A cord blood stem cell lyophilized powder and its use in the preparation of a pharmaceutical or cosmetic product

Abstract

The present application provides a kind of human umbilical cord mesenchymal stem cell freeze-dried powder and its preparation method and application.The freeze-dried powder is prepared by stem cell and a composite protective agent containing trehalose, human serum albumin and recombinant Oligosarcus microlepidotus III type antifreeze protein mutant (rZaAFPIII-M) by specific programmed freeze-drying process. Among them, the rZaAFPIII-M is obtained by site-directed amino acid replacement, with improved stability and solubility. The technical scheme makes the obtained freeze-dried stem cells not only have high cell survival rate after recovery, but also can significantly maintain the ability of key paracrine repair factors and the biological activity to skin target cells, and the comprehensive effect is comparable to traditional cryopreservation. The product has good storage stability under refrigerated conditions, can be conveniently used in the preparation of skin tissue repair drugs or cosmetics with anti-aging and moisturizing effects, and provides a new solution for the standardization and industrialization of stem cell preparations.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a human umbilical cord mesenchymal stem cell freeze-dried powder and its application in the preparation of drugs or cosmetics. Background Technology

[0002] Human umbilical cord mesenchymal stem cells (hUC-MSCs) have attracted widespread attention in the field of regenerative medicine due to their ease of acquisition, rapid proliferation, low immunogenicity, and multi-lineage differentiation potential and paracrine properties. These cells can secrete various bioactive substances such as vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), and have been proven to effectively promote angiogenesis, regulate local immune responses, and stimulate collagen synthesis by skin fibroblasts. Therefore, they show clear therapeutic potential in wound healing, scar repair, and skin rejuvenation.

[0003] Currently, the conventional strategy for long-term preservation of hUC-MSCs is cryopreservation, which involves using a cryoprotectant containing dimethyl sulfoxide (DMSO) or glycerol, and storing the cells in a liquid nitrogen environment at -196°C after programmed cooling. Although this method can maintain cell viability well, its application has significant limitations: First, the long-term reliance on liquid nitrogen tanks to maintain the low-temperature environment is costly, and there are safety hazards such as liquid nitrogen leakage and container damage during storage and transportation; second, chemical protectants such as DMSO are potentially toxic to cells, requiring complex and time-consuming washing to remove residues after thawing, which is inconvenient and may affect cell state; third, cryopreserved cell preparations cannot be transported at room temperature and used immediately, making it difficult to meet the commercialization needs of "ready-to-use" biological products or standardized active ingredients.

[0004] Freeze-drying technology offers a potential solution to the aforementioned bottlenecks. This process transforms cells into a solid dry powder that can be stably stored at room temperature or 4°C by directly sublimating the water in a cell suspension under low temperature and low pressure conditions. This greatly simplifies the storage, transportation, and end-use processes. However, successfully applying freeze-drying to structurally and functionally complex live cells such as hUC-MSCs still faces significant challenges. Conventional freeze-drying protection systems often rely on combinations of sugars (such as trehalose and sucrose) and proteins (such as human serum albumin, HSA), the principle of which is to form a glassy state to stabilize the cell membrane and intracellular proteins. However, practice has shown that such basic formulations have limited protective efficacy for cells. Physicochemical damage such as ice crystal formation and growth, drastic changes in osmotic pressure, and protein denaturation during freeze-drying often leads to a significant reduction in cell survival rate after thawing (often below 70%). Furthermore, cell adhesion, proliferation, and, more importantly, therapeutic paracrine function are severely impaired, greatly limiting its practical application value.

[0005] To enhance the protective effect of freeze-drying, related research has attempted to introduce exogenous antifreeze proteins (AFPs). For example, existing literature has disclosed the use of plant-derived antifreeze proteins. However, the extraction process of plant antifreeze proteins is usually quite complicated, the product composition is complex and difficult to standardize, and there is a risk of introducing plant allergens or exogenous contaminants, making it difficult to meet the strict quality control requirements of pharmaceutical or high-end cosmetic raw materials. Some academic studies have used synthetic polymers, such as polyvinylpyrrolidone (PVP), as ice crystal inhibitors. Although PVP components are well-defined and readily available, its interaction mechanism with cell biomembranes is still unclear, its long-term biosafety needs to be fully assessed, and its potential impact on specific functional lineages of stem cells lacks in-depth research and data support. More importantly, most existing technical solutions only focus on the primary indicator of cell count and viability after freeze-drying, lacking a systematic and quantitative assessment of the core biological functions after cell revival—especially its unique paracrine factor lineage and ability to induce tissue regeneration. This lack of functional evaluation makes it impossible to guarantee the actual efficacy of many frozen stem cell products, thus hindering the industrialization of this technology.

[0006] Therefore, developing a novel freeze-drying technology with clear composition, high protection efficiency, maximum preservation of hUC-MSCs activity and functionality, and stable and controllable process is of vital importance for promoting hUC-MSCs formulations from laboratory research to clinical treatment and consumer market application. Summary of the Invention

[0007] The purpose of this invention is to overcome the above-mentioned defects and provide a human umbilical cord mesenchymal stem cell freeze-dried powder with clearly defined components, excellent protective effect, and the ability to maintain the activity of hUC-MSCs to the maximum extent, as well as its preparation method and application.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] The first invention relates to a recombinant mutant (rZaAFPIII-M) obtained by site-directed mutagenesis of the wild-type American blenny type III antifreeze protein through rational design. The wild-type amino acid sequence corresponds to SEQ ID NO:1, and the mutant amino acid sequence corresponds to SEQ ID NO:2. The core mutation sites are N14D and A16S. The N14D mutation replaces neutral asparagine with negatively charged aspartic acid, optimizing the protein surface charge distribution and improving structural stability in aqueous environment. The A16S mutation replaces alanine with serine, introducing an additional hydroxyl group, strengthening the interaction with solvents and trehalose in the complex protective agent, laying the structural foundation for synergistic protection. The mutant was prepared using an E. coli expression system. The specific procedure is as follows: wild-type and mutant genes optimized with E. coli codons were synthesized by a commissioned team, cloned into the Nde I and Xho I restriction sites of the pET-30a+ vector, respectively, and the recombinant expression plasmid was constructed and transformed into E. coli strain BL21DE3. Single clones of the strain were cultured in LB medium containing kanamycin at 37°C until the OD600 reached approximately 0.6. Then, 0.4 mM IPTG was added, and expression was induced at 18°C ​​for 20 hours. Soluble expression analysis showed that the mutant's soluble expression level reached 45±3 mg / L of culture, significantly higher than the wild-type's 15±2 mg / L, with a statistically significant difference. The protein was enriched by Ni-NTA affinity chromatography, digested with enterokinase at 4°C for 16 hours to remove the N-terminal 6×histidine tag, and then finely purified by Superdex 75 Increase gel filtration chromatography to obtain the target protein with a purity higher than 95%, which meets the requirements for subsequent compound protective agent formulation.

[0010] Secondly, this invention provides a human umbilical cord mesenchymal stem cell lyophilized powder. The core innovation of this formulation lies in its composite lyophilization protectant system, which consists of three components: trehalose, human serum albumin, and a recombinant American blenny type III antifreeze protein mutant (rZaAFPIII-M). These three components work synergistically within a specific concentration range to jointly protect cells from ice crystal damage, osmotic stress, and protein denaturation during dehydration.

[0011] Thirdly, this invention provides a method for preparing the freeze-dried powder. The key to this method is mixing stem cells in their optimal growth state with the aforementioned composite protective agent and processing them through an optimized, programmed freeze-drying process. This process precisely controls the pre-freezing rate to form fine ice crystals, uses physical methods such as pressure rise testing during the first drying stage to accurately determine the drying endpoint to avoid over- or under-drying, and ensures that the residual moisture content of the product meets requirements after the second drying. Finally, the product is sealed under an inert atmosphere to obtain a solid powder that can be stored at room temperature.

[0012] Fourthly, this invention relates to the application of the aforementioned freeze-dried powder. Based on the fact that the freeze-dried powder can effectively maintain the paracrine function of stem cells (such as the continuous secretion of growth factors like VEGF and HGF) after thawing, it can be used to prepare drugs that promote skin wound healing and accelerate tissue regeneration. Simultaneously, it can also be used as a highly effective bioactive ingredient, incorporated into cosmetic formulations, to develop skincare products that enhance skin hydration, repair the skin barrier, and improve signs of aging.

[0013] The beneficial effects of this invention are:

[0014] 1. Excellent Cell Protection: The innovative ternary composite protective agent system of this invention produces a synergistic effect. The hUC-MSCs lyophilized powder prepared using this system achieves a cell viability rate of over 86% after thawing, showing no significant difference compared to liquid nitrogen cryopreservation. Functional experiments confirm that the cell's ability to secrete growth factors per unit volume and the conditioned medium's pro-repair activity on skin fibroblasts are highly preserved after thawing, achieving dual protection from "cell quantity" to "cell quality."

[0015] 2. Technological Innovativeness: Compared to existing freeze-drying technologies that use conventional polymers (such as PVP) as protective agents, the specific bio-derived antifreeze protein mutant complex system of this invention brings unexpected technological advancements under the same experimental conditions. This system not only has advantages in cell recovery rate but also exhibits a significant, albeit not obvious, improvement in the unit functional output of cells after thawing (such as an increase of over 50% in VEGF secretion).

[0016] 3. High Product Practicality: The freeze-dried powder prepared by this invention has a stable morphology and low residual moisture content. Accelerated stability studies have shown that after 6 months of storage under normal refrigeration conditions at 2-8°C, the core activity decreases by less than 15%, greatly reducing storage and logistics costs. This morphology allows it to be easily added to topical formulations like ordinary raw materials. In vitro and preliminary human trials have demonstrated the safety of its formulations and its potential in improving skin hydration and barrier function, paving the way for industrial application. Attached Figure Description

[0017] Figure 1SDS-PAGE results of recombinant American blimp type III antifreeze protein (rZaAFPIII) and its mutant protein (rZaAFPIII-M), where 1 is rZaAFPIII and 2 is rZaAFPIII-M, both with a molecular weight of approximately 7 kDa. Detailed Implementation

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0019] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0020] Example 1: Preparation and testing of recombinant American blimp type III antifreeze protein (rZaAFPIII) and its mutant protein (rZaAFPIII-M)

[0021] 1. Mutant Design and Construction

[0022] Based on the analysis of the crystal structure of the type III antifreeze protein (rZaAFPIII, whose amino acid sequence is shown in SEQ ID NO:1) (UniProtKB: P19614) from the American blenny (Zoarces americanus), its ice crystal binding surface is mainly composed of a conserved Thr / Ala array, and the protein's stability is related to its surface charge distribution. To explore the possibility of improving its freeze-drying protective properties, we conducted a rational design:

[0023] The mutant rZaAFPIII-M: Based on the wild-type sequence, two site-directed mutations, N14D and A16S, were introduced. N14D was chosen to replace neutral asparagine with negatively charged aspartic acid, aiming to optimize the protein surface charge distribution and enhance its stability in aqueous solution. A16S was chosen to replace alanine with serine, introducing an additional hydroxyl group, which may enhance its interaction with sugars in solvents and protective agents. Its amino acid sequence is shown in SEQ ID NO:2.

[0024] Gene synthesis and cloning: GenScript was commissioned to synthesize the wild-type gene (rZaAFPIII-wt) and mutant gene (rZaAFPIII-M) optimized with E. coli codons. Both were cloned into the Nde I and Xho I sites of the expression vector pET-30a(+) to construct recombinant plasmids pET30a-rZaAFPIII-wt and pET30a-rZaAFPIII-M, respectively. Sequence accuracy was verified by DNA sequencing. Both constructs had a cleavable 6×His tag fused to the N-terminus of the target protein.

[0025] 2. Protein Expression and Purification

[0026] The plasmids were transformed into *E. coli* BL21(DE3). Single colonies were picked and cultured in LB medium containing kanamycin at 37°C until OD500. 600 ≈0.6, add 0.4 mM IPTG, and induce expression at 18℃ for 20 hours.

[0027] Soluble expression analysis: Equal amounts of bacterial cells were collected, sonicated, and centrifuged. The supernatant (soluble fraction) was then subjected to SDS-PAGE and grayscale analysis. The results showed that, under the same culture conditions, the soluble expression level of the rZaAFPIII-M mutant was approximately 45 ± 3 mg / L of the culture (n=3), significantly higher than that of the wild type (15 ± 2 mg / L) (p < 0.05).

[0028] Purification: Both proteins were purified using Ni-NTA affinity chromatography. After elution with buffer containing 250 mM imidazole, the His tag was cleaved using enterokinase at 4°C for 16 hours. The cleaved mixture was passed through a Ni-NTA column again, and the flow-through (i.e., the target protein without the tag) was collected and further purified by Superdex 75 Increase gel filtration chromatography to obtain high-purity proteins (>95%). Figure 1 Protein concentration was determined using the BCA method. The purified wild-type rZaAFPIII-wt and mutant rZaAFPIII-M were aliquoted and stored at -80°C for future research.

[0029] 3. Comparison of in vitro biochemical characteristics

[0030] Thermal hysteresis activity assay: Differential scanning calorimetry was used to determine the thermal hysteresis activity of the protein at different concentrations (0.05, 0.1, 0.2 mM). The results showed that at a concentration of 0.1 mM, the thermal hysteresis activity of rZaAFPIII-M was 0.38 ± 0.02°C, which was not statistically different from that of the wild type (0.35 ± 0.02°C) (p > 0.05). However, the calculated specific activity (thermal hysteresis value / μM protein) showed a consistent trend between the two within the tested concentration range.

[0031] Thermal stability assessment: The thermal denaturation temperature (Tm) was determined by differential scanning fluorometry (nanoDSF). The Tm value of rZaAFPIII-M was 52.1 ± 0.3°C, slightly higher than that of wild type (50.5 ± 0.4°C) (p < 0.05), suggesting that the mutation may have slightly improved the thermal stability of the protein.

[0032] Solubility test: The two proteins were dissolved at 4°C in the base buffer (Ca-free) of the composite lyophilization protectant of this invention. 2 + / Mg 2+ The solution was gradually concentrated in PBS. When the concentration reached 15 mg / mL, the wild-type protein solution began to show slight turbidity; while rZaAFPIII-M remained clear even at a concentration of 25 mg / mL. Measurement of the supernatant protein concentration after centrifugation confirmed that the upper limit of the working concentration of rZaAFPIII-M in this system was significantly higher than that of the wild-type.

[0033] 4. Application verification in stem cell freeze-drying preservation

[0034] Experimental design: Using the same batch of hUC-MSCs, composite protectants containing equimolar concentrations (100 μg / mL, approximately 13.7 μM) of wild-type rZaAFPIII-wt or mutant rZaAFPIII-M (other components: 100 mM trehalose, 1% HSA) were prepared. The protectant group without antifreeze proteins served as a negative control.

[0035] Results: After lyophilization and thawing, trypan blue staining showed that the cell viability of the rZaAFPIII-M group was (88.5 ± 2.1)%, slightly better than that of the rZaAFPIII-wt group (86.3 ± 2.3)% and the negative control group (65.4 ± 3.8)%. Although the difference in viability was not statistically significant, the mutant group showed better cell clustering and morphological retention after lyophilization.

[0036] Conclusion: This preliminary experiment shows that the rZaAFPIII-M mutant maintains a protective capacity comparable to or even slightly better than that of the wild type in actual stem cell freeze-drying applications, and its higher soluble expression level and better solubility in formulations provide potential advantages for large-scale production and formulation of high-concentration protective agents.

[0037] Example 2: Isolation, culture and identification of human umbilical cord mesenchymal stem cells (hUC-MSCs)

[0038] Harvest the umbilical cord of a healthy, full-term newborn delivered by cesarean section (with informed consent), and thoroughly rinse it under sterile conditions with phosphate buffer containing 1% penicillin-streptomycin. The umbilical cord is then longitudinally incised, Wharton's jelly tissue is mechanically dissected, and cut into pieces approximately 1 mm in volume. 3 Tissue blocks were used for primary culture via the tissue block adherence method: tissue blocks were evenly spaced at the bottom of a T25 culture flask, and α-MEM basal medium (HyClone) containing 20% ​​fetal bovine serum (Gibco) and 1% penicillin-dextrose antibody was added. The flasks were cultured at 37°C and 5% CO2. The medium was completely replaced every 3 days. After approximately 7-10 days, spindle-shaped cells were observed migrating from the edges of the tissue blocks. When the primary cells reached 80%-90% confluence, they were digested with 0.25% trypsin-EDTA (Gibco) and passaged at a 1:3 ratio for expansion. The complete culture medium used was α-MEM basal medium supplemented with 10% fetal bovine serum, 1% penicillin-dextrose antibody, and 4 ng / mL recombinant human basic fibroblast growth factor.

[0039] Phenotypic identification was performed on well-grown third-generation cells. Cell surface markers were detected using flow cytometry: Cells were resuspended in staining buffer and incubated at 4°C for 30 minutes in the dark with FITC-labeled mouse anti-human CD73 antibody, APC-labeled mouse anti-human CD90 antibody, PE / Cy7-labeled mouse anti-human CD105 antibody (positive markers), as well as corresponding isotype control antibodies and negative marker antibodies (PE anti-human CD45, PerCP anti-human CD34, APC / Cy7 anti-human HLA-DR). After staining, cells were washed with buffer and analyzed using a BD FACSCanto II flow cytometer and FlowJo software. The identification results of three batches of independently isolated and cultured cells showed that the positive rates of CD73, CD90, and CD105 were all higher than 98.5% (mean ± standard deviation 99.1% ± 0.4%, 99.6% ± 0.3%, and 98.7% ± 0.7%, respectively), while the positive rates of CD34, CD45, and HLA-DR were all lower than 0.5% (0.2% ± 0.1%, 0.2% ± 0.2%, and 0.3% ± 0.1%, respectively).

[0040] Further investigation was conducted to identify its multi-lineage differentiation potential through directed differentiation induction. Osteogenic induction: Cells were inoculated at a rate of 5 × 10⁻⁶ cells / year. 3cells / cm 2 Density seeding was performed, and cells were cultured for 21 days in osteogenic induction medium (complete medium containing 10 mM β-glycerophosphate, 50 μM ascorbic acid, and 0.1 μM dexamethasone), with the medium changed every 3 days. Alizarin Red S staining showed obvious formation of red calcium nodules. Adipogenic induction: The fused cells were induced for 3 days in adipogenic induction medium A (complete medium containing 1 μM dexamethasone, 0.5 mM IBMX, 10 μg / mL insulin, and 200 μM indomethacin), then cultured for 1 day in maintenance medium B (complete medium containing 10 μg / mL insulin), and this cycle was repeated 3 times. Cells were then cultured in medium B for another 7 days. Oil Red O staining showed a large accumulation of red lipid droplets within the cells. The uninduced control group cells showed no corresponding staining. These results confirm that the isolated and cultured cells meet the criteria for mesenchymal stem cells defined by the International Society for Cell Therapy.

[0041] Example 3: Preparation of hUC-MSCs lyophilized powder

[0042] 1. Cell pretreatment

[0043] Cell source and preparation: Fourth-generation (P4) human umbilical cord mesenchymal stem cells (hUC-MSCs) identified phenotypically and functionally in Example 2, or commercially available hUC-MSCs (such as those from Shanghai Hongshun Biotechnology Co., Ltd.), were used. The cells were in the logarithmic growth phase with approximately 80% confluence. Cells were digested with 0.25% trypsin-EDTA (Gibco), neutralized with serum-containing complete culture medium, and collected by centrifugation at 300g for 5 minutes. Cell counting and viability were performed using 0.4% trypan blue staining. Three independent counts showed cell viability of 98.1%, 98.5%, and 98.3%, with an average viability of 98.3 ± 0.2%; the average cell density was 5.2 × 10^6 cells / mL.

[0044] Washing: Use pre-cooled, Ca-free water. 2+ / Mg 2+ Resuspend the cell pellet in phosphate-buffered saline (PBS), centrifuge again at 300g for 5 minutes, and discard the supernatant completely to remove serum and impurities from the culture medium. Repeat this step once.

[0045] Cell recovery rate: After washing, the cells were recounted, and the average cell recovery rate was 95.7 ± 2.1%.

[0046] 2. Formulation of composite lyophilization protectant

[0047] Preparation of the protective agent: Accurately weigh the following reagents: D-(+)-trehalose dihydrate, fatty acid-free human serum albumin (HSA), and rZaAFPIII-M prepared in Example 1. Dissolve them in a Ca2+-free solution.2+ / Mg 2+ A 2× composite protective agent working solution was prepared in PBS buffer, consisting of 200 mM trehalose, 2% (w / v) HSA, and 200 μg / mL rZaAFPIII-M. The solution was gently stirred at 4°C with a magnetic stirrer until completely dissolved. The solution was sterilized by filtration through a 0.22 μm pore size polyethersulfone (PES) syringe filter, aliquoted, and stored at 4°C protected from light. It should be used within 24 hours.

[0048] Protective agent loading: The washed hUC-MSCs cell pellet was gently resuspended by pipetting with an equal volume of pre-cooled 2× composite protective agent working solution (equilibrated to 4°C beforehand), avoiding the formation of air bubbles. At this point, the components of the protective agent were diluted to the target final concentrations: 100 mM trehalose, 1% (w / v) HSA, and 100 μg / mL rZaAFPIII-M. Subsequently, the cell suspension density was precisely adjusted to 1.0 × 10^7 cells / mL using this protective agent solution.

[0049] Quality control: Immediately after mixing, the cell suspension was tested using a pH meter and osmometer. The test results of three independently prepared batches were: pH 7.35 ± 0.05, osmotic pressure 310 ± 5 mOsm / kg, all within the physiological range of cells, indicating that the formulation is stable and has good reproducibility.

[0050] 3. Programmed freeze drying

[0051] Dispensing: The cell suspension was aseptically dispensed into 2R type neutral borosilicate glass molded injection vials (vials) at a volume of 1.0 mL / vial, and half-pressed with siliconized butyl rubber freeze-dried stoppers.

[0052] Freeze-drying program: Use a freeze dryer and execute the optimized program. Specific parameters are as follows:

[0053] (1) Pre-freezing stage: The temperature was lowered from 4°C to -40°C at a linear cooling rate of 1.0 °C / min and held at -40°C for 120 minutes to ensure that the sample was completely solidified.

[0054] (2) First drying (sublimation drying): Start the vacuum pump. Once the chamber pressure stabilizes at < 10 Pa, raise the shelf temperature to -25°C at a rate of 0.2 °C / min and maintain this temperature. Determine the endpoint through the "pressure rise test": Close the main isolation valve between the freeze-drying chamber and the condenser, and record the pressure rise value (ΔP) within 30 seconds. When ΔP is < 3 Pa for three consecutive measurements, the first drying is considered complete. In this operation, the first drying stage lasted for 22 hours, and the ΔP value at the endpoint was 2.1 Pa.

[0055] (3) Secondary drying (desorption drying): The shelf temperature is raised to 25°C at a slow rate of 0.1 °C / min and maintained at this temperature. The vacuum degree is kept below 5 Pa, and drying continues for 8 hours.

[0056] The freeze dryer recorder showed that the total freeze-drying cycle (pre-freezing + drying) lasted approximately 33 hours. Towards the end of the second drying stage, the sample temperature and the shelf temperature essentially reached equilibrium.

[0057] 4. Quality evaluation and sealed storage of freeze-dried final products

[0058] Residual moisture content determination: Immediately after the freeze-drying process, three samples were randomly selected and the moisture content was determined using a Karl Fischer coulometric analyzer. The residual moisture contents of the three samples were found to be 2.65%, 2.72%, and 2.75%, respectively, with an average value of 2.71 ± 0.05%.

[0059] Sealing: Inside the freeze dryer chamber, under nitrogen purging (nitrogen purity ≥ 99.999%), completely stopper the vial to seal it. The operation should be completed quickly at an ambient humidity of < 10%.

[0060] Appearance: After sealing, the product is a white to off-white, loosely structured cake-like substance with no obvious collapse, melting or cracks.

[0061] Storage: After labeling, immediately transfer the product to a freezer at -20°C or store at 2~8°C, away from light and in a dry place.

[0062] Example 4: Experiment on the quality and function evaluation of freeze-dried powder

[0063] 1. Experimental Design and Grouping

[0064] To systematically verify the synergistic protective effect of the composite freeze-drying protectant of the present invention, the following nine experimental groups were established. All groups used the same batch, same passage (P4), and qualified human umbilical cord mesenchymal stem cells (hUC-MSCs) identified by the method in Example 2, and adjusted to uniform cell density and viability before freeze-drying.

[0065] G1 (Invention Group): 100 mM trehalose + 1% HSA + 100 μg / mL rZaAFPIII-M (prepared in Example 1);

[0066] G2 (two-group group - glycoprotein): 100 mM trehalose + 1% HSA;

[0067] G3 (single group - sugar): 100 mM trehalose;

[0068] G4 (single group - protein): 1% HSA;

[0069] G5 (single group - AFP): 100 μg / mL rZaAFPIII-M;

[0070] G6 (two-group group - sugar + AFP): 100 mM trehalose + 100 μg / mL rZaAFPIII-M;

[0071] G7 (two-group group - protein + AFP): 1% HSA + 100 μg / mL rZaAFPIII-M;

[0072] G8 (closest to existing technology group): 100 mM trehalose + 1% HSA + 0.05% PVP K30 (Mw=40kDa);

[0073] G9 (positive control group for cryopreservation): The cells were cryopreserved in liquid nitrogen gas phase for one week using commercial serum-free cell cryopreservation solution CryoStor® CS10 (STEMCELL Technologies) according to the supplier's standard procedure and then thawed.

[0074] 2. Preparation and thawing of freeze-dried powder

[0075] All lyophilized groups (G1-G8) were prepared strictly according to the standardized procedure in Example 3, ensuring that all conditions (cell density, lyophilization procedure, residual moisture, and sealing conditions) were completely consistent except for the protectant formulation. The lyophilized powders from each group were stored at 4°C for one week before being used in experiments.

[0076] During resuscitation, add 1.0 mL of preheated 37°C complete culture medium (α-MEM + 10% FBS) to each bottle of lyophilized powder and gently swirl to completely reconstitute the lyophilized powder within 60 seconds.

[0077] 3. Evaluation of cell resuscitation efficiency and activity

[0078] 3.1 Live cell count and survival rate calculation:

[0079] Procedure: After cell resuscitation in each experimental group, gently mix the suspension immediately. Mix 20 μL of cell suspension with 20 μL of 0.4% trypan blue (Gibco) staining solution and incubate at room temperature for 2 minutes. Count the cells using an automated cell counter or hemocytometer, recording the total number of cells and the number of viable (unstained) cells. Set up 3 technical replicates for each group.

[0080] Total cell density results (×10) 6Cells / mL): G1: 10.52, 10.48, 10.61; G2: 9.85, 9.92, 9.78; G3: 9.10, 9.05, 9.18; G4: 8.75, 8.69, 8.81; G5: 9.45, 9.51, 9.39; G6: 10.05, 10.11, 9.98; G7: 9.60, 9.55, 9.66; G8: 10.20, 10.15, 10.25; G9: 10.01, 9.95, 10.08; Fresh cells (not lyophilized): 10.60, 10.55, 10.65.

[0081] viable cell density results (×10) 6 (cells / mL): G1: 9.08, 9.12, 9.05; G2: 6.20, 6.08, 6.14; G3: 4.85, 4.78, 4.83; G4: 4.05, 3.98, 4.03; G5: 5.25, 5.18, 5.17; G6: 6.65, 6.58, 6.63; G7: 5.60, 5.52, 5.57; G8: 7.10, 7.02, 6.95; G9: 8.78, 8.71, 8.69; Fresh cells: 10.45, 10.40, 10.48.

[0082] Results: The average viable cell density and resuscitation survival rate (viable cell density / total cell density × 100%) of each group were calculated based on the above data. The results are summarized in Table 1.

[0083] Table 1: Viable cell density and survival rate after cell resuscitation (Mean ± SD, n=3)

[0084]

[0085] 4. Evaluation of paracrine function based on isoviviparous cell count

[0086] To ensure a fair comparison of the intrinsic functional activity of cells from different groups, subsequent experiments were all conducted based on an equal number of viable cells.

[0087] 4.1 Preparation of Conditioned Culture Medium (CM)

[0088] Steps: Based on the live cell density data in Table 1, accurately calculate and transfer samples containing 2.0 × 10⁻⁶ cells. 5 Cell suspensions of each group of live cells were seeded into 6-well plates. Complete culture medium was added to each well to a final volume of 2 mL. The cells were then incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0089] Quality control: Six hours after inoculation, cells were observed under a microscope and showed good adherence morphology in all groups with no significant differences.

[0090] 4.2 Growth factor secretion assay (ELISA)

[0091] Procedure: After culturing for 24 hours, collect the supernatant from each well and centrifuge at 300g for 10 minutes at 4℃ to remove cell debris. Aliquot the supernatant and store at -80℃ for later use. Use a commercially available human VEGF and HGF ELISA kit, strictly following the instructions. Each sample should be tested in duplicate. Read the OD450 nm value on a microplate reader and calculate the concentration based on the standard curve.

[0092] Results: The measured factor concentration (pg / mL) multiplied by the culture volume (2 mL), and then divided by the number of viable cells inoculated (2 × 10⁻⁶). 5 ), which is ultimately converted to "every 10 6 The total amount of factors secreted by each living cell (pg). Specific data are shown in Table 2.

[0093] Table 2: Secretion of growth factor per unit of live cells (pg / 10) 6 (cells, Mean ± SD, n=3)

[0094]

[0095] 5. Functional activity verification (scratch test)

[0096] Procedure: Human dermal fibroblasts (HDF) were seeded into 24-well plates to form a dense monolayer. A vertical scratch was made in the center of each well using a 200 μL sterile pipette tip. The cells were gently washed twice with PBS to remove floating cells. The prepared conditioned medium (CM) for each group was diluted 1:1 with fresh complete medium and added to the corresponding wells, 500 μL per well. Wells containing only fresh complete medium served as negative controls. Imaging was performed at 0 and 24 hours post-scratching using an inverted microscope in a fixed position (4× objective lens).

[0097] Results: The scratch area at 0h and 24h was quantitatively analyzed using ImageJ software. The migration closure rate was calculated as: [(0h area - 24h area) / 0h area] × 100%. Three independent wells were analyzed for each experimental group, with three fields of view taken from each well. Specific data are shown in Table 3.

[0098] Table 3: Scratch migration closure rate (%, Mean ± SD, n=9 fields of view)

[0099]

[0100] 6. Experimental Results and Summary

[0101] Synergistic protective effect on cell viability: As shown in Table 1, the cell recovery survival rate of the present invention group (G1) reached 86.4%, which was not statistically different from the gold standard for deep cryopreservation (G9). The survival rate of G1 was significantly higher than that of all single-component (G3-G5) and two-component (G2, G6, G7) control groups, especially about 17.6 percentage points higher than the closest prior art group (G8, PVP formulation). It is worth noting that the protective effect of the binary system (G2, G6, G7) lacking any core component was significantly worse than that of the complete ternary system (G1), and the single-component effect was the worst. This clearly confirms that there is a significant synergistic protective effect among trehalose, HSA and rZaAFPIII-M, rather than a simple additive effect.

[0102] Excellent maintenance and synergistic effect of unit cell functional activity: Under the premise of strict comparison of isoviviparous cell numbers (Table 2), the ability of G1 group units of cells to secrete VEGF and HGF (11480, 5280 pg / 10) 6 There were no statistically significant differences between the G1 group and the cryopreserved group (G9) and the non-lyophilized fresh cell group. In contrast, the functional output per unit cell was significantly decreased in all control groups (G2-G8). In particular, the functional indicators of the two-group groups (G2, G6, G7) were significantly lower than those of the G1 group, while the single-group groups (G3-G5) had the lowest values. This demonstrates that the ternary system also has a synergistic effect in maintaining the intrinsic functional activity of each surviving cell. Compared with the existing technology G8 group, the VEGF and HGF secretion per unit cell in the G1 group increased by approximately 54.7% and approximately 53.0%, respectively, which was highly significant.

[0103] Comprehensive validation of biological function: The scratch assay results (Table 3) are highly consistent with the secretory function data. The conditioned medium derived from G1 group cells showed comparable ability to promote HDF migration (85.2%) to the cryopreserved group (83.9%). This effect is significantly superior to all other lyophilized experimental groups (G2-G8). For example, the migration-promoting ability of G1 was approximately 31.3% higher than that of the G8 (pre-existing technology) group. This directly confirms, from the perspective of target cell response, that the lyophilized powder of this invention can maximally preserve the biological efficacy of stem cell secretory groups.

[0104] Example 5: Accelerated stability study of freeze-dried powder

[0105] 1. Experimental Design and Sample Preparation

[0106] To evaluate the storage stability of the lyophilized powder of the present invention, an accelerated stability study was conducted in accordance with the ICH Q1A(R2) guideline. Three batches of independently prepared lyophilized powder samples (batch numbers: S001, S002, S003, prepared in the same manner as in Example 3) were sealed and stored in a humidity-controlled stability test chamber at (2-8) °C under light-protected conditions.

[0107] 2. Sampling time point and detection indicators

[0108] Three bottles were randomly selected from each batch of samples on day 0 (initial), day 30, day 90, and day 180 of storage for testing. Key testing indicators included:

[0109] Appearance and reconstitution characteristics: Observe the appearance of the freeze-dried cake with the naked eye and record the reconstitution time.

[0110] Residual moisture content: measured using the Karl Fischer coulometric method.

[0111] Cell survival rate after resuscitation: Trypan blue staining method was used.

[0112] Unit cell functional activity (VEGF secretion capacity): based on seeding with an equal number of viable cells, detected by ELISA.

[0113] 3. Experimental Procedures and Results

[0114] Sample reconstitution and cell resuscitation: At each time point, the extracted samples were reconstituted in 1 mL of 37°C complete culture medium as described in Example 4. Subsequent assays were performed immediately.

[0115] Cell resuscitation survival rate assay: After rehydration, the cell suspension was stained with trypan blue for counting, and the viable cell density and viable cell percentage retention rate relative to the initial time point (day 0) were calculated. The original data and calculation results are shown in Table 4.

[0116] Table 4: Cell survival rate data after different storage times (n=3 batches, 3 bottles per batch)

[0117]

[0118] VEGF secretion capacity assay per unit cell: Based on the live cell density measured at each time point, an equal amount of live cells (1×10⁻⁶) was precisely seeded. 5 VEGF concentrations were incubated in 24-well plates (number of cells / well) for 24 hours, and the supernatant was collected. VEGF concentration was detected by ELISA and converted to the amount secreted per unit of viable cells (pg / 10⁻¹⁰). 6 (cells). Calculate the retention rate relative to the initial value. Raw data (secretory volume per unit viable cell, pg / 10). 6 The cells and retention rates are shown in Table 5.

[0119] Table 5: VEGF secretion capacity per unit cell after different storage times (n=3 batches)

[0120]

[0121] Appearance and moisture content: All samples were white, loose, cake-like substances throughout the observation period, with reconstitution times of less than 60 seconds. Residual moisture content was 2.71% at day 0 and 2.95% at day 180, within acceptable limits.

[0122] 4. Experimental Results and Summary: This stability study provides detailed tracking data for three batches of samples stored at 4℃. The results show that:

[0123] The stability trend is clear: with prolonged storage time, the cell recovery survival rate and functional activity of the lyophilized powder show a slow and controllable decline trend (Tables 4 and 5). After 6 months (180 days) of storage, the average viable cell ratio was maintained at 89.5%, and the average VEGF secretion capacity per unit cell was maintained at 87.5%, both of which remained above 85%, indicating that the core performance degradation was limited.

[0124] Good batch-to-batch consistency: The detection data of the three independently prepared samples at each time point showed good consistency and small standard deviation, which proved the stability and reproducibility of the preparation process of the present invention.

[0125] Outstanding functional stability: Most importantly, the rate of decline in functional activity (VEGF secretion capacity) is essentially synchronized with or slightly slower than the rate of decline in cell viability. This indicates that the storage process not only maintains the cell count well but also effectively preserves the functional integrity of surviving cells, which is highly consistent with the design goals of the formulation of this invention.

[0126] In summary, the hUC-MSCs lyophilized powder prepared by this invention exhibits excellent physical and functional stability when stored at 2-8℃ in the dark for 6 months, with a core activity index retention rate of more than 85%, meeting the requirements for storage, transportation and subsequent application as a bioactive raw material or intermediate product.

[0127] Example 6: Application and Preliminary Efficacy Evaluation in Cosmetics

[0128] 1. Preparation of essence samples containing lyophilized stem cell powder

[0129] Basic gel matrix: A stable water-based gel was prepared, comprising: 1.0% (w / w) sodium hyaluronate (blended with different molecular weights), 3.0% (w / w) glycerol, 2.0% (w / w) ceramide NP, 0.5% (w / w) disodium EDTA, and appropriate amount of preservative (phenoxyethanol / ethylhexylglycerin). The pH was adjusted to 5.5-6.0 with citric acid. The gel was sterilized by filtration through a 0.22 μm filter membrane.

[0130] Addition of active ingredients: In a sterile operating room, the lyophilized powder (batch number S001) prepared in Example 3 was pre-reconstituted in sterile PBS to form a high-concentration cell suspension. Then, it was slowly and gently mixed with the pre-cooled base gel matrix at a final concentration of 0.05% (w / w) (based on the mass of the lyophilized powder) in a sterile homogenizer to avoid introducing air bubbles and generating high shear forces that could damage the cells. This yielded "Stem Cell Active Repair Essence (TEST)". Simultaneously, a blank matrix gel (CTRL) without lyophilized powder was prepared as a control.

[0131] 2. In vitro skin irritation assessment

[0132] Experimental system: using the commercially available recombinant human epidermal model EpiDerm TM (EPI-200, MatTek).

[0133] Experimental procedure: After the model was restored to culture, 15 μL of TEST essence was applied to the surface of three tissues, and an equal amount of CTRL gel was applied to the surface of the other three tissues. After exposure for 60 minutes, the tissues were gently rinsed with PBS. After culturing for another 42 hours, tissue viability was measured using the MTT assay. After MTT reduction, the absorbance (OD) at 570 nm was measured. The results are shown in Table 6.

[0134] Table 6: Results of OD value detection for tissue viability in recombinant human epidermal model

[0135]

[0136] Note: According to the standard, if the tissue viability after treatment with the test substance is ≥ 50% (negative control), it is considered non-irritating.

[0137] Results: The average tissue viability after TEST treatment was 98.3%, far exceeding the 50% threshold, and showed no difference compared to the blank matrix control. Conclusion: This "stem cell active repair essence" showed no skin irritation under the test conditions.

[0138] 3. In a small-scale (n=15) open-label trial, volunteers applied the serum twice daily to one corner of their eye for 28 days. Tests showed that the average stratum corneum moisture content of the treated skin increased by 28.5%, transepidermal water loss decreased by 18.2%, and visual assessments showed improvement in fine lines.

[0139] 4. Experimental Results and Summary: This application example successfully integrated the freeze-dried powder of the present invention into a cosmetic matrix, and preliminary safety and efficacy evaluations were conducted.

[0140] Formulation compatibility and safety: The freeze-dried powder is highly compatible with commonly used cosmetic bases (hyaluronic acid-ceramide gel), and the prepared essence showed no irritation in in vitro skin models, indicating that it has a good foundation for local application safety.

[0141] Preliminary efficacy is significant: a 28-day open-label trial showed that the serum containing 0.05% freeze-dried powder significantly increased the stratum corneum moisture content (+28.5%) and strengthened the skin barrier function (TEWL decreased by 18.2%). Expert evaluation also supports its potential to improve the appearance of fine lines around the eyes.

[0142] The feasibility of the application has been verified: This experiment has preliminarily demonstrated the feasibility and effectiveness of using the freeze-dried powder of the present invention as an active ingredient in functional cosmetics, and provided valuable preliminary data for its commercial development in the fields of skin moisturizing, barrier repair and anti-aging.

[0143] In summary, this invention not only provides a method for preparing freeze-dried stem cell powder, but also further demonstrates its enormous potential as a highly active raw material in end products (such as serums). The freeze-dried powder form greatly facilitates its formulation and production in cosmetic factories, and combined with its stability and preliminary efficacy, it shows excellent prospects for translational applications.

[0144] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A recombinant American blenny type III antifreeze protein mutant, characterized in that, The recombinant American blimp type III antifreeze protein mutant is rZaAFPIII-M, and its amino acid sequence is shown in SEQ ID NO:

2.

2. A composite lyophilization protectant containing the recombinant American blenny type III antifreeze protein mutant as described in claim 1, characterized in that, The composite lyophilization protectant, based on its final concentration, contains the following components: 100 mM trehalose, 1% HSA (by weight / volume), and 100 μg / mL rZaAFPIII-M. The solvent is Ca-free. 2+ / Mg 2+ PBS buffer.

3. The composite freeze-drying protectant according to claim 2, characterized in that, The composite freeze-drying protectant is sterilized by filtration through a 0.22-micron polyethersulfone needle filter, stored at 4°C away from light, and used within 24 hours.

4. A freeze-dried powder of human umbilical cord mesenchymal stem cells, characterized in that, The human umbilical cord mesenchymal stem cell freeze-dried powder contains an effective amount of human umbilical cord mesenchymal stem cells and the composite freeze-drying protectant of claim 2 distributed therein.

5. The human umbilical cord mesenchymal stem cell lyophilized powder according to claim 4, characterized in that, The concentration of human umbilical cord mesenchymal stem cells in the cell suspension before lyophilization was 1.0 × 10^7 cells / mL.

6. The human umbilical cord mesenchymal stem cell lyophilized powder according to claim 4, characterized in that, The residual moisture content of the human umbilical cord mesenchymal stem cell freeze-dried powder is no higher than 3%.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises human umbilical cord mesenchymal stem cell lyophilized powder as described in any one of claims 4 to 6 and a pharmaceutically acceptable carrier.

8. A cosmetic composition, characterized in that, The cosmetic composition comprises human umbilical cord mesenchymal stem cell lyophilized powder as described in any one of claims 4 to 6 and a cosmetically acceptable matrix.

9. Use of a human umbilical cord mesenchymal stem cell lyophilized powder as described in any one of claims 4 to 6 in the preparation of a medicament for promoting the repair of skin damage.

10. The use of a human umbilical cord mesenchymal stem cell freeze-dried powder as described in any one of claims 4 to 6 in the preparation of cosmetics for improving skin barrier function, moisturizing or anti-aging.

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