• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
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    • 专利摘要:
    The present invention relates to a gel formulation for maintaining the viability of cryopreserved cells, and a stem cell gel formulation that contains stem cells. The gel formulation comprises the following components in weight percent: 1% -3% sodium alginate, 1% -5% dimethyl sulfoxide, 1% -5% propylene glycol, 1% -5% polyphenol, 2% -10% dextran, 1% -5% human serum albumin and water or an additional phosphoric acid saline solution. This gel formulation is biocompatible and easy to use, it can maintain high cell activity and be cryopreserved directly without traditional cryopreservation to liquid nitrogen. After being resuscitated, the cell gel formulation can still maintain high cell activity and stem cell character, so it can be used for the treatment of skin injury or mucosal injury.
    公开号:FR3057141A1
    申请号:FR1759395
    申请日:2017-10-06
    公开日:2018-04-13
    发明作者:Jie Geng；Lu Liang；Zhongchao Han
    申请人:Beijing Health And Biotech Stem Cell Inst Co Ltd；
    IPC主号:
    专利说明:

    Holder (s): BEIJING HEALTH AND BIOTECH STEM CELL INSTITUTE CO. LTD.
    Extension request (s)
    Agent (s): LAWRENCE AND CHARRAS CABINET.
    FR 3 057 141 - A1 f54) GEL FORMULATION OF STEM CELLS TO MAINTAIN THE VIABILITY OF CRYOCONSERVE CELLS AND ITS USE.
    The present invention relates to a gel formulation for maintaining the viability of cryopreserved cells, and to a stem cell gel formulation which contains stem cells. The gel formulation comprises the following components in percentage by mass: 1% -3% sodium alginate, 1% -5% dimethyl sulfoxide, 1% 5% propylene glycol, 1% -5% polyphenol, 2 % -10% dextran, 1% -5% human serum albumin and water or an additional saline solution of phosphoric acid. This gel formulation is biocompatible and easy to use, it can maintain high cell activity and be cryopreserved directly without traditional cryopreservation with liquid nitrogen. After resuscitation, the cell gel formulation can still maintain high cell activity and stem cell character, so it can be used for the treatment of skin injury or mucosal injury.
    GEL FORMULATION OF STEM CELLS TO MAINTAIN THE VIABILITY OF CRYOPRESERVED CELLS AND ITS USE
    FIELD OF THE INVENTION
    The present invention relates to the field of biotechnology, and relates in particular to a gel formulation for cryopreservation of cells.
    The present invention also relates to a stem cell gel formulation comprising mesenchymal stem cells and the gel formulation, and its use.
    PRIOR ART
    To be a permanent substitute, the tissue engineering support material must be degradable and without immunogenicity, and the decomposed products thereof must have no toxicity or abnormal reaction to biological tissue. Although it has a great affinity for cells, collagen, which generally comes from animal tissue, is immunogenic and poses a high risk of pathogenic microbial contamination. In addition, collagen is also not economical, and has poor mechanical performance, which makes it necessary for crosslinking use. Current skin tissue engineering clinical products still trigger immune rejection and have only a weak treatment effect. The classic treatment for a skin injury is to disinfect the wound with a drug for external use, and then expose or dress on the damaged skin. The effect is not ideal, however. Patients with slight repercussions experience pain in the wound, swelling; patients with severe repercussions generate empyema under the crust and their physical activity is limited, which affects the normal work of patients.
    Sodium alginate is an anionic polysaccharide with a straight chain extracted from brown marine algae, which has great biocompatibility, biodegradability and immunoisolation properties, and is the most commonly used bio-support material. An aqueous solution of sodium alginate, a semi-fluid light yellow colloidal liquid, plays a role in protecting and supporting cells. It can quickly and evenly cover the wound surface, slow the evaporation of surface water, and maintain cellular activity. It is a good support for drugs for external use for the repair of skin tissue. An aqueous solution of sodium alginate may also i
    be used as a cell support material for intestinal tissue repair and uterine tissue repair.
    A mesenchymal cell is one of the best sources of tissue cells due to its advantages of stable genetic history, easy separation, culture and expansion, and the ability to maintain their character. strain after long-term culture in vitro. Mesenchymal stem cells, having low immunogenicity, will not cause an abnormal immune response, and can release a variety of anti-inflammatory cytokines, which inhibit the body's inflammatory response. They can also release a variety of factors to promote repair of tissue injuries and mobilize the body's own stem cells to participate in the repair of tissue injuries. Direct cell contact with inflammatory defects or wounds can maximize the repair and therapeutic effect of a stem cell.
    The most common method of maintaining cell viability in long-term cryopreservation is cryopreservation with liquid nitrogen, which maintains cell viability at more than 80%. However, during the transport and clinical application of cellular products, liquid nitrogen is not practical to transport, transport by cell transfer in liquid nitrogen is subject to strong restrictions. In clinical applications, the place of use should be equipped with a liquid nitrogen storage device and a liquid nitrogen supply subject to local conditions and geography.
    Chinese patent application CN101451124A describes a method for preparing the formulation of mesenchymal stem cells from a human umbilical cord, in which methylcellulose is used as a cell matrix. The formulation contains gentamicin which is not suitable for people allergic to antibiotics. In addition, fetal calf serum of animal origin is produced during the rapid preparation of the formulation for external use, which can cause allergies and risks to animal safety. Rapid preparation of the formulation requires several operational steps, the process of which is complicated, leading to a risk of microbial contamination. A 2-week sterility test cannot be carried out before transport, which constitutes the requirements of the pharmacopoeia. The quality of the product cannot be guaranteed.
    Chinese patent application CN 102670654A describes a stem cell formulation for wound repair. The stem cell formulation contains umbilical cord, placental or amniotic mesenchymal stem cells and a macromolecular stabilizer selected from the group consisting of sodium alginate, sodium hyaluronate, chitosan and hydroxyethyl starch. Cell anti-freezing DMSO, humectant propylene glycol or glycerol may also be contained. Although the formulation has a very good therapeutic effect, it has the disadvantage of stability and cell survival rate.
    SUMMARY OF THE INVENTION
    The object of the present invention is to provide a gel formulation for maintaining the viability of cryopreserved cells, which can maintain cell viability over a long period of time under a condition of -80 ° C without cryopreservation with liquid nitrogen. This puts an end to dependence on liquid nitrogen for traditional cell preservation.
    Another object of the present invention is to provide a stem cell gel formulation comprising mesenchymal stem cells and the gel formulation.
    Yet another object of the present invention is to provide the use of the stem cell gel formulation for the preparation of drugs for skin repair.
    According to one aspect of the invention, a gel formulation for maintaining the activity of cryopreserved cells comprises the following components in percentage by mass: 1% -3% of sodium alginate, 1% -5% of dimethyl sulfoxide , 1% -5% propylene glycol, 1% -5% polyphenol, 2% -10% dextran, 1% -5% human serum albumin and water or additional phosphoric acid saline. The phosphate solution consists of 0.02% potassium chloride, 0.0047% magnesium chloride, 0.1158% hydrogenated disodium phosphate, 0.020%, dihydrogenated sodium phosphate, 0.8% chloride sodium and water for injection. The gel formulation preferably includes the following components by weight percentage: 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenol, 6 % dextran, 2% human serum albumin, and an additional phosphate solution which consists of 0.02% potassium chloride, 0.0047% magnesium chloride, 0.1158% hydrogenated disodium phosphate, 0.020% , dihydrogen phosphate sodium, 0.8% sodium chloride and water for injection.
    With the exception of the prior art sodium alginate, polyphenol, dextran and human serum albumin are added to the gel formulation of the present invention. Polyphenol has a very strong antioxidant activity, which is as high as at least 100 times that of vitamin C and 25 times that of vitamin E. Polyphenol can protect cells and DNA from a pity. Dextran is one of the best plasma substitutes available today, which can significantly improve cell viability and reduce the use of DMSO. The addition of human serum albumin ensures that the gel formulation of the present invention does not contain ingredients derived from animals and improves the safety and stability of the formulation. In addition, the composition of the gel formulation is screened to obtain a gel formulation suitable for maintaining the activity of cryopreserved cells.
    The gel formulation of the present invention can be used for the cryopreservation of various cells, which contain, but are not limited to, various tumor cell lines, animal cells, stem cells and the like.
    According to another aspect of the present invention, there is provided a stem cell gel formulation comprising mesenchymal stem cells and the gel formulation. Preferably, the concentration of mesenchymal stem cells is 0.5 x 10 6 to 3 x 10 6 per 1 ml of the gel formulation. Mesenchymal stem cells can be derived from umbilical, placental and amniotic cord tissue to prepare the stem cell gel formulation. Stem cells are derived not only from the tissues listed above, but also from any other source of mesenchymal stem cells.
    The stem cell gel formulation according to the invention is prepared by the following steps consisting in: (1) dissolving and swelling the sodium alginate powder in ultra pure water or a phosphate solution at room temperature for 24 to 48 hours; (2) stir the mixture until homogeneous, and add the remaining components to the mixture to formulate the gel formulation according to claim 1; (3) allow the gel formulation obtained in step (2) to stand for a certain time, then add mesenchymal stem cells therein; (4) mix uniformly, and the gel formulation rich in stem cells according to the invention is prepared.
    In order to maintain the vitality of the cells in the gel, the optimal size of the sodium alginate in the gel formulation is 200 mesh and the viscosity is 600 to 1000 cps. The pH of the solution is 6.5 to 7.5. The cell-rich gel formulation can be frozen directly between -80 ° C and -60 ° C for long-term storage. The cryopreservation method is unscheduled cooling. The lifespan is as long as a year and a half. The recovered cell-rich gel formulation can be used directly and cell viability can be maintained at 90% or more. The gel formulation can also be stored at 4 ° C if not used immediately, and cell viability can be maintained at 80% or more for
    6 hours.
    According to another aspect of the present invention, there is provided the use of the stem cell gel formulation according to the present invention for the preparation of medicaments for the treatment of skin injury or mucosal injury. The stem cell gel formulation is prepared in a pharmaceutical formulation which is suitable for transport and storage and can be used for skin repair treatment. Skin lesions or mucosal lesions as previously described include, but are not limited to, skin ulcers, bed sores, diabetic foot, ulcerative colitis, Crohn's disease, endometrial injury.
    The gel formulation of the invention has good biocompatibility, and can maintain the high activity of cells without the use of cryopreservation with liquid nitrogen. The gel formulation can be directly stored at low temperature and is easy for transportation and use. The defrosting method is simple and quick, without the washing and centrifugation process of traditional methods. The risk of pollution and the effect on cell viability and performance caused by human intervention are reduced. The cell gel formulation can be used directly after thawing, and still maintains high cell activity and stem character after resuscitation.
    DESCRIPTION OF THE FIGURES
    Figure 1: Effects of different sodium alginate materials on cell viability.
    Figure 2: Effects of different concentrations of DMSO on cell viability.
    Figure 3: Effects of different concentrations of human serum albumin on cell viability.
    Figure 4: Effects of different concentrations of polyphenol on cell viability.
    Figure 5: Effects of different concentrations of dextran on cell viability.
    Figure 6: Effects of the combined use of polyphenols and dextran on cell viability.
    Figure 7: Comparison of the optimized formulation with the original gel formulation.
    Figure 8: Viability of mesenchymal stem cells from different sources in sodium alginate gels.
    Figure 9: Cell viability in gel formulations after long-term cryopreservation.
    Figure 10: Therapeutic effect of the cell gel formulation of the present invention on rat skin lesions.
    DESCRIPTION OF THE PREFERRED EMBODIMENTS
    The examples below illustrate and describe the present invention, but are not limiting.
    Source of materials: All materials are commercially available, unless otherwise indicated.
    Cell Sources: Mesenchymal stem cells are derived from, but not limited to, umbilical cord, placenta, amniotic tissue donated by healthy people, obtained by tissue separation, culture and passage.
    Preparation of a cell suspension: Mesenchymal stem cells were cultured in a conventional manner at 37 ° C., under a condition of 5% of CCF using a DMEM medium containing 10% fetal calf serum (complete medium) until that the confluence of the cells is approximately 90%, digested using 0.25% trypsin, then the digestion was stopped using the complete medium above. The harvested cell suspension was centrifuged and the supernatant was discarded. The cell pellet was suspended in a DMEM medium without serum and the mesenchymal stem cells were adjusted to 0.4 χ 10 6 ~ 2.5 χ 10 7 / ml.
    Calculation of cell viability: Cell viability (%) = total number of living cells / (total number of living cells + total number of dead cells) χ 100% Calculation of cell yield: cell yield (%) = total number of living cells / total number of original living cells x 100%
    EXAMPLE 1 Screening of sodium alginate
    Sodium alginate powders of 200 mesh, 100 mesh and 50 mesh are formulated respectively in 1%, 2%, 3% solution of sodium alginate. 3% dimethyl sulfoxide, 5% propylene glycol, then the reanimated mesenchymal stem cells are added to the sodium alginate solution formulated and well mixed, the formulation is placed in a refrigerator at 4 ° C. Cell viability and cell yield are compared at 0, 1.6 hours. Experimental results show that 200 mesh sodium alginate is the most suitable size for cell survival (Fig. 1).
    EXAMPLE 2 Screening and Optimization of the Gel Formulation
    In order to optimize the best gel formulation to maintain cell viability, the ingredients of the formulation are screened one by one to select the best strategy:
    (1) Screening of the gel concentration of sodium alginate
    A 200 mesh sodium alginate powder is added to the phosphate solution to prepare a 1%, 2%, 3%, 4% aqueous sodium alginate solution to observe the clarity and fluidity of the gel. The components of the phosphate solution here are 0.02% potassium chloride, 0.0047% magnesium chloride, 0.158% hydrogenated sodium phosphate, 0.020% dihydrogenated sodium phosphate, 0.8% sodium chloride. sodium and water for injection. 1% sodium alginate gels,
    2% and 3% show good clarity, and 4% sodium alginate gel shows insolubility problem. The fluidity of the gel decreased with increasing concentration of sodium alginate. A 3% sodium alginate gel shows the lowest fluidity and the best adhesion. After synthesizing the clarity and fluidity results, 3% sodium alginate was ultimately selected.
    (2) Screening of DMSO concentration
    The experiment was divided into four groups:
    Groups Components Sodium alginate Dimethylsulfoxide Propyleneglycol Mesenchymal stem cell 1 3% 0% 5% + 2 3% 1.5% 5% + 3 3% 3% 5% + 4 3% 10% 5% +
    The components of each group are carefully mixed to prepare the stem cell gel formulations. The stem cell gel formulations before being frozen are taken as a day -1 sample for counting and then cryostocked overnight. Three stem cell gel formulations are taken at 1 hour, 6 hours and 24 hours after resuscitation to measure cell viability and cell yield. Experimental results indicate that dimethyl sulfoxide at
    1.5% and 3% in the gel had good cell protection. This formula can reduce the dosage of dimethyl sulfoxide based on the principle of protecting cell viability (Fig. 2).
    (3) Screening of human serum albumin concentration
    The experiment was divided into four groups:
    Groups Components Alginateofsodium Dimethylsulfoxide Propylene glycol Serum albuminhuman Mesenchymal stem cell 1 3% 3% 5% 0% + 2 3% 3% 5% 0.5% + 3 3% 3% 5% 1% + 4 3% 3% 5% 2% +
    The above components are thoroughly mixed to prepare a stem cell gel formulation, and the stem cell gel formulation was taken as day -1 for a count before being frozen. The gel formulations are frozen at -80 ° C using the unscheduled cooling method, then 3 gel formulation samples are resuscitated on days 0, 1, 7 respectively, to detect cell viability and cell yield. Experimental results show that human serum albumin can better protect frozen cells, and ultimately a concentration of 2% is chosen as the concentration of human serum albumin in the gel formulation. (Fig. 3).
    (4) Selection of polyphenols and dextran 10 1) Screening of the polyphenol concentration
    The experiment was divided into five groups:
    Groups Components Alginateofsodium Dimethylsulfoxide Propyleneglycol Serumalbuminhuman polyphenol Mesenchymal stem cell 1 3% 3% 5% 2% 0% + 2 3% 3% 5% 2% 0.5% + 3 3% 3% 5% 2% 1% + 4 3% 3% 5% 2% 2% + 5 3% 3% 5% 2% 5% +
    The above components from each group are mixed thoroughly to prepare a stem cell gel formulation, and the stem cell gel formulation is taken as day -1 for a count before being frozen. The gel formulations are frozen at -80 ° C using the unscheduled cooling method, then 3 gel formulation samples are resuscitated on days 0, 1, 7 respectively, to detect cell viability and cell yield. The experimental results show that polyphenol can better protect frozen cells, and in the end a concentration of 2% is chosen as the concentration of polyphenols (Fig. 4).
    2) Screening of dextran concentration The experiment was divided into five groups:
    Groups Components Sodium alginate Dimethylsulfoxide Propyleneglycol Serumalbuminhuman dextran Cellstrainmesenchymateuse 1 3% 3% 5% 2% 0% + 2 3% 3% 5% 2% 1% + 3 3% 3% 5% 2% 2% + 4 3% 3% 5% 2% 3% + 5 3% 3% 5% 2% 6% +
    The above components are mixed thoroughly to prepare a stem cell gel formulation, and the stem cell gel formulation is taken as day -1 for a count before being frozen. The gel formulations are frozen at -80 ° C using an unscheduled cooling method, then 3 gel formulation samples are resuscitated on days 0, 1, 7 respectively, to detect cell viability and cell yield. The experimental results show that dextran can better protect frozen cells, and in the end a concentration of 6% is chosen as the concentration of dextran (Fig. 5).
    3) Combined use of polyphenol and dextran
    The experience was divided into four groups:
    Components Groups 1 2 3 4 Sodium alginate 3% 3% 3% 3% Dimethylsulfoxide 3% 3% 3% 3% Propylene glycol 5% 5% 5% 5% Serum albuminhuman 2% 2% 2% 2% Stem cell + + + + polyphenol - - + +
    dextran - + - +
    The above components are carefully mixed. Twelve stem cell formulations are prepared in each group. The stem cell gel formulation is taken as on day -1 for a count before cryopreservation, then frozen at -80 ° C. with the non-programmed cooling method. After days 0, 1, 7, a gel formulation is revived in each group respectively, to test cell viability and performance. We can see from the experimental results that the best gel formula to maintain cell viability is 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenols, 6% dextran , 2% albumin from human blood (Fig. 6).
    Example 3 Study on the rate of viability after resuscitation and on the long-term stability of the optimized formulation.
    In order to study the rate of cell viability after resuscitation and the long-term stability of the formulation of the present invention, the control gel formula and the optimized formula are compared. Here, the control gel formula is composed of 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, and the optimized gel formulation of the present invention was composed of 3% sodium alginate, 3 % dimethyl sulfoxide, 5% propylene glycol, 2% polyphenols, 6% dextran, 2% albumin from human blood. The cell gel formulation was taken as day -1 for a count before being frozen, and frozen at -80 ° C with unscheduled cooling methods. On day 0, three gel samples from each group are taken for resuscitation, and then placed at 4 C. 6 hours later, cell viability and cell yield are measured. After 7, 30 and 90 days of cryopreservation, the three gel samples from each group are taken to be resuscitated to measure cell viability and cell yield. The experimental results show that the formulation of the present invention improves the lifespan of cells after resuscitation, and significantly enhances the long-term preservation capacity, allowing cells to be kept alive for a long period under nitrogen-free conditions. liquid (Fig. 7).
    he
    Example 4 Detection of the Viability of Mesenchymal Stem Cells Derived from Different Tissues in Sodium Alginate Gels
    The effects of two gel formulations on the viability of mesenchymal stem cells from different tissues are compared. Formula 1, 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenols, 6% dextran, 2% albumin from human blood, and formula 2, 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 5% polyphenols, 10% dextran, 1% human blood albumin are prepared in a gel solution. The gel solutions are mixed with mesenchymal stem cells derived from bone marrow, derived from umbilical cord and derived from placenta, respectively. After mixing the gel solution and the mesenchymal stem cells are frozen at -80 ° C according to the unscheduled cooling method. After 0, 1 and 15 days of cryopreservation, the viability and the cell yield of the gel samples from each group are respectively detected. The experimental results show that the two formulations of sodium alginate gel are good supports for and can maintain the activity of mesenchymal stem cells from various tissues (Fig. 8).
    Example 5 Detection of cell viability in a gel formulation after long-term cryopreservation
    The effects of two gel formulations on cell stability are compared. The formula 1.3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenols, 6% dextran, 2% albumin from human blood, and formula 2, 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 5% polyphenols, 10% dextran, 1% albumin from human blood, are respectively prepared in gel solutions. After mixing the gel solution and the mesenchymal stem cells, are frozen at -80 ° C. with the non-programmed cooling method. At 0, 1, 3, 6, 15 and 18 months, three cell gel formulations are taken to detect cell viability, cell yield, phenotype and differentiation ability, respectively. The experimental results show that these two cell gel formulations can be stored at -80 ° C for 18 months with stability (Fig. 9).
    Example 6 Therapeutic Effect of the Cell Gel Formulation of the Present Invention on Rat Skin Lesions
    Skin lesion rat models are constructed, and each rat has two sores. The rat models are divided into four groups, including a blank group, a high dose group (3 x 10 6 cells), a medium dose group (1 x 10 6 ) and a low dose group (0.2 x 10 6 ). The wound size is measured every 3 to 4 days to compare the degree of wound recovery in each group. The results show that the cell gel started working on the third day, and the wound healing speed is faster than that of the blank control group, suggesting that a cell gel formulation may promote wound healing. dose-dependent wound (Fig. 10).
    权利要求:
    Claims (10)
    [1" id="c-fr-0001]
    Claims
    1. Gel formulation to maintain the viability of cryopreserved cells, comprising the following components in percentage by mass: 1% -3% sodium alginate, 1% -5% dimethyl sulfoxide, 1% -5% propylene glycol , 1% -5% polyphenol, 2% -10% dextran, 1% -5% human serum albumin, and additional water or a saline solution of phosphoric acid.
    [2" id="c-fr-0002]
    2. Gel formulation according to claim 1, in which the gel formulation comprises: 3% sodium alginate, 3% dimethyl sulfoxide, 5% propylene glycol, 2% polyphenol, 6% dextran, 2% human serum albumin, and additional water or saline phosphoric acid;
    in which the saline phosphoric acid solution consists of 0.02% potassium chloride, 0.0047% magnesium chloride, 0.1158% hydrogenated disodium phosphate, 0.020%, dihydrogenated sodium phosphate, 0.8% sodium chloride and water for injection.
    [3" id="c-fr-0003]
    3. Gel formulation according to claim 1, in which the sodium alginate has a viscosity of 600 to 1000 cps and a particle size of 200 mesh, and the gel formulation has a pH of 6.5 to 7, 5.
    [4" id="c-fr-0004]
    4. A stem cell gel formulation comprising mesenchymal stem cells which are not embryonic human stem cells and the gel formulation according to any one of claims 1 to 3.
    [5" id="c-fr-0005]
    5. A stem cell gel formulation according to claim 4, wherein there are between 0.5 x 10 6 and 3 χ 10 6 mesenchymal stem cells per 1 ml of the gel formulation.
    [6" id="c-fr-0006]
    6. A stem cell gel formulation according to claim 5, wherein the mesenchymal stem cells are derived from umbilical, placental or amniotic tissue.
    [7" id="c-fr-0007]
    7. A stem cell gel formulation according to claim 4, wherein the stem cells can be stored for 18 months under conditions of -80 ° C to -60 ° C by unscheduled cooling, and after resuscitation of the gel formulation. of stem cells, cell viability of these is as high as more than 90%, and when resuscitated stem cells are stored at 4 ° C for 6 hours, cell viability of these is as high as more than 80 %.
    [8" id="c-fr-0008]
    8. Method for preparing the stem cell gel formulation according to claim 4, further comprising the following steps consisting in: (1) dissolving and swelling sodium alginate powder in ultra pure water or a phosphate solution at room temperature for 24 to 48 hours; (2) stir the mixture until homogeneous; and add the remaining components to the mixture to formulate the
    5 gel formulation according to claim 1; (3) leave the gel formulation obtained in step (2) to stand for a certain time, then add mesenchymal stem cells which are not embryonic human stem cells therein; (4) mix uniformly, and the stem cell rich gel formulation according to claim 4 is prepared.
    [9" id="c-fr-0009]
    9. The method of claim 8, wherein the sodium alginate has a viscosity of 600 to 1,000 cps and a particle size of 200 mesh, and the gel formulation has a pH of 6.5 to 7.5 .
    10. Use of the stem cell gel formulation according to claim 4 for the preparation of medicaments for the treatment of a skin injury or a
    [10" id="c-fr-0010]
    15 mucosal lesion.
    3057
    1/4
    Cell viability
    100.00%
    00-00%
    80.00%
    70.00%
    60.00%
    50.00%
    Time (hour)
    50 mesh1%
    50 mesh2%
    50 mesh3%
    1ÜOmeshl%
    10Omesh2%
    10Gmesh3%
    20Omesh1%
    2Q0mesh2%
    200mesh3%
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    申请号 | 申请日 | 专利标题
    CN201610878608.3A|CN106538512B|2016-10-08|2016-10-08|A kind of active stem cell gel preparation of holding freeze-stored cell and its application|
    CN2016108786083|2016-10-08|
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