WO2019015584A1 - Human dental pulp stem cell growth medium and preparation method for human dental pulp stem cells - Google Patents

Human dental pulp stem cell growth medium and preparation method for human dental pulp stem cells 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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French (fr)
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/en

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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

Provided are a human dental pulp stem cell growth medium and a preparation method for human dental pulp stem cells. The human dental pulp stem cell growth medium is based on an α-MEM growth medium and comprises human AB blood serum of 5% to 10% in terms of volume percent, 100 μM ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin sodium, and 100 mg/ml streptomycin.

Description

一种人牙髓干细胞培养基及人牙髓干细胞的制备方法Human dental pulp stem cell culture medium and preparation method of human dental pulp stem cells 技术领域Technical field
本发明涉及干细胞制备技术领域，具体涉及一种人牙髓干细胞培养基及人牙髓干细胞的制备方法。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.
背景技术Background technique
干细胞是具有自我复制和多向分化能力的细胞，可以不断地自我更新，并在特定条件下分化成为一种或多种构成人体组织或器官的细胞，可使组织恢复再生能力，修复其功能。研究者在脱落乳牙的牙髓组织中发现一种新的成体干细胞，将其命名为乳牙牙髓干细胞，至今已有较多实验证明乳牙牙髓干细胞具有高度自我更新、克隆形成以及成骨分化的能力。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.
乳牙牙髓干细胞具有以下优势：1）取材方便，多取自自然脱落和拔除的滞留牙，对供者正常牙伤害较少；2）不受伦理限制，可进行自体移植治疗，最大限度降低免疫排斥和交叉感染的风险；3）具有较强的增殖能力和多向分化潜能。但牙髓中干细胞含量较低，必须在体外进行扩增，以获得足够的细胞数量来满足应用需求。目前牙髓干细胞培养体系主要采用含动物源胎牛血清的培养基，动物源胎牛血清在一定程度上有可能引起人体免疫排斥反应，增加了牙髓干细胞临床应用的风险。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. However, 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. At present, 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.
技术问题technical problem
本发明的目的在于提供一种人牙髓干细胞培养基及人牙髓干细胞的制备方法。本发明提供的培养基制备得到的牙髓干细胞产量高、无人体免疫排斥反应，为临床牙的修复与再生提供可靠的种子细胞。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.
技术解决方案Technical solution
本发明提供了一种人牙髓干细胞培养基，所述人牙髓干细胞培养基以α-MEM培养基为基础，包含体积百分含量为5%~10%的人AB血清、100μM抗坏血酸、2 mM L-谷氨酰胺、100 U/ml青霉素钠和100 mg/ml链霉素。The invention provides a human dental pulp stem cell culture medium, which comprises based on α-MEM medium, comprising human AB serum in a volume percentage of 5% to 10%, 100 μM ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin sodium and 100 mg/ml streptomycin.
本发明还提供了基于上述技术方案所述培养基的人牙髓干细胞的制备方法，包括以下步骤：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:
1）将牙齿原料置于含有青霉素钠和硫酸链霉素的0.9%氯化钠溶液中进行清洗和消毒，得到消毒后的牙齿，所述牙齿原料为脱落的乳牙或智齿；1) The dental material is washed and sterilized 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;
2）从步骤1）所述消毒后的牙齿中取出牙髓组织，置于无血清培养基中，将牙髓组织剪成组织块；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;
3）取步骤2）得到的组织块置于人牙髓干细胞培养基中进行培养，覆盖盖玻片；3) taking the tissue block obtained in step 2) and placing it in human dental pulp stem cell culture medium, covering the cover glass;
4）在培养第4~6d时，更换培养基；在培养第10~12d时，半量换培养基；培养第15~18d时，细胞汇合率达到70~80%，进行传代培养，得到牙髓干细胞。4) When the culture is on the 4th to 6th day, the medium is changed; when the culture is on the 10th to 12th, the medium is changed halfway; when the culture is on the 15th to 18th, the cell confluence rate is 70-80%, and the subculture is carried out to obtain the pulp. stem cell.
优选的是，所述牙齿原料为5~12岁儿童脱落的乳牙或18~29岁成人脱落的智齿。Preferably, 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.
优选的是，所述步骤1）的牙齿原料在清洗和消毒前将牙齿置于牙齿保存液中保存，保存的温度为2~8℃。Preferably, 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.
优选的是，所述牙齿保存液以DMEM培养基为基础，包括100U/ml青霉素钠和100mg/ml硫酸链霉素。Preferably, the dental preservation solution is based on DMEM medium and comprises 100 U/ml sodium penicillin and 100 mg/ml streptomycin sulfate.
优选的是，所述步骤1）中青霉素钠在0.9%氯化钠溶液中的浓度为100U/ml，硫酸链霉素的在0.9%氯化钠溶液中的浓度为100mg/ml。Preferably, the concentration of penicillin sodium in the step 1) in the 0.9% sodium chloride solution is 100 U/ml, and the concentration of streptomycin sulfate in the 0.9% sodium chloride solution is 100 mg/ml.
优选的是，所述步骤2）牙髓组织剪成1mm 3的组织块；每8~10块组织块置于3~5ml人牙髓干细胞培养基中。 Preferably, 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.
优选的是，所述步骤3）的培养在37℃、5%CO 2，饱和湿度条件下进行。 Preferably, the culture of step 3) is carried out at 37 ° C, 5% CO 2 , saturated humidity.
优选的是，培养液完全充盈于盖玻片与组织块之间。Preferably, the culture fluid is completely filled between the coverslip and the tissue block.
优选的是，所述步骤4）培养第4~6d时有细胞长出时，去掉盖玻片。Preferably, in the step 4), when the cells grow on the 4th to 6th day, the coverslip is removed.
有益效果Beneficial effect
本发明提供了一种人牙髓干细胞培养基。本发明培养基中含有100 U/ml的青霉素和100 mg/ml的硫酸链霉素，对牙髓组织和细胞的损伤小，组织成活率高；本发明的人牙髓干细胞培养基中采用的人AB血清能够避免动物源血清等异源性污染，解决免疫排斥反应；100μM抗坏血酸，能促进牙髓干细胞外基质的形成，有利于牙髓干细胞的生长，培养的牙髓干细胞数量更多，活性更高，培养第10天细胞数量可达3.9×105，而常规培养体系培养的牙髓干细胞数量仅为2.2×105。实验结果表明，通过流式细胞术检测，本发明培养基培养的牙髓干细胞纯度高，牙髓干细胞表面标志CD73、CD90、CD105阳性表达率98％以上，阴性指标表达率低于2％。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; 100μM 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%.
附图说明DRAWINGS
图1为本发明实施例3提供的流式细胞术检测牙髓干细胞表型结果图。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.
本发明的实施方式Embodiments of the invention
本发明提供了一种人牙髓干细胞培养基，所述人牙髓干细胞培养基以α-MEM培养基为基础，包含体积百分含量为5%~10%的人AB血清、100μM抗坏血酸、2 mM L-谷氨酰胺、100 U/ml青霉素钠和100 mg/ml链霉素。The invention provides a human dental pulp stem cell culture medium, which comprises based on α-MEM medium, comprising human AB serum in a volume percentage of 5% to 10%, 100 μM ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin sodium and 100 mg/ml streptomycin.
在本发明中，所述人牙髓干细胞培养基优选包括10%的人AB血清。本发明的人牙髓干细胞培养基相比常规培养体系（含10%胎牛血清的α-MEM培养基），采用人AB血清，能够避免动物源血清等异源性污染；本发明的人牙髓干细胞培养基含有100μM的抗坏血酸，能促进牙髓干细胞外基质的形成，有利于牙髓干细胞的生长，培养的牙髓干细胞数量更多，活性更高，培养第10天细胞数量可达3.9×105，而常规培养体系培养的牙髓干细胞数量仅为2.2×105。本发明所述人牙髓干细胞培养基只添加100 U/ml 青霉素和100 mg/ml 链霉素两种抗生素，整个过程不采用75%酒精、甲硝唑、两性霉素等消毒试剂，本发明这两种抗生素的使用能够减少上述试剂对牙髓组织和细胞的损伤，提高组织成活率。In the present invention, 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 (α-MEM medium containing 10% fetal bovine serum); The marrow stem cell culture medium contains 100 μM 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.
本发明还提供了基于上述技术方案所述培养基的人牙髓干细胞的制备方法，包括以下步骤：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:
1）将牙齿原料置于含有青霉素钠和硫酸链霉素的0.9%氯化钠溶液中进行清洗和消毒，得到消毒后的牙齿，所述牙齿原料为脱落的乳牙或智齿；1) The dental material is washed and sterilized 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;
2）从步骤1）所述消毒后的牙齿中取出牙髓组织，置于无血清培养基中，将牙髓组织剪成组织块；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;
3）取步骤2）得到的组织块置于人牙髓干细胞培养基中进行培养，覆盖盖玻片；3) taking the tissue block obtained in step 2) and placing it in human dental pulp stem cell culture medium, covering the cover glass;
4）在培养第4~6d时，更换培养基；在培养第10~12d时，半量换培养基；培养第15~18d时，细胞汇合率达到70~80%，进行传代培养，得到牙髓干细胞。4) When the culture is on the 4th to 6th day, the medium is changed; when the culture is on the 10th to 12th, the medium is changed halfway; when the culture is on the 15th to 18th, the cell confluence rate is 70-80%, and the subculture is carried out to obtain the pulp. stem cell.
本发明将牙齿原料置于含有青霉素钠和硫酸链霉素的0.9%氯化钠溶液中进行清洗和消毒，得到消毒后的牙齿，所述牙齿原料为脱落的乳牙或智齿。在本发明中，所述牙齿原料优选为5~12岁儿童脱落的乳牙或18~29岁成人脱落的智齿。在本发明中，所述牙齿原料优选组织完整，无龋、无牙髓病和牙周病等。本发明在获得牙齿后，优选用生理盐水对牙齿表面进行处理。对牙齿表面进行处理后，本发明优选将牙齿原料置于牙齿保存液中进行保存，所述保存的温度为2~8℃，更优选为4℃。本发明对所述保存的装置没有特殊的限定，采用本领域技术人员熟知的恒温保存装置即可，如恒温冰箱或恒温保存箱。在本发明中，所述牙齿保存液以DMEM培养基为基础，包括100U/ml青霉素钠和100mg/ml硫酸链霉素。在本发明中，所述青霉素钠在0.9%氯化钠溶液中的浓度优选为100U/ml，硫酸链霉素的在0.9%氯化钠溶液中的浓度优选为100mg/ml。在本发明中，所述清洗和消毒的时间优选为2~3min，所述清洗和消毒的作用为去除牙周软组织和血污。在本发明中，所述消毒的次数优选为1~3次，更优选为2次。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. In the present invention, 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. In the present invention, the dental material is preferably tissue intact, free from sputum, pulpless disease, and periodontal disease. In the present invention, after the tooth is obtained, the surface of the tooth is preferably treated with physiological saline. After treating the surface of the tooth, 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. In the present invention, the dental preservation solution is based on DMEM medium and comprises 100 U/ml penicillin sodium and 100 mg/ml streptomycin sulfate. In the present invention, the concentration of the penicillin sodium in the 0.9% sodium chloride solution is preferably 100 U/ml, and the concentration of streptomycin sulfate in the 0.9% sodium chloride solution is preferably 100 mg/ml. In the present invention, 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. In the present invention, the number of times of sterilization is preferably from 1 to 3 times, more preferably two times.
得到消毒后的牙齿后，从所述消毒后的牙齿中取出牙髓组织，置于无血清培养基中，将牙髓组织剪成组织块。在本发明中，所述步骤3）取出牙髓组织的方法包括以下步骤：将牙齿固定，去除牙釉质和牙本质，从牙髓腔中取出牙髓组织。在本发明，所述消毒后的牙齿优选采用无菌纱布进行包裹固定。本发明对去除牙釉质和牙本质的具体方法没有特殊的限定，具体的，本发明优选采取台虎钳将牙齿的牙釉质和牙本质钳开，钳开后，打开无菌纱布，优选采用灭菌的镊子从牙髓腔中取出牙髓组织。本发明的牙髓组织提取方法能够将牙齿内部结构全部展开，将牙齿内部的牙龈组织全部取出，相比于常规牙髓针提取法，获得的牙髓组织更多更完整，能够提高后期细胞培养的成功率。After the sterilized teeth are obtained, 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. In the present invention, 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. In the present invention, 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.
本发明优选将取出后的牙髓组织装入无血清培养基中进行保存。本发明对所述无血清培养基没有特殊的限定，采用本领域技术人员熟知的无血清培养基的常规市售产品即可。如：α-MEM培养基。发明将牙髓组织剪成1.0mm 3的组织块，取剪碎后的组织块直接进行培养，与传统的酶消化法相比，操作简单，成本低廉，污染小，无杂质引入，能够更好地保持细胞活力。而常规酶消化法技术繁琐、成本昂贵，同时还存在对细胞的机械损伤和化学损伤，造成细胞培养周期很长，或者细胞培养失败。 In the present invention, 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: α-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.
得到组织块后，取步骤2）得到的组织块置于人牙髓干细胞培养基中进行培养，覆盖盖玻片。在本发明中，所述人牙髓干细胞培养基的体积为3~5ml。每3~5ml的人牙髓干细胞培养基中，优选放置8~10块组织块进行培养，在本发明中，所述组织块的大小为1mm 3，本发明优选采用无菌眼科剪对其进行剪块。本发明在牙髓组织剪块后采用盖玻片对组织块进行压贴，由于牙髓组织块比较小，本发明方法能够更好地保证牙髓组织块贴壁，相比于传统的组织贴壁法，本发明的方法能够进一步保证组织更好贴壁，避免组织块漂浮，培养失败，本发明的方法能够完全保证组织块贴附，加速牙髓干细胞的萌出和生长，牙髓干细胞在培养第4~6天就有大量萌出，比常规的组织块培养法提前2~3天。 After the tissue block is obtained, 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. In the present invention, the human dental pulp stem cell culture medium has a volume of 3 to 5 ml. Preferably, in each of the 3 to 5 ml human dental pulp stem cell culture medium, 8 to 10 tissue blocks are placed for culture. In the present invention, the size of the tissue block is 1 mm 3 , and 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.
具体的，本发明在牙髓干细胞的培养过程中，优选选取培养皿进行培养，在培养皿中，滴加1滴培养基后，将牙科探针取组织块移入培养液滴中，每滴培养液加入8~10块组织块。本发明在培养液中加入组织块后，优选在培养液上覆盖盖玻片，轻轻加压，固定组织块。在本发明中，培养液完全充盈于盖玻片与组织块之间，避免气泡产生。在本发明中，所述牙髓干细胞的培养优选在37℃、5%CO 2，饱和湿度条件下进行。 Specifically, in the process of culturing the dental pulp stem cells, 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. Add 8~10 pieces of tissue block to the solution. In the present invention, after the tissue block is added to the culture solution, the cover glass is preferably covered on the culture solution, and the tissue block is fixed by light pressure. In the present invention, the culture solution is completely filled between the cover glass and the tissue block to prevent the generation of bubbles. In the present invention, the culture of the dental pulp stem cells is preferably carried out at 37 ° C, 5% CO 2 , and saturated humidity.
在本发明中，牙齿保存液、牙齿清洗和消毒液中只添加100 U/ml 青霉素和100 mg/ml 链霉素两种抗生素，整个过程不采用75%酒精、甲硝唑、两性霉素等消毒试剂，本发明这两种抗生素的使用能够减少上述试剂对牙髓组织和细胞的损伤，提高组织成活率。In the present invention, only 100 U/ml penicillin and 100 are added to the tooth preservation solution, tooth cleaning and disinfecting solution. 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.
本发明在在培养第4~6d时，更换培养基；在培养第10~12d时，半量换培养基；培养第15~18d时，细胞汇合率达到70~80%，进行传代培养，得到牙髓干细胞。在本发明中，所述培养第4~6d时有细胞长出时，去掉盖玻片。本发明所述传代培养的条件没有特殊的限定，采用本领域技术人员熟知的牙髓细胞常规传代培养方法即可。In the present invention, 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. In the present invention, 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.
本发明还提供了上述技术方案所述制备方法得到的牙髓干细胞在牙齿的修复和再生中的应用。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.
下面结合具体实施例对本发明所述的一种人牙髓干细胞培养基及人牙髓干细胞的制备方法做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。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.
实施例1Example 1
在生物安全柜中放置2个10cm无菌培养皿，加入 40ml含双抗（青霉素钠（100U/ml），硫酸链霉素（100mg/ml））的0.9%氯化钠注射液。用无菌镊子取出采集瓶中的牙齿，放入到装有双抗和0.9%氯化钠注射液的培养皿中清洗和消毒2min，充分去除牙周软组织和血液，将消毒后的牙齿放入第二个培养皿中再消毒一次。Two 10 cm sterile Petri dishes were placed in a biosafety cabinet, and 40 ml of a 0.9% sodium chloride injection containing a double antibody (sodium penicillin (100 U/ml), streptomycin sulfate (100 mg/ml)) was added. Remove the teeth in the collection bottle with sterile forceps, put them into a Petri dish containing double-antibody and 0.9% sodium chloride injection for 2 minutes, thoroughly remove the periodontal soft tissue and blood, and place the disinfected teeth. Disinfect the second dish again.
将清洗后的牙齿放在无菌纱布上，然后用纱布包裹牙齿，用台虎钳将牙齿的牙釉质和牙本质钳开，将无菌纱布打开，用镊子从牙髓腔内找出牙髓组织，将其装入含无血清培养基的EP管中。Place the cleaned teeth on sterile gauze, then wrap the teeth with gauze, pliers the teeth enamel and dentin with a vise, open the sterile gauze, and use the tweezers to find the pulp from the pulp cavity. The tissue was placed in an EP tube containing serum-free medium.
在无血清培养基充分浸润条件下，用无菌眼科剪将牙髓组织剪成约1.0mm 3大小的组织块。在直径6cm的培养皿的底壁滴加1滴培养基，培养体系为含5%人AB血清，100μM抗坏血酸，2 mM L-谷氨酰胺，100 U/ml青霉素，100 mg/ml 链霉素的α-MEM培养基。用牙科探针将组织碎块移入培养皿中的培养液滴中，使每滴培养液内含8小块组织。加入培养液放入培养箱中，于37℃、5％CO 2饱和湿度条件下培养。 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. Add 1 drop of medium to the bottom wall of a 6 cm diameter culture dish containing 5% human AB serum, 100 μM ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin α-MEM 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 .
在培养的第5天，更换培养基，当有细胞长出时，去掉盖玻片，在培养10天时培养基进行半量换液，培养至16天时，细胞汇合率达到72％时，进行传代培养。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. .
结果：培养第10天可获得牙髓干细胞数量为2.6×105。流式细胞术检测结果显示牙髓干细胞表面标志CD73、CD90、CD105阳性表达率95％以上，阴性指标表达率低于5％。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%.
实施例2Example 2
在生物安全柜中放置2个10cm无菌培养皿，加入 40ml含双抗（青霉素钠（100U/ml），硫酸链霉素（100mg/ml））的0.9%氯化钠注射液。用无菌镊子取出采集瓶中的牙齿，放入到装有双抗和0.9%氯化钠注射液的培养皿中清洗和消毒3min，充分去除牙周软组织和血液，将消毒后的牙齿放入第二个培养皿中再消毒一次。Two 10 cm sterile Petri dishes were placed in a biosafety cabinet, and 40 ml of a 0.9% sodium chloride injection containing a double antibody (sodium penicillin (100 U/ml), streptomycin sulfate (100 mg/ml)) was added. Remove the teeth in the collection bottle with sterile forceps, put them into a Petri dish containing double-antibody and 0.9% sodium chloride injection for 3 minutes, thoroughly remove the periodontal soft tissue and blood, and place the disinfected teeth. Disinfect the second dish again.
将清洗后的牙齿放在无菌纱布上，然后用纱布包裹牙齿，用台虎钳将牙齿的牙釉质和牙本质钳开，将无菌纱布打开，用镊子从牙髓腔内找出牙髓组织，将其装入含无血清培养基的EP管中。Place the cleaned teeth on sterile gauze, then wrap the teeth with gauze, pliers the teeth enamel and dentin with a vise, open the sterile gauze, and use the tweezers to find the pulp from the pulp cavity. The tissue was placed in an EP tube containing serum-free medium.
在无血清培养基充分浸润条件下，用无菌眼科剪将牙髓组织剪成约1.0mm 3大小的组织块。在直径6cm的培养皿的底壁滴加1滴培养基，培养体系为含8%人AB血清，100μM抗坏血酸，2 mM L-谷氨酰胺，100 U/ml青霉素，100 mg/ml 链霉素的α-MEM培养基。用牙科探针将组织碎块移入培养皿中的培养液滴中，使每滴培养液内约含10小块组织。加入培养液放入培养箱中，于37℃、5％CO 2饱和湿度条件下培养。 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 μM ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin. α-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 .
在培养的第6天，更换培养基，当有细胞长出时，去掉盖玻片，在培养11天时培养基进行半量换液，培养至16天时，细胞汇合率达到80％时，进行传代培养。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. .
结果：培养第10天可获得牙髓干细胞数量为3.1×105。流式细胞术检测结果显示牙髓干细胞表面标志CD73、CD90、CD105阳性表达率97％以上，阴性指标表达率低于3％。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%.
实施例3Example 3
在生物安全柜中放置2个10cm无菌培养皿，加入40ml含双抗（青霉素钠（100U/ml），硫酸链霉素（100mg/ml））的0.9%氯化钠注射液。用无菌镊子取出采集瓶中的牙齿，放入到装有双抗和0.9%氯化钠注射液的培养皿中清洗和消毒2min，充分去除牙周软组织和血液，将消毒后的牙齿放入第二个培养皿中再消毒一次。Two 10 cm sterile petri dishes were placed in a biosafety cabinet, and 40 ml of a 0.9% sodium chloride injection containing a double antibody (sodium penicillin (100 U/ml), streptomycin sulfate (100 mg/ml)) was added. Remove the teeth in the collection bottle with sterile forceps, put them into a Petri dish containing double-antibody and 0.9% sodium chloride injection for 2 minutes, thoroughly remove the periodontal soft tissue and blood, and place the disinfected teeth. Disinfect the second dish again.
将清洗后的牙齿放在无菌纱布上，然后用纱布包裹牙齿，用台虎钳将牙齿的牙釉质和牙本质钳开，将无菌纱布打开，用镊子从牙髓腔内找出牙髓组织，将其装入含无血清培养基的EP管中。Place the cleaned teeth on sterile gauze, then wrap the teeth with gauze, pliers the teeth enamel and dentin with a vise, open the sterile gauze, and use the tweezers to find the pulp from the pulp cavity. The tissue was placed in an EP tube containing serum-free medium.
在无血清培养基充分浸润条件下，用无菌眼科剪将牙髓组织剪成约1.0mm 3大小的组织块。在直径6cm的培养皿的底壁滴加1滴培养基，培养体系为含10%人AB血清，100μM抗坏血酸，2 mM L-谷氨酰胺，100 U/ml青霉素，100 mg/ml 链霉素的α-MEM培养基。用牙科探针将组织碎块移入培养皿中的培养液滴中，使每滴培养液内约含9小块组织。将消毒后的盖玻片缓慢覆盖于组织块上，轻轻加压，以固定组织块，依次将剩余组织块进行同样操作。加入培养液放入培养箱中，于37℃、5％CO 2饱和湿度条件下培养。覆盖时应注意使培养液完全充盈于盖玻片与组织块之间，切勿产生气泡。 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. Add 1 drop of medium to the bottom wall of a 6 cm diameter culture dish containing 10% human AB serum, 100 μM ascorbic acid, 2 mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin α-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 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.
在培养的第5天，更换培养基，当有细胞长出时，去掉盖玻片，在培养10天时培养基进行半量换液，培养至15天时，细胞汇合率达到70％时，进行传代培养。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. .
结果：培养第10天可获得牙髓干细胞数量为3.9×105。流式细胞术检测牙髓干细胞表型结果如图1所示。流式细胞术检测结果显示牙髓干细胞表面标志CD73、CD90、CD105阳性表达率98％以上，阴性指标表达率低于2％。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%.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

Claims (10)

  1. 一种人牙髓干细胞培养基，其特征在于，所述人牙髓干细胞培养基以α-MEM培养基为基础，包含体积百分含量为5%~10%的人AB血清、100μM抗坏血酸、2 mM L-谷氨酰胺、100 U/ml青霉素钠和100 mg/ml链霉素。A human dental pulp stem cell culture medium, characterized in that the human dental pulp stem cell culture medium is based on α-MEM medium, and comprises human AB serum in a volume percentage of 5% to 10%, 100 μM ascorbic acid, 2 mM L-Glutamine, 100 U/ml penicillin sodium and 100 mg/ml streptomycin.
  2. 一种基于权利要求1所述培养基的人牙髓干细胞的制备方法，包括以下步骤：A method for preparing human dental pulp stem cells based on the medium of claim 1, comprising the steps of:
    1）将牙齿原料置于含有青霉素钠和硫酸链霉素的0.9%氯化钠溶液中进行清洗和消毒，得到消毒后的牙齿，所述牙齿原料为脱落的乳牙或智齿；1) The dental material is washed and sterilized 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;
    2）从步骤1）所述消毒后的牙齿中取出牙髓组织，置于无血清培养基中，将牙髓组织剪成组织块；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;
    3）取步骤2）得到的组织块置于人牙髓干细胞培养基中进行培养，覆盖盖玻片；3) taking the tissue block obtained in step 2) and placing it in human dental pulp stem cell culture medium, covering the cover glass;
    4）在培养第4~6d时，更换培养基；在培养第10~12d时，半量换培养基；培养第15~18d时，细胞汇合率达到70~80%，进行传代培养，得到牙髓干细胞。4) When the culture is on the 4th to 6th day, the medium is changed; when the culture is on the 10th to 12th, the medium is changed halfway; when the culture is on the 15th to 18th, the cell confluence rate is 70-80%, and the subculture is carried out to obtain the pulp. stem cell.
  3. 根据权利要求2所述的人牙髓干细胞的制备方法，其特征在于，所述牙齿原料为5~12岁儿童脱落的乳牙或18~29岁成人脱落的智齿。The method for preparing human dental pulp stem cells according to claim 2, wherein the dental material is a deciduous tooth of a child aged 5 to 12 years old or a wisdom tooth of an adult of 18 to 29 years old.
  4. 根据权利要求2所述的人牙髓干细胞的制备方法，其特征在于，所述步骤1）的牙齿原料在清洗和消毒前将牙齿置于牙齿保存液中保存，保存的温度为2~8℃。The method for preparing human dental pulp stem cells according to claim 2, wherein the dental material of the step 1) is stored in a tooth preservation liquid before washing and disinfecting, and the storage temperature is 2-8 ° C. .
  5. 根据权利要求4所述的人牙髓干细胞的制备方法，其特征在于，所述牙齿保存液以DMEM培养基为基础，包括100U/ml青霉素钠和100mg/ml硫酸链霉素。The method for preparing human dental pulp stem cells according to claim 4, wherein the dental preservation solution is based on DMEM medium and comprises 100 U/ml of penicillin sodium and 100 mg/ml of streptomycin sulfate.
  6. 根据权利要求2所述的人牙髓干细胞的制备方法，其特征在于，所述步骤1）中青霉素钠在0.9%氯化钠溶液中的浓度为100U/ml，硫酸链霉素的在0.9%氯化钠溶液中的浓度为100mg/ml。The method for preparing human dental pulp stem cells according to claim 2, wherein the concentration of penicillin sodium in the step 1) is 0.9 U/ml in 0.9% sodium chloride solution, and 0.9% in streptomycin sulfate. The concentration in the sodium chloride solution was 100 mg/ml.
  7. 根据权利要求2所述的人牙髓干细胞的制备方法，其特征在于，所述步骤2）牙髓组织剪成1mm 3的组织块；每8~10块组织块置于3~5ml人牙髓干细胞培养基中。 The method for preparing human dental pulp stem cells according to claim 2, wherein the step 2) the pulp tissue is cut into a tissue block of 1 mm 3 ; each 8 to 10 tissue blocks are placed in a 3 to 5 ml human dental pulp. In stem cell culture medium.
  8. 根据权利要求2所述的人牙髓干细胞的制备方法，其特征在于，所述步骤3）的培养在37℃、5%CO 2，饱和湿度条件下进行。 The method for producing human dental pulp stem cells according to claim 2, wherein the culture of the step 3) is carried out at 37 ° C, 5% CO 2 , and saturated humidity.
  9. 根据权利要求2所述的人牙髓干细胞的制备方法，其特征在于，培养液完全充盈于盖玻片与组织块之间。The method for preparing human dental pulp stem cells according to claim 2, wherein the culture solution is completely filled between the cover glass and the tissue block.
  10. 根据权利要求2所述的人牙髓干细胞的制备方法，其特征在于，所述步骤4）培养第4~6d时有细胞长出时，去掉盖玻片。The method for preparing human dental pulp stem cells according to claim 2, wherein in the step 4), when the cells grow on the 4th to 6th day, the coverslip is removed.
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