CN112655702B - Solution for umbilical cord mesenchymal stem cells, umbilical cord mesenchymal stem cell preparation, preparation method and application - Google Patents
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Abstract
The invention belongs to the field of umbilical cord mesenchymal stem cell application, and particularly relates to a solution for umbilical cord mesenchymal stem cells and application thereof, an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis diseases, and a preparation method and application thereof. The invention provides a solution for umbilical cord mesenchymal stem cells, which comprises clinical-grade DMSO, human serum albumin, low-molecular dextran, trehalose, melatonin and a compound electrolyte solution. The solution can effectively stabilize the cell number of the umbilical cord mesenchymal stem cells subjected to cryopreservation, can also effectively improve the cell activity of the umbilical cord mesenchymal stem cells subjected to cryopreservation, and can maintain the immunosuppressive function of the umbilical cord mesenchymal stem cells subjected to cryopreservation. The solution containing the umbilical cord mesenchymal stem cells is convenient to transport and use for a long distance, and the industrial production and use of the umbilical cord mesenchymal stem cells can be effectively promoted.
Description
Technical Field
The invention belongs to the field of umbilical cord mesenchymal stem cell application, and particularly relates to a solution for umbilical cord mesenchymal stem cells and application thereof, an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis diseases, and a preparation method and application thereof.
Background
Ulcerative Colitis (UC) is a chronic nonspecific inflammatory bowel disease with unknown etiology mainly involving the colon and rectum, and is clinically manifested by diarrhea, intermittent hematochezia or purulent stool, abdominal pain, vomiting, etc. Ulcerative colitis is a chronic intestinal inflammation, the pathogenesis mainly involves immune factors, genetic factors, infection factors, environmental factors and the like, and the current research shows that ulcerative colitis is mainly caused by intestinal endothelial cell injury caused by human immune abnormality, for example, dendritic cells (DC cells) excessively activate Th1 and Th2 cells to produce a large amount of TNF-a or the expression of immune regulatory factors is down-regulated to cause immune abnormal activation. Therefore, the treatment of ulcerative colitis in clinic mainly aims at immune regulation, and the TNF-a inhibitor is a common medicament.
For the treatment of ulcerative colitis, the treatment mainly comprises three main types of medicaments, namely aminosalicylic acid, adrenocortical hormone and immunosuppressant, and the patients who are not resistant to the medicament treatment or have an emergency outbreak can adopt intervention or operation treatment. The traditional means of operations, medicines and the like only relieve surface inflammatory reaction, and treat symptoms and root causes, so the treatment effect is not ideal, the course of disease is long, and the treatment effect is often repeated.
Disclosure of Invention
In order to solve the problems, the invention provides a solution for umbilical cord mesenchymal stem cells and application thereof, an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis diseases and a preparation method and application thereof. The solution for umbilical cord mesenchymal stem cells provided by the invention can effectively stabilize the cell number of the umbilical cord mesenchymal stem cells subjected to cryopreservation and improve the activity of the cells. Meanwhile, the umbilical cord mesenchymal stem cell preparation provided by the invention can reverse the course of ulcerative colitis, help patients get rid of the influence of abdominal pain and diarrhea on life and the toxic and side effects caused by medicines, and thoroughly cure ulcerative colitis.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a solution for umbilical cord mesenchymal stem cells, which comprises clinical-grade DMSO, human serum albumin, low-molecular dextran, trehalose, melatonin and a compound electrolyte solution;
preferably, in the solution for umbilical cord mesenchymal stem cells, the volume percentage content of clinical-grade DMSO is 5-10%, the mass concentration of human serum albumin is 1-6 g/mL, the mass concentration of low-molecular dextran is 1g/L, the mass concentration of trehalose is 1-5 g/L, and the mass concentration of melatonin is 0.05-0.5 g/L.
The invention also provides application of the solution for umbilical cord mesenchymal stem cells in cryopreservation of umbilical cord mesenchymal stem cells.
The invention also provides application of the solution for umbilical cord mesenchymal stem cells in improving the viability of umbilical cord mesenchymal stem cells after cryopreservation.
The invention also provides application of the umbilical cord mesenchymal stem cell solution in preparing an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis.
The present invention also provides an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis disease, comprising: the umbilical cord mesenchymal stem cell solution and the umbilical cord mesenchymal stem cells; the concentration of the umbilical cord mesenchymal stem cells in the preparation is 2 x 1051X 10 per ml7One per ml.
Preferably, the umbilical cord mesenchymal stem cell preparation comprises an injection.
Preferably, the concentration of the umbilical cord mesenchymal stem cells in the umbilical cord mesenchymal stem cell preparation is 2 × 1051X 10 per ml7One per ml.
The invention also provides a preparation method of the umbilical cord mesenchymal stem cell preparation, which comprises the following steps:
and mixing the umbilical cord mesenchymal stem cells with the umbilical cord mesenchymal stem cell solution to obtain the umbilical cord mesenchymal stem cell preparation.
The invention also provides the application of the umbilical cord mesenchymal stem cell preparation or the umbilical cord mesenchymal stem cell preparation prepared by the preparation method in preparing a medicament for treating ulcerative colitis.
The invention provides a solution for umbilical cord mesenchymal stem cells, which comprises clinical-grade DMSO, human serum albumin, low-molecular dextran, trehalose, melatonin and a compound electrolyte solution. The solution can effectively stabilize the cell number of the umbilical cord mesenchymal stem cells subjected to cryopreservation, can also effectively improve the cell activity of the umbilical cord mesenchymal stem cells subjected to cryopreservation, and can maintain the immunosuppressive function of the umbilical cord mesenchymal stem cells subjected to cryopreservation. The preparation containing the umbilical cord mesenchymal stem cells is convenient to transport and use for a long distance, and the industrial production and use of the umbilical cord mesenchymal stem cells can be effectively promoted. Meanwhile, when the solution for umbilical cord mesenchymal stem cells provided by the invention is applied to the frozen storage of umbilical cord mesenchymal stem cells, the solution also has the characteristics of convenience and rapidness, and the umbilical cord mesenchymal stem cell preparation frozen by the solution for umbilical cord mesenchymal stem cells provided by the invention is recovered to normal temperature when in use, and can be used immediately.
The invention also provides an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis diseases, which is used for correcting immune disorder in an ulcerative colitis patient and preventing the autoimmune disorder from continuously damaging intestinal tracts; and meanwhile, a plurality of cell growth factors and repair factors are secreted to promote the healing of the ulcer of the pathological intestinal section, thereby achieving the aim of fundamentally treating the ulcerative colitis. Meanwhile, the umbilical cord mesenchymal stem cell preparation provided by the invention is a ready-to-use preparation, does not need to be eluted after the cells are recovered, can be directly injected and used in clinic, and has the advantages of convenience and quickness. According to the embodiment, after 20 days of treatment of the umbilical cord mesenchymal stem cell preparation provided by the invention, the healing condition of the whole colonic ulcer of the mouse is up to more than 77%.
Drawings
FIG. 1 is a flow chart of the technical solution of the present invention;
FIG. 2 is a microphotograph of umbilical cord mesenchymal stem cells in the example;
FIG. 3 shows the flow test results of the example;
FIG. 4 shows the results of ELISA detection of IL-10 factor in examples;
FIG. 5 shows the results of ELISA detection of TNF-a factor in examples.
Detailed Description
The invention provides a solution for umbilical cord mesenchymal stem cells, which comprises clinical-grade DMSO, human serum albumin, low-molecular dextran, trehalose, melatonin and a compound electrolyte solution. The solution for umbilical cord mesenchymal stem cells provided by the invention can effectively stabilize the cell number of umbilical cord mesenchymal stem cells after long-time cryopreservation, can effectively maintain the cell activity of umbilical cord mesenchymal stem cells after long-time cryopreservation, and can also maintain the immunosuppressive function of umbilical cord mesenchymal stem cells after cryopreservation.
The umbilical cord mesenchymal stem cell solution provided by the invention comprises 5-10% of clinical grade DMSO (dimethyl sulfoxide), and is further preferably 6% of DMSO. In the invention, the clinical grade DMSO is used as a permeable cryoprotectant, so that the freezing point of cells can be lowered, and the formation of ice crystals in the freezing storage process is reduced, thereby reducing the damage to the cells.
The solution for umbilical cord mesenchymal stem cells provided by the invention comprises 1-6 g/mL, more preferably 3g/mL of human serum albumin by mass concentration. In the invention, the quality of the human serum albumin is used as an impermeable cryoprotectant, so that the content of free water in a solution is reduced, and the formation of ice crystals is reduced; meanwhile, because of the large molecular weight, the concentration of electrolyte in the solution is reduced, thereby reducing solute damage and greatly improving the survival rate of cells.
The solution for umbilical cord mesenchymal stem cells provided by the invention comprises 1g/L of low molecular dextran by mass concentration. In the invention, the low molecular dextran has the functions of stabilizing the osmotic pressure of cells and keeping the morphology and activity of the cells.
The solution for umbilical cord mesenchymal stem cells provided by the invention comprises 1-5 g/L of trehalose by mass concentration, and is more preferably 2 g/L. In the invention, the trehalose is used for maintaining the activity of intracellular biomacromolecules such as protein, enzyme, lipid and the like, thereby maintaining the activity of cells.
The solution for umbilical cord mesenchymal stem cells provided by the invention comprises 0.05-0.5 g/L of melatonin by mass concentration, and more preferably 0.1 g/L. In the invention, the melatonin plays a certain role in protecting the stress response of cells in the liquid nitrogen freezing process of the cells and reduces the damage to the cells in the freezing process.
In the invention, the balance of the solution for umbilical cord mesenchymal stem cells is compound electrolyte solution; the compound electrolyte solution has the functions of stabilizing the osmotic pressure of cells, maintaining the cell morphology and being used as a diluent during intravenous infusion of the cells.
The solution for umbilical cord mesenchymal stem cells provided by the invention obtains the technical effects of stabilizing the cell number of umbilical cord mesenchymal stem cells after long-time cryopreservation and maintaining the cell activity of umbilical cord mesenchymal stem cells after long-time cryopreservation by the proportion.
The composition of the solution is not particularly limited in the present invention, and a conventional commercially available product known to those skilled in the art may be used.
The invention also provides application of the solution for umbilical cord mesenchymal stem cells in cryopreservation of umbilical cord mesenchymal stem cells. The application provided by the invention is convenient for the preparation containing the umbilical cord mesenchymal stem cells to carry out long-distance transportation and use, and can effectively promote the industrial production and use of the umbilical cord mesenchymal stem cells.
The invention also provides application of the solution for umbilical cord mesenchymal stem cells in improving the viability of umbilical cord mesenchymal stem cells after cryopreservation. After the solution for umbilical cord mesenchymal stem cells is applied to cryopreservation of umbilical cord mesenchymal stem cells, flow detection shows that the umbilical cord mesenchymal stem cells subjected to long-term cryopreservation still have strong cell dryness, and the activity of the umbilical cord mesenchymal stem cells subjected to cryopreservation can be fully improved.
The invention also provides application of the umbilical cord mesenchymal stem cell solution in preparing an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis. In the present invention, the preparation is preferably an injection. The umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis provided by the invention can effectively improve the repair proportion of ulcerative colitis model mice ulcer, and can reach more than 77% at most.
The present invention also provides an umbilical cord mesenchymal stem cell preparation for treating ulcerative colitis disease, comprising: the solution for umbilical cord mesenchymal stem cells and umbilical cord mesenchymal stem cells are provided. In the present invention, the concentration of the umbilical cord mesenchymal stem cells in the preparation is preferably 2 × 1051X 10 per ml7One per ml. In the preparation provided by the invention, the concentration of the umbilical cord mesenchymal stem cells has safety and effectiveness. The cell concentration range of higher cell activity can be kept when the cells are frozen; the concentration range can avoid ineffectiveness in use and reduce the risk of venous embolism caused by overhigh concentration in use.
The invention also provides a preparation method of the umbilical cord mesenchymal stem cell preparation, which comprises the following steps:
and mixing the umbilical cord mesenchymal stem cells with the umbilical cord mesenchymal stem cell solution to obtain the umbilical cord mesenchymal stem cell preparation.
In the present invention, the preparation method preferably includes:
1. collecting tissues;
2. preparing umbilical cord mesenchymal stem cells;
3. and (3) preparing an umbilical cord mesenchymal stem cell preparation.
The schematic diagram of the preparation method is shown in figure 1.
The invention also provides the application of the umbilical cord mesenchymal stem cell preparation or the umbilical cord mesenchymal stem cell preparation prepared by the preparation method in preparing a medicament for treating ulcerative colitis.
In order to further illustrate the present invention, the following detailed description of the solution for umbilical cord mesenchymal stem cells and its application, the preparation of umbilical cord mesenchymal stem cells for treating ulcerative colitis and its preparation method and application are provided in connection with the accompanying drawings and examples, which should not be construed as limiting the scope of the present invention.
Examples
1. Preparation process of umbilical cord mesenchymal stem cell injection
(1) Collecting the umbilical cord specimen which meets the collection standard, and carrying out cell separation within 48 h. Abandoning the umbilical cord preservation solution, washing with PBS for 2-3 times, transferring the umbilical cord into a phi 10cm culture dish, and intercepting small sections of the umbilical cord in areas with good brightness, no damage and no edema, wherein each section is 2-4 cm. The umbilical cord is longitudinally split by using a tissue scissors, umbilical veins and umbilical arteries are removed, and the umbilical cord is washed for 4-8 times by using PBS. Using special scissors for umbilical cord tissue to cut residual tissue of umbilical cord, namely Wharton's jelly into 1mm3The tissue block is inoculated into a new culture dish of phi 10cm, 4ml of serum-free complete culture medium is supplemented after 24 hours, and half-amount liquid changing operation of supplementing 4ml with 3ml is carried out every 2-4 days. If obvious cell clone formation is found in the culture dish, the tissue blocks are discarded in time, 8ml of serum-free complete culture medium is supplemented, and the culture is continued. When the number of cell clones in each dish is increased to more than 4-8 and the cell fusion degree reaches 80% -90%, cell digestion is carried out.
(2) And (4) discarding the supernatant in the culture dish, and washing the cells at the bottom of the culture dish for 1-2 times by using PBS. The cells were digested with 0.05% Try-EDTA and stopped with fresh serum-free complete medium when the cells became rounder from the long spindle and floated. The cells were collected and mixed well, sampled and counted, and then centrifuged: centrifugation was carried out at 600g for 10min at 4 ℃. After centrifugation, the cell sediment is prepared by using 5-10% of clinical-grade DMSO (dimethyl sulfoxide), 1-6 g/mL of human serum albumin, 1g/L of low molecular dextran, 1-5 g/L of trehalose, 0.05-0.5 g/L of melatonin and 1 × 10 of compound electrolyte solution in proportion by volume6one/ml-5X 106Resuspending at a concentration of 1 × 10, mixing, and freezing to obtain P0 generation cells6Perml/tube 5X 106Per ml/tube. And (4) performing cell cryopreservation by adopting a gradient cooling or program control cooling mode.
(3) The cells were recovered from the P0 passage in liquid nitrogen in a 37 ℃ thermostat water bath. Resuspending the cell suspension and DMEM at a ratio of 1:9, and then carrying out the steps(2) The centrifugation conditions in (1) are centrifugation. Resuspending the mixture in serum-free medium according to 10000-12000/cm2Inoculating the cells at the density of 80-90% and harvesting the cells when culturing for 3-4 days, and obtaining cells of P1, P3 and P5 generations after multiple passages, wherein the frozen concentration of the cells except the cells of P5 generations is the same as that of the cells of P0 generations.
(4) The harvested P5 generation cells are resuspended by 5-10% of clinical grade DMSO in volume ratio, 1-6 g/mL of human serum albumin in mass concentration, 1g/L of low molecular dextran in mass concentration, 1-5 g/L of trehalose in mass concentration, 0.05-0.5 g/L of melatonin in mass concentration and the balance of compound electrolyte solution, and the cell concentration is 2 multiplied by 105 1X 10 per ml7Per ml; and filling the cell suspension into a cell cryopreservation bag with the volume of 30ml, cooling by using a program-controlled cooling instrument, and transferring into liquid nitrogen for long-term storage. Before use, the cell preparation needs to be placed in a 37 ℃ constant-temperature water bath for rapid recovery, and the thawed cell preparation can be used after being rewarmed to room temperature.
The umbilical cord mesenchymal stem cells in the whole preparation process keep high cell activity and proliferation multiple, the cell growth state is good, the cell form is in a long fusiform shape, and a microscopic picture of the umbilical cord mesenchymal stem cells is shown in figure 2.
2. And (3) detecting the phenotype of the umbilical cord mesenchymal stem cells by flow cytometry.
(1) Take 1.5X 106The umbilical cord mesenchymal stem cells (15 tubes can be made or 9 tubes can be equally divided) are centrifuged at 1500rpm for 7 min. The supernatant was discarded, 4ml of PBS was added to resuspend the cells, and the cells were centrifuged at 1500rpm for 7 min. 1.5ml PBS was added to resuspend the cells and the cell suspension was filtered using a nylon mesh.
(2) And taking 9 branch type on-machine test tubes, marking as 1-9, and adding a flow type antibody combination in advance (adding antibodies to different positions at the bottom of the on-machine test tube by using a liquid transfer machine to prevent cross of the flow type antibodies). 100ul of cells are added into each machine test tube (at least 1.0X 10 cells in each tube)5Individual cell), filtering the cell suspension through a 300-mesh cell sieve, fully shaking and uniformly mixing the antibody and the cell suspension, instantaneously centrifuging to enable the cell on the tube wall to sink to the bottom of the tube, adding redundant cells into the first tube, and adjusting and compensating for the later timeThe preparation is used.
(3) Incubate at 4 ℃ for 30min in the dark. 2ml PBS was added to resuspend the cells, and the cells were harvested after centrifugation at 1500rpm for 7 min. And adding 400ul of PBS for resuspension in the negative control tube, adding 100ul of PBS for resuspension in the single-staining compensation tube, supplementing the cells in the 100ul of negative control tube, and adding 200ul of PBS for resuspension in the detection tube.
(4) And (4) performing on-machine detection after the flow pipe is swirled, and performing data analysis. The flow assay results are shown in FIG. 3.
From the flow-through results of fig. 3, it was found that CD44, CD73, CD90 and CD105 were 90% or more, and CD34, CD45 and HLA-DR were 2% or less. The umbilical cord mesenchymal stem cells obtained by the preparation method provided by the invention have strong cell dryness and can fully improve the activity of the umbilical cord mesenchymal stem cells after cryopreservation.
3. And (3) detecting the immunosuppressive capacity of the umbilical cord mesenchymal stem cells by an ELISA method.
(1) Resuscitating the cryopreserved stem cells (at least 1.0X 10 stem cells are needed for each sample)6Individual cells) were seeded in 24-well plates overnight. Resuscitating PBMC were resuspended in 3mL 10% fetal bovine +1640 medium (PBMC at least 1.0X 10)7Individual cells), three wells of a 24-well plate were seeded and cultured overnight as well.
(2) PBMC are collected on the next day, 10% 1640 culture medium is used for metering to 18ml, and PHA with the final concentration of 3 mug/ml is added into a cell culture system for stimulating lymphoproliferation in a TNF-a detection project for detecting TNF-a. The stem cell culture medium was discarded and 1ml of PBMC cell suspension was added per well. Co-cultivation was started for 3 d.
(3) After co-culturing for 3 days, collecting cell culture supernatant, centrifuging for 10min at 1000g, collecting cell supernatant, and storing at-20 deg.C for use.
(4) TNF-a factor and IL-10 factor assays were performed according to ELISA reagent instructions.
Data analysis
Drawing a standard curve: and (3) taking the concentration of the standard substance as an abscissa and taking the corresponding OD value as an ordinate, drawing a linear regression curve (generally selecting four-parameter regression), and calculating the concentration value of each sample according to a curve equation. The concentration values of the samples were plotted and compared, as shown in FIGS. 4 and 5, in which FIG. 4 shows the ELISA detection result of IL-10 factor and FIG. 5 shows the ELISA detection result of TNF-a factor.
As can be seen from FIGS. 4 and 5, the mesenchymal stem cell pair provided by the invention can effectively inhibit TNF-a production and promote the release of IL10 factor. The inhibition of the TNF-a production indicates that the aggravation or canceration of the ulcer surface of the pathological intestinal section can be inhibited, and the promotion of the release of IL10 factor indicates that the healing of the ulcer surface of the pathological intestinal section can be effectively promoted.
4. And (3) carrying out safety and effectiveness test on the mesenchymal stem cells in the mouse model of the ulcerative colitis.
Balb/c mice were selected for 6-8 weeks in the test and randomly grouped according to the control group and the treatment group, with 40 mice per group. Continuously exposing with 2.0% DSS (dextran sulfate sodium) for 5 days, and replacing with new DSS solution every day, wherein each bottle contains 100ml of water; normal water was changed after day 5.
Treatment groups were injected with the cell preparation of the present invention tail vein on days 2 and 5 of DSS exposure. 0.2mL of 5.0X 10 per animal6Cell suspension/mL. And correspondingly injecting an equal volume of cell solvents (namely clinical-grade DMSO with the volume ratio of 5-10%, human serum albumin with the mass concentration of 1-6 g/L, low-molecular dextran with the mass concentration of 1g/L, trehalose with the mass concentration of 1-5 g/L, melatonin with the mass concentration of 0.05-0.5 g/L and compound electrolyte solution) into tail veins of the control group at the administration time point of the treatment group.
10 mice (subgroups) were taken on days 2, 5, 10 and 20 after administration of the mice, sacrificed by cervical dislocation, and the ratio of colonic ulcer repair was counted for 5 days, 10 days and 20 days of treatment, using the average ulcer length 2 days after release of the damaging agent DSS exposure as a basic relative value, and analyzed by t-test, the analysis results are shown in table 1.
TABLE 1 ratio of UC-MSC to ulcerative colitis model mouse ulcer repair
Note: the repair ratio (length of each subgroup ulcer-subgroup mean of 2 days of treatment)/subgroup mean of 2 days of treatment × 100%;
***: p <0.001 treatment vs model control group.
As can be seen from Table 1, the results are statistically significantly different (P <0.05), which indicates that the cell preparation provided by the invention can significantly promote the recovery process of the mouse ulcerative colitis caused by DSS exposure.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. An application of a solution for umbilical cord mesenchymal stem cells in improving the activity of umbilical cord mesenchymal stem cells after cryopreservation;
the solution for umbilical cord mesenchymal stem cells consists of clinical-grade DMSO, human serum albumin, low-molecular dextran, trehalose, melatonin and a compound electrolyte solution;
in the solution for umbilical cord mesenchymal stem cells, the volume percentage content of clinical-grade DMSO is 5-10%, the mass concentration of human serum albumin is 1-6 g/mL, the mass concentration of low-molecular dextran is 1g/L, the mass concentration of trehalose is 1-5 g/L, and the mass concentration of melatonin is 0.05-0.5 g/L.
2. An umbilical cord mesenchymal stem cell preparation for use in the treatment of an ulcerative colitis disease, said umbilical cord mesenchymal stem cell preparation comprising: the solution for umbilical cord mesenchymal stem cells and umbilical cord mesenchymal stem cells for use according to claim 1.
3. The umbilical cord mesenchymal stem cell preparation of claim 2, wherein the umbilical cord mesenchymal stem cell preparation comprises an injection.
4. The method of claim 2The umbilical cord mesenchymal stem cell preparation of (1), wherein the concentration of the umbilical cord mesenchymal stem cells in the umbilical cord mesenchymal stem cell preparation is 2 x 1051X 10 per ml7One per ml.
5. A method of preparing an umbilical cord mesenchymal stem cell preparation according to any one of claims 2 to 4, comprising the steps of:
mixing umbilical cord mesenchymal stem cells with the umbilical cord mesenchymal stem cell solution for the application of claim 1 to obtain the umbilical cord mesenchymal stem cell preparation.
6. Use of the umbilical cord mesenchymal stem cell preparation of any one of claims 2 to 4 or the umbilical cord mesenchymal stem cell preparation prepared by the preparation method of claim 5 in the preparation of a medicament for treating ulcerative colitis.
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