KR100458268B1 - Process for Preparing Collagen Comprising 3-Dimensional Cultivation of Fibroblast - Google Patents

Abstract

The present invention relates to a method for culturing fibroblasts in three dimensions and producing collagen therefrom. Collagen production method using the three-dimensional culture of the fibroblasts of the present invention comprises the steps of first culturing the fibroblasts in a culture dish; Inoculating the primary culture on a support capable of maintaining the three-dimensional form of the synthesized collagen, and second-dimensionally culturing; Inducing secondary culture to synthesize collagen; And obtaining the secondary cultured support, and separating and extracting collagen adhered to the support surface. According to the present invention, the synthesis and extraction of collagen by cells has the advantage that a large number of cells can be obtained according to the cell culture method, and thus it is possible to solve the limitation of the supply brought from collagen extracted from the tissues. It may be widely used in plastic surgery development, such as removal of wrinkles of collagen preparations, skin wound dressings and the like, and tissue engineering development such as collagen support.

Description

Process for Preparing Collagen Using Three-Dimensional Culture of Fibroblasts {Process for Preparing Collagen Comprising 3-Dimensional Cultivation of Fibroblast}

The present invention relates to a method for producing collagen using three-dimensional culture of fibroblasts. More specifically, the present invention relates to a method for culturing fibroblasts three-dimensionally and producing collagen in a form similar to that in vivo.

As the body ages, components such as collagen and elastin are released from the skin due to ultraviolet rays, hormonal changes after childbirth, smoking, pollution, etc., resulting in a decrease in elasticity of the skin, and consequently wrinkles on the skin. Among the methods of removing such wrinkles, the most obvious and fundamental method is to remove wrinkles, but a considerable amount of time and money are required, and thus, simple injection or drug therapy is mainly used. The most common substance used in such injection or drug therapy is collagen, which is known to be effective in removing fine wrinkles around the eyes and around the mouth.

Collagen is a fibrous albuminoid that fills cells between cells in humans and animals, and is present in large amounts of cells, bones, cartilage, blood vessel walls, teeth, and muscles. . In particular, collagen is known as a major component of the extracellular matrix in epithelial tissue. Such collagen has been used for food in the form of gelatin, and the ingested collagen is digested by digestive enzymes in the digestive tract and absorbed in the form of most amino acids, and the ingested collagen improves immune function and regeneration of cells. It promotes to strengthen the joints, it is known to exhibit an excellent effect on skin beauty through the activation of skin metabolism and maintaining the moisturizing power.

Such collagen is mainly produced by extracting from cattle skins, bones, cattle knee joints, etc., or for special purposes, when human collagen is needed from the cadaveric or placental tissues. However, when collagen extracted from animals is used for food, there is no problem, but when used as an injection composition for removing skin wrinkles of humans, there is a possibility of causing an immune rejection reaction. In order to overcome this problem, a method of using human collagen has been used. However, since the extraction of human collagen is not easy, the production amount is insufficient, and thus a high cost is required. In order to overcome these disadvantages, research has been conducted to produce collagen that does not cause an immune rejection reaction in humans using cell culture methods, but the production yield is low, and the produced collagen has a disadvantage in that its stability is lower than that of natural collagen. However, they have not been used commercially, and efforts are being made to overcome them.

Therefore, there is a constant need to develop a method for producing large amounts of collagen.

Thus, the inventors of the present invention to develop a method for producing a large amount of collagen, as a result of research, when using a support for maintaining the three-dimensional form of collagen, when fibroblasts were three-dimensionally cultured to synthesize collagen, It was confirmed that the collagen having the same morphological characteristics as natural collagen can be produced in large quantities through continuous culture of cells, and thus, the present invention was completed.

After all, the main object of the present invention is to provide a method for culturing fibroblasts in three dimensions and producing collagen therefrom.

1A is a micrograph of a cell-support complex cultured for two weeks.

1B is a micrograph of the cell-support complex cultured for 4 weeks.

1C is a micrograph of the cell-support complex cultured for 6 weeks.

2A is a scanning electron micrograph after 2 days of culture.

2B is a scanning electron micrograph after two weeks of culture.

2C is a transmission electron micrograph after two weeks of culture.

3A is an optical micrograph showing the expression of collagen type 1 in a two-week cultured cell-support complex.

3B is an optical micrograph showing the expression of collagen type 1 in a cell-support complex cultured 4 weeks.

3C is an optical micrograph showing the expression of collagen type 1 in a 6-week cultured cell-support complex.

Collagen production method using the three-dimensional culture of the fibroblasts of the present invention comprises the steps of first culturing the fibroblasts in a culture dish; Inoculating the primary culture on a support capable of maintaining the three-dimensional form of the synthesized collagen, and second-dimensionally culturing; Inducing secondary culture to synthesize collagen; And obtaining the secondary cultured support, and separating and extracting collagen adhered to the support surface.

Hereinafter, the production method of collagen of the present invention will be described in more detail by dividing each step.

First step : primary culture

Primary culturing of fibroblasts in a culture dish: Fibroblasts to be cultured together are selected while culturing human epithelial cells obtained by conventional methods, and subcultured with the selected fibroblasts to obtain a primary culture. .

Second Step : Secondary Culture

The primary culture is inoculated onto a support capable of maintaining the three-dimensional form of the synthesized collagen, followed by two-dimensional incubation: The support generally contains a large amount of collagen, which prolongs the incubation period. In this case, since the collagen component contained in the support is released into the culture solution, the support may collapse. In order to overcome this disadvantage, the present inventors overcome the conventional disadvantage by using a support mainly composed of PLGA (poly (D, L-glycolic-co-lactic acid)), which is not a collagen but a glycolic acid derivative. Although the support mainly based on such PLGA is not particularly limited, it is preferable to use one prepared by a method of preparing a foamed salt using an acidic aqueous solution at room temperature, and more preferably a support having pores formed therein. The pores in the support provide sufficient space for the collagen synthesized in the cells to adhere and maintain their three-dimensional shape. For example, PLGA is dissolved in an organic solvent, a polymer aqueous solution is added to obtain a gel-like PLGA precipitate, and then a foamed salt such as ammonium bicarbonate of a certain size is added thereto, and the acidic solution is added to the mold. The support was prepared by adding a salt to foam and removing residual solvent. More preferably, a support having an average pore size of 300-500 μm and having a porosity of 90% is used.

Step 3 : Induce differentiation into collagen

Secondary cultures are induced to synthesize collagen: The method of inducing collagen is not particularly limited, but ascorbic acid is preferably added as is known.

Fourth Step : Extraction of Collagen

The secondary cultured support is obtained, and collagen adhered to the support surface is separated and extracted. At this time, the method of separating the adhered collagen is not particularly limited, but it is preferable to use a method of separating the support and collagen by treating the acid. In addition, the separated collagen may be used in a liquid state or solidified, but considering the ease of storage and distribution, it is preferable to solidify the collagen, and the method of solidifying collagen is not particularly limited, but a freeze-drying method, Precipitation methods and the like can be used, and it is preferable to use a method of treating and precipitating a compound containing sodium ions, followed by drying.

In general, the method of culturing cells three-dimensionally on the support has a disadvantage in that cell growth is slower than the method of two-dimensional culturing using a culture dish, but it is possible to restore the three-dimensional structure of the original cells. There is an advantage. In particular, when applied to the method for producing collagen of the present invention, since the amount of collagen secreted and lost in the culture medium is significantly reduced in three-dimensional culture than in two-dimensional culture, the cell culture method is used. It was confirmed that it can produce a large amount of collagen.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Production of Collagen Using Three-Dimensional Culture

Human fibroblasts are primaryly cultured in a culture dish, inoculated onto a three-dimensional support, then secondaryly cultured to induce differentiation into collagen, then a support is obtained, and collagen adhered to the support is extracted and produced. .

Example 1-1 Primary Culture of Human Fibroblasts

Epithelial spontaneous exudates were obtained from patients who underwent excision due to intraoral disease, from which fibroblasts were isolated in a conventional manner. That is, the primary culture medium, high concentration of glucose DMEM (LG-DMEM, glucose 1g / L, GIBCO, MO, USA) medium and epithelium were mixed, the cells were centrifuged by primary centrifugation, and then Percoll (Percoll, Only nucleated cells were isolated by ultracentrifugation using SIGMA, MD, USA). Incubated in an incubator maintained at 5% CO ₂ , 37.5 ° C, and attempted to isolate and culture only fibroblasts by changing the medium (high concentration DMEM medium containing 10% fetal calf serum) every 2 days. Subsequent subcultures were performed to propagate a sufficient number of cells.

Example 1-2 Secondary Culture of Human Fibroblasts and Synthesis of Collagen

Polymeric scaffold (Innotech., Korea), the main component of PLGA required for three-dimensional culture, was sequentially pretreated using 100, 90, 70, and 10% (v / v) ethanol at 4 ° C, followed by PBS (phosphate buffered saline) The culture medium was washed and then dried. Subsequently, 100 μl of cell suspensions of different cell densities (1 × 10 ⁶ , 2.5 × 10 ⁶ ) were dispensed on the polymer support, and culture was started in a culture dish of 60 mm diameter. After 2 hours, 4 ml of medium was added and cultured using a shake incubator (30 rpm). Then, after 2 days, collagen formation inducer (1 mM L-ascorbic acid-2 was added every 3 days. A new medium supplemented with -phosphate) was supplied and further cultured for 6 weeks.

Example 1-3 Extraction of Collagen

From the secondary culture in which the differentiation was conducted, a support was obtained, washed with PBS, and then ground. The ground support was immersed in 10 ml of 0.2 M acetic acid aqueous solution, shaken at 4 ° C. for 2 days, and then centrifuged at 12,000 × g, 4 ° C. for 2 hours to obtain a supernatant. NaCl was added to 30% (w / v) of the obtained supernatant, and left at 4 ° C. for 1 hour, and then centrifuged at 4,000 × g and 4 ° C. for 30 minutes to obtain a precipitate. . The obtained precipitate was resuspended in 10 ml of 0.05 M acetic acid aqueous solution, and dialyzed three times at 6 ° C. with 0.02 M Na ₂ HPO ₄ aqueous solution at 4 ° C. for 3 minutes, and then at 4,000 × g and 4 ° C. for 30 minutes. Centrifugation gave a precipitate. The precipitate was vacuum lyophilized and stored at 4 ° C.

Example 2 Characterization of Collagen

The collagen obtained in Example 1 was observed with an optical microscope and an electron microscope according to the culture time, and the type 1 collagen generated during the formation of collagen was confirmed by an immunological method.

Example 2-1 : Observation using an Optical Microscope

Each cell-support complex (hBMSCs-scaffolds complex) cultured for 2 weeks, 4 weeks and 6 weeks was fixed in 10% neutral formalin for 8 hours or more, and embedded in paraffin, and then 5 μm serial sections were prepared. Subsequently, H / E staining was performed to observe cell distribution and extracellular matrix deposition in the scaffold (see FIGS. 1A, 1B and 1C). FIG. 1A is a micrograph of the cell-support complex cultured for 2 weeks, FIG. 1B is a micrograph of the cell-support complex cultured for 4 weeks, and FIG. 1C is a micrograph of the cell-support complex cultured for 6 weeks. As shown in Figure 1a to 1c, as the incubation period increases, the amount of collagen increases, it was found that the cells are killed at the same time.

Example 2-2 : Observation using an electron microscope

In order to determine the morphological characteristics of collagen in vivo (scanning electron microscopy (HITACHI S-800) and transmission electron microscopy (transmission electron microscopy, Philips CM-10)) was observed.

First, the extracted samples were pre-fixed with 2% (v / v) glutaraldehyde and paraformaldehyde buffered with 0.1 M PBS (pH 7.4) to perform observation using a transmission electron microscope. Post fixation with 1.33% OsO ₄ was performed. The alcohol-immobilized tissues are dehydrated, replaced with propylene oxide, embedded in an epon mixture, and made into ultra thin sections, which are doubled with uranyl acetate and lead citrate. After staining, it was observed by transmission electron microscopy.

Next, in order to perform observation using a scanning electron microscope, after dehydration with isoamyllactate, after 6 hours, a critical point dry was performed, followed by an ion coater (Eiko IB). -3) was gold coated to a thickness of 300 nm and observed with a scanning electron microscope (see Figures 2a, 2b, 2c). FIG. 2A is a scanning electron micrograph 2 days after cell inoculation, FIG. 2B is a scanning electron micrograph after 2 weeks culture, and FIG. 2C is a transmission electron micrograph after 2 weeks culture. As shown in Figures 2a to 2c, after two weeks of culture, the cells secrete an extracellular matrix rich on the surface of the support, and abundant ribosomes and endoplasmic reticulum and numerous secretory granules are observed in the cytoplasm, thus secreting collagen from the cells. Could know.

Example 2-3 Identification of Type 1 Collagen

To identify the type 1 collagen formed by the cells, Masson's Trichrome, an immunohistochemical staining method, was performed. That is, 5 μm-thick paraffin continuous slices prepared in Example 2-1 were treated with xylene, stepped with alcohol, and treated with 0.5% (v / v) aqueous hydrogen peroxide solution to endogenous peroxidase. After removal, it was treated with goat serum (goat serum) for 30 minutes. To identify the type 1 collagen, the reaction was performed by adding a primary antibody of type 1 collagen, washed three times with PBS for 5 minutes, and then 30 minutes with a secondary antibody (5 μg / ml biotinylated anti-mouse / anti-rabbit IgG). Reacted. Subsequently, the cells were treated with chromophore (3 μg / ml horseradish peroxidase streptoavidin) for 30 minutes, followed by color development using a colorant (DAB, hydrogen peroxide), followed by staining of cells with hematoxylin to express the expression of collagen type I. Observation was made with an optical microscope (see FIGS. 3A, 3B, and 3C). 3A is an optical micrograph showing the expression of type 1 collagen in a two-week cultured cell-support complex, FIG. 3B is an optical micrograph showing the expression of type 1 collagen in a four-week cultured cell-support complex, 3C is an optical micrograph showing the expression of collagen type 1 in a 6-week cultured cell-support complex. As shown in Figures 3a to 3c, it can be seen that as the culture period increases, the expression of type 1 collagen increases.

As described and demonstrated in detail above, the present invention provides a method for culturing human fibroblasts in three dimensions and producing collagen therefrom. According to the present invention, the synthesis and extraction of collagen by cells has the advantage that a large number of cells can be obtained according to the cell culture method, and thus it is possible to solve the limitation of the supply brought from collagen extracted from the tissues. It may be widely used in plastic surgery development, such as removal of wrinkles of collagen preparations, skin wound dressings and the like, and tissue engineering development such as collagen support.

Claims (4)

(Iii) first culturing the fibroblasts in a culture dish; (Ii) inoculating the primary culture onto a support capable of maintaining the three-dimensional form of the synthesized collagen and secondly culturing in three dimensions; (Iii) inducing the secondary culture to synthesize collagen; And, (Iii) obtaining an electro-secondary cultured support and separating and extracting collagen adhered to the surface of the support, the method of producing collagen using a three-dimensional culture of fibroblasts. The method of claim 1, The support is PLGA (poly (D, L-glycolic-co-lactic) which is a glycolic acid derivative. acid)) as the main component, the average pore size is 300-500㎛, Characterized by having a porosity of 90% Method for producing collagen using three-dimensional culture of fibroblasts. The method of claim 1, Synthetic induction of collagen by adding ascorbic acid Characterized in that Method for producing collagen using three-dimensional culture of fibroblasts. Method for producing collagen using a three-dimensional culture of fibroblasts comprising the step of solidifying the collagen produced by the method of claim 1 by lyophilization or precipitation.