STRETCHABLE SKIN-ON-A-CHIP

Abstract

Disclosed is a skin-on-a-chip, which more closely resembles real skin by simulating the repetition of contraction and relaxation due to stretching of skin cells, by embedding a permanent magnet in the skin-on-a-chip. The skin-on-a-chip includes a connector that causes a linear motion in the skin cells of the chip when driven by a linear drive device outside the chip, which provides forward and backward movement, to thereby simulate contraction and relaxation of skin.

Claims

1. (canceled)

2. (canceled)

3. A skin-on-a-chip, comprising: a base layer; a lower layer disposed on the base layer and configured to include a microfluidic channel and a membrane; and an upper layer disposed on the lower layer and configured to include a culture medium chamber, a skin cell culture chamber for three-dimensionally culturing skin cells, and a connector that causes a linear motion in the skin cells of the chip when driven by a linear drive device outside the chip, which provides linear forward and backward movement, wherein the skin cell culture chamber contains a support for three-dimensional culture of skin cells, wherein the connector is at one side of the skin cell culture chamber, wherein the membrane is positioned below the skin cell culture chamber in the upper layer, preventing the skin cells from being the skin cells from being immersed in the culture medium, wherein the microfluidic channel is supplying the culture medium and oxygen to the skin cells and recovering waste materials and carbon dioxide from the skin cells by connection the membrane and the culture medium chamber.

4. The skin-on-a-chip of claim 3, wherein the connector is mechanically, electrically or magnetically connected to the linear drive device outside the chip.

5. (canceled)

6. The skin-on-a-chip of claim 3, wherein the base layer is made of a material comprising or consisting of glass or a transparent synthetic polymer.

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. The skin-on-a-chip of claim 3, wherein at least one of the lower layer and the upper layer is formed of PDMS (polydimethylsiloxane) or a composition including PDMS.

12. (canceled)

13. (canceled)

14. The skin-on-a-chip of claim 3, wherein the support is at least one selected from the group consisting of collagen, gelatin, fucoidan, alginate, chitosan, hyaluronic acid, silk, polyimide, polyamic acid, polycaprolactone, polyetherimide, nylon, polyaramid, polyvinyl alcohol, polyvinyl pyrrolidone, polybenzyl glutamate, polyphenylene terephthalamide, polyaniline, polyacrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethyl methacrylate, polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-co-polyglycolic acid (PLGA), poly{poly(ethylene oxide)terephthalate-co-butylene terephthalate} (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), poly(ortho-ester) (POE), poly(propylene fumarate)diacrylate (PPF-DA), and poly(ethylene glycol)diacrylate (PEG-DA).

15. (canceled)

16. A method of evaluating efficacy of a dermatological composition by intermittently applying a one-way linear motion by a linear drive device outside the skin-on-a-chip of claim 3 to cause relaxation and contraction in skin cells so as to simulate skin cells.

17. The method of claim 16, wherein the dermatological composition is a cosmetic composition, a skin external preparation composition or a toxicity test substance.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0032] FIG. 1 schematically shows a stretchable skin-on-a-chip, in which the upper layer is configured to include a culture medium chamber, a culture chamber and a permanent magnet, and the lower layer includes a microfluidic channel for supplying a culture medium to the culture chamber for culturing skin cells, and a membrane for preventing the skin cells from being immersed in the culture medium and supplying the culture medium from below the culture chamber;

[0033] FIG. 2 shows (a) a graph showing the results of strain depending on the PDMS base:curing agent mixing ratio and the permanent-magnet-to-electromagnet distance at 30 V, and (b) a graph showing the results of strain depending on the PDMS base:curing agent mixing ratio and the permanent-magnet-to-electromagnet distance at 25 V, *- actual conditions used in the experiment (PDMS base:curing agent=35:1, 25 V, about 10% strain);

[0034] FIG. 3 shows images of H/E (hematoxylin and eosin) staining of skin equivalents using pig skin collagen, in which (a), (b) and (c) are samples cultured through air exposure for 3 days, 5 days and 7 days under static conditions, (d), (e) and (f) are samples cultured through air exposure for 3 days, 5 days and 7 days under cyclic stretching conditions of 12 hr/day and 0.01 Hz at 25 V, 1 A, and 10% strain, and (g), (h) and (i) are samples cultured through air exposure for 3 days, 5 days and 7 days under cyclic stretching conditions of 12 hr/day and 0.05 Hz at 25 V, 1 A, and 10% strain;

[0035] FIG. 4 shows images of special staining of skin equivalents using pig skin collagen, in which (a), (b) and (c) are samples cultured through air exposure for 3 days under static conditions, (d), (e) and (f) are samples cultured through air exposure for 3 days under cyclic stretching conditions of 12 hr/day and 0.01 Hz at 25 V, 1 A, and 10% strain, and (g), (h) and (i) are samples cultured through air exposure for 7 days under cyclic stretching conditions of 12 hr/day and 0.05 Hz at 25 V, 1 A, and 10% strain, (a), (d) and (g)H/E staining, (b), (e) and (h) Masson's trichrome staining, (c), (f) and (i)Sirius staining (optical microscope, 200 magnifications);

[0036] FIG. 5 shows immunohistochemical results of skin equivalents using pig skin collagen, in which (a), (b) and (c) are samples cultured through air exposure for 3 days under static conditions, (d), (e) and (f) are samples cultured through air exposure for 3 days under cyclic stretching conditions of 12 hr/day and 0.01 Hz at 25 V, 1 A, and 10% strain, and (g), (h) and (i) are samples cultured through air exposure for 7 days under cyclic stretching conditions of 12 hr/day and 0.05 Hz at 25 V, 1 A, and 10% strain, (a), (d) and (g)fibronectin staining, (b), (e) and (h)collagen IV staining, and (c), (f) and (i)keratin 10 staining (optical microscope, 200 magnifications);

[0037] FIG. 6 shows the results of H/E staining analysis of skin equivalents using rat tail collagen, in which (a), (b) and (c) are samples cultured through air exposure for 3 days, 5 days and 7 days under static conditions (optical microscope, 200 magnifications), (d) shows the enlarged fibroblast shape of the box of (b) (optical microscope, 800 magnification), (e), (f) and (g) are samples cultured through air exposure for 3 days, 5 days and 7 days under cyclic stretching conditions of 12 hr/day and 0.01 Hz at 25 V, 1 A, and 10% strain (optical microscope, 200 magnifications), and (h) shows the enlarged fibroblast shape of the box of (f) (optical microscope, 800 magnification);

[0038] FIG. 7 shows the results of special staining analysis of skin equivalents using rat tail collagen, in which (a), (b) and (c) are samples cultured through air exposure for 7 days under static conditions, and (d), (e) and (f) are samples cultured through air exposure for 7 days under cyclic stretching conditions of 12 hr/day and 0.01 Hz at 25 V, 1 A, and 10% strain, (a) and (d)H/E staining images, (b) and (e)Masson's trichrome staining images, and (c) and (f)Sirius staining images (optical microscope, 200 magnifications);

[0039] FIG. 8 shows the results of immunohistochemical staining analysis of skin equivalents using rat tail collagen, in which (a), (b) and (c) are samples cultured through air exposure for 7 days under static conditions, and (d), (e) and (f) are samples cultured through air exposure for 7 days under cyclic stretching conditions of 12 hr/day and 0.01 Hz at 25 V, 1 A, and 10% strain, (a) and (d)fibroblast staining images, (b) and (e)collagen IV staining images, and (c) and (f)keratin 10 staining images (optical microscope, 200 magnifications);

[0040] FIG. 9 shows the results of comparison of skin equivalents under cyclic stretching conditions of 12 hr/day and 0.01 Hz at 25 V, 1 A, and 10% strain, (a), (d) and (g) illustrating the results of H/E staining of samples subjected to stretching for 3 days, 5 days and 7 days, (b), (e) and (h) illustrating the results of fibronectin staining of samples subjected to stretching for 3 days, 5 days and 7 days, and (c), (f) and (i) illustrating the results of collagen IV staining of samples subjected to stretching for 3 days, 5 days and 7 days (optical microscope, 200 magnifications);

[0041] FIG. 10 is graphs showing the results of qPCR quantitative analysis, (a) illustrating the expression of -actin over time in static culture and in the stretchable skin-on-a-chip at 0.01 Hz and 10% strain (air exposure for 0 day, 1 day, 3 days, 7 days), (b) illustrating the expression of filaggrin over time in static culture and in the stretchable skin-on-a-chip at 0.01 Hz and 10% strain (air exposure for 0 day, 1 day, 3 days, 7 days), (c) illustrating the expression of laminin 5 over time in static culture and in the stretchable skin-on-a-chip at 0.01 Hz and 10% strain (air exposure for 0 day, 1 day, 3 days, 7 days), (d) illustrating the expression of involucrin over time in static culture and in the stretchable skin-on-a-chip at 0.01 Hz and 10% strain (air exposure for 0 day, 1 day, 3 days, 7 days), and (e) illustrating the expression of P53 over time in static culture and in the stretchable skin-on-a-chip at 0.01 Hz and 10% strain (air exposure for 0 day, 1 day, 3 days, 7 days); and

[0042] FIG. 11 shows changes in skin aging-related protein and gene factors for 3 days and 7 days under stretching conditions.

BEST MODE

[0043] The present invention provides a skin-on-a-chip, suitable for use in culturing skin cells by supplying a culture medium to skin cells three-dimensionally arranged on a chip, the skin-on-a-chip including therein a connector that causes linear motion in the skin cells of the chip when driven by a linear drive device outside the chip, which provides linear forward and backward movement, so that the skin cells are stretched to thereby simulate contraction and relaxation of the skin.

[0044] Also, in the present invention, the connector may be mechanically, electrically or magnetically connected to the linear drive device outside the chip.

[0045] In addition, the present invention provides a skin-on-a-chip, comprising:

[0046] a base layer;

[0047] a lower layer disposed on the base layer and configured to include a microfluidic channel and a membrane; and

[0048] an upper layer disposed on the lower layer and configured to include a culture medium chamber, a skin cell culture chamber for three-dimensionally culturing skin cells, and a connector that may be connected to a linear motion drive device outside the chip, which provides linear forward and backward movement. Here, the connector may be mechanically, electrically or magnetically connected to the linear motion drive device outside the chip.

[0049] Furthermore, in the present invention, the linear drive device and the connector may be connected in a variety of connection manners, including a mechanical connection manner between connecting rings, a manner of passing a connecting ring through a through-hole, and the like, in addition to the use of a magnet, a magnetic field or a magnetic object, and the connection manner is not particularly limited so long as it does not interfere with application of a linear motion to the skin cells to cause contraction and relaxation.

[0050] Also, in the skin-on-a-chip of the present invention, the base layer may be made of a material comprising or consisting of glass or a transparent synthetic polymer. The base layer may be manufactured using a material such as glass and/or an optically clear synthetic polymer such as polystyrol, polycarbonate, polysiloxane, polydimethylsiloxane, etc.

[0051] Also, the microfluidic channel of the lower layer may connect the culture medium chamber and the skin cell culture chamber of the upper layer to supply a culture medium to the skin cells.

[0052] Also, in the skin-on-a-chip of the present invention, the membrane of the lower layer may be positioned below the skin cell culture chamber of the upper layer.

[0053] Also, in the skin-on-a-chip of the present invention, the connector may be positioned around the skin cell culture chamber.

[0054] Also, in the skin-on-a-chip of the present invention, at least one connector may be provided.

[0055] Also, in the skin-on-a-chip of the present invention, at least one of the lower layer and the upper layer may be formed of PDMS (polydimethylsiloxane) or a composition including PDMS.

[0056] Also, in the skin-on-a-chip of the present invention, the skin cells may comprise at least one of fibroblasts and keratinocytes.

[0057] Also, in the skin-on-a-chip of the present invention, the skin cells may be added with a support for three-dimensional cell culture.

[0058] Also, in the skin-on-a-chip of the present invention, the support may be at least one biocompatible support selected from the group consisting of collagen, gelatin, fucoidan, alginate, chitosan, hyaluronic acid, silk, polyimide, polyamic acid, polycaprolactone, polyetherimide, nylon, polyaramid, polyvinyl alcohol, polyvinyl pyrrolidone, polybenzyl glutamate, polyphenylene terephthalamide, polyaniline, polyacrylonitrile, polyethylene oxide, polystyrene, cellulose, polyacrylate, polymethyl methacrylate, polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-co-polyglycolic acid (PLGA), poly{poly(ethylene oxide) terephthalate-co-butylene terephthalate} (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), poly(ortho-ester) (POE), poly(propylene fumarate)diacrylate (PPF-DA), and poly(ethylene glycol)diacrylate (PEG-DA).

[0059] Also, in the skin-on-a-chip of the present invention, the skin cells may include endothelial cells, dermal cells and epithelial cells.

[0060] In addition, the present invention provides a method of evaluating the efficacy of a dermatological composition by intermittently applying a one-way linear motion by a linear drive device outside the skin-on-a-chip described above to cause relaxation and contraction in skin cells so as to simulate skin cells.

[0061] Also, the dermatological composition may be a cosmetic composition, a skin external preparation composition or a toxicity test substance.

MODE FOR INVENTION

[0062] A better understanding of the present invention will be given through the following examples, which are merely set forth to illustrate but are not to be construed as limiting the scope of the present invention, as will be apparent to those skilled in the art.

[0063] Cell Culture

[0064] Human fibroblasts were cultured using a DMEM culture medium (containing 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin), and in the experiment, human fibroblasts were mixed with pig skin type 1 collagen (SK Bioland) or type 1 collagen sol extracted from rat tails, cured for 1 hr in a CO.sub.2 incubator, and then cultured for 4 days while the culture medium was replaced every day. The concentration of the fibroblasts was 2.010.sup.4 cells/ml.

[0065] Human keratinocytes were subcultured using a KGM (Lonza) culture medium, and for stratum corneum formation, human keratinocytes (Biosolution Co., Ltd.) were sprayed onto the surface of collagen gel on which fibroblasts were cultured for 4 days and attached for 1 hr in a CO.sub.2 incubator, after which a KGM culture medium was supplied thereto. As such, the concentration of human keratinocytes was 610.sup.6 cells/ml, and culture was performed for 4 days while the culture medium was replaced every day.

[0066] A DMEM culture medium was used for culturing the collagen gel to which human fibroblasts were added, and when fibroblasts and keratinocytes were cultured together, DMEM was supplied to the lower layer along the microfluidic channel and KGM was supplied onto the collagen gel of the culture chamber.

[0067] In order to induce differentiation of keratinocytes through air exposure, culture was carried out using DMEM/Ham's F12 (10 ng/ml of EGF-1, 0.4 g/ml of hydrocortisone, 5 g/ml of insulin, 5 g/ml of transferrin, 210.sup.11 M 3,3,5-triiodol-thyonine sodium salt, 10.sup.10 M cholera toxin, 10% (v/v) fetal bovine serum, and 1% penicillin/streptomycin).

[0068] Manufacture of Stretchable Skin-On-a-Chip

[0069] In the present invention, the culture medium was supplied through the microfluidic channel, whereby human-like skin tissue was cultured three-dimensionally. Furthermore, a structure in which a permanent magnet was inserted into a chip was designed in order to realize a stretchable skin-on-a-chip that provides a physical stimulus without disturbing the supply of culture medium. To this end, the skin-on-a-chip was manufactured so as to include upper and lower layers.

[0070] In order to manufacture the upper layer, taking into consideration the culture space and the position of a permanent magnet, a PDMS (polydimethylsiloxane) base and a curing agent were mixed at a ratio of 35:1, placed in an aluminum mold (CSI Tech), and cured in an oven at 80 C. for 1 hr, after which the mold was removed therefrom. Then, a permanent magnet was inserted thereto, and the PDMS mixed solution was poured again and cured in an oven at 80 C. for 1 hr. Thereafter, the lower layer was manufactured in a manner in which a PDMS base and a curing agent were mixed at a ratio of 10:1, poured onto a master pattern wafer patterned with a channel having a width of 150 m and a height of 50 m through photolithography, and then cured in an oven at 80 C. for 1 hr, thereby obtaining a lower layer having a fine pattern. The process of adhering the base layer, the lower layer and the upper layer was performed using O.sub.2 plasma (FEMTO Science). The inserted permanent magnet was a disc-shaped neodymium magnet having a diameter of 10 mm and a thickness of 1 mm (JL magnet). As an electromagnet, a circular electromagnet having a diameter of 40 mm (JL magnet) was used, and a magnetic-plate oxide-film-treated aluminum mold (CSI Tech) was used.

[0071] Stretching Test

[0072] In the stretching test, 10% contraction-relaxation was repeated for 12 hr at a frequency of 0.01 Hz using an alternating voltage of 1 A and 25 V applied to the electromagnet, and the static state was maintained for 12 hr (FIG. 2). This device was driven through PC control, which is advantageous in making convenient and precise measurement.

[0073] Histology, IHC Staining, Special Staining

[0074] Skin equivalents were fixed in 4% paraformaldehyde and embedded in paraffin. After rehydration, tissue segments (5 mm) were subjected to H/E (hematoxylin and eosin) staining for tissue experiments or immunohistochemistry for certain protein expression studies.

[0075] As primary antibodies for fibronectin, cytokeratin 10, CD34 and collagen IV, ab2413 (abcam), ab6318 (abcam), ab81289 (abcam) and ab6586 (abcam) were used, and as a secondary antibody, a rabbit-specific HRP/DAB (ABC) detection kit (ab64261, abcam) was used.

[0076] For special staining, MT (Massan Trichrome stain kit Procedure, K7228, IMEB INC) and Sirius red/Fast Green staining were used, and fluorescent slides were visualized and recorded using an OLYMPUS IX173.

[0077] qPCR Quantitative Analysis

[0078] For qPCR analysis, mRNA was extracted in a manner in which the sample was treated with 1 ml of a triazole reagent and thus RNA was separated from cells and extracted through RNA precipitation, RNA washing, and RNA resuspension, and mRNA was quantified using a Nanodrop 2000C (Thermo). Thereafter, cDNA was synthesized using an amfiRivert cDNA Synthesis Platinum Master Mix (genDEPOT). The purified cDNA was subjected to qPCR quantification using an AccuPower 2X GreenStar qPCR Master Mix (Bioneer) through an Exicycler 96 (Bioneer). Each primer is given in Table 1 below. The sequences of Table 1 show SEQ ID NOS: 1 to 12.

[0079] Result 1: Strain Comparison of Stretchable Skin-On-a-Chip

[0080] In order to control the chamber strain of the stretchable skin-on-a-chip, the volume ratio upon PDMS preparation, the distance between the permanent magnet and the electromagnet, and the voltage that was applied to the electromagnet were varied. The PDMS base:curing agent mixing ratio was set to 25:1, 30:1 and 35:1, the distance between the permanent magnet and the electromagnet was set to 5, 6, 8 and 10 mm, and the voltage applied to the electromagnet was set to 25 V and 30 V (FIG. 2).

[0081] As shown in FIG. 2, the strain was relatively high, to the level of about 10%, under conditions such that the voltage applied to the electromagnet was 25 V, the PDMS base:curing agent mixing ratio was 35:1 and the permanent-magnet-to-electromagnet distance was 5 mm, and there was little change in strain despite the decreased distance at a ratio of 25:1. The greatest strain was exhibited at a PDMS base:curing agent ratio of 35:1 at 30 V, and about 11% stain appeared even at a distance of 8 mm. In addition, the strain was significantly increased at 30 V compared to 25 V at ratios of 30:1 and 25:1. In order to adopt about 10% strain, a stretchable skin-on-a-chip manufactured at a PDMS base:curing agent ratio of 35:1 at 25 V, in which the permanent magnet and the electromagnet were spaced apart from each other at a distance of 6 mm, was used for experiments.

[0082] Result 2: Tissue Analysis of Skin Equivalents Using Pig Skin Collagen Under Static and Stretching Conditions

[0083] In a comparative group subjected to stretching, culture was carried out through air exposure for 3 days, 5 days and 7 days under cyclic stretching conditions of 12 hr/day and 0.01 Hz and 0.05 Hz at 25 V, 1 A, and 10% strain, and static culture was conducted through air exposure for 3 days, 5 days and 7 days without stretching. The tissue cross-sections were fixed in paraffin and analyzed through H/E staining (FIG. 3).

[0084] As shown in FIG. 3, based on the results of observation of the cross-sections of tissues after H/E staining of the samples cultured under stretching and static conditions, fibroblasts and keratinocytes were found to have different results under individual conditions. Under cyclic stretching of 0.01 Hz and 0.05 Hz, the phenomena by which keratinocytes gradually infiltrated the collagen gel due to stress on the 7th day were similarly shown, and when the stimulus was more frequently applied by shortening the stretching cycle, the number of fibroblasts was decreased and a fragile stratum corneum was formed.

[0085] Result 3: Masson's Trichrome and Sirius Staining of Skin Equivalents Using Pig Skin Collagen Cultured Under Static and Stretching Conditions

[0086] Fibroblasts function to produce extracellular matrix such as collagen, fibronectin, etc. Thus, fibroblasts play an important role in realizing skin elasticity. In order to evaluate whether fibroblasts function properly under stimulation, total collagen production was analyzed through special staining. As special staining, Masson's trichrome staining and Sirius staining were performed. Here, when these two staining results were consistent with each other, the evidence of total collagen production is regarded as valid.

[0087] In the Masson's trichrome staining method, the portion stained in dark blue represents the cell nucleus and the stratum corneum is stained in pink. Also, collagen is stained in light blue. In the Sirius staining method, the portion stained in dark pink represents the cell nucleus, the stratum corneum is stained in blue, and the collagen is stained in pink. As shown in FIG. 4, the Masson's trichrome and Sirius staining results in the samples were consistent, collagen expression was high around the epithelial layer under static conditions, collagen expression was generally high in the dermal layer at 0.01 Hz stimulation, and the expression level was generally low at 0.05 Hz stimulation (FIG. 4).

[0088] Result 4: Immunohistochemical Staining of Skin Equivalents Using Pig Skin Collagen Under Static and Stretching Conditions

[0089] In the dermal layer of the human body, skin produces collagen, various proteins are expressed, and moisture protection and elasticity are increased, and in the stratum corneum, cells die to thus be keratinized, which protects the human body from fungi, bacteria and foreign substances entering the body and prevents moisture loss. For this reason, collagen IV and fibronectin 10 were measured in order to evaluate the collagen production and fibronectin production of fibroblasts in the present invention, and keratin 10 was measured in order to evaluate the normal function of keratinocytes.

[0090] As shown in FIG. 5, in the case of fibronectin and collagen IV, the portion stained in dark brown represents the cell nucleus and the light brown portion is the expression portion of fibronectin and collagen IV. For reference, the portion stained in blue is the stratum corneum. The results of static culture showed that expression occurred in the periphery of the epithelial layer, like the Masson's trichrome and Sirius staining results. However, in the samples cultured at 0.01 Hz stretching, slight expression occurred in the periphery of the epithelial layer. In addition, at 0.05 Hz, fibronectin and collagen IV showed very low expression in a round ring shape, rather than a thread shape. In keratin 10, the keratin in the stratum corneum was stained in dark brown and the cells were stained in blue. Keratin 10 was well expressed in the outer portion, which was exposed to air, in the sample cultured in the static environment. However, in the samples cultured in the stretching environment, slight expression occurred at the top of the collagen layer at 0.01 Hz and expression hardly occurred at 0.05 Hz (FIG. 5).

[0091] Result 5: Tissue Analysis of Skin Equivalents Using Rat Tail Collagen Under Static and Stretching Conditions

[0092] The above experimental results demonstrated that the skin equivalents using rat tail collagen were more suitable for cells, rather than the skin equivalents using pig skin collagen. Therefore, skin equivalents using 0.85 wt % rat tail collagen were used to compare cell changes when a stretching stimulus was applied to skin equivalents using rat tail collagen, which is more suitable for 3D cell culture.

[0093] For tissue analysis, the experiment was carried out under the same culture conditions as in the pig skin collagen results, and using as a support rat tail collagen (0.85 wt %), the samples cultured on the skin-on-a-chip under static conditions and the stretchable skin-on-a-chip under stretching conditions of 0.01 Hz and 10% strain were compared through H/E staining. FIGS. 6(a), (b) and (c) show the cross-sections of the skin equivalents cultured for 3 days, 5 days and 7 days under static conditions, and FIG. 6(d) shows the enlarged image of the rectangular portion of FIG. 6(b). FIGS. 6(e), (f) and (g) show the cross-sections of the skin equivalents cultured for 3 days, 5 days and 7 days under stretching conditions, and FIG. 6(h) shows the enlarged image of the rectangular portion of FIG. 6(f). As for culture through air exposure on the 5th day, the stratum corneum was thicker under static conditions than under stretching conditions, and was confirmed to be well attached to the dermal layer. When compared numerically, the stratum corneum was formed at a thickness of 49.812 m under stretching conditions, which is about 37 m thinner than 86.426 m under static conditions. Interestingly, a great change in the shape of fibroblasts was observed. Upon each kind of culture through air exposure on the 5th day, the fibroblasts of the skin-on-a-chip under static conditions were extended in a long star-like shape to thus have a length of about 5024 m, and the fibroblasts of the stretchable skin-on-a-chip had a round, small elliptical shape with a length of 11.86.8 m.

[0094] Result 6: Masson's Trichrome and Sirius Staining of Skin Equivalents Using Rat Tail Collagen Cultured Under Static and Stretching Conditions

[0095] The samples on the 7th day of air exposure cultured under static conditions and under stretching conditions were subjected to Masson's trichrome staining and Sirius staining and compared.

[0096] As shown in FIG. 7, the results of Masson's trichrome staining and Sirius staining showed the same tendency under the corresponding conditions. Under static conditions, a lot of collagen newly expressed in thread form appeared in the entire dermal layer and the stratum corneum was more darkly stained. However, under stretching conditions, unlike static conditions, the expression level of collagen in thread form was low, and it was difficult to distinguish it from the rat tail collagen used as the conventional support except for the darkly stained portion around the cells. Thereby, it can be concluded that the collagen expression capacity of fibroblasts was significantly decreased upon application of stress.

[0097] Result 7: Immunostaining of Skin Equivalents Using Rat Tail Collagen Cultured Under Static and Stretching Conditions

[0098] In order to compare the results of immunostaining, the samples on the 7th day of air exposure used in the above tissue-staining results were also used, and in order to evaluate the normal functions of the two cells, fibronectin expression and collagen IV expression were compared as representative protein synthesis indicators of fibroblasts. To evaluate the protein expression of keratinocytes, keratin 10 expression was compared.

[0099] FIGS. 8(g), (h) and (i) are images showing the expression of fibronectin, collagen IV, and keratin 10 of the samples cultured under static conditions. In the entire dermal layer, fibronectin and collagen IV appeared to be well expressed in thread form, which is consistent with the results of special staining. It was also confirmed that keratin 10 was well expressed near the stratum corneum on the epidermal cells. Also, FIGS. 8(j), (k) and (1) are images showing the expression of fibronectin, collagen IV, and keratin 10 of the samples cultured under stretching conditions. As such, fibronectin or collagen IV was not expressed in thread form, as in the tissue cultured under static conditions, and appeared in a round ring shape like the use of pig skin collagen, and was stained much less compared to the case of static conditions. In particular, collagen IV was found to be almost absent in the stretching environment, indicating that it was hardly expressed in the results of Masson's trichrome and Sirius staining and was expressed by the existing rat tail collagen. Moreover, the expression level of keratin 10 was confirmed to be very low when compared with the static conditions.

[0100] Result 8: Comparison of Tissue Images of Skin Equivalents Using Rat Tail Collagen Depending on Stretching Time

[0101] The phenomena by which the stratum corneum was thinned and the number of cells decreased over time were confirmed through tissue images. Thus, in order to evaluate changes in protein expression over time, fibronectin and collagen IV of the samples cultured for 3 days, 5 days, and 7 days were compared through H/E staining and immunohistochemical staining. FIGS. 9(a), (b) and (c) show tissue images of the samples cultured for 3 days under stretching conditions, FIGS. 9(d), (e) and (f) show tissue images of the samples cultured for 5 days under stretching conditions, and FIGS. 9(g), (h) and (i) show tissue images of the samples cultured for 7 days under stretching conditions. It was confirmed that the expression of fibronectin appeared as a round ring shape under stretching conditions, and low expression in the 3.sup.rd day samples, higher expression in the 5th day samples, and low expression in the 7th day samples were observed. Moreover, the penetration of keratinocytes on the 7th day of stretching was confirmed through immunohistochemical staining.

[0102] Result 9: Analysis of Protein Expression Genes Derived from Skin Equivalents Using Rat Tail Collagen Cultured Under Static and Stretching Conditions

[0103] In order to quantitatively analyze the aging phenomena observed through tissue analysis, aging-related factors were compared through qPCR. Among them, filaggrin is a protein that is expressed mainly in keratinocytes and is involved in skin protection and moisturization, and tends to decrease upon aging. Laminin 5 is a protein expressed mainly in the basement membrane at the dermis-stratum corneum junction, plays a role in supporting the skin, and decreases upon aging, thereby causing wrinkled skin. Involucrin is involved in skin protection of the stratum corneum and tends to decrease in aged skin [32], and P53 is a gene that repairs mutant cells and induces apoptosis of aged cells or cancer cells, and the expression level of the P53 gene tends to increase in the aged skin. Furthermore, -actin is important to the cytoskeleton and is used as a control in typical experiments, but -actin cannot be used as a suitable control under conditions that cause aging, because there has been reported a decrease in -actin expression upon aging. As shown in FIG. 10(a), -actin expression gradually decreased over time, and in FIGS. 10(b) and (c), the expression of filaggrin decreased drastically at the beginning of stretching and then gradually increased, and the expression of laminin 5 was similar to the static conditions but decreased on the 7.sup.th day. As shown in FIG. 10(d), the expression of involucrin decreased significantly at the beginning of stretching and gradually decreased over time, and as shown in FIG. 10(e), the expression of P53 gradually decreased on the 1st day and the 3.sup.rd day of stretching, and then remarkably increased about 2.5 times on the 7th day compared to the 3.sup.rd day of stretching.

TABLE-US-00001 TABLE1 Forward Reverse 18sRNA 5-GGCGCCCCCTCGAT 5-GCTCGGGCCTGCTTT GCTCTTAG-3 GAACACTCT-3 -actin 5-TTGCTGATCCACAT 5-GGCACCCAGCACAAT CTGCTGGAAG-3 GAAGATCAA-3 Filaggrin 5-GGAGTCACGTGGCA 5-GGTGTCTAAACCCGG GTCCTCACA-3 ATTCACC-3 Involucrin 5-CCGCAAATGAAACA 5-GGATTCCTCATGCTG GCCAACTCC-3 TTCCCAG-3 Laminin5 5-GGAACTTCCGGCAT 5-GGACAGGCACAGCTC ACGGAGA-3 CACATT-3 P53 5-CCGCCCAACAACAC 5-GGCCTGGGCATCCTT CAGCTCCT-3 GAGTTCC-3

INDUSTRIAL APPLICABILITY

[0104] According to the present invention, a stretchable skin-on-a-chip is useful for testing cosmetics, dermatological drugs, and toxic substances because it can simulate a skin state similar to that of living bodies.

Sequence Listing Free Text

[0105] The sequences of the present invention are primers for performing qPCR on aging-related factors.