WO2018131829A1 - Stretchable skin-on-a-chip - Google Patents

Abstract

Problem: There is a problem that skin cells cultured in a static state cannot imitate a stretching environment faced by real skin. Solution: The present invention relates to a skin-on-a-chip, and can provide a skin-on-a-chip which is more similar to real skin by imitating the repetition of contraction and relaxation due to stretching of skin cells, by embedding a permanent magnet in the skin-on-a-chip.

Description

Stretchable skin chips

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a skin-on-a-chip, and includes a connection that causes linear motion to the skin cells of the chip in response to an external linear motion drive device, thereby repeating the contraction and relaxation by stretching the skin cells. It provides a skin chip that can simulate artificial skin closer to the actual skin.

The present invention discloses the assignment unique number NRF-2015R1A4A1041631, which was carried out by the support project of the "3D printing-based human-on-a-chip fusion laboratory" basic laboratory of the Ministry of Science and Technology Information and Communication.

Organ-on-a-chip is a technology that mimics the dynamic and physiological cellular responses as well as the function and properties of organs by culturing the cells that make up a specific organ on the chip on which the electronic circuit is placed. It is expected to study the mechanism of cellular movement and physicochemical reactions of specific organs in detail, and is expected to be used as a model for drug development and toxicity evaluation.

Although electronic circuits have different roles depending on the types of long-term chips, they commonly mimic the microenvironment of organs and detect and display physiological responses as data. In the lung chip, the body fluid and air flow are imitated using the electronic circuit as a culture system, and in the eye chip, the electronic circuit is used to regularly supply tears while partitioning by cell type.

The first long-term chip was a lung chip in which lung cells were cultivated with electronic circuits on a small plastic chip, about 3 cm long, developed by the Harvard-Wyss Institute in the US under the supervision of the University of Pennsylvania biotechnology. The microchip manufacturing method establishes a micro cell culture environment on a chip with electronic circuits and cultures living lung cells and blood vessel cells in a perfusion chamber to exchange oxygen and carbon dioxide with lung lungs and capillaries like a real lung. . This lung chip is infected with lung-related diseases and can simulate the progress of the disease as it is and can be observed in real time. It is even known to be able to simulate complications caused by the side effects of chemotherapy rather than a single disease.

Since lung chips, various organ models such as heart, eyes, arteries, kidneys and skin have been developed, and the mechanisms of organ movement and physicochemical reactions can be studied in detail. It is expected to be used as a model (see Internet Wikipedia).

Since human skin is exposed to various chemicals and biological agents such as cosmetics, detergents, UV rays, pathogens, environmental pollutants and microorganisms, the main function of the skin is to provide physiological barriers to protect organs. Increased levels of these chemicals and biological agents in the skin can cause various reactions such as skin inflammation, itching, allergies, and even tumors. Therefore, it is necessary to filter out the toxicity of such foreign substances, and to increase the effect of the drug used on the skin. For this purpose, millions of animals, including mice, are used in experiments worldwide. Animal testing, however, has two critical limits. The first is an ethical issue, and the second is a significant difference between mouse and human skin in terms of thickness, hair density and appendages. Furthermore, mice have no sweat glands except for the soles of their feet. According to the Humane Society International, nine out of ten drug candidates that have been shown to be safe and effective in animal testing fail when prescribed to humans, and animal testing often fails to predict the results when used in real humans. For this reason, there is a need to build a surrogate in vitro system that mimics human skin as closely as possible. Various in vitro skin models have been developed and commercialized since the first human skin-like constructs were reported in the early 1980s. However, most of these models employ a static culture system based on fibroblasts and keratinocytes that mimic human epithelium only. The complex structure of the skin cannot be copied by these cells alone. Because the skin contains pores, immune cells, melanocytes, Merkel cell complexes, blood vessels, nerve fibers and multilayered structures. Thus, various researchers in industry, clinical and academia are involved in developing in vitro skin models that can mimic skin and skin diseases.

Most 3D-based cell cultures studied in the past have been conducted in a static environment [Y. G. Anissimov et al., Advanced drug delivery reviews, 65, 169-190 (2013). However, the constant stimulus is given inside and outside the human body. In addition, the skin in the human body is always in contact with the external friction and friction, muscle relaxation and contraction is always occurring. Currently reported studies show that stratum corneum differentiation is increased under stretch environment [Nobuhito Mori et al., IEEE MEMS 24-28 January 2016], inhibition of olfactory cover cell expression [Kamble Harshad et al., Biomed microdevice. 19. May (2016)]. Therefore, in the conventional static state of the skin chip, it is not possible to observe the change of the skin due to the relaxation, contraction of the skin.

Accordingly, an object of the present invention is to overcome the limitations of the conventional static skin chip and to provide a skin chip that mimics a condition that is more similar to human skin conditions, that is, a condition in which contraction and relaxation occur repeatedly.

In the present invention, in order to simulate an external stimulus applied to the skin, a skin chip (Skin on a chip) including a connection for generating a linear motion to the skin cells of the chip corresponding to the external linear linear motion drive device was manufactured, the external drive device Cell culture behavior was observed by applying mechanical stimulation to the chip intermittently or periodically during cell culture. In the present invention, by varying the PDMS (Polydimethylsiloxane) compounding ratio (that is, the compounding ratio of PDMS and the curing agent), the shrinkage rate caused by the change of elasticity is confirmed to be designed to correspond to the shrinkage of about 10% and standardized using an aluminum mold. In addition, the plate is designed to be fixed to a square dish size plate so that a constant stimulus is always applied.

In the experiment condition, the human body adopts a method of applying 10% contraction stimulation at 0.01Hz cycle for 12 hours a day in consideration of sleep and rest time. The tissues cultured in the existing static environment and the tissues when the stretch was applied were analyzed by the optical microscope with H / E staining.

When the skin chip was stretched and maintained in a static state, H / E staining confirmed the cross section of the tissue, and the fibroblast and keratinocyte differentiation showed a big difference. When stretching was performed on the skin chip embedded with permanent magnets, it was confirmed that keratinocytes adhered well to the collagen fragments even after a lapse of time. Gradually penetrating into the blast layer was confirmed. Through these results, it is expected that intermittent stretching may be useful for testing cytotoxicity by bringing skin cells closer to real skin in the skin chip culturing three-dimensional skin cells.

"Skin equivalent" in the description of the present invention means skin cells cultured in the stretchable skin-on-a-chip of the present invention.

Fibroblast appearance and ECM protein and β- Actin  Expression change

The present inventors observed tissues in samples using porcine skin collagen and rat tail collagen, and confirmed changes in expression and shape in fibroblasts, and quantitatively confirmed β-actin expression changes through gene analysis. β-actin is an essential protein in living cells and is involved in the cytoskeleton. It is generally used as a control as a housekeeping gene in experiments but is not suitable for use as a control when aging of cells occurs. Therefore, this change in cytoskeleton can be seen as evidence that aging occurred in the skin equivalent of stretching. In addition, the results of H / E tissue staining showed that the elongated fibroblasts observed in the sample under static conditions changed into a round and small oval shape by stretching.

Fibroblasts play a major role in the production of proteins responsible for extracellular matrix (ECM), typically collagen and fibronectin. However, in aging fibroblasts, the extracellular matrix expression ability is lowered, forming weak skin and wrinkled skin. In the present invention, it was confirmed that the expression capacity of the extracellular matrix was significantly reduced in the skin equivalent sample subjected to the stretching for 7 days. In addition, in the experimental group using porcine skin collagen, the number of fibroblasts decreases as the stimulation cycle is shorter. Therefore, given the rapid stimulation of stretching (5.3 mm / s, 0.05 Hz), the cells become difficult to live and die. It can be said to cause, and the expression of collagen and fibronectin is also affected by stimulation. In the experiment using rat tail collagen, it was confirmed that the fibroblasts showed a large change in the stretched experimental group. As shown in FIG. 6 (d), the fibroblasts were elongated in the static condition, and the fibroblasts were rounded oval in the stretching condition as shown in FIG. 6 (h). Under these two conditions, the length of fibroblasts was about 5 times the difference between the static conditions of 49.8 ± 12㎛ and the stretch of 11.8 ± 6.8㎛. It can be seen that the stretching stimulation has a big change in the cytoskeleton itself, and in this regard, the quantitative results of β-actin confirmed that the value gradually decreased as the stretching was applied [FIG. 10 (a)]. Immunohistochemical staining results also showed a significant difference in the production of fibronectin and collagen [FIG. 9]. Under stretch conditions, the newly produced collagen does not extend into a fibrous shape, but is produced as a soft, round ring-shaped skeleton. We confirmed that stretching conditions affect the fibroblast's cytoskeleton and thus decrease the expression of extracellular matrix protein, resulting in softer skin as in aged skin, and thus fibroblasts by physical stimulation. It can be seen that aging is promoted.

Aging phenomenon of the stratum corneum under stretching conditions

In the present invention, when a quick stimulation to the skin equivalents using the stretching device (5.3mm / s), even in the stratum corneum showed a significant difference from the culture in the static environment. As a result of H / E staining, the faster skin irritation in porcine skin collagen, the thinner and softer the stratum corneum, and the stratum corneum were falling.The stratum corneum cells diffused into the dermis layer on the 7th day of stimulation. The appearance was confirmed [FIG. 4]. After comparing keratin 10 expression, it was confirmed that keratin 10 expression was lowered in the case of 0.01 Hz stimulation than in the static environment over 7 days, and in particular, the expression amount was significantly decreased at 0.05 Hz. Based on the above results, we changed the scaffold to rat tail collagen (0.85wt%), which was more suitable for 3D cell culture, compared with static environment and 0.01Hz stretching environment. The experimental study confirmed that the stratum corneum was thinned by 86.4 ± 26㎛ in static condition and 49.8 ± 12㎛ in stretch condition and thinned by approximately 37㎛ during stretching. It was confirmed that the phenomenon [Fig. 6]. This is because the cells go to a safe place due to the stress on the cells, and wrinkles are formed in the skin during this process. In addition, as in the previous experiment, it was confirmed that the result of the keratin 10 expression is significantly reduced in the stretching conditions. This means that the expression level of keratinocytes is reduced.

To demonstrate the phenomenon that the stratum corneum is weakly attached and wrinkled skin is formed under the stretching condition, filaggrin, Laminin α5 and Involucrin, which are important proteins in the stratum corneum, were quantified and compared by qPCR. In the case of filagrin, which is in charge of protecting and moisturizing the skin, the expression decreased very much after one day of stretching, and the expression level gradually increased to a normal level, and the expression decreased by stretching affected the speed of keratinization. It is believed that the stratum corneum was expressed at a level similar to the static environment on day 7 when the stratum corneum was completely produced. Laminin α5, which plays an important role in the basal layer, contributes to decreased expression and wrinkle formation when aging occurs. The level of laminin α5 expression is similar to that of the static environment in stretching conditions for 1 day and 3 days. Laminin α5 expression level was lowered on day 7. This suggests that aging has started in the cells and confirmed by tissue staining, which supports the explanation that the keratinocytes diffuse into the dermal layer and form wrinkled skin on the 7th day and that the aging of the cells has begun. Involuclin is responsible for protecting the skin, and given a stretch, the amount of expression is very low and the expression continues to decrease as the stretch continues. This phenomenon explains the gradual thinning and weakening of the stratum corneum.

Increased P53 Expression in Aged Skin

P53, a gene that repairs mutant cells or cancer cellized cells or induces apoptosis, tends to increase in aged skin. Therefore, we confirmed that the skin is aging through the increase of P53 when the skin equivalent is stimulated through the stretching device. On the first 1 and 3 days, P53 was further away from normal levels, which is thought to be due to a drop in cell activity caused by stress. However, at the 7th day of stimulation, P53 increased 2.5 times compared to the 3rd day and was higher than normal value. This suggests that expression of P53 was increased by mutant cells and cancer cellized cells, and the expression level was greatly increased considering that cell activity was lowered by stress. Therefore, after entering the 7th day, the increase in P53, which is a representative phenomenon in the aged cells, was confirmed. Therefore, it can be seen that skin aging is caused by mechanical stimulation through the stretching device.

Aging Skin Equivalents

We confirmed the skin aging phenomenon in the stretch-stimulated sample, and confirmed the date of the aging skin equivalent was formed by comparing the samples for the 3, 5, 7 days stretch stimulation. After 3 days of stretching, skin equivalents tended to decrease ECM expression, but the stratum corneum did not form completely, laminin α5 expression did not decrease, and P53 expression did not increase. In addition, keratinocytes did not diffuse into the dermal layer and wrinkled skin was formed even in the 5th day of stretching stimulation conditions. However, in the seventh-day stretching stimulation condition, keratinocytes spread into the soft stratum corneum and dermis layer, which confirmed wrinkled skin formation, decreased ECM protein expression, and increased P53. It is believed to be connected. Therefore, it is considered to promote skin aging when given a stimulus of 7 days under fast stimulation conditions (5.3 mm / s) (Fig. 11).

The Takeuchi group, which previously studied skin equivalents through stretching devices, showed thicker stratum corneum in skin equivalents cultured with blood vessels. However, different results were obtained when we applied skin equivalents through our stretching device. The reason for the opposite result in the stretching condition is that the Takeuchi group was slowly modified using clamps, which influenced cell activity and activated cell proliferation. On the other hand, the stretching device fabricated in the present invention is thought to have made a big difference in the stress felt by the cells because the stretching stimulation at a very high speed (5.3 mm / s), so the high-speed stretching acts as a stress on the cells It can be seen that the difference in activity appears and causes the effect of promoting aging on the shape and function of the cells. When the stretching stimulation lasts more than 7 days, it was experimentally confirmed that the formation of wrinkled skin, the protection of the protein, and the expression of the proteins related to the support occur, thereby forming a skin equivalent such as aging skin.

In order to mimic the skin of aging humans, we created a stretching device that operates at a high speed of 5.3mm / s using permanent magnets and electromagnets, and contracted and relaxed the skin equivalents at a frequency of 0.01Hz. Wrinkled skin, weak stratum corneum, decreased expression of protein related to moisturizing and support, increased expression of P53, etc. I could explain.

First, the changes in the dermal layer were reduced by about 1/5 from elongated shape of fibroblasts to rounded shape by stimulation through stretching device. Thus, expression of extracellular matrix proteins collagen and fibronectin was reduced. The qPCR analysis showed a significant decrease, and as stimulation continued, the expression level of β-actin, which is responsible for the cytoskeleton, gradually decreased. These results suggest that fibroblast expression is reduced by stretching stimulation.

Second, in the stratum corneum, the thickness of the stratum corneum, the behavior of keratinocytes, and protein expression decreased. The thickness of the stratum corneum tended to decrease by about 1/2 compared to the static environment, and the behavior of the stratum corneum diffused into the dermis at 7 days of stimulation through the stretching device. This suggests the formation of wrinkled skin, such as the phenomenon that laminin α5 tends to decrease on the 7th day. The expression of keratin 10 tended to decrease as the intensity of stimulation increased, and there was a big difference in expression when comparing the static environment with the stimulation environment of the 0.01 Hz cycle. In addition, qPCR resulted in a decrease in the amount of filaggrin that is involved in skin protection and moisturization, which gradually increased, which is believed to contribute to the slowing of the formation of the stratum corneum. The expression level tends to decrease, and it is thought that a weak and thin stratum corneum was formed by stretching stimulation.

Third is the expression of P53, which is an important aging marker that repairs or induces apoptosis of mutant or cancer cellized cells and increases expression during aging. In the stretching condition, the activity of the dermis and the stratum corneum was very decreased due to stress, and thus, the expression of P53 was decreased by about 1/2 from the static environment in the 1st and 3rd day. However, on the 7th day, it was about 2.5 times higher than the 3rd day. These results suggest that p53 expression has increased considerably in consideration of the reduced activity of the cells. This is an important result showing that the aging of the skin was achieved on the 7th day of the stretching stimulation, as is the result of the keratinocytes spreading to the soft and stratum corneum by the stretching device.

Through experiments, we observed phenomena in aged skin when the skin equivalent was stretched about 10% under conditions of 25V at 0.01Hz and 6mm distance between permanent magnets and electromagnets at a rate of 5.3mm / s for 7 days. The stretchable skin chip of the present invention can be tested in small chips prior to in vivo experiments in cosmetic development, drug testing, etc., and if further research is conducted, it is expected to be a powerful tool to replace the in vivo experiment. do.

When stretching was performed on the skin chip embedded with a permanent magnet according to the present invention, it was confirmed that keratinocytes adhere well to the collagen fragments even after a time elapses, and when the stretch was applied, the keratin was stressed over time. The cells gradually penetrated into the collagen fibroblast layer.

In addition, according to the present invention, the skin chip of the present invention can more closely simulate the skin that is actually aged by stretching compared to the skin cells that grow under the static culture conditions, so that the skin chips of the present invention are cosmetics, skin external medicines, Useful for testing toxic substances.

1 is a schematic view of a stretchable skin chip. The upper layer includes a culture chamber, a culture chamber, and a permanent magnet, and the lower layer contains a microfluidic channel for supplying the culture medium to the skin cell culture chamber, and the culture medium is supplied from below the skin cell culture chamber while the skin cells are not submerged in the culture medium. And a membrane to make it possible.

Figure 2 (a) is a graph comparing the strain according to the PDMS theme: mixing ratio of the curing agent and the distance between the permanent magnet and the electromagnet at 30V. (b) PDMS at 25 V. Subject: Strain ratio with curing agent mixing ratio and permanent magnet-electromagnet distance. Comparative graph * -conditions actually used in the experiment (PDMS topic: hardener = 35: 1, 25V, about 10% strain).

Figure 3 is a photograph of the H / E (Hematoxylin and eosin stain) staining results of the skin equivalent using porcine skin collagen. (a), (b), (c) Samples incubated for 3 days, 5 days, and 7 days under static conditions under static conditions, (d), (e), (f) 25 V, 1 A, 12 h at 10% strain / Day, samples incubated with air exposure for 3 days, 5 days, and 7 days, respectively, at 0.01 Hz repetition cycle conditions, (g), (h), (i) 25V, 1A, 12 h / day at 10% strain, 0.05 Hz Air exposure for 3, 5, and 7 days under repeated cycle conditions.

Figure 4 is a photograph of the special staining results of skin equivalents using porcine skin collagen. (a), (b), (c) Samples incubated with static air for 3 days under static environmental conditions, (d), (e), (f) 25V, 1A, 12 h / day at 10% strain, 0.01 Hz Samples incubated with air exposure for 3 days under repeated cycle conditions, (g), (h), (i). Samples incubated with air exposure at 25 V, 1 A, 10% strain for 12 h / Day, 0.05 Hz cycle for 7 days, (a), (d), (g)-H / E staining, (b), ( e), (h)-Mason Trichrome staining, (c), (f), (i)-Sirius staining (optical microscope, 200x each).

Figure 5 is a photograph of the immunohistochemistry of the skin equivalent of porcine skin collagen. (a), (b), (c) Samples incubated with static air for 3 days under static environmental conditions, (d), (e), (f) 25V, 1A, 12 h / day at 10% strain, 0.01 Hz Samples incubated with air exposure for 3 days under repeated cycle conditions, (g), (h), (i). Samples incubated with air exposure at 25 V, 1 A, 10% strain at 12 h / Day, 0.05 Hz cycle for 7 days, (a), (d), (g)-fibronectin staining, (b), (e) , (h)-collagen IV staining, (c), (f), (i)-keratin 10 staining (optical microscope, 200-fold each).

6 shows the results of H / E staining in skin equivalents using rat tail collagen. (a), (b), (c)-Samples incubated for 3 days, 5 days and 7 days with air exposure in static environment (200 times optical microscope). The appearance of fibroblasts (800x optical microscope) with an enlarged box of (d)-(b). (e), (f), (g)-Samples incubated for 3 days, 5 days, and 7 days each at 25 h, 1 A, 10% strain at 12 h / Day and 0.01 Hz repetition cycles for air exposure (200 times optical microscope) ). (h)-The appearance of fibroblasts (magnification 800 times) with an enlarged box of (f).

7 shows the results of special staining in skin equivalents using rat tail collagen. (a), (b), (c)-Samples incubated for 7 days with air exposure in static environment. (d), (e), (f)-Samples incubated for 7 days at 25V, 1A, 10% strain at 12h / Day, 0.01Hz repetitive cycle air exposure. (a), (d)-H / E stained photographs, (b), (e)-Mason trisomal stained photographs, (c), (f)-Sirius stained photographs (optical microscope, 200 times each).

Figure 8 shows the results of immunohistochemical staining in skin equivalents using rat tail collagen. (a), (b), (c)-Samples incubated for 7 days with air exposure in static environment. (d), (e), (f)-Samples incubated for 7 days with air exposure at 25V, 1A, 10% strain at 12h / Day, 0.01Hz repetition cycle conditions. (a), (d)-fibroblast staining photograph, (b), (e)-collagen IV staining photograph, (c), (f)-keratin 10 staining photograph (optical microscope, 200-fold each).

9 compares skin equivalents at 12 h / Day, 0.01 Hz repetition cycle conditions at 25 V, 1 A, 10% strain. (a), (d), (g). Comparison of H / E staining of samples with stretch stimulation for 3, 5 and 7 days, respectively. (b), (e), (h) Fibronectin staining of samples subjected to stretching stimulation for 3 days, 5 days and 7 days, respectively. (c), (f), (i) Collagen IV staining of samples subjected to stretching stimulation for 3 days, 5 days, and 7 days, respectively (optical microscope 200 times).

10 is a qPCR quantitative comparison graph. (a) Comparison of β-actin expression over time in culture under static conditions and in stretchable skin chips at 0.01 Hz, 10% strain (0, 1, 3, 7 days of air exposure). Comparison with time of filaggrin expression in stretchable skin-on-a-chip at 0.01 Hz, 10% Strain ( incubation 0, 1, 3, 7 days) in phosphorus environment. (c) Comparison with time of expression of laminin α5 in a static skin culture and stretchable skin chips at 0.01 Hz, 10% strain (0, 1, 3, 7 days of air exposure). (d) Incubation in static environment and time-dependent comparison of involukrin expression in stretchable skin chips at 0.01 Hz, 10% strain (0, 1, 3, 7 days of air exposure). (e) Cultivation in static environment and time-dependent comparison of P53 expression in stretchable skin chips at 0.01 Hz, 10% strain (0, 1, 3, 7 days of air exposure).

FIG. 11 is a graph showing changes in proteins and genetic factors related to skin aging and in stretching environments for 3 days and 7 days, respectively.

The present invention provides a skin chip for culturing skin cells by supplying a culture solution to skin cells arranged in three dimensions on a chip, wherein the skin cells of the chip correspond to a linear driving device outside the chip that provides forward and backward movements in a straight line. It provides a skin on a chip characterized in that it includes a connection that causes a linear movement to stretch the skin cells to simulate the contraction and relaxation of the skin.

In addition, the present invention provides a skin chip, characterized in that the connecting portion is mechanically, electrically or magnetically connected to the linear drive outside the chip.

In addition, the present invention

Base layer;

An underlayer arranged on the base layer and having a microfluidic channel and a membrane formed thereon; And

An upper layer arranged on the lower layer, the upper layer including a culture chamber, a skin cell culture chamber in which skin cells are cultured in three dimensions, and a connection part connected to a linear linear motion driving device outside the chip to provide straight forward and backward movements; It provides a skin chip (skin on a chip) comprising a. The connection part may be mechanically, electrically or magnetically connected to a linear motion driving device outside the chip.

In addition, the present invention can be implemented by using a variety of connection methods, such as a method of using a linear drive device and the connecting portion using a magnet or magnetic field, a magnetic object, a mechanical connection method between the connecting ring, a method of passing the connecting ring through the through hole, and the skin There is no particular limitation on the connection method unless a linear linear motion is applied to the cells to prevent contraction and relaxation.

The invention also relates to a skin chip, characterized in that the base layer is made of a material comprising or consisting of glass or transparent synthetic polymers. As the base layer, materials such as optically transparent synthetic polymers such as glass and / or polystyrene, polycarbonate, polysiloxane, polydimethylsiloxane are used.

In addition, the present invention is characterized in that the microfluidic channel of the lower layer connects the culture medium chamber and the skin cell culture chamber of the upper layer to supply the culture solution to the skin cells.

The present invention also provides a skin chip, wherein the membrane of the lower layer is located below the skin cell culture chamber of the upper layer.

In addition, the skin chip of the present invention is characterized in that the connecting portion is located around the skin cell culture chamber.

In addition, the skin chip of the present invention is characterized in that it comprises at least one connection.

In addition, the skin chip of the present invention is characterized in that at least one of the lower layer and the upper layer is made of a composition comprising PDMS (Polydimethylsiloxane) or PDMS.

In addition, the skin chip of the present invention is characterized in that the skin cells are one or more of fibroblasts or keratinocytes.

In addition, the skin chip of the present invention is characterized by adding a support for three-dimensional cell culture to the skin cells.

Also. In the skin chip of the present invention, the support is collagen, gelatin, fucoidan, alginate, chitosan, hyaluronic acid, silk, polyimides, polyamix acid, polycarprolactone, polyetherimide ( polyetherimide, nylon, polyaramid, polyvinyl alcohol, polyvinylpyrrolidone, poly-benzyl-glutamate, polyphenylene terephthalamide , Polyaniline, polyacrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethylmethacrylate, polylactic acid (polylactic acid; PLA), polyglycolic acid (PGA), copolymers of polylactic acid and polyglycolic acid (PLGA), poly {poly (ethylene jade Side) terephthalate-co-butylene terephthalate} (PEOT / PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester { poly (ortho ester; POE}, poly (propylene fumarate) -diacrylate; poly (propylene fumarate) -diacrylate; PPF-DA} and polyethylene glycol diacrylate; PEG-DA} It is characterized in that at least one biocompatible support selected from the group consisting of.

In addition, the skin chip of the present invention is characterized in that the skin cells include endothelial cells, dermal cells and epithelial cells.

In addition, the present invention provides a method for simulating skin cells and evaluating the efficacy of the external composition of the skin by applying intermittent one-way linear motion to the skin chip external linear drive device to cause relaxation and contraction to the skin cells.

In addition, the present invention relates to a method for evaluating the efficacy of an external composition for skin, characterized in that the external composition for cosmetics is a cosmetic composition, an external drug for skin or a toxic test substance.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it will be apparent to those skilled in the art that the scope of the present invention is not limited only to the description of the embodiments.

Cell culture

Human fibroblasts were cultured using DMEM medium (10% (v / v) fetal calf serum, containing 1% penicillin / streptomycin), and in experiments, human fibroblasts and pig skin type 1 collagen (SK Bioland) or rat tail The extracted Type 1 collagen sol was mixed and solidified for one hour in a CO ₂ incubator, and then cultured with replacing the culture medium every day for 4 days. The concentration of fibroblasts was 2.0 x 10 ⁴ cells / ml.

Human keratinocytes are KGM (Lonza) were sub-cultured using a culture solution, forming the stratum corneum is sprayed to give a human keratinocyte (Biosolution Co., Ltd.) on the surface of collagen gel were cultured for 4 days fibroblasts CO ₂ incubator for one hour After attaching at KGM culture was supplied. At this time, the concentration of human keratinocytes was 6 x 10 ⁶ cells / ml, and cultured with replacing the medium every day for 4 days.

When culturing collagen gels into which human fibroblasts were added, DMEM culture was used. When culturing fibroblasts and keratinocytes together, DMEM was supplied along the microfluidic tube and KGM was supplied onto the collagen gel in the culture chamber. .

DMEM / Ham's F12 (EGF-1 10 ng / ml, Hydrocortisone 0.4 μg / ml, Insulin 5 μg / ml, Transferrin 5 μg / ml, 3,3) to induce differentiation of keratinocytes through air exposure. , 5-TriiodoL-thyonine sodium salt 2 x 10 ^-11 M, cholera toxin 10 ^-10 M, 10% (v / v) fetal bovine serum, 1% penicillin / streptomycin).

Stretching skin chips production

In the present invention, the culture fluid is supplied through the microfluidic channel, and the human-like skin tissue is cultured in three dimensions. In addition, the structure of the permanent magnet is inserted into the chip to implement a stretch skin chip that does not interfere with the supply of the culture medium to give physical stimulation. To this end, the skin chip was manufactured by dividing it into an upper layer and a lower layer.

First of all, the PDMS (Polydimethylsiloxane) base: curing agent is mixed at 35: 1 and poured into aluminum mold (CSI Tech) in consideration of the location of the culture space and the permanent magnet for manufacturing the upper layer. The mold was removed after 1 hour solidification at. After inserting the permanent magnet was poured again PDMS liquid mixture was solidified in an oven at 80 ℃. The lower layer was then poured onto the master pattern wafer patterned into channels 150 μm wide and 50 μm wide using photolithography, mixed with 10: 1 ratio of PDMS main: hardener, and solidified in an oven at 80 ° C. for 1 hour. The lower layer in which the pattern was formed was produced. Base layer: Bottom layer: Top layer The bonding process was bonded using O ₂ plasma (FEMTO science). As the inserted permanent magnet, a disk-shaped neodymium magnet with a diameter of 10 mm and a thickness of 1 mm was used (JL magnet). A 40mm diameter circular electromagnet (JL magnet) was used, and an aluminum mold (CSI Tech) treated with a magnetic plate oxide film was used.

Stretching experiment

In the stretching experiment, 10% contraction-relaxation was repeated for 12 hours at an interval of 0.01 Hz using 1A and 25V AC voltages for the electromagnet, and the state was stopped for 12 hours (FIG. 2). The device is driven by PC control and offers the advantage of convenient and precise measurements.

Histology, IHC staining, special staining

Skin equivalents were fixed with 4% paraformaldehyde and embedded with paraffin. After rehydration, tissue sections (5 mm) were subjected to hematoxylin and eosin (H / E) staining for histology or immunohistochemistry for specific protein expression studies.

The primary antibodies against fibronectin, cytokeratin 10, CD34 and collagen IV were ab2413 (abcam), ab6318 (abcam), ab81289 (abcam) and ab6586 (abcam), and rabbit-specific HRP / DAB (ABC) as a variant antibody. ) Detection kit (ab64261, abcam) was used.

MT (Massan Trichrome stain kit Procedure, K7228, IMEB INC) and Sirius red / Fast Green staining were used for specific staining. Fluorescence slides were visualized and recorded with OLYMPUS IX173.

qPCR Quantitative Analysis

qPCR analysis was performed by first treating 1 ml of Trizol reagent in the sample to extract mRNA, separating RNA from cells, RNA extraction, RNA washing, RNA resuspension, and quantifying mRNA through Nanodrop 2000C (Thermo). It was. Then, cDNA was synthesized using amfiRivert cDNA synthesis Platinum Master Mix (GenDEPOT). Purified cENA was qPCR quantified using an Exicycler ™ 96 (Bioneer) device using AccuPower® 2X GreenStar ™ CR Master Mix (Bioneer). Each primer is shown in Table 1. The sequences in Table 1 are SEQ ID NOs: 1-12, respectively.

Outcome 1: comparison of strain on stretchable skin chips

We controlled the volume ratio, distance between permanent magnets and electromagnets, and electromagnet applied voltage in PDMS fabrication to control chamber strain of stretchable skin chips. PDMS theme: Hardener mixing ratios were 25: 1, 30: 1, and 35: 1, respectively, and the distance between permanent magnets and electromagnets was 5, 6, 8, and 10 mm, and the applied voltages of electromagnets were 25V and 30V (Fig. 2)

As shown in FIG. 2, the electromagnet applied voltage 25V, the PDMS theme: the curing agent mixing ratio of 35: 1, the permanent magnet-electromagnet distance of 5mm showed a relatively high strain rate of about 10%, the distance is 25% ratio There was little change in strain even as it neared. At 30V, the highest strain at the PDMS topic: curing agent ratio 35: 1 was shown, with about 11% strain even at 8mm distance. In addition, the strain was significantly increased at 30V compared to 25V even at 30: 1 and 25: 1 ratios. We used a stretchable skin chip with a 35: 1 ratio of PDMS theme: hardener at 25V to adopt about 10% strain, and the distance between the permanent magnet and the electromagnet was 6 mm.

RESULTS 2: Tissue analysis under static and stretching conditions in skin equivalents using porcine skin collagen

In the comparison group, the air exposure was applied for 3 days, 5 days, and 7 days in a repetition cycle of 0.01 Hz and 0.05 Hz at 25 V, 1 A, and 10% strain at 12 h / Day, respectively. In cultivation, the cultures were exposed to air for 3, 5 and 7 days without stretching. Paraffin was fixed for tissue cross-sectional analysis and the cross-section was analyzed by H / E staining (FIG. 3).

As shown in FIG. 3, H / E staining of the sample cultured in the static state and the culture cultured in the stretch state and the cross-sectional view of the tissue showed that fibroblasts and keratinocytes differed according to each condition. Stretching with 0.01Hz and 0.05Hz repetition cycles showed that keratinocytes gradually penetrated into collagen gel by stress on each 7th day, and the fibroblasts were stimulated more frequently by shorter repetition cycles. The number of cells decreased and a soft stratum corneum was formed.

Result 3: Mason Trichrome and Sirius staining of skin equivalents using porcine skin collagen cultured under static and stretching conditions

Fibroblasts function to produce extracellular matrix, such as collagen and fibronectin. Thus, the function of fibroblasts plays an important role in forming elastic skin. In order to determine whether fibroblasts function properly when given these stimuli, total collagen was analyzed by special staining. Special stains were used for Mason's trichrome and Sirius staining, and when the results of the two matched, the evidence for total collagen is justified.

In Masson's trichrome staining, dark blue stains represent the nuclei of cells and the stratum corneum is colored pink. Collagen is also stained light blue. In Sirius staining, dark pink stains represent the nuclei of cells, the stratum corneum is stained blue, and collagen is stained pink. As shown in FIG. 4, the results of Mason Trichrome and Sirius staining in each sample were consistent. Collagen expression was high around the epithelial layer in the static condition, and collagen expression was high in the dermis layer in the 0.01 Hz stimulation condition and 0.05 Hz stimulation. Under the conditions, the expression level was generally low (FIG. 4).

Result 4: immunohistochemical staining under static and stretching conditions in skin equivalents using porcine skin collagen

In the dermis, the skin produces collagen, expresses various proteins, protects moisture and increases elasticity.In the stratum corneum, cells die and become keratinized, which protects the body from mold, bacteria, and foreign substances entering the body, It functions to prevent loss. For this reason, collagen IV and fibronectin 10 were investigated to determine collagen production and fibronectin production of fibroblasts in the present invention, and keratin 10 was identified to examine normal function of keratinocytes.

As shown in Fig. 5, in the case of fibronectin and collagen IV, the dark brown portion is the nucleus of the cell and the light brown portion is the expression portion of fibronectin and collagen IV. For reference, the part dyed in blue is a stratum corneum. As a result of the cultivation of the static environment, it was confirmed that the expression was shown toward the periphery of the epithelial layer, similar to the Mason Trichrome and Sirius staining. However, the samples cultured in the 0.01 Hz stretch environment were lightly expressed around the epithelial layer. Also, at 0.05Hz, the expression of fibronectin and collagen Ⅳ was very low in round rings rather than solid lines. And keratin 10 in the stratum corneum keratin dark brown and cells are stained blue. Samples cultured in the static environment expressed keratin 10 well in air and exposed outer parts. However, the samples cultured in the stretching environment were slightly expressed in the collagen layer at the 0.01 Hz condition and hardly expressed at the 0.05 Hz condition (FIG. 5).

RESULTS 5: Tissue Assay in Static and Stretching Conditions in Rat Tail Collagen-induced Skin Equivalents

We found that the experimental results were more suitable for cells in skin equivalents using rat tail collagen than skin equivalents using pig skin collagen. Therefore, 0.85% by weight of skin equivalents of rat tail collagen were used to compare changes in cells when stretch stimulation was applied in skin equivalents of rat tail collagen, which is more suitable for 3D cell culture.

For tissue analysis, experiments were carried out under the same culture conditions as those of porcine skin collagen, and cultured on the skin chip under static conditions and stretchable skin chip with 0.01Hz and 10% strain using rat tail collagen (0.85% by weight) as a support. One sample was compared via H / E staining. (A), (b) and (c) of FIG. 6 are cross-sections of skin equivalents cultured over 3 days, 5 days, and 7 days under static conditions, respectively, and FIG. 6 (d) is a rectangle in FIG. 6 (b). This is an enlarged photo. Figure 6 (e), (f), (g) is a cross-section of the skin equivalent incubated over 3 days, 5 days, 7 days in stretching conditions, respectively, Figure 6 (h) is a rectangular portion of Figure 6 (f) It is enlarged photograph. It was confirmed that the stratum corneum was thicker than the stretching condition and adhered to the dermis layer in the static condition in the 5th day air exposure, and it was 86.4 ± 26㎛ in the static condition and 49.8 ± 12 in the stretching condition. It was confirmed that a thin stratum corneum layer having a thickness of about 37 μm was formed. Interestingly, a large change in the shape of the fibroblasts was observed. The fibroblasts of the skin chip in the static condition were elongated and elongated in the shape of 50 ~ 24㎛ in each 5th day air exposure. The stretchable skin chip had fibroblasts with round and small oval shape and 11.8 ± 6.8 ㎛ in length.

Result 6: Mason Trichrome and Sirius staining of skin equivalents of rat tail collagen cultured under static and stretching conditions

Air exposed 7-day samples incubated under static and stretching conditions were compared by Mason Trichrome staining and Sirius staining.

As shown in FIG. 7, the Mason Trichrome staining and Sirius staining results were the same according to each condition. Under the static condition, it was confirmed that the newly expressed collagen appeared in the shape of yarn in the whole dermal layer part, and was dyed darker in the stratum corneum part. However, in the stretching condition, the amount of collagen expressed in the form of thread was smaller than that of the static condition, and it was difficult to distinguish it from the mouse tail collagen used as a conventional support except the darkly stained part around the cell. Through this, it was confirmed that the collagen expression ability of the fibroblasts was significantly reduced when stress was applied.

Result 7: Immunostaining of skin equivalents using rat tail collagen cultured under static and stretching conditions

To compare the immunostaining results, the same day 7-day air exposure samples were used for gastric tissue staining. Fibronectin expression and collagen IV expression were used as representative protein synthesis markers of fibroblasts in order to examine the normal function of the two cells. To compare protein expression of keratinocytes, keratin 10 expression was compared.

8 (g), (h) and (i) are photographs showing the expression of fibronectin, collagen IV, and keratin 10 in samples cultured under static conditions. Fibronectin and collagen IV are very well expressed in the shape of yarn throughout the dermis, which is consistent with the results of special staining. It was also confirmed that keratin 10 was well formed in the vicinity of the stratum corneum on the epidermal cells. 8 (j), (k), and (l) are photographs showing the expression of fibronectin, collagen IV, and keratin 10 in a sample cultured under stretching conditions. Here, fibronectin or collagen IV in the form of tissues cultured in the static condition was not expressed, and as in the case of using pig skin collagen, it appeared in a round ring shape, and much smaller amount was stained compared with the static condition. It became. In particular, it was confirmed that collagen IV hardly appeared in the stretching environment. This suggests that it is hardly expressed in the Mason Trichrome, Sirius staining results, and is expressed by conventional rat tail collagen. It was also confirmed that the expression level of keratin 10 was very small compared to the static condition.

Result 8: Comparison of histological images of skin equivalents using rat tail collagen according to stretching time

We have observed that the thickness of the stratum corneum becomes thinner and the number of cells decreases over time through tissue photographs. Therefore, in order to see what changes in protein expression over time, fibronectin and collagen Ⅳ were compared by H / E staining and immunohistochemical staining of samples cultured over 3, 5 and 7 days, respectively. It was. (A), (b) and (c) are samples incubated for 3 days under stretching conditions, and FIGS. 9 (d), (e) and (f) are 5 days, and FIGS. 9 (g) and (h), (i) is a tissue photograph of a sample incubated for 7 days. It was confirmed that fibronectin expression was expressed in a round ring under the stretched condition. On the 3rd day, the low expression, the higher expression on the 5th day and the low expression on the 7th day were confirmed. In addition, the spread of keratinocytes on day 7 of the stretching condition was confirmed by immunohistochemical staining.

Result 9: Analysis of Gene Expression of Skin Equivalent Protein Using Rat Tail Collagen Cultured Under Static and Stretching Conditions

In order to quantitatively analyze the aging phenomenon through tissue analysis, aging-related factors were compared through qPCR. Among them, filaggrin is a protein mainly expressed in keratinocytes and is a protein involved in skin protection and moisturizing and decreases with age. Laminin α5 is a protein mainly expressed in the basement membrane present in the dermal-stratum corneum junction, which is responsible for skin support and decreases during aging and causes wrinkled skin. Involucrin is involved in skin protection of the stratum corneum and has a tendency to decrease in aged skin [32]. The P53 gene repairs mutations when they occur and repairs aged and cancerous cells. Is a gene for apoptosis, the expression of P53 gene tends to increase in aged skin. Finally, β-actin is responsible for the cytoskeleton and is used as a control in general experiments, but β-actin cannot be used as an appropriate control given the conditions that result in a decrease in the expression of β-actin during aging and given aging-inducing conditions. . As shown in FIG. 10 (a), we observed that β-actin expression gradually decreased with time, and in FIG. 10 (b) and (c), filaggrin decreased rapidly at the beginning of stretching and then gradually decreased. Laminin α5 was expressed similarly to the static condition but decreased on the 7th day. In addition, as shown in FIG. 10 (d), involuclin decreased significantly at the beginning of stretching, and its expression gradually decreased over time. As shown in FIG. 10 (e), in the case of P53, Day 1, 3 In the trend of gradually decreasing after the first day, the increase was about 2.5 times more rapidly than the 3rd day of stretching in the 7th day.

The stretchable skin chip of the present invention can be used for testing cosmetics, external skin medications, and toxic substances because it can mimic skin conditions similar to living bodies.

Sequences of the invention are primers for performing qPCR on aging related factors.

Claims (17)

1. In the skin chip for culturing the skin cells by supplying the culture solution and oxygen to the skin cells arranged in three dimensions on the chip,

   Skin-on-a-chip that simulates the contraction and relaxation of the skin by including connections that cause linear movement to the skin cells of the chip in response to linear drives external to the chip that provide forward and backward movement.
2. The method according to claim 1,

   Wherein the connection part is mechanically, electrically or magnetically connected to a linear drive external to the chip.
3. Base layer;

   An underlayer arranged on the base layer and having a microfluidic channel and a membrane formed thereon; And

   Arranged on the lower layer, a linear chamber is applied to the skin cells of the chip in response to a culture chamber, a skin cell culture chamber in which the skin cells are cultured in three dimensions, and a linear drive outside the chip providing forward and backward movement in a straight line. A skin-on-a-chip comprising a top layer comprising a connection.
4. The method according to claim 3,

   Wherein the connection part is mechanically, electrically or magnetically connected to a linear drive external to the chip.
5. The method according to claim 3,

   The connecting portion is a skin chip, characterized in that the through or connecting ring.
6. The method according to claim 3,

   And the base layer is made of a material comprising or consisting of glass or transparent synthetic polymer.
7. The method according to claim 3,

   The microfluidic channel of the lower layer connects the culture medium chamber and the skin cell culture chamber of the upper layer to supply culture medium and oxygen to the skin cells, and recovers waste and carbon dioxide from the skin cells.
8. The method according to claim 3,

   The membrane of the lower layer is a skin chip, characterized in that located below the skin cell culture chamber of the upper layer.
9. The method according to claim 3,

   The connection part is a skin chip, characterized in that located on one side of the skin cell culture chamber.
10. The method according to claim 3,

    Skin chip comprising one or more of the connecting portion.
11. The method according to claim 3,

    At least one of the lower layer and the upper layer is a skin chip, characterized in that consisting of a composition comprising PDMS (Polydimethylsiloxane) or PDMS.
12. The method according to claim 3,

    The skin cell is a skin chip, characterized in that at least one of fibroblasts or keratinocytes.
13. The method according to claim 3,

    Skin chip, characterized in that the support for the three-dimensional cell culture is added to the skin cells.
14. The method according to claim 13,

    The support is collagen, gelatin, fucoidan, alginate, chitosan, hyaluronic acid, silk, polyimides, polyamix acid, polycarprolactone, polyetherimide, nylon (nylon) ), Polyaramid, polyvinyl alcohol, polyvinylpyrrolidone, poly-benzyl-glutamate, polyphenylene terephthalamide, polyaniline, Polyacrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethylmethacrylate, polylactic acid (PLA) , Polyglycolic acid (PGA), copolymer of polylactic acid and polyglycolic acid (PLGA), poly {poly (ethylene oxide) terephthalate Tri-co-butylene terephthalate} (PEOT / PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), polyorthoester {poly (ortho group consisting of ester; POE}, poly (propylene fumarate) -diacrylate; PPF-DA} and polyethylene glycol diacrylate; PEG-DA Skin chip, characterized in that at least one selected from.
15. The method according to claim 3,

    Wherein said skin cells comprise endothelial cells, dermal cells and epithelial cells.
16. A method for simulating skin cells and evaluating the efficacy of an external composition for skin by applying intermittent one-way linear motion to the skin chip external linear drive device of any one of claims 1 to 15 to cause relaxation and contraction to the skin cells.
17. The method according to claim 16,

    The external composition for skin is a method for evaluating the efficacy of the external composition for skin, characterized in that the cosmetic composition, a skin external agent or a toxic test substance.

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