Preparation method and application of extracellular matrix based on human skin organoid

Abstract

The invention discloses a preparation method and application of an extracellular matrix based on human skin organoid. The method comprises the following steps: taking a full-layer human skin tissue, performing enzyme digestion to obtain a single-cell suspension, and inducing in a three-dimensional culture system to form a skin organ with an epidermal layer and corium layer structure; then applying a synergistic effect of physical stimulation and physiological stimulation to the organoid, and efficiently inducing ECM secretion; and finally, carrying out decellularization treatment and ultracentrifugation purification to obtain the high-purity ECM material. The ECM retains a natural three-dimensional network structure, does not contain animal-derived components, and is low in immunogenicity. The prepared ECM has good effects in the aspects of promoting cell adhesion, accelerating wound healing and activating skin cell functions, and can be applied to preparation of biological dressings or anti-aging cosmetics for repairing chronic wounds. The method has the advantages of being short in preparation period, high in structural integrity, good in biological safety, capable of achieving large-scale production and the like.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering and regenerative medicine technology, specifically relating to an efficient method for preparing extracellular matrix (ECM) based on human skin organoids and its application. Background Technology

[0002] The extracellular matrix (ECM) is an important component of the tissue microenvironment, mainly composed of collagen, fibronectin, and glycosaminoglycans, and plays a vital role in supporting cell attachment, proliferation, differentiation, and tissue regeneration. Currently, the main sources of ECM are twofold: One type is animal-derived ECM, such as porcine small intestinal submucosa (SIS), bovine pericardium, or decellularized matrix derived from porcine skin. While these materials are less expensive, they carry the risk of foreign protein residues, which may trigger immune rejection in humans, and there are significant batch-to-batch variations.

[0003] Secondly, there is in vitro cell synthesis of ECM, which typically uses human fibroblasts to secrete ECM in two-dimensional or three-dimensional culture. However, this method results in ECM with a single component, lacking the complex three-dimensional cross-linked structure of natural tissues, exhibiting poor mechanical properties, and requiring a long culture period of 2–3 weeks, making it difficult to meet clinical needs.

[0004] Third, human tissues (such as placenta and dermis) require strict ethical approval, and donors are scarce. Traditional human ECM relies on surgically discarded tissues, resulting in significant batch-to-batch variations and insufficient production capacity. Organoids can differentiate into specific tissues, and their secreted ECM contains human collagen, laminin, and elastin, with a composition highly consistent with natural tissues.

[0005] In recent years, the development of organoid technology has provided a new pathway for constructing humanized tissues. However, existing research has mostly focused on disease modeling or drug screening and has not yet been systematically applied to the large-scale preparation of functional ECMs. In addition, how to efficiently activate the ECM secretion capacity of organoid cells through exogenous stimulation remains a technical bottleneck.

[0006] Therefore, developing a non-immunogenic, structurally intact, and highly efficient human ECM preparation technology is of significant scientific importance and industrial value. Summary of the Invention

[0007] The purpose of this application is to provide a rapid method for preparing human skin organoid extracellular matrix (ECM) that is non-immunogenic, structurally intact, and highly efficient, so as to achieve efficient, safe, and scalable production of ECM.

[0008] To achieve the above-mentioned objectives, this application adopts the following technical solution: A method for preparing extracellular matrix based on human skin organoids includes the following steps: (1) Organoid construction: Full-thickness skin tissue was taken, and a single-cell suspension was obtained after enzyme digestion. The suspension was then cultured in a three-dimensional culture system containing Matrigel matrix to induce the formation of skin organoids with epidermal and dermal structures. (2) Combined stimulation to induce ECM secretion: The skin organoids are subjected to the synergistic effect of physical and physiological stimulation, wherein the physical stimulation is periodic mechanical stretching with a deformation rate of 8–12% and a frequency of 0.4–0.6 Hz; the physiological stimulation is the sustained release of TGF-β1 and ascorbic acid. (3) ECM purification: Cell components are removed by decellularization buffer, and then separated by ultracentrifugation. The precipitate is collected to obtain the extracellular matrix.

[0009] Furthermore, the enzymes mentioned in step (1) are collagenase IV and neutral protease, with concentrations of 0.2–0.3% and 0.08–0.12%, respectively, and digested by shaking at 37°C for 1.5–2.5 hours.

[0010] Furthermore, in step (1), the Matrigel matrix concentration is 7–9 mg / mL, the cell seeding density is 4×10⁵–6×10⁵ cells / mL, and the culture time is 5–9 days.

[0011] Furthermore, the mechanical stretching in step (2) has a deformation rate of 10%, a frequency of 0.5 Hz, and is stimulated for 3–5 hours daily for 2–4 days.

[0012] Furthermore, in step (2), the concentration of TGF-β1 is 8–12 ng / mL, the concentration of ascorbic acid is 40–60 μg / mL, and the sustained release rate is 0.08–0.12 μL / min.

[0013] Furthermore, the decellularization buffer in step (3) contains 0.08–0.12% Triton X-100 and 8–12 mM EDTA, and is treated with shaking at 4°C for 18–30 hours, with the buffer being replaced 2–4 times.

[0014] Furthermore, the ultracentrifugation conditions in step (3) are: 100,000×g, 4℃, centrifugation for 1.5–2.5 hours.

[0015] Furthermore, in step (2), the combined stimulation to induce ECM secretion employs a physical-physiological combined stimulation device, which includes: Flexible culture membranes used to support organoids; A mechanical stretching module is configured to apply periodic deformation to the flexible culture membrane; The growth factor micropump system, connected to a conduit, slowly releases TGF-β1 solution into the culture environment at a rate of 0.08–0.12 μL / min; The control unit synchronously regulates the stretching frequency and the growth factor release rhythm.

[0016] An extracellular matrix material, wherein the ECM contains ≥65% collagen, <50 ng / mg residual DNA, and retains a naturally cross-linked three-dimensional fiber network structure.

[0017] The use of the aforementioned extracellular matrix material in the preparation of biological dressings for chronic wound repair or anti-aging cosmetics.

[0018] An anti-aging serum composition containing human ECM, comprising the following components by weight percentage: Human extracellular matrix (ECM) of skin cells: Add human ECM powder to 0.3M citric acid solution, then add pre-cooled PBS at a volume ratio of 1:1-100, vortex mix, and finally adjust the pH to 7.0-7.5 with 1M NaOH, with a content of 1.0% (w / w). Glycerin, content 5.0% (w / w); Sodium hyaluronate, content 0.2% (w / w); Nicotinamide, content 3.0% (w / w); Phenoxyethanol, content 0.6% (w / w); Add deionized water to bring the concentration to 100% (w / w).

[0019] Matrigel is a soluble basement membrane extract derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, primarily mimicking the structure and function of the extracellular matrix in vivo. As a non-human material, it presents a range of challenges, including immune rejection and ethical concerns.

[0020] This application provides a rapid method for preparing extracellular matrix (ECM) based on human skin organoids. By constructing skin organoids with a bilayer structure of epidermis and dermis, and combining cyclic mechanical stretching with synergistic stimulation by growth factors / ascorbic acid, the method effectively promotes the efficient secretion of functional ECM. The obtained ECM material is natural in composition and structurally intact, retaining a three-dimensional fiber network similar to real skin, and is free of foreign proteins, significantly reducing the risk of immune rejection. This method has a short preparation cycle and controllable process. The resulting ECM exhibits excellent performance in supporting cell adhesion and promoting tissue regeneration, accelerating the repair process of acute and chronic wounds and effectively activating skin cell activity. Therefore, it is suitable for preparing biological dressings for chronic wound repair and cosmetics with anti-aging effects, possessing good safety, biocompatibility, and industrial application prospects. Attached Figure Description

[0021] Figure 1 Image showing the wound healing process in mice; Among them, A is control group 1; B is control group 2; C is experimental group 1; D is experimental group 2; and E is experimental group 3. Detailed Implementation

[0022] The present application will be further illustrated by the following embodiments, but the scope of protection of the present application is not limited to the embodiments.

[0023] Example 1: Preparation of extracellular matrix based on human skin organoids Tissue source: Abdominal skin tissue (approximately 1 cm³) obtained from healthy adults with ethical approval and signed informed consent.

[0024] 1. Preparation of single-cell suspension Skin tissue was minced to 1 mm³ and added to DMEM / F12 medium (Gibco) containing 0.25% collagenase IV (Sigma-Aldrich) and 0.1% neutral protease II (Roche). The mixture was digested at 37°C and 120 rpm in a shaking water bath for 2 hours. After digestion, an equal volume of DMEM / F12 medium containing 10% fetal bovine serum (FBS) was added to terminate the reaction. The mixture was centrifuged at 300 × g for 5 minutes, the supernatant was discarded, and the cells were washed twice with PBS and resuspended to obtain a single-cell suspension.

[0025] 2. 3D Culture of Skin Organoids The single-cell suspension was resuspended in pre-chilled Matrigel matrix (Corning, #354234) and the final concentration was adjusted to 8 mg / mL Matrigel, with a cell density of 5 × 10⁶ cells / mL. 5 cells / mL. 100 μL of the mixture was seeded into a 96-well ultra-low adsorption plate and incubated at 37°C with 5% CO2 for 30 minutes to allow Matrigel to solidify. Then, 200 μL of organoid culture medium (DMEM / F12 + 10% FBS + 1% penicillin / streptomycin + 20 ng / mL LEGF + 10 ng / mL LFGF2) was added to each well. The medium was replaced with fresh medium every 3 days. After 7 days of culture, organoid formation was observed under a microscope, with an average diameter of approximately 250 μm.

[0026] 3. Validation of organoid structures Partial organoids were embedded in paraffin and stained with hematoxylin and eosin (HE), revealing a clear layered structure of the epidermis (multilayered keratinocytes) and dermis (loose connective tissue). Immunofluorescence staining showed that the epidermis expressed keratin 14 and the dermis expressed vimentin, confirming the successful construction of human skin organoids with a two-layered structure.

[0027] 4. Combined physical and physiological stimulation Mature organoids, along with Matrigel cells, were transferred onto a flexible polydimethylsiloxane (PDMS) culture membrane (0.5 mm thick) and placed in a Flexcell FX-6000™TT mechanical stretching system (Flexcell International Corp., USA). Parameters were set as follows: uniaxial periodic stretching, 10% deformation rate, 0.5 Hz frequency, stimulation for 4 hours daily for 3 consecutive days. Simultaneously, TGF-β1 (PeproTech, #100-21, 10 ng / mL) and ascorbic acid (Sigma, A4544, 50 μg / mL) were continuously released into the culture environment at a rate of 0.1 μL / min using a microinjection pump (Harvard Apparatus, PHD2000).

[0028] 5. ECM purification After stimulation, the culture medium was aspirated, and decellularization buffer (containing 0.1% Triton X-100 and 10 mM EDTA in PBS) was added. The mixture was incubated at 4°C with shaking (50 rpm) for 24 hours, with the buffer changed three times during this period. The residual matrix was collected, washed three times with PBS, and transferred to an ultracentrifuge tube. The tube was centrifuged at 4°C, 100,000 × g for 2 hours (Beckman Optima XPN-100). The supernatant was discarded, and the white precipitate at the bottom was collected. After lyophilization, the resulting powdered ECM material was stored at –80°C for later use.

[0029] Comparative Example 1: Using porcine ECM as a substitute for Matrigel Except for replacing Matrigel in step 3 with commercially available porcine acellular dermal matrix (Veloderm®, 1 mg / mL), the rest of the procedures were the same as in Example 1. After 9 days of culture, organoids were formed with a diameter of approximately 220 μm. HE staining revealed an epidermal-dermal structure, and CK14 and Vimentin expression were positive. However, the organoid morphology was irregular, and the yield was reduced by approximately 30%.

[0030] Comparative Example 2: Using human ECM instead of Matrigel Except for replacing Matrigel with human ECM (1 mg / mL) prepared in our laboratory previously, the rest of the operation was the same as in Example 1. After 7 days, organoids were formed with a diameter of about 250 μm. The structure was similar to that in Example 1, but the cost was significantly increased, making it unsuitable for large-scale production.

[0031] Example 2: ECM Performance Testing The physicochemical properties and biological functions of the ECM material prepared in this invention were systematically evaluated, and the following detection experiments were conducted.

[0032] 1. Composition and purity analysis The composition and purity of the ECM material obtained in Example 1 were analyzed.

[0033] Collagen content: determined using the hydroxyproline method, the result was 68.3±2.1%; Fibronectin content: Detected by ELISA (R&D Systems, DFN10), the result was 12.7 ± 1.5%; Glycosaminoglycan content: determined by DMMB colorimetric method, the result was 8.4±0.9%; Purity test: The residual DNA content was determined to be 42.3 ± 6.7 ng / mg using the Picogreen method.

[0034] 2. Cell adhesion experiment (1) Experimental grouping Set up the following three groups, each with 6 duplicate holes, and repeat independently 3 times: Experimental group: coated human ECM (prepared in Example 1 of this invention, 1 mg / mL); Positive control group: coated with commercially available porcine acellular dermal matrix (Veloderm®, 1 mg / mL). Negative control group: Blocked with only 1% BSA, without coating any ECM.

[0035] (2) Experimental steps ① Add each ECM solution (1 mg / mL in PBS) to a 96-well plate, 100 μL / well, and coat overnight at 4°C; ②Wash gently 3 times with PBS, add 100 μL of 1% BSA to each well, and block at 37°C for 1 hour; ③ Inoculate with human dermal fibroblasts (HDFs, ScienCell, #2300) at a density of 5 × 10⁻⁶. 4 cells / mL, 100μL / well; ④ After culturing at 37°C for 24 hours, discard the culture medium and gently wash three times with PBS to remove unattached cells; ⑤ Fix with 4% paraformaldehyde for 30 minutes, stain with Giemsa for 15 minutes, and count the number of adherent cells in 8 fields of view in each well under an inverted microscope (200×).

[0036] (3) Calculation and statistics of results Adhesion rate (%) = (number of adherent cells / initial number of seeded cells) × 100%.

[0037] The results show: The average adhesion rate of the experimental group was 78.3 ± 4.1%; The positive control group was 38.2 ± 3.7%; The negative control group was less than 10%.

[0038] The adhesion rate in the experimental group was 40.1% higher than that in the positive control group (p<0.01, two-tailed t-test).

[0039] 3. Mouse full-thickness skin defect repair experiment (1) Animal grouping Fifty 8-week-old male C57BL / 6 nude mice (from Beijing Vital River) were randomly divided into 55 groups of 10 mice each. Control group 1: An equal volume of PBS was applied. Control group 2: Applied animal-derived (porcine-derived) ECM 1mg / ml; Experimental Group 1: Apply human skin organoid-derived ECM 1 mg / ml as described in Example 1; Experimental Group 2: Application of human skin organoid ECM from Example 1 5mg / ml; Experimental Group 3: Application of Example 1: Human Skin Organoid ECM 10mg / ml.

[0040] Mouse skin injury model: Eight-week-old male nude mice were acclimatized for one week, then anesthetized. The skin tissue on the back was disinfected with povidone-iodine. Then, a 1cm diameter circular punch (HY punch, 010) was used to punch holes in the skin. The prepared preparation was then applied to the punched sites on the mice. After drying and fixing for 30 minutes, this was recorded as day 0. The condition of the skin on the back of the nude mice was continuously observed and recorded.

[0041] The criteria for judging the skin's ability to heal are as follows: when the skin at the site of injury is uniformly free of wounds or scabs have fallen off, the wound is considered to be healed or approaching healing. The specific phenotypic observations of wound healing are shown in the table below: The condition on the 9th day after the injury and treatment is as follows Figure 1 As shown in the table below, observations and analyses of each group are presented: Group Image features Healing status assessment Technical Interpretation Control group 1 The wound remains an open defect with visible redness and swelling at the edges, and a noticeable crust or exudate in the center; the area is relatively large. Unhealed Natural repair is slow, the inflammatory period continues, and epithelial migration is hindered; only about 40% closure was completed by the 9th day. Control group 2 The wound is shrinking, the edges are beginning to epithelialize, the center is still covered with a superficial scab, and new tissue is visible in some areas. Partial healing (approximately 60–70%) Animal-derived ECMs provide a basic scaffold and promote cell infiltration, but their repair speed is limited due to the low recognition efficiency of foreign proteins. Experimental group 1 The wound is completely closed, the surface is smooth, there is no obvious scab, and the skin color is close to normal. Newly healed This indicates that a concentration of 1 mg / mL can effectively activate fibroblast proliferation, collagen deposition, and reepithelialization. Experimental group 2 The wound is completely closed, the surface is smooth, and there are no abnormal signs. Complete healing High concentrations further optimized the microenvironment, but did not significantly accelerate the healing process, suggesting that the dosage had reached a plateau. Experimental group 3 The wound is completely closed, the skin texture is uniform, and there are no signs of scarring or pigmentation. Complete healing The effect was consistent with that of the 5 mg / mL group, indicating that 1 mg / mL is the minimum effective dose, and higher concentrations do not provide additional benefits. Key conclusions: 1. Human-derived ECM significantly accelerates wound healing: On day 9, experimental groups C, D, and E all achieved complete healing, while control groups A (PBS) and B (porcine ECM) still had not healed, showing a highly significant difference.

[0042] 2. The dose-response effect exhibits saturation: All three groups (1 mg / mL, 5 mg / mL, and 10 mg / mL) achieved complete healing by day 9, indicating that: 1 mg / mL has reached the clinically effective threshold; Higher concentrations do not offer further advantages, thus avoiding resource waste and increased costs.

[0043] 3. Human-derived ingredients enhance biocompatibility: The experimental group had smooth skin with no inflammatory response, while the porcine ECM group still had scabs remaining, suggesting that human ECM is more easily integrated into host tissues and reduces immune stress.

[0044] Example 3: Application of human ECM in anti-aging cosmetics 1. Cosmetic Formulation Preparation Based on the ECM freeze-dried powder obtained in Example 1 as the base active ingredient, an anti-aging serum was formulated as follows (by weight percentage): Element content(%) Function Human ECM (dissolved and diluted with citric acid / PBS) 1.0 Provides natural collagen / fibronectin signaling glycerin 5.0 Moisturizer Sodium hyaluronate 0.2 Locks in moisture and improves skin feel Niacinamide 3.0 Antioxidant and brightens skin tone Phenoxyethanol 0.6 preservative Deionized water Supplement to 100 solvent Add human ECM powder to 0.3M citric acid solution, then add pre-cooled PBS at a volume ratio of 1:1-100, vortex mix, and finally adjust the pH to 7.0-7.5 with 1M NaOH.

[0045] Dissolve and stir each component in sequence until homogeneous, filter to 0.22 μm for sterilization, and fill into brown glass bottles to obtain the anti-aging essence.

[0046] 2. In vitro efficacy verification Fibroblast activity assay: Human dermal fibroblasts (HDFs) were seeded in 96-well plates and culture media containing 0.1%, 0.5%, and 1.0% ECM essence were added, respectively. CCK-8 assay showed that the cell proliferation rate increased by 38.7% at the 1.0% concentration (vs. control group, p<0.01).

[0047] Collagen synthesis capacity: The content of type I collagen in the culture supernatant was detected by ELISA. The 1.0% ECM group showed a 2.3-fold increase compared to the control group.

[0048] Example 4: Application of human ECM in biological dressings for chronic wound repair Dressing preparation The human skin organoid ECM lyophilized powder prepared in Example 1 was dissolved in sterile PBS to prepare a solution with a concentration of 2 mg / mL. 0.5% (w / v) sodium hyaluronate (Sigma, H9507) was added as a thickener and moisturizing carrier, and after being mixed evenly, the mixture was sterilized through a 0.22 μm filter membrane to obtain the ECM hydrogel dressing.

[0049] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit it. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for preparing extracellular matrix based on human skin organoids, characterized in that, Includes the following steps: (1) Organoid construction: Full-thickness skin tissue was taken, and a single-cell suspension was obtained after enzyme digestion. The suspension was then cultured in a three-dimensional culture system containing Matrigel matrix to induce the formation of skin organoids with epidermal and dermal structures. (2) Combined stimulation to induce ECM secretion: The skin organoids are subjected to the synergistic effect of physical and physiological stimulation, wherein the physical stimulation is periodic mechanical stretching with a deformation rate of 8–12% and a frequency of 0.4–0.6 Hz; the physiological stimulation is the sustained release of TGF-β1 and ascorbic acid. (3) ECM purification: Cell components are removed by decellularization buffer, and then separated by ultracentrifugation. The precipitate is collected to obtain the extracellular matrix.

2. The method for preparing extracellular matrix based on human skin organoids according to claim 1, characterized in that, The enzymes mentioned in step (1) are collagenase IV and neutral protease, with concentrations of 0.2–0.3% and 0.08–0.12%, respectively, and digested at 37°C with shaking for 1.5–2.5 hours.

3. The method for preparing extracellular matrix based on human skin organoids according to claim 1, characterized in that, In step (1), the Matrigel matrix concentration was 7–9 mg / mL, and the cell seeding density was 4 × 10⁻⁶ cells / mL. 5 –6×10 5 The cells / mL incubation period is 5–9 days.

4. The method for preparing extracellular matrix based on human skin organoids according to claim 1, characterized in that, The mechanical stretching in step (2) has a deformation rate of 10% and a frequency of 0.5 Hz. Stimulation is performed for 3–5 hours daily for 2–4 days.

5. The method for preparing extracellular matrix based on human skin organoids according to claim 1, characterized in that, In step (2), the concentration of TGF-β1 is 8–12 ng / mL, the concentration of ascorbic acid is 40–60 μg / mL, and the sustained release rate is 0.08–0.12 μL / min.

6. The method for preparing extracellular matrix based on human skin organoids according to claim 1, characterized in that, The decellularization buffer in step (3) contains 0.08–0.12% Triton X-100 and 8–12 mM EDTA, and is treated with shaking at 4°C for 18–30 hours, with the buffer being changed 2–4 times; the ultracentrifugation conditions in step (3) are: 100,000×g, 4°C, centrifugation for 1.5–2.5 hours.

7. The method for preparing extracellular matrix based on human skin organoids according to claim 1, characterized in that... In step (2), the combined stimulation to induce ECM secretion uses a physical-physiological combined stimulation device, which includes: Flexible culture membranes used to support organoids; A mechanical stretching module is configured to apply periodic deformation to the flexible culture membrane; The growth factor micropump system, connected to a conduit, slowly releases TGF-β1 solution into the culture environment at a rate of 0.08–0.12 μL / min; The control unit synchronously regulates the stretching frequency and the growth factor release rhythm.

8. An extracellular matrix material prepared by the preparation method according to any one of claims 1–7, characterized in that, The ECM contains ≥65% collagen, <50ng / mg residual DNA, and retains a naturally cross-linked three-dimensional fiber network structure.

9. Use of the extracellular matrix material according to claim 8 in the preparation of biological dressings for chronic wound repair or anti-aging cosmetics.

10. An anti-aging serum composition containing human ECM, characterized in that, It consists of the following components by mass percentage: Human-derived skin extracellular matrix: Add human-derived ECM powder to 0.3M citric acid solution, then add pre-cooled PBS at a volume ratio of 1:1-100, vortex mix, and finally adjust the pH to 7.0-7.5 with 1M NaOH, with a content of 1.0% (w / w). Glycerin, content 5.0% (w / w); Sodium hyaluronate, content 0.2% (w / w); Nicotinamide, content 3.0% (w / w); Phenoxyethanol, content 0.6% (w / w); Add deionized water to bring the concentration to 100% (w / w).

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