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WO2018131829A1 - Peau-sur-pastille étirable - Google Patents

Peau-sur-pastille étirable Download PDF

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Publication number
WO2018131829A1
WO2018131829A1 PCT/KR2017/015720 KR2017015720W WO2018131829A1 WO 2018131829 A1 WO2018131829 A1 WO 2018131829A1 KR 2017015720 W KR2017015720 W KR 2017015720W WO 2018131829 A1 WO2018131829 A1 WO 2018131829A1
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Prior art keywords
skin
chip
cells
stretching
collagen
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PCT/KR2017/015720
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English (en)
Korean (ko)
Inventor
성건용
임호영
송현정
박성수
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한림대학교 산학협력단
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Priority claimed from KR1020170180679A external-priority patent/KR101970125B1/ko
Application filed by 한림대학교 산학협력단 filed Critical 한림대학교 산학협력단
Priority to US16/478,446 priority Critical patent/US20200239857A1/en
Publication of WO2018131829A1 publication Critical patent/WO2018131829A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • 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.
  • 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.
  • 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.
  • mice 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.
  • 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.
  • 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.
  • the PDMS Polydimethylsiloxane
  • 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.
  • the plate is designed to be fixed to a square dish size plate so that a constant stimulus is always applied.
  • 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.
  • Skin equivalent in the description of the present invention means skin cells cultured in the stretchable skin-on-a-chip of the present invention.
  • 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 ⁇ -actin expression changes through gene analysis.
  • ⁇ -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.
  • 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.
  • ECM extracellular matrix
  • the extracellular matrix expression ability is lowered, forming weak skin and wrinkled skin.
  • the expression capacity of the extracellular matrix was significantly reduced in the skin equivalent sample subjected to the stretching for 7 days.
  • 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.
  • the fibroblasts showed a large change in the stretched experimental group.
  • the fibroblasts were elongated in the static condition, and the fibroblasts were rounded oval in the stretching condition as shown in FIG. 6 (h).
  • the length of fibroblasts was about 5 times the difference between the static conditions of 49.8 ⁇ 12 ⁇ m and the stretch of 11.8 ⁇ 6.8 ⁇ m.
  • the stretching stimulation has a big change in the cytoskeleton itself, and in this regard, the quantitative results of ⁇ -actin confirmed that the value gradually decreased as the stretching was applied [FIG. 10 (a)].
  • filaggrin 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 ⁇ 5 which plays an important role in the basal layer, contributes to decreased expression and wrinkle formation when aging occurs.
  • the level of laminin ⁇ 5 expression is similar to that of the static environment in stretching conditions for 1 day and 3 days.
  • Laminin ⁇ 5 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.
  • 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.
  • P53 was further away from normal levels, which is thought to be due to a drop in cell activity caused by stress.
  • 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.
  • 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.
  • 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.
  • the thickness of the stratum corneum decreased in the stratum corneum.
  • 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 ⁇ 5 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.
  • 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.
  • 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.
  • 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.
  • the upper layer includes a culture chamber, a culture chamber, and a permanent magnet
  • 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 3 is a photograph of the H / E (Hematoxylin and eosin stain) staining results of the skin equivalent using porcine skin collagen.
  • H / E Hematoxylin and eosin stain
  • Figure 4 is a photograph of the special staining results of skin equivalents using porcine skin collagen.
  • Figure 5 is a photograph of the immunohistochemistry of the skin equivalent of porcine skin collagen.
  • FIG. 6 shows the results of H / E staining in skin equivalents using rat tail collagen.
  • Figure 8 shows the results of immunohistochemical staining in skin equivalents using rat tail collagen.
  • (d) (e)-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).
  • 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.
  • 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.
  • An underlayer arranged on the base layer and having a microfluidic channel and a membrane formed thereon;
  • the connection part may be mechanically, electrically or magnetically connected to a linear motion driving device outside the chip.
  • 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
  • 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.
  • the base layer materials such as optically transparent synthetic polymers such as glass and / or polystyrene, polycarbonate, polysiloxane, polydimethylsiloxane are used.
  • 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.
  • the skin chip of the present invention is characterized in that the connecting portion is located around the skin cell culture chamber.
  • the skin chip of the present invention is characterized in that it comprises at least one connection.
  • 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.
  • PDMS Polydimethylsiloxane
  • the skin chip of the present invention is characterized in that the skin cells are one or more of fibroblasts or keratinocytes.
  • the skin chip of the present invention is characterized by adding a support for three-dimensional cell culture to the skin cells.
  • 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 ⁇
  • the skin chip of the present invention is characterized in that the skin cells include endothelial cells, dermal cells and epithelial cells.
  • 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.
  • 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.
  • 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
  • DMEM medium 10% (v / v) fetal calf serum, containing 1% penicillin / streptomycin
  • 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.
  • DMEM culture 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 ⁇ g / ml, Insulin 5 ⁇ g / ml, Transferrin 5 ⁇ g / ml, 3,3) to induce differentiation of keratinocytes through air exposure.
  • the culture fluid is supplied through the microfluidic channel, and the human-like skin tissue is cultured in three dimensions.
  • 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.
  • the skin chip was manufactured by dividing it into an upper layer and a lower layer.
  • 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 °C.
  • the lower layer was then poured onto the master pattern wafer patterned into channels 150 ⁇ m wide and 50 ⁇ m 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
  • FEMTO science O 2 plasma
  • 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.
  • 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.
  • 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 TM 96 (Bioneer) device using AccuPower® 2X GreenStar TM CR Master Mix (Bioneer). Each primer is shown in Table 1. The sequences in Table 1 are SEQ ID NOs: 1-12, respectively.
  • the highest strain at the PDMS topic: curing agent ratio 35: 1 was shown, with about 11% strain even at 8mm distance.
  • the strain was significantly increased at 30V compared to 25V even at 30: 1 and 25: 1 ratios.
  • 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.
  • 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).
  • 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.
  • 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.
  • the skin produces collagen, expresses various proteins, protects moisture and increases elasticity.
  • cells die and become keratinized, which protects the body from mold, bacteria, and foreign substances entering the body, It functions to prevent loss.
  • 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.
  • the dark brown portion is the nucleus of the cell and the light brown portion is the expression portion of fibronectin and collagen IV.
  • the part dyed in blue is a stratum corneum.
  • 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.
  • 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).
  • FIG. 6 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 ⁇ m 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 ⁇ m 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 ⁇ m in each 5th day air exposure.
  • the stretchable skin chip had fibroblasts with round and small oval shape and 11.8 ⁇ 6.8 ⁇ m in length.
  • Air exposed 7-day samples incubated under static and stretching conditions were compared by Mason Trichrome staining and Sirius staining.
  • the Mason Trichrome staining and Sirius staining results were the same according to each condition.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIGS. 9 (d), (e) and (f) are 5 days
  • FIGS. 9 (g) and (h) are 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.
  • filaggrin is a protein mainly expressed in keratinocytes and is a protein involved in skin protection and moisturizing and decreases with age.
  • Laminin ⁇ 5 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.
  • ⁇ -actin is responsible for the cytoskeleton and is used as a control in general experiments, but ⁇ -actin cannot be used as an appropriate control given the conditions that result in a decrease in the expression of ⁇ -actin during aging and given aging-inducing conditions. .
  • FIG. 10 (a) we observed that ⁇ -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 ⁇ 5 was expressed similarly to the static condition but decreased on the 7th day.
  • involuclin decreased significantly at the beginning of stretching, and its expression gradually decreased over time.
  • 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.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Le problème à la base de l'invention concerne le fait selon lequel des cellules cutanées cultivées dans un état statique ne peuvent pas imiter un environnement d'étirement auquel la peau réelle est confrontée. La solution selon l'invention porte sur une peau-sur-pastille et concerne une peau-sur-pastille qui est plus similaire à la peau réelle par l'imitation de la répétition de la contraction et du relâchement dû à l'étirement des cellules de la peau, par incorporation d'un aimant permanent dans la peau-sur-pastille.
PCT/KR2017/015720 2017-01-16 2017-12-29 Peau-sur-pastille étirable WO2018131829A1 (fr)

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US16/478,446 US20200239857A1 (en) 2017-01-16 2017-12-29 Stretchable skin-on-a-chip

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KR10-2017-0007160 2017-01-16
KR20170007160 2017-01-16
KR10-2017-0180679 2017-12-27
KR1020170180679A KR101970125B1 (ko) 2017-01-16 2017-12-27 신축 가능한 피부 칩

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5153136A (en) * 1988-07-22 1992-10-06 Vandenburgh Herman H Apparatus for growing tissue specimens in vitro
KR20100067298A (ko) * 2008-12-11 2010-06-21 한국과학기술원 세포자극기, 세포자극 및 배양장치, 세포자극 및 배양시스템
KR20100088297A (ko) * 2009-01-30 2010-08-09 한국과학기술원 세포배양 복합자극챔버와 이를 이용한 세포배양장치
KR20110044226A (ko) * 2008-07-16 2011-04-28 칠드런'즈 메디컬 센터 코포레이션 마이크로채널을 갖는 기관 모방 장치 및 그 사용 및 제조 방법
KR20150032126A (ko) * 2013-09-17 2015-03-25 인제대학교 산학협력단 세포 복합 자극 및 배양 장치, 및 이를 이용한 세포의 배양 방법

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5153136A (en) * 1988-07-22 1992-10-06 Vandenburgh Herman H Apparatus for growing tissue specimens in vitro
KR20110044226A (ko) * 2008-07-16 2011-04-28 칠드런'즈 메디컬 센터 코포레이션 마이크로채널을 갖는 기관 모방 장치 및 그 사용 및 제조 방법
KR20100067298A (ko) * 2008-12-11 2010-06-21 한국과학기술원 세포자극기, 세포자극 및 배양장치, 세포자극 및 배양시스템
KR20100088297A (ko) * 2009-01-30 2010-08-09 한국과학기술원 세포배양 복합자극챔버와 이를 이용한 세포배양장치
KR20150032126A (ko) * 2013-09-17 2015-03-25 인제대학교 산학협력단 세포 복합 자극 및 배양 장치, 및 이를 이용한 세포의 배양 방법

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