NL2034586B1 - Repair injection containing mesenchymal stem cells - Google Patents
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- NL2034586B1 NL2034586B1 NL2034586A NL2034586A NL2034586B1 NL 2034586 B1 NL2034586 B1 NL 2034586B1 NL 2034586 A NL2034586 A NL 2034586A NL 2034586 A NL2034586 A NL 2034586A NL 2034586 B1 NL2034586 B1 NL 2034586B1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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Abstract
Disclosed is a repair injection containing mesenchymal stem cells, and relates to the field of 5 biotechnology. The repair injection includes mesenchymal stem cells and an injection mixture, wherein the injection mixture includes the following components in parts by weight: 3 - 5 parts of human serum albumin, 10 - 15 parts of magnetic nano sustained-release microspheres, 0.05 - 0.1 part of low-molecular-weight heparin calcium, 0.3 - 0.7 part of vitamin C, 0.001 - 0.003 part of Panax notoginseng saponins, 0.01 - 0.02 part of stabilizer and 100 parts of physiological saline; 10 each millilitre of the injection mixture includes 0.5 x 107- 2 x 107 mesenchymal stem cells. After experimental verification, it is found that the repair injection has good stability and may be stored and used for a longer time. Animal experiments have shown that the repair injection may reduce the stimulation of blood vessels and facilitate angiogenesis. Therefore, the repair injection containing mesenchymal stem cells prepared by the present invention has good application 15 prospects.
Description
REPAIR INJECTION CONTAINING MESENCHYMAL STEM CELLS
The invention relates to the field of biotechnology, and in particular to a repair injection containing mesenchymal stem cells.
Mesenchymal stem cells (MSC) are an important member of the stem cell family, which originate from the mesoderm in the early stage of development and belong to pluripotent stem cells. Initially found in bone marrow, MSC has attracted increasing attention because of its multi-directional differentiation potential, hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication.
At present, it has been revealed that human MSC may avoid allogeneic recognition by secreting cytokines, interfere with the functions of dendritic cells and T- cells, and produce local immunosuppressive microenvironment. It is also revealed that when human MSC is exposed to an inflammatory environment characterized by the increase of local level of y-interferon, the immunomodulatory function of cells may be enhanced. In other words, MSC may be used for tissue regeneration and immunotherapy. However, most clinical MSC are used in a form of injections, which is one of the reasons why researchers have studied more and more injections of mesenchymal stem cells in recent years. However, because the active ingredient in mesenchymal stem cell injections is stem cells, the traditional injection is prepared with sodium chloride, DMSO and other excipients to preserve MSC, but there are some problems, such as obviously reducing the activity of MSC and its original function. Therefore, there is an urgent need for an injection with good stability that may ensure the original function of MSC not to be reduced, so as to solve the technical problems existing in the field of MSC injection at present.
The objective of the present invention is to provide a repair injection containing mesenchymal stem cells to solve the problems existing in the prior art, and the repair injection has good stability and may reduce the stimulation to blood vessels.
In order to achieve the above objective, the present invention provides the following schemes.
The invention provides a repair injection containing mesenchymal stem cells, including mesenchymal stem cells and an injection mixture, wherein the injection mixture includes the following components in parts by weight: 3-5 parts of human serum albumin, 10-15 parts of magnetic nano sustained-release microspheres, 0.05-0.1 part of low-molecular-weight heparin calcium, 0.3-0.7 part of vitamin C, 0.001-0.003 part of Panax notoginseng saponins, 0.01-0.02 part of stabilizer and 100 parts of physiological saline;
each millilitre of the injection mixture includes 0.5 x 107-2 x 107 mesenchymal stem cells.
Preferably, the injection mixture includes the following components in parts by weight: 3 parts of human albumin, 12 parts of magnetic nano sustained-release microspheres, 0.06 part of low-molecular-weight heparin calcium, 0.35 part of vitamin C, 0.001 part of Panax notoginseng saponins, 0.01 part of stabilizer and 100 parts of physiological saline.
Preferably, the mesenchymal stem cells are adipose mesenchymal stem cells.
Preferably, a preparation method of the adipose mesenchymal stem cells includes the following steps: (1) cutting the isolated adipose tissue into fat particles with a diameter of 3-5 mm, adding trypsin according to a same volume ratio for digestion, shaking and placing, and sucking a lower liquid; (2) inducing and culturing the lower liquid with an inducing liquid to obtain the adipose mesenchymal stem cells; the induction liquid includes the following components: MSCBM medium + 5% ultroGRO +1 0 mg/ml insulin + 1 uM dexamethasone + 100 uM indomethacin + 500 uM IBMX.
Preferably, in the step (1), digestion conditions are: digesting with 0.1% trypsin at 37°C for 30 min; and shaking conditions are: shaking at 2500 rpm for 15 min, and then placing for 20 min.
Preferably, in the step (2), the lower liquid and the inducing liquid are mixed according to a volume ratio of 1:10.
Preferably, in the step (2), conditions of inducing and culturing are: culturing at a constant temperature of 37°C for 3 - 8 days.
The invention discloses the following technical effects.
The present invention prepares a repair injection by mixing adipose mesenchymal stem cells and an injection mixture. Specifically, by adding magnetic nano sustained-release microspheres to the injection, the loading rate of adipose mesenchymal stem cells and other drug components is improved, and the drug release effect is sustained and the stability is improved. At the same time, because it contains magnetism, it has stronger targeting. Human serum albumin, low molecular weight heparin calcium, vitamin C, and Panax notoginseng saponins not only provide a good microenvironment for adipose mesenchymal stem cells, but also provide antibacterial, anti-inflammatory, and antioxidant properties to lay the foundation for adipose mesenchymal stem cells to have long-term cell viability. After experimental verification, it is found that the repair injection has good stability and may be stored and used for a longer time. Animal experiments have shown that the repair injection may reduce the stimulation of blood vessels and facilitate angiogenesis. Therefore, the repair injection containing mesenchymal stem cells prepared by the present invention has good application prospects.
Various exemplary embodiments of the present invention will now be described in detail, which should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and embodiments of the present invention.
It should be understood that the terms described in the present invention are only for describing specific embodiments, and are not intended to limit the present invention. In addition, as for the numerical range in the present invention, it should be understood that every intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Intermediate values within any stated value or stated range and every smaller range between any other stated value or intermediate values within the stated range are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention relates. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe methods and/or materials related to the documents. In case of conflict with any incorporated documents, the contents of this specification shall prevail.
Without departing from the scope or spirit of the invention, it is obvious to those skilled in the art that many modifications and changes may be made to the specific embodiments of the specification of the invention. Other embodiments derived from the description of the present invention will be apparent to the skilled person. The specification and examples of this application are only exemplary.
As used herein, the terms “including”, “comprising”, “having”, “containing”, etc. are all open terms, and they mean including but not limited to.
Embodiment 1 A repair injection containing mesenchymal stem cells
The repair injection includes mesenchymal stem cells and an injection mixture, wherein the injection mixture includes the following components in parts by weight: 3 parts of human albumin, 10 parts of magnetic nano sustained-release microspheres, 0.05 part of low-molecular- weight heparin calcium, 0.3 part of vitamin C, 0.001 part of Panax notoginseng saponins, 0.01 part of stabilizer and 100 parts of physiological saline; each millilitre of the injection mixture includes 0.5 x 107 mesenchymal stem cells. (1) A preparation method of the adipose mesenchymal stem cells includes: 10 ml of autologous adipose tissue is extracted from the patient, the blood in the adipose tissue is washed with cleaning solution, and the adipose tissue is crushed into fat particles with a diameter of 5 mm with scissors. The fat particles are digested with equal volume of 0.1% trypsin at 37°C for 30 min, then shaken at 2500 rpm with a shaker for 15 min, then placed for 20 min, and a lower liquid is sucked. The sucked lower liquid is mixed with the induced culture solution according to a volume ratio of 1:10, and they are moved to a culture bottle filled with induction culture solution. Then the culture bottle is transferred to a carbon dioxide incubator to culture at 37°C for 3 days. The induced culture solution is changed every two days and passed for three generations continuously to obtain the adipose mesenchymal stem cells for later use. (2) A preparation of the Panax notoginseng saponins: 300 ug of Panax notoginseng saponins powder is dissolved in 100 ml of sterile distilled water, and then sterile filtered to prepare Panax notoginseng saponins solution for later use. (3) human albumin, magnetic nano sustained-release microspheres, low-molecular-weight heparin calcium, vitamin C, Panax notoginseng saponins, stabilizers and physiological saline are evenly mixed according to the proportion of the above formula, and then the prepared adipose mesenchymal stem cells are added to obtain the repair injection containing mesenchymal stem cells.
Embodiment 2 A repair injection containing mesenchymal stem cells
The repair injection includes mesenchymal stem cells and an injection mixture, wherein the injection mixture includes the following components in parts by weight: 3 parts of human albumin, 12 parts of magnetic nano sustained-release microspheres, 0.06 part of low-molecular- weight heparin calcium, 0.35 part of vitamin C, 0.001 part of Panax notoginseng saponins, 0.01 part of stabilizer and 100 parts of physiological saline; each millilitre of the injection mixture includes 1.5 x 107 mesenchymal stem cells. (1) A preparation method of the adipose mesenchymal stem cells includes: 10 ml of autologous adipose tissue is extracted from the patient, the blood in the adipose tissue is washed with cleaning solution, and the adipose tissue is crushed into fat particles with a diameter of 5 mm with scissors. The fat particles are digested with equal volume of 0.1% trypsin at 37°C for 30 min, then shaken at 2500 rpm with a shaker for 15 min, then placed for 20 min, and a lower liquid is sucked. The sucked lower liquid is mixed with the induced culture solution according to a volume ratio of 1:10, and they are moved to a culture bottle filled with induction culture solution. Then the culture bottle is transferred to a carbon dioxide incubator to culture at 37°C for 5 days. The induced culture solution is changed every two days and passed for three generations continuously to obtain the adipose mesenchymal stem cells for later use. (2) A preparation of the Panax notoginseng saponins: 300 ug of Panax notoginseng saponins powder is dissolved in 100 ml of sterile distilled water, and then sterile filtered to prepare Panax notoginseng saponins solution for later use.
(3) human albumin, magnetic nano sustained-release microspheres, low-molecular-weight heparin calcium, vitamin C, Panax notoginseng saponins, stabilizers and physiological saline are evenly mixed according to the proportion of the above formula, and then the prepared adipose mesenchymal stem cells are added to obtain the repair injection containing mesenchymal stem 5 cells.
Embodiment 3 A repair injection containing mesenchymal stem cells
The repair injection includes mesenchymal stem cells and an injection mixture, wherein the injection mixture includes the following components in parts by weight: 5 parts of human albumin, 15 parts of magnetic nano sustained-release microspheres, 0.1 part of low-molecular- weight heparin calcium, 0.7 part of vitamin C, 0.003 part of Panax notoginseng saponins, 0.02 part of stabilizer and 100 parts of physiological saline; each millilitre of the injection mixture includes 2 x 10” mesenchymal stem cells. (1) A preparation method of the adipose mesenchymal stem cells includes: 10 ml of autologous adipose tissue is extracted from the patient, the blood in the adipose tissue is washed with cleaning solution, and the adipose tissue is crushed into fat particles with a diameter of 5 mm with scissors. The fat particles are digested with equal volume of 0.1% trypsin at 37°C for 30 min, then shaken at 2500 rpm with a shaker for 15 min, then placed for 20 min, and a lower liquid is sucked. The sucked lower liquid is mixed with the induced culture solution according to a volume ratio of 1:10, and they are moved to a culture bottle filled with induction culture solution. Then the culture bottle is transferred to a carbon dioxide incubator to culture at 37°C for 8 days. The induced culture solution is changed every two days and passed for three generations continuously to obtain the adipose mesenchymal stem cells for later use. (2) A preparation of the Panax nofoginseng saponins: 300 ug of Panax notoginseng saponins powder is dissolved in 100 ml of sterile distilled water, and then sterile filtered to prepare Panax notoginseng saponins solution for later use. (3) human albumin, magnetic nano sustained-release microspheres, low-molecular-weight heparin calcium, vitamin C, Panax notoginseng saponins, stabilizers and physiological saline are evenly mixed according to the proportion of the above formula, and then the prepared adipose mesenchymal stem cells are added to obtain the repair injection containing mesenchymal stem cells.
Embodiment 4 Detection of cell viability of adipose mesenchymal stem cells after cryopreservation of injection
The repair injections prepared in Embodiment 1-Embodiment 3 of the present invention are placed in liquid nitrogen, frozen for 1 month, 3 months, 6 months, 9 months and 12 months respectively, then taken out of liquid nitrogen, put in a water bath at 37°C, and resuscitated for 1-2 min. The cells are counted by trypan blue staining method, and the survival rates of mesenchymal stem cells are calculated. At the same time, adipose mesenchymal stem cells prepared according to the preparation methods of adipose mesenchymal stem cells in step (1) of Embodiment 1-Embodiment 3 are used as Controls 1-3, respectively. The injection mixture in each embodiment is replaced by the same amount of physiological saline. The results are shown in Table 1.
Table 1 Test results
Injection
Embodiment 1 95.67% 93.21% 91.33% 90.85% 89.97%
Embodiment 2 97.86% 96.34% 93.41% 92.16% 91.02%
Embodiment 3 | 96.35% 95.12% 92.31% 90.89% 90.00%
Control 1 80.78% 70.35% 50.70% 40.33% 35.12% 82.31% 73.11% 60.12% 46.10% 40.45% 80.75% 70.15% 52.12% 40.38% 30.29%
As may be seen from Table 1, compared with Controls 1-3, the repair injection provided by the invention has good stability, and the adipose mesenchymal stem cells may be preserved for a longer time. After 12 months, the survival rate of adipose mesenchymal stem cells all exceeds 89.97%, indicating that the repair injection provided by the invention may be used to preserve adipose mesenchymal stem cells for a long time.
Embodiment 5 Vascular stimulation test
Eight adult healthy rabbits are randomly divided into four groups, namely, group 1, group 2, group 3 and control group. Rabbits in group 1, group 2 and group 3 are injected with the repair injections of Embodiment 1-Embodiment 3 through the ear vein of rabbits, rabbits in the control group are injected with normal saline through the ear vein. They are all injected continuously for 5 days. The reactions (such as congestion, oedema, etc.) of blood vessels and surrounding tissues at the injection site are observed at different times after administration according to the scoring criteria in Table 2. On the fifth day, 48 hours after administration, the rabbits and the injection site are observed and examined many times. Then the animals are killed, and the blood vessels and surrounding tissues at the injection site are taken for histopathological examination. The results of stimulation test are shown in Table 3.
Table 2 Scoring criteria of vascular stimulation reaction
Stimulation reaction
No obvious reaction is observed at the administration site.
Slight congestion, redness, and swelling of the venous ] vessels at the administration site
Moderate congestion, redness, and swelling of the 5 venous vessels at the administration site
Severe congestion of venous vessels and severe redness and swelling of surrounding tissues at the 3 administration site
Table 3 Results of stimulation score of the repair injection
Repair injection
Group 1 of embodiments 0
Group 2 of embodiments 0
Group 3 of embodiments 0
As may be seen from the results in Table 3, the repair injection prepared by the invention may reduce the stimulation of the injection to blood vessels and is beneficial to blood vessel repair.
The above-mentioned embodiments only describe the preferred mode of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, all kinds of modifications and improvements made by ordinary technicians in the field to the technical scheme of the present invention should fall within the protection scope determined by the claims of the present invention.
Claims (7)
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| CN106237313A (en) * | 2016-09-30 | 2016-12-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells injection and its preparation method and application |
| CN108904783A (en) * | 2018-08-08 | 2018-11-30 | 广州杜德生物科技有限公司 | A kind of stem cell drugs preparing treatment gonitis using autologous fat stem cell |
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| CN106237313A (en) * | 2016-09-30 | 2016-12-21 | 广州赛莱拉干细胞科技股份有限公司 | A kind of umbilical cord mesenchymal stem cells injection and its preparation method and application |
| CN108904783A (en) * | 2018-08-08 | 2018-11-30 | 广州杜德生物科技有限公司 | A kind of stem cell drugs preparing treatment gonitis using autologous fat stem cell |
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