KR20070082114A - Method for coculturing fibroblasts and keratinocytes in biocompatible scaffolds - Google Patents

Abstract

A method for coculturing fibroblasts and keratinocytes in biocompatible scaffolds is provided to manufacture keratinocyte-fibroblast-scaffold complex as an artificial skin substitute for treating skin defects. The method for coculturing fibroblasts and keratinocytes in biocompatible scaffolds comprises the steps of: (a) inoculating the fibroblasts and keratinocytes to the biocompatible scaffolds coated with collagen; and (b) coculturing the inoculated fibroblasts and keratinocytes under submerge condition and air-dry condition, sequentially, wherein the biocompatible scaffold is selected from chitosan, glycosaminoglycan, gelatin, hydroxyapatite, poly-d,l-lactic acid, poly-L-lactic acid, polyglycolic acid, d,l-lactic acid and glycolic acid copolymer, L-lactic acid and glycolic acid copolymer, copolyoxalate, polycaprolactone, poly(lactic acid-caprolactone), poly(glycolic acid-caprolactone), hyaluronic acid and its derivatives, chitin, alginate, fibronectin, polyanhydride, polyorthoester, polyurethane and casein.

Description

Methods for Coculturing Fibroblasts and Keratinocytes in Biocompatible Scaffolds

Figure 1 shows the culture of skin-derived fibroblasts in chitosan scaffold not coated with type I collagen. The inoculation concentration is 1 x 10 ⁶ -10 x 10 ⁶ . After fibroblasts were cultured for 7 days, the results of H / E staining (Panel A) and immunohistochemistry for the fibroblast labeling protein bimentin (Panel B) are shown.

Figure 2 shows the culture of skin-derived fibroblasts in chitosan scaffold coated with type I collagen. The inoculation concentration is 1 x 10 ³ -50 x 10 ³ . After fibroblasts were cultured for 7 days, the results of H / E staining (Panel A) and immunohistochemistry for the fibroblast labeling protein bimentin (Panel B) are shown.

Figure 3 shows co-culture of skin-derived fibroblasts and keratinocytes in a chitosan scaffold that is not coated with type I collagen. The inoculation concentration is 1 x 10 ^{7 for} fibroblasts and 3 x 10 ⁵ for keratinocytes. H / E staining after 7 days incubation (Panel A), immunohistochemistry for the fibroblast marker protein, vimentin (Panel B), immunohistochemistry for the proliferative marker PCNA (Panel C) ) And immunohistochemistry for panel-cytokeratin, a marker protein of skin keratinocytes (Panel D).

Figure 4 shows inoculation of skin-derived fibroblasts and keratinocytes in a type I collagen-coated chitosan scaffold, followed by differentiation of keratinocytes through air-drying culture. The inoculation concentration was 5 x 10 ^{4 for} fibroblasts and 3 x 10 ⁵ for keratinocytes. H / E staining after 7 days incubation (Panel A), immunohistochemistry for the fibroblast marker protein, vimentin (Panel B), immunohistochemistry for the proliferative marker PCNA (Panel C) ) And immunohistochemistry for panel-cytokeratin, a marker protein of keratinocytes (Panel D).

Figure 5 shows co-culture of skin-derived fibroblasts and keratinocytes in a type I collagen-free chitosan scaffold. The inoculation concentration is 1 x 10 ^{7 for} fibroblasts and 3 x 10 ⁵ for keratinocytes. H / E staining (Panel A) and immunohistochemistry for the fibroblast marker protein, vimentin (Panel B). Immunohistochemistry results for PCNA (Panel C) and immunohistochemistry results for panel-cytokeratin, a marker protein of keratinocytes (Panel D).

Figure 6 shows inoculation of skin-derived fibroblasts and keratinocytes in a type I collagen-coated chitosan scaffold, followed by differentiation of keratinocytes through air-drying culture. The inoculation concentration was 5 x 10 ^{4 for} fibroblasts and 3 x 10 ⁵ for keratinocytes. H / E staining (Panel A) and immunohistochemistry for the fibroblast marker protein, vimentin (Panel B). Immunohistochemistry results for PCNA (Panel C) and immunohistochemistry results for panel-cytokeratin, a marker protein of keratinocytes (Panel D).

7 is a schematic of coculture conditions.

Figure 8 is a photograph of the immunohistochemical staining to confirm whether the collagen coating on the chitosan scaffold.

Figure 9 is a photograph showing the results of co-cultivation by inoculating fibroblasts and keratinocytes in a collagen-free chitosan scaffold over time.

The present invention relates to a method of co-culture of fibroblasts and keratinocytes, artificial skin substitutes and biocompatible scaffolds.

Successful in vitro regeneration of the skin, consisting of the epidermis and the dermis, requires several types of cells co-cultured with specific elements present in the original skin and an appropriate scaffold structure to receive and support them. Shall be.

In addition to the two main components, the epidermis and the dermis, one of the major organs of the skin is a capillary network that ensures proper perfusion of nutrients and oxygen. Endothelial skin is a joint of three human cell types in the scaffold: keratinocytes, fibroblasts, and endothelial cells, which can be obtained as a full-thickness skin sample of the body. Can be obtained by culturing.

Suitable dermal scaffolds are an essential component of artificial skin substitutes that can permanently compensate for skin defects. Many biocompatible polymeric materials have been developed to meet this need for dermal scaffold materials. Among many biocompatible materials, collagen is considered to be the most noticeable skin substitute in view of its low inflammatory and cytotoxicity and promoting cell growth. When collagen sponges are used for whole skin defect wounds, fibroblasts and capillaries penetrate into the sponges and proliferate at the wound site, and collagen fibers are formed by the synthesis of autologous collagen, thereby constructing dermal tissue. The collagen sponge is then decomposed and absorbed in an extremely pure manner. Thus, this dermal tissue becomes a complete self-dermal tissue. Because the material itself is transformed into living dermal tissue, the sponge is also called a dermal substitute, which has already been commercialized in some and contains various research and clinical reports.

On the other hand, hyaluronic acid (HA: Hyaluronic acid) has been widely used as a base material of artificial skin, and has been used as an injectable tissue expansion solution. However, these biocompatible materials are limited to be used as dermal scaffolds because of their low mechanical strength and easy contamination by bacteria.

Conventional techniques related to such dermal replacements include the following. Korean Patent Application Publication No. 2000-0013701 discloses a porous collagen dual structure wound protective film for dermal regeneration induction consisting of a hard layer composed of collagen, laminin and hyaluronic acid and a porous layer, and Korean Application Publication No. 2000-0007983 No. 1 discloses a biological tissue comprising a reinforced collagen layer made of a collagen of sponges and a mesh and its use, and the living tissue equivalent of Korean Patent Application Publication No. 94-1379 discloses a fiber in a gelled collagen layer. Dermal replacement consisting of a dermal layer composed of collagen lattice form shrunk into blasts is disclosed.

Throughout this specification, many papers and patent documents are referenced and their citations are indicated. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

The present inventors have made efforts to develop artificial skin substitutes that can treat skin defects. As a result, the biocompatible scaffolds are inoculated with fibroblasts, which are the main cells of the dermal layer, and keratinocytes, which are the main cells of the skin epidermal layer, for excellent efficiency. Co-culture has been successfully achieved, and the present invention has been completed by discovering that it is very efficient to prepare an artificial skin substitute.

Accordingly, an object of the present invention is to provide a method for co-culture of skin-derived fibroblasts and keratinocytes.

Another object of the present invention is to provide an artificial skin substitute consisting of biocompatible scaffolds, fibroblasts and keratinocytes.

Another object of the present invention is to provide a collagen-coated biocompatible scaffold.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention comprises the steps of (a) inoculating fibroblasts and keratinocytes in a biocompatible scaffold; And (b) culturing fibroblasts and keratinocytes inoculated into the biocompatible scaffolds.

The present inventors have made efforts to develop artificial skin substitutes that can treat skin defects. As a result, the biocompatible scaffolds can be used to co-operate fibroblasts, which are the main cells of the dermal layer, and keratinocytes, which are the main cells of the skin epidermis, with excellent efficiency. It can be cultured and found to be very efficient in producing artificial skin substitutes.

A surprising finding in the present invention is that biocompatible scaffolds can be used to co-culture fibroblasts and keratinocytes. As used herein, the term “co-culture” refers to the inoculation of at least two cells into a biocompatible scaffold to proliferate and maintain several cells simultaneously. In addition, the inoculation includes not only inoculating several kinds of cells at the same time, but also inoculating with a time difference.

The method of the present invention is described in detail as follows:

(a) inoculating fibroblasts and keratinocytes into a biocompatible scaffold

The biocompatible scaffolds are seeded with fibroblasts and keratinocytes.

As used herein, the term “biocompatible scaffold” refers to a substance that can be degraded in vivo and can serve as a skeleton to which cells adhere well to form a three-dimensional tissue structure. Preferably, the biocompatible scaffolds used in the present invention are immunologically inert so as not to cause inflammatory or foreign body reactions after transplantation, while actively reacting with surrounding tissues and present in the surrounding cells. Can be induced to multiply and enter, and the rate of decomposition is appropriate so that the material of the supporting layer does not decompose after being transplanted due to a foreign body reaction to the implant, but before the role of the transplanted cells and tissues is finished. After a certain period of time, it will not decompose itself and remain as a foreign substance.

According to a preferred embodiment of the present invention, the biocompatible scaffold is coated with collagen. Collagen coated on biocompatible scaffolds is the major structural material of vertebrates and is present in tissues that function mechanically, and there are at least 19 types, all of which have the same tertiary structure. Any type of collagen may be used in the present invention, preferably collagen type I is used.

According to a preferred embodiment of the invention, the biocompatible scaffold is chitosan, glycosaminoglycans, gelatin, hydroxyapatite, poly-d, l-lactic acid, poly-L-lactic acid, polyglycolic acid, mixed d , l-lactic acid and glycolic acid copolymer, L-lactic acid and glycolic acid copolymer, copolyoxalate, polycaprolactone, poly (lactic acid-caprolactone), poly (glycolic acid-caprolactone), hyaluronic acid (hyaluronic acid) acid) and derivatives thereof, chitin, alginate, fibronectin, polyanhydride, polyorthoesters, polyurethane and casein.

According to a more preferred embodiment, chitosan is used as the biocompatible scaffold. Chitosan and its derivatives are, because even if lysozyme is present in an aqueous medium on the in vitro to degrade at a slower rate chitosan sponge is suitable for the present invention because the shape and size can be kept in a stable state during which the cells are cultured. Moreover, chitosan is not immune, resistant to microbial contamination, can be easily obtained by deacetylation of chitin, and glucosamine, which is a microbial degradation product of chitosan and chitosan, is not toxic. have.

According to a preferred embodiment of the present invention, collagen-coated biocompatible scaffolds are used, which greatly increases the efficiency of coculture of fibroblasts and keratinocytes. The term "collagen-coating" as used in connection with a biocompatible scaffold herein means that the collagen is applied to the surface of the biocompatible scaffold and / or the entire portion of the scaffold thickness. That is, collagen is not used in the process of preparing the scaffold's basic skeleton, but it means that the basic skeleton is already applied to the manufactured scaffold.

The amount of fibroblasts and keratinocytes inoculated into the biocompatible scaffold is not particularly limited and is generally inoculated with 1 × 10 ² -1 × 10 ⁸ cells.

According to a modification of the present invention, the cells inoculated into the biocompatible scaffold may further include other cells constituting the skin in addition to fibroblasts and keratinocytes. For example, melanocytes, undifferentiated adipocytes, adipose tissue-derived mesenchymal stem cells, vascular endothelial cells and / or hair follicle-derived undifferentiated cells.

(b) Cultivation of fibroblasts and keratinocytes inoculated into the biocompatible scaffold

In this step, fibroblasts and keratinocytes inoculated in the biocompatible scaffold are co-cultured. In co-culture, the temperature and atmospheric conditions applied may be applied as it is commonly used in the art. For example, the coculture temperature may be about 37 ° C., and the atmospheric conditions may be about 5% CO ₂ .

According to a preferred embodiment of the present invention, the co-culture of fibroblasts and keratinocytes comprises the following sub-steps: (b-1) Incubation of fibroblasts and keratinocytes inoculated into the biocompatible scaffold culturing under submerge conditions; And (b-2) culturing the cultured fibroblasts and keratinocytes under air-dry conditions.

Culture conditions of inoculated fibroblasts and keratinocytes can be largely selected according to air exposure: air-dry conditions, semi-dry conditions and submerged cultures. ) Condition.

Air-drying conditions herein refer to a state in which fibroblasts inoculated into the biocompatible scaffold are present in the liquid and keratinocytes are completely exposed to the air (see FIG. 7A).

Semi-drying conditions as used herein refer to a condition in which fibroblasts and keratinocytes inoculated in a biocompatible scaffold are covered with a culture medium of the minimum height possible (see FIG. 7B). In this condition, the vesicle is covered as thinly as possible just above the cell layer, and the elements affecting the cell layer act through the culture medium and the air.

Intracellular culture conditions herein refers to a state in which fibroblasts and keratinocytes inoculated in a biocompatible scaffold are covered with a culture medium of the maximum possible height (see FIG. 7C). In this condition, factors affecting the cell layer act through the culture medium.

As mentioned above, according to a preferred embodiment of the present invention the initial coculture is carried out under in-liquid culture conditions and the late co-culture is carried out under air-dry conditions. The duration of the initial co-culture is 1-20 days, preferably 2 -10 days, more preferably 3-9 days, most preferably 6-7 days. The period of late coculture is 1-20 days, preferably 2-10 days, more preferably 3-9 days, and most preferably 6-7 days.

The medium used in the co-culture may be any one commonly used in the culture of animal cells in the art. For example, a medium in which FBS is added to a 3: 1 mixed medium of KGM, FGM, DMEM, MEM, F-12, RPMI, and DMEM: F12 may be used. Preferably, initial culture of coculture (cultivation under in-liquid conditions) is carried out using KGM, and late culture (cultivation under air-dry conditions) is added with 10% FBS to a 3: 1 mixed medium of DMEM: F12. Using the prepared medium.

Meanwhile, the present invention is applied not only to biocompatible scaffolds coated with collagen but also to biocompatible scaffolds not coated with collagen. When the present invention is applied to the collagen uncoated scaffold, it is preferably inoculated with fibroblasts and cultured to some extent and then co-cultured with keratinocytes. According to this variant, the present invention comprises the steps of (i) inoculating and culturing fibroblasts into a biocompatible scaffold; (Ii) inoculating the keratinocytes into the scaffold; (Iii) co-culturing the inoculated fibroblasts and the keratinocytes. The culture of step (iii) is preferably in solution culture. The incubation period is 1-20 days, preferably 2-10 days, more preferably 3-9 days, and most preferably 6-7 days. The culture of step (ii) is preferably carried out in liquid culture at the beginning, and is preferably carried out in air-drying conditions. The period of initial coculture is 1-20 days, preferably 2-10 days, more preferably 3-9 days, most preferably 6-7 days. The period of late coculture is 1-20 days, preferably 2-10 days, more preferably 3-9 days, and most preferably 6-7 days.

As a result of culturing in the biocompatible scaffold through the above process, not only the cultivation efficiency of fibroblasts and keratinocytes is very excellent, but also keratinocytes are moved to the upper side of the scaffold. It is shown that the keratinocyte-fibroblast-scaffold complex formed by the method of the invention can be very usefully used as a skin substitute.

According to another aspect of the present invention, the present invention comprises a biocompatible scaffold, fibroblasts and keratinocytes, and provides an artificial skin substitute prepared by the co-culture method.

Since the artificial skin substitute of the present invention is prepared by the co-culture method described above, the common content between the two is omitted in order to avoid excessive complexity of the present specification.

According to a preferred embodiment of the present invention, the artificial skin substitute of the present invention is prepared according to the method of the present invention described above, which is carried out by culturing under submerge conditions and culturing under air-dry conditions. The keratinocytes are located on top of the scaffold.

The artificial skin substitute of the present invention contains a sufficient amount of fibroblasts and keratinocytes to treat skin defects, and is very useful as an artificial skin substitute because it has a three-dimensional structure in which the keratinocytes are located on top.

According to another aspect of the present invention, the present invention provides a collagen-coated biocompatible scaffold coated with collagen on the surface of the biocompatible scaffold.

According to a preferred embodiment of the invention, the biocompatible scaffold is chitosan, glycosaminoglycans, gelatin, hydroxyapatite, poly-d, l-lactic acid, poly-L-lactic acid, polyglycolic acid, mixed d , l-lactic acid and glycolic acid copolymer, L-lactic acid and glycolic acid copolymer, copolyoxalate, polycaprolactone, poly (lactic acid-caprolactone), poly (glycolic acid-caprolactone), hyaluronic acid (hyaluronic acid) acid) and derivatives thereof, chitin, chitosan, alginate, fibronectin, polyanhydride, polyorthoesters, polyurethane and casein. According to a more preferred embodiment of the invention, the biocompatible scaffold is chitosan, most preferably a neutralizing chitosan sponge.

In a preferred embodiment of the invention, the collagen-coated biocompatible scaffold is used for coculture of fibroblasts and keratinocytes.

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

Experiment method

1. Culture of Human Dermal Fibroblast

      Skin tissues obtained immediately after surgery were washed at least 10 times with phosphate buffered saline (PBS) containing 5% PS and most of the adipose tissue was removed with scissors. Sections were cut into 2 mm tissues and treated with 10 units of dispase (dispase, Roche, Mannheim, Germany) at 4 ° C for 16 hours to separate epithelial cell layers, followed by 0.35% cola at 37 ° C for 2 hours. Genomes (collagenase, Roche, Mannheim, Germany) were treated to separate the fibroblasts distributed in the dermal layer into single cells. Fibroblasts were placed in FGM-2 [Fibroblast medium (Cambrex, walkersville, MD, USA) in 2% FBS, 1 ng / ml hFGF-B (human basic fibroblast growth factor), 5 μg / ml insulin, 0.1% gentamicin, Cultured in amphotericin B].

2. human body Of keratinocytes  culture

The excised tissue was washed at least eight times with PBS containing 5% PS to remove coagulated blood and contaminants. In the process, the blood vessels and fat layer on the dermis were removed as cleanly as possible, and the tissue was cut finely to about 5 mm. The cut tissues were transferred to a 50 ml centrifuge tube, and then 10 ml of trypsin solution (0.125% trypsin: versene = 1: 1) was added, followed by physical stimulation (100 rpm) for 1 hour at room temperature using a magnetic bar. The keratinocytes come off. 40 ml of PBS was added to the trypsin solution, followed by centrifugation at 1,200 rpm and 4 ° C. for 5 minutes, and the supernatant was removed. KGM (keratinocyte growth media) containing 20% FBS in keratinocyte sediment (Cambrex, walkersville, MD, USA) 0.4% bovine pituitary extract (BPE), 0.1 ng / ml hEGF (human epithelial growth facotr) , 5 μg / ml insulin, 0.5 μg / ml hydrocortisone, 0.1% gentamycin, amphotericin B] 10 ml were added and centrifuged (1,200 rpm, 4 ° C., 5 minutes). After the centrifugation, 1-4 ml of fresh KGM medium was added to the centrifuge tube from which the supernatant was removed to unify the cells. The cell counts were calculated and dispensed to 3-5 x 10 ⁵ cell numbers per 10 cm culture dish coated with 0.1 mg / ml collagen IV (Sigma, Saint Louis, Missouri, USA).

3. Chitosan Scaffold  I made

1.5% chitosan (15 g), 1% acetic acid (10 mL) and sterile distilled water were added based on the volume of 1 L to prepare a raw material, which was shaken at room temperature for about 12 hours. The resulting solution was filtered through a glass filter and stored overnight in a 4 ° C. refrigerator to remove bubbles. In a mold (9 × 11 cm), 35 g of the support material is poured into a flat, evenly spread mold, and then rapidly frozen in a -80 ° C. freezer overnight, and then taken out of the freezer at -70 ° C. vacuum to 5 torr. And dried for 24 hours. Alcohol washing was performed to ensure no acetic acid remained on the support. Wash twice with 100% alcohol for 1 hour, wash twice with 70% alcohol for 1 hour, wash twice with 50% alcohol for 1 hour, and then wash with tertiary distilled water for 1 hour to remove alcohol. Then left for one night. Subsequently, the mixture was replaced with tertiary distilled water, washed for 30 minutes, and stored in a -80 ° C. freezer overnight to rapidly freeze. Then, it was taken out of the freezer and put into a freeze dryer of -70 ℃, vacuum 5 torr for 24 hours to prepare a chitosan scaffold finally.

4. Chitosan Scaffold  II production

A neutralized chitosan sponge was prepared according to the method disclosed in Korean Patent No. 298846 of the present inventors.

5. Chitosan Scaffold  Coating with Type I Collagen

The support (size of chitosan scaffold prepared above) in the size required for the experiment was added to 60% alcohol, and the chitosan support was gently pressed with an ear wax to remove bubbles to allow alcohol to completely infiltrate the support. Subsequently, after leaving at room temperature for 1-2 hours, alcohol was removed using kimwipes. Then, 0.3% type I collagen (Vitrogen) in a clean bench was suspended in PBS to make 0.1%, then mixed with the chitosan support and shaken at 4 ° C for 2 hours. The coated chitosan scaffold was well spread in a Petri dish and stored in a -80 ° C. freezer overnight to rapidly freeze, and then taken out of the freezer and placed in a freeze dryer of -70 ° C. and a vacuum of 5 torr, and dried for 24 hours. The lyophilized collagen-coated chitosan scaffold was stored at 4 ° C. or soaked in PBS after UV treatment and impregnated in the medium for 3-4 days before use in experiments.

In order to confirm the collagen coating, immunohistochemical staining was performed on type I collagen. As can be seen in Figure 8, it can be seen that the collagen is coated not only on the surface and the end face of the chitosan scaffold, but also on the middle portion of the scaffold thickness.

Chitosan In the chitosan scaffold  Cell Inoculation and Culture

The chitosan scaffold prepared in Experimental Method 4 or the collagen-coated chitosan scaffold of Experimental Method 4 was cut with an 8 mm biopsy punch, and then immersed in FGM-2 medium for 1 hour to be sufficiently washed. After nearly removing the absorbed medium in the 8 mm chitosan scaffold, fibroblasts and keratinocyte suspensions were inoculated to allow absorption. Cells were inoculated and inoculated with fibroblasts at 37 ° C. and 5% CO ₂ for 7 days, and then cultured in KGM medium when inoculated with keratinocytes in FGM-2 medium. In the case of collagen coating, only 1, 5, 10, 50 x 10 ³ fibroblasts were inoculated, or 5 x 10 ⁴ fibroblasts and 3 x 10 ⁵ skin keratinocytes were inoculated and cultured for 7 days. If skin keratinocytes are mixed, submerged culture for 7 days to submerge in culture, peel off the silicon membrane of chitosan scaffold, transfer to millicell, and then remove E-medium [10% FBS, DMEM (dulbecco's). modified eagle medium): F-12 (nutrient mixture) 3: 1 mixed medium] was incubated for 7 days again under air-dry (air-dry) conditions. The group inoculated only with fibroblasts during the incubation was cultured in FGM-2 medium, and the keratinocyte mixed group was cultured in KGM medium.

Using ABC method PCNA , Bimethine , Plate Cytokeratin  Tissue staining

Paraffin blocks were cut to 4 μm thickness and dried to prepare paraffin sections. 3 times for 5 minutes in xylene, 2 minutes in 100% ethanol, 1 minute in 90% ethanol, 1 minute in 80% ethanol, 1 minute in 70% ethanol for the deparaffinization process After the reaction, it was washed for 10 minutes in PBS. For the pretreatment, the reaction was performed for 10 minutes in methanol containing 0.5% hydrogen peroxide, 5 minutes in 0.3% PBST (PBS containing 0.05% of Tween 20), and 10 minutes in a 0.01 M sodium citrate solution boiled at 100 ° C. In order to inhibit the reaction of non-specific proteins, the reaction was performed for 30 minutes in a solution in which 1% normal blocking serum was mixed with PBS. Primary antibodies to vimentin (Dako, Carpinteria, CA, USA), pancytokeratin (Pancytokeratin: Dako, Carpinteria, CA, USA), PCNA (proliferating cell nuclear antigen: (Dako, Carpinteria, CA, USA) Each primary antibody was mixed with 1% normal blocking serum and allowed to react for 40 minutes, followed by reaction of biotin-attached universal secondary antibody (Universal Secondary Antibody, Vector, Burlingame, CA, USA) for 35 minutes. Streptavidin horseradish peroxidase (Vector, Burlingame, CA, USA) was reacted for 30 minutes, and as a colorant, 3.5% hydrogen peroxide in DAB (Diaminobenzidine: Dako, Carpinteria, CA, USA) solution was used. And nickel were mixed in. Counterstain was used as fast red (Sigma, Saint Louis, MO, USA) 10 times in 70% ethanol for 10 seconds and 10 seconds in 80% ethanol. Times, 90% After reacting 10 times in ethanol for 1 second, 2 minutes in 100% ethanol for 2 minutes, and 3 minutes in xylene for 3 minutes, it was mounted with a mountain solution.

H & E Dyeing

Paraffin blocks were cut to 4 μm thickness and dried to prepare paraffin sections. For deparaffinization, the reaction was carried out three times for 5 minutes in xylene, three times for two minutes in 100% ethanol, once for one minute in 90% ethanol, once for one minute in 80% ethanol, and once for one minute in 70% ethanol. Then, it was washed with running water for 10 minutes. After reacting with hematoxylin stain for 10 minutes, washing with running water for 3 minutes, reacting with Eosin staining solution for 10 minutes, and washing with running water until no eosin stain was released. 10 times in 70% ethanol for 1 second, 10 times in 80% ethanol for 1 second, 10 times in 90% ethanol for 1 second, 2 times for 1 minute in 100% ethanol, 3 minutes in xylene, and then Mounting.

Chitosan Scaffold  ( chitosan  fibroblasts in the scaffold) Keratinocyte  Timed inoculation and culture

Chitosan scaffolds were cut with a 10 mm biopsy punch and then immersed in FGM-2 medium for 1 hour to wash thoroughly. The chitosan scaffold used in this experiment is a chitosan scaffold prepared in Experimental Method 3 without collagen coating. After nearly removing the absorbed media on the 10 mm chitosan scaffold, fibroblasts were inoculated by inoculation with 7 × 10 ⁵ per cm ² . Submerged culture for 7 days submerged in FGM-2 culture in 37 ° C., 5% CO ₂ , incubator, stripped off the silicon membrane of chitosan scaffold, transferred to millicell, and then 1 × 10 ⁶ skins Keratinocytes were seeded on top of the scaffold. Incubate for 7 days in an incubator at 37 ° C., 5% CO ₂ , incubated again in 10% FBS, DMEM (Dulbecco's modified eagle medium) medium containing F-12 (nutrient mixture) 3: 1 (Submerged culture) and incubated in the same medium under air-dry conditions for 7 days.

Experiment result

Chitosan On the scaffold  Proliferation and Survival of Human Dermal Fibroblasts

It was confirmed that human dermal fibroblasts can survive and proliferate in three dimensional culture by use of chitosan scaffold. Human dermal fibroblasts were inoculated at a concentration of 1 x 10 ³ , 5 x 10 ³ , 10 x 10 ³ , 50 x 10 ³ in each of the 8 mm diameter annular scaffolds for type I collagen-coated chitosan scaffolds, Chitosan scaffolds that were not coated with type I collagen were inoculated at concentrations of 1 × 10 ⁶ , 3 × 10 ⁶ , 6 × 10 ⁶ , 10 × 10 ^{6 to} each of the 8 mm diameter annular scaffolds. After inoculation, human dermal fibroblasts were cultured under normal cell culture conditions for 7 days using FGM-2 medium. In order to confirm the survival and proliferation of human dermal fibroblasts in chitosan scaffolds, hematoxylin / eosin staining (H / E staining) and immunohistochemistry for the fibroblast marker protein, bimentin, were performed.

Human dermal fibroblasts proliferated in proportion to cell inoculation concentrations in type I collagen coated chitosan scaffolds and uncoated chitosan scaffolds (FIGS. 1 and 2). These results show that chitosan sponges are suitable scaffolds for 3-D culture of human dermal fibroblasts.

Chitosan On the scaffold  Human dermal fibroblast and human body Keratinocyte  Proliferation and Survival

We investigated whether human dermal fibroblasts and mixtures of human dermal fibroblasts and keratinocytes not mixed with human keratinocytes can survive and proliferate in 3-D culture using chitosan scaffold. Fibroblasts and keratinocytes were inoculated into type I collagen-coated 8 mm diameter circular chitosan scaffolds at concentrations of 5 × 10 ⁴ and 3 × 10 ⁵ , respectively (FIG. 4). When the type I collagen was not coated, fibroblasts and keratinocytes were inoculated into 8 mm diameter circular chitosan scaffolds at concentrations of 1 × 10 ⁷ and 3 × 10 ⁵ , respectively (FIG. 3). After inoculation, cells were cultured in FGM-2 medium for 7 days. To differentiate the keratinocytes, the cells were again incubated in air-dried for 7 days.

First, H / E staining and immunohistochemistry for PCNA were performed to confirm cell survival and proliferation in chitosan scaffolds. Second, immunohistochemical reactions were performed on bimentin, a marker protein of fibroblasts, or plate-cytokeratin, a marker protein of keratinocytes, to verify whether the cultured cells were fibroblasts or keratinocytes. In H / E staining and immunohistochemistry for PCNA, cells proliferated well in both type I collagen coated and uncoated chitosan scaffolds (FIGS. 3 and 4).

Thereafter, immunohistochemical reactions were performed to identify cells proliferating in the chitosan scaffold. For chitosan scaffolds that were not coated with type I collagen, most were bimentin positive (+) cells and plate-cytokeratin negative (−) cells (FIGS. 3 and 5). However, in type I collagen-coated chitosan scaffolds, both bimentin positive (+) cells and plate-cytokeratin positive (+) cells could be observed (FIGS. 4 and 6). These results show that type I collagen coated chitosan scaffolds are particularly preferred for co-culture of fibroblasts and keratinocytes.

In addition, surprisingly, keratinocytes, plate-cytokeratin positive (+) cells migrated to the top of the scaffold given air-drying conditions (Panel D of FIG. 6). This migration of keratinocytes more clearly shows its potential as a skin substitute.

Chitosan On the scaffold  Fibroblasts and Keratinocyte  Timed vaccination and coculture

In addition to co-culture with fibroblasts and keratinocytes at the same time, we examined whether two cells can proliferate well in 3-D cultures using chitosan scaffolds. .

When fibroblasts and keratinocytes were simultaneously inoculated into a type I collagen-coated chitosan scaffold, the plate-cytokeratin negative, a label protein of fibroblasts and a label protein of keratinocytes, was used. -) Cells (Figures 3 and 5). However, when fibroblasts were first inoculated into the type I collagen-coated chitosan scaffold and intracellularly cultured for 7 days, and when keratinocytes were inoculated into the upper layer, as shown in FIG. 9, a bimentin positive protein, a marker protein of fibroblasts, was identified. All plate-cytokeratin positive (+) cells, which are (+) cells and marker proteins of keratinocytes, were observed. This is because the type I collagen produced by the fibroblasts helped the engraftment rate of the keratinocytes during 7 days incubation by inoculating fibroblasts into the type I collagen-free chitosan scaffold first.

As described in detail above, the present invention provides a method for co-culture of skin-derived fibroblasts and keratinocytes, artificial skin substitutes and collagen-coated biocompatible scaffolds. According to the method of the present invention, the fibroblasts, which are the main cells of the dermal dermal layer, and the keratinocytes, which are the main cells of the skin epidermal layer, can be co-cultured with excellent efficiency, which is very efficient for producing artificial skin substitutes, and thus manufactured artificial skin. In the substitute, the keratinocytes are moved to the top of the scaffold, and this three-dimensional structure shows that the keratinocyte-fibroblast-scaffold complex of the present invention can be very usefully used as a skin substitute.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

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Claims (12)

A method of co-culturing fibroblasts and keratinocytes comprising the following steps: (a) inoculating fibroblasts and keratinocytes into a biocompatible scaffold; And (b) culturing fibroblasts and keratinocytes inoculated into the biocompatible scaffold. The method of claim 1, wherein the biocompatible scaffolds are coated with collagen. The biocompatible scaffold of claim 1, wherein the biocompatible scaffold comprises chitosan, glycosaminoglycans, gelatin, hydroxyapatite, poly-d, l-lactic acid, poly-L-lactic acid, polyglycolic acid, mixed d, l- Copolymers of lactic acid and glycolic acid, copolymers of L-lactic acid and glycolic acid, copolyoxalates, polycaprolactones, poly (lactic acid-caprolactones), poly (glycolic acid-caprolactones), hyaluronic acid and A derivative thereof, chitin, chitosan, alginate, fibronectin, polyanhydride, polyorthoesters, polyurethane and casein. 4. The method of claim 3 wherein the biocompatible scaffold is chitosan. 3. The method of claim 2, wherein said collagen is type I collagen. The method of claim 1, wherein step (b) comprises the following sub-steps: (b-1) culturing the fibroblasts and keratinocytes inoculated in the biocompatible scaffold under submerge conditions; And (b-2) culturing the cultured fibroblasts and keratinocytes under air-dry conditions. An artificial skin substitute, comprising the biocompatible scaffold, fibroblasts and keratinocytes, prepared by the method of any one of claims 1 to 6. 8. The artificial skin substitute of claim 7, wherein the artificial skin substitute is prepared by the method of claim 6 and wherein the keratinocytes are located on top of the scaffold. A collagen-coated biocompatible scaffold with collagen coated on the surface of the biocompatible scaffold. 10. The method of claim 9, wherein the biocompatible scaffold is chitosan, glycosaminoglycans, gelatin, hydroxyapatite, poly-d, l-lactic acid, poly-L-lactic acid, polyglycolic acid, mixed d, l- Copolymers of lactic acid and glycolic acid, copolymers of L-lactic acid and glycolic acid, copolyoxalates, polycaprolactones, poly (lactic acid-caprolactones), poly (glycolic acid-caprolactones), hyaluronic acid and Collagen-coating, characterized in that it is selected from the group consisting of derivatives thereof, chitin, chitosan, alginate, fibronectin, polyanhydride, polyorthoesters, polyurethane and casein Biocompatible Scaffolds. The collagen-coated biocompatible scaffold of claim 10, wherein the biocompatible scaffold is chitosan. 10. The collagen-coated biocompatible scaffold of claim 9, wherein the collagen-coated biocompatible scaffold is used for coculture of fibroblasts and keratinocytes.

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