Epidermal skin model and construction method and application thereof

Abstract

The invention discloses an epidermal skin model and a construction method and application thereof. The construction method comprises the following steps: performing gas-liquid culture on a transwell membrane on a 3D epidermal organ prepared from epidermal stem cells to prepare an epidermal skin model. The epidermis skin model prepared by the construction method can be rapidly amplified to realize large-scale modeling application in a short time, and the epidermis skin model is complete in differentiation, high in integrity, good in barrier function and beneficial to epidermis-related safety detection or efficacy detection of drugs or cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and in particular to an epidermal skin model and a construction method and application thereof. Background Art

[0002] With the continuous advancement and development of modern medical technology, transdermal drug delivery technology is gaining increasing attention in the international pharmaceutical community. Transdermal drug delivery technology allows drugs to enter the human bloodstream through the skin, preventing them from being destroyed in the gastrointestinal tract. It offers advantages such as reduced fluctuations in blood drug concentrations, fewer toxic side effects, convenient administration, and improved patient compliance. However, the skin itself acts as a barrier, allowing only molecules with certain characteristics to penetrate. Therefore, in actual transdermal applications, adjunctive methods are needed to help drugs bypass the stratum corneum barrier, such as chemical permeation enhancers, liposomes, and microneedles. However, in the search for improved transdermal efficacy, continuous evaluation of transdermal absorption is necessary to support our experimental hypotheses.

[0003] The skin is a physiological barrier that drugs must cross in transdermal delivery systems. Studying the transport and absorption behavior of drugs in the skin is an important method for formulating, evaluating, and predicting the effectiveness of transdermal drug delivery systems. Human skin is the best model for studying transdermal drug absorption. Due to the large differences between human skin samples and the difficulty in obtaining samples themselves, animal models are often used as a substitute in experiments. However, traditional animal skin models are expensive, have significant species differences, and consume a large number of animals, which does not meet the requirements of animal protection and welfare. On the contrary, after nearly 30 years of development, in vitro reconstructed skin model technology has become increasingly mature and has shown great potential in replacing human skin as a new transdermal drug delivery experimental model.

[0004] The development and application of reconstructed skin models have greatly expanded over the past decade. They are considered to be excellent substitutes for human skin and have been widely used in skin corrosion, skin toxicity, skin permeation research, and other aspects. Current epidermal models are mainly made by spreading keratinocytes on a suitable membrane surface for air-liquid culture. Under immersion conditions, the proliferating keratinocytes form a multilayer matrix covering the membrane surface. After proliferation is completed, the keratinocytes differentiate under conditions of contact with air, forming a structure similar to the human epidermis. However, epidermal models built with keratinocytes are difficult to achieve the thickness and differentiation state of the human epidermis, and the demand for keratinocytes to establish the model is huge, making it difficult to carry out large-scale modeling in a short period of time. Summary of the Invention

[0005] To address the shortcomings of existing skin models constructed using keratinocytes, such as thickness, poor differentiation, and prolonged construction time, the present invention provides an epidermal skin model, its construction method, and its application. This epidermal skin model, constructed using 3D epidermal organoids, can be rapidly expanded for large-scale modeling applications within a short period of time. Furthermore, the model exhibits complete differentiation, high integrity, and excellent barrier function, facilitating epidermal safety or efficacy testing for pharmaceuticals and cosmetics.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The present invention provides a method for constructing an epidermal skin model, which comprises the following steps:

[0008] The epidermal skin model is prepared by culturing 3D epidermal organoids made from epidermal stem cells in an air-liquid culture on a transwell membrane.

[0009] In some embodiments, the air-liquid culturing step comprises culturing the 3D epidermal organoids in the form of resuspended droplets on a transwell membrane.

[0010] In some embodiments, the air-liquid culturing step comprises the step of performing air-liquid culturing in an epidermal skin model culture medium.

[0011] In some embodiments, the seeding density of the 3D epidermal organoids is determined according to the area of ​​the epidermal skin model, for example, per 1×10 6 3D epidermal organoids (digested into single cells and counted) were cultured with 30 μL epidermal skin model culture medium and fused into a 0.3 cm 2 of epidermal sheets.

[0012] In some embodiments, the epidermal skin model culture medium is based on Advanced DMEM / F12 culture medium and includes at least the following components: N-ace (N-acetylcysteine), EGF, and Wnt3A.

[0013] In some embodiments, the epidermal skin model culture medium further comprises one or more of BSA, B27, Glutamax, Hepes, Forskolin, Y27632, and MMP.

[0014] In some specific embodiments, the epidermal skin model culture medium is based on Advanced DMEM / F12 as the basic culture medium, and further includes BSA 0.1%, B27 1%, Glutamax 1%, Hepes 10mM, N-ace 1mM, A8301 1μM, Forskolin 10μM, EGF 50ng / mL, Wnt3A 100ng / mL and Y27632 10μM, and the percentages are all weight volume percentages; preferably, it also includes MMP14 10ng / mL.

[0015] In some embodiments, the epidermal skin model culture uses epilife as the basic culture medium and also includes Vitamin C and / or CaCl2.

[0016] In some specific embodiments, the epidermal skin model culture uses epilife as the basic culture medium and also includes Vitamin C 50 μg / mL and CaCl2 1.5 mM.

[0017] In some embodiments, the air-liquid culture step is performed in a well plate with a transwell membrane, such as a 24-well plate. The epidermal skin model culture is based on the addition of the lower chamber of the well plate.

[0018] In some embodiments, the temperature of the gas-liquid culture is 37°C.

[0019] In some embodiments, the gas-liquid culture duration is 7 days.

[0020] In some embodiments, the air-liquid culture step involves replacing the culture medium with fresh epidermal organoid culture medium every other day. Preferably, the epidermal skin model culture is based on adding the 3D epidermal organoids on the day after they are seeded onto the transwell membrane, and then replacing the culture medium with fresh epidermal organoid culture medium every other day.

[0021] In some embodiments, the epidermal stem cells are human epidermal stem cells.

[0022] In some embodiments, the preparation of the 3D epidermal organoid comprises the following steps:

[0023] Epidermal stem cells, epidermal organoid culture medium and matrigel are mixed and solidified, and then 3D culture is performed using the epidermal organoid culture medium to prepare the 3D epidermal organoid.

[0024] In some embodiments, the epidermal skin model culture medium is based on Advanced DMEM / F12 culture medium and includes at least the following components: N-ace, EGF, and Wnt3A.

[0025] In some embodiments, the epidermal skin model culture medium further comprises one or more of BSA, B27, Glutamax, Hepes, Forskolin, and Y27632.

[0026] In some specific embodiments, the epidermal skin model culture medium is based on Advanced DMEM / F12 as the basic culture medium and further includes BSA 0.1%, B27 1%, Glutamax 1%, Hepes 10mM, N-ace 1mM, A8301 1μM, Forskolin 10μM, EGF 50ng / mL, Wnt3A 100ng / mL and Y27632 10μM, and the percentages are all mass volume percentages.

[0027] In some embodiments, in the mixing step, the volume of the matrigal accounts for no less than 40%, for example, 50%, based on the total volume of the epidermal organoid culture medium and matrigal.

[0028] In some embodiments, in the mixing step, the seeding volume of the epidermal stem cells is 1×10 4 -5×10 5 / mL, for example 1.25×10 5 / mL.

[0029] In some embodiments, the solidification time is 40 minutes.

[0030] In some embodiments, the 3D culturing step is performed in a well plate, such as a six-well plate.

[0031] In some embodiments, the temperature of the 3D culture is 37°C.

[0032] In some embodiments, the 3D culture duration is 7 days.

[0033] In some embodiments, during the 3D culture step, the epidermal organoid culture medium is replaced with fresh culture medium every other day.

[0034] In some embodiments, after the 3D culture step is completed, the steps of removing liquid, digestion and separation are further included.

[0035] In some embodiments, the digestion step is performed using dispase. Preferably, the amount of dispase used is 1.5 mL and the concentration is 1 U / mL.

[0036] In some embodiments, the temperature of the digestion is 37°C.

[0037] In some embodiments, the digestion time is 1 h.

[0038] In some embodiments, the separation step is centrifugation, for example, centrifugation at 300 g for 5 min, and removing the supernatant. Preferably, the separation step further includes a step of resuspending and washing the precipitate with PBS.

[0039] In some embodiments, the 3D epidermal organoid is a 3D epidermal organoid after primary 3D culture, or a 3D epidermal organoid after passage 3D culture; wherein the number of passages is 1-20 times; and the passage is performed every 7 days.

[0040] In some embodiments, the epidermal stem cells are obtained by the following steps:

[0041] The human skin is cut into pieces, digested, the epidermis is separated, the skin is digested again, filtered, centrifuged, and the precipitate is collected to obtain the product.

[0042] In some embodiments, the digestion step is performed using dispase. Preferably, the amount of dispase used is 5 mL and the concentration is 2.5 U / mL.

[0043] In some embodiments, the temperature of the digestion is 37°C.

[0044] In some embodiments, the digestion time is 2 hours.

[0045] In some embodiments, the secondary digestion step is performed using a digestion solution containing pancreatic enzymes, and the digestion solution does not contain EDTA. Preferably, the amount of the digestion solution is 5 mL and the concentration is 0.25%.

[0046] In some embodiments, the temperature of the secondary digestion is 37°C.

[0047] In some embodiments, the secondary digestion time is 26 minutes.

[0048] In some embodiments, the filtering step is performed using a sieve, such as a stainless steel sieve, and the mesh number of the sieve is preferably 100 mesh.

[0049] In some embodiments, the centrifugation is performed at 300 g for 10 min. Preferably, after the supernatant is removed by centrifugation, the method further comprises the steps of resuspending the solution in PBS and then centrifuging again to remove the supernatant.

[0050] The present invention also provides an epidermal skin model, which is obtained by the construction method as described above.

[0051] In some embodiments, the epidermal skin model includes structures of the stratum corneum (SC), stratum granulosum (GR), stratum spinosum (SP), and stratum basale (SB), wherein each structure is identified by immunofluorescence.

[0052] In some embodiments, the thickness of the epidermal skin model is 50-150 μm. The thickness of the epidermal skin model gradually decreases from the center to the edge, where the thickness refers to the thickness distribution range of the epidermal skin model from the center to the edge, and those skilled in the art will know its specific meaning.

[0053] The present invention also provides a use of the aforementioned epidermal skin model in epidermal-related safety testing or efficacy testing of drugs or cosmetics.

[0054] In some embodiments, the epidermis-related safety tests include skin irritation tests, skin barrier repair tests, moisturizing tests, and anti-inflammatory efficacy tests;

[0055] In some embodiments, the efficacy test includes transdermal absorption test, whitening efficacy test, and sun protection efficacy test.

[0056] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0057] The reagents and raw materials used in the present invention are commercially available.

[0058] The positive progress effect of the present invention is:

[0059] The epidermal skin model constructed using epidermal organoids in the present invention has the following effects:

[0060] 1. The epidermal skin model is approximately 100μm thick, similar to the human epidermis. It also displays the well-differentiated structures of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. The stratum corneum is firm and provides a good barrier effect, making it suitable for epidermal safety or efficacy testing of pharmaceuticals and cosmetics, such as transdermal drug absorption evaluation.

[0061] 2. The epidermal skin model uses human epidermal organoids, which can be passaged indefinitely, avoiding the problem of keratinocyte aging during passage, and can achieve rapid expansion of about 20-30 times each time. At the same time, the epidermal organoid plate can mature and be used after one week of culture, which means that the organoid epidermal skin model can be modeled and applied in large quantities in a short period of time.

[0062] 3. The epidermal skin model uses human epidermal organoids, which can be directly derived from patients, ensuring specificity while avoiding differences due to different species. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is an electron micrograph of the epidermal organoid constructed in Example 1 of the present invention.

[0064] Figure 2 This is a HE section image of the epidermal organoid constructed in Example 1 of the present invention.

[0065] Figure 3 This is a HE slice image of the epidermal skin model constructed in Example 2 of the present invention.

[0066] Figure 4 This is the fusion result of epidermal organoids when constructing the epidermal skin model in Example 2 of the present invention.

[0067] Figure 5 This is the differentiation status identification result of the epidermal skin model constructed in Example 2 of the present invention.

[0068] Figure 6 This is the result of the changes in the basal layer when constructing the epidermal skin model in Example 2 of the present invention.

[0069] Figure 7 This is a HE slice image of the epidermal skin model constructed in Example 3 of the present invention.

[0070] Figure 8 This is a HE slice image of the epidermal skin model constructed in Example 4 of the present invention.

[0071] Figure 9 This is a standard curve diagram of the caffeine standard after gradient dilution in Example 1 of the present invention.

[0072] Figure 10 This is a curve diagram of the cumulative transdermal amount of caffeine in the epidermal skin model according to Example 1 of the present invention. DETAILED DESCRIPTION

[0073] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0074] Example 1

[0075] This example provides a method for rapid proliferation of human epidermal organoids by passage, using the following steps:

[0076] Step 1: Establishment of epidermal organoids

[0077] (1) Preparation of organoid culture medium: In Advanced DMEM / F12 basal culture medium (Gibco lot: 2660006), add BSA 0.1%, B27 1%, Glutamax 1%, Hepes 10mM, N-ace 1mM, A8301 1μM, Forskolin 10μM, EGF 50ng / mL, Wnt3A 100ng / mL, and Y27632 10μM to complete the preparation of human epidermal organoid culture medium. The percentages are all weight-volume percentages.

[0078] (2) Organoid plate culture: take 4×10 4 After digestion, epidermal stem cells were centrifuged at 300 g for 10 min and the excess liquid was discarded. Then, 150 μL of epidermal organoid culture medium and 150 μL of matrigel were added to the epidermal stem cell pellet and the epidermal organoid pellet was thoroughly mixed. The cell mixture was seeded into one well of a six-well plate (i.e., 300 μL per well) using an ice-cold pipette tip at a volume of 50 μL per droplet. After placing it in an incubator to solidify for 40 min, 800 μL of epidermal organoid culture medium was added and the medium was changed every other day. The epidermal organoids can be mature and used after 7 days.

[0079] Step 2: Passaging Epidermal Organoids

[0080] Epidermal organoids can be passaged on the seventh day of growth. First, discard the excess culture medium, then add 3 mL of Advanced DMEM / F12 to each well and thoroughly pipette. Then, collect the organoid culture medium mixture in a 15 mL centrifuge tube, centrifuge at 300 g for 5 minutes, discard the upper layer of culture medium with a pipette, leave the organoid, add 3 mL of TrypLE and mix thoroughly, then place in the incubator for digestion for 12 minutes. After removing the organoid and thoroughly pipetting into single cells, add 3.5 mL of 10% DMEM to stop digestion, and count the organoids (the number of organoids at this time is generally 1×10 6 , that is, each passage can achieve 25-fold expansion of organoids) and then repeat the above organoid plate culture process.

[0081] like Figure 1-2 The following are electron micrographs and HE sections of the epidermal organoids constructed in this example at the seventh day of culture in the first generation:

[0082] Hematoxylin and eosin (H&E) staining results showed the morphology of epidermal organoids (mPEOs), with large, flat basal-like cells and hardened keratinized material in the center of the epidermal organoid structure.

[0083] Transmission electron microscopy (TEM) results further confirmed the multilayered phenotype of epidermal organoids. Their structure includes the basal layer (SB), a layer of short, columnar basal cells; the spinous layer (SP), 4–10 layers of polygonal, larger spinous cells with numerous oval, membrane-covered lamellar granules; the granular layer (GR), 3–5 layers of flat, spindle-shaped cells with numerous transparent keratin granules (KG) in the cytoplasm; and the stratum corneum (SC), multiple layers of flat, fully cornified keratinocytes without nuclei or cytoplasm. The SB is located on the exterior of the epidermal organoids, while the SP, GR, and SC are distributed in the center of the epidermal organoids.

[0084] The passage test showed that the epidermal organoids of the present invention can be passaged at least 20 times, and the proliferation state does not weaken after each passage. The number of cells was counted based on the microscopic photos taken at 7 days after each passage. According to the six-well plate (300 μL) per well, the number of cells that can be grown from 4×10 4 1.3×10 6 That is, each passage can achieve a large-scale expansion effect. The inventors' research has shown that the biological properties of epidermal organoids after passage are consistent with those of first-generation epidermal organoids. The epidermal skin model of the present invention can be constructed based on first-generation epidermal organoids or epidermal organoids after multiple passages, with no significant differences in performance parameters.

[0085] Example 2

[0086] This example provides a method for constructing a novel in vitro reconstructed epidermal skin model using human epidermal organoids, using the following steps:

[0087] Step 1: Preparation of epidermal organoid and epidermal skin model culture medium (same formula)

[0088] To Advanced DMEM / F12 basal culture medium, add BSA 0.1%, B27 1%, Glutamax 1%, Hepes 10mM, N-ace 1mM, A8301 1μM, Forskolin 10μM, EGF 50ng / mL, Wnt3A 100ng / mL, and Y27632 10μM to complete the preparation of the culture medium.

[0089] Step 2: Epidermal stem cell isolation

[0090] The human skin was placed in a culture dish, soaked in 75% alcohol for 3 minutes, and quickly washed in a culture dish containing PBS. The skin tissue was cut into 0.2cm×0.5cm long strips with scissors, and then washed twice with PBS. 5mL2.5U / mL dispase digestion solution was added. After digestion in a 37°C incubator for 2h, the dermis and epidermis were mechanically separated with tweezers. The epidermal tissue was then collected in a new culture dish and cut into pieces. 5mL 0.25% trypsin digestion solution (without EDTA) was added to the shredded epidermis. After digestion at 37°C for 26min, the digested epidermal single cell solution was filtered out with a 100-mesh stainless steel sieve to remove the fragments. The collected solution was centrifuged at 300g for 10 minutes in a test tube. The supernatant was removed, and the lower cells were resuspended with 10mL PBS and counted. 4×10 4 The cells were centrifuged at 300 g for 10 minutes in a new test tube, and the supernatant was discarded to obtain a cell pellet containing a large number of epidermal stem cells.

[0091] Step 3: Establishment of epidermal organoids

[0092] Add 150 μL of epidermal organoid culture medium and 150 μL of matrigel to the cell pellet, mix and resuspend the cell pellet, and use an ice-cold pipette tip to plant the cell mixture into one well of a six-well plate at a volume of 50 μL per droplet (i.e., 300 μL per well). Place in an incubator to solidify for 40 minutes, then add 800 μL of epidermal organoid culture medium and change the medium every other day. The epidermal organoids can be mature and used after 7 days.

[0093] Step 4: Epidermal skin model establishment

[0094] After the epidermal organoids matured, the epidermal organoid culture medium was discarded, and 1.5 mL of 1 U / mL dispase was added to the 6-well plate in which the epidermal organoids were cultured. The cells were digested at 37°C for 1 h. The digested epidermal organoids were collected into a 15 mL centrifuge tube and centrifuged at 300 g for 5 min. The epidermal organoids were then resuspended and washed with 10 mL of PBS and centrifuged at 300 g for 5 min. The supernatant was discarded and 1 × 10 6 The density of epidermal organoids was set, and droplets of the mixed epidermal organoids were spotted on the transwell membrane of a 24-well plate. On the next day, 300 μL of epidermal skin model culture medium was added to the lower chamber. The medium was changed every other day. After two days, the epidermal sheets were fused and matured for application.

[0095] like Figure 3 The figure shows a HE slice of the epidermal skin model constructed in this example. As can be seen from the figure, the side close to the liquid is composed of dense, short columnar cells, with granular cells in the middle becoming increasingly sparse and larger in shape, and the side close to the gas is composed of a layer of anucleated keratinocytes.

[0096] like Figure 4 Figure 1 shows the fusion results of epidermal organoids used in the epidermal skin model constructed in this example. Microscopic images taken at 0 hours, 5 hours, 10 hours, 15 hours, 3 days, and 7 days show that during air-liquid culture in high-density droplets, the epidermal organoids initially formed independent spheres, then gradually separated and merged into sheets.

[0097] like Figure 5 The following figure shows the differentiation status of the epidermal skin model constructed in this embodiment. In the figure, K14 is the basal layer and K10 is the spinous layer. It can be seen that the model has an excellent differentiation state, just like the human epidermis.

[0098] like Figure 6 The figure shows the changes in the basal layer during the construction of the epidermal skin model in this embodiment. As can be seen from the figure, during the 7-day fusion process, the basal layer gradually transitioned from a spherical shape to a flat sheet.

[0099] Example 3

[0100] This example provides a method for constructing a novel in vitro reconstructed epidermal skin model using human epidermal organoids, using the following steps:

[0101] Step 1: Preparation of epidermal organoid and epidermal skin model culture medium:

[0102] In Advanced DMEM / F12 basal culture medium, add BSA 0.1%, B27 1%, Glutamax 1%, Hepes 10mM, N-ace 1mM, A8301 1μM, Forskolin 10μM, EGF 50ng / mL, Wnt3A 100ng / mL, and Y27632 10μM respectively to complete the preparation of human epidermal organoid culture medium.

[0103] To the epilife basal culture medium (Gibco lot: MEPI500CA), add 50 μg / mL of Vitamin C and 1.5 mM of CaCl2 to complete the preparation of the epidermal skin model culture medium.

[0104] Step 2: Epidermal stem cell isolation

[0105] The human skin was placed in a culture dish, soaked in 75% alcohol for 3 minutes, and quickly washed in a culture dish containing PBS. The skin tissue was cut into 0.2cm×0.5cm long strips with scissors, and then washed twice with PBS. 5mL2.5U / mL dispase digestion solution was added. After digestion in a 37°C incubator for 2h, the dermis and epidermis were mechanically separated with tweezers. The epidermal tissue was then collected in a new culture dish and cut into pieces. 5mL 0.25% trypsin digestion solution (without EDTA) was added to the shredded epidermis. After digestion at 37°C for 26min, the digested epidermal single cell solution was filtered out of the fragments with a 100-mesh stainless steel sieve. The collected solution was centrifuged at 300g for 10 minutes in a test tube. The supernatant was removed, and the lower cells were resuspended with 10mL PBS and counted. 4×10 4 The cells were centrifuged at 300 g for 10 minutes in a new test tube, and the supernatant was discarded to obtain a cell pellet containing a large number of epidermal stem cells.

[0106] Step 3: Establishment of epidermal organoids

[0107] Add 150 μL of epidermal organoid culture medium and 150 μL of matrigel to the cell pellet, mix and resuspend the cell pellet, and use an ice-cold pipette tip to plant the cell mixture into one well of a six-well plate at a volume of 50 μL per droplet (i.e., 300 μL per well). Place in an incubator to solidify for 40 minutes, then add 800 μL of epidermal organoid culture medium and change the medium every other day. The epidermal organoids can be mature and used after 7 days.

[0108] Step 4: Epidermal skin model establishment

[0109] After the epidermal organoids matured, the epidermal organoid culture medium was discarded, and 1.5 mL of 1 U / mL dispase was added to the 6-well plate in which the epidermal organoids were cultured. The cells were digested at 37°C for 1 h. The digested epidermal organoids were collected into a 15 mL centrifuge tube and centrifuged at 300 g for 5 min. The epidermal organoids were then resuspended and washed with 10 mL of PBS and centrifuged at 300 g for 5 min. The supernatant was discarded and 1 × 10 6 The density of epidermal organoids was set, and droplets of the mixed epidermal organoids were spotted on the transwell membrane of a 24-well plate. On the next day, 300 μL of epidermal skin model culture medium was added to the lower chamber. The medium was changed every other day. After two days, the epidermal sheets were fused and matured for application.

[0110] like Figure 7 The figure shows a HE slice of the epidermal skin model constructed in this example. As can be seen from the figure, the side close to the liquid is composed of dense, short columnar cells, with granular cells in the middle gradually becoming sparse, large, and flat. The side close to the gas is composed of a layer of anucleated keratinocytes.

[0111] Example 4

[0112] This example provides a method for constructing a novel in vitro reconstructed epidermal model using human epidermal organoids, using the following steps:

[0113] Step 1: Preparation of epidermal organoid and epidermal skin model culture medium

[0114] In Advanced DMEM / F12 basal culture medium, add BSA 0.1%, B27 1%, Glutamax 1%, Hepes 10mM, N-ace 1mM, A8301 1μM, Forskolin 10μM, EGF 50ng / mL, Wnt3A 100ng / mL, and Y27632 10μM respectively to complete the preparation of human epidermal organoid culture medium.

[0115] Add MMP14 at a concentration of 10 ng / mL to the epidermal organoid culture medium to complete the preparation of the epidermal model culture medium.

[0116] Step 2: Epidermal stem cell isolation

[0117] The human skin was placed in a culture dish, soaked in 75% alcohol for 3 minutes, and quickly washed in a culture dish containing PBS. The skin tissue was cut into 0.2cm×0.5cm long strips with scissors, and then washed twice with PBS. 5mL2.5U / mL dispase digestion solution was added. After digestion in a 37°C incubator for 2h, the dermis and epidermis were mechanically separated with tweezers. The epidermal tissue was then collected in a new culture dish and cut into pieces. 5mL 0.25% trypsin digestion solution (without EDTA) was added to the shredded epidermis. After digestion at 37°C for 26min, the digested epidermal single cell solution was filtered out of the fragments with a 100-mesh stainless steel sieve. The collected solution was centrifuged at 300g for 10 minutes in a test tube. The supernatant was removed, and the lower cells were resuspended with 10mL PBS and counted. 4×10 4 The cells were centrifuged at 300 g for 10 minutes in a new test tube, and the supernatant was discarded to obtain a cell pellet containing a large number of epidermal stem cells.

[0118] Step 3: Establishment of epidermal organoids

[0119] Add 150 μL of epidermal organoid culture medium and 150 μL of matrigel to the cell pellet, mix and resuspend the cell pellet, and use an ice-cold pipette tip to plant the cell mixture into one well of a six-well plate at a volume of 50 μL per droplet (i.e., 300 μL per well). Place in an incubator to solidify for 40 minutes, then add 800 μL of epidermal organoid culture medium and change the medium every other day. The epidermal organoids can be mature and used after 7 days.

[0120] Step 4: Epidermal skin model establishment

[0121] After the epidermal organoids matured, the epidermal organoid culture medium was discarded, and 1.5 mL of 1 U / mL dispase was added to the 6-well plate in which the epidermal organoids were cultured. The cells were digested at 37°C for 1 h. The digested epidermal organoids were collected in a 15 mL centrifuge tube and centrifuged at 300 g for 5 min. The epidermal organoids were then resuspended and washed with 10 mL of PBS and centrifuged at 300 g for 5 min. The supernatant was discarded and 1 × 10 6 The density of epidermal organoids was set, and droplets of the mixed epidermal organoids were spotted on the transwell membrane of a 24-well plate. On the next day, 300 μL of epidermal skin model culture medium was added to the lower chamber. The medium was changed every other day. After two days, the epidermal sheets were fused and matured for application.

[0122] like Figure 8 Figure 2 shows an HE slice of the epidermal skin model constructed in this example. As can be seen, the side near the liquid is composed of dense, short columnar cells, with increasingly sparse, large, and flat granular cells in the middle. The gas-side is composed of a single layer of anucleated keratinized cells, demonstrating excellent stratum corneum thickness.

[0123] Effect Example 1

[0124] 1. Based on the reconstructed epidermal skin models prepared in Examples 2, 3, and 4, a caffeine transdermal experiment was conducted using a transwell and quantitative loop device, using the following steps:

[0125] (1) Preparation of transdermal test substances: Prepare a caffeine propylene glycol solution with a caffeine concentration of 1.94 mg / mL as test substance 1, take 50 μL of test substance 1 and add 1 μg of oleic acid powder as test substance 2, and at the same time take a certain amount of test substance 1 for serial dilution to obtain a series of standard curve samples with concentrations of 2, 8, 20, 40, 200, and 400 μg / mL, respectively.

[0126] (2) Two reconstructed epidermal skin models that meet the test criteria in Example 2 (the epidermal skin models in Examples 3 and 4 can be verified by referring to the same steps) were placed in a 6-well plate, numbered ①② respectively, and installed with a silicone quantitative ring with an inner diameter of 7 mm, an outer diameter of 24 mm, and a thickness of 1 mm.

[0127] (3) Add 2.5 mL of normal saline as the receiving solution to the lower chambers of the epidermal skin model ① and ②, respectively. Be careful to add it slowly along the edge of the well plate to prevent the generation of bubbles. Slowly add 26 μL of test substance 1 to the upper chamber of the epidermal skin model ①, and slowly add 26 μL of test substance 2 to the upper chamber of the epidermal skin model ②.

[0128] (4) Place the experimental system in a cell culture incubator and use a shaker to allow the test substance to fully penetrate the skin.

[0129] (5) 1 mL of saline solution was taken out from the lower chamber at 0.5 h, 1.5 h, 3 h, 5 h, 10 h, 15 h, and 24 h after adding the test sample for caffeine content determination, and the missing saline solution in the lower chamber was replenished in time to maintain system balance.

[0130] (6) After the 24-hour experiment, the quantitative loop, culture chamber, and epidermal model surface were thoroughly rinsed with 5 mL of normal saline, and samples were collected for calculation of the content of samples that did not penetrate the skin. At the same time, the epidermal tissue sample and transwell membrane were removed and placed in 3 mL of methanol solution. After mixing, they were placed in a tissue homogenizer and ground at a frequency of 60 Hz for 30 seconds. This was repeated three times, and then the sample was removed and mixed. Finally, the caffeine sample was fully extracted by ultrasonication for 10 minutes. After filtering through a 100-mesh sieve, it was used for high-performance liquid chromatography content determination.

[0131] 2. Transdermal absorption test sample determination and result processing

[0132] The sample HPLC detection conditions are shown in Table 1:

[0133] Table 1. HPLC detection conditions of samples

[0134]

[0135] Preparation of standard curve for sample standards:

[0136] The corresponding concentration (C, X) was weighted by the peak area (As, Y) of caffeine (1 / X 2 ) linear regression to obtain the standard curve of caffeine solution. The results are as follows Figure 9 shown.

[0137] The calculation formula for the cumulative transdermal dose (Q) of the sample is as follows:

[0138] Q=[Cn×V+∑Ci×V0]

[0139] (Where Q is the cumulative permeation volume; V is the volume of the receiving fluid in the receiving chamber; V0 is the volume of each sampling; Ci is the drug concentration in the receiving fluid from the first sampling to the last sampling; Cn is the drug concentration in the receiving fluid at the time of the current sampling.)

[0140] The cumulative permeation of caffeine samples in the two epidermal skin models over time was determined as follows: Figure 10 As shown in Table 2:

[0141] Table 2. Cumulative caffeine permeation in the epidermal skin model in the caffeine permeation test

[0142] 0.5h 1.5h 3h 5h 10h 15h 24h Model ① 3.85 μg 14.32 μg 27.60 μg 39.38 μg 48.57 μg 50.79 μg 49.44 μg Model ② 6.65 μg 23.44 μg 38.10 μg 51.82 μg 50.64 μg 52.01 μg 52.87 μg

[0143] The calculation formula for skin absorption rate and caffeine recovery rate is as follows:

[0144] Skin absorption rate = Q + N / sample amount × 100%

[0145] Caffeine recovery rate = Q + N + W / sample amount × 100%

[0146] (Where Q is the cumulative total amount of permeation, N is the total amount of caffeine inside the epidermis and on the surface of the transwell, and W is the total amount of caffeine that did not permeate the quantitative loop and the upper part of the epidermal model)

[0147] The calculated absorption rate and recovery rate are shown in Table 3:

[0148] Table 3. Skin absorption rate and caffeine recovery rate in the epidermal skin model in the caffeine transdermal test

[0149] Skin model number Skin absorption rate (%) Caffeine recovery rate (%) ① 98.14 99.18 ② 104.97 106.44

[0150] In summary, the caffeine transdermal test was conducted using oleic acid, a typical transdermal enhancer in pharmaceutical preparations. The results showed that the addition of oleic acid significantly promoted caffeine transdermal penetration, indicating that the epidermal model of the present invention can replace human skin to a certain extent.

[0151] In addition, compared with real skin, the permeability of the epidermal skin model is much higher than that of the real epidermis. For substances and drugs that cannot penetrate or are difficult to penetrate the human epidermis, the epidermal skin model can be used to find linear relationships and then adjust the formulation of the preparation.

Claims

1. A method for constructing an epidermal skin model, characterized in that: It includes the following steps: The epidermal skin model is prepared by culturing 3D epidermal organoids made from epidermal stem cells in an air-liquid culture on a transwell membrane.

2. The construction method according to claim 1, wherein The air-liquid culture step comprises culturing the 3D epidermal organoids in the form of resuspended droplets on a transwell membrane in an air-liquid culture; And / or, the air-liquid culture step includes the step of performing air-liquid culture using an epidermal skin model culture medium; Preferably, the seeding density of the 3D epidermal organoids is 1×10 6 Each 3D epidermal organoid was cultured with 30 μL of the epidermal skin model culture medium; And / or, the epidermal skin model culture medium is based on Advanced DMEM / F12 culture medium, and at least includes the following components: N-ace, EGF and Wnt3A; preferably, the epidermal skin model culture medium further includes one or more of BSA, B27, Glutamax, Hepes, Forskolin, Y27632 and MMP; Alternatively, the epidermal skin model culture uses epilife as the basic culture medium and also includes Vitamin C and / or CaCl2; More preferably, in the air-liquid culture step, the culture medium is replaced with fresh epidermal organoid culture medium every other day; Further preferably, the epidermal skin model culture is based on adding the 3D epidermal organoids on the second day after they are seeded on the transwell membrane, and then the culture medium is replaced with fresh epidermal organoid culture medium every other day.

3. The construction method according to claim 2, wherein: The epidermal skin model culture medium is based on Advanced DMEM / F12 culture medium, and further includes BSA 0.1%, B27 1%, Glutamax 1%, Hepes 10mM, N-ace1mM, A8301 1μM, Forskolin 10μM, EGF 50ng / mL, Wnt3A 100ng / mL and Y27632 10μM, wherein the percentages are all weight volume percentages; preferably, it further includes MMP14 10ng / mL; Alternatively, the epidermal skin model culture uses epilife as the basic culture medium and also includes Vitamin C 50 μg / mL and CaCl2 1.5 mM.

4. The construction method according to claim 1, wherein The preparation of the 3D epidermal organoids comprises the following steps: Epidermal stem cells, epidermal organoid culture medium and matrigal are mixed and solidified, and then 3D culture is performed using the epidermal organoid culture medium to prepare the 3D epidermal organoid.

5. The construction method according to claim 4, wherein: The epidermal skin model culture medium is based on Advanced DMEM / F12 culture medium and includes at least the following components: N-ace, EGF and Wnt3A; Preferably, the epidermal skin model culture medium further comprises one or more of BSA, B27, Glutamax, Hepes, Forskolin and Y27632; More preferably, the epidermal skin model culture medium is based on Advanced DMEM / F12 as the basic culture medium, and further includes BSA 0.1%, B27 1%, Glutamax 1%, Hepes 10mM, N-ace 1mM, A8301 1μM, Forskolin 10μM, EGF 50ng / mL, Wnt3A 100ng / mL and Y2763210μM, where the % are all mass volume percentages.

6. The construction method according to claim 4, wherein: In the mixing step, the volume of the matrigal accounts for no less than 40% of the total volume of the epidermal organoid culture medium and matrigal; And / or, in the mixing step, the inoculation amount of the epidermal stem cells is 1×10 4 -5×10 5 / mL; and / or, in the 3D culture step, the culture medium is replaced with fresh epidermal organoid culture medium every other day; And / or, after the 3D culture step is completed, the steps of removing liquid, digesting and separating are also included; And / or, the 3D epidermal organoid is a 3D epidermal organoid after primary 3D culture, or a 3D epidermal organoid after passage 3D culture.

7. An epidermal skin model, characterized in that: The method is obtained by the construction method according to any one of claims 1 to 6.

8. The epidermal skin model according to claim 7, wherein The epidermal skin model includes the structures of stratum corneum, stratum granulosum, stratum spinosum and stratum basale; And / or, the thickness of the epidermal skin model is 50-150 μm.

9. Use of the epidermal skin model according to claim 7 or 8 in epidermal-related safety testing or efficacy testing of drugs or cosmetics.

10. The use according to claim 9, characterized in that The epidermis-related safety tests include skin irritation test, skin barrier repair test, moisturizing test and anti-inflammatory efficacy test; And / or, the efficacy test includes transdermal absorption test, whitening efficacy test and sun protection efficacy test.

Images