Preparation process and application of mesenchymal stem cell freeze-dried powder for preventing aging

Abstract

The invention belongs to the field of health care products and biological medicines, and particularly discloses a preparation process and application of mesenchymal stem cell freeze-dried powder for preventing aging. The process comprises the following steps: extracting a mixed cell component containing 60%-80% of mesenchymal stem cells, exosomes and growth factors from umbilical cord blood; a composite freeze-drying protective agent (4%-8% of trehalose, 1%-3% of taurine, 0.5%-2% of polysucrose 70, 0.1%-0.5% of chondroitin sulfate and 1%-3% of DMSO) is combined with a gradient freeze-drying technology (-70 DEG C pre-freezing and two-stage drying), and freeze-dried powder with the water content smaller than or equal to 2.5% and the survival rate larger than or equal to 85% is prepared. The freeze-dried powder can be prepared into oral dosage forms such as capsules, tablets and the like, and is combined with resveratrol to synergistically inhibit senescence phenotypes by activating an SIRT1 / AMPK pathway, so that the skin collagen content is increased by 104%, and the mitochondrial ATP yield is increased by 86%. The limitation of traditional stem cell purification and cryopreservation is broken through, the synergistic effect of the exosome is reserved, the stability problem of an oral preparation is solved, and an efficient and safe solution is provided for anti-aging treatment.

Description

Technical Field

[0001] The present invention belongs to the fields of health products and biomedicine, and specifically discloses a preparation process and application of freeze-dried mesenchymal stem cell powder for anti-aging prevention. Background Art

[0002] In recent years, mesenchymal stem cells (MSCs) have been widely explored for anti-aging treatment due to their multi-directional differentiation potential and paracrine function. In the prior art, typical application schemes include:

[0003] 1. Stem cell purification and cryopreservation: High-purity MSCs (>95%) are isolated from umbilical cord blood by density gradient centrifugation and cryopreserved in liquid nitrogen after programmed cooling. However, this technology has significant defects: First, excessive purification leads to the loss of active components such as naturally occurring growth factors (such as VEGF, FGF) and exosomes in umbilical cord blood, weakening the supporting role of the stem cell microenvironment; Second, traditional cryopreservation relies on the liquid nitrogen system, with high costs for transportation and long-term storage, and repeated freezing and thawing result in a decrease in cell viability (the reported viability <70%); Third, high-purity MSCs are prone to inactivation due to detachment from the natural matrix during the freeze-drying process and require protection with a high concentration of DMSO (5%-10%), which poses a risk of cytotoxicity.

[0004] 2. Stem cell freeze-drying technology: Existing freeze-drying processes mostly use a single cryoprotectant (such as trehalose or DMSO), but there are the following problems: 1) The cryoprotectant combination is not optimized for the membrane structure characteristics of MSCs, resulting in low cell viability after freeze-drying (<80%) and slow recovery of adhesion function after rehydration; 2) The freeze-drying parameters (such as pre-freezing temperature, drying gradient) are not designed in coordination with the cryoprotectant components, leading to ice crystal damage and protein denaturation.

[0005] 3. Anti-aging combination scheme: Existing patents mostly focus on the combination of stem cells and chemical drugs (such as rapamycin, metformin), but there are limitations: Chemical drugs require prescription supervision, restricting their application scenarios as health products; The combination mechanism is single, and the synergistic effect of natural components is not fully utilized. Summary of the Invention

[0006] In view of the above problems, the present invention proposes a preparation process and application of freeze-dried mesenchymal stem cell powder for anti-aging prevention. The freeze-dried powder and combination scheme of the present invention can be widely applied to prevent aging-related symptoms such as skin aging, muscle atrophy, and metabolic function decline, and at the same time adapt to the dual-track supervision system of health products and drugs, with significant industrialization value.

[0007] The object of the present invention is achieved by the following technical solutions.

[0008] A preparation process of freeze-dried mesenchymal stem cell powder for anti-aging prevention, comprising the following steps:

[0009] (1) Extract a mixed cell component containing mesenchymal stem cells from umbilical cord blood. The mixed cell component contains mesenchymal stem cells and growth factors, exosomes, and cytokines naturally present in umbilical cord blood, where the proportion of mesenchymal stem cells is 60% - 80%;

[0010] (2) After washing the mixed cell component obtained in step (1) with phosphate buffer, prepare a cell suspension with a concentration of 1×10 6 ~1×10 7 cells / mL;

[0011] (3) Add a composite freeze-drying protectant accounting for 8% - 12% of the volume of the cell suspension to the cell suspension. By mass percentage, the composite freeze-drying protectant contains the following components:

[0012] Trehalose 4% - 8%;

[0013] Taurine 1% - 3%;

[0014] Ficoll 70 0.5% - 2%;

[0015] Chondroitin sulfate 0.1% - 0.5%;

[0016] DMSO 1% - 3%;

[0017] The balance is PBS;

[0018] (4) Pre-freeze the mixed solution at -70°C ± 5°C for 3 - 4 hours, and then perform gradient freeze-drying. In the first stage, dry at -25°C to -15°C and a vacuum degree ≤ 8 Pa for 16 hours. In the second stage, raise the temperature to 0°C to 5°C and a vacuum degree ≤ 5 Pa and dry for 20 - 30 hours to obtain the freeze-dried powder of mesenchymal stem cells, and the water content of the freeze-dried powder ≤ 2.5%.

[0019] Further, in the above preparation process, the mixed cell component in step (1) is obtained by the following method:

[0020] a. Take fresh umbilical cord blood and mix it with heparin sodium anticoagulant at a final concentration of 10 - 20 U / mL. Stack it on Ficoll-Paque PLUS at a volume ratio of 1:1 - 2, and centrifuge at 400 - 800 g for 30 - 60 min at a temperature below 4°C. Aspirate the mononuclear cell layer (PBMC layer);

[0021] Centrifuge the separated supernatant at 12000 - 15000 g for 1 - 2 h at a temperature below 4°C to collect the precipitated exosomes;

[0022] b. Co-culture:

[0023] Mix the mononuclear cells and exosomes at 1×10 6Cells: Mixed with 10 - 20 μg of exosome protein, inoculated into a culture flask containing α-MEM basal medium, and the culture conditions are 37°C and 5% CO 2 ;

[0024] The first medium change time is 72 h after inoculation. Remove non-adherent cells, and then change the medium every 3 days until the cell confluence reaches 80% - 90%;

[0025] c. Screening and harvesting:

[0026] Use 0.25% trypsin-EDTA to digest adherent cells for 2 - 3 minutes, and add an equal volume of serum-containing medium to terminate digestion;

[0027] Centrifuge the cell suspension at 200 g for 5 min, discard the supernatant, and resuspend it in the cryopreservation solution, 90% FBS + 10% DMSO, to obtain a mixed cell component;

[0028] Detect CD73 + 、CD90 + 、CD105 + and CD34 - 、CD45 - , and harvest the cells when the proportion of mesenchymal stem cells is 65% - 75%.

[0029] Furthermore, in the above preparation process, the chondroitin sulfate described in step (3) is low molecular weight chondroitin sulfate with a molecular weight of 5 - 10 kDa.

[0030] Furthermore, in the above preparation process, the PBS described in step (3) also contains 0.05 - 0.15 mM calcium chloride and has a pH value of 7.2 - 7.4.

[0031] The present invention discloses a freeze-dried powder of mesenchymal stem cells for anti-aging, obtained by any of the above preparation processes.

[0032] The present invention discloses a health product or pharmaceutical composition, comprising the above freeze-dried powder of mesenchymal stem cells and pharmaceutical excipients, and the dosage form is selected from one of the following:

[0033] a. Capsules: The freeze-dried powder accounts for 40% - 50% of the content mass, and the excipients include microcrystalline cellulose, hypromellose, and silicon dioxide;

[0034] b. Tablets: The freeze-dried powder accounts for 30% - 40% of the total tablet mass, and the excipients include lactose, croscarmellose sodium, and magnesium stearate;

[0035] c. Pills or granules: The freeze-dried powder accounts for 45% - 60% of the core mass, and the excipients include polyethylene glycol 6000 and mannitol.

[0036] The present invention discloses the application of the above-mentioned mesenchymal stem cell freeze-dried powder in combination with resveratrol in the preparation of a drug for anti-aging.

[0037] Furthermore, in the above application, in the said drug,

[0038] the daily dose of the freeze-dried powder is 100 - 300 mg, and the daily dose of resveratrol is 50 - 200 mg.

[0039] Compared with the existing technology, the present invention has the following advantages and beneficial effects:

[0040] 1. Preparation of highly active freeze-dried powder:

[0041] Using a composite freeze-drying protectant (trehalose + taurine + polysucrose 70 + low molecular weight chondroitin sulfate), synergistically reducing the DMSO dosage to 1% - 3%. After freeze-drying, the cell survival rate ≥ 85% (only 68% in Comparative Example 1), and the adhesion efficiency is increased by 40% (82% in Example 2 vs 55% in Comparative Example 3);

[0042] The gradient freeze-drying process (-70°C pre-freezing + two-stage drying) combined with calcium ion-modified PBS (Example 4) significantly reduces ice crystal damage, and the membrane fluidity polarization value is reduced to 0.22 (0.28 in Example 2), and the apoptosis rate of cells decreases by 33%.

[0043] 2. Bionic advantages of mixed cell components:

[0044] Through the exosome co-culture technology (monocytes: exosome protein = 1×10 6 cells: 10 - 20 μg), the natural growth factors and exosomes in umbilical cord blood are retained, and the exosome protein content in the freeze-dried powder reaches 25 μg / mg (not detected in Comparative Example 2), synergistically enhancing cell functions:

[0045] The osteogenic differentiation efficiency is increased by 94% (the calcium nodule area in Example 2 is 18.5% vs 9.5% in Comparative Example 3);

[0046] The secretion amount of the SASP factor IL-6 is reduced by 52% (120 pg / mL vs 250 pg / mL), and the collagen synthesis amount is increased by 104% (45 μg / mg vs 22 μg / mg).

[0047] 3. Safe and efficient combination scheme:

[0048] The first combination with a natural ingredient (resveratrol), through activating the SIRT1 / AMPK pathway, achieves synergistic anti-aging in an aging mouse model:

[0049] The skin hydroxyproline content increased by 26% compared with the single - use group (48 μg / mg vs 38 μg / mg), and the mitochondrial ATP production increased by 86% (15.8 nmol / mg vs 8.5 nmol / mg);

[0050] The serum IL - 6 level in the combined - use group (120 pg / mL) was significantly lower than that in the chemical - drug combination regimen (about 150 pg / mL in the rapamycin combination group).

[0051] 4. Breakthrough in the stability of oral dosage forms:

[0052] The enteric - coated capsule (hydroxypropyl methylcellulose coating) protected the survival rate of the lyophilized powder ≥85% in the simulated gastric acid environment, and the survival rate remained 85% after 3 - month accelerated stability test, solving the technical problem of the easy inactivation of the oral stem - cell preparation;

[0053] Tablets, pills, granules and capsules can meet the needs of different populations, and the proportion of the lyophilized powder reaches 30% - 50%, which is better than traditional dry - powder preparations (usually ≤20%).

[0054] 5. Industrial application prospects:

[0055] The process parameters (such as centrifugal force 400 - 800g, freeze - drying time ≤48 hours) are suitable for large - scale production, and the cost is reduced by 60% compared with liquid - nitrogen cryopreservation;

[0056] The combination regimen covers the dual - track markets of health products and drugs, and is potentially applicable to aging - related diseases such as skin aging, muscle atrophy, and neurodegenerative diseases. Description of the Drawings

[0057] Figure 1 Comparison of the cell survival rate (%) of the lyophilized powder of Examples 1 - 3 and Comparative Examples 1 - 3;

[0058] Figure 2 Comparison of the cell attachment efficiency (%) of the lyophilized powder of Examples 1 - 3 and Comparative Examples 1 - 3;

[0059] Figure 3 Comparison of the osteogenic differentiation efficiency (%) of the lyophilized powder of Examples 1 - 3 and Comparative Examples 1 - 3;

[0060] Figure 4 Comparison of the adipogenic differentiation efficiency (%) of the lyophilized powder of Examples 1 - 3 and Comparative Examples 1 - 3. Detailed Description of the Invention

[0061] To make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the scope of the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention. All raw materials in the embodiments of the present invention can be obtained through commercial channels.

[0062] It should be noted that, without conflict, the embodiments in this application and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0063] The main reagents and instruments in the embodiments of the present invention are shown in Table 1.

[0064]

[0065] Example 1

[0066] A preparation process of mesenchymal stem cell freeze-dried powder for anti-aging, comprising the following steps:

[0067] (1) Extract a mixed cell component containing mesenchymal stem cells from umbilical cord blood. The mixed cell component contains mesenchymal stem cells and growth factors, exosomes and cytokines naturally present in umbilical cord blood, and the proportion of mesenchymal stem cells is 60%;

[0068] (2) After washing the mixed cell component obtained in step (1) with phosphate buffer solution, prepare a cell suspension with a concentration of 1×10 6 cells / mL;

[0069] (3) Add a composite freeze-drying protectant accounting for 8% of the volume of the cell suspension to the cell suspension. By mass percentage, the composite freeze-drying protectant contains the following components:

[0070] Trehalose 4%,

[0071] Taurine 1%,

[0072] Ficoll 70 0.5%,

[0073] Chondroitin sulfate 0.1%,

[0074] DMSO 1%,

[0075] The balance is PBS;

[0076] (4) Pre-freeze the mixed solution at -70°C ± 5°C for 3 to 4 hours, and then perform gradient freeze-drying. In the first stage, dry at -25°C to -15°C and a vacuum degree ≤ 8 Pa for 16 hours. In the second stage, raise the temperature to 0°C to 5°C and a vacuum degree ≤ 5 Pa and dry for 20 to 30 hours to obtain freeze-dried mesenchymal stem cells, with the water content of the freeze-dried powder ≤ 2.5%;

[0077] The mixed cell components described in step (1) are obtained by the following method:

[0078] a. Take fresh umbilical cord blood and mix it with heparin sodium anticoagulant at a final concentration of 10 - 20 U / mL. Stack it on Ficoll-Paque PLUS according to a volume ratio of 1:1 - 2, and centrifuge at 400 - 800g for 30 - 60 minutes at a temperature below 4°C. Aspirate the mononuclear cell layer (PBMC layer);

[0079] Centrifuge the separated supernatant at 12000 - 15000g for 1 - 2 hours at a temperature below 4°C, and collect the precipitated exosomes;

[0080] b. Mixed culture:

[0081] Mix monocytes and exosomes at a ratio of 1×10 6 cells : 10 - 20 μg exosome protein, inoculate into a culture flask containing α-MEM basal medium, and the culture conditions are 37°C, 5% CO 2 ;

[0082] The first medium change time is 72 hours after inoculation. Remove the non-adherent cells, and then change the medium every 3 days until the cell confluence reaches 80% - 90%;

[0083] c. Screening and harvesting:

[0084] Digest the adherent cells with 0.25% trypsin-EDTA for 2 - 3 minutes, and add an equal volume of serum-containing medium to terminate the digestion;

[0085] Centrifuge the cell suspension at 200g for 5 minutes, discard the supernatant, and resuspend it in the cryopreservation solution, 90% FBS + 10% DMSO, to obtain the mixed cell components;

[0086] Detect CD73 + , CD90 + , CD105 + and CD34 - , CD45 - by flow cytometry, and harvest the cells when the proportion of mesenchymal stem cells is 60%.

[0087] The chondroitin sulfate described in step (3) is low molecular weight chondroitin sulfate with a molecular weight of 5 - 10 kDa.

[0088] Example 2

[0089] A preparation process of mesenchymal stem cell lyophilized powder for anti - aging, comprising the following steps:

[0090] (1) Extract a mixed cell component containing mesenchymal stem cells from umbilical cord blood. The mixed cell component contains mesenchymal stem cells and growth factors, exosomes and cytokines naturally present in umbilical cord blood, and the proportion of mesenchymal stem cells is 70%;

[0091] (2) After washing the mixed cell component obtained in step (1) with phosphate - buffered saline, prepare a cell suspension with a concentration of 0.5×10 7 cells / mL;

[0092] (3) Add a composite freeze - drying protectant accounting for 10% of the volume of the cell suspension to the cell suspension. By mass percentage, the composite freeze - drying protectant contains the following components:

[0093] Trehalose 6%,

[0094] Taurine 2%,

[0095] Ficoll 70 1%,

[0096] Chondroitin sulfate 0.3%,

[0097] DMSO 2%,

[0098] The balance is PBS;

[0099] (4) Pre - freeze the mixture at - 70°C ± 5°C for 3 - 4 hours, and then perform gradient freeze - drying. In the first stage, dry at - 25°C to - 15°C and a vacuum degree ≤ 8 Pa for 16 hours. In the second stage, raise the temperature to 0°C to 5°C and a vacuum degree ≤ 5 Pa and dry for 20 - 30 hours to obtain mesenchymal stem cell lyophilized powder, and the water content of the lyophilized powder ≤ 2.5%;

[0100] The mixed cell component described in step (1) is obtained by the following method:

[0101] a. Mix fresh umbilical cord blood with heparin sodium anticoagulant at a final concentration of 10 - 20 U / mL, stack it on Ficoll - Paque PLUS according to a volume ratio of 1:1 - 2, centrifuge at 400 - 800g for 30 - 60 min at a temperature below 4°C, and aspirate the mononuclear cell layer (PBMC layer);

[0102] Centrifuge the separated supernatant at 12,000 - 15000g for 1 - 2 h at a temperature below 4°C to collect the precipitated exosomes;

[0103] b. Mixed culture:

[0104] Mix monocytes and exosomes at a ratio of 1×10 6 cells : 10 - 20 μg of exosome protein, and inoculate into a culture flask containing α-MEM basal medium. The culture conditions are 37°C and 5% CO 2 ;

[0105] The first medium change is at 72 h after inoculation. Remove non-adherent cells, and then change the medium every 3 days until the cell confluence reaches 80% - 90%;

[0106] c. Screening and harvesting:

[0107] Digest adherent cells with 0.25% trypsin-EDTA for 2 - 3 minutes, and add an equal volume of serum-containing medium to terminate digestion;

[0108] Centrifuge the cell suspension at 200 g for 5 min, discard the supernatant, and resuspend it in the cryopreservation solution, 90% FBS + 10% DMSO, to obtain a mixed cell component;

[0109] Detect CD73 + , CD90 + , CD105 + and CD34 - , CD45 - by flow cytometry, and harvest the cells when the proportion of mesenchymal stem cells is 70%.

[0110] The chondroitin sulfate described in step (3) is low molecular weight chondroitin sulfate with a molecular weight of 5 - 10 kDa.

[0111] Example 3

[0112] A preparation process of freeze-dried powder of mesenchymal stem cells for anti-aging, comprising the following steps:

[0113] (1) Extract a mixed cell component containing mesenchymal stem cells from umbilical cord blood. The mixed cell component contains mesenchymal stem cells and growth factors, exosomes, and cytokines naturally present in umbilical cord blood, and the proportion of mesenchymal stem cells is 80%;

[0114] (2) Wash the mixed cell component obtained in step (1) with phosphate buffer, and prepare it into a cell suspension with a concentration of 1×10 7 cells / mL;

[0115] (3) Add a composite freeze-drying protectant accounting for 8% - 12% of the volume of the cell suspension to the cell suspension. By mass percentage, the composite freeze-drying protectant contains the following components:

[0116] Trehalose 8%;

[0117] Taurine 3%;

[0118] Ficoll 70 2%;

[0119] Chondroitin sulfate 0.5%;

[0120] DMSO 3%;

[0121] The balance is PBS;

[0122] (4) Pre-freeze the mixture at -70°C ± 5°C for 3 - 4 hours, and then perform gradient freeze-drying. In the first stage, dry at -25°C to -15°C and a vacuum degree ≤ 8 Pa for 16 hours. In the second stage, raise the temperature to 0°C to 5°C and a vacuum degree ≤ 5 Pa and dry for 20 - 30 hours to obtain freeze-dried mesenchymal stem cells, and the water content of the freeze-dried powder ≤ 2.5%;

[0123] The mixed cell components described in step (1) are obtained by the following method:

[0124] a. Take fresh cord blood and mix it with heparin sodium anticoagulant at a final concentration of 10 - 20 U / mL, stack it on Ficoll-Paque PLUS according to a volume ratio of 1:1 - 2, centrifuge at 400 - 800g at 4°C or below for 30 - 60 min, and aspirate the mononuclear cell layer (PBMC layer);

[0125] Centrifuge the separated supernatant at 12000 - 15000g for 1 - 2 h at 4°C or below to collect the precipitated exosomes;

[0126] b. Mixed culture:

[0127] Mix the mononuclear cells and exosomes at a ratio of 1×10 6 cells : 10 - 20 μg exosome protein, inoculate them into a culture flask containing α-MEM basal medium, and the culture conditions are 37°C and 5% CO 2 ;

[0128] The first medium change time is 72 h after inoculation, remove the non-adherent cells, and then change the medium every 3 days until the cell confluence reaches 80% - 90%;

[0129] c. Screening and harvesting:

[0130] Digest the adherent cells with 0.25% trypsin-EDTA for 2 - 3 minutes, and add an equal volume of serum-containing medium to terminate the digestion;

[0131] Centrifuge the cell suspension at 200 g for 5 min. After discarding the supernatant, resuspend it in the cryopreservation solution, 90% FBS + 10% DMSO, to obtain a mixed cell component;

[0132] Detect CD73 by flow cytometry + , CD90 + , CD105 + and CD34 - , CD45 - , and harvest the cells when the proportion of mesenchymal stem cells is 80%;

[0133] The chondroitin sulfate described in step (3) is low-molecular-weight chondroitin sulfate with a molecular weight of 5-10 kDa.

[0134] Example 4

[0135] Compared with Example 2, the PBS in this example also contains 0.1 mM calcium chloride and has a pH value of 7.2-7.4.

[0136] Comparative Example 1

[0137] The lyoprotectant is an existing conventional lyoprotectant, and the formula is as follows:

[0138] Trehalose 8 wt%;

[0139] Mannitol 3 wt%;

[0140] DMSO 5 wt%;

[0141] The balance is PBS;

[0142] The rest is the same as in Example 2.

[0143] Comparative Example 2

[0144] In step (1), cord blood is directly used without extracting the mixed cell component containing mesenchymal stem cells.

[0145] The rest is the same as in Example 2.

[0146] Comparative Example 3

[0147] Use an existing process to purify freeze-dried powder of mesenchymal stem cells with a purity of more than 95% from cord blood.

[0148] It includes the following steps:

[0149] 1. Stem cell purification:

[0150] Centrifuge cord blood by density gradient centrifugation with Ficoll-Paque PLUS (1.077 g / mL) (800×g, 30 minutes, 4°C), and collect the mononuclear cell layer (PBMC);

[0151] PBMCs were inoculated into α-MEM medium containing 10% FBS, and the medium was changed every 3 days. Non-adherent cells were removed, and the cells were passaged to the 3rd generation to obtain MSCs with a purity ≥ 95% (flow cytometry detection of CD73 + / CD90 + / CD105 + ≥ 95%, CD34 - / CD45 - ≤ 2%).

[0152] 2. Freeze-drying process:

[0153] Cell suspension (1×10 6 cells / mL) was added with cryoprotectant (8% trehalose + 5% DMSO, w / v);

[0154] Pre-freezing (-80°C, 4 hours), freeze-drying (-50°C to 25°C, 48 hours), and the water content of the freeze-dried powder was ≤ 5%.

[0155] Test Example 1

[0156] Cell viability and functional recovery of the freeze-dried powder

[0157] Test groups: Examples 1-3, Comparative Examples 1-3

[0158] Method:

[0159] 1. Viability detection (trypan blue staining method)

[0160] Steps: The freeze-dried powder was reconstituted in 1 mL of normal saline. 10 μL of the cell suspension was mixed with an equal volume of 0.4% trypan blue staining solution and allowed to stand for 3 minutes. The number of live cells (unstained) and dead cells (blue) was counted using a hemocytometer.

[0161] Standard: Viability (%) = (number of live cells / total number of cells) × 100%, and the average value was taken after repeating 3 times.

[0162] 2. Adhesion efficiency detection

[0163] Steps: The reconstituted cells were inoculated into a 6-well plate at 1×10 4 cells / well. After culturing for 24 hours, the non-adherent cells were removed by washing with PBS, and the number of adherent cells was counted after trypsin digestion.

[0164] Standard: Adhesion efficiency (%) = (number of adherent cells / number of inoculated cells) × 100%.

[0165] 3. Differentiation ability detection

[0166] Osteogenic induction: α-MEM medium containing 10% FBS, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 50 μM ascorbic acid was used. After 14 days of induction, alizarin red staining (pH 4.2) was performed on calcium nodules, and the area percentage was quantified using ImageJ.

[0167] Adipogenic induction: Medium containing 10% FBS, 1 μM insulin, 0.5 mM IBMX, and 0.1 μM dexamethasone was used. After 14 days of induction, oil red O staining was performed on lipid droplets, and the number of lipid droplets per field (200×) was counted under a microscope.

[0168] The results are shown in Table 2 and Figures 1 - 4 .

[0169]

[0170] In Examples 1 - 3, a composite lyoprotectant (containing taurine, ficoll 70, etc.) was used. By reducing ice crystal damage and stabilizing the cell membrane structure, the survival rate (≥85%) was significantly higher than that of Comparative Examples 1 - 3 (≤75%). Exosomes in the mixed cell components (Example 2) promoted cell adhesion and differentiation functions, and the osteogenic differentiation efficiency was increased by 94% compared to Comparative Example 3 (15.2% vs 9.5%), verifying the necessity of retaining natural components.

[0171] Test Example 2

[0172] Exosome activity and secretion of anti-aging factors

[0173] Test groups: Example 2, Comparative Example 2 (without exosomes), Comparative Example 3 (pure stem cells)

[0174] Methods:

[0175] 1. Exosome protein quantification (BCA method)

[0176] Steps: Dissolve 10 mg of freeze-dried powder in PBS, centrifuge at 12,000×g for 30 minutes to remove cell debris, filter the supernatant through a 0.22 μm filter membrane, and measure the protein concentration using a BCA kit.

[0177] Standard: The exosome protein content is expressed as μg / mg of freeze-dried powder.

[0178] 2. Detection of SASP factors (ELISA)

[0179] Steps: After reconstituting the cells and culturing for 48 hours, collect the supernatant and operate according to the instructions of the IL-6 (product number: D6050, R&D Systems) and TNF-α (product number: DTA00C, R&D Systems) ELISA kits.

[0180] Standard: The concentration was calculated by the standard curve method, with the unit of pg / mL.

[0181] 3. Detection of collagen content (hydroxyproline method)

[0182] Steps: The extracellular matrix was hydrolyzed with 6 M HCl (110 °C, 24 hours), and after neutralization, it was reacted with chloramine T / p-dimethylaminobenzaldehyde. The absorbance was measured at 562 nm, and the hydroxyproline content was calculated by referring to the standard curve and converted to the collagen content (the proportion of hydroxyproline was 13.4%).

[0183] The results are shown in Table 3

[0184]

[0185] In Example 2, the exosome protein content in the freeze-dried powder reached 25 μg / mg, which was significantly higher than that in Comparative Examples 2 - 3 (not detected), proving that the mixed cell components effectively retained exosomes. The miRNA-21 carried by exosomes inhibited the NF-κB pathway, reducing the IL-6 level (120 pg / mL) by 52% compared with Comparative Example 3 (250 pg / mL), and increasing the collagen secretion by 104% (45 μg / mg vs 22 μg / mg), confirming the synergistic anti-aging mechanism of exosomes.

[0186] Test Example 3

[0187] Synergistic anti-aging effect in combination with resveratrol

[0188] Test groups:

[0189] Freeze-dried powder of Example 2 (100 mg / day)

[0190] Resveratrol (100 mg / day)

[0191] Example 2 + Resveratrol (100 mg + 100 mg / day)

[0192] Freeze-dried powder of Comparative Example 3 (100 mg / day) + Resveratrol (100 mg / day)

[0193] Model: D-galactose-induced senescent mice (8 weeks old, n = 10 / group)

[0194] Test methods and steps

[0195] 1. Establishment of animal model

[0196] Steps: 8-week-old C57BL / 6 mice were subcutaneously injected with D-galactose (150 mg / kg / d, for 8 weeks) to establish a senescence model.

[0197] Group treatment: model group, single-agent group of Example 2 (gavage at 100 mg / kg), single-agent group of resveratrol (gavage at 100 mg / kg), combination group (100 mg / kg + 100 mg / kg), combination group of Comparative Example 3, continuous administration for 6 weeks.

[0198] 2. Skin aging assessment

[0199] VISIA wrinkle index: The back skin of mice was photographed using the VISIA-CR system, and the proportion of the wrinkled area (%) was analyzed.

[0200] Hydroxyproline detection: Skin tissues were taken and measured by the method of Test Example 2.

[0201] Mitochondrial function detection (ATP content)

[0202] Steps: Skeletal muscle tissues were taken, lysed, and then the ATP concentration was measured by the luciferase method using an ATP detection kit (product number: A22066, Thermo).

[0203] The results are shown in Table 4

[0204]

[0205] The combination group (Example 2 + resveratrol) significantly increased mitochondrial ATP production (15.8 nmol / mg vs 8.5 nmol / mg in the model group) by double-activating the SIRT1 / AMPK pathway, and the skin hydroxyproline content (48 μg / mg) was 26% higher than that of the single-agent group (38 μg / mg in the single-agent group of Example 2). The combination group of Comparative Example 3 was significantly less effective than the present invention due to the lack of exosome synergy (collagen content 33 μg / mg vs 48 μg / mg).

[0206] Test Example 4

[0207] Effect of calcium-modified PBS on cell membrane stability

[0208] Test groups: Example 2, Example 4 (containing 0.1 mM Ca 2+ )

[0209] Test methods and steps

[0210] Apoptosis detection (Annexin V / PI double staining)

[0211] Steps: The resuspended cells were washed with Binding Buffer, Annexin V-FITC (5 μL) and PI (5 μL) were added, incubated in the dark for 15 minutes, and the apoptosis rate was detected by flow cytometry.

[0212] Membrane fluidity detection (DPH fluorescence polarization method)

[0213] Procedure: Incubate the cell suspension with 1,6-diphenyl-1,3,5-hexatriene (DPH, 1 μM) for 30 minutes, and measure the polarization value (P) at an excitation wavelength of 360 nm and an emission wavelength of 430 nm using a fluorescence spectrophotometer.

[0214] The results are shown in Table 5

[0215]

[0216] Example 4 (containing 0.1 mM Ca 2+ ) Calcium ions bind to cell membrane phospholipids, reducing the polarization value of membrane fluidity to 0.22 (0.28 in Example 2), and the apoptosis rate decreases from 12% to 8%, indicating that calcium ions enhance freeze-drying tolerance by stabilizing the membrane lipid bilayer structure.

[0217] Test Example 5

[0218] Oral dosage form stability

[0219] Test group: Capsules, tablets, pills, and granules prepared in Example 2

[0220] 1. Preparation of capsules

[0221] Procedure:

[0222] Mix the mesenchymal stem cell lyophilized powder prepared in Example 2 evenly with excipients (microcrystalline cellulose: hypromellose: silica = 70:25:5, mass ratio);

[0223] Use wet granulation (5% PVP ethanol solution as the binder), pass through a 20-mesh sieve for granulation, and dry at 60 °C until the moisture content ≤ 3%;

[0224] Fill into an enteric gelatin capsule shell (model DRcaps™, Lonza), the mass of the capsule content is 500 mg, and the lyophilized powder accounts for 40%.

[0225] 2. Preparation of tablets

[0226] Procedure:

[0227] Mix the lyophilized powder in Example 2 with excipients (lactose: croscarmellose sodium: magnesium stearate = 80:15:5, mass ratio);

[0228] Direct compression (pressure 10 - 15 kN, punch die diameter 8 mm), tablet weight 600 mg, lyophilized powder accounts for 35%;

[0229] Coat with a film coating (Opadry® II, containing hypromellose).

[0230] 3. Preparation of Pills

[0231] Steps:

[0232] Mix the lyophilized powder of Example 2 with excipients (PEG 6000:mannitol = 60:40, mass ratio).

[0233] Prepare pill cores by the dropping method (aperture 1.2 mm, dropping speed 20 drops / minute), and coat with gelatin film after solidification (weight gain 5%).

[0234] The core mass of the pill is 300 mg, and the lyophilized powder accounts for 50%.

[0235] 4. Preparation of Granules

[0236] Steps:

[0237] Mix the lyophilized powder of Example 2 with excipients (mannitol:silica = 85:15, mass ratio), and granulate by wet granulation with 5% hydroxypropyl methylcellulose ethanol solution as the binder.

[0238] Screen the granules through a 16-mesh sieve, dry under reduced pressure at low temperature (<10 °C) until the moisture content ≤ 4%, and pack into 1 g / bag.

[0239] The lyophilized powder accounts for 30% in the granules.

[0240] Testing Method:

[0241] Accelerated Stability Test

[0242] Steps: Place the capsules, tablets, pills and granules in a constant temperature and humidity chamber at 25 °C / 75% RH for 3 months, and sample and redissolve for detecting the survival rate every month.

[0243] Simulated Gastric Acid Experiment

[0244] Steps: Take the dosage form samples and place them in gastric juice at pH 1.2 (containing 0.2% NaCl, 0.32% pepsin), incubate with shaking at 37 °C for 2 hours, and detect the survival rate after neutralization with PBS.

[0245] The results are shown in Table 6

[0246]

[0247] The dissolution rate of hydroxypropyl methylcellulose enteric-coated film (capsules) in the gastric acid environment < 5%, protecting the survival rate of the lyophilized powder ≥ 85%, while the survival rate of tablets drops to 75% due to direct exposure. This proves the necessity of enteric design for oral application.

[0248] Examples 1-4 of the present invention and comparative tests show that:

[0249] 1. Freeze-drying process optimization: The composite freeze-drying protectant (Example 2) enables the cell survival rate to reach 89±2%, and the adhesion efficiency to reach 82±4%, which is significantly higher than that of the traditional formula (the survival rate of Comparative Example 1 is 68±5%);

[0250] 2. Synergy of mixed components: The level of IL-6 secreted by the mixed cell components (Example 2) that retain exosomes (120±15 pg / mL) is 52% lower than that of pure stem cells (Comparative Example 3), and the collagen synthesis amount is increased by 104%;

[0251] 3. Synergistic enhancement in combination: After being combined with resveratrol, the hydroxyproline content in the skin of aging mice reaches 48±5 μg / mg, and the mitochondrial ATP is increased by 86%, which is superior to the chemical drug combination scheme;

[0252] 4. Dosage form stability: The enteric-coated capsule has the best gastric acid protection effect (survival rate 85±4%), and the survival rates of pills and granules are 78±4% and 72±4% respectively, which also meet the requirements of oral preparations;

[0253] 5. Calcium ion strengthening: Adding 0.1 mM Ca 2+ (Example 4) reduces the apoptosis rate of cells to 8±1% and increases the membrane fluidity by 21%.

[0254] In summary, through the full-chain innovation of process-component-dosage form-combination, the present invention has achieved breakthroughs in the high activity, stability and safety of the stem cell anti-aging preparation.

[0255] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Therefore, based on the innovative concept of the present invention, any changes and modifications made to the embodiments described herein, or equivalent structural or equivalent process transformations made using the content of the specification of the present invention, and directly or indirectly applying the above technical solutions to other related technical fields, are all included in the protection scope of the present invention patent.

Claims

1. A process for preparing freeze-dried mesenchymal stem cell powder for preventing aging, characterized in that: The following steps are involved: (1) extracting a mixed cell component containing mesenchymal stem cells from umbilical cord blood, wherein the mixed cell component comprises mesenchymal stem cells and growth factors, exosomes and cytokines naturally present in umbilical cord blood, wherein the mesenchymal stem cells account for 60% to 80%; (2) The mixed cell fraction obtained in step (1) was washed with phosphate buffer and prepared into a solution with a concentration of 1×10 6 ~1×10 7 cells / mL of cell suspension; (3) Adding a composite lyophilization protective agent in an amount of 8% to 12% of the volume of the cell suspension to the cell suspension, wherein the composite lyophilization protective agent comprises the following components by mass percentage: Trehalose 4%~8%; Taurine 1%~3%; Polysucrose 70 0.5%~2%; Chondroitin sulfate 0.1%~0.5%; DMSO 1%~3%; The remainder is PBS; (4) The mixture was pre-frozen at -70°C ± 5°C for 3 to 4 hours, and then subjected to gradient freeze-drying. The first stage was dried at -25°C to -15°C and a vacuum degree of ≤8 Pa for 16 hours. The second stage was dried at 0°C to 5°C and a vacuum degree of ≤5 Pa for 20 to 30 hours to obtain mesenchymal stem cell freeze-dried powder with a water content of ≤2.5%.

2. The preparation process according to claim 1, characterized in that: The mixed cell components in step (1) are obtained by the following method: a. Mix fresh umbilical cord blood with anticoagulant sodium heparin at a final concentration of 10-20 U / mL, overlay on Ficoll-Paque PLUS at a volume ratio of 1:1-2, centrifuge at 400-800g for 30-60min at below 4°C, and aspirate the mononuclear cell layer and PBMC layer; The separated supernatant was centrifuged at 12000-15000 g for 1-2 h below 4°C to collect the precipitated exosomes; b. Mixed culture: Monocytes and exosomes were collected at a rate of 1×10 6 cells: 10-20 μg of exosome protein were mixed and inoculated into a culture bottle containing α-MEM basal medium at 37°C and 5% CO2; The first medium change time is 72 hours after inoculation to remove non-adherent cells, and the medium is changed every 3 days thereafter until the cell confluence reaches 80%-90%; c. Screening and harvesting: Use 0.25% trypsin-EDTA to digest the adherent cells for 2 to 3 minutes, and add an equal volume of serum-containing medium to terminate the digestion; The cell suspension was centrifuged at 200 g for 5 min, the supernatant was discarded and resuspended in freezing buffer, 90% FBS + 10% DMSO, to obtain a mixed cell fraction; Detection of CD73 by flow cytometry + 、CD90 + 、CD105 + and CD34 - 、CD45 - , so that the cells are harvested when the proportion of mesenchymal stem cells is 60%~80%.

3. The preparation process according to claim 1, characterized in that: The chondroitin sulfate in step (3) is low molecular weight chondroitin sulfate with a molecular weight of 5-10 kDa.

4. The preparation process according to claim 1, characterized in that: The PBS in step (3) further contains 0.05-0.15 mM calcium chloride and has a pH value of 7.2-7.

4.

5. A mesenchymal stem cell freeze-dried powder for preventing aging, characterized in that: Obtained by the preparation process described in any one of claims 1 to 4.

6. A health product or pharmaceutical composition, characterized in that: The composition comprises the mesenchymal stem cell freeze-dried powder according to claim 5 and pharmaceutical excipients, wherein the dosage form is selected from one of the following: a. Capsules: freeze-dried powder accounts for 40% to 50% of the content, and excipients include microcrystalline cellulose, hypromellose and silicon dioxide; b. Tablets: Lyophilized powder accounts for 30% to 40% of the total tablet weight, and excipients include lactose, cross-linked sodium carboxymethyl cellulose and magnesium stearate; c. Pills or granules: Lyophilized powder accounts for 45% to 60% of the core mass. Excipients include polyethylene glycol 6000 and mannitol.

7. Use of the mesenchymal stem cell freeze-dried powder according to claim 5 in combination with resveratrol in the preparation of a drug for preventing aging.

8. The use according to claim 7, characterized in that: In the drug, the daily dosage of the lyophilized powder is 100-300 mg, and the daily dosage of resveratrol is 50-200 mg.

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