KR102452585B1 - Wrinkle forming apparatus for artificial skin, producing method of wrinkled artificial skin using the same, the wrinkled artificial skin and screening method of compound for improving skin wrinkle using the wrinkled artificial skin - Google Patents

Abstract

a mold fixing frame and a mold mounted on the mold fixing frame, wherein the mold includes a mounting part mounted on the mold fixing frame and a stamp connected to the mounting part, the stamp having an upper surface directly coupled to the mounting part and an upper surface facing the upper surface Artificial skin wrinkle forming apparatus including a lower surface, wherein the lower surface includes one or more irregularities, a method for manufacturing wrinkled artificial skin using the same, a wrinkled artificial skin prepared by the manufacturing method, and a skin wrinkle improvement compound screening method using the wrinkled artificial skin provides

Description

Artificial skin wrinkle forming device, wrinkled artificial skin manufacturing method using the same, wrinkled artificial skin prepared by the above manufacturing method, and skin wrinkle improving compound screening method using the wrinkled artificial skin {WRINKLE FORMING APPARATUS FOR ARTIFICIAL SKIN, PRODUCING METHOD OF WRINKLED ARTIFICIAL SKIN USING THE SAME, THE WRINKLED ARTIFICIAL SKIN AND SCREENING METHOD OF COMPOUND FOR IMPROVING SKIN WRINKLE USING THE WRINKLED ARTIFICIAL SKIN}

The present disclosure relates to an artificial skin wrinkle forming device, a wrinkled artificial skin manufacturing method using the same, wrinkled artificial skin prepared by the manufacturing method, and a skin wrinkle improving compound screening method using the wrinkled artificial skin.

The skin is an organ that covers the outside of the body and consists of three layers: the epidermis, the dermis, and the subcutaneous fat layer from the outside. The epidermis is mostly composed of keratinocytes of the stratified squamous epithelium. The dermis, composed of matrix proteins such as collagen fibers and elastic fibers, is located below the epidermis, and the dermis contains blood vessels, nerves, and sweat glands. The subcutaneous fat layer is composed of fat cells. The skin maintains its shape by interacting with various cells and constituents as described above, and exhibits various functions such as body temperature regulation and a function as a barrier to the external environment.

Artificial skin is a three-dimensionally reconstructed skin as described above using skin cells and skin components such as collagen and elastin. Because of its functional properties, it is also called skin equivalent or reconstructed skin. Artificial skin is not only used for replacement (permanent engraftment type) or regeneration (temporary covering type) of damaged skin such as burns and trauma, but also is used in various fields such as skin physiology research, skin irritation evaluation, and skin efficacy evaluation. .

However, unlike real skin, artificial skin has a flat structure in the dermal-epidermal junction (DEJ) layer, so there is a limit to conducting anti-aging experiments using only artificial skin. This is because the interface between the dermal layer and the epidermis layer is curved rather than flat in real skin.

Unfortunately, a lot of research has not been done actively on a method for manufacturing artificial skin that mimics the curves or wrinkles of the skin, and this is because the various wrinkle-improving cosmetics currently on the market have a wrinkle-improving effect compared to the actual advertisements. is one cause of the large drop.

Accordingly, the present inventors have developed a device capable of manufacturing a new wrinkled artificial skin that has not been reported until now, after a long study, and have used this to manufacture wrinkled artificial skin, and through this, a skin wrinkle improving material can be easily obtained can be screened.

The modification of the artificial skin model so far mainly relies on cell biological methods that change cells or add or remove specific substances, and these methods wrinkle the structure of the interface between the dermal and epidermal layers constituting the artificial skin. As it is not the case, it has limitations in several aspects to apply the experimental results of artificial skin models up to now to actual skin as it is. One embodiment is to provide an artificial skin wrinkle forming device for making artificial skin have wrinkles like real skin.

Another embodiment is to provide a method for manufacturing wrinkled artificial skin using the artificial skin wrinkle forming device.

Another embodiment is to provide a wrinkled artificial skin prepared by the above manufacturing method.

Another embodiment is to provide a method for screening a skin wrinkle improving compound using the wrinkled artificial skin.

According to one embodiment, a mold fixing frame and a mold mounted on the mold fixing frame are included, wherein the mold includes a mounting part mounted on the mold fixing frame and a stamp connected to the mounting part, wherein the stamp is an upper surface directly coupled to the mounting part and a lower surface facing the upper surface, wherein the lower surface provides an artificial skin wrinkling device including one or more irregularities.

The stamp may have a width of 3mm to 12mm.

The concave-convex portion may have a triangular shape.

The lower surface of the stamp may include one or more triangular-shaped irregularities, and the width and length of the triangular-shaped irregularities may be the same or different from each other.

The stamp including the concave-convex part of the triangular shape may satisfy Equation 1 below.

[Equation 1]

(w + h) x 2 ≤ S ≤ (w + h) x 5

In Equation 1 above,

S is the width of the stamp,

w is the average width of the concave-convex portion included in the lower surface of the stamp,

h is the average length of the uneven portion included in the lower surface of the stamp.

While the lower surface of the stamp includes one or more triangular-shaped irregularities, the stamp may satisfy Equation 2 below.

[Equation 2]

G x 2 < S

In Equation 2 above,

S is the width of the stamp,

G is the average distance between one or more irregularities included in the lower surface of the stamp.

The concavo-convex portion may have a semicircular shape.

The lower surface of the stamp may include one or more semi-circular concavo-convex portions, and the width and length of the semi-circular concave-convex portions may be the same or different from each other.

While the lower surface of the stamp includes one or more semicircular irregularities, the stamp may satisfy Equation 3 below.

[Equation 3]

w x 24 < S < w x 45

In Equation 3 above,

S is the width of the stamp,

w is the average width of the concave-convex portion included in the lower surface of the stamp.

The lower surface constituting the stamp includes three or more concavo-convex portions, the concave-convex portion includes a first concave-convex portion, a second concave-convex portion, and a third concave-convex portion, and the first concave-convex portion and the third concave-convex portion have widths and lengths equal to each other, the width of the second concave-convex portion is two ? It may be positioned between the third concavo-convex portions.

The artificial skin wrinkle forming device may further include a shoulder attachment device to which the mold fixing frame is attached.

The mold fixing frame and the mold may each independently be made of acrylonitrile-butadiene-styrene (ABS) resin or polylactic acid (PLA) resin.

According to another embodiment, injecting an artificial dermal mixture comprising collagen and fibroblasts to the shoulder attachment device; stamping so that the concave-convex portion of the artificial skin wrinkling device comes into contact with the non-hardened mixture before the mixture hardens; forming a wrinkled artificial dermal layer by hardening the mixture; and forming an epidermal layer on the wrinkled artificial dermal layer.

In the step of injecting the artificial dermal mixture into the shoulder attachment device, the artificial dermal mixture may be introduced in a volume of 400 μl to 800 μl.

The step of forming a wrinkled artificial dermal layer by hardening the mixture may be a step of leaving the mixture in an incubator for cell culture for 1 hour to 5 hours after the stamping.

After the step of forming the artificial dermal layer, before the formation of the epidermal layer, the method may further include putting a fibroblast culture medium on the artificial dermal layer and culturing.

The forming of the epidermal layer may include: seeding keratinocytes on the wrinkled artificial dermal layer, and culturing them in a keratinocyte culture medium; And after the culturing, it may include the step of culturing it in an epidermal layer production medium.

After seeding the keratinocytes on the wrinkled artificial dermal layer, the step of culturing them in a keratinocyte culture medium may be performed for 1 to 3 days.

The step of culturing in the epidermal layer production medium may be a step of culturing in the epidermal layer production medium without exposure to air, and then exposing it to air again and culturing.

According to another embodiment, there is provided wrinkled artificial skin prepared by the method for manufacturing wrinkled artificial skin.

According to another embodiment, a skin wrinkle improvement compound screening method comprising the step of treating the wrinkled artificial skin with a compound and checking whether the wrinkles in the artificial skin disappear over time after the compound treatment provides

Using the artificial skin wrinkle forming apparatus according to an embodiment, it is possible to manufacture wrinkled artificial skin in the form most similar to real skin having wrinkles at the interface between the dermal layer and the epidermal layer. And, since the wrinkled artificial skin has a shape very similar to that of real skin, a skin wrinkle improving compound can be easily screened using this.

1 and 2 are each independently a photograph of an artificial skin wrinkling device (an artificial skin wrinkling device attached to the shoulder attachment device) according to an embodiment.
3 is a photograph of a mold fixing frame constituting an artificial skin wrinkle forming device according to an embodiment.
4 is a photograph of an artificial skin wrinkle forming device (a mold fixing frame and a mold mounted thereon) according to an embodiment.
5 is a photograph showing the state of forming wrinkles in the artificial dermal layer by supporting the artificial dermal layer that is not wrinkled on the shoulder attachment device, and then stamping with the artificial skin wrinkle forming device.
6, 10, 16, 24 and 35 are each independently stamped cross-sectional views of a mold constituting the artificial skin wrinkle forming device according to an embodiment.
7 is a photograph of wrinkled artificial skin (Example 1) manufactured using a mold including the stamp having the cross section of FIG. 6 .
FIG. 8 is a confocal laser scanning microscope (CLS microscope) picture obtained by magnifying the red box of FIG. 7 (x10).
9 is an optical coherence tomography (OCT) photograph of a cross-section of wrinkled artificial skin manufactured using a mold including the stamp having the cross-section of FIG. 6 .
11 is a photograph of wrinkled artificial skin (Example 2) manufactured using a mold including the stamp having the cross section of FIG. 10 .
FIG. 12 is a confocal laser scanning microscope (CLS microscope) photograph obtained by magnifying the red box of FIG. 11 (x10).
13 is an optical coherence tomography (OCT) photograph of a cross-section of wrinkled artificial skin manufactured using a mold including the stamp having the cross-section of FIG. 10 .
14 is a photograph of a conventional artificial skin (Comparative Example 1) in which the interface between the dermal layer and the epidermal layer is flat.
FIG. 15 is a confocal laser scanning microscope (CLS microscope) photograph obtained by magnifying the red box of FIG. 14 (x10).
17 is a photograph of wrinkled artificial skin (Example 3) manufactured using a mold including the stamp having the cross section of FIG. 16 .
18 is an optical coherence tomography (OCT) photograph of a cross-section of wrinkled artificial skin manufactured using a mold including the stamp having the cross-section of FIG. 16 .
19 is a photograph after treating the wrinkled artificial skin of FIG. 17 with retinoic acid, which is well known as a wrinkle-improving material.
20 is an optical coherence tomography (OCT) photograph of a cross-section of wrinkled artificial skin manufactured using a mold including a stamp having a cross-section of jaw 19;
21 is a micrograph showing the cross-section of real skin after H&E tissue staining.
22 is a micrograph showing a cross-section of the conventional artificial skin after H&E tissue staining.
23 is a schematic view showing the step of forming a wrinkled artificial dermal layer.
25 to 33 are photographs of wrinkled artificial skin according to Examples 4 to 12, respectively.
35 is a schematic diagram showing a method of screening whether the compound has skin wrinkle improvement effect by treating a specific compound on wrinkled artificial skin prepared using a mold including the stamp having the cross section of FIG. 34 .

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art to which the present invention pertains can easily implement them. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

As used herein, the term "artificial skin" refers to a three-dimensional reconstitution of skin using skin cells and collagen, which is a component of the skin, and a polymer complex exhibiting structural and functional properties similar to those of actual skin. It is the broadest meaning that includes all ramen noodles.

As used herein, the term "stamping" may mean an operation of applying a force so that the concave-convex part of the artificial skin wrinkling device comes into contact with the imitation layer such as the artificial dermal layer, and after applying the force, the artificial skin wrinkling device is removed again work may not be included.

In the present specification, when a part such as a layer, film, region, plate, etc. is "on" another part, it includes not only the case where the other part is "directly on" but also the case where there is another part in the middle. Conversely, when we say that a part is "just above" another part, we mean that there is no other part in the middle.

Hereinafter, an artificial skin wrinkle forming device according to an embodiment will be described.

In real skin, the interface between the epidermal layer and the dermis layer is not flat, but has curved wrinkles (refer to the circle indicated by the red dotted line in FIG. 21). Reference), since the shape of the real skin and the conventional artificial skin are different, it was difficult to apply the experimental results with the artificial skin to the real skin as it is. In fact, most of the substances judged to be effective in improving skin wrinkles through artificial skin test results are not actually effective in improving skin wrinkles. This is because it is not an appropriate model for judging the improvement effect.

Even if the wrinkle improvement effect of the skin cannot be easily confirmed with the conventional artificial skin model, it is not possible to conduct animal testing according to the Cosmetic Animal Testing Ban Act. There was a problem in that it was difficult to easily predict the wrinkle improvement effect on the actual skin.

In addition, in order to solve the above problems, conventional techniques for forming wrinkles on artificial skin by stretching artificial skin have been introduced. It was impossible to manufacture artificial skin, and above all, there is a problem in that it is not easy to easily form wrinkles in conventional artificial skin by stretching.

However, since the artificial skin wrinkle forming device according to an embodiment forms wrinkles by a stamping method rather than a stretching method, it does not form wrinkles as a follow-up operation on the already manufactured artificial skin, but reduces wrinkles from the initial stage of artificial skin manufacturing. Since the forming step can be carried out and the stamped shape or form can be easily changed, artificial skin having a desired shape of wrinkles can be easily produced in large quantities.

The artificial skin wrinkle forming apparatus according to an embodiment includes a mold fixing frame and a mold mounted on the mold fixing frame, wherein the mold includes a mounting unit mounted on the mold fixing frame and a stamp connected to the mounting unit, wherein the stamp is the mounting unit and a lower surface directly coupled to the upper surface and a lower surface facing the upper surface, wherein the lower surface includes one or more concavo-convex portions.

3 is a photograph of the mold fixing frame, and by inserting the mounting part of the mold into the center hole of the mold fixing frame (see FIG. 4 ), an artificial skin wrinkle forming device composed of a mold and a mold fixing frame can be manufactured. The artificial skin wrinkle forming device having such a shape may adjust the height by pulling or pushing the mounting part of the mold, and may form wrinkles on the artificial skin by pushing the mounting part in the stamp direction.

More specifically, the artificial skin wrinkle forming apparatus according to an embodiment may further include a shoulder attachment device to which the mold fixing frame is shoulder attached (refer to FIGS. 1 and 2 ), at this time, the artificial skin, specifically, in the shoulder attachment device An artificial dermal layer may be supported. That is, after the artificial dermal layer is loaded in the shoulder attachment device, the mold fixing frame of the artificial skin wrinkle forming device composed of the mold and the mold fixing frame is attached to the shoulder attachment device to fix the artificial skin wrinkling device, and then the mounting part of the mold After adjusting the height by pulling or pushing, by pushing the mounting part of the mold in the direction in which the artificial dermal layer is supported, wrinkles of a desired shape can be easily formed in the artificial dermal layer. (See FIG. 5) For example, Transwell ^® sold by Corning may be used as the shoulder attachment device, but the type of the shoulder attachment device is not necessarily limited thereto.

For example, the stamp may have a width of 3mm to 12mm. When the width of the stamp is less than 3 mm, it is difficult to form an uneven portion in the stamp, and when stamping, collagen constituting the artificial dermal mixture may protrude around the stamp. In this case, since the stamp is not easily inserted into the shoulder attachment device, the stamping cannot be performed at a desired height, which may be undesirable as it makes it difficult to form fine wrinkles in the artificial dermal layer.

For example, the concave-convex portion may have various shapes, for example, a polygonal or semicircular shape such as a triangle, a square, a pentagon, a hexagon, and the like. They may have a width and a length, or they may have different widths and lengths.

On the other hand, depending on the shape of the concavo-convex part, the wrinkle can be easily formed on the artificial dermal layer only when the concave-convex part included in the lower surface of the stamp satisfies certain parameters.

Specifically, when the concave-convex portion has a triangular shape, the stamp may satisfy Equation 1 below.

[Equation 1]

(w + h) x 2 ≤ S ≤ (w + h) x 5

In Equation 1 above,

S is the width of the stamp,

w is the average width of the concave-convex portion included in the lower surface of the stamp,

h is the average length of the uneven portion included in the lower surface of the stamp.

More specifically, when the uneven portion has a triangular shape and the width S of the stamp is 10 μm or more, for example, 10 μm to 12 μm, the stamp may satisfy Equation 1-1 below.

[Equation 1-1]

(w + h) x 2.2 ≤ S ≤ (w + h) x 4

In Equation 1-1,

S is the width of the stamp,

w is the average width of the concave-convex portion included in the lower surface of the stamp,

h is the average length of the uneven portion included in the lower surface of the stamp.

When the stamp including the concave-convex part of the triangular shape satisfies Equation 1, or when the stamp including the concave-convex part of the triangular shape and has a width of 10 μm or more satisfies Equation 1-1, the stamp is easily artificial Not only can wrinkles be formed on the dermal layer, but also the shape of fine wrinkles can be reproduced.

In addition, when the concave-convex portion has a triangular shape, the stamp may include one or more, for example, two or more concave-convex portions on the lower surface, while simultaneously satisfying Equation 2 below.

[Equation 2]

G x 2 < S

In Equation 2 above,

S is the width of the stamp,

G is the average distance between one or more, for example, two or more irregularities included in the lower surface of the stamp.

When the stamp including the concave-convex part of the triangular shape satisfies Equation 2 above, the stamp may include a plurality of concavo-convex parts, so that more various types of wrinkle shapes can be formed on the artificial dermal layer.

On the other hand, when the concave-convex portion has a semi-circular shape rather than a triangular shape, the stamp may include one or more, for example, two or more semi-circular concave-convex portions on the lower surface, while simultaneously satisfying Equation 3 below.

[Equation 3]

w x 24 < S < w x 45

In Equation 3 above,

S is the width of the stamp,

w is the average width of the concave-convex portion included in the lower surface of the stamp.

When the concave-convex portion has a round semicircular shape rather than a polygonal shape such as a triangle, it may be easy to form wrinkles in the artificial dermal layer by including several concave-convex portions on the lower surface of the stamp. For example, when the relationship between the width of the stamp and the average width of the semicircular concavo-convex portion satisfies Equation 3, fine and various wrinkles can be reproduced in the artificial dermal layer at the same level as when the concave-convex portion has a polygonal shape.

For example, the lower surface constituting the stamp includes three or more concave-convex portions, the concave-convex portion is composed of a first concave-convex portion, a second concave-convex portion and a third concave-convex portion, and the first concave-convex portion and the third concave-convex portion include a width and a The lengths are the same, the width of the second concavo-convex portion is two to two greater than the width of the first concavo-convex portion, the length of the second concave-convex portion is longer than the length of the first concavo-convex portion, and the second concavo-convex portion is the first concavo-convex portion It may be located between the part and the third concavo-convex part (see FIG. 35 ). In this case, it is possible to form wrinkles in a shape very similar to that of actual human skin on the artificial skin, and an artificial skin wrinkle forming device having a stamp having a lower surface including the first uneven portion, the second uneven portion, and the third uneven portion. When the skin wrinkle improving compound is treated on the wrinkled artificial skin prepared using It can be very useful for screening wrinkle-improving compounds (see FIG. 36 ). Specifically, as a compound having a skin wrinkle improvement effect, 'wrinkled artificial skin manufactured using an artificial skin wrinkle forming device having a stamp having a lower surface including the first uneven part, the second uneven part and the third uneven part' In the case of processing, the wrinkles formed by the first and third uneven portions are not deep enough to be easily restored (wrinkled), but the wrinkles formed by the second uneven portions are deep and not easily restored In bar, by comparing the degree of restoration of the wrinkles formed by the first uneven portion and the third uneven portion with the restoration degree of the wrinkles formed by the second uneven portion, it can be easily determined whether the treated specific compound has the skin wrinkle improvement effect. can be screened.

For example, the mold fixing frame and the mold may each independently be made of acrylonitrile-butadiene-styrene (ABS) resin or polylactic acid (PLA) resin. When the mold fixing frame and the mold are made of the above type of resin, it may be easy to reproduce even fine wrinkles without contaminating the artificial dermal layer even when in direct contact with the artificial dermal layer. Furthermore, the above type of resin is a material that can be manufactured even with a 3D printer, and it may be possible to mass-produce the artificial skin wrinkle forming device according to an embodiment at a low price.

Wrinkled artificial skin manufacturing method according to another embodiment comprises the steps of injecting an artificial dermal mixture containing collagen and fibroblasts to the shoulder attachment device; stamping so that the concave-convex portion of the artificial skin wrinkling device comes into contact with the non-hardened mixture before the mixture hardens; forming a wrinkled artificial dermal layer by hardening the mixture; and forming an epidermal layer on the wrinkled artificial dermal layer.

The dermis is a layer of skin beneath the epidermis made of connective tissue, which acts as a buffer and protects the body from stress and strain. The dermal layer is tightly connected to the epidermis through the basement membrane. The dermal layer provides a matrix that supports various appendages such as blood vessels and nerves that are inherent in the structure.

The artificial dermal layer may be formed using a material mixed with fibroblasts and collagen from the viewpoint of human correlation with the actual skin. The artificial dermal layer may consist of one or two or more layers, and may include, for example, a first dermal layer containing collagen and a second dermal layer containing fibroblasts, and may be composed of collagen and fibroblasts. It may be composed of a single dermal mimic layer containing In this case, the contraction of the dermis of the artificial skin can be further alleviated.

The first dermal layer may contain collagen, and the extracellular matrix constituting the dermis, such as elastin, chitosan, glycosaminoglycans (GAGs; glycosaminoglycans), and hyaluronic acid (HA). extracellular matrix) may be further contained.

The second dermal layer may be located on top of the first dermal layer, contains fibroblasts, elastin, chitosan, glycosaminoglycans (GAGs; glycosaminoglycans), hyaluronic acid (HA; hyaluronic acid) ) and may further contain an extracellular matrix constituting the dermis.

The single artificial dermal layer may contain collagen and fibroblasts, and the collagen and fibroblasts are as described above.

The collagen used may be a collagen of bovine origin, from rat tail or from fish, or any other origin of natural collagen or collagen produced by genetic engineering, which is capable of contracting in the presence of fibroblasts.

The thickness of the artificial dermal layer is generally at least 0.05 cm, in particular between approximately 0.05 cm and 2 cm, but may be increased or decreased as long as the advantageous properties of the artificial skin according to the present invention are not impaired.

In the step of injecting the artificial dermal mixture into the shoulder attachment device, the artificial dermal mixture may be introduced in a volume of 400 μl to 800 μl. When the artificial dermal mixture is added in the volume range, the stamped wrinkles in the artificial dermal layer can be firmly generated.

When the artificial dermal mixture containing the collagen and fibroblasts is put into the shoulder attachment device, the artificial dermal mixture is gelated over time. Thereafter, the mixture is hardened to form a wrinkled artificial dermal layer. For example, after the stamping, by leaving the stamped mixture in an incubator for cell culture (37° C., 5% CO ₂ ) for 1 to 5 hours, the mixture can be hardened to form a wrinkled artificial dermal layer. Specifically, the stamped mixture may refer to a mixture in which a stamp is stamped in the mixture (a mixture in which the concave-convex portion of the wrinkle forming device is in contact with the mixture). When the stamped mixture hardens and gelation is completed, the wrinkled artificial dermal layer can be obtained by removing the wrinkling device.

After the step of forming the artificial dermal layer, before forming the epidermal layer, a fibroblast culture medium (M106, Gibco) is put on the artificial dermal layer and cultured, for example, about 5 to 9 days, about 6 days to about 8 days, Or it may further include the step of culturing for about 7 days. Before forming the epidermal layer, by culturing a fibroblast culture medium on the artificial dermal layer, the artificial dermal layer can be further stabilized.

When the artificial dermal layer is formed as described above, keratinocytes are applied thereon and then cultured. That is, the artificial epidermal layer is formed on the artificial dermal layer by placing and culturing keratinocytes on the artificial dermal layer. The keratinocytes constitute about 80% to 90% of the cells of the epidermal layer, and are formed through mitosis in the basal layer, which is the deepest layer of the epidermal layer, and then ascend to the upper layer toward the skin surface.

Keratinocytes as used herein may be, for example, keratinocytes derived from humans. Any commercially available keratinocytes may be used as the keratinocytes, and keratinocytes derived from other cells as well as keratinocytes directly isolated or isolated from humans and cultured may be used. Commercially available human keratinocytes include NHEK-Neo, Pooled (Neonatal Normal Human Epidermal Keratinocytes, Pooled: Catalog No. 00192906, Tissue Acquisition No. P867, Caucasians), NHEK-Neo (Product No. 00192907, Tissue) provided by Lonza. acquisition number: 20647, white), NHEK-Adult (product number 00192627, tissue acquisition number: 21155, white), and NHEK-Neo (product number 00192907, organization acquisition number: 18080, black). and HEKn (Human Epidermal keratinocytes, neonatal: product number C0015C, tissue acquisition number 1781129, white) and HEKn (product number C0015C, tissue acquisition number 1803827, black) provided by Thermo Fisher. In order to maintain the property of keratinocytes to attach to the base membrane and proliferate, the dish is preferably coated with 0.1% to 0.2% gelatin or 0.1 mg/ml to 0.2 mg/ml collagen type IV. The cells may be cultured in an incubator at 35° C. to 37° C., 5% to 10% CO ₂ and subcultured when the density is about 70% to 80%.

The keratinocytes may be applied on the artificial dermal layer by, for example, seeding, and the culture period may be, for example, 1 to 4 days, 1 to 3 days, or 1 to 2 days, but is not limited thereto.

After the step of applying and culturing the keratinocytes, the step of culturing in an artificial epidermal layer production medium is performed.

Here, "artificial epidermal layer generating medium" means a medium for generating an artificial epidermal layer in artificial skin, and any medium known in the art may be used. For example, the artificial epidermal layer production medium is a bovine pitutary extract (BPE), human epidermal growth factor (hEGF), bovine insulin (bovine Insulin), hydrocortisone (Hydrocortisone), and gentamicin and It may be a medium containing amphotericin-B (GA-1000 (Gentamicin, Amphotericin-B)). In addition, it may be a medium further containing epinephrine and transferrin in the above components. A commercially available example of the medium is CnT-3D-PR (CellnTEC).

For example, forming the epidermal layer may include: seeding keratinocytes on the wrinkled artificial dermal layer, and culturing them in a keratinocyte culture medium; And after the culturing, it may include the step of culturing it in an epidermal layer production medium.

After seeding the keratinocytes on the wrinkled artificial dermal layer, the step of culturing them in a keratinocyte culture medium (EpiLife, Gibco) may be performed for 1 to 3 days.

The step of culturing in the epidermal layer generating medium is culturing in the epidermal layer generating medium (CnT-3D-PR, CellnTEC) without exposure to air for 1 to 2 days, and then exposing it to air again for 13 to 15 days. It may be a step of culturing. As described above, when the artificial dermal layer forming step is performed while exposed to air, the artificial epidermal layer can be well formed without causing any shape change on the already wrinkled artificial dermal layer. In addition, natural tissue contraction occurs during the epidermal layer culture process, and thus artificial skin having wrinkles very similar to that of real skin may be manufactured.

That is, in order to manufacture wrinkled artificial skin using the artificial skin wrinkle forming device according to an embodiment, the shape of the concave-convex part included in the lower surface of the stamp constituting the aforementioned artificial skin wrinkling device and whether specific parameters are satisfied according to the shape , wrinkled artificial skin can be well manufactured in a desired shape depending on the amount of the artificial dermal layer supported (or the amount of collagen loaded) and whether the artificial epidermal layer is exposed to air and cultured. That is, not only the shape of the stamp constituting the artificial skin wrinkle forming device is important, but also the artificial dermal layer loading volume and the artificial epidermal layer formation condition along with the shape of the stamp have an important effect on the shape of the finally manufactured wrinkled artificial skin. can inflict

According to another embodiment, there is provided a wrinkled artificial skin prepared by the above-described wrinkled artificial skin manufacturing method.

According to another embodiment, there is provided a screening method for a compound for improving skin wrinkles using the aforementioned wrinkled artificial skin. For example, the skin wrinkle-improving compound screening method includes the steps of treating the wrinkled artificial skin with a compound and checking whether wrinkles in the artificial skin disappear over time after the compound treatment.

The embodiments of the present invention described above will be described in more detail through the following examples. However, the following examples are for illustrative purposes only and do not limit the scope of the present invention.

Manufacture of artificial skin wrinkle forming device

Preparation Example 1

The ABS resin was molded with a 3D printer (Printed by Fortus 250mc, Stratasys), and an artificial skin wrinkle forming device including a stamp having the same structure as the figure described in FIG. 6 was manufactured.

Preparation 2

By molding ABS resin with a 3D printer (Printed by Fortus 250mc, Stratasys), an artificial skin wrinkle forming device including a stamp having the same structure as the figure described in FIG. 10 was manufactured.

Preparation 3

By molding ABS resin with a 3D printer (Printed by Fortus 250mc, Stratasys), an artificial skin wrinkle forming device including a stamp having the same structure as the figure described in FIG. 16 was manufactured.

The shapes of the stamps constituting the artificial skin wrinkle forming apparatus according to Preparation Examples 1 to 3 are shown in Table 1 below.

(Unit: mm) Preparation Example 1 Preparation 2 Preparation 3 Stamp width (S) 10 10 12 Average width of concavo-convex part (w) One 2 One Average length of concavo-convex part (h) 2.325 2.5 3 Average distance between uneven parts (G) 0 0 2

Preparation 4

By molding the ABS resin with a 3D printer (Printed by Fortus 250mc, Stratasys), an artificial skin wrinkle forming device including a stamp having the same structure as the figure shown in Table 2 and FIG. 24 was prepared.

Preparation Examples 5 to 12

An artificial skin wrinkling device was manufactured in the same manner as in Preparation Example 4, except that the stamp shape constituting the artificial skin wrinkling device was set to the numerical values shown in Tables 2 to 4 below.

(Unit: mm) Preparation 4 Preparation 5 Preparation 6 Stamp width (S) 6 8 7 Average width of concavo-convex part (w) 0.25 0.25 0.25 Average length of concavo-convex part (h) One 2 3 Average distance between uneven parts (G) 0 0 0

(Unit: mm) Preparation 7 Preparation 8 Preparation 9 Stamp width (S) 3 8 7 Average width of concavo-convex part (w) 0.5 0.5 0.5 Average length of concavo-convex part (h) One 2 3 Average distance between uneven parts (G) 0 0 0

(Unit: mm) Preparation 10 Preparation 11 Preparation 12 Stamp width (S) 8 8 8 Average width of concavo-convex part (w) One One One Average length of concavo-convex part (h) One 2 3 Average distance between uneven parts (G) 0 0 0

Manufacture of wrinkled artificial skin

Example 1

After loading a mixture of collagen (Nutragen, Advanced Biomatrix) and fibroblasts (NDFn, ThermoFisher) in Transwell ^® (Corning) in a volume of 600 μl, the artificial skin wrinkling device of Preparation Example 1 was applied to the Transwell ^® ( Corning), the stamp constituting the artificial skin wrinkle forming device was stamped so as to touch the artificial dermal layer. Cell culture at 37 ° C., 5% CO ₂ In an incubator, the stamp was left in contact with the mixture for 2 hours to harden the mixture (gelation), and then the artificial skin wrinkling device was removed to prepare a wrinkled artificial dermal layer. . Thereafter, a fibroblast culture medium (M106, Gibco) was put on the artificial dermal layer and cultured for 7 days to stabilize the artificial dermal layer. Next, keratinocytes (HEKn, ThermoFisher) were seeded on the artificial dermal layer and cultured for 1 day in keratinocyte culture medium (EpiLife, Gibco), followed by epidermal layer generation medium (CnT-3D- Wrinkled artificial skin was prepared by culturing for 1 day in PR, (CellnTEC) and then culturing in the same epidermal layer production medium for 14 days in a state of exposure to air to form an artificial epidermal layer, which is shown in FIG. 7 .

Example 2

A wrinkled artificial skin was prepared in the same manner as in Example 1, except that the artificial skin wrinkling device of Preparation Example 2 was used instead of the artificial skin wrinkling device of Preparation Example 1, which is shown in FIG. 11 .

Example 3

A wrinkled artificial skin was prepared in the same manner as in Example 1, except that the artificial skin wrinkling device of Preparation Example 3 was used instead of the artificial skin wrinkling device of Preparation Example 1, which is shown in FIG. 17 .

Example 4

After loading a mixture of collagen (Nutragen, Advanced Biomatrix) and fibroblasts (NDFn, ThermoFisher) in Transwell ^® (Corning) in a volume of 600 μl, the artificial skin wrinkling device of Preparation Example 4 was applied to the Transwell ^® ( Corning), the stamp constituting the artificial skin wrinkle forming device was stamped so as to touch the artificial dermal layer. Cell culture at 37 ° C., 5% CO ₂ In an incubator, the stamp was left in contact with the mixture for 2 hours to harden the mixture (gelation), and then the artificial skin wrinkling device was removed to prepare a wrinkled artificial dermal layer. . Thereafter, a fibroblast culture medium (M106, Gibco) was put on the artificial dermal layer and cultured for 7 days to stabilize the artificial dermal layer to prepare a wrinkled artificial skin dermal layer, which is shown in FIG. 25 .

Example 5

A wrinkled artificial skin dermal layer was prepared in the same manner as in Example 4, except that the artificial skin wrinkling device of Preparation Example 5 was used instead of the artificial skin wrinkling device of Preparation Example 4, which is shown in FIG. 26 .

Example 6

A wrinkled artificial skin dermal layer was prepared in the same manner as in Example 4, except that the artificial skin wrinkling device of Preparation Example 6 was used instead of the artificial skin wrinkling device of Preparation Example 4, which is shown in FIG. 27 .

Example 7

A wrinkled artificial skin dermal layer was prepared in the same manner as in Example 4, except that the artificial skin wrinkling device of Preparation Example 7 was used instead of the artificial skin wrinkling device of Preparation Example 4, which is shown in FIG. 28 .

Example 8

A wrinkled artificial skin dermal layer was prepared in the same manner as in Example 4, except that the artificial skin wrinkling device of Preparation Example 8 was used instead of the artificial skin wrinkling device of Preparation Example 4, which is shown in FIG. 29 .

Example 9

A wrinkled artificial skin dermal layer was prepared in the same manner as in Example 4, except that the artificial skin wrinkling device of Preparation Example 9 was used instead of the artificial skin wrinkling device of Preparation Example 4, which is shown in FIG. 30 .

Example 10

A wrinkled artificial skin dermal layer was prepared in the same manner as in Example 4, except that the artificial skin wrinkling device of Preparation Example 10 was used instead of the artificial skin wrinkling device of Preparation Example 4, which is shown in FIG. 31 .

Example 11

A wrinkled artificial skin dermal layer was prepared in the same manner as in Example 4, except that the artificial skin wrinkling device of Preparation Example 11 was used instead of the artificial skin wrinkling device of Preparation Example 4, which is shown in FIG. 32 .

Example 12

A wrinkled artificial skin dermal layer was prepared in the same manner as in Example 4, except that the artificial skin wrinkling device of Preparation Example 12 was used instead of the artificial skin wrinkling device of Preparation Example 4, which is shown in FIG. 33 .

Comparative Example 1

Except that the artificial skin wrinkle forming device was not used, in the same manner as in Example 1, non-wrinkled artificial skin was prepared, which is shown in FIG. 14 .

Evaluation 1-1: Confirmation of wrinkles through the naked eye

It was visually confirmed whether wrinkles were formed in the artificial skin (or artificial skin dermal layer) of Examples 1 to 12 and Comparative Example 1 (see FIGS. 7, 11, 14, 17 and 25 to 33). ). From this, it can be seen that the artificial skin (or artificial skin dermal layer) of Examples 1 to 12 has dents compared to the artificial skin of Comparative Example 1, and thus the artificial skin wrinkle forming device according to an embodiment is used. Thus, it can be confirmed that wrinkled artificial skin (or artificial skin dermal layer) can be obtained. Specifically, according to the width and length of the stamp constituting the artificial skin wrinkle forming device according to an embodiment, and the width and length of the concavo-convex part included in the lower surface of the stamp, whether it is possible to implement wrinkled artificial skin similar to real human skin is determined. can be inferred. More specifically, the artificial skin wrinkle forming apparatus including a stamp satisfying Equation 1 on the lower surface is a wrinkled artificial skin similar to real human skin compared to the artificial skin wrinkle forming apparatus including a stamp not satisfying Equation 1 on the lower surface. It can be seen that the implementation of the skin is easier. In the case of Examples 4 and 7, dents occur in the artificial skin, but the degree is insignificant and does not look like wrinkles that exist in actual human skin. It can be seen that it does not look like the wrinkles that exist in human skin.

Evaluation 1-2: Wrinkle check through OCT cross-sectional image

The artificial skin of Example 1, Example 2 and Comparative Example 1 was imaged using a confocal laser scanning microscope (CLS microscope; LSM 710, Zeiss), and the results are shown in FIGS. 8 and 12 and FIG. 15 . In addition, the artificial skin of Examples 1 to 3 was subjected to optical coherence tomography (OCT) to obtain OCT cross-sectional images, and the results are shown in FIGS. 9, 13 and 18 below.

8, 9, 12, 13, 15 and 18, it can be seen that the artificial skin of Examples 1 to 3 has wrinkles, but the artificial skin of Comparative Example 1 has wrinkles. It can be confirmed that it is not formed.

Evaluation 2: Confirmation of Wrinkle Improvement Substance Screening

After treating the wrinkled artificial skin of Example 3 with retinoic acid, which is well known as a wrinkle improving material, it was checked whether the wrinkles of the artificial skin were actually improved. 19 is a photograph after retinoic acid treatment on the wrinkled artificial skin of Example 3, and FIG. 20 is an OCT cross-sectional image after retinoic acid treatment on the wrinkled artificial skin. It can be seen that there has been a definite improvement. Therefore, by using a candidate substance that is judged to have other skin wrinkle improvement effects instead of the retinoic acid, it is possible to easily screen whether it has an effect on wrinkle improvement.

Although preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention defined in the following claims are also present. It belongs to the scope of the right of the invention.

Claims (23)

A mold fixing frame, a mold mounted on the mold fixing frame, and a shoulder attachment device to which the mold fixing frame is attached,
The mold is inserted into the center hole of the mold fixing frame and includes a mounting part that can be pulled or pushed and a stamp connected to the mounting part,
The stamp includes an upper surface directly coupled to the mounting portion and a lower surface facing the upper surface,
The lower surface includes one or more uneven parts
Artificial skin wrinkle forming device. In claim 1,
The stamp is an artificial skin wrinkle forming device having a width of 3mm to 12mm. In claim 1,
The concave-convex part has a triangular shape artificial skin wrinkle forming device. In claim 3,
The lower surface of the stamp includes one or more triangular-shaped irregularities, and the width and length of the triangular-shaped irregularities are identical to each other. In claim 3,
The lower surface of the stamp includes one or more triangular-shaped irregularities, and the width and length of the triangular-shaped irregularities are different from each other. In claim 3,
The stamp is an artificial skin wrinkle forming device that satisfies the following Equation 1:
[Equation 1]
(w + h) x 2 ≤ S ≤ (w + h) x 5
In Equation 1 above,
S is the width of the stamp,
w is the average width of the concave-convex portion included in the lower surface of the stamp,
h is the average length of the uneven portion included in the lower surface of the stamp. In claim 3,
While the lower surface of the stamp includes one or more irregularities, the stamp is an artificial skin wrinkle forming device that satisfies the following Equation 2:
[Equation 2]
G x 2 < S
In Equation 2 above,
S is the width of the stamp,
G is the average distance between one or more irregularities included in the lower surface of the stamp. In claim 1,
The concave-convex part is an artificial skin wrinkle forming device having a semicircular shape. In claim 8,
The lower surface of the stamp includes one or more semi-circular concavo-convex portions, and the width and length of the semi-circular concave-convex portions are identical to each other. In claim 8,
The lower surface of the stamp includes one or more semicircular irregularities, and the width and length of the semicircular irregularities are different from each other. In claim 8,
While the lower surface of the stamp includes one or more uneven parts, the stamp is an artificial skin wrinkle forming device that satisfies the following Equation 3:
[Equation 3]
wx 24 < S < wx 45
In Equation 3 above,
S is the width of the stamp,
w is the average width of the concave-convex portion included in the lower surface of the stamp. In claim 1,
The lower surface includes three or more uneven parts,
The concave-convex portion is composed of a first concave-convex portion, a second concave-convex portion and a third concave-convex portion,
The width and length of the first concave-convex portion and the third concave-convex portion are the same as each other,
The width of the second concave-convex portion is two?
The length of the second concavo-convex portion is longer than the length of the first concave-convex portion,
The second concave-convex portion is an artificial skin wrinkle forming device positioned between the first and third concave-convex portions. delete In claim 1,
The mold fixing frame and the mold are each independently made of acrylonitrile-butadiene-styrene (ABS) resin or polylactic acid (PLA) resin. Injecting an artificial dermal mixture containing collagen and fibroblasts to the shoulder attachment device;
Before the mixture hardens, stamping the uneven portion of the artificial skin wrinkling device according to any one of claims 1 to 12 and 14 to contact the non-solidified mixture;
forming a wrinkled artificial dermal layer by hardening the mixture; and
Forming an epidermal layer on the wrinkled artificial dermal layer
Wrinkled artificial skin manufacturing method comprising a. In claim 15,
In the step of injecting the artificial dermal mixture into the shoulder attachment device, the artificial dermal mixture is injected in a volume of 400 μl to 800 μl. In claim 15,
The step of forming a wrinkled artificial dermal layer by hardening the mixture is a step of leaving the mixture in an incubator for cell culture for 1 hour to 5 hours after the stamping. In claim 15,
The wrinkled artificial skin manufacturing method further comprises the step of culturing a fibroblast culture medium on the artificial dermal layer after forming the artificial dermal layer, before forming the epidermal layer. In claim 15,
The forming of the epidermal layer may include: seeding keratinocytes on the wrinkled artificial dermal layer, and culturing them in a keratinocyte culture medium; and after the culturing, culturing it in an epidermal layer production medium. In paragraph 19,
The step of culturing in the keratinocyte culture medium is a wrinkled artificial skin manufacturing method performed for 1 to 3 days. In paragraph 19,
The step of culturing in the epidermal layer production medium is a step of culturing without exposure to air in the epidermal layer production medium, and then exposing it to air again to culture the wrinkled artificial skin manufacturing method. delete delete

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