Dermal dermal papilla cell spheroids and in vitro culture methods, vesicles and uses thereof

Abstract

The present application belongs to the field of biological materials and regenerative medicine, and particularly relates to a dermal dermal papilla cell sphere, an in-vitro culture method thereof, vesicles and uses. The dermal dermal papilla cell sphere is prepared according to the following method: extracting primary dermal dermal papilla cells from an in-vitro dermal dermal papilla tissue, subculturing for 3-5 generations; inoculating the subcultured dermal dermal papilla cells into an SLF-3D culture system for culture, collecting semi-suspended spheres when the dermal dermal papilla cells grow to a fusion degree of 90-100%, separating the spheres into single cells, and re-inoculating the single cells into the original SLF-3D culture system for culture for 3-5 days, and collecting the formed suspension spheres, which are the dermal dermal papilla cell spheres. The dermal dermal papilla cell spheres and vesicles provided by the present application have better therapeutic effects on scarless healing of a wound surface.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials and regenerative medicine, specifically relating to a dermal papilla cell spheroid, its in vitro culture method, vesicles, and uses. Background Technology

[0002] Full-thickness skin defects often fail to regenerate their tissue structure, resulting in scarring. Scarring not only affects appearance but also leads to a lack of skin appendages, excessive collagen deposition, disordered fiber arrangement, and poor tissue extensibility and tensile strength, severely impacting patients' quality of life. Despite significant advancements in wound healing treatment strategies, the quality of wound healing remains unsatisfactory. Therefore, developing an effective treatment method to accelerate and improve wound healing is crucial. Recent studies have found that hair follicles and related stem cells can reduce scar formation after wound healing. In adult mice, the skin tissue surrounding newly formed hair follicles closely resembles normal skin, suggesting that hair follicles may be key to skin regeneration. Clinical studies using human hair follicle unit transplantation to repair autologous refractory wounds have demonstrated that transplanted hair follicle units can rapidly and effectively repair severe wounds in patients.

[0003] Dermal papilla cells (DPCs) are specialized mesenchymal stem cells located at the base of hair follicles. They possess strong stem cell characteristics and can differentiate into various cell types under specific conditions. DPCs regulate cell proliferation, hair follicle development, and angiogenesis by secreting various growth factors and cytokines, playing a crucial role in epithelial-mesenchymal interactions and hair follicle regeneration. They have become the most important dermal-derived stem cells for hair follicle regeneration. Currently, research on the role of hair follicles and related stem cells in reducing scar formation during wound healing is still in its early stages. Compared with other mesenchymal stem cells such as adipose-derived stem cells and bone marrow stem cells, DPCs, in addition to promoting angiogenesis, reducing inflammation, and accelerating healing, can also improve post-operative scarring. Furthermore, DPCs can be obtained from body hair, beards, and other parts of the body. Compared to other sources of mesenchymal stem cells, they are easier to obtain, have abundant sources, and are not subject to ethical restrictions. They have broad application prospects in tissue regeneration, wound repair, and targeted therapy carrier cells, and are considered key seed cells for scarless wound healing. However, there are no research reports or related products on the role of 3D dermal papillary cell spheres (tdDPCs) in promoting wound healing, especially in scar treatment. Summary of the Invention

[0004] In view of the above technical problems, the present invention provides the following technical solution:

[0005] The first objective of this invention is to provide a method for in vitro culture and isolation of dermal dermal papilla cell spheroids, comprising the following steps:

[0006] Primary dermal dermal papilla cells were extracted from isolated dermal dermal papilla tissue and passaged for 3 to 5 generations.

[0007] After passage, dermal papilla cells were seeded into the SLF-3D culture system and cultured. When the dermal papilla cells grew to a confluence of 90-100%, the semi-suspended spheres were collected, and the spheres were separated into single cells. The single cells were then re-seeded into the original SLF-3D culture system and cultured for 3-5 days. The resulting suspended spheres were collected, which are the dermal papilla cell spheres.

[0008] The SLF-3D culture system uses DEME / F-12 as a solvent, and based on the total volume of the SLF-3D culture system, it also includes the following raw materials: 2-3% fetal bovine serum. 、 Human basic fibroblast growth factor 4–6 ng / mL, human epidermal growth factor 0.8–3 ng / mL, human platelet-derived growth factor 4–6 ng / mL, heparin 1–3 ug / mL, L-ascorbic acid-2-phosphate sesquisodium hydrate 30–60 ug / mL, penicillin 80–120 U / mL.

[0009] Preferably, the seeding density of the dermal papilla cells in SLF-3D culture medium is 5–6 × 10⁻⁶. 4 pcs / cm 2 .

[0010] Preferably, based on the total volume of the SLF-3D culture system, the SLF-3D culture system comprises the following raw materials: 3% fetal bovine serum. 、 Human basic fibroblast growth factor 5 ng / mL, human epidermal growth factor 2 ng / mL, human platelet-derived growth factor 5 ng / mL, heparin 2 ug / mL, L-ascorbic acid-2-phosphate sesquisodium hydrate 50 ug / mL, penicillin 100 U / mL.

[0011] Preferably, the primary dermal papilla cells are extracted from the hair bulb at the end of isolated mouse whiskers using a combination of microscopic separation and modified enzyme digestion.

[0012] Preferably, the specific culture process of the passage culture is as follows: the primary dermal papilla cells are first seeded into Dulbecco modified Eagle medium containing 20% ​​(v / v) fetal bovine serum and cultured. ， When the growth confluence reaches 80-90%, the culture is passaged, and then the medium is replaced with Dulbecco modified Eagle medium containing 10% fetal bovine serum for continued culture.

[0013] A second objective of this invention is to provide a dermal papilla cell sphere obtained according to the above method.

[0014] The third objective of this invention is to provide an extracellular vesicle of the dermal papilla cell sphere, which is prepared by the following method: the dermal papilla cell sphere is suspended and cultured in an ultra-low adhesion culture dish containing DMEM / F-12 culture medium for 24 hours, and the culture supernatant is collected and centrifuged to obtain the vesicle.

[0015] A fourth objective of this invention is to provide the use of the dermal dermal papilla cell spheres or the extracellular vesicles in the preparation of products that inhibit scar formation and / or promote the regeneration of skin appendages and / or promote angiogenesis.

[0016] Preferably, the dermal papilla cell spheroids or the extracellular vesicles are used to prepare products that promote the proliferation and / or migration of keratinocytes;

[0017] The dermal dermal papilla cell spheres or the extracellular vesicles are used to prepare products that promote fibroblast proliferation and / or migration.

[0018] The dermal papilla cell spheres or the extracellular vesicles are used to prepare products that promote the proliferation and / or migration of vascular endothelial cells.

[0019] The fifth objective of this invention is to provide a product that inhibits scar formation and / or promotes the regeneration of skin appendages and / or promotes angiogenesis, characterized in that it comprises the dermal papilla cell spheres and / or the extracellular vesicles.

[0020] Preferably, the product is a drug or cosmetic.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] 1. This invention utilizes a self-feeding layer 3D mesenchymal stem cell culture system (i.e., SLF-3D culture system) to successfully culture novel dermal papilla cell spheroids (tdDPCs). Transcriptome sequencing was used to compare the genetic differences between DPCs and tdDPCs. The results showed that, compared with DPCs, genes related to scar inhibition, hair follicle regeneration, and angiogenesis were significantly upregulated in tdDPCs. Animal experiments confirmed that tdDPCs have a better therapeutic effect on scarless wound healing than DPCs.

[0023] 2. This invention further extracted extracellular vesicles from tdDPCs and DPCs. Compared with DPCs, tdDPCs had a higher vesicle yield (approximately 5 times) and smaller vesicle size (approximately half that of DPCs; studies have shown that smaller vesicles are easier to load for drug delivery and penetration). In vivo and in vitro experiments confirmed that tdDPCs vesicles have a significant advantage over DPCs vesicles in scarless wound healing. Cellular and animal experiments showed that, compared with DPCs vesicles, tdDPCs vesicles promoted wound angiogenesis and vascular maturation, promoted wound epithelialization, promoted the regeneration of wound skin appendages, reduced the expression of scar-related markers, and resulted in a regular and orderly collagen arrangement after wound healing. Finally, the histopathological results of the wound skin tissue after tdDPCs vesicle treatment showed no significant difference from the surrounding normal skin.

[0024] 3. The dermal papillary cell spheres and vesicles provided by this invention have a good healing effect on wounds with full-thickness skin damage. tdDPCs and their EVs can promote epithelial repair of wounds by promoting the proliferation and migration of keratinocytes, promote the reconstruction of the dermal layer of the wound by promoting the proliferation and migration of fibroblasts and inhibiting the transformation of fibroblasts into scar fibroblasts, and accelerate the wound healing process and skin appendage regeneration by promoting angiogenesis and vascular maturation. The coordinated action of the above processes promotes scarless wound healing. Attached Figure Description

[0025] Figure 1 This is a diagram showing the culture process of dermal dermal papilla cell spheres (tdDPCs); A, the process of isolating and extracting primary DPCs; B, the morphological differences between DPCs and tdDPCs after they have been cultured; the left and right images in the DPCs group show DPCs with different degrees of fusion under a 50x field of view; the left image in the tdDPCs group is a direct view of cell culture, and the right image is a 200x magnified view of the left image.

[0026] Figure 2 This is an immunofluorescence double labeling method for detecting ALP and LEF1 markers in DPCs and tdDPCs; the right image in the same group is an enlarged view of the part framed in the left image.

[0027] Figure 3 It is a flow cytometry analysis of surface markers of DPCs;

[0028] Figure 4 It is a flow cytometry analysis of surface markers of tdDPCs;

[0029] Figure 5 It is a test of the adipogenic and osteogenic orientation induction ability of DPCs and tdDPCs;

[0030] Figure 6This is a comparison of the therapeutic effects of DPCs and tdDPCs on full-thickness skin lesions in mice; A. Actual photographs of wound healing; B. H&E staining of skin pathological sections from the wound 14 days later;

[0031] Figure 7 The method involves using transmission electron microscopy (TEM) and Western blotting to identify DPC-EVs and tdDPC-EVs. A) TEM is used to observe the morphology of DPC-EVs and tdDPC-EVs. B) Western blotting is used to identify the specific surface marker proteins of DPC-EVs and tdDPC-EVs.

[0032] Figure 8 This involves particle size identification of DPC-EVs and tdDPC-EVs; A, DPC-EVs; B, tdDPC-EVs;

[0033] Figure 9 The effects of DPC-EVs and tdDPC-EVs on the proliferation of HaCaT cells and fibroblasts; A. HaCaT cells; B. fibroblasts;

[0034] Figure 10 DPC-EVs and tdDPC-EVs promote HaCaT migration; A. Scratched image; B. Cell migration statistics;

[0035] Figure 11 DPC-EVs and tdDPC-EVs promote fibroblast migration; A. Scratched photograph; B. Cell migration statistics;

[0036] Figure 12 The expression of α-SMA in scar fibroblasts after stimulation by DPC-EVs and tdDPC-EVs was detected by immunofluorescence.

[0037] Figure 13 The immunofluorescence assay was used to detect type I collagen expression in scar fibroblasts stimulated by DPC-EVs and tdDPC-EVs.

[0038] Figure 14 The effect of Ki67 immunofluorescence staining on the proliferation of HUVECs was detected by DPC-EVs and tdDPC-EVs.

[0039] Figure 15 The effect of DPC-EVs and tdDPC-EVs on HUVECs cell proliferation was detected by CCK-8 assay.

[0040] Figure 16 This study examines the effects of DPC-EVs and tdDPC-EVs on HUVECs cell migration using a scratch healing assay. The results include: A) photographing the scratch healing process; and B) statistical analysis of the healing rate.

[0041] Figure 17 The crystal violet staining assay was used to detect the effects of DPC-EVs and tdDPC-EVs on HUVECs cell migration; A. Scratch healing photography; B. Cell migration count;

[0042] Figure 18 The effects of DPC-EVs and tdDPC-EVs on the tubule-forming ability of HUVECs;

[0043] Figure 19 The effects of DPC-EVs and tdDPC-EVs on the number of tubules formed in HUVECs; A. wound healing rate; B. cell migration number;

[0044] Figure 20 The effects of the DPC-EVs group and the tdDPC-EVs group on wound healing; A. Wound healing status; B. Wound healing rate;

[0045] Figure 21 These are Doppler signal images used to assess blood perfusion during wound healing in each group; the left image for the same period in each group is the peri-wound blood flow map, and the right image is the epidermal appearance map;

[0046] Figure 22 The analysis involved immunofluorescence to assess angiogenesis in the wounds of each group.

[0047] Figure 23 These are H&E and Masson stainings of skin tissue pathological sections from each group of wounds; the left image for each group with the same staining is a 50x field of view, and the right image is a 200x field of view. Detailed Implementation

[0048] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0049] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0050] The present invention will now be further illustrated with specific examples. The following embodiments are only for explaining the present invention and do not constitute a limitation thereof. The test samples and test procedures used in the following embodiments include the following: If the specific experimental conditions are not specified in the embodiments, they are generally performed according to conventional conditions or the conditions recommended by the reagent company; unless otherwise specified, the reagents, consumables, etc. used in the following embodiments can be obtained commercially.

[0051] The antibodies used in the embodiments of this invention include: anti-LEF1 (2230, Cell Signaling Technology; 1:200), anti-ALPL (AF2910, R&D System; 1:500), anti-Ki67 (SAB5700770, Sigma; 1:100), anti-CD31 (GB11063-2, Servicebio; 1:400), anti-α-SMA (AB124964, Abcam; 1:200), anti-Calnexin (Ab22595, Abcam; 1:1000), anti-TSG101 (ab125011, Abcam; 1:1000), and anti-CD9 (ab92). 726, Abcam; 1:1000), anti-CD81 (ab9201, Abcam; anti-type I collagen (ab96723, Abcam; 1:200), anti-KLF4 (ab214666, Abcam; 1:1000), anti-VEGFA (ab214424, Abcam; 1:1000), horseradish peroxidase-conjugated goat anti-rabbit antibody (D110058, Sangon Biotech; 1:3000), DyLight488-conjugated affinity-pure rabbit antibody (BA1124, Boster; 1:200), DyLight594-conjugated goat anti-rabbit antibody (BA1127, Boster; 1:200).

[0052] Cell source and culture method used in the embodiments of this invention:

[0053] Human keratinocyte cell line (HaCaT cells) was purchased from Beyotime Biotechnology Co., Ltd. (C6282) and cultured in RPMI 1640 medium containing 10% fetal bovine serum.

[0054] Primary human umbilical vein endothelial cells (HUVECs) were obtained from the laboratory cell bank of the Ophthalmology Center of Xijing Hospital, Air Force Medical University, and cultured in endothelial cell-specific medium (ECM, 1001, Sciencecell) containing 1% endothelial cell growth supplement (ECGS, 1052, ScienceCell).

[0055] With the approval of the Ethics Committee of Xijing Hospital, Air Force Medical University, and after obtaining informed consent from the patients, excised human hypertrophic scar tissue and adjacent full-thickness normal skin tissue were used to extract primary fibroblasts and scar fibroblasts. Both fibroblasts and scar fibroblasts were cultured in DMEM containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0056] All data in this invention embodiment were statistically analyzed using GraphPadPrism 8.0. Results are expressed as mean ± standard error (SEM) of the mean. Analysis of variance was used to calculate the significance of differences; a p-value < 0.05 was considered statistically significant.

[0057] Example 1

[0058] Isolation, extraction, identification and characterization of DPCs and tdDPCs

[0059] 1. Extraction

[0060] The whisker pads of 6-week-old male C57BL / 6 mice were excised. Under a microscope, the hair bulb at the end of the whisker was cut from the hair shaft and transferred to a sterile centrifuge tube. Excess connective tissue in the hair bulb was removed by type I collagenase digestion to obtain the dermal dermal papilla. This papilla was then inoculated into a sterile culture dish containing 3 ml of Dulbecco modified Eagle medium (DMEM, SH30022.01, HyClone) and 20% fetal bovine serum (FBS, BioIndustries, the mass concentration of fetal bovine serum in the culture dish mixture was 20%). The specific operation procedure was referred to "Gledhill, K., Gardner, A., and Jahoda, CA (2013). Isolation and establishment of hair follicle The method described in “dermalpapilla cell cultures.MethodsMolBiol989,285-292.10.1007 / 978-1-62703-330-5_22” is followed.

[0061] Primary DPCs from passages 1-3 were cultured for 2-3 days in high-glucose DMEM containing 10% fetal bovine serum (FBS). The culture temperature was 37°C and the CO2 concentration was 5%. After passage 3, the cells were cultured using StemPro. TM Accuase TM Primary DPCs were isolated using reagent (A1110501, Gibco) for 5 min, and a single-cell suspension was prepared. Then, the suspension was prepared with 5–6 × 10⁻⁶ cells. 4 pcs / cm 2The DPCs were seeded at a density of 2% in the SLF-3D culture system. Based on the total volume of the SLF-3D culture system, it contained 2% fetal bovine serum, human basic fibroblast growth factor (bFGF, 5 ng / mL, PeproTech), human epidermal growth factor (EGF 2 ng / mL, PeproTech), human PDGF (5 ng / mL, PeproTech), heparin (2 μg / mL Sigma, Mississaua), L-ascorbic acid-2-phosphate sesquisodium hydrate (50 μg / mL Sigma), and 100 U / mL penicillin. When the DPCs reached 90-100% confluence, the adherent DPCs formed hemispherical spheres, which were tdDPCs. Figure 1 B). When the density of spheres increases to 10 spheres / cm³ 2 Primary tdDPC spheres were collected using vigorous shaking and centrifugation. StemPro was used to collect the spheres. TM Accuase TM The reagents were used to separate primary tdDPC spheroids into individual tdDPC cells, which were then passaged. The individual tdDPC cells were seeded into the culture dishes where the primary tdDPC spheroids had originally been collected, with the original adherent DPCs serving as a scaffold. After 3–5 days of culture, these individual tdDPC cells formed large, round, suspended 3D spheroids, which were the secondary tdDPCs. In subsequent culture and passage, the separated individual tdDPC cells exhibited stable proliferation and passage characteristics in standard DMEM / F-12 medium containing 10% fetal bovine serum.

[0062] like Figure 1 As shown, primary DPCs can be easily observed migrating around the dermal papilla on day 5. Figure 1 A). A large number of tdDPCs are semi-suspended on SLF-3D ( Figure 1 B).

[0063] 2. Identification and characterization

[0064] (1) Immunofluorescence double labeling method for the detection of specific markers such as alkaline phosphatase (ALP) and lymphocyte enhancement factor 1 (LEF1).

[0065] DPCs and tdDPCs were seeded at a confluence of 10–20% into 24-well plates covered with slides. Cells adhering to the slides were washed twice with PBS, fixed with 4% paraformaldehyde, infiltrated with 0.1% Triton X-100, and blocked with 4% BSA. Cells were incubated overnight with primary antibody at 4°C, and the next day incubated with fluorescently conjugated secondary antibody for 1 hour and mounted on a DAPI fluorometer-G (0100-20, Southern Biotech, USA). Slides were placed in EVOS. TMObserved under a FLAuto 2 microscope.

[0066] The results showed that alkaline phosphatase (ALP) and lymphocyte enhancement factor 1 (LEF1) were both expressed in DPCs and tdDPCs. Figure 2 ).

[0067] (2) Flow cytometry analysis of cell surface markers

[0068] DPCs and tdDPCs were incubated with PE-bound anti-CD29 antibody (112207, eBioscience; 1:20), PE-bound anti-CD34 antibody (800504, BioLegend; 1:20), PE-bound anti-CD44 antibody (103007, BioLegend; 1:20), PE-bound anti-CD90 antibody (140307, ​​BioLegend; 1:20), PE-bound anti-CD105 antibody (120407, BioLegend; 1:20), and FITC-bound anti-CD45 antibody (157213, BioLegend; 1:20), respectively, at 4°C for 30 min. The percentage of positive cells was analyzed using a Coulter EPICS XL flow cytometer (Beckman Coulter, USA) and an EXPO32 ADC analyzer (Beckman Coulter, USA).

[0069] Flow cytometry results showed that DPCs ( Figure 3 ) and tdDPCs( Figure 4 It highly expresses CD29, CD44, CD90, and CD105, but not CD34 and CD45.

[0070] (3) Detection of stem cell adipogenic and osteogenic orientation induction ability

[0071] DPCs and tdDPCs of the same generation were seeded into 6-well plates and cultured routinely. When the cell confluence reached 80% and 100%, the medium was replaced with osteogenic induction medium and adipogenic induction medium, respectively. The medium was changed every 2 days. On day 14, the cells in the adipogenic induction medium were stained with Oil Red O. On day 28, the cells in the osteogenic induction medium were stained with alkaline phosphatase and Alizarin Red S to compare the adipogenic and osteogenic directional differentiation abilities of the two cell types.

[0072] Alizarin Red and Oil Red O (ORO) staining showed that both cell types possessed osteogenic and adipogenic differentiation potential. Figure 5 ).

[0073] Example 2

[0074] Treatment of full-thickness skin lesions in mice by DPCs and tdDPCs

[0075] 1. Animal model construction

[0076] Fifteen healthy 6-week-old male C57BL / 6 mice were purchased from the Experimental Animal Center of Air Force Medical University. All procedures were approved by the Ethics Committee of Air Force Medical University. After hair removal from the backs of the mice, two full-thickness skin wounds (5 mm in diameter) were made on both sides of the spine using a surgical perforator. Medical adhesive was used to attach silicone rings around the wounds to restrict wound contraction.

[0077] 2. Method

[0078] Mice were randomly divided into three groups using a random number table: PBS, DPCs, and tdDPCs. Immediately after modeling, DPCs (1 million cells / 100 μL PBS), tdDPCs (1 million cells / 100 μL PBS), or an equal volume of PBS were injected subcutaneously into the wound, and the wound was covered with a sterile medical VSD film. Wound healing was assessed on days 0, 7, and 14. Mice were sacrificed on day 14, and skin tissue samples were collected for subsequent experiments.

[0079] 3. Results

[0080] 3.1 Wound healing

[0081] Fourteen days after modeling, the healing outcomes in the tdDPCs treatment group were significantly better than those in the DPCs or PBS treatment groups. Figure 6 A).

[0082] 3.2 Eosin (H&E) staining of skin tissue pathological sections from wounds

[0083] Mouse skin tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections. The sections were stained with H&E and imaged using a panoramic 250 Flash series digital scanner (3DHISTECH, Hungary).

[0084] H&E staining results showed that the tdDPCs group had abundant regeneration of skin appendages after wound healing, and the skin tissue tended to be normal. The DPCs group had thickened dermis and skin fibrosis, with some new skin appendages visible underneath. The PBS group had no new skin appendages and lower wound healing quality. Figure 6 B).

[0085] The above results indicate that tdDPCs are significant in promoting and improving wound healing. Compared with DPCs and PBS, tdDPCs can significantly improve the speed and quality of wound healing.

[0086] Example 3

[0087] Extraction and identification of DPC-EVs and tdDPC-EVs

[0088] Following the method described in "Hu, S., Li, Z., Lutz, H., Huang, K., Su, T., Cores, J., Dinh, PC, and Cheng, K. (2020). Dermal exosomes containing miR-218-5p promote hair regeneration by regulating β-catenin signaling. Sci Adv 6, eaba 1685.10.1126 / sciadv.aba1685.", the DPCs and tdDPCs extracted in Example 1 were cultured for 24 h in DMEM / F12 medium (51448C, HyClone) containing 10% serum to remove EVs. Then, DPC-EVs and tdDPC-EVs were separated from the culture supernatant by ultracentrifugation.

[0089] DPC-EVs and tdDPC-EVs were quantified at 562 nm using a BCA protein assay kit (Thermo Science, Waltham, MMA, USA). The expression of surface markers in DPC-EVs and tdDPC-EVs was analyzed by Western blotting. Specifically, EVs were lysed in radioimmunoprecipitation (RIPA) buffer containing 1× protease inhibitor. Protein concentration was determined using a BCA kit (P0012, Beyotime). Equal volumes of protein were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane, incubated overnight with primary antibody. The next day, the PVDF membrane was incubated with HRP-conjugated secondary antibody. Protein expression was observed using ECL substrate (WBKLS0050, Millipore).

[0090] The morphology of DPC-EVs and tdDPC-EVs was observed using transmission electron microscopy. The particle concentration and particle size distribution of DPC-EVs and tdDPC-EVs were determined by nanoflow cytometry.

[0091] DPC-EVs and tdDPC-EVs were identified by transmission electron microscopy, Western blotting, and nanoflow cytometry. Figure 7-8 The morphology of DPC-EVs and tdDPC-EVs was observed using transmission electron microscopy, showing a spherical or biconcave disk shape. Figure 7A). Furthermore, both DPC-EVs and tdDPC-EVs expressed EV-specific surface marker proteins TSG101, CD81, or CD9, while calnexin expression was negative in both cases. Figure 7 B). Particle size analysis showed that the average particle sizes of DPC-EVs and tdDPC-EVs were 148.80 nm and 77.44 nm, respectively. Figure 8 A, Figure 8 B). The particle concentrations of DPC-EVs and tdDPC-EVs were 5.56 × 10⁻⁶. 9 Particles / mL and 3.14 × 10 10 Particles / mL.

[0092] Example 4

[0093] In vitro effects of DPC-EVs and tdDPC-EVs on wound epithelialization and dermal repair

[0094] 1. Cell proliferation capacity detection

[0095] HaCaT cells and fibroblasts were seeded into 96-well culture plates (1×10⁶ cells / well). 4 Four replicates were prepared in each well. After cell adhesion, the cells were starved for 24 hours, and then cultured in the corresponding medium containing 10 μg / ml DPC-EVs and tdDPC-EVs and an equal volume of PBS for 12–72 hours. The absorbance was measured at 450 nm using the CCK-8 assay kit (C0038, Beyotime) according to the manufacturer's instructions to detect changes in cell proliferation.

[0096] The results showed that, compared with DPC-EVs, tdDPC-EVs were more effective in promoting the proliferation of HaCaT cells and fibroblasts. Figure 9 A, Figure 9 B).

[0097] 2. Cell migration ability detection

[0098] 5×10 5 HaCaT cells and fibroblasts were seeded into 6-well culture plates. Once the cells reached 100% confluence, the cell surface was scratched using a 200 μL sterile pipette tip in a crisscross pattern. Floating cells were removed with PBS, and serum-free culture medium was added. Three groups were established: two groups were supplemented with DPC-EVs (10 μg / mL) and tdDPC-EVs (10 μg / mL), respectively, with an equal volume of PBS as a control. The scratches were photographed at 0, 24, and 48 hours using an inverted microscope (Zeiss, Germany).

[0099] The results showed that DPC-EVs significantly promoted the migration of HaCaT ( Figure 10 ), while tdDPC-EVs showed a greater migration-promoting effect than DPC-EVs. Figure 10 Furthermore, compared to DPC-EVs, tdDPC-EVs also have a stronger effect in promoting fibroblast migration. Figure 11 ).

[0100] 3. Detection of scar marker expression

[0101] Scar fibroblasts were seeded at 10–20% confluence into 24-well plates covered with slides, with three groups. Two groups received DPC-EVs (10 μg / mL) and tdDPC-EVs (10 μg / mL), respectively, and an equal volume of PBS was used as a control. Cells adhering to the slides were washed twice with PBS, fixed with 4% paraformaldehyde, infiltrated with 0.1% Triton X-100, and blocked with 4% BSA. Scar fibroblasts were incubated overnight at 4°C with anti-α-SMA and anti-type I collagen, respectively. The next day, they were incubated with fluorescently conjugated secondary antibodies for 1 hour and then incubated for 5 minutes using a DAPI fluorometer-G (0100-20, Southern Biotech). The slides were then incubated in EVOS. TM Observed under a FLAuto2 microscope.

[0102] The results showed that, compared with DPC-EVs, tdDPC-EVs stimulated α-SMA in scar fibroblasts. Figure 12 ) and decreased immunofluorescence intensity of type I collagen ( Figure 13 This suggests that tdDPC-EVs can promote post-traumatic dermal reconstruction by reversing the transdifferentiation of fibroblasts into myofibroblasts.

[0103] The above results indicate that tdDPC-EVs can accelerate wound epithelialization, improve dermal reconstruction, and reduce skin fibrosis.

[0104] Example 5

[0105] In vitro effects of DPC-EVs and tdDPC-EVs on angiogenesis

[0106] 1. Detection of HUVECs cell proliferation capacity

[0107] HUVECs were seeded at 20–30% confluence into 24-well plates covered with glass slides, with three groups. Two groups were incubated with DPC-EVs (10 μg / mL) and tdDPC-EVs (10 μg / mL), respectively, for 24 h, with an equal volume of PBS as a control. After 24 h, HUVECs were subjected to Ki67 immunofluorescence staining.

[0108] The results showed that tdDPC-EVs significantly enhanced the proliferation of HUVECs compared to DPC-EVs and PBS. Figure 14 ).

[0109] HUVECs were seeded in 96-well culture plates (1×10⁶). 4 Four replicates were prepared in each well. After cell adhesion, the cells were starved for 24 hours, and then cultured in the corresponding medium containing 10 μg / ml DPC-EVs and tdDPC-EVs and an equal volume of PBS for 48 hours. The absorbance was measured at 450 nm using the CCK-8 assay kit (C0038, Beyotime, China) according to the manufacturer's instructions to detect changes in cell proliferation.

[0110] The results showed that tdDPC-EVs were more effective than DPC-EVs in promoting the proliferation of HUVECs. Figure 15 ).

[0111] 2. HUVECs cell migration ability detection

[0112] 5×10 5 HUVECs were seeded into 6-well culture plates. Once the cells reached 100% confluence, the cell surface was scratched using a 200 μL sterile pipette tip in a crisscross pattern. Floating cells were removed with PBS, and serum-free culture medium was added. Three groups were established: two groups were supplemented with DPC-EVs (10 μg / mL) and tdDPC-EVs (10 μg / mL), respectively, with an equal volume of PBS as a control. The scratches were photographed at 0 and 24 hours using an inverted microscope (Zeiss, Germany).

[0113] 5×10 4 HUVECs were seeded in the upper chamber of a Transwell 24-well plate (3422, Corning, USA). The lower chamber was filled with 600 μL of serum-free and ECGS-free medium as a nutrient inducer. Three groups were set up, with two groups containing DPC-EVs (10 μg / mL) and tdDPC-EVs (10 μg / mL), respectively, and an equal volume of PBS as a control. After 24 h of culture, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 0.5% crystal violet for 30 min. Non-invasive or non-migrating cells were removed using clean cotton swabs. Five fields of view from each well were randomly photographed under a bright-field microscope.

[0114] The results showed that, compared with PBS, although DPC-EVs significantly enhanced cell migration, tdDPC-EVs further enhanced HUVEC migration. Figure 16 and Figure 17 ).

[0115] 3. Detection of HUVECs' ability to form tubules

[0116] Polymerized matrix gel was prepared by mixing matrix gel and ECM medium at a rate of 200 μL / well in a 48-well plate and incubated at 37°C for 30 min. HUVECs (1×10⁻⁶) were then added. 5 (e.g., 1 / cell) were suspended in 200 μL of serum-free ECM medium and inoculated onto polymerized matrix gels. Three groups were set up, with two groups receiving DPC-EVs (10 μg / mL) and tdDPC-EVs (10 μg / mL), respectively, and an equal volume of PBS group serving as a control. After incubation at 37°C for 3 h and 6 h, the cells were inoculated onto EVOS. TM Capillary formation was detected under a FLAuto 2 microscope. Three experiments were conducted under each condition. ImageJ software was used to analyze capillary length and branching points.

[0117] The results showed that tdDPC-EVs further increased the number of tubules formed in HUVECs. Figure 18-19 This indicates that tdDPC-EVs have a greater pro-angiogenic effect on HUVECs than DPC-EVs.

[0118] Example 6

[0119] DPC-EVs and tdDPC-EVs improve wound healing speed and quality in vivo.

[0120] 1. Animal model construction

[0121] Fifteen healthy 6-week-old male C57BL / 6 mice were purchased from the Experimental Animal Center of Air Force Medical University. All procedures were approved by the Ethics Committee of Air Force Medical University. After hair removal from the backs of the mice, two full-thickness skin wounds (5 mm in diameter) were made on both sides of the spine using a surgical perforator. Medical adhesive was used to attach silicone rings around the wounds to restrict wound contraction.

[0122] 2. Method

[0123] Mice were randomly divided into three groups using a random number table: PBS, DPC-EVs, and tdDPC-EVs. Immediately after modeling, DPC-EVs (50 μg / 100 μL PBS), tdDPC-EVs (50 μg / 100 μL PBS), or an equal amount of PBS were applied to the wound, and the wound was covered with a sterile medical VSD dressing. Wound healing and blood perfusion were assessed on days 0, 7, and 14 using laser Doppler blood flow imaging and an animal gross specimen imaging system. Mice were sacrificed on day 14, and skin tissue specimens were collected for subsequent experiments.

[0124] 3. Results

[0125] 3.1 Wound healing

[0126] ImageJ software was used to analyze and quantify the wound area. The wound healing rate (WHR) was calculated as follows: WHR = [(A0 - A...] t ) / A0]×100%, where A0 represents the initial wound size on day 0, A t Indicates the wound size at a specified time point.

[0127] Fourteen days after modeling, the healing outcomes in the tdDPC-EVs treatment group were significantly better than those in the DPC-EVs or PBS treatment groups. Figure 20 A). On day 7, the degree of epithelialization in wounds treated with tdDPC-EVs was statistically higher than that in wounds treated with DPC-EVs or PBS (A). Figure 20 B).

[0128] 3.2 Imaging and assessment of skin blood perfusion in wounds

[0129] Blood flow in the wound and surrounding area was detected using a laser Doppler perfusion imaging (LDPI) analyzer (PerimedAB, Sweden). The laser Doppler was fixed at a distance of 8–10 cm above the wound. Perfusion of the wound area (ROI1) and adjacent skin was detected at a distance of 2 cm from the wound boundary (ROI1). The average perfusion rate was calculated as blood perfusion rate = ROI1 / ROI2.

[0130] Doppler signals detected at the wound edge to assess blood perfusion during wound healing indicated that, although blood perfusion was significantly increased in the DPC-EVs group compared to the PBS group, wound blood flow in the tdDPC-EVs group was significantly higher than that in the DPC-EVs group. Figure 21 ).

[0131] Considering the significant improvement in wound blood perfusion after tdDPC-EVs treatment, we performed immunofluorescence analysis of the endothelial marker CD31 and the mature vessel marker α-SMA on tissue sections on day 14 to assess angiogenesis. In wounds treated with tdDPC-EVs, more mature large vessels and thicker vessel walls were observed, while wounds in the DPC-EVs or PBS groups were covered by numerous small vessels. Figure 22 ).

[0132] 3.3. Eosin (H&E) and Masson staining of skin tissue pathological sections from wounds

[0133] To examine morphological differences during skin regeneration, wound tissue on day 14 was stained with H&E and Masson staining.

[0134] Mouse skin tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections. The sections were stained with H&E or Masson trichrome and imaged using a panoramic 250 Flash series digital scanner (3DHISTECH, Hungary).

[0135] H&E staining results showed that, compared with the DPC-EVs group and the PBS group, the tdDPC-EVs group had intact epithelialization of the epidermal layer, uniform dermal thickness, a significant increase in new skin appendages, and obvious skin and muscle regeneration after wound healing.

[0136] Masson staining results showed that after wound healing, the collagen fibers in the skin of mice treated with DPC-EVs and PBS exhibited a tightly wavy and disordered arrangement, while the tdDPC-EVs treatment group showed improved collagen arrangement, with relatively loose and orderly collagen deposition as shown in the figure. Figure 23 ).

[0137] The above results indicate that tdDPC-EVs are more effective in promoting and improving wound healing. Compared with DPC-EVs and PBS, tdDPC-EVs can significantly improve the speed and quality of wound healing and help promote scarless wound healing.

[0138] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for in vitro culture of dermal dermal papilla cell spheroids, characterized in that, Includes the following steps: Primary dermal dermal papilla cells were extracted from isolated dermal dermal papilla tissue and passaged for 3-5 generations. The primary dermal dermal papilla cells were extracted from the hair bulb at the end of isolated mouse whiskers using a combination of microscopic separation and modified enzyme digestion. After passage, dermal papilla cells were seeded into the SLF-3D culture system. When the dermal papilla cells grew to a confluence of 90-100%, the semi-suspended spheres were collected, and the spheres were separated into single cells. The single cells were then re-seeded into the original SLF-3D culture system and cultured for 3-5 days. The resulting suspended spheres were collected, which are the dermal papilla cell spheres. The SLF-3D culture system is a self-feeding layer 3D mesenchymal stem cell culture system, which uses DEME / F-12 as a solvent. Based on the total volume of the SLF-3D culture system, the SLF-3D culture system also includes the following raw materials: 2-3% fetal bovine serum, human basic fibroblast growth factor 4-6 ng / mL, human epidermal growth factor 0.8-3 ng / mL, human platelet-derived growth factor 4-6 ng / mL, heparin 1-3 ug / mL, L-ascorbic acid-2-phosphate sesquisodium hydrate 30-60 ug / mL, and penicillin 80-120 U / mL.

2. The method according to claim 1, characterized in that, The seeding density of the dermal papilla cells in the SLF-3D culture system was 5~6×10⁶. 4 pcs / cm 2 .

3. The method according to claim 2, characterized in that, Based on the total volume of the SLF-3D culture system, the SLF-3D culture system comprises the following raw materials: 2% fetal bovine serum, human basic fibroblast growth factor 5 ng / mL, human epidermal growth factor 2 ng / mL, human platelet-derived growth factor 5 ng / mL, heparin 2 ug / mL, L-ascorbic acid-2-phosphate sesquisodium hydrate 50 ug / mL, and penicillin 100 U / mL.

4. The method according to claim 1, characterized in that, The specific culture process of the passage culture is as follows: First, the primary dermal papilla cells are seeded into Dulbecco modified Eagle medium containing 20% ​​fetal bovine serum and cultured. When the growth confluence reaches 80-90%, the cells are passaged. After that, the medium is replaced with high-glucose DMEM containing 10% fetal bovine serum for continued culture.

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