A skin liquid dressing and a method of making the same

Abstract

This invention relates to a liquid dressing for skin and its preparation method. The film-forming material of the liquid dressing comprises glial cell supernatant. The glial cells used in this invention are derived from neural stem cells that have achieved long-term in vitro culture and expansion. This allows for the production of culture supernatant with advantages such as large-scale production, stable quality, and mature technology. It can provide a large quantity of liquid dressing products with consistent quality standards in a short time. Furthermore, the supernatant exhibits good film-forming properties and safety, and contains a large number of biocompatible, wound-healing, and bioactive human cell components, making it suitable for large-scale mass production and application.

Description

Technical Field

[0001] This invention relates to the field of surgical supplies such as medical bandages and dressings, particularly to the field of liquid bandages and dressings, and specifically to a liquid dressing for skin and its preparation method. Background Technology

[0002] The skin, including its appendages such as hair, nails, sebaceous glands, and sweat glands, is the largest organ in the human body. The skin is the body's first line of defense against the natural world, its primary function being protection, preventing the invasion of pathogenic microorganisms from the environment and the loss of bodily fluids. Simultaneously, the skin also functions in sensation, exchange, and thermoregulation. Structurally, the skin can be divided into three layers from the outside in: the epidermis, the dermis, and the subcutaneous tissue.

[0003] Normal skin (tissue) will be damaged by external injury factors such as surgery, external force, heat, electric current, chemical substances, low temperature, as well as internal factors such as local blood supply disorders. This is often accompanied by the destruction of skin integrity and the loss of a certain amount of normal tissue. At the same time, the normal function of the skin is impaired, which is also called a wound or trauma.

[0004] Dressings refer to auxiliary materials used in addition to the main material of an item, primarily referring to hemostatic gauze (usually medical degreased gauze). Traditionally, dressings were bandages, mainly dry gauze and oil-soaked gauze. Modern wound dressings include various forms such as interactive wound dressings, calcium alginate dressings, silver dressings, foam dressings, liquid dressings, and hydrogel dressings. An important function of modern wound dressings is that they replace the essential functions of damaged skin and continue to act until wound healing and skin lesion healing, including resisting mechanical factors (such as dirt, impact, inflammation, etc.), resisting contamination and chemical irritation; preventing secondary infection; preventing dryness and fluid loss (electrolyte loss); preventing heat loss; in addition to providing comprehensive protection for the wound, they can also actively influence the wound healing process through debridement and create a microenvironment that promotes wound healing.

[0005] Liquid dressings are medical devices that form a protective layer on the surface of skin wounds through spraying or application, acting as a physical barrier. They are used for the care of small wounds, abrasions, cuts, and other non-chronic or superficial wounds and surrounding skin. Traditionally, people believed that wounds should be kept dry to prevent inflammation. However, medical research has found that a moist environment promotes wound healing and can also reduce pain. Therefore, liquid dressings have gradually replaced traditional bandaging methods, making them more acceptable and convenient to use.

[0006] In medical devices, liquid dressings are usually made of film-forming materials that cannot be absorbed by the human body, such as polyethylene glycol, polyvinyl alcohol, sodium alginate, sodium carboxymethyl cellulose, polyvinylpyrrolidone, carbomer, etc., supplemented with glycerin, purified water, etc.

[0007] Numerous patent documents concerning liquid dressings have been disclosed in the prior art. For example:

[0008] Chinese patent document CN109568218A discloses a cosmetic liquid dressing, which comprises the following raw materials: carbomer, hydroxyethyl cellulose modified product, growth factor, honeysuckle extract, Dipsacus asper extract, moisturizing additive, and water. The moisturizing additive is at least one of amphiphilic carboxymethyl chitosan octadecyl ammonium chloride and quaternary ammonium hyaluronic acid.

[0009] Chinese patent document CN103301501A discloses a medical liquid dressing and its preparation method. The medical liquid dressing contains 0.02-1% solid components, with the remainder being water. The solid components include 10-80% chitosan quaternary ammonium salt, 20-90% organosilicon quaternary ammonium salt, and 6-30% excipients.

[0010] Chinese patent document CN111084902A discloses a liquid dressing and its preparation method. By weight, the liquid dressing raw materials consist of 55-70 parts of anhydrous ethanol, 7-22 parts of polyacrylic acid resin IV, 5-25 parts of ethyl acetate, and 0.1-3 parts of lubricant.

[0011] These liquid dressings are mostly composed of exogenous natural or synthetic ingredients, not human-derived components. When applied to human skin, they may cause rejection by the body's systems, affecting their biocompatibility. They may also contain exogenous cytokines and other active ingredients, and the instability of their bioactivity can impair their fundamental function as a skin barrier.

[0012] Chinese patent document CN118649284A discloses a liquid dressing containing mesenchymal stem cell exosomes, its preparation method, and its application. Each 1L of the liquid dressing contains 3-10% polyvinyl alcohol, 1-3% glycerol, 1-2.5% pharmaceutical-grade ethanol, and 1×102 mesenchymal stem cell exosomes. 8 ~3×10 9 The liquid dressing contains exosomes per ml, with the remainder being deionized water. The liquid dressing prepared by this invention has a simple composition, free of pigments, fragrances, preservatives, and other additives. This liquid dressing adheres better to the wound, providing a good sterile environment for cell and tissue regeneration while avoiding secondary tearing damage to the wound caused by ordinary adhesive bandages during replacement. The mesenchymal stem cell exosomes it contains can participate in wound repair and promote wound healing.

[0013] The method for preparing mesenchymal stem cell exosomes disclosed in this literature is as follows: Fresh umbilical cord tissue is harvested, cleaned by soaking in saline containing antibiotics and acetic acid, and the blood vessels and Wharton's jelly within the umbilical cord are removed. The tissue is then minced into a paste-like consistency in a centrifuge tube and evenly spread in a culture flask. MSCBM medium containing 5% blood is added until it just covers the tissue, and the flask is incubated at 37°C in a CO2 incubator for 5–7 days. Spindle-shaped cells are observed crawling out of the tissue mass, which are then identified as mesenchymal stem cells. The cultured mesenchymal stem cells are passaged at least three times, and the cell culture supernatant is collected after 3–4 days of culture. The mesenchymal stem cell exosome suspension is then extracted by filtration, concentration, and ultracentrifugation.

[0014] Although this technology uses mesenchymal stem cell exosomes obtained from the organism itself as the main component of the liquid dressing, improving biocompatibility, it cannot guarantee batch quality differences in large-scale production and cannot be used as an industrialized medical device product. The reasons are as follows: First, the expansion and passage capacity of mesenchymal stem cells vary greatly from person to person. They are prone to differentiation and death in vitro, and may lose their stem cell characteristics after a few passages, with fundamental changes in their basic properties and greatly weakened expansion capacity. Mesenchymal stem cells need to be obtained from the umbilical cord each time, which limits the source and yield. Second, this technology only uses a very small amount of exosomes from the cell supernatant, while discarding most of the cells themselves and the rest of the culture supernatant, further limiting its yield and making it impossible to achieve large-scale application.

[0015] Similarly, Chinese patent document CN111569145A discloses a liquid dressing for antibacterial repair of skin wounds, its preparation method, and its application. Its components include pure natural antimicrobial peptides, β-glucan, purified water, butylene glycol, and amniotic mesenchymal stem cell exosomes. The exosomes used also suffer from the aforementioned drawbacks, and the addition of antimicrobial peptides further increases the requirements for bioactivity stability, making it unsuitable for use as a liquid dressing for skin barrier purposes. Summary of the Invention

[0016] To address the aforementioned technical problems, this invention provides a liquid dressing for skin, wherein the film-forming material of the liquid dressing comprises glial cell supernatant. Using the entire glial cell culture supernatant as the basic component of the liquid dressing demonstrates extremely high biocompatibility for human skin applications. Furthermore, the liquid dressing leverages the basic functions of glial cells—supporting, nourishing, protecting, and repairing—to enhance its skin barrier function. Simultaneously, the supernatant also contains a large number of natural bioactive factors beneficial for the repair of human skin injuries.

[0017] Preferably, the glial cell supernatant is the supernatant obtained by filtering the glial cell culture medium through a 0.45-micron filter membrane. Filtration removes most microorganisms, dead cells, cell debris, and other components that affect the performance of the liquid dressing, while completely retaining all beneficial components from the cell culture medium.

[0018] More preferably, the glial cell culture medium contains glial cells at a concentration of 1-2 × 10⁻⁶. 6 The density of / ml was obtained after incubation in an incubator for 3-4 days. The components in the culture medium obtained after incubation under these conditions best meet the barrier protection requirements of liquid dressings and provide suitable biological activity.

[0019] More preferably, the glial cells are derived from neural stem cells. The inventors' team has previously solved the technical challenges of easy differentiation and death of neural stem cells in vitro; using this method for long-term, large-scale in vitro culture of human neural stem cells can solve the problem of large-scale culture.

[0020] Preferably, the viscosity of the glial cell supernatant is 30-50 mPa·s. Using a cell supernatant of suitable viscosity contributes to the film-forming properties required by the liquid dressing.

[0021] Most preferably, the liquid dressing comprises: 0.5-2% collagen, 1-5% glycerol, and 0.5-2% trehalose per 1L of liquid dressing, with the remainder being the glial cell supernatant, by volume percentage. Using a liquid dressing with a suitable composition helps to achieve its skin barrier protection function.

[0022] The present invention also provides a method for preparing the aforementioned liquid dressing for skin, comprising the following steps:

[0023] 1) After seeding glial progenitor cells, add them to complete culture medium containing differentiation additives and culture until the cell confluence reaches 80%-90%. Digest the adherent cells, centrifuge and discard the supernatant. Resuspend the cells in complete culture medium and adjust the cell concentration to 1-2 × 10⁻⁶ cells / year. 6 After passing the culture at a concentration of / ml for at least 3 passages, glial cells can be obtained.

[0024] 2) Collect the culture medium of glial cells after culture. Filter the collected glial cell culture medium through a 0.45-micron filter membrane, discard the solid precipitate, and keep the supernatant for later use.

[0025] 3) The obtained glial cell supernatant is mixed with the other components of the liquid dressing to obtain the liquid dressing.

[0026] Preferably, the differentiation additive is 2% (v / v) serum, and the digestion is performed using 0.25% TP-EDTA solution for 3-5 minutes. Using this differentiation additive helps to differentiate glial cells, and using this digestion solution allows adherent cells to be digested into the culture medium.

[0027] This invention also provides the application of glial cell supernatant in liquid dressings.

[0028] Preferably, the glial cell supernatant is the supernatant obtained by filtering glial cell culture medium through a 0.45-micron filter membrane. Filtration removes most microorganisms, dead cells, cell debris, and other components that affect the performance of the liquid dressing, while completely retaining all beneficial liquid components from the cell culture medium. This invention has the following beneficial effects:

[0029] Our team of inventors, utilizing previously patented technology, has solved the technical challenges of easy differentiation and death of neural stem cells in vitro through a separation and passage culture method using a stem cell sphere cutter and a rationally formulated neural stem cell culture medium. This allows for long-term, large-scale in vitro culture of human neural stem cells without any damage to the cells themselves. Furthermore, the human neural stem cells cultured using this method are genetically stable and of controllable quality, providing an important and inexhaustible source of neural stem cells for widespread clinical applications. Multiple experimental results demonstrate that the neural stem cell culture medium of this technology enables long-term (>30 months) in vitro culture and expansion of human neural stem cells, and the genetic stability of the expanded neural stem cells is maintained.

[0030] The glial cells used in this invention are derived from the neural stem cells that have been cultured and expanded in vitro for a long period of time. This results in the culture supernatant obtained from the supernatant having the advantages of large-scale production, stable quality, and mature technology. It can provide a large number of liquid dressing products with stable and consistent quality standards in a short period of time, making it suitable for large-scale mass production applications.

[0031] Those skilled in the art know that the nervous system is mainly composed of neurons and glial cells, with glial cells making up the vast majority. The main functions of the latter are to support, nourish, protect, and repair neurons. This invention uses the entire supernatant from glial cell culture as the basic component of the liquid dressing. This allows the liquid dressing to fulfill its skin barrier function by leveraging the basic supportive, nourishing, protective, and repairing functions of glial cells, without requiring the introduction of exogenous bioactive ingredients such as exogenous cytokines.

[0032] This invention uses the entire supernatant of glial cell culture as the basic component of the liquid dressing; therefore, water remains the primary component of the liquid dressing. Experimental results of this invention show that the resulting liquid dressing exhibits good film-forming properties, meets the definition of a liquid dressing, and can provide a barrier protection function for the skin.

[0033] Furthermore, the glial cells used in this invention are derived from human neural stem cells, thus exhibiting extremely high biocompatibility for use in liquid dressings on human skin. Experimental results of this invention demonstrate that the resulting liquid dressing can promote skin cell proliferation, aid in skin wound healing, and has no toxic side effects or other safety concerns.

[0034] Instruction manual illustrations

[0035] Figure 1 The results of microscopic observation of glial cells according to the present invention are shown.

[0036] Figure 2 The image shows an immunofluorescence staining photograph of glial cells according to the present invention.

[0037] Figure 3 The results of film formation observation of the glial cell supernatant of the present invention are shown.

[0038] Figure 4 The data on skin cell proliferation in the MTT assay of this invention are shown.

[0039] Figure 5 The effects of the liquid dressing of the present invention on the skin healing of actual wounds are demonstrated. Detailed Implementation

[0040] The following detailed description provides further details through specific embodiments. However, it should be noted that the embodiments described below are merely for illustrating the content of the invention and do not represent that the invention is limited to the described embodiments. Therefore, non-essential improvements and adjustments made to the implementation schemes by those skilled in the art based on the above-described invention still fall within the protection scope of the invention, and the scope of protection of the appended claims shall prevail.

[0041] Those skilled in the art will understand that liquid dressings are Class II medical devices as defined in the Medical Device Classification Catalog, and are typically solutions (excluding gels). Their basic requirements include: the components must not have pharmacological effects; the components must not be absorbed by the human body; and they must have a certain viscosity and corresponding film-forming properties. Liquid dressings act as a physical barrier by forming a protective layer on the wound surface, and are used for the care of small wounds, abrasions, cuts, and other non-chronic or superficial wounds and surrounding skin.

[0042] Neural stem cells (NSCs) are cell populations that exist in the nervous system and have the potential to differentiate into neurons, astrocytes, and oligodendrocytes, thereby generating a large number of brain and neural tissue cells, and can self-renew, providing a large number of brain and neural tissue cells.

[0043] The human neural stem cells and their long-term in vitro expansion and culture technology used in this invention refer to the technology disclosed in the previously patented Chinese patent document CN105062972B, "A Neural Stem Cell Culture Medium and a Method for Long-Term In Vitro Culture and Expansion of Human Neural Stem Cells Using the Medium." In this technology, the inventors, through years of research, discovered a rationally formulated neural stem cell culture medium combined with a stem cell sphere cutter for separation and passage culture, which causes no damage to the cells themselves, making it extremely advantageous for long-term culture of neural stem cells. The neural stem cell culture medium of this invention enables human neural stem cells to be cultured and expanded in vitro for extended periods (>30 months), and the neural stem cells remain genetically stable after large-scale expansion. This invention solves the technical problems of easy differentiation and death of neural stem cells in in vitro culture, achieving the goal of long-term, large-scale culture of human neural stem cells. Moreover, the human neural stem cells cultured using this method are genetically stable and of controllable quality, providing an important and inexhaustible supply of neural stem cells for widespread clinical application.

[0044] The specific cultivation method is described in Example 1 of the document, and the culture medium used is described in Examples 6 and 7 of the document. The entire contents of this document are incorporated herein by reference as part of this specification, and the techniques described and disclosed herein are not repeated herein.

[0045] Neuroglial cells, also known simply as glial cells or neuroglial cells, are another major type of cell in neural tissue besides neurons. They also have processes, but lack the distinction between dendrites and axons, and are widely distributed in the central and peripheral nervous systems. In mammals, the ratio of glial cells to neurons is approximately 10:1. The main types of glial cells in the central nervous system (CNS) include astrocytes, oligodendrocytes (collectively referred to as macroglia along with the former), and microglia. Glial cells can differentiate from neural stem cells.

[0046] The supernatant from cell culture is called culture supernatant or supernatant. During cell culture, cells secrete various active components, including cytokines, which are crucial signaling molecules for cell activity. The supernatant is the culture medium harvested after cell culture. It contains exosomes, cell metabolites, cell debris, and dead cells, but its main components are liquids derived from the culture medium, such as water. Most of the solid components, including microorganisms, cell debris, and dead cells, can be removed through centrifugation or filtration to obtain a pure liquid supernatant.

[0047] Existing technologies allow the purchase of filter membranes with pore sizes ranging from 0.01 micrometers to 20 micrometers, with main specifications including 0.01 micrometers, 0.03 micrometers, 0.22 micrometers, 0.45 micrometers, and 0.8 micrometers. This invention uses filter membrane products with a 0.45-micrometer specification, but this does not mean that only a fixed pore size of 0.45 micrometers is allowed. It merely indicates that 0.45-micrometer products can be selected from commercially available products, and also includes filter membranes with pore sizes close to 0.45 micrometers that are prepared in-house.

[0048] Exosomes are messenger particles naturally released from cells, responsible for intercellular communication. They transport genetic information and proteins to cells throughout the body, creating pathways for intercellular communication. These messenger cells have the ability to release growth factors and other beneficial processes. Exosomes constitute only a very small fraction of the supernatant after cell culture.

[0049] Unless otherwise specified in this invention, any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose may be used. Even if the technologies, instruments, equipment, and materials used in this invention are specifically specified, it does not mean that this invention can only use these technologies, instruments, equipment, and materials, but merely represents the preferred solution of this invention. Those skilled in the art can still use any other technologies, instruments, equipment, and materials known to those skilled in the art that can achieve the same purpose.

[0050] Example 1: Obtaining Human Neural Stem Cells

[0051] Based on the technical solution disclosed in the previous work of the inventors' team, namely Chinese patent document CN105062972B "A Neural Stem Cell Culture Medium and a Method for Long-Term In Vitro Culture and Expansion of Human Neural Stem Cells Using the Medium", the following steps are performed to obtain:

[0052] Take the prepared fetal brain-derived hippocampal neural stem cells, at a ratio of 1-2 × 10⁻⁶ 6 / ml of cells were seeded in cell culture flasks. Neural stem cell culture medium (specific composition as described in Example 1 of this literature) was used for culture and expansion. The cell flasks were placed in a 5% CO2, 37°C incubator. Depending on cell proliferation, half of the cell culture medium was replaced every 3-4 days.

[0053] Neural stem cells are cultured in a suspension neurosphere format in neural stem cell culture medium, with half the medium replaced every 3-4 days, for a total of 20-30 days. When the neural stem cell spheres reach a diameter of approximately 500-1000 micrometers, the culture medium containing the spheres is drawn into a 200ml syringe and cut using a stem cell sphere cutter (stainless steel mesh), dividing each large sphere into 3-8 smaller pieces (approximately 30-200 micrometers). These smaller stem cell clusters are then placed in new culture flasks, and half of the freshly prepared culture medium is added. The flasks are then passaged at a ratio of 1:2 or 1:4 for continued culture. This passage expansion method allows for the continuous culture and expansion of human neural stem cells for more than two years.

[0054] Example 2: Acquisition and Identification of Glial Cells

[0055] Neuroglial progenitor cells (human neural stem cells cultured in Example 1) were added to cell culture medium at a concentration of 1-2 × 10⁻⁶. 6 Cells were seeded at a density of / ml in 24-well plates. The cell culture medium contained DMEM / F12 (Hyclone, USA), 2% fetal bovine serum (Zhejiang Tianhang Biotechnology) as differentiation agent, and B27 neural cell culture additive diluted 1:50 (Gibco, USA). Cells were incubated at 37°C in a 5% CO2 incubator. Cell growth was observed and photographed for recording. Figure 1 As shown. The culture medium should be updated as needed during this period.

[0056] When the cell confluence was observed to be 80%-90% under a microscope, the supernatant was discarded, and the cells were washed with D-Hank's solution (0.4g KCl, 0.06g KH2PO4, 0.12g Na2HPO4·12H2O, 0.22g MgSO4·7H2O, 0.14g CaCl2, 8.0g NaCl, 0.35g NaHCO3, and 1.00g glucose, dissolved in water, diluted to 1000mL, and the pH was adjusted to 7.0-7.2; obtained by 0.1μm filtration).

[0057] Digest cells with 0.25% TP-EDTA solution (Gibco, USA) for 3-5 minutes, centrifuge at 663g for 5 minutes, discard the supernatant, resuspend the cells in complete culture medium, and adjust the cell concentration to 1-2 × 10⁻⁶ cells / mL. 6 After passing the solution at a concentration of 1 / ml, the cells are seeded into a cell culture flask and then cultured again in a 37°C, 5% CO2 incubator. After passing the cells through three or more passages, glial cells are obtained.

[0058] The obtained glial cells were identified. The glial cells, after further culture, were digested with 0.25% TP-EDTA solution to prepare a single-cell suspension. Cells were counted and then subjected to a 5 × 10⁻⁶ cell line. 4The slides were seeded onto poly-L-lysine-coated glass coverslips, and immunofluorescence staining was performed 8 days later. The primary antibody solution used for staining was O4 or GFAP (Wuhan Boster Biological Products), and the secondary antibody solution was Cy3 (Wuhan Boster Biological Products). The results were observed and photographed using an inverted fluorescence microscope. Figure 2 As shown.

[0059] GFAP is a type III intermediate filament protein that exists independently and is specifically expressed in large quantities in activated astrocytes. O4 is a specific marker in the differentiation of neural stem cells into oligodendrocytes. Figure 2 As shown, under a microscope, the morphology of the differentiated cells was not significantly different from that of astrocytes and oligodendrocytes, and both GFAP and O4 tests were positive, indicating that the identified cells were astrocytes and oligodendrocytes.

[0060] Example 3: Preparation of Glial Cell Liquid Dressing Product

[0061] The glial cells obtained in Example 2 were cultured in an incubator under the specified conditions, and the cell culture medium was collected after 3 days of culture. The collected cell culture medium was filtered through a 0.45-micron (Millipore) filter membrane, and the solid precipitate was discarded. The resulting supernatant was kept for later use.

[0062] Take a 2L beaker, add trehalose and an appropriate amount of glial cell supernatant, and stir to dissolve. Then, add collagen peptides in portions, stirring constantly, and replenish the glial cell supernatant as needed. Stir until completely dissolved, then add glycerol and stir until homogeneous. Pour the homogeneous solution into a blue-capped bottle and inactivate it in a 60°C water bath for 60 minutes. After the inactivated solution cools, filter it through a 500-mesh filter. Add the remaining glial cell supernatant and mix well to obtain the glial cell liquid dressing of this invention.

[0063] Each 1L of the liquid dressing contains 1% collagen, 1.28% glycerol, 1% trehalose, and 96.72% glial cell supernatant, by volume.

[0064] Example 4: Performance Test of Glial Cell Liquid Dressing Product

[0065] 1. Film-forming properties

[0066] Take a clean glass slide, divide it into two areas, and place it in a constant temperature incubator at 37±1℃ for 5 minutes. Take 0.5 ml of the supernatant prepared in Example 3 and apply it to one area. Take 0.5 ml of water and drop it onto the other area as a control group. Then place the glass slide back into the constant temperature incubator, and observe after the water evaporates: a thin film can form on the surface of the area coated with the supernatant, such as... Figure 3 As shown. The film formation time is 5-10 minutes.

[0067] 2. Skin cell proliferation experiment

[0068] Take a 96-well plate and apply 5-6 × 10 5 / ml of skin cells were inoculated. The 96-well plates were divided into 9 groups, with 6 wells in each group (n=6). The nerve cell fluid dressing was added to each group as follows (final total volume for each well: 100μL):

[0069] A: DMEM / F12+2%FBS, set as zero adjustment group

[0070] B: DMEM / F12 + 2% FBS + skin cells, set as blank control group.

[0071] C: DMEM / F12 + 10% FBS + skin cells, set as the standard control group.

[0072] D: DMEM / F12 + 2% FBS + skin cells + 0.078μL liquid dressing

[0073] E: DMEM / F12 + 2% FBS + skin cells + 0.156μL liquid dressing

[0074] F: DMEM / F12 + 2% FBS + skin cells + 0.313μL liquid dressing

[0075] G: DMEM / F12 + 2% FBS + skin cells + 0.625μL liquid dressing

[0076] H: DMEM / F12 + 2% FBS + skin cells + 1.250μL liquid dressing

[0077] I: DMEM / F12 + 2% FBS + skin cells + 2.500μL liquid dressing

[0078] The 96-well plate was incubated at 37°C. After 2 days, it was removed and an MTT assay was performed. The experimental results are as follows: Figure 4 As shown in the figure, the glial cell liquid dressing can promote the proliferation of skin cells, and within the above dosage range, cell proliferation accelerates with increasing dosage, without toxicity to cells.

[0079] 3. Adhesion

[0080] Take 1 ml of the supernatant prepared in Example 3 and measure its viscosity using a standard rotational viscometer according to the method and conditions described in GB / T 222235-2008 "Determination of Viscosity of Liquids".

[0081] The viscosity of the supernatant of this invention was measured to be 41.5 mPa·s. This indicates that the liquid dressing of this invention has a high viscosity.

[0082] 4. Skin wound healing experiment

[0083] Select healthy, adult domestic rabbits weighing approximately 4 pounds, ensuring they are free from skin diseases, infectious diseases, and other systemic diseases. Place the rabbits in a temperature-controlled environment, feed them their usual rabbit feed, and provide them with plenty of clean drinking water.

[0084] Two suitable, adjacent experimental areas were selected on either side of the rabbit's spine (one as the experimental group and one as the control group). The hair in these areas was shaved using an electric shaver. The shaving area should be larger than the area to be created for the wound, generally 5cm x 5cm, to avoid contaminating the wound site with hair. The shaved skin was then disinfected by wiping it from the inside out with 75% alcohol swabs, covering an area at least 10cm in diameter. This disinfection was repeated three times to ensure thorough disinfection. Surgical instruments, including scalpels, were sterilized by autoclaving and kept for later use. After the alcohol had evaporated and dried, a 2.5cm x 2cm section of skin tissue was removed from the disinfected area using a sterile scalpel, reaching the subcutaneous fascia to avoid damaging the muscle layer and major blood vessels. If bleeding occurred, gentle pressure was applied with sterile gauze for 3-5 minutes until the bleeding stopped.

[0085] The wound site was treated once a day. In the experimental group, the nerve cell liquid dressing described in Example 3 of this invention was applied to the wound site with sterile cotton swabs until it was evenly applied. In the control group, physiological saline was applied evenly. The treatment was continued for 5 days.

[0086] Observe the experimental rabbits' mental state, appetite, activity level, and weight changes at fixed times each day, and record any abnormal behaviors such as lethargy, refusal to eat, or fever to avoid health conditions that may affect the experimental results. Record the skin tissue growth at the wound edge and the scab formation on the wound surface, including the time of scab formation, thickness, and integrity, and take photos of the wound site for subsequent comparative analysis. Measure the change in wound area and calculate the wound healing rate (healing rate = (initial area - unhealed area) / initial area × 100%).

[0087] Experimental results are as follows Figure 5 As shown, according to Figure 5 The wound healing rate data calculated from the tested wound area, compared to the control group at the same time point, are shown in Table 1 below. It can be seen that in the experimental group, more than 75% of the wound area had healed and gradually scabbed over after 7 days, and by day 12, the wound was basically completely healed. In contrast, in the control group, nearly half of the wound area remained unhealed after 7 days, and there was no significant subsequent healing progress. This demonstrates that the nerve cell liquid dressing has a certain skin wound healing effect.

[0088] Table 1. Wound healing rate data

[0089] Day 1 Day 4 Day 7 experimental group 0％ 28.0％ 76.0％ control group 0％ 20.8％ 61.6％

[0090] The above descriptions are merely embodiments of the present invention. Commonly known technical knowledge in the solutions is not described in detail here. Those skilled in the art are aware of all common technical knowledge in the field prior to the filing date, are aware of all existing technologies in that field, and have the ability to apply conventional experimental methods prior to that date. Those skilled in the art can, under the guidance of this application, improve and implement this solution in combination with their own capabilities. Some typical well-known technologies should not be obstacles for those skilled in the art to implement this application. It should be noted that those skilled in the art can make several adjustments and improvements without departing from the structure of the present invention. These should also be considered within the scope of protection of the present invention, and will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A liquid dressing for skin, characterized in that, The liquid dressing is composed of: 0.5-2% collagen, 1-5% glycerol, and 0.5-2% trehalose per 1L of liquid dressing, with the remainder being glial cell supernatant, by volume percentage; the viscosity of the glial cell supernatant is 30-50 mPa·s; the glial cells are differentiated from neural stem cells.

2. The liquid dressing for skin according to claim 1, characterized in that, The glial cell supernatant is the supernatant obtained by filtering the glial cell culture medium through a 0.45-micron filter membrane.

3. The liquid dressing for skin according to claim 2, characterized in that, The glial cell culture medium contains glial cells at a concentration of 1-2 × 10⁶ cells / mL. 6 The density of / ml was obtained after incubation in an incubator for 3-4 days.

4. A method for preparing the liquid dressing for skin according to any one of claims 1-3, characterized in that, Includes the following steps: 1) After seeding glial progenitor cells, add them to complete culture medium containing differentiation additives and culture until the cell confluence reaches 80%-90%. Digest the adherent cells, centrifuge and discard the supernatant. Resuspend the cells in complete culture medium and adjust the cell concentration to 1-2 × 10⁻⁶ cells / year. 6 After passing the culture at a concentration of / ml for at least 3 passages, glial cells can be obtained. 2) Collect the culture medium of glial cells after culture. Filter the collected glial cell culture medium through a 0.45-micron filter membrane, discard the solid precipitate, and keep the supernatant for later use. 3) The obtained glial cell supernatant is mixed with the other components of the liquid dressing to obtain the liquid dressing.

5. The preparation method according to claim 4, characterized in that, The differentiation additive is 2% v / v fetal bovine serum, and the digestion is performed using 0.25% TP-EDTA solution for 3-5 minutes.

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