Stem cell preparation for skin repair and use thereof

Abstract

This invention provides a stem cell preparation for skin repair and its application, belonging to the field of biomedical technology. The stem cell preparation is obtained by inducing stem cells in a composite culture medium containing sodium butyrate, L-carnosine, and quercetin, followed by short-term pulsed hypoxia treatment, ultrasonic disruption, deep freeze-thaw rehydration, and vacuum freeze-drying to obtain a lyophilized stem cell powder. Experimental results show that the stem cell preparation of this invention significantly promotes the proliferation of human skin fibroblasts and significantly promotes the migration of human keratinocytes. In a mouse full-thickness skin defect model, local application after reconstitution in PBS buffer containing 0.1% sodium hyaluronate (concentration ≥100 μg / mL) significantly accelerates wound closure. In summary, the preparation of this invention exhibits high activity, strong stability, and broad prospects for clinical translation.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a stem cell preparation for skin repair and its application. Background Technology

[0002] As the largest organ in the human body, the skin covers the entire surface of the body. It is not only the first physical barrier against external environmental damage, but also undertakes multiple physiological functions such as thermoregulation, sensory conduction, immune defense, endocrine metabolism, and aesthetic appearance. However, skin tissue is extremely susceptible to various injuries in daily life and clinical practice, including trauma, burns, ulcers, radiation damage, diabetic foot, pressure sores, and autoimmune diseases. These injuries can lead to the destruction of skin integrity, causing serious consequences such as infection, fluid loss, scar formation, and even endangering life.

[0003] In recent years, with the rapid development of regenerative medicine, stem cell technology has provided a brand-new treatment approach for skin repair. Stem cells are a type of primitive cell with self-renewal capacity and multi-directional differentiation potential. They can differentiate into various skin cell types and release bioactive substances such as growth factors, cytokines, and exosomes through paracrine mechanisms, thereby regulating the local microenvironment, promoting angiogenesis, inhibiting inflammatory responses, and stimulating the proliferation and migration of endogenous cells.

[0004] Despite the immense potential of stem cells in skin repair, existing stem cell formulations still face numerous challenges in clinical application: low cell survival rates, difficulties in post-transplantation colonization, uncontrollable release of active factors, poor formulation stability, and a lack of standardized preparation procedures all hinder their clinical translation. Therefore, developing a standardized stem cell formulation with high cell activity, stable function, and ease of use is of great significance for promoting the clinical adoption of stem cell therapy. Summary of the Invention

[0005] The purpose of this invention is to provide a stem cell preparation for skin repair and its application, thereby significantly improving the rapid repair effect of stem cell freeze-dried powder on skin damage through the synergistic effect of specific inducing components and short-term pulsed hypoxia treatment.

[0006] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for preparing freeze-dried stem cell powder for skin repair, comprising the following steps: (1) Induction culture Stem cells were seeded into an induction culture medium for induction pretreatment; the induction culture medium was based on a serum-free basal medium and supplemented with a compound induction component containing sodium butyrate, L-carnosine and quercetin. (2) Pulsed hypoxia treatment The pretreated stem cells were replaced with serum-free basal culture medium and subjected to short-term pulsed hypoxia treatment. The short-term pulsed hypoxia treatment was performed by placing the cells in a hypoxic environment for a time T1, and then restoring them to a normoxic environment for a time T2. This was repeated several times as one cycle. (3) Acquisition of all components Collect the processed stem cells and culture supernatant, break the stem cells by physical means, remove cell debris by centrifugation, and collect the mixed supernatant containing intracellular active factors and extracellular secretory components. (4) Preparation of freeze-dried stem cell powder The mixed supernatant was subjected to vacuum freeze-drying to obtain stem cell freeze-dried powder.

[0007] Preferably, the stem cells are human bone marrow mesenchymal stem cells.

[0008] Preferably, the final concentrations of the composite inducing components in the induction medium are: sodium butyrate 0.5-0.75 mM, L-carnosine 8-10 mM, and quercetin 7.5 μM.

[0009] Preferably, the induction pretreatment time is 12 hours.

[0010] Preferably, the specific parameters of the pulsed hypoxia treatment are as follows: the O2 concentration in the hypoxic environment is 1-3%, and the maintenance time T1 is 3 hours; the O2 concentration in the normoxic environment is 21%, and the maintenance time T2 is 21 hours; the treatment is carried out continuously for 3 days with 24 hours as one cycle.

[0011] Preferably, the method for physically disrupting stem cells is as follows: The mixed suspension was placed in an ice bath for ultrasonic disruption, followed by multiple deep freeze-thaw cycles; the ultrasonic disruption parameters were 200W, 3s operation, 5s interval, and 10 cycles. The deep-freezing temperature is -80°C, and the thawing temperature is 37°C; The deep freeze-thaw cycle is repeated 3 times.

[0012] Secondly, the present invention provides a stem cell preparation prepared by the above-described preparation method, characterized in that the stem cell preparation has a significant proliferative effect on human skin fibroblasts and a significant migration-promoting effect on human keratinocytes.

[0013] Thirdly, the present invention provides the application of the above-mentioned stem cell preparation in the preparation of drugs for rapid repair of skin wounds.

[0014] Preferably, in the drug, the stem cell preparation is a lyophilized stem cell powder, which is reconstituted in PBS buffer containing hyaluronic acid to prepare a liquid drug solution with a concentration ≥100μg / mL.

[0015] Fourthly, the present invention provides a drug for rapid repair of skin wounds, the drug being composed of the above-mentioned stem cell preparation and a liquid solvent, wherein the stem cell preparation is a lyophilized stem cell powder, and the liquid solvent is a PBS buffer containing 0.1% sodium hyaluronate. In the drug, the concentration of the lyophilized stem cell powder is ≥100μg / mL.

[0016] The beneficial effects of this invention are as follows: First, the preparation method of this invention has an excellent synergistic effect. This invention innovatively combines the chemical induction of "sodium butyrate + L-carnosine + quercetin" with the physical stress of "short-term pulsed hypoxia". Experimental data shows that Example 1 is significantly better than each single-factor treatment group in terms of promoting proliferation and migration. This non-linear superposition effect proves that there is a very strong synergistic effect between the process parameters.

[0017] Secondly, the formulation provided by this invention possesses high stability and advantages in clinical translation. This invention employs serum-free culture combined with vacuum freeze-drying technology and adds specific protective agents to ensure the purity and long-term stability of the formulation. Furthermore, it allows for convenient reconstitution and precise concentration control, providing practical technical support for the clinical standardization of stem cell therapy for skin lesions. Attached Figure Description

[0018] Figure 1 This is a comparative bar chart showing the effect of freeze-dried stem cell powder obtained in the embodiments and comparative examples of the present invention on the relative proliferation rate of human skin fibroblasts (HSF); Figure 2 This is a comparative bar chart showing the effect of freeze-dried stem cell powder obtained in the embodiments and comparative examples of the present invention on the scratch healing rate (24h) of human keratinocytes (HaCaT). Figure 3 The figures (a) and (b) show the results of wound closure rate on the 7th day after surgery in Example 1 and the core comparative example of the present invention in a mouse full-thickness skin defect model. Detailed Implementation

[0019] Example 1 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Prepare induction culture medium Add the following compound to serum-free DMEM / F12 basal medium: Sodium butyrate (CAS No.: 156-54-7): Final concentration 0.75mM; L-Carnosine (CAS No.: 305-84-0): Final concentration 8mM; Quercetin (CAS No.: 117-39-5): Final concentration 7.5 μM; The induction medium was obtained.

[0020] 3. Short-term pulse hypoxia treatment After 24 hours of incubation, the medium was replaced with induction medium and treated for 12 hours. Remove the induction medium and replace it with serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator, adjust the O2 concentration to 2%, and maintain for 3 hours; Adjust the O2 concentration back to 21% and maintain it for 21 hours. Repeat the above short-term pulse hypoxia treatment of 3 hours of low oxygen + 21 hours of normoxic oxygen for 3 consecutive days. 4. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0021] Example 2 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Prepare induction culture medium Add the following compound to serum-free DMEM / F12 basal medium: Sodium butyrate (CAS No.: 156-54-7): Final concentration 0.5mM; L-Carnosine (CAS No.: 305-84-0): Final concentration 10mM; Quercetin (CAS No.: 117-39-5): Final concentration 7.5 μM; The induction medium was obtained.

[0022] 3. Short-term pulse hypoxia treatment After 24 hours of incubation, the medium was replaced with induction medium and treated for 12 hours. Remove the induction medium and replace it with serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator, adjust the O2 concentration to 3%, and maintain for 3 hours; Adjust the O2 concentration back to 21% and maintain it for 21 hours. Repeat the above short-term pulse hypoxia treatment of 3 hours of low oxygen + 21 hours of normoxic oxygen for 3 consecutive days. 4. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0023] Example 3 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Prepare induction culture medium Add the following compound to serum-free DMEM / F12 basal medium: Sodium butyrate (CAS No.: 156-54-7): Final concentration 0.5mM; L-Carnosine (CAS No.: 305-84-0): Final concentration 10mM; Quercetin (CAS No.: 117-39-5): Final concentration 7.5 μM; The induction medium was obtained.

[0024] 3. Short-term pulse hypoxia treatment After 24 hours of incubation, the medium was replaced with induction medium and treated for 12 hours. Remove the induction medium and replace it with serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator, adjust the O2 concentration to 1%, and maintain for 3 hours; Adjust the O2 concentration back to 21% and maintain it for 21 hours. Repeat the above short-term pulse hypoxia treatment of 3 hours of low oxygen + 21 hours of normoxic oxygen for 3 consecutive days. 4. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0025] Comparative Example 1 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Routine treatment After 24 hours of incubation, the cells were replaced with serum-free DMEM / F12 and treated for another 12 hours. Remove the culture medium and replace it with new serum-free DMEM / F12 basal medium, and culture for 3 days; 3. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0026] Comparative Example 2 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Prepare induction culture medium Add the following compound to serum-free DMEM / F12 basal medium: Sodium butyrate (CAS No.: 156-54-7): Final concentration 0.75mM; L-Carnosine (CAS No.: 305-84-0): Final concentration 8mM; Quercetin (CAS No.: 117-39-5): Final concentration 7.5 μM; The induction medium was obtained.

[0027] 3. Normal oxygen treatment After 24 hours of incubation, the medium was replaced with induction medium and treated for 12 hours. Remove the induction medium and replace it with serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator and incubate continuously for 3 days under 21% O2 conditions; 4. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0028] Comparative Example 3 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Short-term pulse hypoxia treatment After culturing for 24 hours, the culture medium was replaced with serum-free DMEM / F12 basal medium and treated for 12 hours. Remove the culture medium and replace it with new serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator, adjust the O2 concentration to 2%, and maintain for 3 hours; Adjust the O2 concentration back to 21% and maintain it for 21 hours. Repeat the above short-term pulse hypoxia treatment of 3 hours of low oxygen + 21 hours of normoxic oxygen for 3 consecutive days. 3. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0029] Comparative Example 4 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Prepare induction culture medium Add the following compound to serum-free DMEM / F12 basal medium: Sodium butyrate (CAS No.: 156-54-7): Final concentration 0.75mM; L-Carnosine (CAS No.: 305-84-0): Final concentration 8mM; Quercetin (CAS No.: 117-39-5): Final concentration 7.5 μM; The induction medium was obtained.

[0030] 3. Conventional hypoxia treatment After 24 hours of incubation, the medium was replaced with induction medium and treated for 12 hours. Remove the induction medium and replace it with serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator, adjust the O2 concentration to 2%, and maintain for 9 hours; Adjust the O2 concentration back to 21% and continue culturing for 63 hours; 4. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0031] Comparative Example 5 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Prepare induction culture medium Add the following compound to serum-free DMEM / F12 basal medium: Sodium butyrate (CAS No.: 156-54-7): Final concentration 0.75mM; L-Carnosine (CAS No.: 305-84-0): Final concentration 8 mM; The induction medium was obtained.

[0032] 3. Short-term pulse hypoxia treatment After 24 hours of incubation, the medium was replaced with induction medium and treated for 12 hours. Remove the induction medium and replace it with serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator, adjust the O2 concentration to 2%, and maintain for 3 hours; Adjust the O2 concentration back to 21% and maintain it for 21 hours. Repeat the above short-term pulse hypoxia treatment of 3 hours of low oxygen + 21 hours of normoxic oxygen for 3 consecutive days. 4. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0033] Comparative Example 6 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Prepare induction culture medium Add the following compound to serum-free DMEM / F12 basal medium: Sodium butyrate (CAS No.: 156-54-7): Final concentration 0.75mM; Quercetin (CAS No.: 117-39-5): Final concentration 7.5 μM; The induction medium was obtained.

[0034] 3. Short-term pulse hypoxia treatment After 24 hours of incubation, the medium was replaced with induction medium and treated for 12 hours. Remove the induction medium and replace it with serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator, adjust the O2 concentration to 2%, and maintain for 3 hours; Adjust the O2 concentration back to 21% and maintain it for 21 hours. Repeat the above short-term pulse hypoxia treatment of 3 hours of low oxygen + 21 hours of normoxic oxygen for 3 consecutive days. 4. Acquisition of culture components The cells were scraped off using a cell scraper and suspended in the culture supernatant to obtain a mixed suspension; The mixed suspension was placed in an ice bath and treated with an ultrasonic disruptor at 200W for 3 seconds, followed by a 5-second interval, for 10 cycles to obtain a disrupted suspension. The broken suspension was rapidly frozen at -80°C and then rapidly thawed at 37°C. This process was repeated three times to obtain the reconstituted suspension. Centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris and obtain the supernatant; Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0035] Comparative Example 7 1. Cell preparation: Human bone marrow mesenchymal stem cells (BMSCs) passaged to passage P5 and in the logarithmic growth phase were seeded in 10 mL of cell suspension into T75 culture flasks at a density of 5 × 10⁶ cells / mL. 4 cells / mL; 2. Prepare induction culture medium Add the following compound to serum-free DMEM / F12 basal medium: Sodium butyrate (CAS No.: 156-54-7): Final concentration 0.75mM; L-Carnosine (CAS No.: 305-84-0): Final concentration 8 mM; Quercetin (CAS No.: 117-39-5): Final concentration 7.5 μM; The induction medium was obtained.

[0036] 3. Short-term pulse hypoxia treatment After 24 hours of incubation, the medium was replaced with induction medium and treated for 12 hours. Remove the induction medium and replace it with serum-free DMEM / F12 basal medium; Place the T75 culture flask in a three-gas incubator, adjust the O2 concentration to 2%, and maintain for 3 hours; Adjust the O2 concentration back to 21% and maintain it for 21 hours. Repeat the above short-term pulse hypoxia treatment of 3 hours of low oxygen + 21 hours of normoxic oxygen for 3 consecutive days. 4. Acquisition of culture components Collect the culture supernatant, centrifuge at 12,000 rpm, 4°C for 20 minutes to remove large cell debris, and obtain the supernatant. Add 5% mannitol as a preservative and perform vacuum freeze-drying to obtain freeze-dried stem cell powder.

[0037] Example 4 1. Cell resuscitation: Remove the HSF (human skin fibroblast) cell cryopreservation tube from liquid nitrogen and quickly place it in a 37°C water bath; After melting, transfer to a biosafety cabinet, wipe the tube opening with 75% alcohol, transfer to a centrifuge tube, add pre-warmed DMEM complete culture medium, and centrifuge to collect cells; 2. Cell expansion: Cells were seeded in T75 culture flasks at a density of 5 × 10⁶ cells / year. 5 Add 10 mL of complete culture medium to each vial; Place in a cell culture incubator and culture to P5 generation by passage every 2-3 days; 3. Cell treatment: After digestion, the cells were seeded into 96-well cell culture plates at a density of 5 × 10⁶ cells / well.3 Each sample is filled with 100 μL of ... Remove the 96-well plate, use a pipette to remove the old culture medium, wash with PBS, and add the sample working solution: Control group: DMEM complete culture medium was added; Positive control group: DMEM complete medium containing VEGF 10 ng / mL + EGF 10 ng / mL was added; Example 1 group: DMEM complete culture medium containing 100 μg / mL of the lyophilized powder prepared in Example 1 was added; Example 2 group: DMEM complete culture medium containing 100 μg / mL of the lyophilized powder prepared in Example 2 was added; Example 3 group: DMEM complete culture medium containing 100 μg / mL of the lyophilized powder prepared in Example 3 was added; Comparative Example 1: DMEM complete medium containing 100 μg / mL of the lyophilized powder prepared in Comparative Example 1 was added. Comparative Example 2: DMEM complete medium containing 100 μg / mL of the lyophilized powder prepared in Comparative Example 2 was added. Comparative Example 3: DMEM complete medium containing 100 μg / mL of the lyophilized powder prepared in Comparative Example 3 was added. Comparative Example 4: DMEM complete medium containing 100 μg / mL of the lyophilized powder prepared in Comparative Example 4 was added. Comparative Example 5: DMEM complete medium containing 100 μg / mL of the lyophilized powder prepared in Comparative Example 5 was added. Comparative Example 6: DMEM complete medium containing 100 μg / mL of the lyophilized powder prepared in Comparative Example 6 was added. Comparative Example 7: DMEM complete medium containing 100 μg / mL of the lyophilized powder prepared in Comparative Example 7 was added. All culture media were preheated at 37°C; 4. Experimental Testing: Prepare the CCK-8 working solution according to the ratio of CCK-8 reagent to serum-free culture medium of 1:10; Remove the 96-well plate, discard the old culture medium, and add 100 μL of CCK-8 working solution to each well; After incubation in the dark for 2.5 hours, the absorbance at 450 nm was measured, and the relative proliferation rate was calculated. The results are as follows: Figure 1 As shown.

[0038] The relative proliferation rate of the positive control group was 178.25±11.31%, the relative proliferation rate of the Example 1 group was 232.59±13.96%, the relative proliferation rate of the Example 2 group was 199.94±15.64%, and the relative proliferation rate of the Example 3 group was 215.72±12.71%. The relative proliferation rates of Comparative Example 1 were 111.15±2.62%, Comparative Example 2 were 135.67±5.56%, Comparative Example 3 were 146.08±4.62%, Comparative Example 4 were 172.26±8.24%, Comparative Example 5 were 167.58±7.31%, Comparative Example 6 were 162.45±8.49%, and Comparative Example 7 were 147.34±8.32%.

[0039] The results above show that the control group (Comparative Example 1, untreated BMSCs lysate) exhibited basic proliferative activity (111.15±2.62%), which may be related to the growth factors secreted by the BMSCs themselves. The positive control group (recombinant VEGF+EGF) showed a relative proliferation rate of 178.25±11.31%, demonstrating a significant proliferative effect.

[0040] Surprisingly, the proliferation-promoting effects of Examples 1-3 of this invention were significantly better than those of the positive control. Example 1 showed a proliferation rate of 232.59 ± 13.96%, an increase of approximately 30.5% compared to the positive control (P < 0.001) and an increase of 109.3% compared to the control group (P < 0.001). This result indicates that the proliferation-promoting activity of BMSCs was significantly enhanced after specific induction and pulsed hypoxia treatment, and the effect surpassed that of the recombinant growth factor combination.

[0041] The results of the comparative studies showed that the effects of simple induction (Comparative Study 2, 135.67%) or simple hypoxia (Comparative Study 3, 146.08%) were significantly lower than those of the examples; although a single long-term hypoxia treatment (Comparative Study 4, 172.26%) was close to the positive control, it was still significantly lower than the pulsed treatment (P<0.05). The effects of the default single component (Comparative Studies 5-6, 162-168%) or using only the supernatant (Comparative Study 7, 147.34%) were significantly reduced, demonstrating the necessity of the three-component synergistic and cell lysis process used in this invention.

[0042] Example 5 1. Cell resuscitation: Remove the HaCaT (human immortalized keratinocyte) cell cryopreservation tube from liquid nitrogen and quickly place it in a 37°C water bath; After melting, transfer to a biosafety cabinet, wipe the tube opening with 75% alcohol, transfer to a centrifuge tube, add pre-warmed DMEM complete culture medium, and centrifuge to collect cells; 2. Cell expansion: The cells were seeded in T75 culture flasks at a density of 5 × 10⁵ cells / flask, and 10 mL of complete culture medium was added. Place in a cell culture incubator and culture to P5 generation by passage every 2-3 days; 3. Cell treatment: After digestion, the cells were seeded into 6-well cell culture plates at a density of 2 × 10⁶ cells / well. 5 Cells per well, 2 mL in volume, mixed using the cross-hatching method, and then placed in a cell culture incubator (37℃, 5% CO2) for static culture until the cell monolayer confluence reaches more than 90%. Remove the 6-well plate, discard the old culture medium, and gently wash the cells once with pre-warmed PBS; Take out the mitomycin C stock solution at -20℃ and dilute it to a final concentration of 10 μg / mL using pre-warmed serum-free DMEM; Add 2 mL of culture medium containing 10 μg / mL mitomycin C to each well, place in a cell culture incubator, and incubate in the dark for 2 hours; After incubation, discard the culture medium containing mitomycin C and gently wash the cells three times with pre-warmed PBS. Gently scrub the bottom of the plate with a 200 μL pipette tip perpendicular to the plate, and wash twice with PBS to remove suspended cells and cell debris. Following the treatment method of Example 4, 2 mL of preheated DMEM complete culture medium was added to each treatment group; 4. Experimental Testing: Cells were photographed at 0 hours and 24 hours of culture, and then ImageJ was used to analyze the cell migration rate of different treatment groups. The results are as follows. Figure 2 As shown.

[0043] The cell migration rates were as follows: control group: 18.42±2.26%; positive control group: 52.37±4.39%; Example 1 group: 88.53±4.32%; Example 2 group: 71.12±3.45%; Example 3 group: 78.58±2.53%; Comparative Example 1 group: 23.43±1.55%; Comparative Example 2 group: 34.12±2.60%; Comparative Example 3 group: 36.67±2.75%; Comparative Example 4 group: 52.73±2.95%; Comparative Example 5 group: 48.85±3.79%; Comparative Example 6 group: 39.67±2.68%; Comparative Example 7 group: 55.08±2.85%.

[0044] The results showed that, compared with the positive control group, the cell migration ability of the Example 1-3 groups was significantly enhanced (P<0.001). Among them, the Example 1 group performed the best, with a cell migration rate of 88.53±4.32%, which was 69.1% higher than the positive control group and close to the level of complete healing, showing a strong ability to promote epithelialization.

[0045] Comparative Example 2 (induction + normoxic) and Comparative Example 3 (hypoxia without induction) showed significantly lower results than Example 1, demonstrating that a single factor is insufficient to achieve the optimal migration-promoting effect of Example 1.

[0046] The effects of Comparative Example 4 (single long-term hypoxia) were also significantly lower than those of Example 1, indicating that short-term pulsed hypoxia treatment is an indispensable treatment method for achieving synergistic induction and hypoxia.

[0047] The results of Comparative Examples 5 and 6 indicate that simultaneous treatment with sodium butyrate, carnosine, and quercetin is necessary during induction.

[0048] The results of Comparative Example 7 demonstrate that the intracellular active components in the cell lysate are indispensable for the multi-dimensional synergistic optimization of the lyophilized powder of the present invention.

[0049] Example 6 Stem cell preparations were prepared using the lyophilized stem cell powder from Example 1 as an example. (1) Transfer the freeze-dried stem cell powder stored at -80℃ to a 4℃ refrigerator and place it in the dark for 30 minutes; (2) Add 1 mg of lyophilized stem cell powder to 10 mL of PBS buffer containing 0.1% sodium hyaluronate, place on a horizontal shaker, and shake at 30 rpm until completely dissolved; (3) Let stand at 4℃ for 15 minutes to obtain stem cell preparation A.

[0050] Using the same method, stem cell preparation a containing the lyophilized powder of Comparative Example 1, stem cell preparation b containing the lyophilized powder of Comparative Example 2, and stem cell preparation c containing the lyophilized powder of Comparative Example 3 were prepared.

[0051] Example 7 (1) Healthy male BALB / c mice aged 6-8 weeks were acclimatized for 7 days at a temperature of 22±2℃ and a humidity of 50–60%, with a 12-hour light / dark cycle, and were allowed free access to standard feed and sterilized drinking water; (2) Weigh and record the mice before the experiment. The mice were randomly divided into 4 groups according to their weight and random number table method: Comparative Example 1, Comparative Example 2, Comparative Example 3, and Example 1, with 6 mice in each group. (3) Anesthetize mice with 1% sodium amobarbital, shave the hair on their backs, and use depilatory cream for fine hair removal. Disinfect the skin on their backs with 75% alcohol. Then, use a sterile punch with a diameter of 10 mm to remove the full-thickness skin on both sides of the spine on the back. Prepare two wounds for each mouse. (4) 50 μL of stem cell preparation a, stem cell preparation b, stem cell preparation c and stem cell preparation A were evenly applied to the center of the wound in Comparative Examples 1-3 and Example 1, respectively. (5) After administration, cover the mice with sterile breathable dressings and fix them with elastic bandages. Then, house the mice individually in a clean, dry, and warm environment. (6) On the 7th day after surgery, mice were anesthetized again with sodium amobarbital, the dressing was removed, and the skin around the wound was gently cleaned with physiological saline. The wound was photographed using a graduated ruler, and the wound area and wound closure rate were calculated using ImageJ software. The results are as follows: Figure 3 As shown.

[0052] exist Figure 3 In the comparison, the wound closure rate of Comparative Example 1 was 36.86±1.49%, the wound closure rate of Comparative Example 2 was 41.25±1.80%, the wound closure rate of Comparative Example 3 was 50.64±3.50%, and the wound closure rate of Example 1 was 73.95±3.04%.

[0053] The results show that the wound closure rate in Comparative Example 1 was slightly higher than the body's natural healing level, indicating that at a concentration of 100 μg / mL, some factors secreted by stem cells themselves have a slight promoting effect on wound repair. Comparative Example 2 showed an 11.9% increase compared to Comparative Example 1, demonstrating that induction treatment can enhance the secretory capacity of stem cells to some extent, but the effect is relatively limited. Comparative Example 3 showed a 37.4% increase compared to Comparative Example 1, indicating that the lyophilized stem cell powder obtained after hypoxia treatment has a stronger wound healing ability.

[0054] The wound closure rate of Example 1 was 100.6% higher than that of Comparative Example 1, 46.0% higher than that of Comparative Example 3, and 79.3% higher than that of Comparative Example 2. This effect is not only significantly better than that of each single-factor treatment group, but also exceeds the simple additive effect, proving that the stem cell preparation obtained by chemical induction and pulsed hypoxia in this invention has a synergistic effect.

Claims

1. A method for preparing a stem cell lyophilized powder for skin repair, characterized by, Includes the following steps: (1) Induction culture Stem cells were seeded into an induction culture medium for induction pretreatment; the induction culture medium was based on a serum-free basal medium and supplemented with a compound induction component containing sodium butyrate, L-carnosine and quercetin. (2) Pulsed hypoxia treatment The pretreated stem cells were replaced with serum-free basal culture medium and subjected to short-term pulsed hypoxia treatment. The short-term pulsed hypoxia treatment was performed by placing the cells in a hypoxic environment for a time T1, and then restoring them to a normoxic environment for a time T2. This was repeated several times as one cycle. (3) Acquisition of all components Collect the processed stem cells and culture supernatant, break the stem cells by physical means, remove cell debris by centrifugation, and collect the mixed supernatant containing intracellular active factors and extracellular secretory components. (4) Preparation of freeze-dried stem cell powder The mixed supernatant was subjected to vacuum freeze-drying to obtain stem cell freeze-dried powder.

2. The production method according to claim 1, characterized by, The stem cells mentioned are human bone marrow mesenchymal stem cells.

3. The method of claim 2, wherein, The final concentrations of the composite inducing components in the induction medium were: sodium butyrate 0.5-0.75 mM, L-carnosine 8-10 mM, and quercetin 7.5 μM.

4. The production method according to claim 3, characterized by, The induction pretreatment time is 12 hours.

5. The preparation method according to claim 4, characterized in that, The specific parameters of the pulsed hypoxia treatment are as follows: the O2 concentration in the hypoxic environment is 1-3%, and the maintenance time T1 is 3 hours; the O2 concentration in the normoxic environment is 21%, and the maintenance time T2 is 21 hours; the treatment is carried out continuously for 3 days with 24 hours as one cycle.

6. The preparation method according to claim 5, characterized in that, The method for physically disrupting stem cells is as follows: The mixed suspension was placed in an ice bath for ultrasonic disruption, followed by multiple deep freeze-thaw cycles; the ultrasonic disruption parameters were 200W, 3s operation, 5s interval, and 10 cycles. The deep-freezing temperature is -80°C, and the thawing temperature is 37°C; The deep freeze-thaw cycle is repeated 3 times.

7. A stem cell preparation obtained by the preparation method according to any one of claims 1-6, characterized in that, The stem cell preparation has a significant proliferative effect on human skin fibroblasts and a significant migration-promoting effect on human keratinocytes.

8. The use of the stem cell preparation as described in claim 7 in the preparation of a drug for rapid repair of skin wounds.

9. The application according to claim 8, characterized in that, In the drug, the stem cell preparation is a lyophilized stem cell powder, which is reconstituted in PBS buffer containing hyaluronic acid to prepare a liquid drug solution with a concentration ≥100μg / mL.

10. A medicine for rapid repair of skin wounds, characterized in that, The drug is composed of the stem cell preparation as described in claim 7 and a liquid solvent, wherein the stem cell preparation is a lyophilized stem cell powder and the liquid solvent is a PBS buffer containing 0.1% sodium hyaluronate. In the drug, the concentration of the lyophilized stem cell powder is ≥100μg / mL.

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