KR20230169028A - Producing method of artificial skin and artificial skin - Google Patents

Abstract

Preparing a dermal simulant layer by mixing fibroblasts, collagen, and a collagen gel strength regulator; Applying keratinocytes on the dermal simulant layer; And a step of culturing, wherein the dermal simulant layer includes a first dermal layer and a second dermal layer located on top of the first dermal layer, the first dermal layer includes collagen, and the second dermal layer includes fibroblasts. The culturing step is a step in which calcium ions are added while being performed in a dish exposed to air, and the dish is coated with 0.1% to 0.2% gelatin or 0.1 mg/ml to 0.2 mg/ml collagen type IV. Provided is a method for manufacturing artificial skin and artificial skin manufactured accordingly.

Description

Artificial skin manufacturing method and artificial skin {PRODUCING METHOD OF ARTIFICIAL SKIN AND ARTIFICIAL SKIN}

This description relates to a method of manufacturing artificial skin using a collagen gel strength regulator for artificial skin and artificial skin manufactured according to the manufacturing method.

The skin is an organ that covers the outside of the body and is composed of three layers from the outside: the epidermis, dermis, and subcutaneous fat. The epidermis is comprised mostly of keratinocytes of stratified squamous epithelium. The dermis, which is made up of matrix proteins such as collagen fibers and elastic fibers, is located under the epidermis, and 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 components as described above, and exhibits various functions such as regulating body temperature and functioning as a barrier to the external environment.

Artificial skin is a three-dimensional reconstruction of the skin described above using skin cells and skin components such as collagen and elastin. It is composed of living fibroblasts and keratinocytes and has a structure similar to real skin. Because it exhibits functional properties, it is also called skin equivalent or reconstructed skin. Artificial skin is mainly a polymer composite that exhibits similar physical properties to skin in terms of elasticity, strength, and material penetration, but is different in that it does not exhibit the same vital phenomena as skin. Artificial skin is not only used to replace (permanent engraftment type) or regenerate (temporary covering type) damaged skin such as burns or trauma, but is also used in various fields such as skin physiology research, skin irritation evaluation, and skin efficacy evaluation. .

The dermal layer that makes up our skin contains collagen gel, and the strength of the dermal layer varies depending on the strength of the collagen gel. The strength of the skin is changing due to various diseases, aging, etc., and if it is possible to manufacture a new artificial skin that mimics the strength of the skin, it can be applied as personalized artificial skin and used as an aging research platform. It can dramatically improve the problem of loss of skin elasticity due to aging.

Modifications of artificial skin models to date mainly rely on cell biological methods to change cells or add or remove specific substances. Accordingly, technologies that can change the physical characteristics of the artificial skin model, such as tissue strength, have not yet been reported, so there are limitations in expanding the artificial skin model in various ways. In one embodiment, artificial skin is made using a polymer containing a specific structural unit, which can directly control the strength of the collagen gel, and a new skin simulation model can be manufactured by adjusting the strength of the artificial skin model.

According to one embodiment, a collagen gel strength regulator comprising a structural unit represented by the following Chemical Formula 1 is provided.

[Formula 1]

In Formula 1,

R ¹ is a hydrogen atom or a substituted or unsubstituted C1 to C20 alkyl group,

L ¹ is a substituted or unsubstituted C1 to C20 alkylene group,

m and n are each independently integers from 1 to 10000.

The structural unit represented by Formula 1 may satisfy Equation 1 below.

[Equation 1]

0 < n/(n+m) x 100 ≤ 5

The collagen gel strength regulator may further include calcium ions.

The collagen gel strength regulator may have a weight average molecular weight of 10,000 g/mol to 1,000,000 g/mol.

According to another embodiment, preparing a dermal simulant layer by mixing fibroblasts, collagen, and the collagen gel strength regulator according to any one of claims 1 to 4; Applying keratinocytes on the dermal simulant layer; It provides a method for manufacturing artificial skin including the step of culturing.

The step of applying keratinocytes on the dermal simulant layer may be a step of placing the keratinocytes on the dermal simulant layer and culturing them for two days.

The culturing step may be performed while exposed to air.

Another embodiment provides artificial skin containing the collagen gel strength regulator.

Another embodiment provides artificial skin manufactured according to the artificial skin manufacturing method.

The collagen gel strength regulator according to one embodiment includes a polymer containing a specific structural unit. When the polymer is added to a collagen solution, the strength of the collagen gel constituting the dermal layer of artificial skin can be adjusted to various desired levels. .

Figure 1 is a schematic diagram showing the structure of a collagen gel strength regulator according to one embodiment.
Figure 2 is a flowchart showing a method for manufacturing artificial skin according to one embodiment.
Figures 3 and 4 each independently show NMR data of compounds according to Comparative Example 1 and Example 1.
Figure 5 is a diagram showing the strength control mechanism for controlling the strength of collagen gel according to one embodiment.
Figures 6 and 7 each independently show pyrene fluorescence analysis data of the compounds according to Example 1 and Example 2.
Figures 8 and 9 are schematic diagrams each independently showing the structures of compounds according to Examples 1 and 2.
Figure 10 is a graph showing collagen gel strength according to the content of a collagen gel strength regulator according to one embodiment.
Figure 11 is a graph showing collagen gel strength according to the type of compound used as a collagen gel strength regulator.
Figure 12 is a scanning electron microscope (SEM) photograph (scale bar = 5㎛) of the internal structure of the dermis layer according to the content of the collagen gel strength regulator.
Figure 13 is a graph showing changes in fibroblast behavior according to the addition of a collagen gel strength regulator according to one embodiment.
Figure 14 is a photograph of the dermal layer and the epidermal layer of an artificial skin model manufactured by adding a collagen gel strength regulator according to one embodiment.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. However, the present invention may be implemented in many different forms and is not limited to the embodiments described herein.

As used herein, “artificial skin” refers to a three-dimensional reconstruction of skin using skin cells and collagen, a skin component, and is a polymer complex that exhibits structural and functional characteristics similar to real skin. It has the broadest meaning that includes all ramen.

Unless otherwise specified herein, “alkyl group” refers to a C1 to C20 alkyl group, “alkenyl group” refers to a C2 to C20 alkenyl group, and “cycloalkenyl group” refers to a C3 to C20 cycloalkenyl group. , “Heterocycloalkenyl group” refers to a C3 to C20 heterocycloalkenyl group, “aryl group” refers to a C6 to C20 aryl group, “arylalkyl group” refers to a C6 to C20 arylalkyl group, and “alkylene group” means a C1 to C20 alkylene group, “arylene group” means a C6 to C20 arylene group, “alkylarylene group” means a C6 to C20 alkylarylene group, and “heteroarylene group” means a C3 to C20 heteroarylene group. It means an arylene group, and “alkoxylene group” means a C1 to C20 alkoxylene group.

Unless otherwise specified herein, “substitution” means that at least one hydrogen atom is replaced by a halogen atom (F, Cl, Br, I), a hydroxy group, a C1 to C20 alkoxy group, a nitro group, a cyano group, an amine group, or an imino group. , azido group, amidino group, hydrazino group, hydrazono group, carbonyl group, carbamyl group, thiol group, ester group, ether group, carboxyl group or its salt, sulfonic acid group or its salt, phosphoric acid or its salt, C1 to C20 alkyl group, C2 to C20 alkenyl group, C2 to C20 alkynyl group, C6 to C20 aryl group, C3 to C20 cycloalkyl group, C3 to C20 cycloalkenyl group, C3 to C20 cycloalkynyl group, C2 to C20 heterocycloalkyl group, It means substituted with a substituent of a C2 to C20 heterocycloalkenyl group, a C2 to C20 heterocycloalkynyl group, a C3 to C20 heteroaryl group, or a combination thereof.

Also, unless otherwise specified herein, “hetero” means that at least one hetero atom of N, O, S, and P is included in the chemical formula.

Also, unless otherwise specified in the specification, “(meth)acrylate” means that both “acrylate” and “methacrylate” are possible, and “(meth)acrylic acid” means “acrylic acid” and “methacrylic acid.” "It means that both are possible.

Unless otherwise specified herein, “combination” means mixing or copolymerization.

Unless otherwise defined in the chemical formulas in this specification, if a chemical bond is not drawn at a position where a chemical bond should be drawn, it means that a hydrogen atom is bonded at that position.

In this specification, when a part of a layer, membrane, region, plate, etc. is said to be “on” another part, this includes not only the case where it is “right on” the other part, but also the case where there is another part in between. Conversely, when a part is said to be “right on top” of another part, it means that there is no other part in between.

Hereinafter, a collagen gel strength regulator according to one embodiment will be described.

The collagen gel strength regulator according to one embodiment is represented by the following formula (1).

[Formula 1]

In Formula 1,

R ¹ is a hydrogen atom or a substituted or unsubstituted C1 to C20 alkyl group,

L ¹ is a substituted or unsubstituted C1 to C20 alkylene group,

m and n are each independently integers from 1 to 10000.

The collagen gel strength regulator represented by Formula 1 includes both a repeating unit of m moles and a repeating unit of n moles. Unlike the repeating unit of m moles, the n moles of repeating units include a grafted acrylate group. , a Michael addition reaction occurs between the amino group of the collagen fiber and the grafted acrylate group, so that the collagen gel strength regulator represented by Formula 1 can be attached to the collagen fiber, and thus the strength of the collagen gel can be easily adjusted. .

For example, the structural unit represented by Formula 1 may satisfy Equation 1 below.

[Equation 1]

0 < n/(n+m) x 100 ≤ 5

The collagen gel strength regulator according to one embodiment is a type of graft copolymer containing the structural unit represented by Formula 1, and depending on how much the acrylate group is grafted to the backbone, the desired effect is achieved, that is, the desired effect is achieved. The ability to adjust the level of intensity may vary. In other words, the degree of substitution (DS) of the acrylate group, which is a grafting group, to the main chain is important. When the structural unit represented by Formula 1 has a grafting degree of 5 mol% or less (more than 0 mol%), , the strength can be controlled to a desired level, and when the grafting degree exceeds 5 mol%, a self-assembly phenomenon occurs (self-assembly formation), which leads to a decrease in strength control. Equation 1 is a formula that represents the degree of grafting. In other words, the degree of grafting refers to the number of repeating units of n moles per 100 polymer units composed only of the structural units represented by Formula 1.

In general, the greater the number of acrylate groups grafted to the main chain in order to increase the Michael addition reaction rate with collagen fibers, the better the strength control can be expected. However, pyrene as a strength regulator with a grafting degree of 5 mol% or less is used. Looking at FIG. 6, which is the (pyrene) fluorescence analysis data, the I ₃ /I ₁ value is almost constant, but looking at FIG. 7, which is the pyrene fluorescence analysis data of the strength regulator with a grafting degree exceeding 5 mol%, the I ₃ /I ₁ value is It can be seen that it increases in a sigmoidal form. This is because the polarity of the microenvironment of pyrene molecules decreases, and the more acrylate groups it contains, the more evidence shows that the formation of self-assembly begins in an aqueous solution.

In other words, because the acrylate group has weak hydrophobicity, when the degree of grafting increases, the structural unit represented by Formula 1 self-assembles in an aqueous solution, so the acrylate group that must undergo a Michael addition reaction with collagen fibers self-assembles. It becomes trapped inside, and eventually the Michael addition reaction rate with collagen fibers decreases, inevitably leading to a decrease in strength control. On the other hand, when the degree of grafting is low, below 5 mol%, the number of sites capable of ionic cross-linking increases relatively due to the addition of calcium ions, etc., which will be described later, thereby improving the physical ionic cross-linking ability and ultimately strength. Control is bound to be excellent.

Figures 8 and 9 are both schematic diagrams showing the structural unit represented by Formula 1. Figure 8 shows the case where the degree of grafting is 5 mol% or less, but does not form self-assembly, but shows the case where the degree of grafting is more than 5 mol%. Figure 9 shows that a self-assembly is formed.

The collagen gel strength regulator may further include calcium ions. The calcium ion is capable of ionic bonding with the main chain of Formula 1, helping additional bonding between collagen fibers and controlling the strength of the collagen gel.

From Figure 5, the collagen gel strength regulator containing the structural unit represented by Formula 1 is introduced into collagen fibers to control the strength of the collagen gel, and calcium ions are additionally added to the collagen gel, thereby forming the collagen gel through ionic crosslinking. It can be seen that intensity control becomes easier. In particular, calcium ions can have an excellent effect in controlling the strength of the dermal layer in artificial skin through ionic cross-linking when manufacturing artificial skin.

The collagen gel strength regulator may have a weight average molecular weight of 10,000 g/mol to 1,000,000 g/mol. If the molecular weight of the collagen gel strength regulator is less than 10,000 g/mol, crosslinking is difficult, and if it is more than 1,000,000 g/mol, problems of increased viscosity and decreased solubility may occur.

Meanwhile, the collagen gel strength modifier is used as an additive, but does not adversely affect fibroblast behavior. That is, the collagen gel strength regulator according to one embodiment is cell-friendly.

Another embodiment includes preparing a dermal simulant layer by mixing fibroblasts, collagen, and the collagen gel strength regulator; Applying keratinocytes on the dermal simulant layer; It provides a method for manufacturing artificial skin including the step of culturing.

Referring to Figure 2, the collagen gel strength regulator according to one embodiment in the collagen fiber causes bonding between collagen fibers through a Michael addition reaction between the amino group in the collagen fiber and the acrylate group, which is a grafting group in the collagen gel strength regulator, Due to the calcium ions added during the air exposure culture process after keratinocyte seeding, additional bonding occurs between collagen fibers due to ionic bonding between the main chain constituting the collagen gel strength regulator and the calcium ions, It can be schematically confirmed that artificial skin is manufactured.

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

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

The first dermal layer may contain collagen, and the extracellular matrix that constitutes the dermis, such as elastin, chitosan, glycosaminoglycans (GAGs), and hyaluronic acid (HA). It may contain more extracellular matrix.

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

The single dermal simulant layer may contain collagen and fibroblasts, the collagen and fibroblasts being as described above.

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

The thickness of the dermal replica layer is generally at least 0.05 cm, especially approximately 0.05 cm to 2 cm, but can be increased or decreased without impairing the advantageous properties of the artificial skin according to the invention.

After producing a dermal simulant layer using a mixture of fibroblasts and collagen, it can be cultured for, for example, about 5 to 9 days, about 6 to about 8 days, or about 7 days.

Once the dermal simulant layer is formed, keratinocytes are applied on top of it and then cultured. That is, the step of applying keratinocytes on the dermal simulant layer may be a step of placing the keratinocytes on the dermal simulant layer and culturing them for two days.

The keratinocytes make up about 80% to 90% of the cells of the epidermis and are formed through mitosis in the basal layer, the deepest layer of the epidermis, and then rise to the upper layer toward the skin surface.

The step of culturing the keratinocytes after applying them is the first step in forming the epidermal simulant layer of artificial skin. The keratinocytes used here may be, for example, keratinocytes derived from humans. The keratinocytes can be any commercially available keratinocytes, and keratinocytes derived from other cells as well as keratinocytes directly isolated from humans or cultured after isolation can also be used. Commercially available human keratinocytes include NHEK-Neo, Pooled (Neonatal Normal Human Epidermal Keratinocytes, Pooled: product number 00192906, tissue acquisition number P867, Caucasians), NHEK-Neo (product number 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, tissue acquisition number: 18080, black). And HEKn (Human Epidermal keratinocytes, neonatal: product number C0015C, tissue accession number 1781129, white) and HEKn (product number C0015C, tissue accession number 1803827, black) provided by Thermo Fisher. In order to maintain the property of keratinocytes to adhere to and proliferate on the basement membrane, it is preferable to use dishes coated with 0.1% to 0.2% gelatin or 0.1 mg/ml to 0.2 mg/ml collagen type IV. The cells can be cultured in an incubator at 35°C to 37°C and 5% to 10% CO ₂ and can be subcultured when the density reaches about 70% to 80%.

The keratinocytes may be applied on the dermal simulant layer, for example, by 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 going through the application and culturing steps of the keratinocytes, a step of culturing them in an epidermal layer formation medium is performed.

Here, “epidermal layer formation medium” refers to a medium for forming the epidermal layer in artificial skin, and any medium known in the art can be used. For example, the epidermal layer formation medium contains bovine pitutary extract (BPE), human epidermal growth factor (hEGF), bovine insulin, hydrocortisone, gentamicin, and ampholyte. It may be a medium containing terisin-B (GA-1000 (Gentamicin, Amphotericin-B)). Additionally, the medium may further contain epinephrine and transferrin. A commercially available example of the medium is CnT-3D-PR (CellnTEC).

The epidermal layer formation medium may be installed to contact the lower surface of the dermis replica layer, and the surface opposite to the surface on which the epidermal layer formation medium is installed may be exposed to air. Cultivation in this epidermal layer formation medium may be carried out for, for example, 3 to 20 days, 4 to 18 days, 5 to 15 days, or 7 to 14 days, but is not limited thereto.

By going through the above steps, it is possible to obtain an artificial skin structure in which an epidermal simulant layer is formed on a dermis simulant layer.

When manufacturing artificial skin according to the above-described method, not only can an artificial skin model substantially similar to that found in real human skin be obtained, but also the strength of the artificial skin can be controlled to a desired level by the collagen gel strength regulator. You can.

In other words, it is possible to control the strength to the desired level without using any conventional cross-linking agents or regulators, and to implement an artificial skin model that does not adversely affect the behavior of fibroblasts.

According to another embodiment, artificial skin containing the above-described collagen gel strength regulator is provided.

According to another embodiment, artificial skin manufactured according to the above-described manufacturing method is provided.

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

Collagen gel strength regulator synthesis

Example 1

Alginic acid (Alginic acid sodium salt, Sigma) was added at 1.0% (w/w/%) in 0.1 M 2-(N-morpholino) ethanesulfonic acid (2-(N-morpholino) ethanesulfonic acid, MES, Sigma) buffer solution at pH 6.4. v) and sufficiently dissolved at 30-40°C while stirring. Once completely dissolved, 1-hydroxybenzotriazole (HOBt, Sigma), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1-ethyl-3-(3) -dimethylaminopropyl) carbodiimide (EDC, Sigma) and 2-aminoethyl methacrylate (AEMA, Sigma) were slowly added and reacted with stirring for 18 hours. After the reaction was completed, the reaction solution was dialyzed with distilled water for 3 days and then freeze-dried to obtain a molecule represented by the following formula E-1 to which methacrylate was grafted. The synthesized molecule was dissolved in D ₂ O and its structure was analyzed by ¹ H-NMR (Avance II, Bruker Biospin) at 400 MHz, and the results are shown in Figure 4. The degree of grafting (DS) of the synthetic molecule was calculated (5 mol%) by measuring unreacted COOH functional groups (free carboxylate groups) by titration with sodium hydroxide (NaOH, Sigma).

[Formula E-1]

Example 2

The molecule represented by the formula E-1 was obtained in the same manner as in Example 1, except that the grafting degree was set to 7 mol%.

Comparative Example 1

Alginic acid used in Example 1

Assessment 1: Structural Analysis

¹ H-NMR spectra in D ₂ O of the molecules according to Example 1 and Comparative Example 1 are shown in Figures 3 and 4.

Unlike Figure 3 (Comparative Example 1), Figure 4 (Example 1) can be seen to have several different peaks, and it can be inferred that this makes it possible to control the strength of the collagen gel.

Evaluation 2: Changes according to degree of grafting

Pyrene fluorescence analysis was performed on the synthetic molecules according to Examples 1 and 2, and the results are shown in Figures 5 and 6.

From Figures 5 and 6, in the case of Example 1 with a low grafting degree, the I ₃ /I ₁ value is almost constant, but in the case of Example 2 with a high grafting degree, the I ₃ /I ₁ value increases in a sigmodal form. It can be seen that this is because the microenvironment polarity of pyrene molecules decreases, and in the case of Example 2, which contains a lot of acrylate groups, it can be inferred that self-assembly was formed in the aqueous solution. That is, unlike Example 1, in Example 2, it can be inferred that the acrylate group is trapped inside the self-assembly, so that Michael addition reaction with collagen fibers will not occur.

Evaluation 3: Change in strength of collagen gel

(1) To confirm the effect of controlling the strength of the artificial skin dermis, an artificial skin dermis was prepared by adding a synthesized molecule (Example 1). The dermal mixture consisted of collagen solution (Type I, 6 mg/ml, Advaced BioMatrix), P buffer (NaOH, NaHCO ₃ , HEPES in distilled water), and R buffer (Dulbecco's Modified Eagle Medium powder, Ham's F-12 Nutrient Mxiture, Antibiotic- Antimycotic in DI water), 5N NaOH solution was prepared at a ratio of 8:1:1:0.05. The synthetic molecule (Example 1) was prepared in a solution of 3% concentration in phosphate buffer saline (PBS) and added to the dermal mixture at various concentrations. Human dermal fibroblasts (neonatal, Invitrogen) were grown in a 75 cm ² flask in media 106 (Gibco) with low serum growth supplement (LSGS, 50x, Gibco) and 1% penicillin-streptomycin (P/S, 100X, Biowest). was cultured in an environment of 37°C and 5% CO ₂ using the culture medium added. Cultured skin fibroblasts were added to the dermal mixture to encapsulate 5 × ¹⁰ cells per well. 500 μL of the dermis mixture was placed in a 48-well plate and gelled for 1 hour in an incubator at 37°C to prepare artificial skin dermis. Cell stability and Michael addition reaction between collagen and acrylate of the synthetic molecule (Example 1) were induced for about 48 hours after preparation. To induce gelation of the synthetic molecule, the culture medium was replaced with 5mM calcium ions, and after 12 hours, the culture medium was replaced with the existing culture medium, and the results are shown in Figure 10.

As shown in Figure 10, it can be seen that the strength of the collagen gel increases when synthetic molecules are added and as the content of synthetic molecules increases, compared to when synthetic molecules are not added. That is, it can be confirmed that the collagen gel strength regulator according to one embodiment can control the strength of the collagen gel.

(2) To measure the strength of the artificial skin dermis into which synthetic molecules (Example 1) were introduced, artificial skin dermis without cells was prepared. After going through the same culture process, the strength of the artificial skin dermis manufactured on the 6th day of production was tested using a universal testing machine (UTM, DrTech) device. The strength of the manufactured artificial skin dermis was measured by installing a load cell guide to measure the strength without removing it from the 48-well plate. The settings of the universal material testing machine were the same as the measurement distance test ratio of 10%, test speed of 0.5 mm/min, and load range of 1.0 kgf. In measuring compressive strength, the unit area was set to 7 mm, which is the size of the tip of the load cell guide. The results are shown in Figure 11.

From Figure 11, it can be seen that when the acrylate group is not grafted, there is no effect of increasing collagen gel strength.

(3) Cross-linking degree and internal structure of collagen gel when synthetic molecules (Example 1) were not used and when different amounts (0.6 mg, 1.2 mg, 2.4 mg) of synthetic molecules (Example 1) were used. Photographs were taken to confirm structural changes, and the results are shown in Figure 12. (The internal structure (microstructure) of the artificial skin dermis into which synthetic molecules were introduced was observed using a scanning electron microscope (FE-Scanning electron microscope, SIGMA, Carl Ziess). The artificial skin dermis to be observed was freeze-dried and coated with platinum. )

From Figure 12, it can be seen that when synthetic molecules (Example 1) are used, the collagen fibers become thicker and the internal structure becomes more dense as the amount of synthetic molecules added increases, compared to when synthetic molecules (Example 1) are not used.

Evaluation 4: Whether the behavior of fibroblasts changes due to the addition of synthetic molecules

MTT assay was performed to confirm the cell viability of skin fibroblasts in the dermis of artificial skin to which synthetic molecules (Example 1) were added. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide, sigma) was prepared in PBS at a concentration of 5 mg/ml and then added 1:10 to the artificial skin dermis culture medium. After 4 hours, remove the culture medium, add 500 μL lysis buffer (20% Sodium dodecyl sulfate in N,N Dimethylformamid/DI water (1/1), adjusted to pH 4.7 with 1N HCl), and formazan in the dermis of the artificial skin for 12 hours. melted. Afterwards, to measure the concentration of formazan, 100 μL of culture medium was extracted from each well, placed in a 96-well plate, and the optical density (OD) was measured at a wavelength of 570 nm. WST-1 assay and MTT assay were performed on days 1, 3, 5, and 7 after manufacturing the artificial skin dermis. The measurement results are shown in Figure 13.

From Figure 13, it can be seen that the synthetic molecule (Example 1) does not adversely affect fibroblast behavior (cell-friendly). In other words, it can be confirmed that the synthetic molecule (Example 1) can be used in cell experiments such as manufacturing artificial skin.

Artificial skin production

Example 3

(1) Step 1: Manufacturing of dermis replica layer

A dermal simulant layer was produced by mixing fibroblasts (NDFn, ThermoFisher), collagen (Nutragen, Advanced Biomatrix), and synthetic molecules (Example 1), followed by M106 medium containing LSGS (ThermoFisher) and 100 μg/ml ascorbic acid. (TermoFisher) and cultured for 1 week. The concentration of the mixed synthetic molecules (Example 1) was fixed at 1.2 mg.

(2) Step 2: Keratinocyte application and culture step

After step 1, keratinocytes (HEKn, Thermo Fisher) were placed on the dermal simulation layer and cultured for 2 days.

(3) Step 3: Culture step in the first medium

After the second step, the first medium (CnT-3D-PR, CellnTEC) was placed in contact with the lower part of the dermis replica, and the epidermal layer was exposed to air and cultured for 8 days.

A microscopic cross-sectional photograph of the artificial skin model of Example 3 is shown in Figure 14.

Evaluation 5: Cross-sectional observation of artificial skin model

A cross section of the artificial skin model obtained in Example 3 was observed under a microscope.

Figure 14 is a cross-section of the artificial skin obtained in Example 3 and is a micrograph after H&E tissue staining.

Referring to Figure 14, it can be seen that growth of normal dermal and epidermal layers occurs in the artificial skin model to which synthetic molecules (Example 1) were added.

Although the 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 can be made by those skilled in the art using the basic concept of the present invention as defined in the following claims. It falls within the scope of invention rights.

Claims (6)

Preparing a dermal simulant layer by mixing fibroblasts, collagen, and a collagen gel strength regulator;
Applying keratinocytes on the dermal simulant layer; and
Cultivation stage
Including,
The dermal replica layer includes a first dermal layer and a second dermal layer located on top of the first dermal layer,
The first dermal layer includes collagen,
The second dermal layer includes fibroblasts,
The culturing step is a step in which calcium ions are added while being carried out in a dish exposed to air,
The dish is coated with 0.1% to 0.2% gelatin or 0.1 mg/ml to 0.2 mg/ml collagen type IV. In paragraph 1:
The step of applying keratinocytes on the dermal simulant layer is a method of manufacturing artificial skin wherein the keratinocytes are placed on the dermal simulant layer and cultured for two days. In paragraph 1:
The collagen gel strength regulator is a method of producing artificial skin comprising a structural unit represented by the following formula (1):
[Formula 1]

In Formula 1,
R ¹ is a hydrogen atom or a substituted or unsubstituted C1 to C20 alkyl group,
L ¹ is a substituted or unsubstituted C1 to C20 alkylene group,
m and n are each independently integers from 1 to 10000. In paragraph 3,
A method of manufacturing artificial skin where the structural unit represented by Formula 1 satisfies the following Equation 1.
[Equation 1]
0 < n/(n+m) x 100 ≤ 5 In paragraph 1:
The collagen gel strength regulator is a method of producing artificial skin having a weight average molecular weight of 10,000 g/mol to 1,000,000 g/mol. Artificial skin manufactured according to the manufacturing method according to any one of claims 1 to 5.