TW201432048A - Composition containing growth factors and method of preparing thereof - Google Patents

Abstract

The present invention relates to a composition containing growth factors and the method of preparing the composition. The method includes the following steps: obtaining autologous blood; isolating a composition of the blood from the autologous blood; preparing a first cell culture medium by using the composition of the blood; culturing at least one autologous cells for 1 to 3 days, allowing the autologous cells to secrete a plurality of specific growth factors and forming a second cell culture medium; isolating the second cell culture medium; preparing the composition containing growth factors by using the second cell culture medium; wherein the growth factors includes vascular endothelial growth factor, hepatocyte growth factor, and the growth factors further includes keratinocyte growth factor.

Description

Growth factor preparation and production method thereof

The present invention relates to a growth factor preparation and a process for the preparation thereof, and more particularly to a growth factor preparation for promoting skin tissue regeneration.

A healthy human body has a well-established physiological mechanism of self-repair, especially a specific organ or tissue that is frequently metabolized, such as skin tissue or blood. Specific cells in these tissues have active division and replacement ability, which can support damage or aging in a timely manner. The organization is repaired. Among them, the most well-known are stem cells or epithelial cells with distinct differentiation or division ability. The above-mentioned principle is based on the manual method of obtaining autologous tissues of the same kind, such as skin, fat, and stem cells, to achieve clinical treatment or repair. In addition, the use of autologous tissue or materials eliminates the need for other types of somatic cell therapy, such as the infection of infectious diseases caused by allogeneic or xenogeneic therapies, or The risk of sequelae caused by immune rejection.

For the skin, it is an important line of defense against external environmental damage or pathogenic infections. Various types of injuries, such as burns, surgery, inflamed wounds, ulcers or long-lasting wounds (especially common in diabetic patients), Both pose a threat to human health, so accelerating wound healing has been an important medical issue for many years. On the other hand, "aging" is also a type of skin that tends to be weakened and unhealthy. Modern people pay more attention to appearance and often actively seek ways to delay skin aging. One of the ways is to promote the repair of epidermal tissue. Regeneration mechanism.

Early studies have found that in addition to coagulation function, platelets contained in blood plasma have a slightly acidic state from 7.0 to 7.2 to 6.5 to 6.7 in a specific environment (for example, increasing platelet concentration or decreasing pH). ), platelet stimulation can produce a large number of important growth factors required for skin growth and repair. These growth factors can effectively help repair damage, thereby accelerating wound healing, so as to reduce the formation of scar tissue. Therefore, current growth factors have been widely used in Bed treatment and treatment, including surgery, plastic surgery, orthopedics, dentistry and skin grafts. The treatment of autologous plasma is usually based on the principle of "rich in platelets in plasma", and plasma containing high concentrations of platelets is used as a material for treatment.

In addition to plasma, the "serum" in the blood is also of value for medical applications. Medical applications for autologous serum are mainly used in ophthalmic care, especially for ocular surface-related symptoms such as damaged cornea, poor corneal healing, corneal erosion or ulceration, and severe dry eye. The effective mechanism of serum for ophthalmic care is that its acid and alkali are similar to the composition of tears. In addition to basic nutrients such as protein, fat, electrolytes, immune proteins and vitamins, it also contains many growth factors that help the repair or regeneration of eyeball epithelial cells. Factors such as factors and cytokines are safer and more effective than medications (such as steroids or various anti-inflammatory drugs that cause side effects or derivatization adverse reactions).

For medical applications, the difference between plasma and serum is that plasma is rich in platelets, but also contains electrolytes, lipids, proteins, sugars and other components. The electrolyte contains sodium, potassium, calcium, magnesium and phosphorus. And a variety of trace metal ions, these electrolyte components may affect the physiological functions of cells; a variety of proteins in plasma, such as "plasma protein" is commonly used in the collectively called plasma proteins (can be roughly divided into albumin, globulin and fibrinogen ), as well as a series of coagulation factor proteins, fibrinogen and hydrolase related to the blood coagulation mechanism. As for the biggest difference between serum and plasma, the serum must be obtained after blood coagulation, so the serum does not have fibrinogen, and contains a small amount of substances released by platelets during blood coagulation, which is often used clinically as a biochemical value and immune-related test. Test material. In addition, serum is rich in a variety of serum proteins, antibodies, and plays an important role in the individual's immunity, while other trace components such as hormones or protease inhibitors are essential to maintain metabolism and constant.

The mechanism of skin wound healing can be roughly divided into Haemostasis, Inflammation, Proliferation and Remodeling. The cells involved in each period are different, and the role and importance of growth factors are also There are differences. For example, the coagulation phase and the inflammatory phase are mainly involved in platelets, macrophage and neutrophil; the main proliferative phase is macrophage, lymphocytes (Lymphocyte). ), fibroblasts, epithelial cells, and endothelial cells; the main involved in maturity is fibroblasts. Among them, "fibroblasts" play an important role in the mechanism of wound healing, and the wounds produce early platelets and Macrophages secrete chemotactic substances (Cytokine or Chemokine) and growth factors, stimulate mesenchymal cells to differentiate into fibroblasts, and promote fibroblasts to produce collagen and induce angiogenesis (Angiogenesis) . The fibroblasts in turn produce the collagen fibers necessary for wound healing, increasing the elasticity and tension of the wound, forming scar tissue, entering the mature stage, and the collagen fibers in the scar tissue continue to increase and continue to reorganize or regularize, making Scar resilience increases, and the entire maturity process can last for months or even years. On the whole, fibroblasts have the function of being stimulated by growth factors and secreting growth factors, which is an indispensable part of the signal transduction mechanism caused by growth factors.

With regard to the skin aging mechanism, it also involves complex physiological regulation mechanisms, which is a continuous and long process. In the process of skin aging, fibroblasts also play an important role. In short, fibroblasts are an important role in the maintenance of connective tissue health in the skin. The secreted collagen can maintain epidermal growth and repair, which produces degradation. Connective tissue protein lipid-related hydrolase, such as matrix metalloproteinase (MMP), and stimulation of various growth factors causes fibroblasts to secrete connective tissue proteins or hydrolases in a timely manner, and feedback secretory growth factors to regulate peripheral cells. The skin maintains normal hyperplasia and metabolic function. However, as the age increases, the collagen production of fibroblasts gradually decreases, and the proportion of soluble collagen is also reduced. Instead, insoluble collagen or mutated collagen causes the skin to lose its elasticity. Start aging such as wrinkles. Therefore, the current medical and cosmetic industry is generally aimed at "stimulating fibroblast-proliferating collagen" or "slowing down the decomposition of collagen" and actively seeking more effective anti-aging methods.

For the treatment of damaged skin or wounds, supportive therapy has traditionally been used, that is, supplemental nutrition, adequate rest, dressing care, antibiotics, etc., so that patients rely on their own immunity to fight against pathogens and complete repair mechanisms. And healed. With scientific research on growth factors and mechanisms that promote skin regeneration and repair, growth factors are increasingly being used to promote wound healing.

There are a variety of growth factors associated with wound repair or maintenance of normal skin function, including Epidermal Growth Factor (EGF), Platelet Derived Growth Factor (PDGF), and Basic Fibroblast Growth (basic). Fibroblast growth factor (bFGF), insulin-like growth factor (insulin-like growth) Factor, IGF), Keratinocyte Growth Factor (KGF, its alias FGF-7 or FGF-10), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) ) and many other factors, not one by one. The various growth factors produce different influences on the tissue microenvironment of the wound state in different periods, and the final physiological function is to repair the wound and achieve healing effect. In the process of wound healing, a single growth factor cannot achieve the above effects, but must be coordinated by various growth factors to complete.

Among the above growth factors, keratinocyte growth factor (KGF) has an important influence on the epithelialization of wound healing. This phenomenon refers to the proliferative and mature stages of wound healing, and epithelial cells. (Epithelial cell) The phenomenon of proliferation and migration causes the proliferating keratinocytes to adequately cover the wound tissue to form a protective membrane for the wound. According to the research, the secretory source of KGF in wound tissue is mainly keratinocytes, and the message transmission pathways mainly include TGF-α and TGF-β conduction pathways, which promote fibroblasts, myoiibroblasts, masculin fibers. Proto-myofibroblasts synergistically produce an extracellular matrix (ECM).

Vascular endothelial growth factor (VEGF), as its name suggests, is closely related to the "angiogenesis" process during wound repair. It is specific for vascular endothelial cells (Endothelium cells), which can induce angiogenesis and affect lymphangiogenesis and differentiation. It is also an important signal-conducting molecule, which dominates the proliferation, migration, growth and longevity of endothelial cells, and it also regulates vascular permeability. These mechanisms play an important role in the process of wound healing. In addition, more studies have pointed out that VEGF can help treat ischemic heart disease (see Journal of the American College of Cardiology), promote hair follicle health and hair production (see Yano et al., The Journal of Clinical Investigation. 107:409 -417; 2001), treatment of degenerative neurological diseases (see Wang et al., The Journal of Neuroscience. 27 (2): 304-307; 2007 and Yang et al., The Journal of Neurochemistry. 93: 118-127; 2005) Equivalent.

The aforementioned hepatocyte growth factor (HGF) also plays an important role in the wound healing mechanism, which can accelerate the healing process of wounds. In wound tissue, the main source of hepatocyte growth factor includes fibroblasts, while the c-Met receptor of hepatocyte growth factor is mainly distributed in keratinocytes, so hepatocyte growth factor The mechanism is mainly to attract neutrophils, monocytes and mast cells to the wound, thereby promoting the migration of endothelial cells to the wound and promoting the migration and proliferation of keratinocytes. To speed up the progress of the repair mechanism. It is noteworthy that hepatocyte growth factor differs in the mechanism of action between chronic and acute wounds (see Current Signal Transduction Therapy. 6(2): 152-155; 2011), especially in chronic wounds. This phenomenon shows that hepatocyte growth factor is more important for the repair mechanism of chronic wounds (common in diabetic patients). The study also pointed out that hepatocyte growth factor is a necessary growth factor for wound healing process, which has obvious effects on the repair and regeneration of neovascularization and Granulation tissue (see Yoshida et al., Journal of Investigative Dermatology. 120(2): 335-43; 2003). In addition to the above functions, more studies have pointed out the role of hepatocyte growth factor in other physiological repair mechanisms. For example, hepatocyte growth factor can help slow the progression of emphysema by inhibiting alveolar cell death and promoting alveolar cell regeneration ( See Shigemura et al., Circulaton. 111: 1407-1414; 2005); hepatocyte growth factor can slow chronic renal fibrosis by inhibiting the activation of myo-fibroblast and antagonizing the TGF-β signaling pathway. The process (see Liu, American Joumal of Physiology-Renal Physiology); hepatocyte growth factor can reduce the spinal cord nerve damage caused by autoimmunity by regulating mesenchymal stem cells, and has the potential to treat multiple sclerosis; Recent studies have also reported that hepatocyte growth factor can effectively ameliorate subcutaneous ischemic ulcers caused by peripheral arterial occlusive disease (see Makino et al., Arteriosclerosis, Thrombosis, and Vascular Biology 32: 2503-2509; 2012).

Fibroblasts are found throughout the body and can be obtained from skin tissue. They have the property of being easy to culture and secrete collagen, which is very important for maintaining skin health. In addition to secreting collagen, fibroblasts also have the ability to secrete growth factors, such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) transforming growth factor (Transformation growth factor-β, TGF). -β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), etc., and retains this property under in vitro culture conditions.

Adipose derived stem cells (ADSC) are a group of stem cells isolated from fat cells. They have significant multipotent and can differentiate into many other cells other than fat cells, such as hard bone, cartilage, nerve cells, etc. ,because This is widely used in tissue repair and cell regeneration. Studies have reported that adipose-derived stem cells have the effect of inhibiting inflammatory response, promoting angiogenesis, promoting fibroblast proliferation and secreting collagen, and adipose-derived stem cells can differentiate into epithelial cells and secrete various growth factors, which can initiate or strengthen physiological responses of cells. Including proliferation or migration to promote wound healing and repair skin aging (wrinkles).

In response to the above-mentioned demand for growth factors, a large number of human growth factors must be obtained, and at present, genetic engineering methods are mainly used to produce growth factors. that is. The gene of the growth factor is transferred into a vector expressing the corresponding growth factor protein, and then purified and obtained. However, although the above genetic engineering method can reduce the cost of producing growth factors, it has the following disadvantages: (1) the number of genes that can be loaded and expressed by the current gene transfer method (ie, the number of genes inserted into the vector) and the scale (the gene fragments are Limited nucleotide length): If a single vector is used to express multiple growth factor genes, the difficulty in operation is significantly improved. For example, when three or more genes are simultaneously expressed, it is difficult to ensure their normal performance and corresponding activity; The expression of growth factors makes the process of plastid preparation and protein expression more complicated. (2) When the above-mentioned transgenic genes use prokaryotic or xenogeneic organisms, the protein of the growth factor protein produced by them has poor activity or different purity, which weakens the practical effect of the growth factor, and even worse. If the protein extract of the growth factor protein contains endotoxin or other impurities, it will not only affect the efficacy, but also most likely cause the negative effects of allergic reactions.

Due to the shortcomings of the above-mentioned methods for obtaining growth factors, people are gradually turning to a more desirable source of growth factors, that is, naturally occurring growth factors for the human body, among which materials derived from autogenous sources are preferred. Under the premise of safe operation, autologous biomaterials can avoid the immune rejection caused by allogeneic biological materials, and avoid the risk of infectious diseases (such as hepatitis and AIDS). The most readily available biological material in the human body is "blood". Therefore, the current pharmaceutical industry often uses blood to obtain a source of growth factors and uses growth factors to promote wound healing and anti-aging.

In view of the above-mentioned lack of conventional methods and needs, the present invention provides a method for producing a high concentration growth factor preparation from blood and a growth factor preparation prepared by the method.

The present invention provides a method for producing a growth factor preparation, which comprises The following steps: (1) obtaining an autologous blood; (2) separating a blood composition from the autologous blood; (3) preparing a first cell culture solution from the blood composition; (4) first The cell culture medium cultures at least one autologous cell, and the culture time is between 20 and 72 hours, so that the autologous cells secrete a plurality of growth factors into the first cell culture solution to form a second cell culture solution; (5) Taking out the second cell culture solution; and (6) preparing the second cell culture solution into a growth factor preparation, the growth factor comprising at least one vascular endothelial growth factor and one hepatocyte growth factor. The concentration of vascular endothelial growth factor contained in the second cell culture medium is between 150 and 1000 pg/ml; and the concentration of hepatocyte growth factor is between 800 and 3000 pg/ml.

The blood composition in the above production method of the growth factor preparation contains serum, and the component of the first cell culture solution contains 90% or more of serum. In the above method for producing a growth factor preparation, the growth factor secreted by the autologous cell may further comprise a keratinocyte growth factor, and the concentration of the keratinocyte growth factor contained in the second cell culture solution is between 20 and 45 pg/ml.

The autologous cells used in the above method for producing a growth factor preparation may be selected from autologous fibroblasts derived from the same individual, autologous adipose stem cells, or mixed cells composed of autologous fibroblasts and autologous stem cells. .

The filtration step may be further included after the step (5) or the step (6) of the above production method of the growth factor preparation.

A growth factor preparation prepared by the method for producing a growth factor preparation according to any one of the foregoing, the growth factor preparation comprising a plurality of growth factors, the growth factor comprising at least a vascular endothelial growth factor and a hepatocyte growth factor, The concentration of vascular endothelial growth factor is between 150 and 1000 pg/ml; the concentration of hepatocyte growth factor is between 800 and 3000 pg/ml.

The present invention provides a growth factor preparation prepared by the aforementioned growth factor preparation production method, the growth factor preparation comprising a plurality of growth factors, the growth factor comprising a vascular endothelial growth factor and a hepatocyte growth factor, wherein The concentration of vascular endothelial growth factor is between 150 and 1000 pg/ml; and the concentration of hepatocyte growth factor is between 800 and 3000 pg/ml.

The above growth factor preparation may further comprise a keratinocyte growth factor, and the keratinocyte growth factor concentration is between 20 and 45 pg/ml.

The invention proposes a combination system for promoting skin tissue regeneration And a growth factor preparation prepared by the method for producing a growth factor preparation, comprising a plurality of growth factors, wherein at least one vascular endothelial growth factor and one hepatocyte growth factor; a buffer; and a nutrient, The composition comprises a glucose and a plurality of electrolytes.

Based on the spirit of the above-described growth factor preparation production method, growth factor preparation and combined preparation for promoting skin tissue regeneration, one of the objects of the present invention is to provide a simple and effective method for producing a growth factor preparation. The growth factor preparation prepared by the method, and the combined preparation for promoting skin tissue regeneration by using the aforementioned growth factor preparation, having various high concentrations of natural growth factors, and the above growth factor preparation and its derivative products (for example, The combined preparations are safer than genetically engineered growth factors.

Another object of the present invention is that the material used in the production method of the growth factor preparation of the present invention is an Autologous material, so that the risk of infection and immune rejection caused by allogeneic transplantation can be eliminated.

Another object of the present invention is to produce a growth factor preparation method according to the present invention, which uses an autologous stimulator (including autologous serum or related preparation) to culture autologous cells, and promotes autologous cells to secrete various growth factors containing autologous sources. The self-contained growth factor is produced by simulating the natural human environment, and is prepared into a preparation, so that the preparation can highly exert a good healing or delay aging effect without causing adverse reactions such as toxicity and allergy due to the purity of the preparation or individual differences.

A further object of the present invention is that the preparation prepared by the method for producing a growth factor preparation of the present invention can be injected into the body to achieve a better healing or repairing effect, thereby achieving repair or protection. Anti-aging target.

According to the above object of the present invention, a further object of the present invention is to provide a preparation prepared by the method for producing a growth factor preparation of the present invention, based on the simple preparation process, high safety, and the advantages of using the injection to give the patient self-body. And based on the characteristics of the specific growth factor, further applying the growth factor preparation production method and the growth factor preparation to a condition other than skin tissue wound or aging, such as nerve damage repair, kidney repair or treatment for ischemic heart disease .

In combination with the technical features and the object of the invention provided by the present invention, the present invention provides a growth factor preparation component and a production method thereof, which can autologously culture autologous cells to cause autologous serum to stimulate a large amount of growth of autologous cells. Factor, especially vascular endothelium Long-factor, hepatocyte growth factor, and keratinocyte growth factor concentrations are most prominent, and growth factor preparations rich in autologous growth factors are produced to obtain highly safe medical or cosmetic products.

In addition, according to the spirit of the present invention, a buffer or a nutrient may be further added according to the above-mentioned growth factor preparation according to a wider range of uses and needs to produce a combined preparation for promoting skin tissue regeneration, providing a wider medical treatment. Use with beauty care.

1 is a data chart showing changes in growth factor content in a growth preparation prepared according to the method for producing a growth factor preparation of the present invention, showing a first embodiment of the present invention using autologous adipose stem cells, and a production method for a growth factor preparation according to the present invention. A change in the concentration of a particular growth factor in the prepared growth factor formulation.

Fig. 2 is a graph showing the change in the concentration of vascular endothelial growth factor in a growth factor preparation prepared by using autologous adipose stem cells through the growth factor preparation production method of the present invention, according to the data sheet of Experimental Example 1 of the present invention.

Fig. 3 is a graph showing the change in the concentration of hepatocyte growth factor in a growth factor preparation prepared by using autologous adipose stem cells through the growth factor preparation production method of the present invention, according to the data sheet of Experimental Example 2 of the present invention.

Fig. 4 is a graph showing the change in the concentration of keratinocyte growth factor in a growth factor preparation prepared by using autologous adipose stem cells through the growth factor preparation production method of the present invention, according to the data sheet of Experimental Example 3 of the present invention.

Figure 5 is a data plot showing changes in growth factor content in a growth preparation prepared according to the method for producing a growth factor preparation of the present invention, showing the second embodiment of the present invention using autologous fibroblasts, produced by the growth factor preparation of the present invention. The variation of specific growth factor concentration in the growth factor preparation prepared by the method.

Fig. 6 is a graph showing the change in the concentration of vascular endothelial growth factor in a growth factor preparation prepared by using autologous fibroblasts through the growth factor preparation production method of the present invention, according to the data sheet of Experimental Example 4 of the present invention.

Fig. 7 is a graph showing the change in the concentration of hepatocyte growth factor in a growth factor preparation prepared by using autologous fibroblasts through the growth factor preparation production method of the present invention, according to the data sheet of Experimental Example 5 of the present invention.

The present invention will be understood by those skilled in the relevant art, and the present invention will be described in the following description. The preferred embodiments are further illustrated. The drawings referred to in the following texts are illustrative of the features of the present invention and are not required to be completely drawn according to the actual situation; and the description of the embodiments of the present invention relates to the technical contents well known to those skilled in the art, nor Let me repeat them and explain them first.

The functions and applications based on the present invention are mainly for medical and cosmetic purposes, so the object to be sampled is referred to as "patient", and the material from the same patient (ie, the same individual) is regarded as "Autologous" material.

The present invention provides a growth factor preparation and a method of producing the same. The method for producing a growth factor preparation comprises the following steps:

(1) Obtain an autologous blood.

(2) A blood component is separated from the autologous blood.

(3) Preparing a first cell culture fluid with a blood composition.

(4) cultivating at least one autologous cell in the first cell culture medium, the culture time is between 20 and 72 hours, allowing the autologous cells to secrete a plurality of growth factors into the first cell culture solution to form a second cell culture solution. .

(5) Take out the second cell culture solution.

(6) preparing a second cell culture solution into a growth factor preparation, the growth factor comprising at least a vascular endothelial growth factor and a hepatocyte growth factor.

The following steps are explained one by one for the above steps: firstly, according to steps (1) and (2), autologous blood is obtained from the patient's vein by blood drawing, and the whole blood drawing process is facilitated to obtain a specific blood composition, ie, serum. And the blood drawing equipment, do not use any anticoagulant, so that the blood taken out of the body naturally initiates the blood coagulation mechanism to complete the process of blood agglutination. The volume of blood drawn at this stage is preferably between 150 and 200 ml, in order to obtain the charge without affecting the immediate health status of the patient. The amount of blood in the foot. After obtaining autologous blood, the blood was dispensed into a sterile 50 ml centrifuge tube and allowed to stand at room temperature for 1 hour to allow the blood to be fully completed. Next, the blood is transferred to an environment of 4 ° C for storage, and the storage time is preferably within 48 hours, that is, the blood must be treated continuously within 48 hours to avoid affecting the preparation effect.

Next, blood was taken out from the environment at 4 ° C, and centrifuged at a centrifugal speed of 1000 g, and the centrifugation time was 10 minutes. Blood stratification is more clearly observed after the centrifugation procedure is completed. Continue to separate the supernatant according to the stratification, and obtain the blood composition used in the production method of the growth factor preparation of the present invention, that is, serum (hereinafter referred to as "autologous serum"), and the volume thereof accounts for about the volume of the original blood volume. 50%.

After obtaining the autologous serum, the serum may be initially treated according to the subsequent application requirements. The more commonly used method is to filter the filter device or membrane with a pore size of 0.22 μm to remove the pathogen which may be present in the serum or The impurities produced during the separation of the supernatant are maintained in a sterile state. Therefore, according to the above objectives, other sterilization means can replace the filtering equipment of the present invention as long as the target of removing impurities or sterility can be achieved, provided that the sterilization means which may significantly affect the composition and function of the serum should be avoided.

The autologous serum obtained in the above steps (1) and (2) is further subjected to the step (3), and the first cell culture solution is prepared from the autologous serum. Specifically, the first cell culture fluid used herein can be used for primary culture and subculture of autologous cells; however, the first cell culture fluid can not only fully maintain the physiological function of the autologous cells, but also A more important function is to provide a nutrient-rich and stimulating culture environment to stimulate autologous cells to secrete or produce a specific type of growth factor in response to external stimuli.

The first cell culture solution used in the present invention is composed of a certain proportion of autologous serum and a specific cell culture medium or an isotonic buffer solution, and the autologous serum can be used as an isotonic buffer according to the needs of the user (as shown in Table 1). The column buffer is diluted or added with the addition to obtain the first cell culture solution described in the step (3), so that the components of the first cell culture solution contain 90% or more of autologous serum, wherein the whole serum is cultured. For the preferred embodiment. Further, the present invention is not particularly limited to components other than autologous serum, and is preferably an isotonic buffer or a serum-free cell culture medium which is complete and safe, and the medium is selected according to the type of autologous cells. Different.

According to the step (4), the first cell culture medium is cultured in the autologous cells. The first cell culture solution is added to a culture flask containing the autologous cells, and cultured, so that the autologous cells have sufficient time and stimulation conditions to secrete a plurality of specific growth factors into the first cell culture solution to A second cell culture medium enriched with a specific growth factor is formed. The above-mentioned culture process, if the overall time is too short, will not be sufficient for the autologous cells to fully secrete growth factors; but the excessive culture time is easy to accumulate cell metabolites in the second cell culture medium, and may derive growth factor drop. The problem of solution or decay, so the preferred culture time is between 20-72 hours. Wherein, before the addition of the first cell culture solution, the fullness of the culture of the autologous cells is preferably 5 to 7 percent, which is a period in which the growth of the autologous cells is relatively stable and the physiological function is active, so that the cells are accepted. After the culture of the first cell culture medium, growth can be continued, and at the same time, a large amount of specific growth factors of high quality are sufficiently secreted.

The above autologous cell line is selected from cells rich in growth factor-producing cells, such as various stem cells, without departing from the scope of the present invention. In the present invention, autologous fibroblasts or autologous adipose stem cells are preferred embodiments. In addition, the scope of the present invention also covers a mixed cell population co-cultured by autologous fibroblasts and autologous adipose stem cells, and the mixing ratio thereof can be adjusted according to the application needs of the user.

In the above culture process, the autologous cells will be stimulated by the first cell culture medium to densely and largely secrete specific growth factors, and are released into the liquid culture environment. Therefore, according to the step (5), the culture solution after the culture is collected to obtain a second cell culture solution rich in a specific growth factor.

According to the step (6), the growth factor preparation can be prepared directly by using the second cell culture solution, or the growth factor concentration can be raised again after the second cell culture solution is passed through the concentration program according to the requirement, and then the growth factor preparation is prepared. By "concentration" herein is meant a means of increasing the concentration of growth factors in a second cell culture medium. In a broad sense, it may also cover specific purification means, such as removal of metabolites or potential pathogens by filtration or dialysis. Wait. With regard to the concentration method, for example, column chromatography purification and concentration or other dialysis methods which are readily available to those skilled in the art can be applied at this stage. Meanwhile, after the foregoing step (5) or (6), a filtering step may be added according to the user's demand to maintain or enhance the quality of the final produced growth factor preparation.

The growth factor prepared by the above steps is mainly rich in growth factors including vascular endothelial growth factor and hepatocyte growth factor. However, depending on the use of different types of autologous cells, vascular endothelial growth factors and hepatocyte growth factors such as keratinocyte growth factors can be produced. The vascular endothelial growth factor and hepatocyte growth factor produced by the method of the present invention are significantly increased with the culture time to obtain a growth factor preparation rich in vascular endothelial growth factor and hepatocyte growth factor. For example, a second cell culture fluid obtained by the method for producing a growth factor preparation of the present invention, which comprises vascular endothelial growth factor The concentration ranged from 150 to 1000 pg/ml and the concentration of hepatocyte growth factor ranged from 800 to 3000 pg/ml, which was much higher than the concentration of the above two long factors in autologous serum.

The growth factor component of the growth factor reagent obtained by the growth factor preparation production method of the present invention is related to the selected autologous cells, and according to different kinds of autologous cells, a growth factor reagent rich in various growth factors can be prepared. . Therefore, according to the foregoing steps, the second cell culture solution prepared by the method for producing a growth factor preparation of the present invention may further comprise keratinocyte growth factor at a concentration of 20 to 45 pg/ml.

According to the above production method of the growth factor preparation, a growth factor preparation which is rich in various growth factors can be obtained. Specifically, when autologous adipose stem cells are used, a growth factor preparation particularly rich in vascular endothelial growth factor, hepatocyte growth factor and keratinocyte growth factor can be produced; and when the autologous fibroblast is used to carry out the invention A growth factor preparation production method for producing a growth factor preparation particularly rich in vascular endothelial growth factor and hepatocyte growth factor. The concentration of vascular endothelial growth factor in the above growth factor preparation is between 150 and 1000 pg/ml; the concentration of hepatocyte growth factor is between 800 and 3000 pg/ml; and the concentration of keratinocyte growth factor is between 20 and 45 pg/ml.

After obtaining the above growth factor preparation, it can be directly stored in a frozen manner until use, and the method can be administered by injection (including local injection or intravenous drip) or external application to a patient for treatment; and the growth factor can also be concentrated and stored after lyophilization; The growth factor preparation can be further processed into a preparation combination, so that the growth factor preparation production method and the corresponding growth factor preparation proposed by the invention can be more widely used in medical and cosmetic maintenance.

Therefore, the growth factor preparation prepared according to the above production method of the growth factor preparation is widely used, so that the user can select not only the above-mentioned growth factor preparation to medical or cosmetic maintenance use, but also the above-mentioned growth factor preparation. Concentration, purification, or various processing procedures are performed to produce a combined preparation for promoting skin tissue regeneration. The composition of the combined preparation for promoting skin tissue regeneration comprises the above growth factor preparation, buffer and nutrient. Wherein, the growth factor preparation comprises at least vascular endothelial growth factor and hepatocyte growth factor, and may further comprise keratinocyte growth factor. The buffer of the present invention is not particularly limited as long as it is a body fluid compatible buffer (especially an isotonic solution) can be used in the combined preparation of the present invention; It mainly contains glucose and a plurality of electrolytes, wherein the electrolyte contains sodium, potassium, calcium, magnesium, etc., and the preferred glucose concentration is The weight ratio is 5%. The average osmotic pressure range of human body fluid is about 300 mOsm/L, and the exact measurement value varies slightly with individual physiological state (such as electrolyte loss) (positive and negative deviation value is about 30). For example, the buffers/infusions or other buffers similar to those listed in Table 1 are suitable for the combined preparation for promoting skin tissue regeneration according to the present invention.

In order to fully explain the method of the present invention, the following examples are given and described below.

First embodiment:

A growth factor agent production method is produced by using Adipose derived stem cells (ADSC) according to the growth factor preparation production method of the present invention. First, the flow of obtaining autologous adipose stem cells will be described. The adipose stem cells according to the present invention generally refer to multifunctional cells which are isolated from adipocytes and have significant differentiation ability. The method for obtaining autologous adipose stem cells according to the present invention adopts an adipocyte obtained from a patient's own body, and the source thereof is mainly through fat extraction surgery. The overall process of obtaining autologous adipose stem cells should be carried out in an aseptically operated environment.

Specifically, after obtaining a lipoaspirate in which a variety of cells and connective tissues are mixed (about 300 ml is sufficient), the blood and adipose tissue are sufficiently layered after standing, and then adipose tissue is obtained and a rinsing process is performed to HBSS buffer (add antibiotics or antifungal agents as needed) to repeatedly rinse the adipose tissue by adding enough HBSS buffer and letting it stand for 1 to 5 minutes to float the adipose tissue in the HBSS buffer, then extract it with a pipette. Remove the HBSS and repeat the above steps several times. The main purpose of this rinsing procedure is to wash away the blood cells in the extract first.

Then, the freshly prepared collagen hydrolase (collagenase) solution is added to the washed adipose tissue and mixed slightly, and the connective tissue is subjected to digestion for about 30 to 60 minutes at 37 ° C to separate the mixture. Processed lipoaspirate cells (PLA cells). The cell mixture obtained by hydrolysis (the appearance is in an emulsified state) is also often referred to as a stromal vascular fraction (hereinafter referred to as SVF), which comprises autologous adipose stem cells. Next, after the above SVF was centrifuged at 400 g for 10 minutes, the adipose tissue which was not sufficiently hydrolyzed was removed. The SVF was then dispensed and the SVF was again treated with red blood cell lysate to adequately remove red blood cells. Continue to remove cell clumps and impurities by multiple centrifugation procedures, and The cells were initially isolated by gradient cell separation. Since the cells obtained at this stage are still mixed with other cells such as endothelial cells or white blood cells, the cell sorting method is continued, and specific cell type markers such as CD31 are used for endothelial cells. It is labeled for its cells; white blood cells are labeled with CD45 and identified using specific antibody antigen binding methods, thereby isolating and removing endothelial cells or white blood cells. The autologous adipose stem cells referred to in the present invention can be obtained by the above method. After obtaining the autologous adipose stem cells, the normal cell subculture method is adopted to maintain the normal growth of the autologous adipose stem cells, and the autologous adipose stem cells are used to prepare and grow the growth factor preparation production method according to the present invention. Factor preparation.

Before performing the growth factor preparation production method of the present invention, the autologous adipose stem cells of the same patient should be obtained in advance according to the above-described procedure for obtaining autologous adipose stem cells. According to the autologous adipose stem cells obtained by the above-mentioned embodiment, the preparation process of the growth factor preparation is as follows: 200 ml of venous blood is collected from the patient, and the device is placed in a 50 ml centrifuge tube, and left at room temperature for 1 hour, and then stored in a refrigerator at 4 ° C. spare. Next, the blood was taken out and centrifuged at 1000 g for 10 minutes to obtain a layered blood sample. The supernatant is taken from the blood sample and is serum. Depending on the needs of use, the serum is diluted with an appropriate buffer or as a serum stock solution, and used as a first cell culture solution, and used in subsequent steps. The above first cell culture solution was filtered through a filter bowl having a pore size of 0.22 μm. Next, an autologous adipose stem cell cultured in a cell culture flask was taken, and the cell growth degree was 5-7 minutes. The culture medium of the autologous adipose stem cells is replaced with the first cell culture medium by adding a sufficient amount of the first cell culture medium to the culture flask (for example, a 175T culture flask uses about 30 ml of the first cell culture solution). Continue to culture for 1-3 days to obtain a second cell culture medium. When undiluted serum is used as the first culture solution, serum (second cell culture solution) rich in vascular endothelial growth factor, hepatocyte growth factor, and keratinocyte growth factor is obtained at this time. After the second cell culture solution is obtained, it can be directly used as a growth factor preparation, and stored in a freeze-drying manner; or a buffer solution and a nutrient can be further added to the growth factor preparation to prepare a combined preparation for promoting skin tissue regeneration.

The growth factor preparation prepared by fully producing the growth factor preparation production method of the present invention contains a large amount of specific growth factors, and the specific growth factor content of the growth factor preparation is determined by enzyme immunoassay (EILISA).

Determination of vascular endothelial growth factor, hepatocyte growth factor and keratinocyte growth factor (hereinafter referred to as VEGF, HGF and KGF, respectively)

The VEGF test kit, the HGF test kit and the KGF test kit produced by R&D Company were used to determine the contents of VEGF, HGF and KGF in the growth factor preparation prepared by the growth factor preparation production method of the present invention. The main method used is the Sandwich ELISA. The detection principle and implementation details are well known to those skilled in the art and will not be described in detail. Briefly, a 96-well plate for detection is prepared: a primary antibody solution is prepared by diluting a capture antibody to an appropriate concentration, and the primary antibody solution is added to the well plate in a volume of 100 μl, and reacted at room temperature. Overnight, the primary antibody was fully coated in the well. After completion, the excess antibody is washed away, and blocking is performed with a blocking reagent to prevent subsequent non-specific binding. After obtaining the 96-well plate with the primary antibody, the sample can be reacted and detected: 100 μl of the sample and standards are added to each well in the well plate and reacted at room temperature for 2 hours; One hundred microliters of the detection antibody was reacted at room temperature for 2 hours; after the reaction was completed, a rinsing procedure was performed to sufficiently remove unbound antibodies or various impurities that may cause interference. Add 100 μl of horseradish peroxidase (HRP)-labeled streptavidin to each well and allow to react at room temperature for 20 minutes in the dark. Stop the reaction with Stop Solution and finally detect each by spectrometer. The absorbance reading of the sample at a wavelength of 405 nm reflects the growth factor (VEGF, HGF, and KGF).

Since the present invention uses the "self-source" material, it may cause individual differences due to individual differences, so all the following data showing the achievable effects of the present invention are based on the control data of the same individual source. Make comparisons and analysis instructions for objectiveness.

Referring to Fig. 1, there is shown a variation of a specific growth factor concentration in a growth factor preparation prepared by the autologous adipose stem cells through the growth factor preparation production method of the present invention, as described in the first embodiment of the present invention. The data presentation method shown in Fig. 1 is based on the first, second, and third day after the culture, and the samples of the second cell culture solution and the control group (i.e., 100% autologous serum) are respectively obtained and measured. Among them, each growth factor data. Then, after the detection data of the control group is added up, the average value is taken as the "background value of blank controls". Next, the data obtained from the second cell culture liquid sample at the first, second, and third day time points are processed and analyzed by the above-mentioned control average value, and each sample is known relative to the control. The average value (i.e., the control data described below) was used to fold to blank control.

Therefore, it can be seen from Fig. 1 that the content of VEGF, HGF and KGF in the growth factor preparation (the second culture solution) prepared by the growth factor preparation production method according to the first embodiment of the present invention is significantly increased, and The culture time is prolonged, and the contents of VEGF, HGF and KGF are also increased. Among them, the increase of KGF was the most significant, which was 3.8 times higher than that of the control group on the first day of culture, and the concentration continued to increase with the culture time. By the third day of culture, the concentration of KGF was higher than that of the control group. 6.6 times. Secondly, the content of VEGF increased significantly to about 3 times that of the control group on the second day of culture, and was nearly 6 times higher than that of the control group on the third day of culture. As for the content of HGF, the increase was also observed (2.4 times) from the second day of culture, and it was 3.18 times higher than that of the control group on the third day of culture. Therefore, according to the first figure, the second cell culture solution rich in VEGF, HGF and KGF can be successfully produced by the method of the first embodiment of the present invention, and the user can take different culture time points according to the demand (at least culture The second cell culture solution, which is preferably 2 days, is concentrated or purified to prepare a growth factor preparation, or further processed to produce a combined preparation.

In order to explain in more detail the effects achieved by the method for producing a growth factor preparation of the present invention, and the characteristics of the growth factor preparation prepared according to the method superior to those of other growth factor preparations, the following experimental examples are given, together with the corresponding data results.

Experimental example 1

According to the method described in the first embodiment of the present invention, a second cell culture solution produced using autologous adipose stem cells (hereinafter referred to as ADSC) was obtained, and samples were taken on days 1, 2, and 3 of the culture process, respectively, and the ELISA was performed as described above. The reagent kit was used to determine the content of VEGF. In the control group, the first cell culture solution (i.e., 100% autologous serum used in the first embodiment) which was not cultured with ADSC was used to compare the effects achieved by the production method of the growth factor preparation of the present invention. For the results, please refer to Figure 2 (in the figure, the legend indicates that W/O ADSC represents the control sample without cultured ADSC; the legend with ADSC indicates the second cell culture sample obtained by cultured ADSC, the following is no longer Repeatedly explained) the case where the VEGF concentration significantly increased with the culture time. The concentration of VEGF was increased to 2 times (11.6 pg/ml) in the control group on the first day of culture, and the concentration of VEGF increased significantly with the increase of culture time. By the third day, The concentration has reached 859.3 pg/ml, which is much higher than 8.8 times of the control group on the third day. It is obvious that the production method of the growth factor preparation of the present invention can significantly stimulate the secretion of VEGF by ADSC. In the culture environment, the VEGF content of the growth factor preparation was significantly improved.

Experimental example 2

According to the method described in the first embodiment of the present invention, the second cell culture liquid produced by using ADSC is obtained, and samples are taken on the first, second, and third days in the culture process, and the HGF content is determined by the aforementioned ELISA reagent kit. . In the control group, the first cell culture solution (i.e., 100% autologous serum used in the first embodiment) which was not cultured with ADSC was used to compare the effects achieved by the production method of the growth factor preparation of the present invention. The results are shown in Figure 3, which shows a significant increase in HGF concentration with incubation time. The concentration of HGF increased slightly on the first day of culture, and with the increase of culture time, the concentration of HGF increased significantly to 2230.7 pg/ml on the second day of culture, reaching 2.65 times of the control group on the second day. On the third day of culture, the concentration reached 2906.9 pg/ml, which was much higher than 3.1 times of the control group. It is obvious that the production method of the growth factor preparation of the present invention can significantly stimulate the secretion of HGF from the ADSC into the culture environment, and the growth factor preparation is obviously improved. HGF content.

Experimental example 3

According to the method described in the first embodiment of the present invention, the second cell culture solution produced by using ADSC is obtained, and samples are taken on the first, second, and third days in the culture process, and the KGF content is determined by the aforementioned ELISA reagent kit. . In the control group, the first cell culture solution (i.e., 100% autologous serum used in the first embodiment) which was not cultured with ADSC was used to compare the effects achieved by the production method of the growth factor preparation of the present invention. The results are shown in Figure 4, which shows a significant increase in HGF concentration with incubation time. When the KGF concentration was increased on the first day of culture, the concentration was increased to 4.7 times (24.5 pg/ml) of the control group on the first day, and the KGF concentration was significantly increased as the culture time increased to the second day of culture. 35.0 pg/ml, much higher than 10 times of the control group on the second day. By the third day of culture, the concentration reached 42.0 pg/ml, and the number of the control group was 4 times on the third day. The factor preparation production method can significantly stimulate the secretion of KGF from ADSC into the culture environment, and significantly increase the KGF content of the growth factor preparation.

Second embodiment:

Growth factor reagents are produced according to the growth factor preparation production method of the present invention using autologous fibroblasts (FB). First, the process of obtaining autologous fibroblasts is described. The autologous fibroblasts described in the second embodiment refer to fibroblasts isolated from the patient's autologous skin tissue, and the source thereof is mainly a skin sample, after sampling. Skin sample The whole process of obtaining the autologous fibroblasts in the transport liquid should be carried out in an aseptically operated environment.

First, prepare a plurality of 6-cm sterile cell culture dishes, and add 8 ml of PBS and antibiotic (gentamicin). The skin samples are taken out from the transport liquid by a sterile dropper, and placed in a sterile cell culture dish. The skin sample was transferred to the next sterile cell culture dish and the cleaning procedure was repeated three times. Next, the skin sample was transferred to another empty sterile cell culture dish, and the skin sample was cut into 0.5 mm-1 mm cubic skin pieces by a sterile scalpel blade. The skin fragments are transferred to a culture flask containing a freshly prepared enzyme buffer. The configuration of the enzyme reaction solution is: 4 ml of fibroblast culture solution (containing antibiotic gentamicin), 0.5 ml of 10X collagenase, 0.5 ml of 10X dispase. The culture flask was transferred to a 37 ° C cell culture incubator to allow sufficient hydrolysis for 18-24 hours. Next, the separated sample and the enzyme reaction solution were taken out as a whole, and centrifuged at 1000 rpm for 5 minutes. The supernatant after centrifugation is removed, and the lower layer (ie, cells and cell mass) is gently aspirated by a pipette to sufficiently disperse the cells to obtain a fibroblast solution, and the fibroblast solution is maintained at 2-5 ml. .

Carefully remove the supernatant with a sterile tube and take 1.5 ml of the fibroblast culture medium to repeatedly vomit to break up the cells and skin fragments.

Maintain the volume of the cell culture medium in the range of 2-5 ml, so that the fibroblasts can grow stably and stably, and routinely update or add the cell culture medium, and the cell growth density can reach 90% after about 1-2 weeks after the culture. At this time, a subculture method can be routinely performed, and the fibroblasts are subcultured, and the growth factor preparation is prepared by the growth factor preparation production method of the present invention in a state in which the number of cells is stably grown and sufficient.

Prior to performing the growth factor preparation production method of the present invention, autologous adipose-derived stem cells of the same patient should be obtained in advance according to the procedure for obtaining autologous fibroblasts as described above. According to the autologous fibroblast obtained by the above-mentioned embodiment, the procedure for preparing the growth factor preparation is as follows: 200 ml of venous blood is collected from the patient, and the device is placed in a 50 ml centrifuge tube, and left at room temperature for 1 hour, and stored at 4 ° C. Refrigerator spare. Next, the blood was taken out and centrifuged at 1000 g for 10 minutes to obtain a layered blood sample. The supernatant is taken from the blood sample and is serum. Depending on the needs of use, the serum is diluted with an appropriate buffer or as a serum stock solution, and used as a first cell culture solution, and used in subsequent steps. The first cell culture solution is at a pore size of 0.22 μm. The filter bowl is filtered. Next, an autologous fibroblast cultured in a cell culture flask was taken, and the cell growth fullness was 5-7 minutes. The culture medium of autologous fibroblasts is replaced with a first cell culture medium by adding a sufficient amount of the first cell culture medium to a culture flask (for example, a 175T culture flask uses about 30 ml of the first cell culture solution). Continue to culture for 1-3 days to obtain a second cell culture medium. When undiluted serum is used as the first culture solution, serum (second cell culture solution) rich in vascular endothelial growth factor and hepatocyte growth factor is obtained at this time. After the second cell culture solution is obtained, it can be directly used as a growth factor preparation, and stored in a freeze-drying manner; or a buffer solution and a nutrient can be further added to the growth factor preparation to prepare a combined preparation for promoting skin tissue regeneration.

The second embodiment of the present invention differs from the first embodiment mainly in that the autologous cells are different, and the remaining embodiments (including the growth factor detecting method) are substantially the same as the first embodiment.

Referring to Fig. 5, there is shown a variation of a specific growth factor concentration in a growth factor preparation prepared by autologous fibroblasts via the growth factor preparation production method of the present invention, as described in the second embodiment of the present invention. The data presented in Figure 5 is based on the first, second, and third day after the culture, and the samples of the second cell culture solution and the control group (ie, 100% autologous serum) are obtained. Among them, each growth factor data. Then, after the detection data of the control group is added up, the average value is taken as the "background value of blank controls". Next, the data obtained from the second cell culture solution sample at the first, second, and third day time points are processed and analyzed by the average value of the above-mentioned control, and the average value of each sample relative to the control is obtained (ie, the next The control group data) fold to blank control.

Therefore, as can be seen from FIG. 5, the growth factor preparation (this refers to the second culture solution) prepared by the growth factor preparation production method according to the second embodiment of the present invention has a significant increase in the content of VEGF and HGF, and with the culture time. With extension, the levels of VEGF and HGF also increased. Among them, the increase of HGF was the most significant, which was 6.64 times higher than that of the control group on the first day of culture, and the concentration continued to increase with the culture time. By the third day of culture, the concentration of HGF was higher than that of the control group. 21.9 times. Secondly, the content of VEGF increased significantly to about 2.4 times of the control group on the first day of culture, and its concentration continued to increase with the culture time. By the third day of culture, it was 3.77 times higher than that of the control group. Therefore, according to the first figure, the second cell culture solution rich in VEGF, HGF and KGF can be successfully produced by the method of the second embodiment of the present invention, and the user can, according to the needs, The second cell culture solution at different culture time points (at least 1 day for culture) is taken, concentrated or purified to prepare a growth factor preparation, or further processed to produce a combined preparation.

Similarly, according to the contents of the second embodiment, the effects achieved by the growth factor preparation production method of the present invention will be described in more detail, and the growth factor preparation prepared according to the method is superior to other growth factor preparations, and continues to be enumerated. The following experimental examples and corresponding data results are described.

Experimental example 4

According to the method described in the second embodiment of the present invention, a second cell culture solution produced using autologous fibroblasts (hereinafter referred to as FB) is obtained, and samples are taken on the first, second, and third days in the culture process, respectively, and The ELISA kit was used to determine the content of VEGF. In the control group, the first cell culture solution (i.e., 100% autologous serum used in the second embodiment) which was not cultured with FB was used to compare the effects achieved by the production method of the growth factor preparation of the present invention. The results are shown in Figure 6 (in the figure, the legend indicates that W/O FB represents the control sample without culturing FB; the legend with FB indicates the second cell culture solution sample obtained by culturing FB, the following is no longer Repeatedly explained) the case where the VEGF concentration significantly increased with the culture time. When the concentration of VEGF was increased on the first day of culture, the concentration (187.9 pg/ml) was increased to 2 times in the control group on the first day, and the concentration of VEGF continued to increase slightly with the increase of culture time. On the third day, The concentration has reached 291.7 pg/ml, which is much higher than 3.7 times of the control group on the third day. It is obvious that the production method of the growth factor preparation of the present invention can significantly stimulate the secretion of VEGF into the culture environment by FB, and significantly increase the VEGF content of the growth factor preparation.

Experimental example 5

According to the method described in the second embodiment of the present invention, the second cell culture solution produced by using FB is obtained, and samples are taken on the first, second, and third days in the culture process, and the HGF content is determined by the aforementioned ELISA reagent kit. . In the control group, the first cell culture solution (i.e., 100% autologous serum used in the first embodiment) which was not cultured FB was used for comparison to compare the effects achieved by the production method of the growth factor preparation of the present invention. The results are shown in Figure 7, which shows a significant increase in HGF concentration with incubation time. The concentration of HGF increased significantly to 869.5 mg/ml on the first day of culture, which was 12.7 times of that of the control group on the first day. As the culture time increased, the concentration of HGF continued to increase significantly on the second day of culture. To 1919.3 pg/ml, reaching 14.7 times of the control group on the second day, the concentration reached 2871.3 pg/ml on the third day of culture, much higher than the control. The number of groups is 14.9 times. It is obvious that the production method of the growth factor preparation of the present invention can significantly stimulate the secretion of HGF by FB into the culture environment, and the HGF content of the growth factor preparation is obviously improved.

Claims (10)

A method for producing a growth factor preparation, the method comprising the steps of: (1) obtaining an autologous blood; (2) separating a blood component from the autologous blood; and (3) preparing a first composition of the blood composition a cell culture solution; (4) cultivating at least one autologous cell in the first cell culture medium, the culture time is between 20 and 72 hours, and the autologous cell secretes a plurality of growth factors into the first cell culture solution, Forming a second cell culture solution; (5) removing the second cell culture solution; and (6) preparing the second cell culture solution to prepare a growth factor preparation, the growth factors comprising at least one vascular endothelial growth factor and one The hepatocyte growth factor; wherein the concentration of the vascular endothelial growth factor contained in the second cell culture medium is between 150 and 1000 pg/ml, and the concentration of the hepatocyte growth factor is between 800 and 3000 pg/ml. The method for producing a growth factor preparation according to the above aspect of the invention, wherein the blood composition is a serum. The method for producing a growth factor preparation according to claim 1, wherein the component of the first cell culture solution contains 90% or more of the blood composition. The method for producing a growth factor preparation according to the above aspect of the invention, wherein the growth factor further comprises a keratinocyte growth factor, and the concentration of the keratinocyte growth factor contained in the second cell culture solution is between 20 ~45 pg/ml. The method for producing a growth factor preparation according to claim 1, wherein the autologous cell line is selected from an autologous fibroblast, an autologous adipose stem cell, or an autologous fibroblast and an autologous fat. A group of stem cell combinations. The method for producing a growth factor preparation according to claim 1, wherein the step (5) or the step (6) may further comprise a filtration step. A growth factor preparation, which is prepared by the method for producing a growth factor preparation according to any one of claims 1 to 6, wherein the growth factor preparation contains a plurality of growth factors, The growth factor system comprises a vascular endothelial growth factor and a hepatocyte growth factor, the concentration of the vascular endothelial growth factor is between 150 and 1000 pg/ml; and the concentration of the hepatocyte growth factor is between 800 and 3000 pg/ml. . According to the growth factor preparation of claim 7, the growth factor further comprises a keratinocyte growth factor having a concentration of 20 to 45 pg/ml. A combination preparation for promoting the regeneration of skin tissue, characterized in that the combination preparation for promoting the regeneration of skin tissue comprises: a growth factor preparation according to any one of claims 7 or 8 of the patent application, The invention comprises a plurality of growth factors, the growth factor preparation comprising at least a vascular endothelial growth factor and a hepatocyte growth factor; a buffer; and a nutrient comprising a glucose and a plurality of electrolytes. According to the combined preparation for promoting skin tissue regeneration according to the scope of claim 9, the weight ratio of the glucose is 5%.