Preparation method of external vesicles of hair papilla
By culturing hair papillary cells in three-dimensionally and recycling external vesicles in hair papillary, the problem of reduced hair regeneration ability caused by two-dimensional subculture is solved, the number of hair papillary cells amplification and the maintenance of stem cell characteristics is achieved, and the hair growth ability is enhanced.
Patent Information
- Application Number
- CN202510566443.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-30
- Publication Date
- 2025-07-29
AI Technical Summary
Two-dimensional subculture of hair papillary cells leads to a decrease in hair regeneration ability and makes it difficult to expand the number of hair papillary cells.
Using a three-dimensional culture method, the hair papillary cells are spread over the surface of the substrate for non-passion proliferation culture. After meeting specific conditions, the outer vesicles of the hair papillary are recovered. The culture medium contains basic fibroblast growth factor and insulin, and the substrate is coated with cell adhesion components.
Efficiently expand hair epithelial stem cells, maintain their stem cell characteristics, and enhance hair growth ability.
Smart Images
Figure CN120384044A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to an extracellular vesicle extraction technology, in particular to a method for preparing dermal papilla extracellular vesicles. Background Art
[0002] Dermal papilla cells (DPCs) are the core regulatory cells at the bottom of the hair follicle, and maintain the activity of hair follicle stem cells and promote hair growth by secreting growth factors and regulating signal pathways. In order to expand the number of dermal papilla cells, the dermal papilla cells are repeatedly subcultured two-dimensionally. However, the hair regeneration ability of the two-dimensionally subcultured dermal papilla cells will be greatly reduced, which is common technical knowledge. In order to overcome the problem of reduced hair regeneration ability in two-dimensional subculture, the dermal papilla cells are cultured three-dimensionally, that is, the dermal papilla cells are cultured in layers. When culturing, although the hair regeneration ability of the dermal papilla can be restored to a certain extent, it is difficult to expand the number of dermal papilla cells. Summary of the Invention
[0003] The purpose of the present invention is to provide a method for preparing dermal papilla extracellular vesicles, including: Step S100, laying dermal papilla cells containing at least one hair growth-related gene on the surface of a substrate until it reaches a confluent state; Step S200, adding and replacing the culture medium for non-passage proliferation culture, and continuously culturing for a period of time after reaching the proliferation end condition, satisfying any one of the following conditions, the proliferation ends: (1) The cell layer cultured and proliferated reaches the set condition; (2) The culture time reaches the set condition; (3) The cell density of the cells reaches the set condition; (4) The expression level of one or more hair growth-related genes of the cells increases to the set condition; (5) The number of dermal papilla cells contained in the cells reaches the set condition; Step S300, recovering the dermal papilla extracellular vesicles in the supernatant formed during the proliferation culture.
[0004] Further, all or part of the surface of the substrate is coated with a cell adhesion component.
[0005] Further, the confluent state in step S100 is that the cell density is between (3.5×10 4 cells / cm 2 , 7×10 4 cells / cm 2 ).
[0006] Further, in step S200, the culture medium components include basic fibroblast growth factor, or a mixture of basic fibroblast growth factor and insulin.
[0007] Further, regarding condition (1) where the cell layer proliferates to 6 layers, compared to the first cell layer, the covering area of each layer is as follows: The covering area of the second cell layer is more than 60%, preferably 100%; The covering area of the third cell layer is more than 25%, preferably more than 95%; The covering area of the fourth cell layer is more than 15%, preferably more than 65%; The covering area of the fifth cell layer is more than 15%, preferably more than 55%; The covering area of the sixth cell layer is more than 15%, preferably more than 55%.
[0008] Further, regarding condition (2), the culture time is [180h, 3600h], preferably [600h, 800h].
[0009] Further, regarding condition (3), the culture is carried out until the cell density range is [7×10 4 cells / cm 2 , 30×10 4 cells / cm 2 , preferably [11×10 4 cells / cm 2 , 14×10 4 cells / cm 2 ; or the cell density is N times that of the original cells, and the value range of N is [2, 10], preferably 4 times.
[0010] Further, regarding condition (4), the expression level of one or more hair growth-related genes of dermal papilla cells increases to M times that of the original cells, and the value range of M is [1.2, 100], preferably [3.6, 10].
[0011] Further, regarding condition (5), the proportion of the number of dermal papilla cells contained in the dermal papilla cells accounts for [5%, 100%).
[0012] Through long-term culture and specific culture conditions, the present invention can efficiently expand hair epithelial stem cells while maintaining their stem cell characteristics. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 It is a schematic diagram of cell counting on different days.
[0014] Figure 2Schematic diagram of relative expression levels of ALP for different days.
[0015] Figure 3 Schematic diagram for comparison of relative expression levels of ALP between this example and the control group. Detailed implementation manner
[0016] The dermal papilla cells (DPCs) involved in this example are a type of specialized cells present in the dermal papilla (DP) at the bottom of the hair follicle. The dermal papilla (DP) is a small dermal structure rich in capillaries and signal regulatory factors, located in the center of the hair bulb of the hair follicle, directly contacting the hair follicle epithelial cells, and playing a key role in the gene regulation of the hair cycle.
[0017] The exosomes involved in this example are a type of nano - scale extracellular vesicles, usually with a diameter between 30 - 150 nm, which are generated by cells through the endocytosis - lysosome pathway and released into the extracellular environment. Exosomes are widely present in body fluids such as blood, urine, cerebrospinal fluid, breast milk, etc., and play important roles in physiological processes such as intercellular communication, tissue repair, and immune regulation. Dermal papilla cells secrete exosomes (DPC - Exosomes), which are rich in bioactive molecules such as proteins, miRNAs, mRNAs, and lipids, and can act as carriers for intercellular signal transmission, playing a key role in the regulation of the hair follicle growth cycle, activation of hair follicle stem cells, angiogenesis, etc. Exosomes derived from dermal papilla cells (DPCs) have been proven to be able to activate hair follicle stem cells, regulate the hair growth cycle, and show great application potential in the field of hair loss treatment and hair regeneration.
[0018] Combined with Figure 1 , a method for preparing dermal papilla exosomes, in which the hair follicle mesenchymal cells adhered to the surface of the substrate are cultured without sub - passage from a confluent state, specifically including the following steps: Step S100, confluently plating dermal papilla cells on the surface of the substrate; Step S200, adding a culture medium for non - sub - passage culture and proliferation, and continuing to culture for a period of time after reaching the proliferation conditions; Step S300, recovering the dermal papilla exosomes in the supernatant formed during the proliferation process.
[0019] The dermal papilla cells described in step S100 are mesenchymal cells with hair regeneration ability. The dermal papilla cells transplanted into the organism promote hair growth at the site where the dermal papilla cells are transplanted by secreting hair growth-promoting factors (such as Wnt). The dermal papilla cells can be follicle-derived cells collected from the organism, or cells obtained by in vitro differentiation induction of cells derived from tissues other than follicles. Further, the dermal papilla cells can be derived from the dermal papilla and / or dermal root sheath contained in the follicles collected from the organism, or can be derived from the dermal papilla and / or dermal sheath cup of the hair bulb. In this embodiment, it is preferably derived from the dermal papilla. Further, the cells used in the differentiation induction of the dermal papilla cells are cells that have the ability to differentiate into the dermal papilla cells in vitro. For example, they are preferably selected from pluripotent stem cells, stem cells other than the pluripotent stem cells, or mesenchymal cells derived from the skin of a fetus / newborn; pluripotent stem cells such as iPS cells (induced Pluripotent Stem Cells), ES cells (Embbryonic Stem Cells), Muse cells (Multilineage-differentiating stress-enduring Cells), and EG cells (Embryonic Germ Cells), and more than 1 type of them; stem cells other than pluripotent stem cells such as stem cells obtained by reprogramming differentiated cells and mesenchymal stem cells (such as mesenchymal stem cells derived from adipose tissue), and more than 1 type of them; mesenchymal cells derived from the skin of a fetus / newborn such as mesenchymal cells derived from the dermis of the skin of a fetus or newborn, and more than 1 type of them. In this embodiment, the dermal papilla cells are selected as follicle-derived cells from the human body, especially dermal papilla cells, and the prepared extracellular vesicles of the dermal papilla will also be transplanted onto the same individual source.
[0020] In step S100, the dermal papilla cells preferably express one or more hair growth-related genes, which can be one or more selected from the group consisting of alkaline phosphatase (ALP) gene, Versican gene, LEF1 gene, WNT5A gene, NOG gene, BMP4 gene, Sox2 gene, αMSA gene, and GREM2 gene. The ALP gene is a gene that is strongly expressed in dermal papilla cells. The αMSA gene is a gene that is strongly expressed in dermal root sheath cells of the hair follicle (including dermal root sheath cells in the hair bulb). The GREM2 gene is a gene that is strongly expressed in dermal root sheath cells in the hair bulb. In this embodiment, the dermal papilla cells express one or more mesenchymal cells selected from the ALP gene, αMSA gene, and GREM2 gene, more preferably one or more mesenchymal cells selected from the ALP gene and GREM2 gene, and particularly preferably mesenchymal cells expressing the ALP gene.
[0021] The material on the surface of the substrate in step S100 can be composed of one or more materials selected from resins (such as polystyrene or polypropylene), glass, metals, and ceramics. The surface of the substrate can be non-porous or porous. The surface of the substrate can be treated to improve cell adhesion (such as coating with cell adhesion components), or such treatment can be not performed. The cell adhesion components that can be coated on the surface of the substrate can be compounds artificially introduced with extracellular matrix (such as collagen or fibronectin) and / or cell adhesion sites (such as amino acid sequences containing RGD sequences). If there are adhesive treatment areas and non-adhesive treatment areas on the surface of the substrate, the adhesive treatment area is used as the surface of the substrate for culturing dermal papilla cells. The surface of the substrate can be the surface of a culture carrier (such as a particulate culture carrier like microcarrier beads) placed on the bottom surface or suspended in the culture medium in a culture container. The shape of the surface of the substrate can be a flat surface (such as the flat bottom surface of a culture dish, culture flask, or the well of a multi-well plate), or a curved surface (such as the inner surface of a roller bottle and / or the surface of a particulate culture carrier like microcarrier beads); specifically, the surface of the substrate can be the bottom surface of a culture flask (the bottom surface of the first stage in the case of a multi-stage flask), the inner surface of a roller bottle, the bottom surface of a culture dish, the bottom surface of one well of a multi-well plate, a single cell adhesion surface surrounded by a surface non-adhesive to dermal papilla cells, or the surface of a single particle of a particulate carrier (such as a microcarrier bead).
[0022] The confluent state described in step S100 can be determined by the cell density of dermal papilla cells on the surface of the substrate (the density obtained by dividing the total number of dermal papilla cells on one substrate surface by the area of the one substrate surface). The dermal papilla cells being in a confluent state can be that the cell density of dermal papilla cells on the surface of the substrate is 3.5×10 4 cells / cm 2 or more and 7×104 cells / cm 2 The method for calculating the cell density of dermal papilla cells on the substrate surface is as follows: A measurement kit containing a fluorescent enzyme and a precursor compound that is converted into the fluorescent enzyme by living cells is used for measurement. Specifically, a measurement kit containing luciferase as the fluorescent enzyme and a cell membrane-permeable precursor compound that is converted into the substrate of luciferase in the cytoplasm of living cells is used. First, luciferase and the precursor compound are added to the culture solution containing dermal papilla cells on the substrate surface. As a result, the precursor compound penetrates from the culture solution into the cytoplasm of living dermal papilla cells, and in the cytoplasm, it is converted into the substrate of luciferase by the reducing ability of the dermal papilla cells; the generated cell membrane-permeable substrate leaks from the cytoplasm of the dermal papilla cells into the culture solution, and in the culture solution, it is converted into a luminescent substance by luciferase; the intensity of the fluorescence (e.g., absorbance at a specific wavelength) derived from the luminescent substance is measured, and based on the measured fluorescence intensity and a pre-made standard curve (a graph showing the relationship between fluorescence intensity and the number of cells), the number of dermal papilla cells on the substrate surface is calculated. Among them, the standard curve is made, for example, using dermal papilla cells at the time of inoculation on the substrate surface; then, the calculated number of dermal papilla cells is divided by the area of the substrate surface, thereby calculating the cell density of the dermal papilla cells on the substrate surface.
[0023] The culture solution components described in step S200 include one or more of bFGF (basic fibroblast growth factor) and insulin, especially a culture solution containing bFGF. The culture medium is changed every certain period (usually 48 hours), and the dermal papilla extracellular vesicles are obtained from the changed culture medium (i.e., the supernatant).
[0024] The proliferation end condition in step S200 is selected from any of the following conditions: (1) The cell layer of cultured and proliferated cells reaches the set condition; (2) The culture time reaches the set condition; (3) The cell density of the cells reaches the set condition; (4) The expression level of one or more hair growth-related genes of the cells increases to the set condition; (5) The number of dermal papilla cells contained in the cells reaches the set condition.
[0025] Regarding condition (1), the cell layer preferably proliferates to 6 layers. Compared with the first cell layer, the covering area of each layer is as follows: The covering area of the second cell layer is 60% or more, preferably 100%; The covering area of the third cell layer is 25% or more, preferably 95% or more; The coverage area of the fourth cell layer is more than 15%, preferably more than 65%; The coverage area of the fifth cell layer is more than 15%, preferably more than 55%; The coverage area of the sixth cell layer is more than 15%, preferably more than 55%.
[0026] Since the proliferation of dermal papilla cells may not be uniform throughout the substrate surface, the number of layers contained in the cell layer formed on the substrate surface may also vary on the substrate surface. For example, a first cell layer containing a first number of dermal papilla cell layers (e.g., 1, 2, or 3 dermal papilla cell layers) is formed on a part of the substrate surface, and a second cell layer containing a number of dermal papilla cell layers that is 1 or more, 2 or more, or 3 or more greater than the number of the first cell layer (e.g., 4 or more, 5 or more, or 6 or more dermal papilla cell layers) is formed on another part of the substrate surface.
[0027] Regarding condition (2), the culture time is in the range of [180 h, 3600 h], preferably in the range of [600 h, 800 h].
[0028] Regarding condition (3), the culture is carried out until the cell density range is [7×10 4 cells / cm 2 , 30×10 4 cells / cm 2 , preferably [11×10 4 cells / cm 2 , 14×10 4 cells / cm 2 . Or the cell density of dermal papilla cells is N times that of the initially confluent cells, and the value range of N is [2, 10], preferably 4 times.
[0029] Regarding condition (4), the expression level of one or more hair growth-related genes of dermal papilla cells is increased to M times that of the initially confluent cells, and the value range of M is [1.2, 100], preferably [3.6, 10].
[0030] Regarding condition (5), the proportion of the number of dermal papilla cells contained in dermal papilla cells to the total number of dermal papilla cells is in the range of [5%, 100%).
[0031] In step S200, the dermal papilla cells that have ended proliferation are continuously cultured for 2 - 10 days under a high-density condition. Since after the cells have just formed a multi-layer structure, compared to when the number of cells in the confluent state is less than that in the initial state, there are many other cells around the cells in the multi-layer structure, and each cell absorbs oxygen, thus forming a microenvironment with relatively less oxygen. In an environment with scarce oxygen, the dermal papilla cells will regain their activity, and continuous culturing for a period of time is equivalent to allowing the cells to recover their cell functions for a longer period in a hypoxic environment.
[0032] In step S300, the supernatant formed during the cell proliferation culture is taken, and ultracentrifugation is used to extract dermal papilla extracellular vesicles.
[0033] For the amplified dermal papilla cells, by performing enzymatic treatment on the amplified dermal papilla cells adhered to the surface of the substrate, the amplified dermal papilla cells are detached and dispersed from the surface of the substrate, and the amplified dermal papilla cells dispersed in the culture medium are recovered. The enzymatic treatment preferably uses a protease such as trypsin. By breaking or detaching the cell layer of the amplified dermal papilla cells formed on the surface of the substrate, one or more cell layer sheets dispersed in the culture medium are recovered. For one cell layer sheet, a cell scraper or other breaking tool can be used to break the cell layer of the amplified dermal papilla cells covering the surface of the substrate, thereby recovering multiple cell layer sheets dispersed in the culture medium formed by the breaking of the cell layer; or a cell spatula or other peeling tool can be used in the culture medium to peel only the peripheral part of the cell layer in the culture medium, thereby promoting the subsequent peeling and aggregation of the cell layer from the peripheral part, and finally detaching the aggregate of the cell layer from the surface of the substrate; or dermal papilla cells can be cultured on the surface of a cell-adhesive substrate coated with a temperature-responsive polymer to form a cell layer, and then the temperature-responsive polymer is solubilized by changing the temperature, thereby detaching the cell layer from the surface of the substrate; or an electrochemical method or an ultracentrifuge can be used to extract the amplified dermal papilla cells. For multiple cell layer sheets realized by multiple adhesion treatment regions on the surface of the substrate, multiple cell layers of amplified dermal papilla cells can be formed on the surface of the substrate, and then the multiple cell layers are respectively detached from the surface of the substrate, thereby recovering multiple cell layer sheets dispersed in the culture medium. Multiple regions that are adhesive to dermal papilla cells and are respectively surrounded by surfaces that are non-adhesive to dermal papilla cells are formed on the surface of the substrate, and dermal papilla cells are cultured in each of the adhesive regions on the surface of the substrate to form a cell layer. Examples
[0034] To verify the function of the extracellular vesicles of the multi-layer cultured dermal papilla on the restoration of cell stemness, 1×10 4Dermal papilla cells were added with 500 μL of culture medium and cultured for 5 days, after which they were divided into an experimental group and a control group. 50 mL of the supernatant of dermal papilla cells cultured for 10 days and 30 days was respectively collected, re-suspended with 5 mL of phosphate buffer solution (PBS), and made into an extracellular vesicle solution for standby. 20 μL of the above-prepared extracellular vesicle solution was added to the experimental group; 20 μL of PBS solution was added to the control group. Then, the cells were collected on the fifth day of culture, and the relative expression levels of gene transcripts were measured. The results are shown in Table 1 and Figure 3 as follows.
[0035] Table 1
[0036] Culture conditions Relative ALP expression Standard deviation Control group 1.0005 0.045 Day 10 2.585 0.142 Day 30 2.476 0.32
[0037] As shown in Table 1 and Figure 3 as follows, the extracellular vesicles obtained by the present invention can significantly enhance the ability of cell stemness recovery.
Claims
1. A method for preparing dermal papilla extracellular vesicles, characterized in that, include: Step S100, paving dermal papilla cells containing at least one hair growth-related gene on the surface of a substrate until the cells are fully confluent; Step S200: Add and replace the culture medium to perform non-subculture proliferation culture. After reaching the proliferation end condition, continue culturing for a period of time. If any of the following conditions is met, the proliferation ends: (1) The cultured and proliferated cell layer reaches the set conditions; (2) The culture time reaches the set conditions; (3) The cell density reaches the set condition; (4) The expression level of one or more germinative genes in the cell increases to the set condition; (5) The number of dermal papilla cells contained in the cells reaches the set condition; Step S300, recovering the extradermal papilla vesicles in the supernatant formed during the proliferation culture process.
2. The method according to claim 1, characterized in that, The surface of the substrate is fully or partially coated with a cell-adhesive component.
3. The method according to claim 2, characterized in that The confluence state in step S100 is when the cell density is (3.5×10 4 cells / cm 2 ,7×10 4 cells / cm 2 )between.
4. The method according to claim 1, characterized in that: In step S200 , the culture solution comprises basic fibroblast growth factor, or a mixture of basic fibroblast growth factor and insulin.
5. The method according to claim 1, characterized in that Regarding condition (1), when the cell layer proliferates to 6 layers, the area covered by each layer compared to the first cell layer is as follows: The second cell layer covers an area of more than 60%, preferably 100%; The third cell layer covers an area of more than 25%, preferably more than 95%; The fourth cell layer covers an area of more than 15%, preferably more than 65%; The fifth cell layer covers an area of more than 15%, preferably more than 55%; The coverage area of the sixth cell layer is greater than 15%, preferably greater than 55%.
6. The method according to claim 1, wherein Regarding condition (2), the culture time is [180 h, 3600 h], preferably [600 h, 800 h].
7. The method according to claim 1, wherein Regarding condition (3), culturing until the cell density range is [7×10 4 cells / cm 2 , 30×10 4 cells / cm 2 , preferably [11×10 4 cells / cm 2 , 14×10 4 cells / cm 2 ; or the cell density is N times the initial cells, and the value range of N is [2, 10], preferably 4 times.
8. The method according to claim 1, characterized in that Regarding condition (4), the expression level of one or more hair growth-related genes in dermal papilla cells is increased to M times that of the initial cells, and the value range of M is [1.2, 100], preferably [3.6, 10].
9. The method according to claim 1, characterized in that: Regarding condition (5), the ratio of the number of dermal papilla cells contained in the dermal papilla cells to the total number of dermal papilla cells is [5%, 100%).