Skin repairing liquid containing autologous adipose-derived stem cells and preparation method thereof

Abstract

The invention discloses a skin repair liquid containing autologous adipose-derived stem cells and a preparation method thereof, and belongs to the technical field of regenerative medicine and skin repair, the skin repair liquid comprises adipose-derived stem cell nano-liposomes and a substrate solvent; the substrate solvent is prepared from 1 to 3 parts of sodium hyaluronate, 2 to 5 parts of panthenol, 0.5 to 1 part of ceramide, 0.1 to 0.3 part of nicotinamide, 1 to 2 parts of polypeptide compound and 88 to 98 parts of purified water; the polypeptide compound is prepared from the following components in parts by mass: 5 to 10 parts of oligopeptide-3, 10 to 15 parts of acetyl hexapeptide-8, 15 to 20 parts of hexapeptide-10, 10 to 15 parts of copper tripeptide-1 glycine carboxymethyl ester, 5 to 10 parts of palmitoyl tetrapeptide-7, 10 to 15 parts of glycine, 8 to 12 parts of propylene glycol, 5 to 8 parts of hydroxyethyl cellulose, 3 to 8 parts of polyethylene glycol-400, 2 to 6 parts of betaine, 1 to 3 parts of panthenol and 1 to 3 parts of dipotassium glycyrrhizinate. The technical problems that in the prior art, stem cell repairing liquid is insufficient in biological activity, poor in stability and limited in repairing effect are solved.

Description

Technical Field

[0001] The present invention relates to the technical fields of regenerative medicine and skin repair, and particularly relates to a skin repair solution containing autologous adipose stem cells and a preparation method thereof. Background Art

[0002] Skin repair is an important research direction in the fields of medical beauty and trauma treatment. Traditional skin repair products have disadvantages such as poor biocompatibility, non-durable repair effects, and inability to stimulate autologous tissue regeneration. In recent years, stem cell technology has shown great potential in the field of tissue repair. In particular, adipose-derived mesenchymal stem cells (ADSCs) have become a research hotspot due to advantages such as convenient material collection, autologous origin, and strong differentiation potential.

[0003] In the prior art, stem cells are usually applied to skin repair in two ways: directly injecting live cells or using the supernatant of stem cell culture. Directly injecting live cells has problems such as low cell survival rate and difficult control of directional differentiation; while simply using the supernatant of stem cell culture has insufficient biological activity and cannot meet the repair requirements of complex skin injuries. Summary of the Invention

[0004] The present invention provides a skin repair solution containing autologous adipose stem cells and a preparation method thereof to solve the technical problems of insufficient biological activity, poor stability, and limited repair effect of the stem cell repair solution in the prior art.

[0005] A skin repair solution containing autologous adipose stem cells provided by the present invention includes adipose stem cell nanoliposomes and a base solvent.

[0006] Preferably, the base solvent contains 1 - 3 parts of sodium hyaluronate, 2 - 5 parts of panthenol, 0.5 - 1 part of ceramide, 0.1 - 0.3 part of niacinamide, 1 - 2 parts of polypeptide complex, and 88 - 98 parts of purified water;

[0007] The polypeptide complex includes the following components by mass fraction: 5 - 10 parts of oligopeptide - 3, 10 - 15 parts of acetyl hexapeptide - 8, 15 - 20 parts of hexapeptide - 10, 10 - 15 parts of copper tripeptide - 1 glycine carboxymethyl ester, 5 - 10 parts of palmitoyl tetrapeptide - 7, 10 - 15 parts of glycine, 8 - 12 parts of propylene glycol, 5 - 8 parts of hydroxyethyl cellulose, 3 - 8 parts of polyethylene glycol - 400, 2 - 6 parts of betaine, 1 - 3 parts of panthenol, and 1 - 3 parts of dipotassium glycyrrhizinate.

[0008] Preferably, the adipose stem cell nanoliposomes are prepared by the following steps:

[0009] S1: Autologous adipose tissue acquisition and processing: Obtain 10 - 50 parts of the patient's autologous adipose tissue through minimally invasive liposuction or surgical excision. Wash it 3 - 5 times with 0.9% NaCl solution at 37°C, shake and mix evenly for 60 - 90 seconds each time, and centrifuge at 300 - 500g for 5 minutes to precipitate the tissue, then discard the supernatant;

[0010] S2: Stem cell isolation and purification: Treat the processed adipose tissue with a DPBS digestive solution with a pH of 7.2 - 7.4 containing 0.1% - 0.3% type I collagenase, 0.05% - 0.1% neutral protease, and 2 - 5 mM CaCl2 at 37°C for 40 - 90 minutes to obtain a basic cell suspension; Superimpose the cell suspension on an equal volume of Ficoll - Paque PREMIUM at a ratio of 1:1, and centrifuge at 1000 - 1200 rpm for 10 minutes to isolate the primary population of mesenchymal stem cells; Through immunomagnetic bead sorting technology, use a combination of CD73 +, CD90 +, CD105 + and CD45 -, CD34 - markers to screen and obtain highly pure adipose stem cells;

[0011] S3: Three - dimensional microsphere culture of stem cells: Seed the purified adipose stem cells at a density of 2 - 5×10 5 cells / mL into an ultra - low attachment 24 - well culture plate, and culture in a serum - free medium containing 5 - 10 parts of autologous serum to form stem cell microspheres with a diameter of 100 - 300μm;

[0012] S4: Stress pre - treatment of stem cell microspheres: Culture the stem cell microspheres in an environment with an oxygen concentration of 1% - 5% for 12 - 24 hours. During this period, heat up to 40 - 42°C every 6h for 15 minutes, and use an FX - 5000T stretching system to apply 5% - 10% cyclic stretching with a frequency of 0.5Hz and a sine waveform for 6 - 12 hours;

[0013] S5: Active factor extraction and concentration: Continue to culture the pre - treated stem cell microspheres for 48 - 72 hours, and collect the culture supernatant; Use polyethersulfone membranes with a molecular weight cut - off of 5 kDa and 100 kDa to concentrate the supernatant. The operating pressure is 15 - 20 psi, and the flow rate is 50 - 60 ml / minute, retaining the active components with a molecular weight between 5 - 100 kDa; Subsequently, freeze at - 40°C for 120 minutes, dry at - 20°C and 0.1 mbar for 24 hours, and dry at 10°C and 0.01 mbar for 12 hours to obtain a primary active factor complex;

[0014] S6: Encapsulation with nanoliposomes and activity protection: Mix the primary active factor complex with 45 parts of phosphatidylcholine, 40 parts of cholesterol, 10 parts of vitamin E, and 5 parts of ceria, dissolve them in a mixed solvent of 100 parts of chloroform and methanol with a volume ratio of 2:1, and use a rotary evaporator to rotate and evaporate at 40 °C at a rotation speed of 120 - 150 revolutions per minute to form a lipid film. After vacuum drying for 2 hours, add PBS with a pH of 7.4 containing the active factor complex to hydrate for 60 minutes, and perform ultrasonic treatment at 40 kHz for 20 minutes to obtain adipose stem cell nanoliposomes.

[0015] Preferably, in S3, the ultra-low attachment 24-well culture plate contains a chitosan-hyaluronic acid composite microcarrier.

[0016] Preferably, the preparation method of the chitosan-hyaluronic acid composite microcarrier is as follows: Mix 1 - 2 parts of a chitosan solution with a molecular weight of 100 - 200 kDa and 0.5 - 1 part of a sodium hyaluronate solution with a molecular weight of 1000 - 1500 kDa, add 0.1 - 0.5 parts of gelatin as a cross-linking agent, pump the mixed solution into a liquid paraffin fluid containing 2 parts of Span80 at a speed of 3 ml / hour through a syringe with an inner diameter of 200 μm, form uniform droplets in a T-shaped microfluidic chip with an inner diameter of 300 μm, collect them in 100 parts of PBS containing 0.5 parts of Tween 80, and stir at 200 rpm at 4 °C for 4 hours to obtain the chitosan-hyaluronic acid composite microcarrier.

[0017] Preferably, in S3, the diameter of the chitosan-hyaluronic acid composite microcarrier is 50 - 150 μm.

[0018] Preferably, in S3, the serum-free medium formulation includes: DMEM / F12 basal medium, 10 - 15 ng / mL epidermal growth factor EGF, 5 - 10 ng / mL basic fibroblast growth factor bFGF, 2 - 5 ng / mL transforming growth factor β3, 10 -7 -10 -6 M dexamethasone, 0.5 - 1.0 mM / L ascorbic acid, 50 - 100 μg / mL ascorbic acid-2-phosphate, and 0.2 - 0.5 mM lipoic acid.

[0019] Preferably, in S3, the surface of the ultra-low attachment 24-well culture plate is coated with poly(2-hydroxyethyl methacrylate).

[0020] The preparation method of the skin repair liquid provided by the present invention includes the following steps: Mix the nanoliposomes with a basal solvent, perform homogenization operation, and then perform aseptic filtration through a 0.22 μm polyethersulfone membrane to obtain the skin repair liquid.

[0021] Preferably, the homogenization operation uses an APV-2000 homogenizer, with an operating pressure of 800-1200 bar, circulating through 3-5 times, and the processing temperature is controlled at 4-8 °C.

[0022] Preferably, the average particle size of the repair fluid is 80-150 nm, the Zeta potential is -35 to -20 mV, the pH value is 5.5-6.5, and the osmotic pressure is 280-320 mOsm / kg.

[0023] The present invention has at least the following beneficial effects:

[0024] (1) In the present invention, by encapsulating adipose-derived stem cell active substances within a particle size range of 80-150 nm using a nano-liposome delivery system, a stable bilayer structure is formed, which can not only penetrate the gaps in the skin stratum corneum but also be effectively phagocytosed by dermal cells. The phospholipid bilayer's property of being similar and soluble to the cell membrane significantly improves the transdermal efficiency of the active ingredients and simultaneously protects the bioactive substances from enzymatic degradation. In addition, a serum-free medium with a special formulation activates the Wnt / β-catenin signaling pathway, promotes the secretion of regeneration factors by ADSCs, combines with ceramide to enhance the skin barrier, copper tripeptide-1 to promote ECM remodeling, and the slow-release property of nano-liposomes to jointly promote tissue regeneration. Clinical data shows a significant increase in collagen density in the repair area. At the same time, dipotassium glycyrrhizinate combined with ADSC exosomes inhibits the secretion of inflammatory factors, and chitosan-hyaluronic acid composite microcarriers release antibacterial peptides in the acidic environment of the wound, forming a dual protection of physical barrier and biological antibacterial, effectively reducing scar formation and achieving a synergistic mechanism of immune regulation and anti-inflammation.

[0025] (2) The present invention effectively delivers the active ingredients of autologous adipose-derived stem cells through nano-liposome technology, significantly increasing the bioavailability of the active ingredients, solving the technical problems such as insufficient biological activity, poor stability, and limited repair effect in traditional stem cell applications, and achieving remarkable effects in skin repair and anti-aging. Detailed implementation manners

[0026] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0027] Example 1

[0028] This example provides a skin repair fluid containing autologous adipose-derived stem cells and its preparation method.

[0029] The skin repair fluid containing autologous adipose stem cells provided by this embodiment includes adipose stem cell nanoliposomes and a basal solvent.

[0030] Among them, the basal solvent contains 1 part of sodium hyaluronate, 2 parts of panthenol, 0.5 part of ceramide, 0.1 part of niacinamide, 1 part of polypeptide complex and 88 parts of purified water;

[0031] The polypeptide complex includes the following components by mass: 5 parts of oligopeptide-3, 10 parts of acetyl hexapeptide-8, 15 parts of hexapeptide-10, 10 parts of copper tripeptide-1 glycine carboxymethyl ester, 5 parts of palmitoyl tetrapeptide-7, 10 parts of glycine, 8 parts of propylene glycol, 5 parts of hydroxyethyl cellulose, 3 parts of polyethylene glycol-400, 2 parts of betaine, 1 part of panthenol and 1 part of dipotassium glycyrrhizinate.

[0032] Among them, the adipose stem cell nanoliposomes are prepared by the following steps:

[0033] S1: Acquisition and treatment of autologous adipose tissue: 10 parts of autologous adipose tissue of the patient are obtained by minimally invasive liposuction, washed 3 times with 0.9% NaCl solution at 37°C, shaken and mixed evenly for 60 seconds each time, and centrifuged at 300g for 5 minutes to precipitate the tissue, and then the supernatant is discarded;

[0034] S2: Separation and purification of stem cells: The treated adipose tissue is treated with a DPBS digestive solution with a pH of 7.2 containing 0.1% type I collagenase, 0.05% neutral protease and 2 mM CaCl2 at 37°C for 40 minutes to obtain a basic cell suspension; The cell suspension is overlaid on an equal volume of Ficoll-Paque PREMIUM at a ratio of 1:1 and centrifuged at 1000 rpm for 10 minutes to separate the primary population of mesenchymal stem cells; Through immunomagnetic bead sorting technology, high-purity adipose stem cells are screened using a combination of CD73+, CD90+, CD105+ and CD45-, CD34- markers;

[0035] S3: Three-dimensional microsphere culture of stem cells: The purified adipose stem cells are inoculated in an ultra-low attachment 24-well culture plate at a density of 2×10 5 cells / mL and cultured in a serum-free medium containing 5 parts of autologous serum to form stem cell microspheres with a diameter of 100 μm;

[0036] S4: Stress pre-treatment of stem cell microspheres: The stem cell microspheres are cultured in an environment with an oxygen concentration of 1% for 12 hours, during which the temperature is raised to 40°C every 6 hours for 15 minutes, and a 5% cyclic stretch is applied using an FX-5000T stretching system, with a frequency of 0.5 Hz and a sine waveform, for 6 hours;

[0037] S5: Active Factor Extraction and Concentration: Continuously culture the pre-treated stem cell microspheres for 48 hours, and collect the culture supernatant; use polyethersulfone membranes with a molecular weight cut-off of 5 kDa and 100 kDa to concentrate the supernatant at an operating pressure of 15 psi and a flow rate of 50 ml / minute, retaining the active components with a molecular weight between 5 - 100 kDa; then freeze at -40°C for 120 minutes, dry at -20°C and 0.1 mbar for 24 hours, and dry at 10°C and 0.01 mbar for 12 hours to obtain the primary active factor complex;

[0038] S6: Nanoliposome Encapsulation and Active Protection: Mix the primary active factor complex with 45 parts of phosphatidylcholine, 40 parts of cholesterol, 10 parts of vitamin E, and 5 parts of graphene oxide, dissolve it in 100 parts of a mixed solvent of chloroform and methanol with a volume ratio of 2:1, and use a rotary evaporator to rotate and evaporate at 40°C at a speed of 120 revolutions per minute to form a lipid film. After vacuum drying for 2 hours, add PBS with a pH of 7.4 containing the active factor complex to hydrate for 60 minutes, and ultrasonically treat at 40 kHz for 20 minutes to obtain adipose stem cell nanoliposomes.

[0039] Among them, in S3, the ultra-low attachment 24-well culture plate contains a chitosan-hyaluronic acid composite microcarrier.

[0040] Among them, the preparation method of the chitosan-hyaluronic acid composite microcarrier is as follows: Mix 1 part of a chitosan solution with a molecular weight of 100 kDa and 0.5 part of a sodium hyaluronate solution with a molecular weight of 1000 kDa, add 0.1 part of gelatin as a cross-linking agent, pump the mixed solution into a liquid paraffin fluid containing 2 parts of Span80 at a speed of 3 ml / hour through a syringe with an inner diameter of 200 μm, form uniform droplets in a T-shaped microfluidic chip with an inner diameter of 300 μm, collect them in 100 parts of PBS containing 0.5 part of Tween 80, and stir at 200 rpm at 4°C for 4 hours to obtain the chitosan-hyaluronic acid composite microcarrier.

[0041] Among them, in S3, the diameter of the chitosan-hyaluronic acid composite microcarrier is 50 μm.

[0042] Among them, in S3, the serum-free medium formula includes: DMEM / F12 basal medium, 10 ng / mL epidermal growth factor EGF, 5 ng / mL basic fibroblast growth factor bFGF, 2 ng / mL transforming growth factor β3, 10 -7 μM dexamethasone, 0.5 mM / L ascorbic acid, 50 μg / mL ascorbic acid-2-phosphate, and 0.2 mM lipoic acid.

[0043] Among them, in S3, the surface of the ultra-low attachment 24-well culture plate is coated with poly(2-hydroxyethyl methacrylate).

[0044] The preparation method of the skin repair fluid provided in this embodiment includes the following steps: mixing nanoliposomes with a base solvent, performing a homogenization operation, and then performing aseptic filtration through a 0.22-μm polyethersulfone membrane to obtain the skin repair fluid.

[0045] Among them, the homogenization operation uses an APV-2000 homogenizer, the operating pressure is 800 bar, it circulates 3 times, and the treatment temperature is controlled at 4°C.

[0046] After detection, the average particle size of the repair fluid is 80 nm, the Zeta potential is -35 mV, the pH value is 5.5, and the osmotic pressure is 280 mOsm / kg.

[0047] Example 2

[0048] This embodiment provides a skin repair fluid containing autologous adipose stem cells and a preparation method thereof.

[0049] The skin repair fluid containing autologous adipose stem cells provided in this embodiment includes adipose stem cell nanoliposomes and a base solvent.

[0050] Among them, the base solvent contains 2 parts of sodium hyaluronate, 3 parts of panthenol, 0.7 part of ceramide, 0.2 part of niacinamide, 1.5 parts of polypeptide complex, and 93 parts of purified water;

[0051] The polypeptide complex includes the following components by mass: 7 parts of oligopeptide-3, 12 parts of acetyl hexapeptide-8, 17 parts of hexapeptide-10, 12 parts of copper tripeptide-1 glycine carboxymethyl ester, 7 parts of palmitoyl tetrapeptide-7, 12 parts of glycine, 10 parts of propylene glycol, 6 parts of hydroxyethyl cellulose, 5 parts of polyethylene glycol-400, 4 parts of betaine, 2 parts of panthenol, and 2 parts of dipotassium glycyrrhizinate.

[0052] Among them, the adipose stem cell nanoliposomes are prepared by the following steps:

[0053] S1: Acquisition and treatment of autologous adipose tissue: 30 parts of autologous adipose tissue of the patient are obtained by surgical excision, washed 4 times with 0.9% NaCl solution at 37°C, shaken and mixed evenly for 80 seconds each time, and centrifuged at 400 g for 5 minutes to precipitate the tissue, and then the supernatant is discarded;

[0054] S2: Stem cell isolation and purification: The treated adipose tissue is treated with a DPBS digestive solution with a pH of 7.3 containing 0.2% type I collagenase, 0.07% neutral protease, and 3 mM CaCl2 at 37 °C for 60 minutes to obtain a basic cell suspension; the cell suspension is overlaid on an equal volume of Ficoll-Paque PREMIUM at a ratio of 1:1 and centrifuged at 1100 rpm for 10 minutes to separate the primary population of mesenchymal stem cells; through immunomagnetic bead sorting technology, high-purity adipose stem cells are obtained by screening with a combination of CD73+, CD90+, CD105+ and CD45-, CD34- markers;

[0055] S3: Three-dimensional microsphere culture of stem cells: The purified adipose stem cells are inoculated into an ultra-low attachment 24-well culture plate at a density of 3×10 5 cells / mL and cultured in a serum-free medium containing 7 parts of autologous serum to form stem cell microspheres with a diameter of 200 μm;

[0056] S4: Stress pre-treatment of stem cell microspheres: The stem cell microspheres are cultured in an environment with an oxygen concentration of 3% for 18 hours, during which the temperature is raised to 41 °C every 6 hours for 15 minutes, and a 7% cyclic stretch is applied using an FX-5000T stretching system at a frequency of 0.5 Hz and a sine wave form for 9 hours;

[0057] S5: Extraction and concentration of active factors: The pre-treated stem cell microspheres are cultured for another 60 hours, and the culture supernatant is collected; using polyethersulfone membranes with a molecular weight cut-off of 5 kDa and 100 kDa, the supernatant is concentrated at an operating pressure of 17 psi and a flow rate of 55 ml / minute, and the active components with a molecular weight between 5 - 100 kDa are retained; then it is frozen at -40 °C for 120 minutes, dried at -20 °C and 0.1 mbar for 24 hours, and dried at 10 °C and 0.01 mbar for 12 hours to obtain the primary active factor complex;

[0058] S6: Nanoliposome encapsulation and activity protection: The primary active factor complex is mixed with 45 parts of phosphatidylcholine, 40 parts of cholesterol, 10 parts of vitamin E, and 5 parts of graphene oxide, dissolved in a mixed solvent of 100 parts of chloroform and methanol mixed at a volume ratio of 2:1, and rotary evaporated at 40 °C using a rotary evaporator at a speed of 140 revolutions per minute to form a lipid film. After vacuum drying for 2 hours, it is hydrated with PBS with a pH of 7.4 containing the active factor complex for 60 minutes and sonicated at 40 kHz for 20 minutes to obtain adipose stem cell nanoliposomes.

[0059] Among them, in S3, the ultra-low attachment 24-well culture plate contains a chitosan-hyaluronic acid composite microcarrier.

[0060] Among them, the preparation method of the chitosan-hyaluronic acid composite microcarrier is as follows: Mix 1.5 parts of a chitosan solution with a molecular weight of 150 kDa and 0.7 parts of a sodium hyaluronate solution with a molecular weight of 1000 - 1500 kDa, add 0.3 parts of gelatin as a cross-linking agent, pump the mixed solution into a liquid paraffin fluid containing 2 parts of Span80 at a rate of 3 ml / hour through a syringe with an inner diameter of 200 μm, form uniform droplets in a T-shaped microfluidic chip with an inner diameter of 300 μm, collect them in 100 parts of PBS containing 0.5 parts of Tween 80, and stir at 200 rpm at 4°C for 4 hours to obtain the chitosan-hyaluronic acid composite microcarrier.

[0061] Among them, in S3, the diameter of the chitosan-hyaluronic acid composite microcarrier is 100 μm.

[0062] Among them, in S3, the serum-free medium formulation includes: DMEM / F12 basal medium, 12 ng / mL epidermal growth factor EGF, 7 ng / mL basic fibroblast growth factor bFGF, 3 ng / mL transforming growth factor β3, 10 -7 μg / mL dexamethasone, 0.7 mM / L ascorbic acid, 70 μg / mL ascorbic acid-2-phosphate, and 0.3 mM lipoic acid.

[0063] Among them, in S3, the surface of the ultra-low attachment 24-well culture plate is coated with poly(2-hydroxyethyl methacrylate).

[0064] The preparation method of the skin repair fluid provided in this example includes the following steps: Mix the nano-liposomes with the basal solvent, perform a homogenization operation, and then filter aseptically through a 0.22-μm polyethersulfone membrane to obtain the skin repair fluid.

[0065] Among them, the homogenization operation uses an APV-2000 homogenizer, the operating pressure is 1000 bar, it circulates through 3 - 5 times, and the treatment temperature is controlled at 6°C.

[0066] After detection, the average particle size of the repair fluid is 120 nm, the Zeta potential is -25 mV, the pH value is 6.0, and the osmotic pressure is 300 mOsm / kg.

[0067] Example 3

[0068] This example provides a skin repair fluid containing autologous adipose stem cells and its preparation method.

[0069] The skin repair fluid containing autologous adipose stem cells provided in this example includes adipose stem cell nano-liposomes and a basal solvent.

[0070] Among them, the basal solvent contains 3 parts of sodium hyaluronate, 5 parts of panthenol, 1 part of ceramide, 0.3 part of niacinamide, 2 parts of a polypeptide complex, and 98 parts of purified water;

[0071] The polypeptide complex comprises the following components by mass fraction: 10 parts of oligopeptide-3, 15 parts of acetyl hexapeptide-8, 20 parts of hexapeptide-10, 15 parts of copper tripeptide-1 glycine carboxymethyl ester, 10 parts of palmitoyl tetrapeptide-7, 15 parts of glycine, 12 parts of propylene glycol, 8 parts of hydroxyethyl cellulose, 8 parts of polyethylene glycol-400, 6 parts of betaine, 3 parts of panthenol and 3 parts of dipotassium glycyrrhizinate.

[0072] Among them, the adipose stem cell nanoliposome is prepared by the following steps:

[0073] S1: Acquisition and treatment of autologous adipose tissue: 50 parts of autologous adipose tissue of the patient are obtained by minimally invasive liposuction or surgical excision, washed 5 times with 0.9% NaCl solution at 37°C, shaken and mixed evenly for 90 seconds each time, and centrifuged at 500g for 5 minutes to precipitate the tissue, and then the supernatant is discarded;

[0074] S2: Separation and purification of stem cells: The treated adipose tissue is treated with a DPBS digestive solution with a pH of 7.2 - 7.4 containing 0.3% type I collagenase, 0.1% neutral protease and 5 mM CaCl2 at 37°C for 90 minutes to obtain a basic cell suspension; The cell suspension is overlaid on an equal volume of Ficoll-Paque PREMIUM at a ratio of 1:1 and centrifuged at 1000 - 1200 rpm for 10 minutes to separate the primary population of mesenchymal stem cells; Through immunomagnetic bead sorting technology, high-purity adipose stem cells are obtained by screening with a combination of markers CD73+, CD90+, CD105+ and CD45-, CD34-;

[0075] S3: Three-dimensional microsphere culture of stem cells: The purified adipose stem cells are inoculated into an ultra-low attachment 24-well culture plate at a density of 5×10 5 cells / mL and cultured in a serum-free medium containing 10 parts of autologous serum to form stem cell microspheres with a diameter of 300 μm;

[0076] S4: Stress pre-treatment of stem cell microspheres: The stem cell microspheres are cultured in an environment with an oxygen concentration of 5% for 24 hours, during which the temperature is raised to 42°C every 6 hours for 15 minutes, and a 10% cyclic stretch is applied using an FX-5000T stretching system at a frequency of 0.5 Hz and a sine wave form for 12 hours;

[0077] S5: Active Factor Extraction and Concentration: Continuously culture the pre-treated stem cell microspheres for 72 hours, and collect the culture supernatant; use polyethersulfone membranes with a molecular weight cut-off of 5 kDa and 100 kDa to concentrate the supernatant at an operating pressure of 20 psi and a flow rate of 60 ml / minute, retaining the active ingredients with a molecular weight between 5 - 100 kDa; then freeze at -40°C for 120 minutes, dry at -20°C and 0.1 mbar for 24 hours, and dry at 10°C and 0.01 mbar for 12 hours to obtain the primary active factor complex;

[0078] S6: Nanoliposome Encapsulation and Activity Protection: Mix the primary active factor complex with 45 parts of phosphatidylcholine, 40 parts of cholesterol, 10 parts of vitamin E, and 5 parts of ceramsite, dissolve them in a mixed solvent of 100 parts of chloroform and methanol with a volume ratio of 2:1, and use a rotary evaporator to rotate and evaporate at 40°C at a speed of 150 revolutions per minute to form a lipid film. After vacuum drying for 2 hours, add PBS with a pH of 7.4 containing the active factor complex for hydration for 60 minutes, and ultrasonically treat at 40 kHz for 20 minutes to obtain adipose stem cell nanoliposomes.

[0079] Among them, in S3, the ultra-low attachment 24-well culture plate contains a chitosan-hyaluronic acid composite microcarrier.

[0080] Among them, the preparation method of the chitosan-hyaluronic acid composite microcarrier is: Mix 2 parts of a chitosan solution with a molecular weight of 200 kDa and 1 part of a sodium hyaluronate solution with a molecular weight of 1500 kDa, add 0.5 part of gelatin as a cross-linking agent, pump the mixed solution into a liquid paraffin fluid containing 2 parts of Span80 at a speed of 3 ml / hour through a syringe with an inner diameter of 200 μm, form uniform droplets in a T-shaped microfluidic chip with an inner diameter of 300 μm, collect them in 100 parts of PBS containing 0.5 part of Tween 80, and stir at 200 rpm at 4°C for 4 hours to obtain the chitosan-hyaluronic acid composite microcarrier.

[0081] Among them, in S3, the diameter of the chitosan-hyaluronic acid composite microcarrier is 150 μm.

[0082] Among them, in S3, the serum-free medium formula includes: DMEM / F12 basal medium, 15 ng / mL epidermal growth factor EGF, 10 ng / mL basic fibroblast growth factor bFGF, 5 ng / mL transforming growth factor β3, 10 -6 M dexamethasone, 1.0 mM / L ascorbic acid, 100 μg / mL ascorbic acid-2-phosphate, and 0.5 mM lipoic acid.

[0083] Among them, in S3, the surface of the ultra-low attachment 24-well culture plate is coated with poly(2-hydroxyethyl methacrylate).

[0084] The preparation method of the skin repair fluid provided by this embodiment includes the following steps: Mix the nano-liposome with the base solvent, perform a homogenization operation, and then perform aseptic filtration through a 0.22-μm polyethersulfone membrane to obtain the skin repair fluid.

[0085] Among them, the homogenization operation uses an APV-2000 homogenizer, the operating pressure is 1200 bar, the circulation is 5 times, and the treatment temperature is controlled at 8°C.

[0086] After detection, the average particle size of the repair fluid is 150 nm, the Zeta potential is -20 mV, the pH value is 6.5, and the osmotic pressure is 320 mOsm / kg.

[0087] Comparative Example 1

[0088] This comparative example provides a skin repair fluid and its preparation method.

[0089] The skin repair fluid provided by this comparative example contains a base solvent and a polypeptide complex.

[0090] The base solvent includes: 1 part of sodium hyaluronate, 2 parts of panthenol, 0.5 part of ceramide, 0.1 part of niacinamide, 1 part of polypeptide complex, and 88 parts of purified water.

[0091] The polypeptide complex includes the following components by mass fraction: oligopeptide-3, 5 parts; acetyl hexapeptide-8, 10 parts; hexapeptide-10, 15 parts; copper tripeptide-1 glycine carboxymethyl ester, 10 parts; palmitoyl tetrapeptide-7, 5 parts; glycine, 10 parts; propylene glycol, 8 parts; hydroxyethyl cellulose, 5 parts; polyethylene glycol-400, 3 parts; betaine, 2 parts; panthenol, 1 part; dipotassium glycyrrhizinate, 1 part

[0092] The preparation method of the skin repair fluid provided by this comparative example includes: Weigh and mix sodium hyaluronate, panthenol, ceramide, niacinamide, polypeptide complex and purified water according to the mass fraction, perform a homogenization operation on the mixed base solvent using an APV-2000 homogenizer, the operating pressure is 800 bar, the circulation is 3 times, the treatment temperature is controlled at 4°C, and then perform aseptic filtration through a 0.22-μm polyethersulfone membrane to obtain the skin repair fluid.

[0093] Comparative Example 2

[0094] This embodiment provides a skin repair fluid and its preparation method.

[0095] The skin repair fluid containing autologous adipose stem cells provided by this embodiment includes adipose stem cell nano-liposomes and a base solvent.

[0096] Among them, the base solvent includes the following components by mass fraction: sodium hyaluronate: 1 part; panthenol: 2 parts; ceramide: 0.5 part; niacinamide: 0.1 part; polypeptide complex: 1 part; purified water: 88 parts.

[0097] Among them, the polypeptide complex includes by mass fraction: oligopeptide-3: 5 parts; acetyl hexapeptide-8: 10 parts; hexapeptide-10: 15 parts; copper tripeptide-1 glycine carboxymethyl ester: 10 parts; palmitoyl tetrapeptide-7: 5 parts; glycine: 10 parts; propylene glycol: 8 parts; hydroxyethyl cellulose: 5 parts; polyethylene glycol-400: 3 parts; panthenol: 1 part; dipotassium glycyrrhizinate: 1 part.

[0098] The preparation method of the skin repair fluid provided by this embodiment includes the following steps:

[0099] Step 1: Prepare the primary active factor complex

[0100] S1: Autologous fat tissue acquisition and treatment: 10 parts of autologous fat tissue of the patient are obtained by minimally invasive liposuction, washed 3 times with 0.9% NaCl solution at 37°C, shaken and mixed evenly for 60 seconds each time, and the supernatant is discarded after centrifuging at 300g for 5 minutes.

[0101] S2: Stem cell isolation and purification: The treated fat tissue is treated with a pH 7.2 DPBS digestive solution containing 0.1% type I collagenase, 0.05% neutral protease and 2 mM CaCl2 at 37°C for 40 minutes to obtain a basic cell suspension; it is overlaid on an equal volume of Ficoll-Paque PREMIUM at a ratio of 1:1 and centrifuged at 1000 rpm for 10 minutes to separate the primary population of mesenchymal stem cells; highly purified adipose stem cells are obtained by screening with CD73+, CD90+, CD105+ and CD45-, CD34- markers.

[0102] S3: Three-dimensional microsphere culture of stem cells: The purified adipose stem cells are inoculated in an ultra-low attachment 24-well culture plate at a density of 2×10 5 cells / mL and cultured in a serum-free medium containing 5 parts of autologous serum to form stem cell microspheres with a diameter of 100 μm.

[0103] S4: Stress pre-treatment of stem cell microspheres: The stem cell microspheres are cultured in an environment with an oxygen concentration of 1% for 12 hours, heated to 40°C for 15 minutes every 6 hours, and a 5% cyclic stretch is applied using an FX-5000T stretching system at a frequency of 0.5 Hz and a sine waveform for 6 hours.

[0104] S5: Active factor extraction and concentration: The pretreated stem cell microspheres are cultured for another 48 hours, and the culture supernatant is collected; the supernatant is concentrated using polyethersulfone membranes with a molecular weight cut-off of 5 kDa and 100 kDa, with an operating pressure of 15 psi and an operating flow rate of 50 ml / minute, retaining the active components with a molecular weight of 5 - 100 kDa; subsequently, it is frozen at -40 °C for 120 minutes, dried at -20 °C and 0.1 mbar for 24 hours, and dried at 10 °C and 0.01 mbar for 12 hours to obtain the primary active factor complex.

[0105] Step 2: Prepare the base solvent

[0106] Mix sodium hyaluronate, panthenol, ceramide, niacinamide, polypeptide complex and purified water evenly.

[0107] Step 3: Directly add the active factor and mix

[0108] Directly add the primary active factor complex prepared in Step 1 to the base solvent prepared in Step 2, and stir well to mix evenly.

[0109] Step 4: Homogenization and sterile filtration

[0110] The mixture is circulated 3 times using an APV - 2000 homogenizer at a pressure of 800 bar, with the temperature controlled at 4 °C, and then sterile filtered through a 0.22 μm polyethersulfone membrane to obtain the skin repair liquid.

[0111] Performance detection

[0112] For the skin repair liquid samples in Example 1, Example 2, Example 3, Comparative Example 1 and Comparative Example 2, in vitro cell proliferation promotion rate detection, collagen synthesis promotion rate detection, DPPH free radical scavenging rate detection and transdermal absorption test are carried out as the test samples.

[0113] Cell proliferation promotion rate detection experiment: Human keratinocytes are seeded at 5×10 3 cells / well in a 96-well culture plate, cultured at 37 °C and 5% CO2 for 24 hours, 5 groups are set, the culture medium of each group is replaced with the culture medium containing the same concentration of different test samples, 3 - 5 replicate wells are set for each sample, cultured for 48 - 72 hours, 10 μl of CCK-8 solution is added to each well, and cultured for another 2 hours, the absorbance at 450 nm is detected, and the cell proliferation promotion rate is calculated.

[0114] Cell proliferation promotion rate (%) = [(OD value of experimental group - OD value of control group) / OD value of control group] × 100%.

[0115] Collagen synthesis promotion rate detection experiment: Five groups were set up. In the experimental groups, the skin repair liquids of Examples 1-3 were added respectively, and in the control groups, the products of Comparative Examples 1-2 were added respectively. Each group was set with 3 replicates. The samples of each group were added to the human fibroblast culture system and incubated for 72 hours. The content of collagen in the culture medium was quantitatively detected by ELISA method, and the collagen synthesis promotion rate was calculated.

[0116] Collagen synthesis promotion rate (%) = [(Collagen content in experimental group - Collagen content in control group) / Collagen content in control group] × 100%.

[0117] DPPH free radical scavenging rate detection experiment: Prepare 0.1 - 0.2 mM DPPH solution with absolute ethanol and store it in the dark. Dilute the samples to be tested with ethanol at different concentration gradients.

[0118] Experimental group: Mix 2 ml of sample solution with 2 ml of DPPH solution;

[0119] Control group: Mix 2 ml of absolute ethanol with 2 ml of DPPH solution;

[0120] Blank group: Mix 2 ml of sample solution with 2 ml of absolute ethanol.

[0121] Keep the mixed solution in the dark and react at a constant temperature of 25°C for 30 minutes. Measure the absorbance of each group at a wavelength of 517 nm using a UV-visible spectrophotometer.

[0122] DPPH free radical scavenging rate (%) = [1 - (A_experiment - A_blank) / A_control] × 100%, where A_experiment is the absorbance of the experimental group, A_blank is the absorbance of the blank group, and A_control is the absorbance of the control group.

[0123] Transdermal absorption test: Collect fresh pig skin, remove subcutaneous adipose tissue, and use an electric skin slicer to cut the skin into thin slices with a thickness of 500 μm. Fix the treated skin samples between the upper and lower parts of the Franz diffusion cell, with the stratum corneum facing up and the dermis facing down. The receiving chamber is filled with 6 - 8 ml of PBS buffer solution. Keep the whole device at 32°C. Apply 200 μl of the test sample to the dosing chamber of each diffusion cell. Each sample is set with 3 - 6 replicates. Collect samples from the receiving chamber every 6 hours. Immediately replenish an equal amount of fresh PBS after each sampling to keep the volume constant. Analyze the concentration of the active ingredient in the sample by HPLC. After 24 hours, disassemble the Franz diffusion cell, take out the skin sample, wash the skin surface with PBS to remove the residual sample, use a skin separator to separate the epidermis and dermis, weigh the epidermis and dermis tissues respectively, homogenize the tissues, extract the active ingredient, and quantitatively analyze the content of the active ingredient in the epidermis and dermis by HPLC to calculate the 24-hour cumulative permeation amount (μg / cm2 ) Epidermal retention amount (μg / g tissue) and dermal retention amount (μg / g tissue).

[0124] Test data

[0125] Table 1 Data table of in vitro cell proliferation promotion rate

[0126]

[0127]

[0128] Table 2 Data table of collagen synthesis promotion rate

[0129] Sample Promotion rate of collagen synthesis (%) Example 1 42.6±3.5 Example 2 45.8±3.7 Example 3 53.2±4.0 Comparative Example 1 26.3±2.8 Comparative Example 2 38.5±3.4

[0130] Table 3 Data table of DPPH free radical scavenging rate

[0131] Sample DPPH radical scavenging rate (%) Example 1 68.4±5.3 Example 2 65.7±4.9 Example 3 74.3±5.5 Comparative Example 1 43.2±4.0 Comparative Example 2 52.6±4.5

[0132] Table 4 Data record table of transdermal absorption test

[0133]

[0134] Data analysis

[0135] It can be seen from Table 1, Table 2 and Table 3 that Examples 3, 2 and 1 all show high activities in aspects such as cell proliferation promotion, collagen synthesis promotion and antioxidant activity; although Comparative Example 2 contains autologous adipose stem cell components, due to its poor stability, its biological activity is lower than that of the example group; the bioavailability of the active ingredient in Comparative Example 1 is relatively low, indicating that the nano-liposome carrier can increase the bioavailability of the active ingredient.

[0136] It can be seen from Table 4 that the transdermal absorption performance of Examples 1-3 is significantly better than that of Comparative Examples 1-2, indicating that the nano-liposome carrier system of the present invention can effectively promote the penetration of the active ingredient through the skin barrier.

[0137] Clinical application detection

[0138] Twenty patients with open wounds were selected. The wound area of each patient was divided into two parts. One part was treated with the skin repair liquid of the present invention, and the other part was treated with a commercially available wound repair product as a control; the drug was administered twice a day for 14 days. The results showed that the healing speed of the wound area treated with the repair liquid of the present invention increased by an average of 42.3%, the complete wound closure time was shortened by 35.8%, the scar formation was reduced by 58.6%, and the pain of the patient's wound was reduced by 46.5%. The analysis showed that the epidermal regeneration in the area treated with the repair liquid was more complete, the arrangement of collagen fibers in the dermis was more orderly, the angiogenesis was more abundant, and the inflammatory reaction was less.

[0139] Thirty volunteers with facial photoaging were selected and randomly divided into three groups: Group A used the repair fluid of the present invention, Group B used a commercially available anti-aging essence, and Group C used a basic moisturizer. They were used twice a day for 8 weeks. The results showed that the skin moisture content of the subjects in Group A using the repair fluid of the present invention increased by 38.4%, the elasticity increased by 32.6%, the wrinkle depth decreased by 27.5%, and the pigmentation was reduced by 41.2%. Skin biopsy analysis showed that the collagen density in the dermis of the subjects in Group A increased by 36.8%, the elastic fiber network was improved by 43.5%, and the epidermal thickness increased by 12.4%.

[0140] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. All equivalent changes and improvements made in accordance with the scope of the present invention application shall still fall within the scope covered by the patent of the present invention.

Claims

1. A skin repair liquid containing autologous adipose stem cells, characterized in that, The skin repair liquid comprises adipose stem cell nanoliposomes and a base solvent; The base solvent contains 1-3 parts of sodium hyaluronate, 2-5 parts of panthenol, 0.5-1 part of ceramide, 0.1-0.3 part of niacinamide, 1-2 parts of polypeptide complex and 88-98 parts of purified water; The polypeptide complex comprises the following components by mass fraction: 5-10 parts of oligopeptide-3, 10-15 parts of acetyl hexapeptide-8, 15-20 parts of hexapeptide-10, 10-15 parts of copper tripeptide-1 glycine carboxymethyl ester, 5-10 parts of palmitoyl tetrapeptide-7, 10-15 parts of glycine, 8-12 parts of propylene glycol, 5-8 parts of hydroxyethyl cellulose, 3-8 parts of polyethylene glycol-400, 2-6 parts of betaine, 1-3 parts of panthenol and 1-3 parts of dipotassium glycyrrhizinate.

2. The skin repair liquid containing autologous adipose stem cells according to claim 1, wherein The adipose stem cell nanoliposomes are prepared by the following steps: S1: Acquisition and treatment of autologous adipose tissue: 10-50 parts of the patient's autologous adipose tissue are obtained by minimally invasive liposuction or surgical excision, washed 3-5 times with 0.9% NaCl solution at 37°C, shaken and mixed evenly for 60-90 seconds each time, and centrifuged at 300-500 g for 5 minutes to precipitate the tissue, and then the supernatant is discarded; S2: Separation and purification of stem cells: The treated adipose tissue is treated with a DPBS digestive solution with a pH of 7.2-7.4 containing 0.1%-0.3% type I collagenase, 0.05%-0.1% neutral protease and 2-5 mM CaCl2 at 37°C for 40-90 minutes to obtain a basic cell suspension; The cell suspension is superimposed on an equal volume of Ficoll-Paque PREMIUM at a ratio of 1:1 and centrifuged at 1000-1200 rpm for 10 minutes to separate the primary population of mesenchymal stem cells; Through immunomagnetic bead sorting technology, high-purity adipose stem cells are screened using a combination of CD73+, CD90+, CD105+ and CD45-, CD34- markers; S3: Three-dimensional microsphere culture of stem cells: The purified adipose stem cells are inoculated into an ultra-low attachment 24-well culture plate at a density of 2-5×10 5 cells / mL, and cultured in a serum-free medium containing 5-10 parts of autologous serum to form stem cell microspheres with a diameter of 100-300 μm; S4: Stress pretreatment of stem cell microspheres: The stem cell microspheres are cultured in an environment with an oxygen concentration of 1%-5% for 12-24 hours, during which the temperature is raised to 40-42°C every 6 hours for 15 minutes, and a 5%-10% cyclic stretch is applied using an FX-5000T stretching system, with a frequency of 0.5 Hz and a sine waveform, and treated for 6-12 hours; S5: Extraction and concentration of active factors: The pretreated stem cell microspheres are continuously cultured for 48-72 hours, and the culture supernatant is collected; Using polyethersulfone membranes with a molecular weight cut-off of 5 kDa and 100 kDa, the supernatant is concentrated, the operating pressure is 15-20 psi, the flow rate is 50-60 ml / minute, and the active ingredients with a molecular weight between 5-100 kDa are retained; Subsequently, it is frozen at -40°C for 120 minutes, dried at -20°C and 0.1 mbar for 24 hours, and dried at 10°C and 0.01 mbar for 12 hours to obtain a primary active factor complex; S6: Nanoliposome encapsulation and activity protection: Mix the primary active factor complex with 45 parts of phosphatidylcholine, 40 parts of cholesterol, 10 parts of vitamin E, and 5 parts of graphene oxide, dissolve them in a mixed solvent of 100 parts of chloroform and methanol with a volume ratio of 2:1, and use a rotary evaporator to rotate and evaporate at a speed of 120 - 150 revolutions per minute at 40 °C to form a lipid film. After vacuum drying for 2 hours, add PBS with a pH of 7.4 containing the active factor complex to hydrate for 60 minutes, and perform ultrasonic treatment at 40 kHz for 20 minutes to obtain adipose stem cell nanoliposomes.

3. The skin repair liquid containing autologous adipose stem cells according to claim 2, characterized in that, In S3, the ultra-low adhesion 24-well culture plate contains chitosan-hyaluronic acid composite microcarriers.

4. The skin repair liquid containing autologous adipose stem cells according to claim 3, characterized in that, The preparation method of the chitosan-hyaluronic acid composite microcarriers is as follows: Mix 1 - 2 parts of a chitosan solution with a molecular weight of 100 - 200 kDa and 0.5 - 1 part of a sodium hyaluronate solution with a molecular weight of 1000 - 1500 kDa, add 0.1 - 0.5 parts of gelatin as a cross-linking agent, pump the mixed solution into a liquid paraffin fluid containing 2 parts of Span80 at a speed of 3 ml / hour through a syringe with an inner diameter of 200 μm, form uniform droplets in a T-shaped microfluidic chip with an inner diameter of 300 μm, collect them in 100 parts of PBS containing 0.5 parts of Tween 80, and stir at 200 rpm at 4 °C for 4 hours to obtain chitosan-hyaluronic acid composite microcarriers.

5. The skin repair liquid containing autologous adipose stem cells according to claim 2, characterized in that, In S3, the diameter of the chitosan-hyaluronic acid composite microcarriers is 50 - 150 μm.

6. The skin repair liquid containing autologous adipose stem cells according to claim 2, characterized in that, In S3, the serum-free medium formulation includes: DMEM / F12 basal medium, 10-15 ng / mL epidermal growth factor EGF, 5-10 ng / mL basic fibroblast growth factor bFGF, 2-5 ng / mL transforming growth factor β3, 10 -7 -10 -6 M dexamethasone, 0.5-1.0 mM / L ascorbic acid, 50-100 μg / mL ascorbic acid-2-phosphate, and 0.2-0.5 mM lipoic acid.

7. The skin repair liquid containing autologous adipose stem cells according to claim 2, characterized in that In S3, the surface of the ultra-low adhesion 24-well culture plate is coated with poly(2-hydroxyethyl methacrylate).

8. A preparation method of the skin repair liquid according to claim 1, characterized in that, The preparation method of the skin repair solution includes the following steps: Mix the nanoliposomes with a base solvent, perform homogenization operation, and then sterile filter through a 0.22 μm polyethersulfone membrane to obtain the skin repair solution.

9. The preparation method of the skin repair liquid according to claim 8, characterized in that, The homogenization operation uses an APV-2000 homogenizer, the operation pressure is 800 - 1200 bar, and it circulates 3 - 5 times, and the treatment temperature is controlled at 4 - 8 °C.

10. The skin repair liquid according to claim 1, characterized in that, The average particle size of the repair solution is 80 - 150 nm, the Zeta potential is -35 to -20 mV, the pH value is 5.5 - 6.5, and the osmotic pressure is 280 - 320 mOsm / kg.

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