WO2019015584A1 - Milieu de croissance de cellules souches de pulpe dentaire humaine et procédé de préparation de cellules souches de pulpe dentaire humaine - Google Patents

Milieu de croissance de cellules souches de pulpe dentaire humaine et procédé de préparation de cellules souches de pulpe dentaire humaine Download PDF

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WO2019015584A1
WO2019015584A1 PCT/CN2018/095978 CN2018095978W WO2019015584A1 WO 2019015584 A1 WO2019015584 A1 WO 2019015584A1 CN 2018095978 W CN2018095978 W CN 2018095978W WO 2019015584 A1 WO2019015584 A1 WO 2019015584A1
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dental pulp
pulp stem
tissue
stem cells
medium
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PCT/CN2018/095978
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English (en)
Chinese (zh)
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姜舒
张芸
纪惜銮
杨顺
罗朝霞
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深圳市茵冠生物科技有限公司
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Publication of WO2019015584A1 publication Critical patent/WO2019015584A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0664—Dental pulp stem cells, Dental follicle stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • the invention relates to the technical field of stem cell preparation, in particular to a human dental pulp stem cell culture medium and a preparation method of human dental pulp stem cells.
  • Stem cells are cells with self-replication and multi-directional differentiation. They can continuously self-renew and differentiate into one or more cells that make up human tissues or organs under specific conditions, which can restore the ability of tissues to repair and repair their functions.
  • the researchers found a new adult stem cell in the pulp tissue of the deciduous deciduous teeth, which was named as the deciduous dental pulp stem cell. So far, many experiments have shown that the deciduous dental pulp stem cells have high self-renewal, colony formation and osteogenic differentiation. ability.
  • the deciduous dental pulp stem cells have the following advantages: 1) easy to take, more from the natural detachment and removal of the retained teeth, less damage to the donor's normal teeth; 2) without ethical restrictions, autologous transplantation can be performed to minimize immunity Risk of rejection and cross-infection; 3) Strong proliferative capacity and multi-directional differentiation potential.
  • the stem cells in the pulp have a low content and must be expanded in vitro to obtain a sufficient number of cells to meet the application requirements.
  • the dental pulp stem cell culture system mainly adopts a medium containing animal-derived fetal bovine serum.
  • the animal-derived fetal bovine serum may cause human immunological rejection to a certain extent, and increases the risk of clinical application of dental pulp stem cells.
  • the object of the present invention is to provide a human dental pulp stem cell culture medium and a method for preparing human dental pulp stem cells.
  • the dental pulp stem cells prepared by the culture medium provided by the invention have high yield and no human immunological rejection, and provide reliable seed cells for clinical tooth repair and regeneration.
  • the invention provides a human dental pulp stem cell culture medium, which comprises based on ⁇ -MEM medium, comprising human AB serum in a volume percentage of 5% to 10%, 100 ⁇ M ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin sodium and 100 mg/ml streptomycin.
  • the invention also provides a preparation method of human dental pulp stem cells based on the medium described in the above technical solution, comprising the following steps:
  • step 2) removing the pulp tissue from the disinfected tooth in step 1), placing it in a serum-free medium, and cutting the pulp tissue into tissue pieces;
  • step 3 taking the tissue block obtained in step 2) and placing it in human dental pulp stem cell culture medium, covering the cover glass;
  • the dental material is a deciduous tooth of a child aged 5 to 12 years old or a wisdom tooth of an 18 to 29 year old adult.
  • the dental material of the step 1) is stored in the tooth preservation solution before washing and disinfecting, and the storage temperature is 2-8 ° C.
  • the dental preservation solution is based on DMEM medium and comprises 100 U/ml sodium penicillin and 100 mg/ml streptomycin sulfate.
  • the concentration of penicillin sodium in the step 1) in the 0.9% sodium chloride solution is 100 U/ml
  • the concentration of streptomycin sulfate in the 0.9% sodium chloride solution is 100 mg/ml.
  • the step 2) pulp tissue is cut into 1 mm 3 tissue blocks; each 8-10 tissue blocks are placed in 3 to 5 ml human dental pulp stem cell culture medium.
  • the culture of step 3) is carried out at 37 ° C, 5% CO 2 , saturated humidity.
  • the culture fluid is completely filled between the coverslip and the tissue block.
  • the coverslip is removed.
  • the invention provides a human dental pulp stem cell culture medium.
  • the medium of the present invention contains 100 U/ml of penicillin and 100 The mg/ml streptomycin sulfate has less damage to the pulp tissue and cells, and has a high tissue survival rate;
  • the human AB serum used in the human dental pulp stem cell culture medium of the present invention can avoid heterogeneous contamination of animal-derived serum, Resolve immune rejection;
  • 100 ⁇ M ascorbic acid can promote the formation of extracellular matrix of dental pulp stem cells, which is beneficial to the growth of dental pulp stem cells.
  • the number of cultured dental pulp stem cells is more and the activity is higher.
  • the number of cells on the 10th day of culture can reach 3.9 ⁇ 105.
  • the number of dental pulp stem cells cultured in the conventional culture system was only 2.2 ⁇ 105.
  • the experimental results showed that the purity of dental pulp stem cells cultured in the medium of the present invention was high by flow cytometry, and the positive expression rate of dental stem stem cell surface markers CD73, CD90 and CD105 was over 98%, and the negative index expression rate was less than 2%.
  • Example 1 is a graph showing the results of phenotype of dental pulp stem cells detected by flow cytometry according to Example 3 of the present invention.
  • the invention provides a human dental pulp stem cell culture medium, which comprises based on ⁇ -MEM medium, comprising human AB serum in a volume percentage of 5% to 10%, 100 ⁇ M ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin sodium and 100 mg/ml streptomycin.
  • the human dental pulp stem cell culture medium preferably comprises 10% human AB serum.
  • the human dental pulp stem cell culture medium of the present invention can avoid heterogeneous contamination of animal-derived serum and the like by using human AB serum compared to a conventional culture system ( ⁇ -MEM medium containing 10% fetal bovine serum);
  • the marrow stem cell culture medium contains 100 ⁇ M ascorbic acid, which can promote the formation of extracellular matrix of dental pulp stem cells, which is beneficial to the growth of dental pulp stem cells.
  • the number of cultured dental pulp stem cells is more and the activity is higher.
  • the number of cells on the 10th day of culture can reach 3.9 ⁇ . 105, while the number of dental pulp stem cells cultured in the conventional culture system is only 2.2 ⁇ 105.
  • the human dental pulp stem cell culture medium of the present invention only adds 100 U/ml penicillin and 100 Mg/ml streptomycin two antibiotics, the whole process does not use 75% alcohol, metronidazole, amphotericin and other disinfecting agents, the use of these two antibiotics can reduce the damage of the above-mentioned reagents on pulp tissue and cells, Improve the survival rate of the organization.
  • the invention also provides a preparation method of human dental pulp stem cells based on the medium described in the above technical solution, comprising the following steps:
  • step 2) removing the pulp tissue from the disinfected tooth in step 1), placing it in a serum-free medium, and cutting the pulp tissue into tissue pieces;
  • step 3 taking the tissue block obtained in step 2) and placing it in human dental pulp stem cell culture medium, covering the cover glass;
  • the present invention cleans and sterilizes the dental material in a 0.9% sodium chloride solution containing penicillin sodium and streptomycin sulfate to obtain a sterilized tooth which is a deciduous deciduous tooth or wisdom tooth.
  • the dental material is preferably a deciduous tooth that has fallen from a child of 5 to 12 years old or a wisdom tooth that has fallen off from an adult of 18 to 29 years old.
  • the dental material is preferably tissue intact, free from sputum, pulpless disease, and periodontal disease.
  • the surface of the tooth is preferably treated with physiological saline.
  • the present invention preferably stores the dental material in a dental preservation solution at a temperature of 2 to 8 ° C, more preferably 4 ° C.
  • the present invention is not particularly limited to the device to be stored, and may be a thermostatic storage device well known to those skilled in the art, such as a thermostat refrigerator or a thermostat storage box.
  • the dental preservation solution is based on DMEM medium and comprises 100 U/ml penicillin sodium and 100 mg/ml streptomycin sulfate.
  • the concentration of the penicillin sodium in the 0.9% sodium chloride solution is preferably 100 U/ml
  • the concentration of streptomycin sulfate in the 0.9% sodium chloride solution is preferably 100 mg/ml.
  • the cleaning and disinfecting time is preferably 2 to 3 minutes, and the cleaning and disinfecting function is to remove periodontal soft tissue and blood stains.
  • the number of times of sterilization is preferably from 1 to 3 times, more preferably two times.
  • the pulp tissue is taken out from the sterilized teeth, placed in a serum-free medium, and the pulp tissue is cut into tissue pieces.
  • the step 3) of removing the pulp tissue includes the steps of: fixing the teeth, removing the enamel and dentin, and removing the pulp tissue from the pulp cavity.
  • the sterilized teeth are preferably wrapped and secured with sterile gauze.
  • the specific method for removing the enamel and the dentin of the present invention is not particularly limited. Specifically, the present invention preferably uses a vise to clamp the enamel and dentin of the tooth, and after clamping, the sterile gauze is opened, preferably by using The scorpion of the bacterium removes the pulp tissue from the pulp cavity.
  • the dental pulp tissue extraction method of the invention can fully unfold the internal structure of the tooth and take out the gingival tissue inside the tooth. Compared with the conventional pulp needle extraction method, the obtained pulp tissue is more complete and can improve the cell culture in the later stage. Success rate.
  • the extracted pulp tissue is preferably placed in a serum-free medium for storage.
  • the serum-free medium of the present invention is not particularly limited, and a conventional commercially available product of a serum-free medium well known to those skilled in the art may be used. Such as: ⁇ -MEM medium.
  • the invention cuts the pulp tissue into 1.0 mm 3 tissue block, and directly adopts the cut tissue block to culture. Compared with the traditional enzyme digestion method, the operation is simple, the cost is low, the pollution is small, and no impurities are introduced, which is better. Keep your cells alive.
  • the conventional enzymatic digestion technique is cumbersome and expensive, and there are also mechanical damage and chemical damage to the cells, resulting in a long cell culture cycle or failure of cell culture.
  • the tissue block obtained in the step 2) is placed in a human dental pulp stem cell culture medium and cultured to cover the coverslip.
  • the human dental pulp stem cell culture medium has a volume of 3 to 5 ml.
  • 8 to 10 tissue blocks are placed for culture.
  • the size of the tissue block is 1 mm 3
  • the present invention preferably uses a sterile ophthalmic scissors. Cut the block.
  • the invention adopts a cover slip to press the tissue block after the pulp tissue is cut, and the dental pulp tissue block is better to ensure the adhesion of the pulp tissue block, compared to the traditional tissue paste.
  • the wall method, the method of the invention can further ensure that the tissue is better adhered, avoids the floating of the tissue block, and the culture fails.
  • the method of the invention can completely ensure the attachment of the tissue block, accelerate the eruption and growth of the dental pulp stem cells, and the dental pulp stem cells are cultured. On the 4th to 6th day, there was a large amount of eruption, which was 2 to 3 days earlier than the conventional tissue block culture method.
  • the culture dish is preferably selected from the culture dish, and after adding a drop of the medium in the culture dish, the dental probe is taken into the culture droplets, and each drop is cultured.
  • the cover glass is preferably covered on the culture solution, and the tissue block is fixed by light pressure.
  • the culture solution is completely filled between the cover glass and the tissue block to prevent the generation of bubbles.
  • the culture of the dental pulp stem cells is preferably carried out at 37 ° C, 5% CO 2 , and saturated humidity.
  • the medium is replaced at the 4th to 6th day of culture; when the culture is 10th to 12th, the medium is replaced by a half amount; when the culture is 15th to 18th, the cell confluence rate is 70-80%, and subculture is performed to obtain a tooth.
  • Medullary stem cells when cells are grown on the 4th to 6th day of the culture, the coverslip is removed.
  • the conditions of the subculture of the present invention are not particularly limited, and a conventional subculture method of dental pulp cells well known to those skilled in the art may be employed.
  • the invention also provides the application of the dental pulp stem cells obtained by the preparation method of the above technical solution in the repair and regeneration of teeth.
  • a method for preparing a human dental pulp stem cell culture medium and a human dental pulp stem cell according to the present invention will be further described in detail below with reference to specific embodiments.
  • the technical solutions of the present invention include, but are not limited to, the following examples.
  • the pulp tissue was cut into tissue pieces of about 1.0 mm 3 size with a sterile ophthalmic scissors under conditions sufficient to infiltrate the serum-free medium.
  • the tissue fragments were transferred into the culture droplets in the Petri dish using a dental probe so that each drop contained 8 small pieces of tissue.
  • the culture solution was added to an incubator, and cultured at 37 ° C under a saturated humidity of 5% CO 2 .
  • the medium On the fifth day of culture, the medium was changed. When the cells were grown, the coverslips were removed, and the medium was changed for half a day after the culture for 10 days. When the cells were cultured for 16 days, the cell confluence rate reached 72%, and subculture was carried out. .
  • results The number of dental pulp stem cells was 2.6 ⁇ 105 on the 10th day of culture.
  • the results of flow cytometry showed that the positive expression rate of CD73, CD90 and CD105 on dental pulp stem cells was over 95%, and the expression rate of negative indicators was less than 5%.
  • the pulp tissue was cut into tissue pieces of about 1.0 mm 3 size with a sterile ophthalmic scissors under conditions sufficient to infiltrate the serum-free medium.
  • One drop of medium was added to the bottom wall of a 6 cm diameter culture dish.
  • the culture system contained 8% human AB serum, 100 ⁇ M ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin. ⁇ -MEM medium.
  • the tissue fragments were transferred into the culture droplets in the Petri dish using a dental probe so that each drop contained approximately 10 small pieces of tissue.
  • the culture solution was added to an incubator, and cultured at 37 ° C under a saturated humidity of 5% CO 2 .
  • the medium On the 6th day of culture, the medium was changed. When the cells were grown, the coverslips were removed, and the medium was changed for half a day after the culture for 11 days. When the cells were cultured for 16 days, the cell confluence rate reached 80%, and subculture was carried out. .
  • results The number of dental pulp stem cells obtained on the 10th day of culture was 3.1 ⁇ 105.
  • the results of flow cytometry showed that the positive expression rate of CD73, CD90 and CD105 on dental pulp stem cells was over 97%, and the expression rate of negative indicators was less than 3%.
  • the pulp tissue was cut into tissue pieces of about 1.0 mm 3 size with a sterile ophthalmic scissors under conditions sufficient to infiltrate the serum-free medium.
  • the tissue fragments were transferred into the culture droplets in the Petri dish using a dental probe so that each drop contained approximately 9 small pieces of tissue.
  • the sterilized coverslip is slowly covered on the tissue block, gently pressed to fix the tissue block, and the remaining tissue blocks are sequentially subjected to the same operation.
  • the culture solution was added to an incubator, and cultured at 37 ° C under a saturated humidity of 5% CO 2 . When covering, care should be taken to completely fill the culture solution between the coverslip and the tissue block. Do not create air bubbles.
  • the medium On the fifth day of culture, the medium was changed. When the cells were grown, the coverslips were removed, and the medium was changed for half a day after the culture for 10 days. When the cells were cultured for 15 days, the cell confluence rate reached 70%, and subculture was carried out. .
  • results The number of dental pulp stem cells obtained on the 10th day of culture was 3.9 ⁇ 105.
  • the phenotypic results of dental pulp stem cells detected by flow cytometry are shown in Figure 1.
  • the results of flow cytometry showed that the positive expression rate of CD73, CD90 and CD105 on dental pulp stem cells was over 98%, and the expression rate of negative indicators was less than 2%.

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Abstract

La présente invention concerne un milieu de croissance de cellules souches de pulpe dentaire humaine et un procédé de préparation de cellules souches de pulpe dentaire. Le milieu de croissance de cellules souches de pulpe dentaire humaine est basé sur un milieu de croissance α-MEM et comprend du sérum de sang AB humain de 5 % à 10 % en termes de pourcentage en volume, de l'acide ascorbique à 100 µM, de la L-glutamine à 2 mM, de la pénicilline sodique à 100 U/ml, et 100 mg/ml de streptomycine.
PCT/CN2018/095978 2017-07-20 2018-07-17 Milieu de croissance de cellules souches de pulpe dentaire humaine et procédé de préparation de cellules souches de pulpe dentaire humaine WO2019015584A1 (fr)

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CN201710596515.6 2017-07-20
CN201710596515.6A CN107177546A (zh) 2017-07-20 2017-07-20 一种人牙髓干细胞培养基及人牙髓干细胞的制备方法

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CN107177546A (zh) * 2017-07-20 2017-09-19 深圳市茵冠生物科技有限公司 一种人牙髓干细胞培养基及人牙髓干细胞的制备方法
CN110004114A (zh) * 2019-04-02 2019-07-12 浙江优牙生物科技有限公司 一种牙髓干细胞的无血清培养基
CN110468098A (zh) * 2019-07-05 2019-11-19 北京中瑞联合生物科技有限公司 一种牙髓干细胞制备方法
CN115521906A (zh) * 2021-06-24 2022-12-27 东莞宣冠干细胞再生医学有限公司 一种制备高品质人源牙髓干细胞的方法
CN113699102A (zh) * 2021-06-29 2021-11-26 上海赐道医疗投资管理有限公司 牙髓干细胞采样的含组合抗生素的保护液
CN113846057A (zh) * 2021-11-17 2021-12-28 贺函 一种牙髓干细胞培养基及其制备方法

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