived from adult tissues of mammals including humans, preferably humans. For example, various stem cells such as bone marrow, blood, brain, skin, fat, umbilical cord blood, etc. Can be derived from adult cells.

The human adipose-derived stem cells are a kind of adult stem cells isolated from human adipose tissue. Acquisition of adipose tissue can be obtained incidentally in the process of liposuction, which is conventionally performed, and thus has an advantage of easily obtaining and culturing a sufficient amount of stem cells, and is also superior in safety to bone marrow harvesting.

The human adipose derived stem cells may be cultured in a conventional manner using a stem cell culture medium, for example, a serum medium. Preferably, human adipose derived stem cells are cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum, and finally cultured continuously in a 1: 1 mixed medium of serum-free DMEM and Ham's F-12 nutrient mixtures. By doing so, it is possible to increase the protein content in the culture broth obtained. The serum-free medium step minimizes the differentiation by exposing the stem cells isolated from adipose tissue to a specific extreme environment and allows the recovery of as much protein as possible from the culture medium.

The present invention also provides a method for promoting migration, proliferation or keratin synthesis of keratinocytes, characterized in that the composition is topically applied or intradermally injected to the skin. The dosage form may preferably be topical application or intradermal injection, but is not limited thereto.

The present invention also provides an intractable skin disease treatment agent comprising the composition as an active ingredient. The refractory skin disease may be severe atopic dermatitis, but is not limited thereto.

Pharmaceutically acceptable carriers included in the treatment of refractory skin diseases of the present invention are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin , Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, However, it is not limited thereto.

The refractory skin disease treatment agent of the present invention is formulated using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those skilled in the art in the unit dosage form. It may be prepared or incorporated into a multi-dose container, and may further include a dispersant or stabilizer.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1: Obtaining Adipose Stem Cells

Consent was obtained from a patient who had undergone a subcutaneous fat removal procedure, and the adipose tissue was removed from the patient by liposuction. After aseptic homogenization of the adipose tissue, the top oil layer was removed by centrifugation at 1200 × g for 5 minutes. The process of homogenizing the isolated adipose tissue with PBS (phosphate buffered saline) and centrifugation to obtain a stromal vascular fraction (SVF) was repeated several times. The collected vascular fractions were collected, washed and suspended in PBS, and other tissues were removed through a 70 μm nylon cell filter. Then, histopaque-1077 (Sigma, St. Louis, MO, USA) was used to separate cell debris and mononuclear cells including red blood cells. .

Isolated mononuclear cells were cultured with DMEM (Dulbecco's Modified Eagle's Medium; Lonza, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA), 1% penicillin-streptomycin (Gibco, USA). After incubation for 24 hours in a 37 ℃, 5% CO ₂ incubator, the non-adhesive cells were removed to isolate the fat stem cells.

Example 2: Culture of Stem Cells

Adipose stem cells 1.25 × 10 ⁵ cells obtained in Example 1 were obtained with 1000 mg / L of D glucose, 584 mg / L of L-glutamine, 110 mg / L of sodium pyruvate, 10% fetal bovine serum, It was added to DMEM medium containing 1% penicillin-streptomycin and passaged for 30 days in a 5% CO ₂ incubator under about 90% humidity and about 37 ° C. temperature conditions. The culture medium was removed from the passaged culture using a pipette and washed three times with PBS, containing 365 mg / L of L-glutamine, 15 mM HEPES, and 55 mg / L of sodium pyruvate. Inoculated at a concentration of 1.2 × 10 ⁶ cells / dish or 1.2 × 10 ⁸ cells / cell factory in a 1: 1 mixed medium (DMEM / F-12) of DMEM and Ham's F-12 nutrient mixtures, under hypoxia conditions. Incubated for a time ~ 1 week.

Example 3: Culture and Concentration of Stem Cells

500 mL of the culture solution finally obtained in Example 2 was centrifuged at 300 x g for 5 minutes to remove stem cells that had sunk into pellets. The resulting supernatant was filtered with a 0.22 μm syringe filter to remove residual stem cells and unknown macromolecules. The obtained solution (hereinafter referred to as AAPE) was divided into small portions, dispensed and lyophilized, or divided in half and concentrated under reduced pressure to obtain a concentrated solution of 5 mL. The resulting concentrate was lyophilized and stored at -70 ° C.

Example 4: normal oxygen condition and Hypoxia Comparison of culture patterns of human adipose derived stem cells under conditions

Human adipose derived stem cells were cultured in normal oxygen conditions (normoxia) and hypoxia conditions (hypoxia), and then the pattern was observed under a microscope. Figure 1 shows that the differentiation or division of the stem cells in hypoxic conditions is suppressed cells are focused on the production of secretory material.

Human adipose derived stem cells are secured up to 4 passages, and then AAPE is obtained in the final 5 passages serum free medium phase.

Example 5: On keratinocytes About AAPE Wound healing effect

The keratinocytes were divided into AAPE treated and untreated (control) groups, and microtips were used to artificially scratch the center of the cell mass. Then, the treated group was incubated with the addition of 1.22 μg / mL of AAPE, and only the culture medium was added to the control group for 24 hours.

As a result, the migration of keratinocytes was significantly increased in the AAPE treated group compared to the control group (FIG. 2). The results show that AAPE has a healing effect on skin wounds by promoting migration of keratinocytes.

Example 6: AAPE By processing In keratinocytes Analysis of Genes with Increased Expression

After AAPE was treated to keratinocytes, genes with increased expression were identified using a DNA chip (Agilent DNA microarray).

Table 1 shows that AAPE in keratinocytes is effective for repairing damaged outermost skin keratin layers by promoting the synthesis of keratin molecules important for skin barrier formation. That is, AAPE has an effective molecular function in reconstructing the damage of the skin curtain by epidermal dermatitis.

Table 2 shows the results of analysis of genes with increased expression in AAPE-treated keratinocytes using DNA chips (Agilent DNA microarray). The results indicate that AAPE promotes cell proliferation against keratinocytes.

Therefore, the treatment of AAPE can induce the active cell proliferation and migration of keratinocytes to fill the damaged epidermal layer to restore the skin barrier layer, and may have a therapeutic effect against various epidermal diseases.

Example 7: AAPE Available protein groups present in AAPE 44K-8

The group of effective proteins present in AAPE was traced back by examining the gene expression patterns of producing cells. Total RNA was extracted from adipose derived stem cells (ADSC) in the serum-free medium culture step and converted to DNA, followed by fluorescent labeling, and then analyzed by a microarray method that hybridizes the human genomic DNA chip (Agilent).

Table 3 shows that AAPE is composed of molecules that can contribute to the regeneration of skin tissue. AAPE 44K-8 is composed of 8 molecules including CTGF (connective tissue growth factor), FGF7 (keratinocyte growth factor), SPARC (secreted protein acidic and rich in cystein), etc. It is thought to be a functional molecular composition that can directly affect cell proliferation of the dermal layer. Therefore, the AAPE may be effective for the treatment of refractory skin diseases such as severe atopic dermatitis.

After culturing human adipose derived stem cells (ADSC) under normal oxygen conditions (normoxia) and hypoxia conditions (hypoxia), the expression pattern of AAPE 44K-8 molecular group was analyzed using a DNA chip (Agilent DNA microarray). 3 and 4 show an increase in the expression of the AAPE 44K-8 molecular group required for regeneration of skin tissue in hypoxia cultures than in normal oxygen condition (normoxia) cultures. In other words, human adipose-derived stem cells more effectively express a major molecular group required for cell tissue regeneration of refractory skin diseases in a hypoxic culture environment.

Claims

Human fat-derived stem cells are subcultured in serum medium and serially cultured in serum-free medium, and the composition for promoting migration, proliferation, or keratin synthesis of keratin cells obtained by filtering the stem cell culture medium or its concentrate as an active ingredient. .

The method of claim 1, wherein the stem cell culture or its concentrate
Subcultured human adipose derived stem cells in serum medium followed by continuous culture in serum-free medium;
Applying any one or more physical stimuli selected from low oxygen culture, ultraviolet irradiation, low frequency treatment, nutrient deprivation, and mechanical friction to the cultured stem cells;
Culturing by adding one or more vitamins selected from vitamin A, vitamin B, vitamin C and vitamin D to the culture medium, and
Composition for promoting migration, proliferation or keratin synthesis of keratinocytes, characterized in that prepared by filtering the culture solution.

The composition for promoting migration, proliferation or keratin synthesis of keratin cells according to claim 1, wherein the serum-free medium is a mixed medium of DMEM and Ham's F-12 nutrient mixture.

A method for promoting migration, proliferation or keratin synthesis of keratinocytes, wherein the composition of claim 1 is applied topically or intradermally to the skin.

Refractory skin disease treatment comprising the composition of claim 1 as an active ingredient.

The method of claim 5, wherein the refractory skin disease is a treatment for intractable skin disease, characterized in that severe atopic dermatitis.