KR101293762B1 - Culture media having enhanced wound healing activity and a method for preparing the same - Google Patents

Abstract

The present invention is treated with lysophosphatidic acid (hereinafter, abbreviated as 'LPA') to a stem cell culture medium before or in culture, after the stem cell culture prepared by improving stem cell culture, It relates to a composition for improving and treating wounds containing the stem cell culture as an active ingredient and a method for producing the stem cell culture. Stem cell culture of the present invention has the effect of promoting the regeneration of skin wounds through the regeneration of epithelial tissue, the promotion of angiogenesis, the infiltration of immune cells in wound animal model as well as no side effects and can be provided at low cost It can be used as a pharmaceutical composition for improving or treating wounds.

Description

{Culture media having enhanced wound healing activity and a method for preparing the same}

The present invention relates to a stem cell culture solution with improved wound healing ability and a method for producing the same.

In human life, wounds are indispensable, and research on the rapid healing of wounds is actively conducted, and the market for wound treatment in 2012 for the treatment of wounds is expected to reach $ 12.5 billion annually. (The Global Market for Advanced Wound Care Products 2008; Espicom Business Intelligence).

Wound healing is a tissue response to injury and is a process of tissue repair, including chemotaxis, differentiation and replication of cells, synthesis of matrix proteins, angiogenesis, and reconstruction of wounds. And complex biological processes including (Steed, et al ., Clin. Plast. Surg . 25: 397-405, 1998). Growth factors are one of the representative substances that appear early in the wound healing process and regulate subsequent processes. They have a powerful effect throughout the wound healing process, controlling cell growth, differentiation, metabolism, and the like. Since the environment is also regulated, the development of treatments using it is steadily in progress.

Among them, epidermal growth factor (EGF, Carpenter et al ., Exp. Cell Res. , 164: 1-10, 1986), acidic and basic FGF, McGee et al ., J Surg. Res ., 45: 145-153, 1988), growth factors such as transforming growth factors (TGF-α and TGF-β) and insulin-like growth factors (IGF-I and II) (Kris et al ., Nat. Biotechnol ., 3: 135-140, 1985), adhesion factors such as fibronectin, laminin, vitronectin, TGF-β-induced protein 3, and the like. Various proteins are known, including (Perham et al ., Ann. Rev. Biochem ., 52: 961-1004, 1983).

Skin regeneration or wound healing occurs through a number of processes, including inflammation, reepithelialization, granulation, granulation, fibroplasia, and contraction of wound tissue (Freedberg). et al ., J. Clin. Psychol ., 57: 433-455, 2001). A series of processes of skin regeneration are carried out by the cooperation of several cells, such as keratinocytes, fibroblasts, endothelial cells, macrophages, platelets, and the like. Complex processes such as, infiltration, proliferation, and differentiation are regulated by various growth factors, cytokines, and chemokines, so the effectiveness of the above mentioned factors or chemicals is known.

However, wound healing techniques using the growth factor and cytokines are expensive to produce and isolate these proteins, and when only one type of protein is used in the wound healing process, these proteins are combined to act as a wound healing agent. This provides partial wound relief, but there is a problem in actual commercialization due to its ineffectiveness such as long healing period and poor response to treatment.

Mesenchymal stem cells (MSCs) are cell lines with self-renewal ability, high viability and potential differentiation into various cells. This regenerative capacity is more influenced by indirect factors such as cytokines and growth factors secreted from mesenchymal stem cells than by the differentiation ability of mesenchymal stem cells directly into tissue cells (Fox et al . , Br. J. Haematol ., 137: 491-502, 2007). Therefore, mesenchymal stem cell culture has attracted attention as a therapeutic material for regeneration of wound tissue. However, in the case of inactivated mesenchymal stem cells, the culture medium of the inactivated mesenchymal stem cells is not suitable for use as a composition for the treatment of wounds and cosmetics due to the low amount of cytokines and growth factors secreted from the cells.

An object of the present invention is to provide a stem cell culture with improved wound healing ability by activating the culture of the inactivated stem cells.

Another object of the present invention is to provide a composition for treating wounds containing the stem cell culture as an active ingredient.

Still another object of the present invention is to provide a method for preparing the stem cell culture.

The final object of the present invention is to provide a method of increasing the collagen content by activating the stem cell culture.

The present inventors have a high cost in producing and isolating growth factors and cytokines that exhibit conventional wound healing effects, and when only one type of such separated protein is used, the effect is partial and the healing period is ineffective. As a result of our efforts to develop a low-cost wound treatment product containing high concentrations of proteins that can be combined with wound treatment to solve these problems, treatment of LPA (lysophosphatidic acid) on stem cells has a wound healing effect. The present invention was completed by confirming that a medium containing a high concentration of cytokines and growth factors can be obtained from mesenchymal stem cells at low cost.

The present invention has been made to achieve the above object, according to one embodiment of the present invention, lysophosphatidic acid (hereinafter, abbreviated as 'LPA') to the stem cell culture medium before or in culture Provided is a stem cell culture solution having improved wound healing ability prepared by culturing stem cells after treatment.

At this time, the LPA is preferably treated at a concentration of 0.1 to 100 μM, but is not limited thereto. The treatment period is preferably 1 to 4 days, but is not limited thereto. The stem cell culture medium may include collagen and fibronectin. The content of extracellular matrix protein is markedly increased compared to before LPA treatment.

Preferably, the stem cells are embryonic stem cells or adult stem cells, but are not limited thereto. The adult stem cells are preferably hematopoietic stem cells or mesenchymal stem cells, and more preferably, mesenchymal stem cells. It doesn't happen. The mesenchymal stem cells may be umbilical cord blood-derived stem cells, placental stem cells, adipose-derived stem cells, bone marrow-derived stem cells or peripheral blood-derived stem cells, cord blood-derived stem cells, adipose-derived stems, depending on the degree of invasiveness in harvesting More preferably, the cells or peripheral blood-derived stem cells are not limited thereto.

According to another aspect of the present invention, there is provided a composition for wound treatment and skin regeneration comprising the stem cell culture solution with improved wound healing ability as an active ingredient.

The composition may additionally include a pharmaceutically acceptable carrier, excipient or diluent, and the formulation of the upper limb composition is preferably, but not limited to, an external preparation.

According to still another aspect of the present invention,

Iii) treating LPA to the stem cell culture medium before or during the culture;

Ii) culturing stem cells in a culture medium treated with LPA; And

Iii) there is provided a method of producing a stem cell culture with increased wound healing ability, including terminating further culture and recovering the stem cell culture.

At this time, the LPA is preferably treated at a concentration of 0.1 to 100 μM, but is not limited thereto, and further culture after LPA is preferably performed for 1 to 4 days, but is not limited thereto. Meanwhile, a pretreatment step of removing serum components from the culture medium may be added prior to step iii. Between steps ii and iii, after a certain time after LPA treatment, the culture medium containing LPA is replaced with a medium not containing LPA. Removing the LPA from the culture medium may further include, but is not limited thereto.

The culture medium is preferably serum-free medium, but is not limited thereto.

Preferably, the stem cells are embryonic stem cells or adult stem cells, but are not limited thereto. The adult stem cells are preferably hematopoietic stem cells or mesenchymal stem cells, and more preferably, mesenchymal stem cells. It doesn't happen. The mesenchymal stem cells may be umbilical cord blood-derived stem cells, placental stem cells, adipose-derived stem cells, bone marrow-derived stem cells or peripheral blood-derived stem cells, cord blood-derived stem cells, adipose-derived stems, depending on the degree of invasiveness in harvesting More preferably, the cells or peripheral blood-derived stem cells are not limited thereto.

In addition, according to another embodiment of the present invention,

Iii) treating LPA to the stem cell culture medium before or during the culture; And

Ii) there is provided a method of increasing collagen content in a stem cell culture comprising culturing stem cells in a culture medium treated with LPA.

At this time, the LPA is preferably treated at a concentration of 0.1 to 100 μM, but is not limited thereto, and further culture after LPA is preferably performed for 1 to 4 days, but is not limited thereto. Meanwhile, a pretreatment step of removing serum components from the culture medium may be added prior to step iii, and after step ii, the LPA culture medium is exchanged by replacing the LPA-containing culture medium with a medium not containing LPA at a certain point after the LPA treatment. The step of removing from may further include, but is not limited to.

The culture medium is preferably serum-free medium, but is not limited thereto.

Preferably, the stem cells are embryonic stem cells or adult stem cells, but are not limited thereto. The adult stem cells are preferably hematopoietic stem cells or mesenchymal stem cells, and more preferably, mesenchymal stem cells. It doesn't happen. The mesenchymal stem cells may be umbilical cord blood-derived stem cells, placental stem cells, adipose-derived stem cells, bone marrow-derived stem cells or peripheral blood-derived stem cells, cord blood-derived stem cells, adipose-derived stems, depending on the degree of invasiveness in harvesting More preferably, the cells or peripheral blood-derived stem cells are not limited thereto.

As a result of animal experiments, the present inventors confirmed that LPA-treated stem cell cultures were more effective for wound treatment and skin improvement than untreated cultures (see FIGS. 2 and 3). In addition, it was confirmed that the result of increasing the content of collagen (see Fig. 4).

Therefore, the LPA-treated stem cell culture medium of the present invention can be supplied at an economical price without side effects, and it is not only concentrated in collagen and fibronectin, which are effective proteins for wound treatment, but also shows excellent effects in actual wound treatment. It can be seen that it can be effectively used as a wound healing agent.

According to the present invention, the cell culture solution obtained after treating LPA to stem cells not only shows an excellent effect on the treatment of wounds, but also has no side effects and can be supplied at an economical price. The collagen and fibronectin, which are effective proteins for wound treatment, are concentrated. By using the culture solution, re-epithelialization, angiogenesis, infiltration of inflammatory cells, etc., which are important processes in the wound regeneration process, are promoted. Therefore, the cell culture solution isolated after treating LPA to stem cells can be used as a wound treatment and cosmetic composition for skin improvement.

1 is a photomicrograph of mesenchymal stem cells derived from fat.
Figure 2 is a picture (A) and a graph showing the wound area (A) and the wound area is rapidly recovered when the culture of LPA-induced mesenchymal stem cells in the animal skin wound model.
Figure 3 is a control medium (top), which is a culture medium of mesenchymal stem cells not treated with LPA and culture medium (bottom) of mesenchymal stem cells induced by LPA after treatment in an animal skin wound model and photographed by taking the tissue of the wound site The tissue analysis picture.
4 is a result of Western blot analysis showing that the content of collagen type 1, an extracellular matrix protein, is increased in the cell lysate (A) and culture medium (B) of mesenchymal stem cells induced by LPA.

Definition of Terms

Hereinafter, terms used in the present document will be described.

Stem cells are the cells that form the basis of cells or tissues that make up an individual, and are referred to as undifferentiated cells that have the ability to differentiate into any specific or multiple functional cells and self-replicating ability to produce the same cells repeatedly. Can be. Stem cells can be largely divided into embryonic stem cells (ES cells) and adult stem cells (adult stem cells) depending on the possibility of differentiation.

Embryonic stem cells are stem cells isolated from the inner cell mass (ICM) that will develop into the fetus in the blastocyst embryo, which is a very early stage after the embryo is formed and before implantation into the endometrium. Endoderm, ectoderm and mesoderm It may be a pluripotent cell capable of differentiating into cells of all tissues produced by the three germ layers of.

On the other hand, ' adult stem cells ' are stem cells specific to each organ, which are obtained from the placenta at the stage where the embryonic process of adult development or embryo development progresses and each organ of the embryo is formed. It is a multipotent cell capable of only limited differentiation. These adult stem cells remain in most organs after adulthood and may serve to compensate for the loss of normal or pathological cells. Representative adult stem cells may be mesenchymal stem cells that are differentiated into hematopoietic stem cells present in bone marrow and connective tissue cells other than blood cells. Hematopoietic stem cells can be differentiated into various blood cells, such as red blood cells and white blood cells, and mesenchymal stem cells can be differentiated into osteoblasts, chondrocytes, adipocytes and myoblasts. Mesenchymal stem cells may be isolated from bone marrow, which is an important reservoir of mesenchymal stem cells, but may be difficult to harvest, and may be isolated and cultured in adipose tissue and the like. In the present invention, mesenchymal stem cells may be any cell having stem cell capacity, that is, differentiation capacity and proliferation capacity.

In the present invention, LPA is produced from activated platelets in wound tissue. LPA has been reported to play a role in promoting wound regeneration by increasing angiogenesis, fibroblast migration, and the like.

' Wound ' refers to a condition in which a living body is damaged, and pathological in which tissues that make up an internal or external surface of the living body, such as skin, muscle, nerve tissue, bone, soft tissue, internal organs or vascular tissue, are divided or destroyed. It covers the state. Examples of wounds include, but are not limited to, non-healing traumatic wounds, destruction of tissue by irradiation, abrasion, osteonecrosis, laceration, avulsion, and penetrating wounds. penetrated wounds, gun shot wounds, cuts, burns, frostbite, contusion or bruise, pressure sores, dry skin, keratosis, cracking, bursting, dermatitis, dermatitis Pain, surgical wounds, vascular disease wounds, corneal wounds, bedsores, ulcers, conditions related to diabetes and poor circulation such as diabetic skin erosion, chronic ulcers, sutures after plastic surgery, spinal injury wounds, gynecological wounds And chemical damage and acne, and may include damage to any part of the individual.

Extracellular matrix (ECM) is a complex collection of biopolymers that fills the intracellular or extracellular space and is present in large amounts in connective tissue, composed of molecules synthesized and secreted and accumulated by cells. Examples of the constituent molecules include fibrous proteins such as collagen and elastin, proteoglycan, glycosaminoglycna complex protein, fibronectin, laminin, and vitronectin. cell-attached glycoproteins such as (vitronectin).

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the present invention will be described in more detail.

The present invention provides a stem cell culture solution having improved wound healing ability prepared by culturing stem cells after treating LPA to a culture medium of stem cells before or during culture.

The stem cells are preferably obtained from humans.

In addition, the stem cells of the present invention include all cells having the potential to differentiate, and whether the stem cells are derived from an adult, embryo or derived from the fetus, preferably adult stem cells It may be, more preferably may be mesenchymal stem cells.

Unlike embryonic stem cells, adult stem cells are extracted from growing autologous body tissues, which not only avoids bioethical problems, but there is also no fear of immune rejection reactions. It has the advantage of being differentiated from embryonic stem cells. In addition, it is possible to perform self-renewal to recreate and store undifferentiated stem cells, which are required later, and to have tissue-specific differentiation ability to differentiate itself according to characteristics of surrounding tissues. can do.

The mesenchymal stem cells of the present invention is irrelevant to tissues or organs derived therefrom, preferably, may be extracted from fat, bone marrow, peripheral blood or umbilical cord blood, and more preferably, depending on the degree of invasiveness of the sampling method, It may be extracted from fat, peripheral blood or umbilical cord blood, most preferably from fat.

This suggests that adipose stem cells have almost the same or better multipotency and proliferative capacity as compared to bone marrow-derived stem cells, and are economical because they can obtain and culture sufficient stem cells in small amounts of adipose tissue from actual clinical utility. There is an advantage, and the actual procedure is painless and safe to compare with bone marrow harvesting. In addition, local anesthesia during the actual procedure can reduce the risk of general anesthesia, and the wound area of the patient heals within a week (Ogawa et al ., Curr. Stem Cell Res. Ther ., 1: 13-20, 2006) can also act as a factor in maximizing utilization and excellence of adipose stem cells.

Adipose stem cells of the present invention can be obtained by any method known to those skilled in the art. For example, adipose tissue may be obtained by liposuction (syringe or power-assisted liposuction), or by lipostomy, such as suction-assisted lipoplasty, ultrasound-assisted lipoplasty and extraction. Because stromal stem cells can be isolated from fat that has been removed from the patient and discarded by excisional lipectomy or a combination thereof, its usefulness is increased because no additional invasive procedure is required, and it is safe and inexpensive to separate stem cells. Can be.

In the present invention, the concentration of LPA to be treated in the stem cells is not particularly limited, but is preferably treated at a concentration of 0.1 to 100 μM. When treated below the concentration, the amount of effective protein in the cell culture may not show a wound healing effect, and when treated above the concentration, the concentration may be appropriate because LPA may affect the survival of stem cells. .

In the present invention, the treatment of stem cells with LPA is not particularly limited, but may be preferably performed for 1 to 4 days. If the treatment is less than one day, the secretion of protein in the cell culture may be less effective for wound healing, and treatment for more than four days may cause the death of stem cells, so it may be desirable to treat with the same number of days.

Inactivated mesenchymal stem cells lack the concentration of proteins that promote wound healing such as collagen and fibronectin secreted from the cells, and thus are not suitable for use as a wound healing composition, whereas stem cells treated with LPA of the present invention Using a culture of the concentration of the wound healing protein containing the protein is concentrated can exhibit a wound healing effect as soon as possible. That is, since the concentration of proteins that promote wound healing, such as collagen and fibronectin, is increased in the culture medium of stem cells treated with LPA, re-epithelialization and blood vessels, which are important processes in the wound regeneration process, are applied to the wound site. Angiogenesis, infiltration of inflammatory cells, etc. may be promoted to promote regeneration of skin wounds.

The wound of the present invention refers to a state in which a living body is damaged, and may include damage to any part of an individual such as tissues forming an internal or external surface of the living body.

The present invention also provides a composition for improving or treating wounds comprising the stem cell culture as an active ingredient. The composition can be used for pharmaceutical use or functional cosmetic use.

The active ingredient in the composition of the present invention may be contained in 0.1 to 100% by weight based on the total weight of the composition.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, the preparation of the pharmaceutical composition may be used as additives for the preparation of solid or liquid. The preparation additive may be either organic or inorganic.

Examples of the excipient include lactose, sucrose, saccharin, glucose, corn starch, starch, talc, sorbit, crystalline cellulose, dextrin, kaolin, calcium carbonate and silicon dioxide. Examples of the binder include polyvinyl alcohol, polyvinyl ether, ethylcellulose, methylcellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropylcellulose, hydroxypropylmethylcellulose, calcium citrate, Dextrin, pectin, and the like. Examples of the lubricant include magnesium stearate, talc, polyethylene glycol, silica, and hydrogenated vegetable oil. Any coloring agent may be used as long as it is usually allowed to be added to pharmaceuticals. These tablets and granules can be suitably coated with sugar (sugar coating), gelatin coating, and others as required. If necessary, preservatives, antioxidants and the like may be added.

The pharmaceutical compositions of the present invention may be prepared in any formulation commonly prepared in the art (e.g., Remington's Pharmaceutical Science, latest edition; Mack Publishing Company, Easton PA), and the form of the formulation is not particularly limited, Preferably it may be an external preparation. The external preparation of the present invention includes a sheet, a liquid coating, a spray, a lotion, a cream, a pape, a powder, a penetrating pad, a spray, a gel including a hydrogel, a pasta, a liniment, an ointment, an aerosol, a powder, a suspension. Formulations may include conventional external preparations such as transdermal absorbents. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania 18042 (Chapter 87: Blaug, Seymour), a prescription generally known for all pharmaceutical chemistry.

In one example of the invention, the composition may be applied directly to the wound. That is, it may be scattered on the wound site. In the form of a sheet, it is applied to the wound site, where appropriate dressing is applied to the applied site to protect the wound while preventing the therapeutic effect of the active ingredient from decreasing. The dressing can be any commercially available or commonly known. Examples of commercially available dressings include Compeel, Duoderm, Tagaderm and Opsite.

In the external preparations of the present invention, pharmaceutically acceptable carriers include hydrocarbons such as petrolatum, liquid paraffin, gelled hydrocarbons (also called plastibases), depending on the formulation thereof; Animal and vegetable oils such as medium chain fatty acid triglyceride, lard, hard fat and cacao butter; Higher fatty acid alcohols and fatty acids such as cetanol, stearyl alcohol, stearic acid and isopropyl palmitate and esters thereof; Water-soluble bases such as macrogol (polyethylene glycol), 1,3-butylene glycol, glycerol, gelatin, white sugar and sugar alcohol; Emulsifiers such as glycerin fatty acid esters, polyoxyl stearic acid and polyoxyethylene-cured castor oil; Pressure-sensitive adhesives such as acrylate ester and sodium alginate; Propellants such as liquefied petroleum gas and carbon dioxide; Preservatives, such as paraoxybenzoic acid ester, etc. are mentioned, The external preparation of this invention can be manufactured according to a conventional method using these. In addition to these, stabilizers, flavors, colorants, pH adjusters, diluents, surfactants, preservatives, antioxidants and the like may be blended as necessary. The external preparation of the present invention can be applied to the topical wounds by conventional methods.

In addition, the external preparation according to the present invention may be used by being adhered onto a solid support such as a wound peeling cover of a conventional band-aid. In an aspect of the present invention, the solid support is first coated with an adhesive layer to improve adhesion of the culture solution to the solid support. Examples of the pressure-sensitive adhesive may include polyacrylate and cyanoacrylate.

Many formulations of this type are commercially available, such as Smith & Nephew Ltd. with a non-adhesive wound release cover in the form of perforated plastic films, thin strips, patches, BAND-AIDs in the form of spots, plastic strips; Curity CURAD (Ouchless) Band-Aid from Colgate-Palmolive Co. (Kendall); And STIK-TITE elastic strips from American White Cross Laboratories, Inc .;

In one example of the present invention, the pharmaceutical composition according to the present invention may be formulated in the form of a liquid coating agent by mixing the culture powder and physiological saline in a constant volume ratio. As an example of the present invention, the pharmaceutical composition according to the present invention may be formulated in the form of an ointment by mixing the culture solution powder of the present invention and a water-soluble ointment and adding physiological saline thereto.

The pharmaceutically effective amount of the present invention may vary depending on the type of wound, the site of application, the number of treatments, the treatment time, the formulation, the condition of the patient, the type of adjuvant, and the like. The amount of use is not particularly limited, but usually The daily effective amount of the pharmaceutical composition of the present invention may be 1 to 50 μl / cm ² when the cell culture is applied to the wound, preferably 5 to 20 μl / cm ^2. have. The daily dose may be administered once a day or divided into two to three times a day at appropriate intervals, or may be administered intermittently at intervals of several days.

However, the amount of the pharmaceutical composition according to the present invention may be used in terms of the route of administration, the age, sex, weight of the patient, the severity of the patient, the type of wound, the site of application, the number of treatments, the treatment time, the dosage form, the condition of the patient, the type of supplement, and the like. The effective amount is not to be understood as limiting the scope of the present invention in any aspect because it is determined in view of various related factors.

In addition,

Iii) treating LPA to the stem cell culture medium before or during the culture;

Ii) culturing stem cells in a culture medium treated with LPA; And

Iii) providing a method for producing a stem cell culture with increased wound healing ability, including terminating further culture and recovering the stem cell culture.

The stem cell-derived species is not particularly limited in kind, but may preferably be derived from humans.

In addition, the stem cells are not particularly limited in kind, but may preferably be adult stem cells, more preferably mesenchymal stem cells.

In one example of the present invention, the mesenchymal stem cells may be extracted from fat, bone marrow, peripheral blood or umbilical cord blood. Among these, fat-derived stem cells extracted from fat are excellent in pluripotency and proliferative ability, a safe procedure, and economically sufficient stem cells can be obtained from a small amount of adipose tissue.

In one embodiment of the present invention, the method of isolating and culturing the stem cells, collagenase (collagenase), preferably after treatment of the discarded adipose tissue with HBSS (Hank's balanced salt solution) in patients undergoing subcutaneous fat removal Preferably it can be treated with 0.075% collagenase at 37 ° C. for 1 to 2 hours, preferably 30 minutes. After centrifugation, the pellet is resuspended by adding cell culture solution (alpha-minimum essential medium with 1-20% fetal bovine serum, 50-200 U / ml penicillin, and 50-200 μg / ml streptomycin). The cells isolated from the adipose tissue by collagenase can be obtained by passing through a nylon mesh. Cells that have passed through the nylon network may be plated in a culture dish to remove unattached cells after 18 to 36 hours, and the attached cells may be cultured while exchanging the cell culture solution.

As an example of the present invention, as a method for isolating and culturing the stem cells, the bone marrow obtained from the patient is centrifuged using a Ficoll-Hypaque gradient (density 1.077 g / cm ³ , Sigma, USA). After separation, the plate is plated in a culture dish, and then the cells attached to the culture dish can be proliferated and cultured while exchanging the cell culture solution.

In addition, the concentration of LPA to be treated in the stem cells of the present invention is not particularly limited, it may be preferred that the treatment at a concentration of 0.1 to 100 μM. When the concentration is less than the concentration of the effective protein in the cell culture solution may not exhibit a wound healing effect, the treatment may be appropriate because the concentration of the stem cells may be reduced by LPA when treated above the concentration.

In addition, the treatment period of the stem cells with the LPA is not particularly limited, but may be preferably treated for 1 to 4 days.

The present invention may further comprise the step of removing the serum components from the stem cells before the process of treating the stem cells with LPA.

Removing the serum component (serum component) in the stem cells may be carried out by a method commonly performed in the art, for example, the serum component can be removed by washing with HBSS (Hank's balanced salt solution).

In addition, the present invention may add the step of removing LPA from the culture medium by replacing the LPA-containing culture medium with the LPA-containing culture medium between the steps ii and iii, which is related to tumor formation and metastasis. In order to minimize side effects caused by LPA remaining in the medium.

On the other hand, in the method of the present invention, the culture medium used is preferably serum-free medium, but is not limited thereto. The use of serum-free medium is preferred, in the case of fetal bovine serum (FBS) contained in the medium may be a problem of prion infection that causes mad cow disease, undesired by the presence of non-human proteins contained in fetal bovine serum This is because an allergic reaction can be caused.

In addition, the method of the present invention may further include the step of removing the stem cells or stem cell debris remaining in the stem cell culture by centrifuging the recovered stem cell culture after step iii.

Furthermore, the present invention

Iii) treating LPA to the stem cell culture medium before or during the culture; And

Ii) providing a method of increasing collagen content in a stem cell culture comprising culturing the stem cells in a culture medium treated with LPA.

In the above method, the stem cell-derived species is not particularly limited in kind, but may preferably be derived from humans.

In addition, the stem cells are not particularly limited in kind, but may preferably be adult stem cells, more preferably mesenchymal stem cells.

In one example of the present invention, the mesenchymal stem cells may be extracted from fat, bone marrow, peripheral blood or umbilical cord blood. Among these, fat-derived stem cells extracted from fat are excellent in pluripotency and proliferative ability, a safe procedure, and economically sufficient stem cells can be obtained from a small amount of adipose tissue.

In one embodiment of the present invention, the method of isolating and culturing the stem cells, collagenase (collagenase), preferably after treatment of the discarded adipose tissue with HBSS (Hank's balanced salt solution) in patients undergoing subcutaneous fat removal Preferably it can be treated with 0.075% collagenase at 37 ° C. for 1 to 2 hours, preferably 30 minutes. After centrifugation, the pellet is resuspended by adding cell culture solution (alpha-minimum essential medium with 1-20% fetal bovine serum, 50-200 U / ml penicillin, and 50-200 μg / ml streptomycin). The cells isolated from the adipose tissue by collagenase can be obtained by passing through a nylon mesh. Cells that have passed through the nylon network may be plated in a culture dish to remove unattached cells after 18 to 36 hours, and the attached cells may be cultured while exchanging the cell culture solution.

As an example of the present invention, as a method for isolating and culturing the stem cells, the bone marrow obtained from the patient is centrifuged using a Ficoll-Hypaque gradient (density 1.077 g / cm ³ , Sigma, USA). After separation, the plate is plated in a culture dish, and then the cells attached to the culture dish can be proliferated and cultured while exchanging the cell culture solution.

In addition, the concentration of LPA to be treated in the stem cells of the present invention is not particularly limited, it may be preferred that the treatment at a concentration of 0.1 to 100 μM. When the concentration is less than the concentration of the effective protein in the cell culture solution may not exhibit a wound healing effect, the treatment may be appropriate because the concentration of the stem cells may be reduced by LPA when treated above the concentration.

In addition, the treatment period of the stem cells with the LPA is not particularly limited, but may be preferably treated for 1 to 4 days.

The method of the present invention may further include a pretreatment step of removing serum components from stem cells prior to step iii.

Removing the serum component (serum component) in the stem cells may be carried out by a method commonly performed in the art, for example, the serum component can be removed by washing with HBSS (Hank's balanced salt solution).

In addition, the present invention may add a step of removing the LPA from the culture medium by replacing the LPA-containing culture medium with the LPA-containing culture medium between the steps iii and ii, which is the LPA and the tumor formation and metastasis Because it is related, it is to minimize the side effects that can be caused by LPA remaining in the medium.

On the other hand, in the method of the present invention, the culture medium used is preferably serum-free medium, but is not limited thereto. The use of serum-free medium is preferred, in the case of fetal bovine serum (FBS) contained in the medium may be a problem of prion infection that causes mad cow disease, undesired by the presence of non-human proteins contained in fetal bovine serum This is because an allergic reaction can be caused.

The culture medium is concentrated protein that promotes wound healing, such as collagen, fibronectin, VEGF, SDF-1, the mass concentration of these proteins can be produced at a low cost by the production method of the present invention, which was conventionally produced at high production cost There is an advantage that it can.

In addition, when the culture solution prepared by the present invention is applied to the wound site, re-epithelialization, angiogenesis, infiltration of inflammatory cells, etc., which are important processes in the wound regeneration process, are promoted, so that the regeneration of the skin wound is excellent. Burn tissue, postoperative infection, surgical wound destruction, internal ulcers, bleeding, bone ulcers, pressure sores, ulcers, compromised cuts, non-healing traumatic wounds, cremation, after shaving, dental work, chronic ulcers (diabetes, varicose veins) , After stroke), destruction of tissue by irradiation, spinal injury wound, gynecological wound, chemical wound, vascular disease wound, diabetic skin erosion, diabetic foot, physical trauma, suture after plastic surgery, sunburn (sunburn) Or episiotomy, etc., but may be useful for improving or treating wounds in animals and humans for a variety of conditions.

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example  One: Intermediate lobe  Isolation of Stem Cells

Mesenchymal stem cells included in the composition of the present invention can be extracted from human fat or bone marrow. The present inventors isolated the stromal stem cells from the adipose tissue obtained from a patient who had undergone a fat removal surgery at Pusan National University Hospital. The isolated adipose tissue was washed with HBSS, and the adipose tissue was thinly cut using tweezers and scissors, and then treated with 0.075% collagenase at 37 ° C. for 30 minutes. After centrifugation, the pellet was resuspended by adding cell culture solution (alpha-minimum essential medium with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin), and then resuspended in 100 μm nylon mesh. Was passed to obtain cells isolated from adipose tissue by collagenase. Cells passed through the nylon net were plated in a culture dish to remove unattached cells after 24 hours. The cells attached to the culture dish were cultured while exchanging the cell culture solution (FIG. 1). Optionally, to separate the mesenchymal stem cells from the bone marrow, the bone marrow obtained from the patient was isolated by centrifugation using a picol-high park concentration gradient (density = 1.077 g / cm ³ , Sigma, USA), and then plate culture plates. After that, the cells attached to the culture dish can be proliferated and cultured while exchanging the cell culture solution.

Example 2: LPA Treatment and Isolation of Cell Cultures

Mesenchymal stem cells were proliferated and cultured in two different 15 cm plates to sub-confluent stages, and then washed twice with HBSS to remove serum components. Each mesenchymal stem cell was incubated with a culture medium (alpha minimum essential medium) containing 10 μM LPA and a culture medium (control) not included. After 48 hours, each culture was recovered and centrifuged at 3000 rpm for 10 minutes to remove cell debris.

Example 3: Experiment with Animal Wound Models

Six-week-old rats (Sprague Dawley Rats) were divided into two groups, respiratory anesthesia with 2% concentration of Enflurane, and the back waist hairs were removed using an electric clipper. Subsequently, the exposed area was disinfected with 70% ethanol, the wound was induced with an 8 mm biopsy punch, and the wound surface was cut out with scissors so as not to damage the muscle layer. Induced wound sites of the two groups of animals were treated with 10 μl of mesenchymal stem cell culture obtained by treating HBSS and LPA, respectively. On the 9th day, the wound site | part was visually observed (A of FIG. 2), and the area of the wound was measured (B of FIG. 2).

As a result, as shown in Figure 2, it was found that re-epithelialization occurred most actively in the group treated with LPA-induced culture. This result suggests that LPA-induced culture of mesenchymal stem cells promotes re-epithelialization of skin wounds.

Example 4: Histological Analysis

Rats subjected to the analysis in Example 3 were sacrificed using CO ₂ gas, and tissues around the wound were collected to perform histological analysis. Specifically, 4 μM-thick sections were made through general tissue processing and analyzed by hematoxylin and eosin (hematoxylin & eosin, H & E) staining and immunohistochemical staining. In order to confirm the degree of re-epithelialization, the results of H & E staining were taken under a microscope to measure the distance between both ends of the epithelial layer (FIG. 3).

As a result, as shown in Figure 3, compared to the H & E control group, the re-epithelialization of the wound tissue and the increase in the generation of granulation tissue in the wound tissue was more active under the conditions treated with the culture medium of LPA-induced mesenchymal stem cells. Could know.

Example 5: Collagen Expression Analysis in Stem Cells

In order to confirm the mechanism by which the wound treatment effect of the culture medium of LPA-induced mesenchymal stem cells such as Examples 3 and 4 is expressed, the mesenchymal stem cell lysate and the protein for the culture medium Expression patterns were analyzed. Specifically, after treating the mesenchymal stem cells with LPA for 48 hours, the stem cell lysate and the culture supernatant were recovered. Dilute stem cell lysate and supernatant proteins are electrophoresed according to the molecular weight of proteins using SDS-PAGE electrophoresis system, and then the separated proteins are transferred to nitrocellulose membranes (NC membranes) using electric pressure. After the membrane was reacted with 0.1% Tween-20 containing skim milk powder at a concentration of 5%, PBS solution (PBST) was added to block the protein-free membrane surface. Subsequently, in order to measure the content of collagen type 1 in the stem cell lysate and the culture medium, the type 1 collagen-specific antibody (Rockland, USA) was diluted 1: 1,000 and reacted with the membrane for 1 hour, followed by PBST solution. After washing three times, horseradish peroxidase-bound secondary antibody was reacted for 1 hour and washed three times with PBST solution. After adding a light-emitting reagent of wasabi peroxidase bound to the NC membrane, it was developed on an X-ray film to detect type 1 collagen in stem cell lysate and culture medium (FIG. 4). At this time, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as an internal standard protein.

As shown in FIG. 4, the amount of type 1 collagen in the stem cell culture solution (FIG. 4A) as well as in the stem cell culture medium (FIG. 4B) was increased compared to the control group. And it was found.

These results indicate that the improvement of wound healing ability of stem cell culture medium by LPA treatment is directly involved in wound treatment as well as the large secretion of substances such as growth factors that promote mesenchymal stem cell proliferation by LPA treatment. This suggests that expression of collagen, which is a major component of the extracellular matrix, and the promotion of secretion into the culture medium.

Claims (17)

Lysophosphatidic acid (hereinafter, abbreviated as 'LPA') was treated by mesenchymal stem cell culture medium before or during incubation, followed by culturing the mesenchymal stem cells. Mesenchymal stem cell culture with improved wound healing ability than mesenchymal stem cell culture medium (control). The method of claim 1,
The mesenchymal stem cells are human-derived mesenchymal stem cell culture. delete delete The method of claim 1,
The mesenchymal stem cells are extracted from fat, bone marrow, peripheral blood, or umbilical cord blood, mesenchymal stem cell culture. A wound improvement or treatment composition comprising the mesenchymal stem cell culture of any one of claims 1, 2 and 5 as an active ingredient. The method according to claim 6,
The composition is formulated in an external preparation form. Iii) treating LPA to the mesenchymal stem cell culture medium before or during the culture;
Ii) culturing the mesenchymal stem cells in LPA-treated culture medium; And
Iii) a method for producing mesenchymal stem cell culture with increased wound healing ability than LPA-treated mesenchymal stem cell culture medium (control), which comprises the step of terminating further culture and recovering the mesenchymal stem cell culture. The method of claim 8,
And removing serum components from the mesenchymal stem cell culture medium before step iii. The method of claim 8,
And removing mesenchymal stem cells or mesenchymal stem cell debris from the culture medium recovered after step iii. The method of claim 8,
The LPA is treated at a concentration of 0.1 to 100 μM, manufacturing method. The method of claim 8,
The LPA is treated for 1 to 4 days. The method of claim 8,
The mesenchymal stem cells are derived from human, manufacturing method. delete delete The method of claim 8,
The mesenchymal stem cells are extracted from fat, bone marrow, peripheral blood, or umbilical cord blood. Iii) treating LPA to the mesenchymal stem cell culture medium before or during the culture; And
Ii) a method of increasing collagen content in mesenchymal stem cell culture comprising culturing the mesenchymal stem cells in a culture medium treated with LPA.

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