In-vitro dermal scaffold, in-vitro 3D melanoma model and preparation method thereof

Abstract

The invention provides an in-vitro dermal scaffold, an in-vitro 3D melanoma model and a preparation method thereof. The in-vitro dermal scaffold comprises a basement membrane and an extracellular matrix layer which are connected with each other, and the first in-vitro skin is subjected to hypertonic dehydration to remove cells of the first in-vitro skin and completely reserve the basement membrane and the extracellular matrix layer to form the basement membrane and the extracellular matrix layer which are connected with each other. According to the in-vitro dermal scaffold, the first in-vitro skin is subjected to hypertonic dehydration so as to remove cells of the first in-vitro skin, and the first in-vitro skin does not need to be subjected to thermal expansion and cold contraction so as to completely retain a basement membrane and an extracellular matrix; the operation is simple, the complete basilar membrane can promote proliferation and differentiation of keratinocytes, the interaction of melanoma cells and keratinocytes in real cells in vivo is better reduced, and the microenvironment formed by melanoma in vivo is truly and objectively simulated.

Description

Technical Field

[0001] The present disclosure relates to the technical field of biological tissue engineering, and in particular to an in vitro dermal scaffold, an in vitro 3D melanoma model and a preparation method thereof. Background Art

[0002] Melanoma is a malignant tumor caused by the canceration of melanocytes. The incidence of melanoma is relatively low among skin cancers, but the mortality rate of melanoma is very high due to its highly invasive nature. Although there are many clinical treatment drugs or methods for various tumors including melanoma, so far, no drug or treatment method with good therapeutic effect and low toxicity has been achieved for the treatment of melanoma.

[0003] At present, one of the main factors hindering the development of melanoma drugs is the lack of a suitable in vitro melanoma model. At present, the in vitro culture methods of melanoma include 2D cell culture system, 2D culture model, animal model and 3D spheroid melanoma model. Compared with 2D cell culture system, 2D culture model and animal model, 3D spheroid melanoma model can provide a certain three-dimensional structure for melanoma, so as to realize the co-culture of melanoma cells and 3D spheroid melanoma model. However, 3D spheroid melanoma model generally uses polymer biomaterials, which makes it impossible for such melanoma model to truly and objectively simulate the microenvironment formed by melanoma in vivo.

[0004] Based on this, some scholars have proposed an in vitro three-dimensional skin dermis model. For example, Chinese patent document No. CN107001830B discloses an in vitro three-dimensional skin dermis model. However, its dermal scaffold is composed of a membrane formed by fibroblasts and the extracellular matrix secreted by them, and lacks the basement membrane that is crucial for cell proliferation and differentiation in the epidermis. As a result, the dermal scaffold still cannot truly simulate the microenvironment formed by melanoma in vivo. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an in vitro dermal scaffold, an in vitro 3D melanoma model and a preparation method thereof that do not require thermal expansion and contraction operations to completely retain the basement membrane and extracellular matrix, which is not only simple to operate, but the completely retained basement membrane can more realistically and objectively simulate the microenvironment formed by melanoma in vivo.

[0006] The purpose of this disclosure is achieved through the following technical solutions:

[0007] An in vitro dermal scaffold comprises a basement membrane and an extracellular matrix layer connected to each other, wherein a first ex vivo skin is subjected to hypertonic dehydration to remove cells of the first ex vivo skin, while the basement membrane and the extracellular matrix are completely retained to form the basement membrane and the extracellular matrix layer connected to each other.

[0008] A method for preparing an in vitro 3D melanoma model comprises the following steps:

[0009] obtaining a mixed cell suspension, wherein the mixed cell suspension comprises keratinocytes and melanoma cells;

[0010] Placing the mixed cell suspension on the in vitro dermal scaffold described in any one of the above embodiments for inoculation treatment, so that the mixed cell suspension is randomly attached to one side of the basement membrane;

[0011] The mixed cell suspension after the inoculation treatment is subjected to a unilateral parallel culture operation, so that a cell culture environment is formed on one side of the basement membrane, and the mixed cell suspension is in the cell culture environment, so that the mixed cell suspension is promoted to proliferate in parallel on one side of the basement membrane to form a cell layer, thereby obtaining a pre-treated 3D melanoma model;

[0012] The pretreated 3D melanoma model is subjected to an air-liquid interface culture operation, so that the pretreated 3D melanoma model forms a cell culture environment in the thickness direction, prompts the cell layer to form a new epidermis layer, and prompts the melanoma cells to invade the extracellular matrix layer.

[0013] An in vitro 3D melanoma model is prepared by using the method for preparing an in vitro 3D melanoma model described in any of the above embodiments.

[0014] An application of an in vitro 3D melanoma model, wherein the in vitro 3D melanoma model of any of the above embodiments is applied to the analysis of melanoma pathology, the screening of anti-melanoma drugs, the evaluation of anti-melanoma drugs, the screening of anti-melanoma therapies or the evaluation of anti-melanoma therapies.

[0015] Compared with the prior art, the present invention has at least the following advantages:

[0016] 1) The first ex vivo skin is subjected to hypertonic dehydration to remove cells of the first ex vivo skin, and there is no need to perform thermal expansion and contraction on the first ex vivo skin, so as to completely retain the basement membrane and the extracellular matrix; not only is the operation simple, but the connected basement membrane and extracellular matrix layer are formed, which is closer to the structure of the real skin in vivo, so that the intact basement membrane can promote the proliferation and differentiation of keratinocytes, and better restore the interaction between melanoma cells and keratinocytes in real cells in vivo, so as to more realistically and objectively simulate the microenvironment formed by melanoma in vivo.

[0017] 2) The mixed cell suspension of keratinocytes and melanoma cells is placed on an in vitro dermal scaffold for inoculation treatment, and then the mixed cell suspension after inoculation treatment is subjected to a unilateral parallel culture operation, so that the mixed cell suspension is attached to the side of the basement membrane away from the extracellular matrix layer and proliferates in parallel to form a cell layer, so that the keratinocytes and melanoma cells can randomly adhere to one side of the basement membrane, so as to ensure that the melanoma cells are randomly distributed on the in vitro 3D melanoma model; and effectively avoid a small part of the mixed cell suspension of keratinocytes and melanoma cells from infiltrating into the basement membrane and failing to truly simulate the microenvironment formed by melanoma cells in vivo; finally, the pretreated 3D melanoma model is subjected to an air-liquid interface culture operation, so that the pretreated 3D The dermis of the melanoma model is fully exposed to the air, ensuring that the growth environment of the pre-treated 3D melanoma model is close to the growth environment of the real skin in vivo, so that the pre-treated 3D melanoma model forms a cell culture environment in the thickness direction, prompting the cell layer to form a new epidermis containing keratinocytes and melanoma cells, and also prompting melanoma cells to break through the basement membrane and invade the extracellular matrix layer, so as to more realistically and objectively simulate the microenvironment formed by melanoma in vivo, so as to maximize and objectively simulate the pathological process and physiological changes of melanoma in vivo, and is suitable for the analysis of melanoma pathology, screening of anti-melanoma drugs, evaluation of anti-melanoma drugs, screening of anti-melanoma therapies, or evaluation of anti-melanoma therapies.

[0018] 3) Since the hollow tube is placed on the basement membrane, the hollow tube and the basement membrane are sealed and abutted to form a semi-closed inoculation culture channel; then the mixed cell suspension is placed in the inoculation culture channel through the inoculation end, and is cast along the inoculation culture channel to one side of the basement membrane, and the culture fluid is added to the semi-closed inoculation culture channel at the same time, so that the hollow tube forms a restricted cell culture environment with the inoculation culture channel as the area on one side of the basement membrane, thereby ensuring that the mixed cell suspension can randomly settle and adhere to the outermost layer of the basement membrane in the restricted cell culture environment for parallel proliferation, so as to ensure that the mixed cell suspension can be quickly and uniformly generated on the basement membrane in the restricted cell culture environment, while ensuring that the melanoma cells are randomly distributed in the in vitro 3D melanoma model, so as to more realistically simulate the microenvironment formed by the melanoma cells in vivo, and also ensure the efficiency of the in vitro 3D melanoma model modeling.

[0019] 4) Since the modified culture medium adds fibroblast growth factor, transforming growth factor and epidermal growth factor, it is ensured that the prepared keratinocytes have good proliferation and differentiation capabilities, thereby ensuring that during subsequent inoculation, the keratinocytes in the mixed cell suspension can not only adhere well to one side of the basement membrane; it is also ensured that during the subsequent mixed cell suspension air-liquid interface culture operation, the keratinocytes can proliferate to form a complete new epidermis layer, thereby ensuring the efficiency of the in vitro 3D melanoma model modeling. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present disclosure and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is a flow chart of a method for preparing an in vitro 3D melanoma model according to an embodiment of the present invention;

[0022] Figure 2 It is a comparison diagram of the structural staining microscope images of the basement membrane of Example 1 of the present invention and Comparative Example 1;

[0023] Figure 3 It is a comparison diagram of the structural staining microscope images of the basement membranes of Example 1 of the present invention and Comparative Example 2;

[0024] Figure 4 It is a comparison diagram of the structural staining microscope images of the basement membranes of Example 1 of the present invention and Comparative Example 3;

[0025] Figure 5 It is a comparison diagram of the structural staining microscope images of the basement membrane of Example 1 of the present invention and Comparative Example 5;

[0026] Figure 6 It is a comparison diagram of the structural staining microscope images of the basement membranes of Example 1 of the present invention and Comparative Example 6;

[0027] Figure 7 This is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 1;

[0028] Figure 8 This is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 2;

[0029] Fig. 9 This is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 3;

[0030] Fig.10 This is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 5;

[0031] Fig.11 This is a comparison chart of cell viability of keratinocytes in Example 1 of the present invention and Comparative Example 7;

[0032] Fig.12It is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 10;

[0033] Fig.13 It is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 11;

[0034] Fig.14 It is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 12;

[0035] Fig.15 It is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 13;

[0036] Fig.16 This is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 15;

[0037] Fig.17 It is a comparison diagram of the structural staining microscope images of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 16;

[0038] Fig.18 It is a diagram of the culture process of the in vitro 3D melanoma model of Example 1 of the present invention and Comparative Example 16;

[0039] Fig.19 This is a real picture of the hollow metal ring and the in vitro dermal scaffold inoculated in one embodiment of the present invention;

[0040] Fig. 20 This is a schematic structural diagram of a hollow metal ring in one direction according to another embodiment of the present invention;

[0041] Fig.21 This is a diagram of the gas-liquid interface culture state of the in vitro 3D melanoma model of Comparative Example 16 of the present invention;

[0042] Fig. 22 It is a schematic structural diagram of a metal mesh support in one direction according to an embodiment of the present invention. DETAILED DESCRIPTION

[0043] In order to facilitate the understanding of the present disclosure, the present disclosure will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present disclosure are given in the drawings. However, the present disclosure can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present disclosure more thoroughly and comprehensively understood.

[0044] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be a central element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be a central element at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only and do not represent the only implementation method.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present disclosure. The terms used herein in the specification of the present disclosure are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0046] The present disclosure provides an in vitro dermal scaffold, which includes a basement membrane and an extracellular matrix layer connected to each other, wherein a first ex vivo skin is subjected to hypertonic dehydration to remove cells of the first ex vivo skin, and the basement membrane and the extracellular matrix are completely retained to form a basement membrane and an extracellular matrix layer connected to each other.

[0047] The above-mentioned in vitro dermal scaffold performs hypertonic dehydration on the first ex vivo skin to remove the cells of the first ex vivo skin, and there is no need to perform thermal expansion and contraction on the first ex vivo skin to completely retain the basement membrane and extracellular matrix; it is not only simple to operate, but also forms a connected basement membrane and extracellular matrix layer that is closer to the structure of real skin in vivo, so that the intact basement membrane can promote the proliferation and differentiation of keratinocytes, better restore the interaction between melanoma cells and keratinocytes in real cells in vivo, so as to truly and objectively simulate the microenvironment formed by melanoma in vivo.

[0048] In order to better understand the technical solutions and beneficial effects of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0049] In an embodiment of the in vitro dermal scaffold, the in vitro dermal scaffold comprises a basement membrane and an extracellular matrix layer connected to each other, wherein the first ex vivo skin is subjected to hypertonic dehydration to remove cells of the first ex vivo skin, while the basement membrane and the extracellular matrix are completely retained to form a basement membrane and an extracellular matrix layer connected to each other.

[0050] It should be noted that in some early in vitro melanoma models, such as the method for preparing artificial skin melanoma tissue disclosed in Chinese patent document No. CN101392238A, a skin specimen of a healthy adult is taken, routinely disinfected, and the fat tissue layer is removed, and then the remaining tissue is made into a 1x1 cm' skin slice and immersed in a 56°C PBS solution for 30 minutes; then the epidermis is carefully torn off with tweezers after being taken out; the dermal tissue without the epidermis is placed in a polyethylene plastic bottle, and it is quickly placed in liquid nitrogen for 5 minutes, and then placed at room temperature for 30 minutes after being taken out, and then placed in liquid nitrogen for 5 minutes, and after 10 consecutive cycles in this way, the HDED matrix material is obtained; the cell components in the HDED after this treatment are killed, and the basement membrane components are contained.

[0051] However, after 10 consecutive cycles of liquid nitrogen freezing and room temperature storage, the HDED matrix material not only has the problem of complicated operation and high cost, but also the basement membrane is affected by thermal expansion and contraction, resulting in deformation or damage of the toughness and elasticity of the basement membrane, such as changes in the mechanical properties and permeability of the basement membrane, that is, the structure of the basement membrane cannot be preserved completely. For details, please refer to Figure 5 The structural staining microscope image of the basement membrane of Comparative Example 5 cannot provide keratinocytes with better proliferation and differentiation capabilities, that is, it cannot truly restore the interaction between melanoma cells and keratinocytes in the body, and thus cannot truly and objectively simulate the microenvironment formed by melanoma in the body.

[0052] Therefore, in the present disclosure, the cells of the first ex vivo skin are removed by performing hypertonic dehydration on the first ex vivo skin, and there is no need to perform thermal expansion and contraction on the first ex vivo skin, so as to completely retain the basement membrane and the extracellular matrix; for details, please refer to Figures 2 to 6 , making the structure of the basement membrane disclosed in the present invention closer to the structure of the basement membrane in a normal body; in this way, not only is the operation simple, but the connected basement membrane and extracellular matrix layer formed are closer to the structure of the real skin in vivo, so that the intact basement membrane can promote the proliferation and differentiation of keratinocytes, and better restore the interaction between melanoma cells and keratinocytes in real cells in vivo, so as to truly and objectively simulate the microenvironment formed by melanoma in vivo.

[0053] It is worth mentioning that in the study of basement membrane, we generally use immunohistochemistry to observe the staining expression of collagen IV and laminin in the basement membrane to evaluate the integrity of the basement membrane structure. If the staining expression is stronger, it proves that there are more basement membrane components and the structure is more complete; if the staining expression is very weak, it means that the integrity of the basement membrane is poor; if there is no staining expression, it means that the basement membrane does not exist.

[0054] In one embodiment, a hypertonic dehydrating agent is used to perform hypertonic dehydration on the first ex vivo skin to effectively remove cells of the first ex vivo skin, completely retain the basement membrane and the extracellular matrix, and better retain the original structure of the basement membrane and the extracellular matrix, so as to better restore the real cell interaction between melanoma cells and keratinocytes in vivo, so as to realistically and objectively simulate the microenvironment formed by melanoma in vivo.

[0055] In one embodiment, the hypertonic dehydrating agent includes at least one of a high-concentration salt solution and a high-concentration organic solution. It can be understood that since the high-concentration salt solution and the high-concentration organic solution have a high osmotic pressure, they can attract and absorb the water around the cells, thereby effectively reducing the water content in the cells, so as to achieve hypertonic dehydration of the first ex vivo skin, thereby completely retaining the basement membrane and the extracellular matrix layer.

[0056] It is understandable that the user can select the corresponding high-concentration salt solution and high-concentration organic solution according to the actual use situation. For example, in some embodiments, the high-concentration salt solution can select one or more of a high-concentration sodium chloride solution, a high-concentration potassium chloride solution, a high-concentration sodium sulfate solution, a high-concentration potassium nitrate solution, and a high-concentration calcium chloride solution to achieve high-concentration salt solution The hypertonic dehydration of the first ex vivo skin, thereby completely retaining the basement membrane and the extracellular matrix layer. For example, in some other embodiments, the high-concentration organic solution can select one or more of a high-concentration glucose solution, a high-concentration mannitol solution, and a high-concentration sorbitol solution. Of course, those skilled in the art can also select a high-concentration salt solution or a high-concentration organic solution with a similar hypertonic dehydration effect, so they are not listed one by one in the present disclosure.

[0057] It can be understood that if the concentration of the high-concentration sodium chloride solution is lower than 0.8 mol / L, the osmotic pressure is too low to achieve hypertonic dehydration of the first ex vivo skin, and if the concentration of the high-concentration sodium chloride solution is greater than 2.0 mol / L, the osmotic pressure is too high to cause excessive damage to the first ex vivo skin, and the basement membrane and the extracellular matrix cannot be completely preserved. Therefore, in a preferred embodiment, the concentration of the high-concentration sodium chloride solution is controlled to be 0.8 mol / L to 2.0 mol / L to ensure that the high osmotic pressure of the high-concentration sodium chloride solution is more appropriate, thereby ensuring that the high-concentration sodium chloride solution can perform hypertonic dehydration on the first ex vivo skin to completely preserve the basement membrane and the extracellular matrix.

[0058] In some other embodiments, the concentration of the high concentration potassium chloride solution is 1.4 mol / L to 2.2 mol / L.

[0059] In some other embodiments, the concentration of the high concentration glucose solution is 1.4 mol / L to 2.8 mol / L.

[0060] In some other embodiments, the concentration of the high concentration mannitol solution is 1.1 mol / L to 1.7 mol / L.

[0061] It can be understood that the high osmotic pressure of the potassium chloride solution, glucose solution and mannitol solution is more suitable to achieve hypertonic dehydration of the first ex vivo skin and to completely preserve the basement membrane and extracellular matrix.

[0062] In one embodiment, the first ex vivo skin is subjected to hypertonic dehydration using a hypertonic dehydrating agent, and the specific operation is as follows: the first ex vivo skin is immersed in the hypertonic dehydrating agent to cause hypertonic dehydration of cells on the first ex vivo skin.

[0063] Specifically, in one embodiment, the first ex vivo skin is immersed in a hypertonic dehydrating agent for 12 hours to 16 hours at a temperature of 28° C. to 38° C. to achieve hypertonic dehydration of cells on the first ex vivo skin.

[0064] In one embodiment, the size of the first ex vivo skin is 4 cm 2 ~6cm 2 To ensure that the area of ​​the first ex vivo skin is more appropriate, thereby ensuring that the area of ​​the prepared in vitro dermal scaffold is relatively large, which is conducive to subsequent inoculation operations.

[0065] In one embodiment, the shape of the first ex-vivo skin is any one of a triangle, a quadrilateral, a polygon, a circle or a sector, so as to realize the preparation of the first ex-vivo skin sample.

[0066] In a preferred embodiment, the first ex vivo skin is 4 cm 2 square. Since 4cm 2 The square size is moderate, which is convenient for subsequent further processing and use on the one hand, and is convenient for subsequent formation of a well-sealed restricted cell culture environment with the hollow tube on the other hand, which is conducive to the random adhesion of the mixed cell suspension on the basement membrane of the restricted cell culture environment.

[0067] In one embodiment, the first ex vivo skin is soaked in a hypertonic dehydrating agent, so that the first ex vivo skin is immersed in the hypertonic dehydrating agent, and the hypertonic dehydrating agent can fully and completely remove the cells on the first ex vivo skin, thereby completely retaining the basement membrane and the extracellular matrix layer.

[0068] In one embodiment, before performing the hypertonic dehydration operation on the first ex vivo skin, the following operation is also included: performing a fat removal operation on the first ex vivo skin to remove the fat tissue on the first ex vivo skin.

[0069] In one embodiment, large pieces of fat tissue on the first ex vivo skin are first removed to quickly remove the fat tissue under the dermis; then, the fat tissue fragments on the first ex vivo skin are removed to completely remove the fat tissue under the dermis to obtain a connected epidermis and dermis, i.e., a skin sample.

[0070] In one embodiment, the specific operation of fat removal on the first ex vivo skin is as follows: first, use scissors to cut off large pieces of fat tissue in the subdermal layer of the first ex vivo skin, and then use a scraper to completely scrape off the fat tissue fragments remaining in the subdermal layer of the first ex vivo skin; then use PBS antibacterial agent to rinse the first ex vivo skin to further kill the microorganisms on the first ex vivo skin.

[0071] In one embodiment, before performing the fat removal operation on the first ex vivo skin, the following operation is also included: performing a sterilization operation on the first ex vivo skin to kill microorganisms on the first ex vivo skin to ensure that a non-contaminated and sterile in vitro dermal scaffold is prepared.

[0072] In one embodiment, the specific operation of sterilizing the first ex vivo skin is as follows: the first ex vivo skin is immersed in the collection liquid for 30 minutes to 40 minutes to kill the microorganisms on the first ex vivo skin and reduce the contamination of the first ex vivo skin by the microorganisms.

[0073] Specifically, in one embodiment, the collection liquid includes at least one of PBS antimicrobial agent, three antibiotics and gentamicin. The above-mentioned PBS antimicrobial agent can effectively kill and inhibit common bacteria, fungi and other microorganisms; the three antibiotics can kill and inhibit a variety of bacteria, fungi and other microorganisms; gentamicin can inhibit gram-negative rods, gram-positive cocci and other microorganisms.

[0074] In a preferred embodiment, the collection fluid includes PBS antibacterial agent, three antibiotics and gentamicin to more comprehensively kill microorganisms such as bacteria, fungi, Gram-negative bacilli, Gram-positive cocci, etc. that inhibit the first excised skin.

[0075] The present disclosure provides a method for preparing an in vitro dermal scaffold, which comprises the following steps: firstly obtaining a first ex vivo skin; then, soaking the first ex vivo skin in a collection solution (PBS antibacterial agent, three antibiotics and gentamicin) for half an hour to kill microorganisms in the first ex vivo skin; then, taking out the first ex vivo skin from the collection solution, cutting off large pieces of fat tissue on the first ex vivo skin with scissors, and then completely scraping off the fat tissue fragments with a scraper to obtain a skin sample, and then washing the skin sample with a PBS antibacterial agent; immersing the skin sample in a hypertonic dehydrating agent, such as a high-concentration sodium chloride solution of 0.8 mol / L to 2.0 mol / L, for 12 h to 16 h to remove cells in the skin sample to obtain an in vitro dermal scaffold.

[0076] The in vitro dermal scaffold preparation method does not require thermal expansion and contraction operations to completely preserve the basement membrane and extracellular matrix. Not only is the operation simple, but the intact basement membrane promotes the proliferation and differentiation of keratinocytes, and better restores the interaction between melanoma cells and keratinocytes in real cells in the body, so as to truly and objectively simulate the microenvironment formed by melanoma in the body.

[0077] The present disclosure also provides a method for preparing an in vitro 3D melanoma model, comprising obtaining a mixed cell suspension, wherein the mixed cell suspension comprises keratinocytes and melanoma cells; placing the mixed cell suspension on an in vitro dermal scaffold of any of the above-mentioned embodiments for inoculation treatment, so that the mixed cell suspension is randomly attached to one side of a basement membrane; performing a unilateral parallel culture operation on the mixed cell suspension after the inoculation treatment, so that a cell culture environment is formed on one side of the basement membrane, and the mixed cell suspension is in the cell culture environment, so that the mixed cell suspension is promoted to proliferate in parallel on one side of the basement membrane to form a cell layer, thereby obtaining a pretreated 3D melanoma model; performing an air-liquid interface culture operation on the pretreated 3D melanoma model, so that the pretreated 3D melanoma model forms a cell culture environment in the thickness direction, so that the cell layer is promoted to form a new epidermis layer, and the melanoma cells are promoted to invade the extracellular matrix layer.

[0078] The above-mentioned method for preparing an in vitro 3D melanoma model simultaneously inoculates a mixed cell suspension of keratinocytes and melanoma cells onto an in vitro dermal scaffold to ensure that keratinocytes and melanoma cells proliferate in parallel from the side of the basement membrane away from the extracellular matrix layer to form a cell layer, thereby prompting the cell layer to form a new epidermal layer containing keratinocytes and melanoma cells, and at the same time prompting the melanoma cells to break through the basement membrane and invade the extracellular matrix layer, thereby forming an obvious three-dimensional invasion phenomenon on the in vitro 3D melanoma model, so as to efficiently, effectively, objectively and to the greatest extent simulate the pathological process and physiological changes of melanoma in vivo, so as to truly simulate the interaction between cells under the real physiological state of melanoma cells in vivo, so as to be closer to the microenvironment formed by melanoma cells in vivo in real life.

[0079] See also Figure 1 In order to better understand the technical solutions and beneficial effects of the present disclosure, the present disclosure is further described in detail below in conjunction with specific embodiments. The preparation method of an in vitro 3D melanoma model in one embodiment includes some or all of the following steps:

[0080] S110, obtaining a mixed cell suspension, wherein the mixed cell suspension includes keratinocytes and melanoma cells.

[0081] It is understandable that in the general traditional preparation of melanoma tissue, such as disclosed in Chinese patent document No. CN101392238A, keratinocytes are usually inoculated first for incubation and then melanoma cells are inoculated, which will affect the growth of melanoma cells in the dermal scaffold in vitro, resulting in no obvious melanoma being observed in the in vitro 3D melanoma model. Fig.14 The structural staining microscope image of the in vitro 3D melanoma model of Comparative Example 12 is shown; if melanoma cells are inoculated first and then keratinocytes are inoculated, as shown in FIG. Fig.15 As shown, although the obtained in vitro 3D melanoma model can see the growth of melanoma cells in the in vitro dermal scaffold, no complete new epidermis can be observed in the in vitro 3D melanoma model.

[0082] Therefore, in the present disclosure, keratinocytes and melanoma cells are mixed to obtain a mixed cell suspension, so as to ensure that the keratinocytes and melanoma cells interact with each other before inoculation, thereby ensuring that the keratinocytes and melanoma cells can randomly adhere to the outermost layer of the basement membrane during subsequent inoculation, so as to ensure that the melanoma cells are randomly distributed on the in vitro 3D melanoma model; Fig.16 The structural staining microscope image of the in vitro 3D melanoma model of Example 1 is shown. In this way, while ensuring that the in vitro 3D melanoma model of the present invention has obvious three-dimensional invasion phenomenon, it also ensures that the in vitro 3D melanoma model has a complete stratum corneum, so as to be closer to the real physiological state of melanoma cells in vivo.

[0083] S120, placing the mixed cell suspension on the in vitro dermal scaffold of any of the above embodiments for inoculation treatment, so that the mixed cell suspension is randomly attached to one side of the basement membrane.

[0084] It can be understood that by inoculating the mixed cell suspension on the in vitro dermal scaffold, keratinocytes and melanoma cells can randomly adhere to one side of the basement membrane, so as to ensure that the melanoma cells of the subsequently prepared in vitro 3D melanoma model can be randomly distributed in the new epidermis, so as to objectively reflect the invasion of melanoma cells in the new epidermis, and truly simulate the interaction between melanoma cells and keratinocytes under the real physiological state in vivo, so as to be closer to the microenvironment formed by melanoma cells in the real body.

[0085] S130, performing a unilateral parallel culture operation on the mixed cell suspension after the inoculation treatment, so that a cell culture environment is formed on one side of the basement membrane. The mixed cell suspension is in the cell culture environment, and the mixed cell suspension is promoted to proliferate in parallel on one side of the basement membrane to form a cell layer, thereby obtaining a pretreated 3D melanoma model.

[0086] It can be understood that by performing a unilateral parallel culture operation on the mixed cell suspension, it is ensured that the keratinocytes and melanoma cells in the mixed cell suspension can randomly adhere to one side of the basement membrane and form a cell layer, which effectively avoids a small amount of keratinocytes and melanoma cells The mixed cell suspension penetrates into the basement membrane and cannot truly simulate the microenvironment formed by melanoma cells in vivo.

[0087] S140, performing an air-liquid interface culture operation on the pretreated 3D melanoma model, so that the pretreated 3D melanoma model forms a cell culture environment in the thickness direction, promotes the cell layer to form a new epidermis layer, and promotes the melanoma cells to invade the extracellular matrix layer.

[0088] It can be understood that by performing an air-liquid interface culture operation on the pretreated 3D melanoma model, the new epidermis of the pretreated 3D melanoma model is fully exposed to the air, ensuring that the growth environment of the pretreated 3D melanoma model is close to the growth environment of the real skin in vivo, so that the pretreated 3D melanoma model forms a cell culture environment in the thickness direction, prompting the cell layer to form a new epidermis containing keratinocytes and melanoma cells; at the same time, it also prompts melanoma cells to break through the basement membrane and invade the extracellular matrix layer, so as to efficiently, objectively and to the greatest extent possible simulate the pathological process and physiological changes of melanoma in vivo.

[0089] The above-mentioned in vitro 3D melanoma model preparation method firstly places a mixed cell suspension of keratinocytes and melanoma cells on an in vitro dermal scaffold for inoculation treatment, and then performs a unilateral parallel culture operation on the mixed cell suspension after the inoculation treatment, so that the mixed cell suspension is attached to the side of the basement membrane away from the extracellular matrix layer and proliferates in parallel to form a cell layer, so that the keratinocytes and melanoma cells can randomly adhere to one side of the basement membrane, so as to ensure that the melanoma cells are randomly distributed on the new epidermis, so as to be closer to the real physiological state of melanoma cells in vivo; and effectively avoid a small part of the mixed cell suspension of keratinocytes and melanoma cells from infiltrating into the basement membrane and failing to truly simulate the melanoma cells. The microenvironment formed by cells in vivo; finally, the pretreated 3D melanoma model is subjected to air-liquid interface culture operation, so that the dermis layer of the pretreated 3D melanoma model is fully exposed to the air, ensuring that the growth environment of the 3D melanoma model to be cultured is close to the growth environment of the real skin in vivo, so that the pretreated 3D melanoma model forms a cell culture environment in the thickness direction, prompting the cell layer to form a new epidermis layer containing keratinocytes and melanoma cells, and at the same time, it also prompts melanoma cells to break through the basement membrane and invade the extracellular matrix layer, so as to form an obvious three-dimensional invasion phenomenon on the in vitro 3D melanoma model, so as to efficiently, objectively and to the greatest extent simulate the pathological process and physiological changes of melanoma in vivo. Fig.17 As shown, the microenvironment formed by melanoma in vivo is simulated more realistically and objectively.

[0090] In one embodiment, a mixed cell suspension is obtained by mixing keratinocytes and melanoma cells at a concentration ratio of (3-20):1.

[0091] It is understandable that if the concentration ratio of keratinocytes to melanoma cells is higher than 20:1 or lower than 3:1, it will affect the modeling effect of the in vitro 3D melanoma model, such as Fig.11 and Fig.12 As shown; therefore, in the present disclosure, by controlling the concentration ratio of keratinocytes and melanoma cells to be (3-20):1, it is ensured that the inoculation concentration of keratinocytes and melanoma cells is more appropriate. In this way, while ensuring that the in vitro 3D melanoma model has obvious three-dimensional invasion phenomenon, it is also ensured that the in vitro 3D melanoma model has a complete stratum corneum, thereby ensuring that the in vitro 3D melanoma model can efficiently, objectively and realistically simulate the pathological process and physiological changes of melanoma in vivo.

[0092] In one embodiment, the expanded keratinocytes and melanoma cells are digested with 0.125% trypsin, respectively, and then subjected to high-speed centrifugation to collect the keratinocytes and melanoma cells respectively; then the original culture medium is discarded, and FG culture medium is added to resuspend the keratinocytes and melanoma cells respectively, and then the densities of the keratinocytes and melanoma cells are counted to obtain the concentrations of the keratinocytes and melanoma cells respectively; then, according to the total volume added (100 μL to 500 μL), the two types of cells are mixed in a ratio of (3 to 20): 1, and finally the mixed keratinocytes and melanoma cells are placed in a 10 mL to 15 mL centrifuge tube for mixing to obtain a mixed cell suspension for standby use.

[0093] It should be noted that if conventional general inoculation conditions are used, such as directly inoculating the mixed cell suspension onto the surface of the basement membrane with an inoculation loop or dropper under sterile conditions, a small portion of the mixed cell suspension will infiltrate the basement membrane and fail to adhere to the basement membrane surface, thereby causing a small portion of melanoma cells or keratinocytes to directly enter the basement membrane and fail to truly simulate the microenvironment formed by melanoma cells in vivo. In addition, some conventional 3D melanoma models currently take a relatively long time to model, such as Chinese patent document number CN101392238A, which usually takes 12 to 15 days, and cannot rapidly analyze and study the pathology of melanoma cells to affect the screening of melanoma cell drugs, drug evaluation, and screening of therapies.

[0094] Therefore, in one of the embodiments, in the operation of placing the mixed cell suspension on an in vitro dermal scaffold for inoculation treatment, the specific operation is as follows: first, a hollow tube is obtained, wherein an inoculation culture channel is formed through the hollow tube, one end of the hollow tube is an abutment end, and the abutment end is used to seal and abut with the basement membrane, and the other end of the hollow tube is an inoculation end, and the inoculation end is convenient for the user to inoculate; then, the hollow tube is placed on the basement membrane, and because the hollow tube has a certain weight, the hollow tube placed on the basement membrane can better press the in vitro dermal scaffold, so that the hollow tube and the basement membrane are sealed and abutted to form a semi-closed inoculation culture channel, so that the hollow tube forms a restricted cell culture environment with the inoculation culture channel as the area on one side of the basement membrane, such as Fig.19 As shown, the mixed cell suspension is placed in the inoculation culture channel through the inoculation end, and flows along the inoculation culture channel to one side of the basement membrane, so that the mixed cell suspension can be added to the inoculation culture channel from the inoculation end. In this way, on the one hand, it ensures that the added mixed cell suspension can be randomly distributed on the basement membrane at the bottom of the semi-enclosed inoculation culture channel, so as to be closer to the real physiological state of melanoma cells in vivo; on the other hand, it reduces the exchange of the mixed cell suspension with the non-restricted cell culture environment, so as to ensure that the mixed cell suspension can grow quickly and uniformly on the basement membrane at the bottom of the semi-enclosed inoculation culture channel, so as to ensure that the mixed cell suspension can grow quickly and uniformly on the basement membrane at the bottom of the semi-enclosed inoculation culture channel; to ensure that the pathological process and physiological changes of melanoma cells invading the extracellular matrix layer can be observed after the pre-treated 3D melanoma model is cultured at the air-liquid interface for 5 to 8 days, so as to achieve rapid pathological analysis and research on melanoma in an efficient, objective and maximum extent, which will help to accelerate the screening of drugs for melanoma cells, drug evaluation, and screening of therapies. It is worth mentioning that Fig.19 The in vitro dermal scaffolds were purple, which was mainly affected by the purple culture medium.

[0095] In one embodiment, under sterile conditions, the mixed cell suspension is dripped into the inoculation culture channel from the inoculation end through a dropper. Specifically, the inoculation volume of the mixed cell suspension is 100 μL to 500 μL.

[0096] In one embodiment, before placing the mixed cell suspension on the in vitro dermal scaffold for inoculation, the following operation is also included: placing the in vitro dermal scaffold on a well plate to complete the subsequent inoculation operation. Specifically, the well plate can be 6-well, 24-well, 96-well, etc., and the user can choose to use it according to the actual situation.

[0097] In order to ensure that the hollow tube can better compact and seal the in vitro dermal scaffold, in one embodiment, the hollow tube is a hollow metal ring. The weight of the hollow metal ring is higher than that of the hollow plastic ring, so that the hollow metal ring can better compact and seal the in vitro dermal scaffold by its own weight, better limit the exchange of the mixed cell suspension and the non-restricted cell culture environment, thereby ensuring that the mixed cell suspension can grow quickly and uniformly on the basement membrane at the bottom of the semi-enclosed inoculation culture channel.

[0098] It can be understood that although the use of a heavy hollow metal ring can better compact the extracorporeal dermal stent, the abutting end of the hollow metal ring is easy to damage the extracorporeal dermal stent. Therefore, in one embodiment, the hollow tube also includes an elastic closed base, which is detachably arranged at the abutting end, and a blocking channel connected to the inoculation culture channel is formed in the elastic closed base, so that the blocking channel of the added elastic closed base can be connected to the inoculation culture channel, and the blocking channel is also used to seal and abut with the basement membrane, so that the mixed cell suspension in the inoculation culture channel can enter the basement membrane from the blocking channel, and the blocking channel can also block the exchange of the mixed cell suspension with the non-restricted cell culture environment; the added elastic closed base can reduce the damage to the in vitro dermal scaffold on the one hand, and on the other hand, the elastic closed base can enhance the sealing with the in vitro dermal scaffold, so that, while reducing the damage to the in vitro dermal scaffold, the mixed cell suspension can also settle and adhere to the basement membrane at the bottom of the semi-closed inoculation culture channel more quickly and randomly, thereby ensuring that the mixed cell suspension can grow more uniformly on the basement membrane at the bottom of the semi-closed inoculation culture channel.

[0099] Furthermore, since the elastic closed base is detachably arranged at the abutting end, it is convenient for the user to disassemble and assemble the elastic closed base, which not only facilitates the user to replace a damaged elastic closed base, but also facilitates the user to clean the hollow tube and the elastic closed base.

[0100] Specifically, in one of the embodiments, the elastic closed base includes any one of a silicone base, a polyurethane base or a thermoplastic elastomer base. In a preferred embodiment, the elastic closed base is a silicone base.

[0101] In one of the embodiments, the dimensions of the hollow metal ring are: outer diameter 16mm~34mm; height 5mm~15mm; diameter of the inoculation and culture channel is 5mm~11mm, so as to ensure that the weight of the hollow metal ring is relatively appropriate, especially with the height of the elastic closed base of 1mm~5mm and the diameter of the barrier channel of 5mm~11mm; to ensure that the height distribution of the hollow metal ring and the elastic closed base is more appropriate. In this way, on the premise that the hollow tube has a more appropriate weight, the hollow tube can achieve better sealing of the in vitro dermal stent and reduce damage to the in vitro dermal stent.

[0102] like Fig. 20 As shown, in one embodiment, a wedge-shaped portion is formed at one end of the elastic closed base away from the hollow tube, so that the added wedge-shaped portion can better seal and compact the extracorporeal dermal support, and the wedge-shaped portion can also better reduce damage to the extracorporeal dermal support.

[0103] In one embodiment, the elastic closed base is connected to one end of the hollow tube by a snap-fit ​​connection; or, the elastic closed base is connected to one end of the hollow tube by a threaded connection, so as to realize the detachable connection between the elastic closed base and the hollow tube. Of course, the detachable connection between the elastic closed base and the hollow tube can also be other connection methods used by those skilled in the art.

[0104] In one embodiment, the mixed cell suspension after inoculation is subjected to a unilateral parallel culture operation, and the specific operation is as follows: first, a culture fluid is added to the semi-enclosed inoculation culture channel to form a restricted cell culture environment with the inoculation culture channel as the area on one side of the basement membrane; the added culture fluid can better infiltrate the basement membrane, and at the same time, with the use of an elastic closed base, the infiltration of the basement membrane not only reduces the friction with the elastic closed base, but also improves the sealing with the elastic closed base, so that the mixed cell suspension can more quickly and randomly settle and adhere to the outermost layer of the basement membrane; and the added culture fluid can better wash away the mixed cell suspension adhering to the inoculation culture channel, so that the mixed cell Under the action of gravity, the suspension can quickly and randomly settle and adhere to the basement membrane that restricts the cell culture environment. Then, the mixed cell suspension after adding the culture medium is subjected to a static culture operation, so that the mixed cell suspension that randomly settles and adheres to the basement membrane can proliferate in parallel on one side of the basement membrane to form a cell layer, so as to ensure that the melanoma cells of the subsequently prepared in vitro 3D melanoma model can be randomly distributed in the new epidermis, basement membrane or dermis, so as to more objectively reflect the invasion phenomenon of melanoma cells in vivo, to truly simulate the interaction between melanoma cells and keratinocytes under the real physiological state in vivo, so as to be closer to the microenvironment formed by melanoma cells in the real body.

[0105] It is understandable that keratinocytes have a slightly lower adhesion than melanoma cells. Therefore, by adding the culture medium to the mixed cell suspension and performing a static culture operation, it is ensured that the keratinocytes can adhere well to the basement membrane, and the problem of keratinocytes falling off during the subsequent transfer pretreatment of the 3D melanoma model is effectively avoided. Specifically, the static culture operation is 1d to 2d to ensure that the keratinocytes can adhere to the basement membrane more comprehensively.

[0106] In one embodiment, in the operation of adding culture fluid to the inoculation culture channel, the added culture fluid is made to fill the inoculation culture channel, so that the filled culture fluid can completely wash away the mixed cell suspension adhering to the inoculation culture channel, thereby ensuring that the inoculated mixed cell suspension can completely and randomly settle and adhere to the outermost layer of the basement membrane that restricts the cell culture environment, effectively avoiding the mixed cell suspension from affecting the normal adhesion of keratinocytes due to the incomplete filling of the culture fluid, thereby avoiding the phenomenon that no melanoma cells and a complete new epidermis can be observed in the finally prepared in vitro 3D melanoma model, such as Fig.16 shown.

[0107] In one embodiment, in the operation of performing air-liquid interface culture operation on the pre-treated 3D melanoma model, the specific operation is as follows: first, the hollow cylinder of the pre-treated 3D melanoma model is removed to expose the cell layer, thereby obtaining the 3D melanoma model to be cultured; then, the 3D melanoma model to be cultured is placed on a well plate with a support frame; then, a culture fluid is added around the well plate, such as Fig.21 As shown, the liquid level of the culture fluid is not higher than the surface of the 3D melanoma model to be cultured, so that the cell layer is exposed to the air, ensuring that the growth environment of the 3D melanoma model to be cultured is close to the growth environment of the real skin in vivo, and then the 3D melanoma model to be cultured is subjected to air-liquid interface culture for 5 to 8 days, and then the pathological process and physiological changes of melanoma cells invading the extracellular matrix layer can be seen, which effectively shortens the time for modeling the in vitro 3D melanoma model.

[0108] It should be noted that adding culture medium around the well plate can effectively prevent the culture medium from dispersing the adhered cell layer and affecting the in vitro 3D melanoma model's real effect of simulating melanoma in vivo.

[0109] In one embodiment, when adding culture fluid to the periphery of the well plate, the liquid level of the culture fluid is 0 mm to 5 mm below the surface of the 3D melanoma model to be cultured, so as to ensure that the growth environment of the 3D melanoma model to be cultured is close to the growth environment of the real skin in vivo.

[0110] In a preferred embodiment, the liquid surface of the culture fluid is flush with the surface of the 3D melanoma model to be cultured, so as to ensure that the outermost surface of the 3D melanoma model to be cultured just contacts the air. In this way, while ensuring that sufficient nutrients can be provided to the 3D melanoma model to be cultured during air-liquid interface culture, it provides effective guarantees for efficient modeling of the in vitro 3D melanoma model, and also ensures that the growth environment of the 3D melanoma model to be cultured is close to the growth environment of the real skin in vivo.

[0111] like Fig.18 and Fig.21As shown, in one embodiment, the support frame is a metal mesh support frame. Since the metal mesh support frame has a certain porosity, the culture fluid can more comprehensively infiltrate the bottom of the 3D melanoma model to be cultured, so as to ensure that the 3D melanoma model to be cultured has sufficient nutrients, so as to ensure that the melanoma cells can better break through the basement membrane and invade the extracellular matrix layer, and ensure that the finally prepared in vitro 3D melanoma model can clearly see the pathological process and physiological changes of melanoma cell invasion.

[0112] Specifically, in a preferred embodiment, the dimensions of the metal mesh support are: 16 mm to 24 mm in length, 16 mm to 24 mm in width, 2 mm to 10 mm in height, and 2 mm to 5 mm in aperture.

[0113] In one embodiment, obtaining keratinocytes comprises the following operations: first, digesting and decomposing the epidermis of the second ex vivo skin, so that undifferentiated keratinocytes remain above the dermis layer of the second ex vivo skin; then, isolating and culturing the undifferentiated keratinocytes to obtain keratinocytes.

[0114] In one embodiment, in the operation of digesting and decomposing the epidermis of the second ex vivo skin, the specific operation is as follows: soaking the second ex vivo skin with a digestive enzyme to digest and decompose the epidermis of the second ex vivo skin.

[0115] Specifically, in one embodiment, the second ex vivo skin is soaked in digestive enzymes for 12 hours to 16 hours at a temperature of 2° C. to 8° C. to digest and decompose the epidermis. Furthermore, the digestive enzymes are pancreatic enzymes at a concentration of 0.01% to 0.5%.

[0116] In one of the embodiments, in the operation of isolating and culturing undifferentiated keratinocytes, the specific operation is as follows: first, the undifferentiated keratinocytes remaining above the dermis of the second ex vivo skin are prepared into a suspension containing undifferentiated keratinocytes; then, the suspension containing undifferentiated keratinocytes is centrifuged and filtered to obtain undifferentiated keratinocytes; finally, the undifferentiated keratinocytes are amplified and cultured using a modified culture medium, so that the prepared keratinocytes have good proliferation and differentiation abilities, thereby ensuring that the subsequent in vitro 3D melanoma model can form a complete new epidermis layer, and ensuring the high efficiency of the in vitro 3D melanoma model modeling.

[0117] It can be understood that compared with the traditional general FG culture medium, the cell growth cycle is longer and cannot have good proliferation and differentiation capabilities, which results in that during the subsequent mixed inoculation, keratinocytes with poor proliferation and differentiation capabilities cannot quickly adhere to the surface of the basement membrane. Therefore, in one embodiment, the modified culture medium includes a basal culture medium, additives and a variety of growth factors, and the multiple growth factors include the following components: 170μL to 1000μL of fibroblast growth factor; 100μL to 500μL of transforming growth factor; 1μL to 17μL of epidermal growth factor, and the added fibroblast growth factor, transforming growth factor and epidermal growth factor ensure that the prepared keratinocytes have good proliferation and differentiation capabilities, such as Fig.11 As shown, this ensures that keratinocytes can adhere randomly to the outermost layer of the basement membrane during subsequent inoculation; it also ensures that keratinocytes can proliferate to form a complete new epidermis layer to ensure the efficiency of in vitro 3D melanoma modeling.

[0118] In one embodiment, the basal culture medium includes at least one of a DMEM culture medium and a HAMS culture medium. In a preferred embodiment, the basal culture medium includes a DMEM culture medium and a HAMS culture medium.

[0119] In one embodiment, the additives include fetal bovine serum, double antibody, L-glutamine, adenine, L-thyroxine sodium salt, transferrin, non-essential amino acid solution and insulin to ensure that the prepared keratinocytes have good proliferation and differentiation capabilities.

[0120] Specifically, in one embodiment, DMEM medium 280mL~350mL; HAMS medium 100mL~110mL; fetal bovine serum 45mL~55mL; double antibody 1mL~5mL; L-glutamine 1mL~5mL; adenine 1mL~5mL; L-thyronine sodium salt 200μL~1000μL; transferrin 200μL~1000μL; non-essential amino acid solution 200μL~1000μL; insulin 200μL~1000μL; fibroblast growth factor 200μL~1000μL; transforming growth factor 100μL~500μL; epidermal growth factor 1μL~20μL.

[0121] In one embodiment, the expansion culture temperature is 28° C. to 38° C. and the time is 5 to 14 days to ensure that the prepared keratinocytes have good proliferation and differentiation capabilities.

[0122] In one embodiment, the first ex vivo skin and the second ex vivo skin are derived from the same raw material to reduce the problem of keratinocytes from different sources being seeded on an in vitro dermal scaffold and causing greater rejection and thus affecting adhesion.

[0123] In one embodiment, the same ex vivo skin is obtained, and the collection liquid is used to soak the same ex vivo skin, and then the fat tissue is removed, and then the same ex vivo skin is cut into 4 cm 2 The ex vivo skin is cut into square pieces to obtain the first ex vivo skin; and the ex vivo skin fragments cut off are the second ex vivo skin, so as to effectively reduce the problem of keratinocytes from different sources being inoculated on the in vitro dermal scaffold and causing greater rejection and affecting adhesion.

[0124] The present disclosure also provides an in vitro 3D melanoma model, which is prepared by the method for preparing an in vitro 3D melanoma model of any of the above embodiments.

[0125] It can be understood that by placing the hollow tube on the basement membrane, the hollow tube and the basement membrane are sealed and abutted to form a semi-closed inoculation culture channel; then the mixed cell suspension is placed in the inoculation culture channel through the inoculation end, and flows along the inoculation culture channel to one side of the basement membrane, and at the same time, the culture fluid is added to the semi-closed inoculation culture channel, so that the hollow tube forms a restricted cell culture environment with the inoculation culture channel as the area on one side of the basement membrane, thereby ensuring that the mixed cell suspension can randomly settle and adhere to the outermost layer of the basement membrane in the restricted cell culture environment for parallel proliferation, thereby ensuring that the melanoma cells are randomly distributed on the in vitro 3D melanoma model to form an obvious three-dimensional invasion phenomenon, so as to efficiently, objectively and to the greatest extent possible simulate the pathological process and physiological changes of melanoma under the real physiological state in vivo.

[0126] The present disclosure also provides an application of an in vitro 3D melanoma model, and the in vitro 3D melanoma model of any of the above embodiments is applied to the analysis of melanoma pathology, the screening of anti-melanoma drugs, the evaluation of anti-melanoma drugs, the screening of anti-melanoma therapies, or the evaluation of anti-melanoma therapies, so as to simulate the pathological process and physiological changes of melanoma in vivo to the greatest extent, which is conducive to promoting the treatment research of melanoma.

[0127] Some specific examples are given below, and if % is mentioned, it means percentage by weight. It should be noted that the following examples do not exhaust all possible situations, and the materials used in the following examples can be obtained from commercial sources unless otherwise specified.

[0128] Example 1

[0129] S1. Fabrication of in vitro dermal scaffolds:

[0130] S11. Obtain discarded skin samples after surgery;

[0131] S12, place the skin sample in the collection solution (PBS antimicrobial agent, triple antibody and gentamicin) for half an hour;

[0132] S13, taking out the skin sample from the collection fluid, cutting off the large pieces of fat tissue in the dermis layer on the skin sample with scissors, and then further scraping off the fat tissue fragments with a scraper to obtain a skin sample;

[0133] S14, rinse the cortical sample with PBS antiseptic;

[0134] S15. Cut the cortical sample into 4 cm 2 to obtain the first ex vivo skin; and the small pieces cut off are the second ex vivo skin for later use.

[0135] S16. Immerse the first ex vivo skin in a 1.0 mol / L sodium chloride solution for 16 hours to obtain an in vitro dermal scaffold, namely, a DED dermal scaffold.

[0136] S2. Acquisition of keratinocytes

[0137] S21, soaking the second ex vivo skin in the S15 operation in 0.25% pancreatic enzyme for 14 hours at a soaking temperature of 8° C. to obtain residual undifferentiated keratinocytes above the dermis;

[0138] S22, preparing a suspension containing undifferentiated keratinocytes by using the undifferentiated keratinocytes remaining above the dermis layer of the second ex vivo skin;

[0139] S23, centrifuging and filtering the suspension containing undifferentiated keratinocytes to obtain undifferentiated keratinocytes;

[0140] S24. Use modified culture medium to expand and culture undifferentiated keratinocytes to obtain keratinocytes. The expansion temperature is 37°C and the time is 5 days. Among them, 300 mL of DMEM culture medium; 105 mL of HAMS culture medium; 50 mL of fetal bovine serum; 3 mL of double antibody; 3 mL of L-glutamine; 3 mL of adenine; 600 μL of L-thyronine sodium salt; 600 μL of transferrin; 600 μL of non-essential amino acid solution; 600 μL of insulin; 600 μL of fibroblast growth factor; 300 μL of transforming growth factor; and 10 μL of epidermal growth factor.

[0141] S3. Preparation of mixed cell suspension

[0142] Keratinocytes and melanoma cells were mixed at a concentration ratio of 3:1 in a 15 mL sterile centrifuge tube to obtain a mixed cell suspension.

[0143] S4. Inoculation operation

[0144] S41, placing the in vitro dermal scaffold obtained by operation S16 in a 24-well plate;

[0145] S42, pressing the hollow metal ring onto the DED dermal support, wherein the abutting end of the hollow metal ring is provided with a silicone base, and forms a semi-enclosed inoculation and culture channel with the DED dermal support, such as Fig.19 As shown;

[0146] S43, using a dropper, add 100 μL of the mixed cell suspension along the inoculation end of the semi-enclosed inoculation culture channel, so that the mixed cell suspension will adhere to the basement membrane of the DED dermal scaffold along the inoculation culture channel;

[0147] S44, adding a cell-free culture solution to the semi-enclosed inoculation culture channel, and filling the inoculation culture channel with the cell-free culture solution;

[0148] S45, after two days of static culture, the mixed cell suspension is allowed to adhere to the DED dermal scaffold to obtain a pretreated 3D melanoma model;

[0149] S5. Air-Liquid Interface Culture Operation for Pre-treated 3D Melanoma Model

[0150] S51, removing the hollow metal ring to obtain a 3D melanoma model to be cultured;

[0151] S52. Use tweezers to transfer the 3D melanoma model to be cultured to the stainless steel metal mesh support of a suitable well plate; the size of the stainless steel metal mesh support is 20*20*6mm. 2 , the small hole of the stainless steel metal mesh bracket is 4mm 2 ;

[0152] S53, adding cell culture fluid around the well plate in step S52, making the liquid level of the cell culture fluid 0 mm below the surface of the 3D melanoma model to be cultured, maintaining the culture at the air-liquid interface level for 7 days, and obtaining an in vitro 3D melanoma model.

[0153] Example 2

[0154] The difference from Example 1 is that the 1.0 mol / L sodium chloride solution in the S16 operation of Example 1 is replaced by a 1.5 mol / L potassium chloride solution, and the rest remains unchanged.

[0155] Example 3

[0156] The difference from Example 1 is that the 1.0 mol / L sodium chloride solution in the S16 operation of Example 1 is replaced by a 1.6 mol / L glucose solution, and the rest remains unchanged.

[0157] Example 4

[0158] The difference from Example 1 is that the 1.0 mol / L sodium chloride solution in the S16 operation of Example 1 is replaced by a 2.0 mol / L sodium chloride solution, and the rest remains unchanged.

[0159] Example 5

[0160] The difference from Example 1 is that the 1.0 mol / L sodium chloride solution in the S16 operation of Example 1 is replaced by a 0.8 mol / L sodium chloride solution, and the rest remains unchanged.

[0161] Example 6

[0162] The difference from Example 1 is that 16h in the S16 operation of Example 1 is replaced by 12h, and the rest remains unchanged.

[0163] Example 7

[0164] The difference from Example 1 is that the amount of modified culture medium used in the S24 operation is different. The modified culture medium of Example 7 includes 280 mL of DMEM culture medium; 100 mL of HAMS culture medium; 45 mL of fetal bovine serum; 1 mL of double antibody; 1 mL of L-glutamine; 1 mL of adenine; 200 μL of L-thyronine sodium salt; 200 μL of transferrin; 200 μL of non-essential amino acid solution; 200 μL of insulin; 1000 μL of fibroblast growth factor; 500 μL of transforming growth factor; and 20 μL of epidermal growth factor.

[0165] Example 8

[0166] The difference from Example 1 is that the amount of modified culture medium used in the S24 operation is different, that is, the modified culture medium of Example 8 includes 350 mL of DMEM culture medium; 110 mL of HAMS culture medium; 55 mL of fetal bovine serum; 5 mL of double antibody; 5 mL of L-glutamine; 5 mL of adenine; 1000 μL of L-thyronine sodium salt; 1000 μL of transferrin; 1000 μL of non-essential amino acid solution; 1000 μL of insulin; 500 μL of fibroblast growth factor; 500 μL of transforming growth factor; and 20 μL of epidermal growth factor.

[0167] Example 9

[0168] The difference from Example 1 is that the concentration ratio in the S3 operation of Example 1 is replaced with 10:1, and the rest remains unchanged.

[0169] Example 10

[0170] The difference from Example 1 is that the concentration ratio in the S3 operation of Example 1 is replaced with 20:1, and the rest remains unchanged.

[0171] Embodiment 11

[0172] The difference from Example 1 is that the distance in which the liquid level of the cell culture medium is lower than the surface of the 3D melanoma model to be cultured in the S53 operation of Example 1 is set to 0 mm, the 0 mm for maintaining the culture at the air-liquid interface level for 7 days is replaced by 3 mm, and 7 days is replaced by 8 days, while the rest remains unchanged.

[0173] Example 12

[0174] The difference from Example 1 is that the distance of making the liquid surface of the cell culture medium lower than the surface of the pre-treated 3D melanoma model in the S53 operation of Example 1 is replaced by 5 mm from 0 mm, and 7 d is replaced by 8 d, and the rest remains unchanged.

[0175] Comparative Example 1

[0176] The difference from Example 1 is that the 1.0 mol / L sodium chloride solution in the S16 operation of Example 1 is replaced by a 3.2 mol / L sodium chloride solution, and the rest remains unchanged.

[0177] Comparative Example 2

[0178] The difference from Example 1 is that the 1.0 mol / L sodium chloride solution in the S16 operation of Example 1 is replaced by a 0.5 mol / L sodium chloride solution, and the rest remains unchanged.

[0179] Comparative Example 3

[0180] The difference from Example 1 is that 16h in the S16 operation of Example 1 is replaced by 18h, and the rest remains unchanged.

[0181] Comparative Example 4

[0182] The difference from Example 1 is that 16h in the S16 operation of Example 1 is replaced by 10h, and the rest remains unchanged.

[0183] Comparative Example 5

[0184] The difference from Example 1 is that the S16 operation of Example 1 directly adopts 10 consecutive liquid nitrogen freezing and room temperature cycle operations of Chinese patent document No. CN101392238A.

[0185] Comparative Example 6

[0186] The difference from Example 1 is that the operation steps S11 to S16 of Example 1 are omitted, and the normal untreated ex vivo skin is directly stained to observe the structure of the normal basement membrane in vivo.

[0187] Comparative Example 7

[0188] The difference from Example 1 is that the fibroblast growth factor in the modified culture medium in operation S24 of Example 1 is omitted.

[0189] Comparative Example 8

[0190] The difference from Example 1 is that the transforming growth factor for modifying the culture medium in operation S24 of Example 1 is omitted.

[0191] Comparative Example 9

[0192] The difference from Example 1 is that the epidermal growth factor in the modified culture medium in operation S24 of Example 1 is omitted.

[0193] Comparative Example 10

[0194] The difference from Example 1 is that the concentration ratio in the S3 operation of Example 1 is replaced with 22:1, and the rest remains unchanged.

[0195] Comparative Example 11

[0196] The difference from Example 1 is that the concentration ratio in the S3 operation of Example 1 is replaced with 2:1, and the rest remains unchanged.

[0197] Comparative Example 12

[0198] The difference from Example 1 is that the S3 operation of Example 1 is omitted, and in the S4 inoculation operation, keratinocytes are inoculated first and then melanoma cells are inoculated, and the rest remain unchanged.

[0199] Comparative Example 13

[0200] The difference from Example 1 is that the S3 operation of Example 1 is omitted, and in the S4 inoculation operation, melanoma cells are inoculated first and then keratinocytes are inoculated, and the rest remain unchanged.

[0201] Comparative Example 14

[0202] The difference from Example 1 is that the hollow metal ring and the silicone base in the S42 operation of Example 1 are omitted, and the rest remain unchanged.

[0203] Comparative Example 15

[0204] The difference from Example 1 is that the cell-free culture solution in the S44 operation of Example 1 is omitted and the inoculation culture channel is not filled, and the rest remains unchanged.

[0205] Comparative Example 16

[0206] The difference from Example 1 is that the operations of S2, S3 and S4 of Example 1 are omitted, that is, a blank experiment, and the rest remain unchanged.

[0207] The DED dermal scaffolds prepared in the above Examples 1 to 12 and Comparative Examples 1 to 6 were subjected to immunohistochemical analysis;

[0208] Among them, the method of immunohistochemical detection and analysis is as follows: collect the samples to be tested and fix them in 4% paraformaldehyde for 24 to 48 hours, then slice them to obtain samples with a thickness of 4μm; incubate the antibody (collagen type IV, Biyuntian AG8299) with the sample at 4°C overnight. Then wash it three times with PBS solution, and then incubate it with the corresponding secondary antibody at a constant temperature of 37°C for 1h. Wash the sample three times with PBS, and then develop it with DAB color developing solution; then dehydrate and make it transparent with anhydrous ethanol and xylene, and finally observe the staining under a microscope; and the brown staining indicated by the red arrow is the expression of collagen IV, representing the basement membrane at the top of the dermis.

[0209] As can be seen from the comparison between Examples 1 to 12 and Comparative Example 6, the hypertonic pressure of the hypertonic dehydrating agent used in Examples 1 to 12 is relatively suitable, and the immersion method of the hypertonic dehydrating agent is used to remove the cells of the first ex vivo skin, and the basement membrane and the extracellular matrix are completely retained. Figures 2 to 5 As shown, among them, the structural staining microscope image of the basement membrane of Example 1 is the strongest, proving that Example 1 has a good ability to retain the integrity of the basement membrane. The brown staining indicated by the red arrow is the expression of collagen IV, representing the basement membrane at the top of the dermis; and from Figures 1 to 6 It can be seen that the structural staining microscopic image of the basement membrane of Example 1 is closest to the structural staining microscopic image of Comparative Example 6, which proves that the structure of the basement membrane prepared in Examples 1 to 12 is closer to the real basement membrane structure in vivo.

[0210] Furthermore, from the comparison between Example 1 and Comparative Example 1, it can be seen that since Comparative Example 1 uses a 3.2 mol / L sodium chloride solution, the osmotic pressure is high and the damage to the basement membrane is relatively large, so that the structural staining microscope image of the basement membrane of Comparative Example 1 is significantly weaker than that of Example 1; this proves that Comparative Example 1 has a poor ability to retain the integrity of the basement membrane. For details, please refer to Figure 2 .

[0211] From the comparison between Example 1 and Comparative Example 2, it can be seen that since Comparative Example 2 uses 0.5 mol / L sodium chloride solution, the osmotic pressure is low and the basement membrane cannot be separated well, resulting in the basement membrane structure staining microscope image of Comparative Example 2 being significantly weaker than that of Example 1, proving that Comparative Example 2 has poor ability to retain the integrity of the basement membrane. For details, please refer to Figure 3 .

[0212] From the comparison between Example 1 and Comparative Examples 3 and 4, it can be seen that the basement membrane is damaged greatly due to the long immersion time of Comparative Example 3, resulting in extremely weak collagen IV staining in Comparative Example 3, which proves that Comparative Example 3 has poor ability to retain the integrity of the basement membrane. Please refer to Figure 4 Since the immersion time of Comparative Example 4 was too short, the basement membrane could not be separated well, resulting in the structural staining microscope image of the basement membrane of Comparative Example 4 being significantly weaker than that of Example 1, proving that Comparative Example 4 has poor ability to retain the integrity of the basement membrane.

[0213] From the comparison between Example 1 and Comparative Example 5, it can be seen that since Comparative Example 5 uses multiple cycles of liquid nitrogen and room temperature, the structural damage to the basement membrane is relatively large, resulting in the intensity of the structural staining microscope image of the basement membrane of Comparative Example 5 being significantly weaker than that of Example 1, proving that Comparative Example 5 has a poor ability to retain the integrity of the basement membrane. For details, please refer to Figure 5 .

[0214] The in vitro 3D melanoma models prepared in the above Examples 1 to 12 and Comparative Examples 1 to 16 were subjected to immunohistochemical analysis;

[0215] Since Examples 1 to 12 completely retain the basement membrane structure and cooperate with the special inoculation method of the hollow metal ring, the mixed cell suspension can randomly settle and adhere to the outermost layer of the basement membrane in the restricted cell culture environment for parallel proliferation, thereby ensuring that the melanoma cells are randomly distributed on the in vitro 3D melanoma model to form an obvious three-dimensional invasion phenomenon, so as to maximize and objectively simulate the pathological process and physiological changes of melanoma in vivo. For details, please refer to Fig.17 Example 1 can clearly observe the trend of melanoma cells proliferating to the dermis and neoepidermis in the in vitro 3D melanoma model. At the same time, it can also be clearly seen that the melanoma cells break through the basement membrane and invade the dermis downward, and the efficiency of in vitro 3D melanoma model building is improved. Among them, the comprehensive indicators of Example 1 are the best.

[0216] Specifically, it can be seen from the comparison between Example 1 and Comparative Examples 1 to 5 that Figure 7 to Figure 10 As shown, due to the poor integrity of the basement membranes of Comparative Examples 1 to 5, the in vitro 3D melanoma models of Comparative Examples 1 to 5 cannot form a complete epidermis and cannot observe the characteristics of melanoma cell aggregation and erosion. For example, in the in vitro 3D melanoma model of Comparative Example 5, only sporadic melanoma cells and an incomplete new epidermis can be observed, and the phenomenon of melanoma cells aggregation and invasion of the dermis or the new epidermis cannot be observed. That is, Comparative Examples 1 to 5 cannot efficiently, objectively and obviously observe the invasion phenomenon of melanoma, and thus cannot truly simulate the pathological process and physiological changes of melanoma in vivo.

[0217] From the comparison between Examples 1, 7, and 8 and Comparative Examples 7 to 9, it can be seen that since fibroblast growth factor, transforming growth factor, and epidermal growth factor are added simultaneously in Examples 1, 7, and 8, the keratinocytes prepared in Examples 1, 7, and 8 have good proliferation and differentiation abilities. For details, refer to Fig.11 The cell viability comparison chart of keratinocytes of Example 1 and Comparative Example 7 is shown to ensure that the in vitro 3D melanoma model of Example 1 can efficiently, objectively and clearly observe the pathological process and physiological changes of melanoma in vivo.

[0218] From the comparison between Examples 1, 8, 9 and Comparative Examples 10-11, it can be seen that when the ratio of keratinocytes to melanoma cells in the cell mixed suspension of Comparative Example 10 is 22:1, Fig.11 As shown, the invasion phenomenon of the in vitro 3D melanoma model of Example 1 is more obvious than that of the in vitro 3D melanoma model of Comparative Example 10; when the ratio of keratinocytes to melanoma cells in the cell mixed suspension of Comparative Example 11 is 2:1, as Fig.13 As shown, although the invasion phenomenon of the in vitro 3D melanoma model of Comparative Example 11 is more obvious than that of the in vitro 3D melanoma model of Example 2, the stratum corneum of Comparative Example 11 is also damaged, such as the defective stratum corneum in the upper right corner of the structural staining microscope image of the in vitro 3D melanoma model of Comparative Example 11, which proves that this ratio cannot ensure a complete stratum corneum. Therefore, when the concentration ratio of keratinocytes to melanoma cells is (3-20):1, while ensuring that the in vitro 3D melanoma model has obvious three-dimensional invasion phenomenon, it also ensures that the in vitro 3D melanoma model has a complete stratum corneum, thereby ensuring that Examples 1, 8, and 9 can efficiently, objectively, and realistically simulate the pathological process and physiological changes of melanoma in vivo.

[0219] From the comparison between Example 1 and Comparative Example 12, it can be seen that since Comparative Example 12 is inoculated with keratinocytes first and then with melanoma cells, Fig.14 As shown, no obvious melanoma was observed in the in vitro 3D melanoma model of Comparative Example 12. Therefore, if keratinocytes are inoculated first and then melanoma cells are inoculated, the growth of melanoma cells in the dermal scaffold in vitro will be affected; from the comparison between Example 1 and Comparative Example 13, it can be seen that since Comparative Example 13 is inoculated first with melanoma cells and then with keratinocytes, Fig.15As shown, although the in vitro 3D melanoma model of Comparative Example 13 can see the growth of melanoma cells in the in vitro dermal scaffold, no complete new epidermis can be observed in the in vitro 3D melanoma model. Therefore, when the keratinocytes in the cell mixed suspension and the melanoma cells are inoculated simultaneously in Example 1, it is ensured that the keratinocytes and the melanoma cells start the cell-to-cell interaction at the beginning, thereby ensuring that the in vitro 3D melanoma model of Example 1 has obvious three-dimensional invasion phenomenon, and also ensures that the in vitro 3D melanoma model has a complete stratum corneum, thereby ensuring that Example 1 can efficiently, objectively and realistically simulate the pathological process and physiological changes of melanoma in vivo, so as to be more realistically close to the microenvironment formed by melanoma in vivo.

[0220] From the comparison between Example 1 and Comparative Example 14, it can be seen that since Comparative Example 14 does not add a silicone base, the surface of the in vitro dermal scaffold of Comparative Example 14 shows traces of slight damage, which affects the growth of the new epidermis layer of the in vitro 3D melanoma model, resulting in no obvious invasion phenomenon in the in vitro 3D melanoma model.

[0221] From the comparison between Example 1 and Comparative Example 15, it can be seen that since the culture solution added in Comparative Example 15 does not fill the inoculation culture channel, it will affect the normal adhesion of the cell mixed suspension to the basement membrane of the in vitro dermal scaffold, resulting in only a very thin layer of keratinocytes being observed in Comparative Example 15, while no melanoma cells and a complete new epidermis layer can be observed. Fig.16 shown.

[0222] The above embodiments only express several implementation methods of the present disclosure, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the disclosed patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present disclosure, and these all belong to the protection scope of the present disclosure. Therefore, the protection scope of the disclosed patent shall be subject to the attached claims.

Claims

1. An in vitro dermal scaffold, characterized in that: The in vitro dermal scaffold includes a basement membrane and an extracellular matrix layer connected to each other, wherein the first ex vivo skin is subjected to hypertonic dehydration to remove cells of the first ex vivo skin, while the basement membrane and the extracellular matrix are completely retained to form the basement membrane and the extracellular matrix layer connected to each other.

2. The in vitro dermal scaffold according to claim 1, characterized in that: The first ex vivo skin is subjected to hypertonic dehydration using a hypertonic dehydrating agent.

3. The in vitro dermal scaffold according to claim 1, characterized in that: The hypertonic dehydrating agent includes at least one of a high-concentration salt solution and a high-concentration organic solution.

4. The in vitro dermal scaffold according to claim 3, characterized in that: The high-concentration salt solution includes at least one of a high-concentration sodium chloride solution, a high-concentration potassium chloride solution, a high-concentration sodium sulfate solution, a potassium nitrate solution and a calcium chloride solution.

5. The in vitro dermal scaffold according to claim 3, characterized in that: The high-concentration organic solution includes at least one of a high-concentration glucose solution, a high-concentration mannitol solution, and a high-concentration sorbitol solution.

6. The in vitro dermal scaffold according to claim 4, characterized in that: The concentration of the high-concentration sodium chloride solution is 0.8 mol / L to 2.0 mol / L.

7. The in vitro dermal scaffold according to claim 1, characterized in that: The first ex vivo skin is subjected to hypertonic dehydration using a hypertonic dehydrating agent, and the specific operation is as follows: The first ex vivo skin is immersed in a hypertonic dehydrating agent to hypertonic dehydrate the cells on the first ex vivo skin.

8. The in vitro dermal scaffold according to claim 7, characterized in that: Using a hypertonic dehydrating agent to soak the first ex vivo skin for 12 hours to 16 hours; and / or, The first ex vivo skin is immersed in a hypertonic dehydrating agent at a temperature of 28° C. to 38° C.; and / or, The size of the first ex vivo skin is 4 cm 2 ~6cm 2 and / or, The shape of the first ex vivo skin is any one of a triangle, a quadrilateral, a polygon, a circle or a sector; and / or, The first ex vivo skin is soaked in a hypertonic dehydrating agent so that the first ex vivo skin is immersed in the hypertonic dehydrating agent.

9. The in vitro dermal scaffold according to claim 1, characterized in that: Before performing the hypertonic dehydration operation on the first ex vivo skin, the method further includes performing a fat removal operation on the first ex vivo skin to remove fat tissue on the first ex vivo skin.

10. The in vitro dermal scaffold according to claim 9, characterized in that: The first ex vivo skin is subjected to a fat removal operation, and the specific operation is as follows: The large pieces of fat tissue on the first ex-vivo skin are removed, and then the fat tissue fragments on the first ex-vivo skin are removed, and then the first ex-vivo skin is rinsed with an antibacterial agent.

11. The in vitro dermal scaffold according to claim 10, characterized in that: The first ex vivo skin is subjected to a fat removal operation, and the specific operation is as follows: The large pieces of fat tissue on the first excised skin are cut off, and then the fat tissue fragments on the first excised skin are scraped off, and then the first excised skin is rinsed with PBS antibacterial agent.

12. The in vitro dermal scaffold according to any one of claims 9 to 11, characterized in that: Before performing the fat removal operation on the first ex-vivo skin, the method further includes the following operation: performing a sterilization operation on the first ex-vivo skin.

13. The in vitro dermal scaffold according to claim 11, characterized in that: The first ex vivo skin is sterilized, and the specific operation is as follows: The first ex vivo skin is soaked in the collection liquid to kill microorganisms on the first ex vivo skin.

14. The in vitro dermal scaffold according to claim 13, characterized in that: The collection solution includes at least one of PBS antibacterial agent, triple antibiotic and gentamicin.

15. A method for preparing an in vitro 3D melanoma model, characterized in that: The steps include: obtaining a mixed cell suspension, wherein the mixed cell suspension comprises keratinocytes and melanoma cells; Placing the mixed cell suspension on the in vitro dermal scaffold according to any one of claims 1 to 14 for inoculation treatment, so that the mixed cell suspension is randomly attached to one side of the basement membrane; The mixed cell suspension after the inoculation treatment is subjected to a unilateral parallel culture operation, so that a cell culture environment is formed on one side of the basement membrane, and the mixed cell suspension is in the cell culture environment, so that the mixed cell suspension is promoted to proliferate in parallel on one side of the basement membrane to form a cell layer, thereby obtaining a pre-treated 3D melanoma model; The pretreated 3D melanoma model is subjected to an air-liquid interface culture operation, so that the pretreated 3D melanoma model forms a cell culture environment in the thickness direction, prompts the cell layer to form a new epidermis layer, and prompts the melanoma cells to invade the extracellular matrix layer.

16. The method for preparing an in vitro 3D melanoma model according to claim 15, characterized in that: To obtain a mixed cell suspension, follow these steps: The keratinocytes and the melanoma cells are mixed at a concentration ratio of (3-20):1 to obtain the mixed cell suspension.

17. The method for preparing an in vitro 3D melanoma model according to claim 15, characterized in that: In the operation of placing the mixed cell suspension on the in vitro dermal scaffold for inoculation, the specific operation is as follows: Obtain a hollow tube, wherein the hollow tube is penetrated to form an inoculation and culture channel, one end of the hollow tube is an abutment end, and the other end of the hollow tube is an inoculation end; Placing the hollow tube on the basement membrane so that the abutting end of the hollow tube is in sealing abutment with the basement membrane to form a semi-enclosed inoculation and culture channel; The mixed cell suspension is placed in the inoculation culture channel through the inoculation end, and flows along the inoculation culture channel to one side of the basement membrane.

18. The method for preparing an in vitro 3D melanoma model according to claim 17, characterized in that: The hollow tube also includes an elastic closed base, which is detachably arranged at the abutting end, and a blocking channel connected to the inoculation and culture channel is formed in the elastic closed base, and the blocking channel is used for sealing and abutting with the basement membrane.

19. The method for preparing an in vitro 3D melanoma model according to claim 18, characterized in that: The hollow cylinder is a hollow metal ring.

20. The method for preparing an in vitro 3D melanoma model according to claim 19, characterized in that: The dimensions of the hollow metal ring are: outer diameter 16 mm to 34 mm; height 5 mm to 15 mm; the diameter of the inoculation and culture channel is 5 mm to 11 mm; and / or, The height of the elastic closed base is 1 mm to 5 mm; and / or, The diameter of the blocking channel is 5 mm to 11 mm; and / or, The elastic closed base includes any one of a silicone base, a polyurethane base or a thermoplastic elastomer base; and / or, A wedge-shaped portion is formed at one end of the elastic closed base away from the hollow cylinder.

21. The method for preparing an in vitro 3D melanoma model according to claim 18, characterized in that: The elastic closed base is connected to one end of the hollow tube by snap-fitting; or, The elastic closed base is threadedly connected to one end of the hollow tube.

22. The method for preparing an in vitro 3D melanoma model according to any one of claims 17 to 21, characterized in that: The mixed cell suspension after the inoculation treatment is subjected to a unilateral parallel culture operation, and the specific operation is as follows: Adding culture fluid to the semi-enclosed inoculation culture channel to form a restricted cell culture environment with the inoculation culture channel as the area on one side of the basement membrane; The mixed cell suspension after adding the culture fluid is subjected to a static culture operation, so that the mixed cell suspension is allowed to settle and adhere to one side of the basement membrane that restricts the cell culture environment and proliferate in parallel.

23. The method for preparing an in vitro 3D melanoma model according to claim 22, characterized in that: In the operation of adding culture solution to the inoculation culture channel, the added culture solution is made to fill the inoculation culture channel.

24. The method for preparing an in vitro 3D melanoma model according to claim 22, characterized in that: In the operation of performing the air-liquid interface culture operation on the pretreated 3D melanoma model, the specific operation is as follows: removing the hollow cylinder of the pretreated 3D melanoma model to obtain a 3D melanoma model to be cultured; Placing the 3D melanoma model to be cultured on a well plate with a support frame; Adding culture fluid around the well plate so that the liquid level of the culture fluid is not higher than the surface of the 3D melanoma model to be cultured; The 3D melanoma model to be cultured is cultured at the air-liquid interface for 5 to 8 days.

25. The method for preparing an in vitro 3D melanoma model according to claim 24, characterized in that: In the operation of adding culture solution to the periphery of the well plate, the liquid level of the culture solution is 0 mm to 5 mm below the surface of the 3D melanoma model to be cultured.

26. The method for preparing an in vitro 3D melanoma model according to claim 24, characterized in that: The support frame is a metal mesh support frame.

27. The method for preparing an in vitro 3D melanoma model according to claim 26, characterized in that: The dimensions of the metal mesh bracket are: length 16mm-24mm, width 16mm-24mm, height 2mm-10mm, and aperture 2mm-5mm.

28. The method for preparing an in vitro 3D melanoma model according to claim 15, characterized in that: The acquisition of keratinocytes comprises the following operations: performing epidermal digestion and decomposition on the second ex vivo skin, so that undifferentiated keratinocytes remain above the dermis layer of the second ex vivo skin; The undifferentiated keratinocytes are separated and cultured to obtain the keratinocytes.

29. The method for preparing an in vitro 3D melanoma model according to claim 28, characterized in that: In the operation of epidermal digestion and decomposition of the second ex vivo skin, the specific operation is as follows: The second ex vivo skin is soaked with digestive enzymes to digest and decompose the epidermis of the second ex vivo skin.

30. The method for preparing an in vitro 3D melanoma model according to claim 29, characterized in that: soaking the second ex vivo skin with digestive enzymes for 12 to 16 hours; and / or, The second ex vivo skin is soaked with digestive enzymes at a temperature of 2°C to 8°C; and / or, The digestive enzyme is 0.01% to 0.5% pancreatic enzyme.

31. The method for preparing an in vitro 3D melanoma model according to claim 28, characterized in that: In the operation of isolating and culturing the undifferentiated keratinocytes, the specific operation is as follows: preparing a suspension containing undifferentiated keratinocytes by using the undifferentiated keratinocytes remaining above the dermis layer of the second ex vivo skin; Centrifugally filtering the undifferentiated keratinocyte suspension to obtain the undifferentiated keratinocytes; The undifferentiated keratinocytes are expanded and cultured using a modified culture medium.

32. The method for preparing an in vitro 3D melanoma model according to claim 31, wherein the modified culture medium comprises a basal culture medium, additives and a plurality of growth factors, wherein: The multiple growth factors include the following components: Fibroblast growth factor 170 μL to 1000 μL; Transforming growth factor 100 μL to 500 μL; Epidermal growth factor 1μL~17μL.

33. The method for preparing an in vitro 3D melanoma model according to claim 32, characterized in that: The basic culture medium includes at least one of DMEM culture medium and HAMS culture medium; and / or, The additives include fetal bovine serum, double antibody, L-glutamine, adenine, L-thyroxine sodium salt, transferrin, non-essential amino acid solution and insulin.

34. The method for preparing an in vitro 3D melanoma model according to claim 31, characterized in that: The temperature of the amplification culture is 28°C to 38°C; and / or, The amplification culture time is 5 to 14 days.

35. The method for preparing an in vitro 3D melanoma model according to claim 28, characterized in that: The first ex vivo skin and the second ex vivo skin are derived from the same raw material.

36. The method for preparing an in vitro 3D melanoma model according to claim 15, characterized in that: The specific steps include: S1. Fabrication of in vitro dermal scaffolds: S11. Obtain discarded skin samples after surgery; S12, placing the skin sample in a collection solution (PBS antibacterial agent, three antibiotics and gentamicin) and soaking for half an hour; S13, taking out the skin sample from the collection liquid, cutting off large pieces of fat tissue in the dermis layer of the skin sample with scissors, and then further scraping off the fat tissue fragments with a scraper to obtain a skin layer sample; S14, washing the cortical sample with PBS antibacterial agent; S15, cut the cortical sample into 4 cm 2 The first ex vivo skin is obtained by cutting the small pieces into squares, and the small pieces are used as the second ex vivo skin for later use; S16, immersing the first ex vivo skin in a 1.0 mol / L sodium chloride solution for 16 hours to obtain an in vitro dermal scaffold; S2. Acquisition of keratinocytes: S21, soaking the second ex vivo skin in the operation of S15 in 0.25% pancreatic enzyme for 14 hours at a soaking temperature of 8° C. to obtain residual undifferentiated keratinocytes above the dermis; S22, preparing a suspension containing undifferentiated keratinocytes by using the undifferentiated keratinocytes remaining above the dermis layer of the second ex vivo skin; S23, centrifuging and filtering the suspension containing undifferentiated keratinocytes to obtain the undifferentiated keratinocytes; S24, using a modified culture medium to amplify and culture the undifferentiated keratinocytes to obtain keratinocytes, the amplification temperature is 37°C, and the time is 5 days, wherein the following ingredients are used: 300 mL of DMEM culture medium; 105 mL of HAMS culture medium; 50 mL of fetal bovine serum; 3 mL of double antibody; 3 mL of L-glutamine; 3 mL of adenine; 600 μL of L-thyronine sodium salt; 600 μL of transferrin; 600 μL of non-essential amino acid solution; 600 μL of insulin; 600 μL of fibroblast growth factor; 300 μL of transforming growth factor; and 10 μL of epidermal growth factor; S3. Preparation of mixed cell suspension: The keratinocytes and the melanoma cells are mixed in a 15 mL sterile centrifuge tube at a concentration ratio of 3:1 to obtain the mixed cell suspension; S4. Inoculation operation: S41, placing the in vitro dermal scaffold obtained by operation S16 in a 24-well plate; S42, pressing the hollow metal ring onto the in vitro dermal scaffold, wherein the abutting end of the hollow metal ring is provided with a silicone base, and forms a semi-closed inoculation and culture channel with the in vitro dermal scaffold; S43, using a dropper to add 100 μL of the mixed cell suspension along the inoculation end of the semi-enclosed inoculation culture channel, so that the mixed cell suspension will adhere to the basement membrane of the in vitro dermal scaffold along the inoculation culture channel; S44, adding a cell-free culture solution to the semi-enclosed inoculation culture channel, and filling the inoculation culture channel with the cell-free culture solution; S45, after two days of static culture, allowing the mixed cell suspension to adhere to the in vitro dermal scaffold to obtain a pretreated 3D melanoma model; S5. Performing air-liquid interface culture operation on the pretreated 3D melanoma model: S51, removing the hollow metal ring to obtain a 3D melanoma model to be cultured; S52, using tweezers to transfer the 3D melanoma model to be cultured to a stainless steel metal mesh support of a suitable well plate; wherein the size of the stainless steel metal mesh support is 20*20*6mm 2 , the small hole of the stainless steel metal mesh bracket is 4mm 2 ; S53, adding cell culture fluid around the well plate in step S52, so that the liquid level of the cell culture fluid is 0 mm below the surface of the pretreated 3D melanoma model, and maintaining the culture at the air-liquid interface level for 7 days to obtain the in vitro 3D melanoma model.

37. An in vitro 3D melanoma model, characterized in that The in vitro 3D melanoma model is prepared by the preparation method of any one of claims 15 to 36.

38. An application of an in vitro 3D melanoma model, characterized in that: The in vitro 3D melanoma model of claim 37 is applied to the analysis of melanoma pathology, the screening of anti-melanoma drugs, the evaluation of anti-melanoma drugs, the screening of anti-melanoma therapies or the evaluation of anti-melanoma therapies.

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