Biological formulation, preparation method therefor, and use thereof

Abstract

The present application belongs to the technical field of biomedicine. Disclosed are a biological formulation, a preparation method therefor, and the use thereof. The biological formulation is a culture solution supernatant obtained by culturing fibroblasts with a special medium (containing a basic medium, a human platelet lysate, vitamin C, vitamin E, cysteine and glutathione) of the present application. The biological formulation can inhibit the expression of inflammatory factors around skin wounds while promoting the healing of the wounds, relieve inflammatory responses, relieve discomfort produced during wound healing and improve the comfort during the healing period of the wounds. In addition, when being used for culturing fibroblasts, the medium provided by the present application can improve the amplification capability of fibroblasts and enhance the expression levels of functional genes, thereby improving secretion of a plurality of growth factors and cytokines that can promote wound healing. The fibroblasts obtained by culture with the medium have a remarkably improved amplification fold and also exhibit obviously upregulated expression levels of functional genes.

Description

A biological preparation and its preparation method and application Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a biological preparation and its preparation method and application. Background Art

[0002] As the body's first line of defense, the skin prevents water loss, protects the body from UV damage, and blocks microbial invasion, thereby protecting the body's internal organs. Once the skin is damaged, especially wounds such as skin breaks, microorganisms can take advantage of the situation and cause bacterial infections and other problems. Therefore, rapid healing of skin breaks is crucial for maintaining homeostasis.

[0003] Skin wound healing is a dynamic regulatory process, with numerous cells, cytokines, and extracellular matrix participating in the healing process in an orderly manner in time and space. The healing process can be roughly divided into four consecutive phases: blood coagulation, inflammation, proliferation, and tissue remodeling. Each phase involves different cells, such as epithelial cells, immune cells, endothelial cells, and fibroblasts. These cells can coordinately regulate the wound healing process by changing their own gene expression and cell phenotype. Among them, fibroblasts are particularly worthy of attention.

[0004] Broadly speaking, fibroblasts reside in the dermis and synthesize and secrete collagen and extracellular matrix. During skin wound healing, fibroblasts release a large amount of extracellular matrix (ECM) containing collagen. Macrophages release inflammatory factors such as platelet-derived growth factor (PDGF), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α), which recruit fibroblasts to the site of inflammation. Fibroblast proteases degrade fibrin clots while simultaneously secreting large amounts of synthesized collagen, providing signaling molecules and supporting epidermal formation for newly formed blood vessels and granulation tissue. Approximately one week later, fibroblasts undergo structural and functional changes under the stimulation of transforming growth factor-β (TGF-β), differentiating into myofibroblasts. The adhesive proteins and glycosaminoglycans secreted by myofibroblasts, as well as the α-smooth muscle actin (α-SMA) expressed by them, can induce contractile pressure from epithelial cells to connect the wound edges, thereby accelerating wound healing. After wound healing, myofibroblasts undergo apoptosis and are cleared from the wound.

[0005] In the related art, for example, the Chinese invention patent with publication number CN108865990A describes a biological preparation for skin damage repair and its preparation method, the preparation method is: resuscitating P3 adipose-derived mesenchymal stem cells, adding DMEM high-glucose complete medium containing 10-12% FBS, culturing until the cell confluence reaches 80-90%, and collecting the supernatant; washing the P3 adipose-derived mesenchymal stem cells, adding DMEM / F12 basal medium containing 50-55 μg / ml β-glucan, culturing for 24-48 hours, and collecting the supernatant; taking the cell culture after collecting the supernatant, adding DMEM / F12 basal medium containing 50-55 μg / ml β-glucan, continuing to culture, collecting the culture fluid, repeating 1-2 times; combining the supernatants to obtain a biological preparation for skin damage repair; the biological preparation prepared by this method can significantly promote the proliferation of skin fibroblasts and collagen secretion, and has a good effect in promoting skin damage repair. However, the extraction and separation of adipose-derived mesenchymal stem cells is difficult, and the study only examined the ability of this biological preparation to promote the proliferation and collagen secretion of human skin fibroblasts when the cells were cultured in vitro.

[0006] For example, the Chinese invention patent with publication number CN110559468A records a medical dressing containing human fibroblast conditioned culture fluid, a preparation method and an application. The medical dressing includes a liposome freeze-dried powder A containing human fibroblast conditioned culture fluid. The preparation method of the liposome freeze-dried powder A containing human fibroblast conditioned culture fluid includes: preparing a liposome suspension containing human fibroblast conditioned culture fluid (dissolving 3-18% soy lecithin, 1-6% cholesterol and 0.1-3% vitamin E in anhydrous ethanol, removing the anhydrous ethanol by reduced pressure rotary evaporation at 0.04-0.06 MPa and 35-40°C to obtain a uniform film; adding 20-50% human fibroblast conditioned culture concentrate, 5-10% trehalose and 5-10% mannitol to a phosphate buffer solution with a mass percentage of 3-65.9%, mixing and using The mixed solution is rotated to clean the film and shaken for 30 to 60 minutes to hydrate. The hydrated mixture is extruded 10 to 20 times through a liposome extruder to obtain human fibroblast conditioned culture fluid liposomes; preparation of lyophilized powder of liposomes encapsulating human fibroblast conditioned culture fluid (pre-freeze the prepared liposome suspension at -40 to -20°C for 3 to 6 hours, freeze-dry for 24 to 48 hours to obtain lyophilized powder of liposomes encapsulating the active ingredients of human fibroblast conditioned culture fluid), and human fibroblast conditioned culture concentrate is obtained by ultrafiltration and concentration of serum-free human fibroblast conditioned culture fluid (supernatant of human fibroblast conditioned culture during passage (P6 to P15) culture). The preparation method of the above-mentioned medical dressing is complicated, and the symptoms applied in this patent are mainly skin tightness, skin redness, skin itching, and skin pain, and do not involve wound healing.

[0007] Furthermore, the wound healing mechanism described above demonstrates that inflammation occurs during the wound healing process. While inflammation is the body's defense mechanism against injury and infection, it can also cause discomfort. For example, TNF and IL-1 can cause fever.

[0008] Therefore, if the inflammatory response can be reduced in the process of promoting wound healing, that is, the expression of inflammatory factors can be inhibited, the discomfort caused by the wound healing process can be alleviated and the comfort during the wound healing period can be improved. Summary of the Invention

[0009] 1. Purpose of the Invention

[0010] The purpose of the invention of this application is to provide a biological preparation and its preparation method and application. The biological preparation is the supernatant of the culture fluid after culturing fibroblasts using the culture medium of this application. While promoting skin wound healing, the biological preparation can inhibit the expression of inflammatory factors around the wound, reduce inflammatory response, relieve discomfort caused by the wound healing process, and improve comfort during the wound healing period.

[0011] 2. Technical solution

[0012] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0013] A method for preparing a biological agent, comprising the following steps:

[0014] S1, fibroblasts are taken and inoculated into medium 1 for culture, and subculture is performed when the cell density reaches 80-90%; the medium 1 comprises basal medium, human platelet lysate (hPL), vitamin C (VC), vitamin E (VE), cysteine ​​(Cys), and glutathione (GSH);

[0015] S2, collecting the culture supernatant of each cell culture generation, mixing and filtering to obtain the biological preparation.

[0016] Furthermore, the above-mentioned basal culture medium includes high-glucose DMEM basal culture medium.

[0017] Furthermore, the concentration of the human platelet lysate (hPL) is 5% to 10% (v / v), wherein the human platelet lysate (hPL) can replace fetal bovine serum, thereby avoiding the introduction of exogenous genes.

[0018] Furthermore, the concentration of the vitamin C (VC) is 50-150 μM. Vitamin C can promote collagen production in tissues and help repair aging and damaged skin. Furthermore, the concentration of the vitamin C (VC) is 100 μM.

[0019] Furthermore, the concentration of the vitamin E (VE) is 0.5 to 1.5 μg / mL. Vitamin E has antioxidant properties that can help protect against free radical damage. By neutralizing free radicals, vitamin E protects cells from oxidative stress. Vitamin E also has a protective effect on the skin, reducing damage from ultraviolet rays and helping maintain skin elasticity and health. Furthermore, the concentration of the vitamin E (VE) is 1 μg / mL.

[0020] Furthermore, the concentration of cysteine ​​(Cys) is 15-25 μg / mL. Cysteine ​​can scavenge free radicals in the body and is rapidly converted into cystine, a stable sulfur-rich amino acid. Furthermore, the concentration of cysteine ​​(Cys) is 20 μg / mL.

[0021] Furthermore, the concentration of glutathione (GSH) is 0.5 to 1.5 μg / mL. Glutathione not only eliminates free radicals in the human body, but also enhances immunity and protects hemoglobin from oxidation by hydrogen peroxide and free radicals, thereby maintaining its ability to transport oxygen. Furthermore, the concentration of glutathione (GSH) is 1 μg / mL.

[0022] Furthermore, the above-mentioned fibroblasts include skin fibroblasts.

[0023] Furthermore, the skin fibroblasts include those obtained by digestion and separation of skin tissue. Furthermore, the skin fibroblasts include those obtained by digestion and separation of foreskin tissue from children under 13 years old. Such skin fibroblasts, isolated and cultured from such tissue, can secrete a variety of growth factors and cytokines that promote wound healing, thereby aiding collagen regeneration in skin tissue and accelerating wound healing.

[0024] Furthermore, the above method for obtaining skin fibroblasts by digestion and separation comprises the following steps:

[0025] Obtain skin tissue and rinse it with sterile PBS buffer;

[0026] Povidone-iodine and ethanol disinfection: Place the skin tissue in a disinfectant containing 5% povidone-iodine for 3-5 minutes, then rinse with PBS. Add 75% ethanol and soak for 3-5 minutes, then rinse with PBS. Using povidone-iodine and ethanol instead of antibiotics avoids interference caused by the use of antibiotics.

[0027] The washed intact skin tissue was placed in a neutral protease solution at 4°C for digestion; after digestion, the epidermal tissue was removed and rinsed with PBS;

[0028] Collagenase solution was added, and then the dermal tissue was cut into 1 mm × 1 mm size using sterile scissors and digested in a 37°C incubator;

[0029] The digested cells were filtered through a 70 μm filter and centrifuged to obtain skin fibroblasts.

[0030] Furthermore, the number of fibroblasts seeded in the above-mentioned culture medium 1 is 2.0-4.0×10 4 viable cells / cm 2 .

[0031] Furthermore, the conditions for culture and subculture in the above-mentioned culture medium 1 are: 37° C., 5% CO 2 .

[0032] Furthermore, the cells were cultured in the above culture medium 1, and subcultured when the cell density reached 90%.

[0033] Furthermore, the collecting of the culture supernatant of each cell culture generation includes collecting the culture supernatant of at least the fifth cell culture generation.

[0034] Furthermore, the collecting of the culture supernatant of each cell culture generation includes collecting the culture supernatant of the 1st to 10th cell cultures.

[0035] Furthermore, the above-mentioned collection of the culture supernatant of each cell culture generation includes collecting the culture supernatant after cell culture from the 1st to the 5th generation, or the 1st to the 6th generation, or the 1st to the 7th generation, or the 1st to the 8th generation, or the 1st to the 9th generation, or the 1st to the 10th generation.

[0036] Furthermore, the collecting of the culture supernatant of each cell culture generation includes collecting the culture supernatant of the first to sixth cell cultures.

[0037] The present application also provides a biological preparation prepared by the above-mentioned method for preparing a biological preparation.

[0038] The present application also provides the use of the above-mentioned method for preparing a biological preparation in the preparation of a drug for promoting skin wound healing and / or inhibiting inflammatory reactions around skin wounds.

[0039] The present application also provides the use of the above-mentioned biological preparation in the preparation of a drug for promoting skin wound healing and / or inhibiting inflammatory response around skin wounds.

[0040] Furthermore, the above-mentioned medicine includes a dressing.

[0041] The present application also provides a pharmaceutical composition for promoting skin wound healing and / or inhibiting inflammatory response around skin wounds, wherein the pharmaceutical composition comprises the above-mentioned biological preparation.

[0042] The present application also provides a culture medium for promoting fibroblast proliferation and collagen synthesis, which culture medium includes a basal culture medium, human platelet lysate (hPL), vitamin C (VC), vitamin E (VE), cysteine ​​(Cys) and glutathione (GSH). When culturing fibroblasts, the culture medium can promote the proliferation of fibroblasts and the synthesis of collagen.

[0043] Furthermore, the above-mentioned basal culture medium includes high-glucose DMEM basal culture medium.

[0044] Furthermore, the concentration of the human platelet lysate (hPL) is 5% to 10% (v / v), wherein the human platelet lysate (hPL) can replace fetal bovine serum, thereby avoiding the introduction of exogenous genes.

[0045] Furthermore, the concentration of the vitamin C (VC) is 50-150 μM. Vitamin C can promote collagen production in tissues and help repair aging and damaged skin. Furthermore, the concentration of the vitamin C (VC) is 100 μM.

[0046] Furthermore, the concentration of the vitamin E (VE) is 0.5 to 1.5 μg / mL. Vitamin E has antioxidant properties that can help protect against free radical damage. By neutralizing free radicals, vitamin E protects cells from oxidative stress. Vitamin E also has a protective effect on the skin, reducing damage from ultraviolet rays and helping maintain skin elasticity and health. Furthermore, the concentration of the vitamin E (VE) is 1 μg / mL.

[0047] Furthermore, the concentration of cysteine ​​(Cys) is 15-25 μg / mL. Cysteine ​​can scavenge free radicals in the body and is rapidly converted into cystine, a stable sulfur-rich amino acid. Furthermore, the concentration of cysteine ​​(Cys) is 20 μg / mL.

[0048] Furthermore, the concentration of glutathione (GSH) is 0.5 to 1.5 μg / mL. Glutathione not only eliminates free radicals in the human body, but also enhances immunity and protects hemoglobin from oxidation by hydrogen peroxide and free radicals, thereby maintaining its ability to transport oxygen. Furthermore, the concentration of glutathione (GSH) is 1 μg / mL.

[0049] The present application also provides the use of the above-mentioned culture medium for promoting fibroblast proliferation and collagen synthesis in fibroblast culture.

[0050] Furthermore, the above application includes the step of isolating the fibroblasts at a rate of 2.0 to 4.0×104 viable cells / cm 2 The cells were inoculated into the above-mentioned culture medium for promoting fibroblast proliferation and collagen synthesis, and cultured at 37° C. and 5% CO 2 .

[0051] Furthermore, the above application also includes subculturing after the cell density reaches 80% to 90%.

[0052] Furthermore, the above-mentioned fibroblasts include skin fibroblasts.

[0053] Furthermore, the skin fibroblasts include those obtained by digestion and separation of skin tissue. Furthermore, the skin fibroblasts include those obtained by digestion and separation of foreskin tissue from children under 13 years old. Such skin fibroblasts, isolated and cultured from such tissue, can secrete a variety of growth factors and cytokines that promote wound healing, thereby aiding collagen regeneration in skin tissue and accelerating wound healing.

[0054] Furthermore, the above method for obtaining skin fibroblasts by digestion and separation comprises the following steps:

[0055] Obtain skin tissue and rinse it with sterile PBS buffer;

[0056] Povidone-iodine and ethanol disinfection: Place the skin tissue in a disinfectant containing 5% povidone-iodine for 3-5 minutes, then rinse with PBS. Add 75% ethanol and soak for 3-5 minutes, then rinse with PBS. Using povidone-iodine and ethanol instead of antibiotics avoids interference caused by the use of antibiotics.

[0057] The washed intact skin tissue was placed in a neutral protease solution at 4°C for digestion; after digestion, the epidermal tissue was removed and rinsed with PBS;

[0058] Collagenase solution was added, and then the dermal tissue was cut into 1 mm × 1 mm size using sterile scissors and digested in a 37°C incubator;

[0059] The digested cells were filtered through a 70 μm filter and centrifuged to obtain skin fibroblasts.

[0060] The present application also provides a method for culturing fibroblasts, the method comprising:

[0061] Fibroblasts were cultured at a rate of 2.0 to 4.0 × 10 4 viable cells / cm 2 Inoculate into the above-mentioned culture medium for promoting fibroblast proliferation and collagen synthesis, and culture at 37°C and 5% CO2;

[0062] When the cell density reaches 80% to 90%, subculture is performed.

[0063] Furthermore, the fibroblasts include skin fibroblasts. Furthermore, the skin fibroblasts include skin fibroblasts obtained by digestion and separation of foreskin tissue from children under 13 years old. The skin fibroblasts obtained by digestion, separation, and culture of this tissue can secrete a variety of growth factors and cytokines that promote wound healing, thereby assisting collagen regeneration in skin tissue and accelerating wound healing.

[0064] Furthermore, the above method for obtaining skin fibroblasts by digestion and separation comprises the following steps:

[0065] Obtain skin tissue and rinse it with sterile PBS buffer;

[0066] Povidone-iodine and ethanol disinfection: Place the skin tissue in a disinfectant containing 5% povidone-iodine for 3-5 minutes, then rinse with PBS. Add 75% ethanol and soak for 3-5 minutes, then rinse with PBS. Using povidone-iodine and ethanol instead of antibiotics avoids interference caused by the use of antibiotics.

[0067] The washed intact skin tissue was placed in a neutral protease solution at 4°C for digestion; after digestion, the epidermal tissue was removed and rinsed with PBS;

[0068] Collagenase solution was added, and then the dermal tissue was cut into 1 mm × 1 mm size using sterile scissors and digested in a 37°C incubator;

[0069] The digested cells were filtered through a 70 μm filter and centrifuged to obtain skin fibroblasts.

[0070] The present application also provides a fibroblast obtained by culturing the above-mentioned fibroblast culture method, wherein the fibroblast has high expression of fibroblast-specific protein-1 (FSP-1), type I collagen α1 (Col1A1), vimentin (Vimentin), and α-smooth muscle actin (α-SMA), and has stronger fibrogenic function.

[0071] The present application also provides the use of the above-mentioned fibroblasts in the preparation of drugs for repairing heart damage, repairing skin damage, treating rheumatoid arthritis, repairing oral mucosa and gingival bone, or treating intervertebral disc damage. 3. Beneficial effects

[0072] Compared with the prior art, the present application has the following advantages:

[0073] (1) The present application provides a biological preparation, a preparation method, and an application thereof. The biological preparation is the supernatant of the culture fluid after culturing fibroblasts using the culture medium of the present application. While promoting skin wound healing, the biological preparation can inhibit the expression of inflammatory factors around the wound, reduce the inflammatory response, alleviate the discomfort caused by the wound healing process, and improve the comfort during the wound healing period.

[0074] (2) The present application provides a culture medium for promoting fibroblast proliferation and collagen synthesis, which includes a basal culture medium, human platelet lysate (hPL), vitamin C (VC), vitamin E (VE), cysteine ​​(Cys) and glutathione (GSH). When used for fibroblast culture, the small molecule additive combination can enhance the proliferation ability of fibroblasts and enhance the expression of fibroblast functional genes, and can secrete a variety of growth factors and cytokines that promote wound healing, which can help collagen regeneration in skin tissue and accelerate wound healing; the proliferation multiples of fibroblasts cultured under such conditions will be significantly improved, and the expression of functional genes will also be significantly upregulated. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 shows the morphology and density of fibroblasts after one generation of cell culture with the addition of small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0076] Figure 2 shows the morphology and density of fibroblasts after culturing the cells for two generations with the addition of small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0077] Figure 3 shows the morphology and density of fibroblasts after culturing cells for three generations with the addition of small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0078] Figure 4 shows the morphology and density of fibroblasts after culturing cells for 4 generations with the addition of small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0079] Figure 5 shows the morphology and density of fibroblasts after culturing cells for 5 generations with the addition of small molecule additives. Note: Control is the blank culture control group, VC: vitamin C small molecule additive group, VE: vitamin E small molecule additive group, GSH: glutathione small molecule additive group, Cys: cysteine ​​small molecule additive group, VC+VE+GSH+Cys: all the above small molecule combination additive group.

[0080] FIG6 is a graph showing cell doubling time (PDL) and total cell number.

[0081] FIG7 is the expression of functional genes detected by q-PCR.

[0082] Figure 8 shows the recovery of mouse skin wounds, where: A is the blank control group; B is the fibroblast group cultured in ordinary culture medium (without small molecule additives); C is the fibroblast group cultured with small molecule additives.

[0083] FIG9 shows the size of mouse skin wounds.

[0084] Figure 10 shows the expression of related inflammatory factors IL-1β, TNF-a, IL-8, and IL-6 in the skin tissue around the mouse skin wound. DETAILED DESCRIPTION

[0085] The present application is further described below with reference to specific embodiments.

[0086] It should be noted that the terms such as "upper", "lower", "left", "right", and "middle" cited in this specification are only for the convenience of description and are not used to limit the scope of implementation. Changes or adjustments to their relative relationships should be regarded as the scope of implementation of this application without substantially changing the technical content.

[0087] Unless defined otherwise, 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 belongs; the term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0088] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0089] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. One skilled in the art can readily determine the degree of flexibility for a particular variable.

[0090] As used herein, the term "at least one of" is intended to be synonymous with "one or more of." For example, "at least one of A, B, and C" explicitly includes only A, only B, only C, and combinations of each thereof.

[0091] Concentration, amount and other numerical data can be presented in range format in this article.Should be understood that such range format is only used for convenience and brevity, and should be flexibly interpreted as not only including the numerical value clearly described as range limit, but also including all independent numerical values ​​or subranges encompassed within the scope, just as each numerical value and subrange are clearly described.For example, the numerical range of about 1 to about 4.5 should be interpreted as not only including the limit value of 1 to about 4.5 clearly described, but also including independent numerals (such as 2,3,4) and subranges (such as 1 to 3,2 to 4 etc.).The same principle is applicable to the scope of only narrating a numerical value, such as "less than about 4.5", which should be interpreted as including all above-mentioned values ​​and scopes.In addition, no matter how the breadth of described scope or feature is, this explanation should be applicable.

[0092] Example 1

[0093] This example provides the preparation of skin fibroblasts.

[0094] In this example, skin fibroblasts were obtained by digesting and isolating skin tissue.

[0095] In this embodiment, the skin tissue is foreskin tissue from children under 13 years old, and the digestion and separation of skin fibroblasts includes the following steps:

[0096] Obtain the foreskin tissue and rinse it 10 times with sterile PBS buffer;

[0097] Disinfection with povidone-iodine and ethanol (place the skin tissue in a disinfectant containing 5% povidone-iodine and soak it in a centrifuge tube for 3 minutes; replace sterile tweezers, take the skin tissue and place it in a new cell culture dish, rinse it three times with PBS, and transfer it to a centrifuge tube containing 75% ethanol and soak it for 5 minutes (excessive disinfection will reduce cell viability). After soaking, rinse it three times with PBS; use povidone-iodine and ethanol instead of antibiotics to avoid interference caused by the use of antibiotics;

[0098] Place the washed intact foreskin tissue in a 15 mL centrifuge tube containing 2 U / mL neutral protease (Cat. No. D4693, Sigma) and digest at 4°C for 12 h. Remove the 15 mL centrifuge tube from the overnight digestion, pour out the digested tissue, remove the epidermal tissue, and rinse 10 times with 20 mL of PBS buffer. Repeat this three times and discard the washing solution.

[0099] Add 0.1 U / mL collagenase NB6 (Nordmark, N0002779) solution, then cut the dermis into 1 mm × 1 mm pieces with sterile scissors and transfer it to a 50 mL centrifuge tube containing foreskin tissue suspension. Seal the tube with parafilm and place it in a 37°C incubator with gentle shaking (70 rpm) for 2 h.

[0100] The digested cells were filtered through a 70 μm sieve and centrifuged at 300 g for 5 min to obtain digested and separated skin fibroblasts.

[0101] Example 2

[0102] This example provides a comparison of cultured skin fibroblasts using culture media supplemented with different small molecule additives.

[0103] The skin fibroblasts isolated and digested in Example 1 were resuspended in 1 mL of high-glucose DMEM basal medium and counted.

[0104] According to 2.0~4.0×10 4 viable cells / cm 2 The cells were inoculated into T25 culture flasks and cultured in a 37°C, 5% CO2 cell culture incubator. The culture medium consisted of high-glucose DMEM basal medium + 10% human platelet lysate, and also included the small molecule additives listed in Table 1.

[0105] Table 1 Small molecule additives and dosage

[0106] After adding small molecule additives, the cell morphology and density after five subcultures are shown in Figures 1-5. After five subcultures, the morphology of skin fibroblasts did not change significantly. At the same time, under the conditions of co-culture with multiple small molecule additives, the cell growth rate increased.

[0107] During passage, cells were collected and plotted for Doubling time (DT), PDL, and total cell number. As shown in Figure 6, the addition of various small molecule additives significantly shortened cell expansion time and increased the total number of cells harvested.

[0108] Sample collection for q-PCR: Use the cell lysis buffer in the Promega Total RNA Extraction Kit (LS1040), add 300 μL to each sample to fully lyse the sample, and store at -20°C.

[0109] Sample RNA extraction for q-PCR: Strictly follow the kit instructions.

[0110] Reverse transcription of RNA samples: Use the Promega RNA Reverse Transcription Kit (LS2052) strictly according to the kit instructions. Each reaction volume is 20 μL, and 400 ng of RNA is added to each reaction. Reverse transcription is performed at 37°C for 15 minutes and 98°C for 5 minutes.

[0111] q-PCR reaction: The reaction was completed strictly according to the SYBR green qPCR Mix kit (D7265) of Biyuntian. The components were: Mix 10 μL, cDNA template 200 ng, primer 0.5 μL, and water to 20 μL.

[0112] q-PCR assays were used to detect functional gene expression, and the results are shown in Figure 7. Among these molecular combinations, the combination of multiple small molecule additives (VC+VE+GSH+Cys) significantly increased the expression of multiple genes, including FSP-1, Col1A1, Vimentin, and a-SMA, significantly upregulating fibroblast gene function. In particular, as shown by Vimentin gene expression, individual small molecule additives had little ability to increase Vimentin gene expression, and even decreased it (in the VE group). However, the synergistic effect of the four small molecule additives significantly upregulated Vimentin gene expression.

[0113] Example 3

[0114] This embodiment provides a biological preparation.

[0115] In Example 2, fibroblasts were cultured in a culture medium containing multiple small molecule additives (VC+VE+GSH+Cys), and the supernatant of each generation (1st to 6th generation) of cells after 3 days of culture was collected. After mixing, the cells were centrifuged at 300g for 5 minutes to obtain the cell culture supernatant. The cell culture supernatant was passed through a 0.22μm filter to obtain the biological preparation.

[0116] At the same time, fibroblasts were cultured with the control culture medium in Example 2, and the supernatant of each generation (1st to 6th generation) of cells after 3 days of culture was collected. After mixing, the cells were centrifuged at 300g for 5 minutes to obtain the cell culture supernatant. The cell culture supernatant was passed through a 0.22μm filter and used as a control biological preparation.

[0117] Example 4

[0118] This embodiment provides the use of the biological preparation of Example 3 in promoting skin wound healing, including:

[0119] Construction of mouse skin wound model:

[0120] Mice aged 5 to 7 weeks were anesthetized, and the hair on their backs near the tails was removed. After disinfection with iodine, a 1×1 cm 2 Use a sterilized scalpel to cut the skin tissue according to the marked area, disinfect the wound with iodine, and bandage the wound with gauze.

[0121] The biological preparation obtained in Example 3 and the control biological preparation were applied to the wounds of mice, 1 mL per mouse, and the wound healing of the mice was measured after 3 weeks.

[0122] The results are shown in Figures 8 and 9. Compared with the wounds of mice coated with the control biological agent (Figure 8B), the wounds of mice coated with the biological agent (Figure 8C) were smaller and healed faster.

[0123] Example 5

[0124] This example provides the use of the biological preparation of Example 3 in inhibiting the expression of inflammatory factors around a wound, including:

[0125] Referring to Example 4, three weeks after the biological agent was applied to the wound, mice were anesthetized with isoflurane. Using autoclaved surgical scissors and forceps, skin tissue surrounding the original wound was excised and minced into small pieces. The pieces were then quickly placed in a cell lysis buffer for RNA extraction. The specific extraction method was the same as in Example 2.

[0126] After the extraction was completed, the expression of related inflammatory factors IL-1β, TNF-a, IL-8, and IL-6 was detected.

[0127] The results are shown in FIG10 . q-PCR studies show that after application of the biological preparation of the present application, the expression of inflammatory factors around the wound is significantly reduced.

[0128] In summary, the function of fibroblasts cultured in the culture medium with the addition of small molecule additives in the present application will be significantly improved. At the same time, the supernatant of the culture fluid of fibroblasts cultured under these conditions can significantly promote wound healing of skin tissue, and at the same time significantly inhibit the expression of inflammatory factors in the tissue around the wound.

Claims

1. A method for preparing a biological agent, characterized in that: The preparation method comprises the following steps: S1, taking fibroblasts, inoculating them into medium 1 for culture, and performing subculture when the cell density reaches 80-90%, wherein the medium 1 comprises basal medium, human platelet lysate, vitamin C, vitamin E, cysteine, and glutathione; S2, collecting the culture supernatant of each cell culture generation, mixing and filtering to obtain the biological preparation.

2. The method for preparing a biological agent according to claim 1, characterized in that: The concentration of the human platelet lysate is 5% to 10% (v / v); and / or the concentration of the vitamin C is 50 to 150 μM; and / or the concentration of vitamin E is 0.5 to 1.5 μg / mL; and / or the concentration of cysteine ​​is 15 to 25 μg / mL; And / or the concentration of glutathione is 0.5-1.5 μg / mL.

3. The method for preparing a biological agent according to claim 2, characterized in that: The number of fibroblasts seeded in the culture medium 1 was 2.0-4.0×10 4 viable cells / cm 2 .

4. The method for preparing a biological agent according to claim 2 or 3, characterized in that: The fibroblasts include skin fibroblasts.

5. A biological preparation prepared by the method for preparing a biological preparation according to any one of claims 1 to 4.

6. A method for preparing a biological preparation according to any one of claims 1 to 4, or use of a biological preparation according to claim 5 in the preparation of a medicament for promoting skin wound healing and / or inhibiting inflammatory reactions around skin wounds.

7. A pharmaceutical composition for promoting skin wound healing and / or inhibiting inflammatory response around skin wounds, characterized in that: The pharmaceutical composition comprises the biological preparation according to claim 5.

8. A culture medium for promoting fibroblast proliferation and collagen synthesis, characterized in that: The culture medium comprises a basal culture medium, human platelet lysate, vitamin C, vitamin E, cysteine ​​and glutathione; The concentration of the human platelet lysate is 5% to 10% (v / v); The concentration of vitamin C is 50 to 150 μM; The concentration of vitamin E is 0.5-1.5 μg / mL; The concentration of cysteine ​​is 15-25 μg / mL; The concentration of the glutathione is 0.5-1.5 μg / mL.

9. Use of the culture medium for promoting fibroblast proliferation and collagen synthesis according to claim 8 in fibroblast culture.

10. A method for culturing fibroblasts, characterized in that: The method comprises: Fibroblasts were cultured at a rate of 2.0 to 4.0 × 10 4 viable cells / cm 2 Inoculate the culture medium for promoting fibroblast proliferation and collagen synthesis according to claim 8, and culture at 37°C and 5% CO2; When the cell density reaches 80% to 90%, subculture is performed.

11. A fibroblast obtained by culturing the fibroblast according to the culturing method of claim 10.

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