WO2022222848A1 - Microsphères de peau, leur procédé de préparation et leur application - Google Patents

Microsphères de peau, leur procédé de préparation et leur application Download PDF

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WO2022222848A1
WO2022222848A1 PCT/CN2022/086934 CN2022086934W WO2022222848A1 WO 2022222848 A1 WO2022222848 A1 WO 2022222848A1 CN 2022086934 W CN2022086934 W CN 2022086934W WO 2022222848 A1 WO2022222848 A1 WO 2022222848A1
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microspheres
skin
dermal
cells
signaling pathway
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PCT/CN2022/086934
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Chinese (zh)
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吴耀炯
谢俊东
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清华大学深圳国际研究生院
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • C12N2503/06—Screening or testing on artificial skin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present application relates to the technical field of tissue engineering, in particular to skin microspheres and their preparation methods and applications.
  • the skin is the largest organ of the human body, mainly composed of epidermis, dermis, subcutaneous tissue and skin appendages. It has functions such as physical barrier, sensing the outside world, maintaining body temperature and moisture. Skin health is closely linked to the state of cellular and matrix components in the epidermis and dermis. Dysfunction of one of these components can lead to skin diseases, such as scarring caused by over-activated fibroblasts.
  • skin drugs and skin care products on the market can often act on different components in the skin to achieve the purpose of treating diseases and improving skin conditions.
  • the discovery of active ingredients relies heavily on in vitro models and screening techniques. Compared with human and animal testing, in vitro models have many advantages, such as low cost and low ethical risk. Therefore, reliable and advanced skin in vitro models are of great significance for skin drug screening.
  • This type of tissue-engineered skin product using a variety of cells is composed of the upper layer of keratinocytes and the lower layer of artificial skin structure, and the upper layer of keratinocytes can be used to test the barrier function of the epidermis after keratinization.
  • the present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a skin microsphere capable of detecting the effect of a drug in the epidermis or dermis, and a preparation method and application thereof.
  • a first aspect of the present application provides skin microspheres comprising:
  • Dermal cell microspheres contain dermal cells, and dermal cells have the first signaling pathway activity reporter system;
  • An epidermis layer covers the surface of the dermal cell microspheres, the epidermis layer contains epidermal cells, and the epidermal cells have a second signaling pathway activity reporter system.
  • the skin microsphere adopts a composite double-layer structure formed by the inner core formed by dermal cells and the outer layer of epidermal cells, which better simulates the skin structure of the human body itself.
  • the signal pathway activity reporting system is carried on the inner core and the outer shell respectively, so that it can respond to the effect of the test product in time, so as to realize the detection of the effect of the test product in the epidermis and/or dermis layer.
  • the signaling pathway activity reporter system refers to a system that can detect the activity of a specific signaling pathway and make a qualitative or quantitative response according to the detection result.
  • the non-limiting way of its realization includes a reporter gene that can detect a specific signaling pathway.
  • the first signaling pathway activity reporter system and the second signaling pathway activity reporter system are only used to distinguish that the signaling pathway activity reporter system is carried by the dermal cells in the dermal cell microspheres or the epidermal cells in the epidermal layer, not To specifically limit the type and number of signaling pathway activity reporter systems.
  • the first signaling pathway activity reporter system includes at least one of the Shh signaling pathway activity reporter system and the MAPK/ERK signaling pathway activity reporter system.
  • Shh plays an important role in regulating the development of various tissues and organs involved in cell structure, proliferation and survival, especially can promote the proliferation of dermal papilla cells leading to hair follicle regeneration and hair growth, and its signaling pathways with similar regulatory roles also include MAPK /ERK, etc.
  • the interaction between drugs and cells can be detected by carrying the above-mentioned signaling pathway reporting system in dermal cells to determine whether it can activate the above-mentioned cellular pathways in dermal cells after penetrating the epidermal barrier, and further, the above-mentioned signaling pathways can also be determined. What is the activity after activation.
  • the dermal cells are transfected with an SRE reporter gene.
  • the MAPK/ERK signaling pathway is a major player in the regulation of cell growth and differentiation.
  • transcription factor substrates form complexes with serum response factors (SRFs) and combine with serum response elements (SREs) to achieve gene expression regulation. Therefore, selecting SRE as the reporter gene in the signaling pathway activity reporter system can effectively detect the activity of MAPK/ERK signaling pathway.
  • the second signaling pathway activity reporter system includes at least one of canonical Wnt signaling pathway activity reporter system and Akt signaling pathway activity reporter system.
  • the Wnt signaling pathway is a complex regulatory network that includes three branches including the canonical Wnt signaling pathway.
  • the canonical Wnt signaling pathway is mainly involved in regulating the pluripotent differentiation of stem cells, the development and regeneration of organs, especially the promotion of cell proliferation and regeneration.
  • the Akt signaling pathway has a similar regulatory role to the canonical Wnt signaling pathway. Therefore, the interaction between the drug and the cell can be detected by the activity reporter system of these signaling pathways carried by the epidermal cells, and the effect on cell proliferation and regeneration can be judged.
  • epidermal cells are transfected with a TCF/LEF1 reporter gene.
  • TCF/LEF is a class of HMG (High Mobility Group) transcription factors.
  • HMG High Mobility Group
  • TCF/LEF participates in the feedback regulation of Wnt signaling pathway.
  • the stable ⁇ -catenin complex combined with this transcription factor will cause the activation of the classical Wnt signaling pathway. Therefore, selecting TCF/LEF1 as the reporter gene in the signaling pathway activity reporter system can more sensitively detect the activity of the canonical Wnt signaling pathway.
  • the epidermal cell reporter system includes key pathways that promote cell proliferation and regeneration, such as the canonical Wnt signaling pathway, Akt pathway, etc.
  • the reporter system of dermal cells selects signaling pathways that promote the formation of hair follicles and dermal papillae and hair follicle regeneration, such as Shh signaling pathway, MAPK/ERK signaling pathway, etc., so that it can be used to observe whether the test product can activate the cell signaling pathway and the degree of activation. Discovery of active pharmaceutical ingredients that promote skin and hair follicle regeneration.
  • the diameter of the skin microspheres ranges from 0.02 to 10 mm.
  • the actual size of the skin microspheres can be determined according to the research needs, but considering the needs of product cryopreservation and recovery, the diameter of the microspheres should be 0.02-10 mm, preferably within 1 mm.
  • the epidermal cells are keratinocytes derived from skin or mucosa, and these keratinocytes can be primary cells, or culture-expanded keratinocytes, or immortalized keratinocytes such as HaCaT .
  • the keratinocytes in the epidermis layer can further be single or multi-layered, for example, when detecting the effect of drugs on the proliferation of keratinocytes, the skin microspheres can use the epidermis layer containing a single layer of keratinocytes; When it is necessary to detect the barrier function of the epidermis and the ability of the drug to penetrate the skin, the keratinocytes can be further differentiated and matured to form the stratum corneum.
  • other cells or substances can also be incorporated according to different application needs.
  • melanocytes can be incorporated to test the effect of drugs on melanocytes in the epidermal layer of the skin; Infiltrate a certain percentage of immune cells (such as Langerhans cells) to study the effect of drugs on skin defense function.
  • immune cells such as Langerhans cells
  • the dermal cells are fibroblasts isolated from the dermis of the skin, and these dermal cells can be primary cells, or culture-expanded fibroblasts, or immortalized fibroblasts ;
  • fibroblasts can also be embryonic stem cells or iPS cells after induced differentiation.
  • the dermal cell microspheres containing fibroblasts can also be used for the detection and screening of drugs or cosmetics targeting dermal cells in addition to the functional support of the epidermal layer wrapped on them.
  • a certain proportion of endothelial cells can be further added to the dermal cell microspheres to detect angiogenic drugs, or a certain proportion of immune cells can be added to conduct immune-related detection or research.
  • the dermal cell microspheres may also contain extracellular matrix macromolecules, such as non-limiting examples of collagen, and may further contain other extracellular matrix molecules such as mucopolysaccharides , which can be hyaluronic acid. Alternatively, some or all of these extracellular matrix molecules may be replaced by synthetic materials.
  • a second aspect of the present application provides a preparation method of the above-mentioned skin microspheres, the preparation method comprising the following steps:
  • the dermal cells are mixed with the hydrogel to obtain a dermal cell mixture
  • the dermal cell microspheres were mixed with epidermal cells and cultured in 3D to obtain the skin microspheres.
  • the microspheres of dermal cells are first formed in the oil phase, and then the skin microspheres obtained by mixing with epidermal cells in 3D culture are compared with the existing products. Therefore, the surface of the dermal cell microspheres is more completely covered.
  • the skin microspheres are used to detect and screen drugs, the drugs cannot directly enter the inner dermal cell microspheres from the pore wall, so that the epidermal shell can be more accurately reflected. Barrier effect and transdermal capacity of drugs.
  • the morphology of the final skin microspheres also makes it easier to sample and analyze, and more convenient for storage and transportation.
  • the dermal cell microspheres before being mixed with epidermal cells, are resuspended in a medium and cultured for 12-36 hours. During this period, as the dermal cells spread, the size of the dermal cell microspheres will decrease slightly. There is reduction, resulting in a more stable structure that facilitates the subsequent adhesion of epidermal cells to its surface.
  • the third aspect of the present application provides the application of the above-mentioned skin microspheres in the detection of drugs and/or cosmetics.
  • the above-mentioned skin microspheres carry corresponding signaling pathway activity reporting systems on the inner core and the outer shell respectively, which can respond in time to the effect of the drug and/or cosmetic to be tested, so as to serve as a better detection model for the detection of drugs and/or cosmetics. Detection of effects in the epidermis as well as the dermis.
  • a fourth aspect of the present application provides a detection method for a product to be tested, the detection method comprising the following steps:
  • the above-mentioned skin microspheres are contacted with the test product, and the changes of the skin microspheres before and after contact are detected.
  • the skin microspheres used in the detection method carry corresponding signal pathway activity reporting systems on the inner core and the outer shell respectively.
  • the detection model reflects the effects of drugs and/or cosmetics in the epidermis and dermis.
  • FIG. 1 is a picture of the skin microspheres prepared in Example 1 of the present application.
  • Figure 2 is the result of the reaction of the skin microspheres to the drug in Example 2 of the present application.
  • FIG. 3 is a schematic diagram of a plasmid loaded with a signaling pathway activity reporter system and a picture of a skin microsphere carrying a signaling pathway activity reporter system in Example 3 of the present application.
  • the meaning of several is more than one, the meaning of multiple is two or more, greater than, less than, exceeding, etc. are understood as not including this number, above, below, within, etc. are understood as including this number. If it is described that the first and the second are only for the purpose of distinguishing technical features, it cannot be understood as indicating or implying relative importance, or indicating the number of the indicated technical features or the order of the indicated technical features. relation.
  • references to the terms “one embodiment,” “some embodiments,” “exemplary embodiment,” “example,” “specific example,” or “some examples”, etc., are meant to incorporate the embodiments
  • a particular feature, structure, material, or characteristic described or exemplified is included in at least one embodiment or example of the present application.
  • schematic representations of the above terms do not necessarily refer to the same embodiment or example.
  • the particular features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
  • This embodiment provides a skin microsphere and a preparation method of the skin microsphere.
  • Rat tail collagen type I (Corning Company, USA, product number 354236); F3100 oil phase (3M Company, USA); Cisplatin (MacLean Company); DMEM (Corning Company, USA); FBS (BI Company, USA).
  • A is a picture of the skin microspheres at a scale of 1000 ⁇ m
  • B is a picture of brightfield, different color channels (GFP and mCherry) and merged at a scale of 200 ⁇ m.
  • GFP and mCherry different color channels
  • the skin microspheres can be completely formed, with regular shape and uniform size, and have a double-layer skin structure, including dermal cell microspheres composed of fibroblasts and extracellular matrix (collagen).
  • Spheres (mCherry labeled), and a composite structure formed by the epidermal layer (green fluorescent protein GFP labeled) wrapped by epidermal cells in the outer layer of dermal cell microspheres, this structure is very similar to the structure of human skin and can be used as a good detection method.
  • the model simulates human skin.
  • mice Sigma, USA
  • mouse anti-E-cadherin antibody Thermofisher, product number 13-1700
  • rabbit anti-Ki67 antibody CST, product number 9662
  • goat anti-rabbit 555 antibody CST, product number 3452
  • goat anti-mouse 647 antibody CST, product number 4410).
  • Example 1 Put the skin microspheres prepared in Example 1 into a non-adherent 24-well plate for culture, add 2ml of culture medium (containing 2% serum) to each well, then add drugs, and put them into a 37-degree incubator to continue culturing 48h.
  • culture medium containing 2% serum
  • the frozen section was taken out from the -20°C refrigerator, placed at room temperature for 15 minutes, and washed twice with PBS for 10 minutes each time. After the slide was wiped dry, an immunohistochemical pen was used to draw a circle along the edge of the sample, and the sample was permeabilized with 0.25% TritonX-100 for 30 min. TritonX-100 was removed by suction, washed twice with PBS, and PBS solution containing 3% bovine serum albumin (BSA) or 10% goat serum was added to block for 30 min at room temperature.
  • BSA bovine serum albumin
  • the primary antibody was aspirated, washed twice with PBS, and a fluorescently labeled secondary antibody with appropriate dilution was added, and incubated at room temperature for one hour.
  • Aspirate the secondary antibody wash twice with PBS, add DAPI staining solution, stain at room temperature for 20 min, wash twice with PBS, mount the slides with mounting medium, dry and observe and take pictures with an inverted fluorescence microscope or confocal microscope.
  • the primary antibodies were mouse anti-E-cadherin antibody and rabbit anti-Ki67 antibody
  • the secondary antibodies were goat anti-mouse 647 antibody and goat anti-rabbit 555 antibody.
  • luciferase reporter plasmids 1 and 2 wherein there is a TCF/LEF reporter gene on the reporter plasmid 1, and this TCF/LEF reporter gene comprises TCF/LEF binding sequence and corresponding luciferase Eluc sequence;
  • an SRE reporter gene on the reporter plasmid 2 there is an SRE reporter gene on the reporter plasmid 2, and the SRE reporter gene includes the SRE binding sequence and the luciferase Fluc sequence.
  • the reporter plasmids 1 and 2 were further provided with a green Renilla luciferase reporter gene (GrRenilla) and a Renilla luciferase reporter gene (Renilla) as internal controls, respectively.
  • Reporter plasmid 1 and reporter plasmid 2 were packaged by lentivirus to infect epidermal cells and dermal cells, respectively, and high-expressing cell lines were selected by flow cytometry. Then, according to the preparation method in Example 1, a skin microsphere with dual luciferase reporter system was constructed. The constructed skin microspheres are shown in B in Figure 3.
  • the epidermis layer includes epidermal cells with the Wnt signaling pathway activity reporter system, and the dermal microspheres includes dermal cells with the MAPK/ERK signaling pathway activity reporter system.
  • the specific effects of the drugs can be determined by detecting the activity of luciferase.
  • the specific detection process is as follows:
  • the canonical Wnt signaling pathway is a key pathway involved in promoting cell proliferation and regeneration
  • the MAPK/ERK signaling pathway is a signaling pathway involved in promoting the formation of hair follicles and hair follicle regeneration. It is used to discover the active ingredients of drugs that can promote the regeneration of skin and hair follicles, so as to achieve the purpose of detection or screening of the test products.

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Abstract

L'invention concerne des microsphères de peau, un procédé de préparation associé, et une application associée. L'invention concerne des microsphères de peau. Les microsphères de peau comprennent les éléments suivants : des microsphères de cellules dermiques, les microsphères de cellules dermiques comprenant des cellules dermiques présentant un premier système de rapport d'activité de voie de signalisation ; une couche épidermique, recouvrant la surface des microsphères de cellules dermiques, et comprenant des cellules épidermiques, les cellules épidermiques présentant un second système de rapport d'activité de voie de signalisation. Les microsphères de peau ont au minimum les effets bénéfiques suivants : les microsphères de peau utilisent une structure composite à double couche formée en enveloppant un noyau interne formé de cellules dermiques et une couche externe de cellules épidermiques, imitant ainsi plus fidèlement la structure de la peau d'un corps humain. En outre, le noyau interne et l'enveloppe externe portent chacun un système de rapport d'activité de voie de signalisation, afin que les microsphères cutanées puissent réagir rapidement à l'effet d'un échantillon à analyser, détectant ainsi l'effet de l'échantillon sur la couche épidermique et/ou la couche dermique.
PCT/CN2022/086934 2021-04-19 2022-04-15 Microsphères de peau, leur procédé de préparation et leur application WO2022222848A1 (fr)

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