CN109722410B

CN109722410B - 3D full-layer skin model, culture medium for forming same and preparation method

Info

Publication number: CN109722410B
Application number: CN201711023350.XA
Authority: CN
Inventors: 宋肖洁; 周春霞; 史晓婷; 吴越
Original assignee: Jala Group Co
Current assignee: Shanghai Natural Hall Group Co ltd
Priority date: 2017-10-27
Filing date: 2017-10-27
Publication date: 2024-01-05
Anticipated expiration: 2037-10-27
Also published as: CN109722410A

Abstract

The invention discloses a 3D full-layer skin model, a culture medium for forming the same and a preparation method. The culture medium comprises a protein preparation, wherein the protein preparation comprises 0.1-99% of albumin, 0.1-99% of globulin and 0.1-99% of fibronectin, and the percentages are mass percentages. The air-liquid phase process for preparing the 3D full-layer skin model by combining the culture medium with the pig type I collagen does not need serum, and the production cost is reduced; the prepared 3D full-layer skin model is complete, the activity and the stability are both obviously improved, the application field of the model is expanded, and the large-scale industrialized production of the skin model is realized.

Description

3D full-layer skin model, culture medium for forming same and preparation method

Technical Field

The present invention relates to the field of skin tissue engineering. In particular to a 3D full-layer skin model, a culture medium for forming the same and a preparation method.

Background

The skin is the largest organ of the human body, is an important barrier between the external environment and the internal organs, can prevent the loss of body moisture, environmental influence, injury or infection, and the like, and is helpful for regulating the body temperature. While the skin barrier is generally self-healing due to its exposure to the outside world, which is susceptible to mechanical, thermal or chemical damage, many factors such as infection, oxidation or excessive wound area can render the skin barrier difficult to repair. In clinical treatment, the inoculation of skin is usually selected, but the inoculation is limited by available sites of healthy donors, such as limited available site area, so that a plurality of skin substitutes, such as artificial synthetic membranes, allogenic skin substitutes and the like, are developed, but the survival time of the skin substitutes at the grafting point is limited, and only a short-time physiological wound surface closure can be provided, so that the application of the skin substitutes is greatly limited. With respect to these limitations, human skin models have been studied and developed in recent years using skin engineering, and are widely used in the fields of research of wound healing, skin cancer, skin biology, and the like. Because of its barrier function, the skin model is an important component for performing relevant tests in place of skin. With the european union regulations no longer permitting the sale of consumer products for animal experiments, it has become a trend to use skin tissue engineering to build skin models to simulate natural skin, replacing animals with related safety tests. In addition, skin models are widely used in other different industries, such as pharmaceutical, where skin models are commonly used to study the mechanism of action and pathogenesis of drugs. Can be used in the cosmetic field to screen actives or evaluate the efficacy and safety of cosmetic materials. Based on this, there is a need to develop artificial skin having a structure simulating a natural skin layer.

Examples of representative methods developed so far for preparing an artificially cultured skin model include a method of preparing a reconstituted epidermis on an epidermis-removed dermis, in which human keratinocytes are three-dimensionally cultured on the epidermis-removed dermis. Specifically, the method comprises (1) culturing fibroblast and epidermal cell (human keratinocyte) respectively; (2) culturing the fibroblast to construct a dermis layer; (3) constructing an epidermis layer using human keratinocytes; (4) The culture of the whole layer of skin is usually gas-liquid culture (CN 104726396A). Among these, the construction of the dermis layer, which is the basis for keratinocyte growth, is fundamentally based on the choice of materials.

Currently, high molecular synthetic materials such as pure high molecular polylactic acid (PLA), polyglycolic acid (PGA), polyurethane and the like or natural materials are used in the construction of the dermis layer. Since natural materials are originally truly present in nature, their composition and structure more closely resemble real skin tissue, and are therefore more widely used in skin model construction. In terms of the use of natural raw materials, there are two main techniques: the first is to apply the technology of mechanics and bioengineering to crosslink biological materials such as chitosan, collagen, chondroitin sulfate, etc. to form porous materials for use as dermis substitutes. Glutaraldehyde crosslinking with human type I, type III collagen, 6-chondroitin sulfate to form a dermal scaffold, such as university of medical science, wang Xu, et al, chinese patent CN1310027A (crosslinking with human type I, type III collagen, 6-chondroitin sulfate). However, this type of technology has the following drawbacks: (1) The stability is poor, the controllability of the finished product is poor due to unavoidable batch variability of natural raw materials, and the pore diameters of the formed porous raw materials are large in variability; (2) The preparation technology has high requirements, the synthesis technology is complex, and the large-scale production cannot be realized. (3) the cost is high. The second is to construct the dermis layer directly using collagen. The earliest development in China is that Wu Jinjin and the like are prepared into cell gel by using rat tail collagen and Fb, and KC is inoculated on the cell gel; artificial Skin (Active Skin) using bovine type I collagen gel, university of army medical science Jin Yan, etc.; meanwhile, patent WO9116 and 010 also discloses that a high-purity nonporous collagen layer is used, wherein the collagen is type I bovine collagen and type III bovine collagen; patent CN106573087a uses native or atelopeptide murine collagen and some studies use human type I, type III collagen. However, the use of bovine, human, murine collagen suffers from the following drawbacks: (1) Human collagen is closest to human skin but of limited origin. (2) mouse collagen, the yield is low and mass production is not easy. (3) Bovine collagen is relatively widely available, but has the potential to transmit viruses. Therefore, collagen with easy material availability, low cost and strong quality control is selected to have great significance for the preparation of skin models.

After the skin model including the dermis layer and the epidermis layer is prepared, 3D full-thickness skin can be obtained by subjecting it to a gas-liquid phase culture method. 3D full-thickness skin is a three-dimensional skin model that contains both epidermis and dermis layers. The selection of a culture system is involved in the gas-liquid phase culture process, and the culture system also affects the stability of the prepared 3D full-layer skin model and the stability among batches. At present, the culture systems of skin models are mainly divided into two types: serum-free culture system and serum-containing culture. The serum-free cell culture medium has relatively clear composition, simple preparation process, capability of avoiding quality variation among serum batches, improvement of repeatability of cell culture and experimental results and contribution to purification of cell products. However, the whole process of serum-free culture has higher cost, and the preservation and application of the culture medium are not convenient as those of the traditional culture medium, such as patent CN105602890A, which uses embryonic stem cell culture solution, and uses Bovine Pituitary Extract (BPE) as a component for replacing serum, and the components are complex. Like serum, BPE presents a lot-to-lot quality variation, and the risk of spreading viruses, and is expensive. On the other hand, in the skin model in the case of serum-free culture, the epidermis is thin, the subsequent continuous culture time is short, and the differentiation failure of the epidermis and the overall morphology are likely to be poor. The whole state of the skin model can be better by using serum for culture, but the skin model is influenced by different fluctuation of serum batches, so that the fluctuation of the quality of the skin model is larger, and the industrial mass production is not facilitated; in addition, the serum components are complex, which is not beneficial to subsequent detection and application. The culture of the skin model is in the final stage of obtaining the full-scale skin model, and the searching of a culture medium which is beneficial to the culture of the skin model is important.

Disclosure of Invention

The invention aims to overcome the defects that the controllability of a skin model provided by the prior art is poor, the stability of a construction method is poor, the cost is high, the synthesis technology is complex, the industrial production is not facilitated, the collagen degradation speed in the construction process is high, and the like, and provides a 3D full-layer skin model, a culture medium for forming the same and a preparation method. The skin model has high stability, long in-vitro survival period and wide application field, and the preparation method has low cost and simple process and is suitable for large-scale production.

The culture medium is used for forming a gas-liquid phase culture stage in the construction process of the 3D full-layer skin model, and is different from the existing serum-free culture system, and the serum-free culture medium is characterized in that a protein combination preparation is used for replacing serum, and the combination comprises albumin, globulin and fibronectin. The three proteins are widely available, wherein albumin, globulin and fibronectin are albumin, globulin and fibronectin in the conventional sense. The invention well avoids quality fluctuation among batches caused by serum or BPE by utilizing the combination of high-purity protein preparations, and the protein preparation with clear components can lead a skin model to be well grown and differentiated, and improves the controllability of the construction process, thereby realizing the large-scale industrialized production of the skin model.

The invention provides a serum-free culture medium for forming a 3D full-layer skin model, which comprises a protein preparation, wherein the protein preparation comprises 0.1-99% of albumin, 0.1-99% of globulin and 0.1-99% of fibronectin, and the percentages are in mass percent.

Wherein the culture medium is preferably a culture medium for gas-liquid phase interface culture of the 3D full-layer skin model; the protein formulation may be conventional in the art, preferably of animal or human origin, more preferably of human origin.

Preferably, the culture medium further comprises a basic culture solution, and the basic culture solution is preferably mixed culture solution of F12 and DMEM in a mass ratio of 1:2.

Preferably, the medium further comprises hydrocortisone or hydrocortisone hydrochloride, and/or insulin in order to obtain better proliferation and differentiation of the epidermal part of the skin model.

In a preferred embodiment of the invention, the medium comprises the following components: 60-69% of DMEM, 30-33% of F12, 1-10% of protein preparation, 0.2-0.6 mu g/ml of hydrocortisone and 1-50 mu g/ml of insulin, wherein the protein preparation comprises 30-80% of albumin, 10-50% of globulin and 5-20% of fibronectin; more preferably, the protein preparation is 10% in content, the protein preparation is composed of 60% albumin, 30% globulin and 10% fibronectin, the concentration of hydrocortisone is 0.5 μg/ml, and the concentration of insulin is 8 μg/ml; the percentages are mass percentages. Wherein, the protein preparation is unfavorable for the growth and proliferation of cells when the percentage content of the protein preparation in the culture medium is lower than 1%; whereas above 10%, cell growth is inhibited.

The invention provides a preparation method of a 3D full-layer skin model, which comprises the step of culturing the skin model comprising a dermis layer and an epidermis layer by a gas-liquid phase interface, wherein a culture medium IV of the gas-liquid phase interface culture is the culture medium containing a protein preparation. The gas-liquid phase interface culture can be conventional in the art, and is preferably performed on the surface of a culture support; the gas-liquid phase interface is cultured for 7 days, and liquid is changed every 2 days until a full-layer skin model is obtained.

In the invention, the dermis layer is prepared by mixing single-cell fibroblasts with porcine type I collagen, culturing for 2-3 hours until solidification, and then adding a culture medium I for culturing; preferably: the density of the pig type I collagen is 2-5 mg/ml, more preferably 3.5mg/ml; when the collagen density is lower than 2mg/ml, the three-dimensional structure required by the model cannot be well formed and maintained; when the density is higher than 5mg/ml, collagen is wasted on the one hand and the growth of fibroblasts in collagen after mixing is also not favored on the other hand.

The medium I may be conventional in the art, preferably comprises 90% DMEM and 10% FBS; more preferably, vitamin C can be added; the percentages are by volume.

The density of the mixed fibroblasts is preferably (0.5 to 3). Times.10 ⁶ The fibroblast density within this range is most suitable for constructing the dermis portion of the skin model, preferably 1.7X10 ⁶ Individual/ml; when the density is lower than 0.5X10 ⁶ When the cell density is too low, it may cause the cells to stop growing and die after mixing, which is detrimental to the living dermis required for the model; when the density is higher than 3×10 ⁶ When collagen degradation is accelerated.

The conditions for culturing the fibroblasts after mixing with porcine type I collagen may be conventional in the art, preferably 5% CO ₂ Culturing in a 37 ℃ incubator for 3-4 days.

The above-mentioned fibroblast is preferably a cultured fibroblast; preferably, the culture is carried out by culturing the fibroblasts at the 4 th to 16 th passages (the passage of the passage number, the growth and proliferation states of the cells are better, the cells grow too low, the cells grow slowly, and the too high cells tend to age and die) in the form of (0.5 to 1.5). Times.10 ⁶ Inoculating the strain into the culture medium I for culturing for 5-7 days.

In the invention, the epidermis layer is prepared by inoculating keratinocytes on the surface of the dermis layer for culture; the density of the keratinocyte cell inoculation is preferably (0.1 to 1.5). Times.10 ⁵ Individual/cm ² . If the density is outside this range, a good epidermis structure is not well formed, and when the density is too low, the epidermis may have voids, and when the density is too high, keratinocytes are wasted. Preferably, the density of the keratinocyte seedingIs 0.8X10 ⁵ Individual/cm ² ；

The culture medium II for the keratinocyte culture may be conventional in the art and preferably comprises the following components: 60% DMEM, 30% F12, 10% FBS, hydrocortisone 0.2-0.6 μg/ml, insulin 1-50 μg/ml, adenine 10-50 μg/ml, isoprenaline 0.1-0.5 μg/ml, triiodothyronine 0.5-2 ng/ml and epidermal growth factor 10-60 ng/ml; the percentages are by volume. Wherein the action of hydrocortisone and insulin is as described above; however, concentrations of adenine, isoproterenol, triiodothyronine and epidermal growth factor below the lower limit of the above-mentioned range are unfavorable for promoting keratinocyte growth, and above the upper limit of the above-mentioned range, costs are wasted and the probability of abnormal keratinocyte growth or differentiation is greatly increased.

The time for culturing the keratinocytes is preferably 5 to 10 days; more preferably 7 days.

The keratinocytes may be conventional in the art, preferably, the 2 nd to 4 th generation keratinocytes; more preferably, the keratinocytes are cultured in Gibco's KSFM medium (Medium III) or Medium II described above.

The invention provides a 3D full-layer skin model prepared by the preparation method.

The invention provides application of the skin model in the fields of cosmetics and medicine research.

It should be understood that the "I", "II", "III" and "IV" in the medium I, medium II, medium III and medium IV are not actually meant to be the only differences between the substances in the different steps or the same terminology.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

The reagents and materials used in the present invention are commercially available.

The invention has the positive progress effects that:

(1) Serum is not needed in the whole gas-liquid phase process of the 3D full-layer skin model construction, the influence of complex serum components and batch fluctuation on stability between model construction batches is eliminated to the greatest extent, the protein preparation with clear components can enable the skin model to be well grown and differentiated, the defect of high collagen degradation speed in the construction process is effectively overcome, and the controllability of the construction process is improved.

(2) The pig type I collagen has wide material sources, greatly reduces the production cost and has strong quality control; the 3D full-skin model prepared by the method has a good DEJ structure, and can be better subjected to expansion production.

(3) According to the invention, the dermis layer is constructed by utilizing the pig type I collagen, and serum-free culture conditions are used in combination in a gas-liquid culture stage, so that only three culture mediums are needed at most in the construction process, the preparation process is simplified, the construction process is shortened, the controllability of the construction process is improved, and the activity and the stability of the skin model are both obviously improved; the epidermis layer of a successful skin model is usually composed of more than 10 layers of epidermal cells and is able to survive in vitro for more than 14 days, with 7-8 layers of stratum corneum being seen in the epidermis, and with more than 31 days of culture after 17 days of harvest, the dermis of the skin is intact.

(4) The model constructed by the invention is complete, can be used for stably replacing animal and cosmetic detection or safety and efficacy detection (barrier permeation related detection) of various active substances (such as cosmetics) developed by human skin tissues, and greatly expands the application field of the model. Thereby realizing the large-scale industrialized production of the skin model.

Drawings

FIG. 1 is a morphological comparison of normal human skin with a 3D skin model; hematoxylin eosin staining was performed using paraffin sections, eyepiece x objective: 10×10.

FIG. 2 is a graph showing the results of growth and differentiation of the epidermis layer of a 3D skin model over time; d17, D18, D19, D20, D24 and D31 represent days 17, 18, 19, 20, 24, 31, respectively, of seeding keratinocytes into the dermis layer (masson trictaining using paraffin sections, eyepiece x objective: 10 x 10).

FIG. 3 shows comparison of immunofluorescence staining of epidermis layer transglutaminase and silk fibroin of normal human skin and 3D skin model.

FIG. 4 shows immunofluorescence staining contrast of collagen type IV and laminin of normal human skin versus basal membrane of 3D skin model.

Fig. 5 is a comparison of dermal layer immunohistochemical staining of normal human skin versus 3D skin model.

FIG. 6 is a 3D skin model aquaporin immunofluorescent staining.

FIG. 7 is a 3D skin model 4-HNE immunofluorescent staining.

FIG. 8 shows immunofluorescent staining of the 3D skin model paphiopedilum.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Culture medium I:90% dmem+10% fbs; other ingredients such as vitamin C (see CN 104726396A) may be added to the culture medium as appropriate;

medium II:60% DMEM+30% F12+10% FBS, hydrocortisone 0.5 μg/ml, insulin 8 μg/ml, adenine 30 μg/ml, isoproterenol 0.3 μg/ml, triiodothyronine 1.7ng/ml and epidermal growth factor 35ng/ml were added;

medium III: KSFM of Gibco, commercially available;

protein preparation (the percentages are mass percent):

example 1: albumin 0.1%, globulin 0.9%, fibronectin 99%;

example 2: albumin 0.9%, globulin 99%, fibronectin 0.1%;

example 3: albumin 99%, globulin 0.1%, fibronectin 0.9%;

example 4: 30% of albumin, 50% of globulin and 20% of fibronectin;

example 5: 80% of albumin, 10% of globulin and 10% of fibronectin;

example 6: albumin 50%, globulin 45%, fibronectin 5%;

example 7: 60% of albumin, 30% of globulin and 10% of fibronectin.

Other protein components which are beneficial to the growth of skin cells, such as other proteins in serum or serum replacement proteins, can also be added to the protein preparation.

Medium IV:66% DMEM, 30% F12, 1-10% protein preparation, hydrocortisone 0.5 μg/ml, insulin 8 μg/ml; optionally, vitamin C may be added to the medium. Wherein the component proteins in the protein formulation were purchased from Sigma. Among them, the present invention attempts to use protein preparations of different contents, as follows;

culture medium IV-1: 10% of protein preparation (selected from the compositions of example 1 above);

culture medium IV-2: 9% of protein preparation (selected from the composition of example 2);

culture medium IV-3: 1% of protein preparation (selected from the compositions of example 3 above);

culture medium IV-4: protein preparation 8% (selected from the above example 4);

culture medium IV-5: 4% of protein preparation (selected from the compositions of example 5 above);

medium IV-6: 6% of protein preparation (selected from the compositions of example 6 above);

medium IV-7: 10% of protein preparation (selected from the compositions of example 7 above).

Example 1 isolated culture of fibroblasts and keratinocytes

Human skin samples were taken from hospitals, transported to a laboratory for removal of fat and connective tissue from the skin, cut into strips, placed in a thermolysin at a concentration of 400 μg/mL, digested overnight at 4 ℃, and then pulled in opposite directions with forceps to separate the epidermis and dermis.

1. Fibroblast extraction and culture

This may be done using prior art techniques such as the method in CN104726396 a.

Placing dermis layer into collagenase A0.4 mg/ml, stirring with rotorDigestion at 37℃for 2-4 h, stopping digestion with enzyme by adding culture medium, filtering with 70 μm cell sieve, centrifuging at 1500r/min for 10min. Removing supernatant, adding culture medium I for cell count, inoculating cells, and culturing fibroblast with density of 2×10 ⁴ ～2×10 ⁵ Individual/cm ² Placing 5% CO ₂ Culturing in an incubator at 37 ℃.

2. Keratinocyte extraction and culture

Placing the epidermis layer into pancreatin, digesting, blowing with a pipette for 2min, digesting for 2min in a water bath at 37 ℃, repeating the operation for 3 times, and collecting cells. The pancreatin digestion was terminated by adding the medium. The cell tissue suspension was filtered through a 70 μm cell screen and then placed in a centrifuge for centrifugation at 1500r/min for 10min. The supernatant was discarded, medium III was added for cell counting and the cells were inoculated. Keratinocyte culture density 2×10 ⁴ ～2×10 ⁵ Individual/cm ² Placing 5% CO ₂ Culturing in an incubator at 37 ℃. And taking 0-4 generation of cells for construction.

Example 2 preparation of dermis layer

The fibroblasts cultured to the 10 th generation in example 1 were mixed with porcine type I collagen (3.5 mg/ml) (Gentishold Co.) which had been dissolved at a concentration of 1.7X10 per ml ⁶ The fibroblasts were mixed on ice and the pH was adjusted to neutral. The mixture of fibroblasts and porcine type I collagen was added to a 6cm cell culture plate in a volume of 8 ml. Placing 5% CO ₂ Culturing in a 37 ℃ incubator for 2-3 h, adding a culture medium I after solidification, and adding 5% CO ₂ Culturing in a 37 ℃ incubator for 3-4 days.

Example 3 preparation of skin layers

The 4 th generation keratinocytes prepared in example 1 were prepared at a ratio of 0.8X10 ⁵ Individual/cm ² Inoculating onto the surface of dermis, adding culture medium II, immersing the surface, and placing in 5% CO ₂ Liquid is changed every 48 hours at 37 ℃ and cultured for 7 days, thus obtaining the double-layer skin.

EXAMPLE 4 preparation of full skin model

Taking out the prepared skin model, transferring to the surface of a culture bracket for gas-liquid phase culture, adding a culture medium IV (the culture medium IV-1 to the culture medium IV-7 respectively correspond to the examples 4 (1) to (7)) under the bracket, and culturing for 7 days, wherein the liquid is changed for 1 time every 2 days, and the whole skin model is obtained after the culture is finished.

EXAMPLE 5H & E staining

The skin model of example 4 (7) was fixed in 10% formalin (purchased from Sigma) and embedded using paraffin (Leica). Sections were taken at 5 μm thickness (sections were purchased from Leica), stained with hematoxylin and eosin (purchased from Sigma), and photographed using a zeiss optical microscope.

From fig. 1, it can be observed that the 3D skin model constructed using chinese human skin cells has a better layered structure on the epidermis structure, including basal layer, spinous layer, granular layer and keratinized layer, has full collagen on the dermis structure, and the cells are uniformly distributed in the dermis layer. From fig. 2, it can be observed that the epidermal layer of the 3D skin model grows and differentiates with the increase of the number of days of seeding keratinocytes. D17, D18, D19, D20, D24 and D31 represent day 17, day 18, day 19, day 20, day 24 and day 31, respectively, when seeding keratinocytes was initiated. Overall, 3D skin models constructed using chinese skin cells were highly similar in morphology to normal human skin, demonstrating successful modeling; and the quality of the skin model is still kept high along with the increase of time, so that the good model quality can be kept in the later research, the stability is strong, and the skin model cannot interfere with experiments.

The skin models of examples 4 (1) to 4 (6) were H & E stained and found to have a 3D full-thickness skin model, and the modeling was successful. The skin model of example 4 (7) was used to conduct the tests of examples 6 to 7 below.

EXAMPLE 6 immunofluorescent staining and immunohistochemical staining to verify expression and distribution of markers associated with the epidermis, basement membrane and dermis layers in skin models

Immunofluorescent staining was performed using 5 μm frozen sections. Transglutaminase (TG-1), COL IV (collagen IV), laminin (Laminin) and microfibrils (fibrinlin) were detected, respectively. The sections were fixed in cold formaldehyde for 10min, washed 2 times in phosphate buffer for 5 min each, blocked with 0.5% BSA/PBS for 1h and diluted antibody was added overnight. The following day was washed 2 times with phosphate buffer for 5 minutes each and 2 antibody treatments were added for 1 hour. Washing with phosphate buffer, sealing, and observing.

Immunohistochemical detection was performed using 5um paraffin sections. For detection of Filaggrin (Filaggrin) and COL I, the sections were immersed in 0.01M sodium citrate buffer (ph 6.0) for 30 min at 100 ℃ and then cooled to room temperature within 30 min. The primary antibody incubation was performed at room temperature for 1 hour, and then stained with Dako strept ABC complex/HRP kit according to the manufacturer's instructions. Nuclei were stained with hematoxylin at room temperature for 5 min.

After the morphological characteristics of the 3D skin model are observed, the expression and distribution of specific biochemical markers related to the growth and differentiation of different skin layers are analyzed. In both normal human cells and 3D skin models, transglutaminase (TG-1) associated with the differentiation between epidermal cells was significantly expressed. Filaggrin (Filaggrin), an important component of keratinocytes, is one of the important markers of keratinocyte terminal differentiation, and is observed to be normally expressed in normal human cells and 3D skin models. Experiments prove that markers related to epidermal growth and differentiation have normal expression similar to normal human skin in a 3D skin model (fig. 3).

The layer of tissue present between the epidermis and dermis is called the basement membrane, an important platform for the exchange of substances and the transduction of cellular signals by the epidermis and dermis, and is mainly composed of molecules such as collagen, laminin, proteoglycans, etc. Collagen type IV (COL IV) located at the junction of the dermis and a Laminin (Laminin), a major component of the basement membrane, were normally expressed in both 3D skin models and normal human skin (fig. 4).

The dermis layer is located under the epidermis layer of the skin, and cells are mainly composed of fibroblasts, and contain a large amount of collagen fibers to maintain the mechanical strength of the skin. Collagen type I (COL I) is the main collagen in the dermis layer, and microfibrils (fibrinlins) constituting skin elastin play a key role in maintaining skin elasticity, as can be seen from fig. 5, it is normally expressed in a 3D skin model relative to normal human skin, therefore, it is inferred that the skin model is constructed relatively successfully, normal human skin can be simulated to a great extent, and further, efficacy detection of cosmetics or raw materials can be performed instead of animal experiments.

Example 7 application of skin model

1. Moisture retention aspect

By constructing the skin model, it can be used to study various effects of cosmetics or raw materials. As a result of examining the moisturizing effect, it was found that the fluorescence expression intensity of aquaporin (AQP 3) was significantly increased in a skin model to which the hot spring water was added (fig. 6) by measuring the expression of a marker related to moisturizing by adding the hot spring water having the moisturizing effect, indicating that the model can be applied to the detection of the moisturizing effect of cosmetics and the like.

2. Antioxidant aspect

The antioxidant efficacy of raw materials or cosmetics can be studied by measuring the fluorescent expression of the marker 4-hydroxynonenal (4-HNE) associated with oxidation. The apparent up-regulation of the expression intensity of 4-HNE in skin models after treatment with oxidizing substances was found by immunofluorescence staining (fig. 7), demonstrating that the models can be used for analysis of antioxidant efficacy of cosmetics or raw materials.

3. Pollution aspect

At present, PM2.5 is seriously out of standard due to serious atmospheric pollution, so that the physical health of people is seriously jeopardized. The construction of this skin model can be used to study the evaluation of anti-air pollution activity. For example, the fluorescent expression of skin barrier functions associated with anti-pollution, such as pappalin (Loricrin), can be determined. In the case of co-use of contaminant treatment, an increase in the lorecrin fluorescence intensity in the skin model can be observed by the addition of the active (fig. 8), indicating an increase in the barrier function of the skin, better protection against contamination by the external atmosphere, and further indicating that the model can be applied to evaluate the efficacy of anti-contaminant.

Claims

1. A preparation method of a 3D full-layer skin model comprises the steps of culturing a skin model comprising a dermis layer and an epidermis layer through a gas-liquid phase interface, and is characterized in that the gas-liquid phase interface is cultured on the surface of a culture bracket; culturing the gas-liquid phase interface for 7 days, and changing the liquid once every 2 days until a full-layer skin model is obtained; the culture medium for gas-liquid phase interface culture is a culture medium IV;

the culture medium IV comprises the following components: 60-66% of DMEM, 30-33% of F12, 1-10% of protein preparation, 0.2-0.6 mu g/ml of hydrocortisone and 1-50 mu g/ml of insulin; the protein preparation consists of 30-80% of albumin, 10-50% of globulin and 5-20% of fibronectin; the percentage is mass percentage;

the dermis layer is prepared by mixing single-cell fibroblasts with porcine type I collagen, culturing for 2-3 hours until solidification, and then adding a culture medium I for culturing;

the culture medium I comprises 90% DMEM and 10% FBS, wherein the percentages are volume percentages.

2. The method of claim 1, wherein the source of the protein preparation is animal or human.

3. The method of claim 1, wherein the protein formulation is present in an amount of 10%, the protein formulation is comprised of 60% albumin, 30% globulin, and 10% fibronectin, the hydrocortisone is present in an amount of 0.5 μg/ml, and the insulin is present in an amount of 8 μg/ml; the percentages are mass percentages.

4. The method of claim 1, wherein the density of porcine type I collagen is 2-5 mg/ml;

the density of the mixed fibroblast is (0.5-3) multiplied by 10 ⁶ Individual/ml;

and/or, the condition of culturing the fibroblast after being mixed with the pig type I collagen is 5% CO ₂ Culturing in an incubator at 37 ℃ for 3-4 days.

5. The method of claim 4, wherein the porcine type I collagen has a density of 3.5mg/ml;

and/or the density of the mixed fibroblast is 1.7X10 ⁶ And each ml.

6. The method of any one of claims 1-5, wherein the fibroblasts are cultured fibroblasts.

7. The method according to claim 6, wherein the culturing is performed by culturing the 4 th to 16 th generation fibroblasts in a ratio of (0.5 to 1.5). Times.10 ⁶ Inoculating the strain/ml strain to the culture medium I for 5-7 days.

8. The method of claim 1, wherein the epidermis is produced by inoculating keratinocytes to the dermis surface for culturing.

9. The method according to claim 8, wherein the keratinocyte cell inoculation density is (0.1 to 1.5) ×10 ⁵ Individual/cm ² ；

The culture medium II for the keratinocyte culture comprises the following components: 60% DMEM, 30% F12, 10% FBS, hydrocortisone 0.2-0.6 μg/ml, insulin 1-50 μg/ml, adenine 10-50 μg/ml, isoprenaline 0.1-0.5 μg/ml, triiodothyronine 0.5-2 ng/ml and epidermal growth factor 10-60 ng/ml; the percentage is volume percentage;

and/or the keratinocyte is cultured for 5-10 days.

10. The method of claim 9, wherein the keratinocyte seeding density is 0.8x10 ⁵ Individual/cm ² ；

And/or, the keratinocyte cell culture is for 7 days.

11. The method of any one of claims 8-10, wherein the keratinocytes are 2 nd-4 th generation keratinocytes.

12. The method of claim 11, wherein the keratinocytes are cultured in Gibco's KSFM medium.

13. The method of claim 9, wherein said keratinocytes are cells cultured in said medium II.