CN102965338A - Extraction and culture method of human umbilical cord mesenchymal stem cells - Google Patents
- ️Wed Mar 13 2013
CN102965338A - Extraction and culture method of human umbilical cord mesenchymal stem cells - Google Patents
Extraction and culture method of human umbilical cord mesenchymal stem cells Download PDFInfo
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- CN102965338A CN102965338A CN 201210510379 CN201210510379A CN102965338A CN 102965338 A CN102965338 A CN 102965338A CN 201210510379 CN201210510379 CN 201210510379 CN 201210510379 A CN201210510379 A CN 201210510379A CN 102965338 A CN102965338 A CN 102965338A Authority
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Abstract
本发明公开了一种人脐带间充质干细胞的提取和培养方法。脐带组织切碎后经I型胶原酶消化后移入含培养基的培养瓶中持续培养。所用培养基为低糖DMEM基础培养基并且添加了胎牛血清、成纤维生长因子、上皮细胞生长因子、细胞转录因子、胆固醇。使用本发明的提取和培养方法可以长期培养人的脐带间充质干细胞，并且维持干细胞活性。本发明解决了目前人脐带间充质干细胞培养中细胞衰老过快以及分化问题，能够长期获得具有干细胞特性的人脐带间充质干细胞。
The invention discloses a method for extracting and culturing human umbilical cord mesenchymal stem cells. The umbilical cord tissue was minced, digested with type I collagenase, and then transferred to a culture bottle containing medium for continuous culture. The medium used is a low-sugar DMEM basal medium supplemented with fetal bovine serum, fibroblast growth factors, epithelial cell growth factors, cell transcription factors, and cholesterol. The human umbilical cord mesenchymal stem cells can be cultured for a long time by using the extraction and culture method of the present invention, and the activity of the stem cells can be maintained. The present invention solves the problem of excessive cell aging and differentiation in the current culture of human umbilical cord mesenchymal stem cells, and can obtain human umbilical cord mesenchymal stem cells with stem cell characteristics for a long time.
Description
技术领域 technical field
本发明涉及一种人脐带间充质干细胞的提取和培养方法。The invention relates to a method for extracting and culturing human umbilical cord mesenchymal stem cells.
背景技术 Background technique
间充质干细胞（mesenchymal stem cells，MSCs）是一种来源于中胚层和外胚层的多潜能干细胞，属于成体干细胞。间充质干细胞具有自我更新的能力，并具有多向分化潜能，在特定诱导条件下能分化为骨骼、软骨、脂肪、神经、心肌等多种组织细胞。间充质干细胞存在于多种组织中，目前已经从骨髓、脂肪、肌肉等组织中成功获得了间充质干细胞。来源于成体组织中的间充质干细胞由于数量有限，且受患者年龄、疾病等因素的影响，限制了其在临床上的应用。脐带以及脐带血以其非侵入式的获得方式，对捐献者不会造成任何损害而成为一种理想的间充质干细胞来源。而与脐带血相比，脐带来源的间充质干细胞更易于获得和培养。每条脐带的间充质干细胞的数目为4×105个，密度为10-15×103个/cm，而每份脐带血中的数目为1-5×103骨髓中的数目为每106个单个核细胞含有1-10个间充质干细胞。Mesenchymal stem cells (MSCs) are pluripotent stem cells derived from mesoderm and ectoderm, and belong to adult stem cells. Mesenchymal stem cells have the ability of self-renewal and multi-directional differentiation potential, and can differentiate into bone, cartilage, fat, nerve, cardiac muscle and other tissue cells under specific induction conditions. Mesenchymal stem cells exist in a variety of tissues, and mesenchymal stem cells have been successfully obtained from bone marrow, fat, muscle and other tissues. Due to the limited number of mesenchymal stem cells derived from adult tissues and the influence of factors such as patient age and disease, their clinical application is limited. Umbilical cord and umbilical cord blood are an ideal source of mesenchymal stem cells because of their non-invasive way of obtaining without any damage to the donor. Compared with umbilical cord blood, umbilical cord-derived mesenchymal stem cells are easier to obtain and culture. The number of mesenchymal stem cells in each umbilical cord is 4×10 5 , and the density is 10-15×10 3 cells/cm, while the number in each umbilical cord blood is 1-5×10 3 in the bone marrow. 10 6 mononuclear cells contain 1-10 mesenchymal stem cells.
脐带的结构从外到内依次为脐带上皮、羊膜下基质、裂隙、血管间基质（华通胶）、血管周围基质、血管（两动脉一静脉）。目前从脐带的不同组织部位都获得了间充质干细胞，而其主要来源是血管间的华通胶基质。脐带组织包含丰富的间充质干细胞，脐带间充质干细胞具有以下特点：（1）增殖能力强：体外培养第7代后可扩增300倍，且无明显衰老迹象。（2）免疫原性低：脐带间充质干细胞可剂量依赖性的抑制激活T淋巴细胞增殖，同时可维持和促进调节性T淋巴细胞扩增。脐带间充质干细胞抑制免疫细胞的增殖，不会引起同种异源或异种免疫细胞的增殖，具有免疫调节作用和较低的免疫原性。（3）致肿瘤风险低：脐带间充质干细胞具有稳定的端粒酶活性，培养多代也不会丧失锚定依赖性和血清依赖性，在维持其增殖性的同时不具有致肿瘤性。The structure of the umbilical cord from outside to inside is umbilical cord epithelium, subamniotic matrix, fissure, intervascular matrix (Huatong glue), perivascular matrix, and blood vessels (two arteries and one vein). At present, mesenchymal stem cells have been obtained from different tissue parts of the umbilical cord, and their main source is the Wharton's jelly matrix between blood vessels. Umbilical cord tissue contains abundant mesenchymal stem cells. Umbilical cord mesenchymal stem cells have the following characteristics: (1) Strong proliferation ability: after the 7th passage in vitro, they can be expanded 300 times, and there is no obvious sign of aging. (2) Low immunogenicity: Umbilical cord mesenchymal stem cells can inhibit the proliferation of activated T lymphocytes in a dose-dependent manner, while maintaining and promoting the expansion of regulatory T lymphocytes. Umbilical cord mesenchymal stem cells inhibit the proliferation of immune cells, will not cause the proliferation of allogeneic or heterogeneous immune cells, and have immunomodulatory effects and low immunogenicity. (3) Low risk of tumorigenesis: Umbilical cord mesenchymal stem cells have stable telomerase activity, and they will not lose anchorage dependence and serum dependence after being cultured for multiple generations, and they are not tumorigenic while maintaining their proliferation.
发明内容 Contents of the invention
技术问题：本发明要解决的技术问题是提供一种能长期维持人脐带间充质干细胞活性的提取和培养方法。使用本发明提取和培养的人脐带间充质干细胞在传至10代后仍能维持干细胞的活性，保持干细胞的增殖和多项分化潜能。Technical problem: The technical problem to be solved by the present invention is to provide an extraction and culture method that can maintain the activity of human umbilical cord mesenchymal stem cells for a long time. The human umbilical cord mesenchymal stem cells extracted and cultivated by the present invention can still maintain the activity of the stem cells after passing on for 10 generations, and maintain the proliferation and multiple differentiation potentials of the stem cells.
技术方案：为解决上述技术问题，本发明公开了一种人脐带间充质干细胞的提取和培养方法，该方法包括如下步骤：Technical solution: In order to solve the above technical problems, the present invention discloses a method for extracting and culturing human umbilical cord mesenchymal stem cells. The method includes the following steps:
步骤1：取健康胎儿脐带切去动脉和静脉，剪碎；Step 1: Take a healthy fetal umbilical cord, cut off the artery and vein, and cut it into pieces;
步骤2：用I型胶原酶消化脐带；Step 2: Digest the umbilical cord with type I collagenase;
步骤3：磷酸盐缓冲液清洗组织块；Step 3: Wash the tissue block with phosphate buffered saline;
步骤4：组织块移入培养瓶中，加入培养基；Step 4: Move the tissue block into a culture bottle and add culture medium;
步骤5：培养瓶放入培养箱中持续传代培养。Step 5: Place the culture bottle into the incubator for continuous subculture.
优选的，步骤1中，脐带去除两天脐动脉和一条脐静脉，并剪碎至0.5mm3-2mm3。Preferably, in step 1, two days of umbilical artery and one umbilical vein are removed from the umbilical cord, and chopped to 0.5mm 3 -2mm 3 .
优选的，步骤2中，脐带使用I型胶原酶消化，I型胶原酶的质量体积比为0.2%-1%，每克脐带组织使用0.5-2毫升I型胶原酶消化，消化时间为1-3小时。Preferably, in step 2, the umbilical cord is digested with type I collagenase, the mass volume ratio of type I collagenase is 0.2%-1%, each gram of umbilical cord tissue is digested with 0.5-2 ml type I collagenase, and the digestion time is 1- 3 hours.
优选的，步骤4中使用的培养基成分包括胎牛血清，人成纤维生长因子，人上皮细胞生长因子，细胞转录因子，胆固醇。Preferably, the medium components used in step 4 include fetal bovine serum, human fibroblast growth factor, human epithelial growth factor, cell transcription factor, and cholesterol.
优选的，培养基的成分配比为胎牛血清5%-10%，人成纤维生长因子5-10ng/ml，人上皮细胞生长因子5-10ng/ml，细胞转录因子1-5ng/ml，胆固醇10-20ng/ml。Preferably, the composition ratio of the medium is 5%-10% of fetal bovine serum, 5-10ng/ml of human fibroblast growth factor, 5-10ng/ml of human epithelial growth factor, and 1-5ng/ml of cell transcription factor, Cholesterol 10-20ng/ml.
优选的，培养基使用低糖DMEM培养基，并且加入了质量体积比0.5%-2%的青霉素和链霉素。Preferably, the medium is low-sugar DMEM medium, and penicillin and streptomycin are added in a mass volume ratio of 0.5%-2%.
有益效果：本发明提供的人脐带间充质干细胞的提取和培养方法能快速获得大量脐带间充质干细胞，并且能长时间维持脐带间充质干细胞的增殖和分化能力，在传代至第10代后获得的细胞仍然具有干细胞活性。Beneficial effects: the method for extracting and culturing human umbilical cord mesenchymal stem cells provided by the present invention can quickly obtain a large number of umbilical cord mesenchymal stem cells, and can maintain the proliferation and differentiation ability of umbilical cord mesenchymal stem cells for a long time. The obtained cells still have stem cell activity.
附图说明 Description of drawings
图1a为第1代人脐带间充质干细胞形态；Figure 1a is the morphology of the first generation human umbilical cord mesenchymal stem cells;
图1b为第5代人脐带间充质干细胞形态；Fig. 1b is the morphology of human umbilical cord mesenchymal stem cells at passage 5;
图1c为第7代人脐带间充质干细胞形态；Figure 1c is the morphology of human umbilical cord mesenchymal stem cells at passage 7;
图1d为第10代人脐带间充质干细胞形态；Figure 1d is the morphology of human umbilical cord mesenchymal stem cells at passage 10;
图2为第3代、第7代、第10代的人脐带间充质干细胞生长曲线；Fig. 2 is the growth curve of human umbilical cord mesenchymal stem cells of the 3rd generation, the 7th generation, and the 10th generation;
图3a为人脐带间充质干细胞表面抗原CD29表达；Figure 3a shows the expression of the surface antigen CD29 of human umbilical cord mesenchymal stem cells;
图3b为人脐带间充质干细胞表面抗原CD44表达；Figure 3b shows the expression of the surface antigen CD44 of human umbilical cord mesenchymal stem cells;
图3c为人脐带间充质干细胞表面抗原CD105表达；Figure 3c shows the expression of the surface antigen CD105 of human umbilical cord mesenchymal stem cells;
图3d为人脐带间充质干细胞表面抗原CD34表达；Figure 3d shows the expression of the surface antigen CD34 of human umbilical cord mesenchymal stem cells;
图3e为人脐带间充质干细胞表面抗原CD45表达；Figure 3e shows the expression of the surface antigen CD45 of human umbilical cord mesenchymal stem cells;
图3f为人脐带间充质干细胞表面抗原HLA-DR表达。Fig. 3f shows the expression of surface antigen HLA-DR of human umbilical cord mesenchymal stem cells.
具体实施方式 Detailed ways
下面结合附图，对本发明做进一步说明。Below in conjunction with accompanying drawing, the present invention will be further described.
本发明提供的人脐带间充质干细胞的提取和培养方法，该方法包括如下步骤：The method for extracting and culturing human umbilical cord mesenchymal stem cells provided by the invention comprises the following steps:
步骤1：取健康胎儿脐带切去动脉和静脉，剪碎；Step 1: Take a healthy fetal umbilical cord, cut off the artery and vein, and cut it into pieces;
步骤2：用I型胶原酶消化脐带；Step 2: Digest the umbilical cord with type I collagenase;
步骤3：磷酸盐缓冲液清洗组织块；Step 3: Wash the tissue block with phosphate buffered saline;
步骤4：组织块移入培养瓶中，加入培养基；Step 4: Move the tissue block into a culture bottle and add culture medium;
步骤5：培养瓶放入培养箱中持续传代培养。Step 5: Place the culture bottle into the incubator for continuous subculture.
步骤1中，脐带去除两天脐动脉和一条脐静脉，并剪碎至0.5mm3-2mm3。In step 1, two days of umbilical artery and one umbilical vein were removed from the umbilical cord, and chopped to 0.5mm 3 -2mm 3 .
步骤2中，脐带使用I型胶原酶消化，I型胶原酶的质量体积比为0.2%-1%，每克脐带组织使用0.5-2毫升I型胶原酶消化，消化时间为1-3小时。In step 2, the umbilical cord is digested with type I collagenase, the mass volume ratio of type I collagenase is 0.2%-1%, and 0.5-2 ml of type I collagenase is used for digestion per gram of umbilical cord tissue, and the digestion time is 1-3 hours.
步骤4中使用的培养基成分包括胎牛血清，人成纤维生长因子，人上皮细胞生长因子，细胞转录因子，胆固醇。The medium components used in step 4 include fetal bovine serum, human fibroblast growth factor, human epithelial growth factor, cell transcription factor, and cholesterol.
培养基的成分配比为胎牛血清5%-10%，人成纤维生长因子5-10ng/ml，人上皮细胞生长因子5-10ng/ml，细胞转录因子1-5ng/ml，胆固醇10-20ng/ml。The composition ratio of the medium is fetal bovine serum 5%-10%, human fibroblast growth factor 5-10ng/ml, human epithelial cell growth factor 5-10ng/ml, cell transcription factor 1-5ng/ml, cholesterol 10- 20ng/ml.
培养基使用低糖DMEM培养基，并且加入了质量体积比0.5%-2%的青霉素和链霉素。The medium uses low-sugar DMEM medium, and adds penicillin and streptomycin at a mass volume ratio of 0.5%-2%.
实施例1：Example 1:
人脐带间充质干细胞培养基配制：在500ml低糖培养基中加入25ml胎牛血清、2.5μg人成纤维生长因子、2.5μg上皮细胞生长因子、0.5μg细胞转录因子、5μg胆固醇、5ml抗生素，0.22μm滤器过滤除菌，4℃储存备用。Human umbilical cord mesenchymal stem cell medium preparation: add 25ml fetal bovine serum, 2.5μg human fibroblast growth factor, 2.5μg epithelial cell growth factor, 0.5μg cell transcription factor, 5μg cholesterol, 5ml antibiotic to 500ml low-glucose medium, 0.22 Sterilize with a μm filter and store at 4°C for later use.
人脐带间充质干细胞提取与培养Extraction and culture of human umbilical cord mesenchymal stem cells
1、取健康、自然顺产的胎儿脐带首先剪为3cm的片段，然后用PBS清洗数次洗掉残留血液，去除两条动脉和一条静脉，剪碎至1mm3左右的小块。1. Take the umbilical cord of a healthy, naturally delivered fetus and cut it into 3cm pieces first, then wash it with PBS several times to wash off the residual blood, remove two arteries and one vein, and cut it into small pieces of about 1mm 3 .
2、加入PBS清洗，800rmp离心5分钟去除上清，重复清洗两遍。2. Add PBS to wash, centrifuge at 800rmp for 5 minutes to remove supernatant, and repeat washing twice.
3、加入20ml I型胶原酶消化2h，消化过程中间断混匀组织块以促进消化。3. Add 20ml type I collagenase to digest for 2 hours, and mix the tissue pieces intermittently during the digestion process to promote digestion.
4、消化完成后800rmp离心5min去除上清，加入PBS清洗，800rmp离心5min清洗两遍。4. After the digestion is completed, centrifuge at 800rmp for 5min to remove the supernatant, add PBS to wash, and centrifuge at 800rmp for 5min to wash twice.
5、消化后的组织块移到3瓶含5ml培养基的25ml培养瓶中放入培养箱37℃,5%CO2条件下培养。5. Transfer the digested tissue pieces to 3 bottles of 25ml culture flasks containing 5ml of culture medium and place them in an incubator at 37°C and 5% CO 2 for cultivation.
6、培养瓶前3天不要移动以使组织块贴壁，第4天半量换液，第7天全量换液，第12天传代，一瓶传3瓶。6. Do not move the culture bottle for the first 3 days to make the tissue block adhere to the wall. Change the medium in half on the 4th day, change the full volume on the 7th day, pass passage on the 12th day, pass 3 bottles per bottle.
实施例2：Example 2:
人脐带间充质干细胞培养基配制：在500ml低糖培养基中加入50ml胎牛血清、5μg人成纤维生长因子、5μg上皮细胞生长因子、1μg细胞转录因子、10μg胆固醇、5ml抗生素，0.22μm滤器过滤除菌，4℃储存备用。Human umbilical cord mesenchymal stem cell medium preparation: add 50ml fetal bovine serum, 5μg human fibroblast growth factor, 5μg epithelial cell growth factor, 1μg cell transcription factor, 10μg cholesterol, 5ml antibiotics to 500ml low-glucose medium, filter with 0.22μm filter Sterilize and store at 4°C for later use.
人脐带间充质干细胞提取与培养Extraction and culture of human umbilical cord mesenchymal stem cells
1、取健康、自然顺产的胎儿脐带9cm首先剪为3节各3cm的片段，然后用PBS清洗数次洗掉残留血液，去除两条动脉和一条静脉，剪碎至1mm3左右的小块。1. Take a 9cm healthy and natural fetal umbilical cord and cut it into 3 sections of 3cm each, then wash it with PBS several times to wash off the residual blood, remove two arteries and one vein, and cut it into small pieces of about 1mm 3 .
2、加入PBS清洗，800rmp离心5分钟去除上清，重复清洗两遍。2. Add PBS to wash, centrifuge at 800rmp for 5 minutes to remove supernatant, and repeat washing twice.
3、加入20ml I型胶原酶消化2h，消化过程中间断混匀组织块以促进消化。3. Add 20ml type I collagenase to digest for 2 hours, and mix the tissue pieces intermittently during the digestion process to promote digestion.
4、消化完成后800rmp离心5min去除上清，加入PBS清洗，800rmp离心5min清洗两遍。4. After the digestion is completed, centrifuge at 800rmp for 5min to remove the supernatant, add PBS to wash, and centrifuge at 800rmp for 5min to wash twice.
5、消化后的组织块移到3瓶含5ml培养基的25ml培养瓶中放入培养箱37℃,5%CO2条件下培养。5. Transfer the digested tissue pieces to 3 bottles of 25ml culture flasks containing 5ml of culture medium and place them in an incubator at 37°C and 5% CO 2 for cultivation.
6、培养瓶前3天不要移动以使组织块贴壁，第4天半量换液，第7天全量换液，第12天传代，一瓶传3瓶。（图1）6. Do not move the culture bottle for the first 3 days to make the tissue block adhere to the wall. Change the medium in half on the 4th day, change the full volume on the 7th day, pass passage on the 12th day, pass 3 bottles per bottle. (figure 1)
实施例3：Example 3:
细胞增殖能力检测：Detection of cell proliferation ability:
1、取第3代、第7代和第10代培养的人脐带间充质干细胞，各加入1ml 0.25%的胰酶消化细胞。1. Take human umbilical cord mesenchymal stem cells cultured at passage 3, passage 7 and passage 10, and add 1ml of 0.25% trypsin to digest the cells.
2、用血球计数板对细胞计数，稀释细胞悬液至1×105个/ml。2. Count the cells with a hemocytometer and dilute the cell suspension to 1×10 5 cells/ml.
3、用3块12孔细胞培养板，在第一块培养板的第一排四个孔接种第3代细胞1×105个/ml，第二排接种第7代细胞1×105个/ml，第三排接种第10代细胞1×105个/ml，每孔加入2ml细胞悬液，其它2块培养板使用相同操作。3. Use three 12-well cell culture plates, inoculate the 3rd generation cells 1×10 5 cells/ml in the first row of four wells of the first culture plate, and inoculate the 7th generation cells 1×10 5 cells in the second row /ml, the third row was inoculated with 1×10 5 cells/ml of the 10th generation cells, and 2ml of cell suspension was added to each well, and the same procedure was used for the other two culture plates.
4、24h后，取出第1块培养板，计数每种细胞的4个孔中的细胞数。4. After 24 hours, take out the first culture plate, and count the number of cells in 4 wells of each type of cells.
5、36h后取出第2块培养板，计数每种细胞的4个孔中的细胞数。5. Take out the second culture plate after 36 hours, and count the number of cells in 4 wells of each type of cells.
6、72h后，取出第3块培养板，计数每种细胞的4个孔中的细胞数。6. After 72 hours, take out the third culture plate, and count the number of cells in 4 wells of each type of cells.
图2结果显示人脐带间充质干细胞在培养至第10代后增殖能力没有明显变化，72h后细胞数达到8.5×105个/ml,与第3代（9.1×105个/ml）和第7代（7.9×105个/ml）没有明显差异，仍然维持较高的增殖能力。The results in Figure 2 show that the proliferation ability of human umbilical cord mesenchymal stem cells did not change significantly after cultured to the 10th passage, and the number of cells reached 8.5×10 5 cells/ml after 72 hours, compared with the third passage (9.1×10 5 cells/ml) and The 7th passage (7.9×10 5 cells/ml) had no significant difference, and still maintained a high proliferation ability.
实施例4：Example 4:
免疫表型检测：Immunophenotyping:
1、取第3代和第10代细胞分别用胰酶消化后移入1.5ml离心管，1500rmp离心5min去除上清。1. Take the 3rd and 10th passage cells and trypsinize them respectively, transfer them into 1.5ml centrifuge tubes, and centrifuge at 1500rmp for 5min to remove the supernatant.
2、每管加入1ml PBS重悬，1500rmp离心5min去除上清。2. Add 1ml PBS to each tube to resuspend, centrifuge at 1500rmp for 5min to remove the supernatant.
3、加入PBS重悬细胞，计数，调整细胞数为1×106个/ml。3. Add PBS to resuspend the cells, count and adjust the cell number to 1×10 6 cells/ml.
4、将细胞悬液分为每管500μl。4. Divide the cell suspension into 500 μl per tube.
5、加入流式抗体，4℃避光孵育30min。5. Add flow cytometry antibody and incubate at 4°C in the dark for 30 minutes.
6、1500rmp离心5min去除上清。6. Centrifuge at 1500rmp for 5min to remove the supernatant.
7、加入1ml PBS重悬，1500rmp离心5min去除上清。7. Add 1ml PBS to resuspend, centrifuge at 1500rmp for 5min to remove the supernatant.
8、加入500μl PBS重悬细胞。8. Add 500μl PBS to resuspend the cells.
9、流式细胞仪检测样品。9. Samples were detected by flow cytometry.
图3经流式细胞术检测，使用本发明方法培养的人脐带间充质干细胞均高表达CD29、CD44、CD73，而CD34、CD45、HLA-DR呈阴性，表明经培养第10代后细胞仍表达间充质干细胞标志抗原，细胞仍保持干细胞特征。Figure 3 is detected by flow cytometry, and the human umbilical cord mesenchymal stem cells cultured using the method of the present invention all highly express CD29, CD44, and CD73, while CD34, CD45, and HLA-DR are negative, indicating that the cells still remain after the 10th generation of culture. Expressing the marker antigen of mesenchymal stem cells, the cells still maintain the characteristics of stem cells.
由以上实施例得，使用本发明提取和培养的人脐带间充质干细胞内长期维持干细胞活性，对传至10代的间充质干细胞进行增殖能力检测和免疫表型分析均无明显变化。这一发明能有效用于人脐带间充质干细胞的科学研究和进一步临床应用。According to the above examples, the human umbilical cord mesenchymal stem cells extracted and cultured by the present invention maintain the activity of stem cells for a long time, and there is no obvious change in the detection of proliferation ability and immunophenotype analysis of the mesenchymal stem cells passed down to 10 generations. This invention can be effectively used in scientific research and further clinical application of human umbilical cord mesenchymal stem cells.
以上所述仅为本发明的较佳实施方式，本发明的保护范围并不以上述实施方式为限，但凡本领域普通技术人员根据本发明所揭示内容所作的等效修饰或变化，皆应纳入权利要求书中记载的保护范围内。The above descriptions are only preferred embodiments of the present invention, and the scope of protection of the present invention is not limited to the above embodiments, but all equivalent modifications or changes made by those of ordinary skill in the art according to the disclosure of the present invention should be included within the scope of protection described in the claims.
Claims (6)
1.一种人脐带间充质干细胞的提取和培养方法，其特征在于：该方法包括如下步骤： 1. A method for extracting and culturing human umbilical cord mesenchymal stem cells, characterized in that: the method may further comprise the steps: 步骤1：取健康胎儿脐带切去动脉和静脉，剪碎； Step 1: Take a healthy fetal umbilical cord, cut off the artery and vein, and cut it into pieces; 步骤2：用I型胶原酶消化脐带； Step 2: Digest the umbilical cord with type I collagenase; 步骤3：磷酸盐缓冲液清洗组织块； Step 3: Wash the tissue block with phosphate buffered saline; 步骤4：组织块移入培养瓶中，加入培养基； Step 4: Move the tissue block into a culture bottle and add culture medium; 步骤5：培养瓶放入培养箱中持续传代培养。 Step 5: Place the culture bottle into the incubator for continuous subculture. 2.根据权利要求1所述的人脐带间充质干细胞的提取和培养方法，其特征在于：步骤1中，脐带去除两天脐动脉和一条脐静脉，并剪碎至0.5mm3-2mm3。 2. The method for extracting and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: in step 1, the umbilical cord is removed for two days from the umbilical artery and an umbilical vein, and shredded to 0.5mm 3 -2mm 3 . 3.根据权利要求1所述的人脐带间充质干细胞的提取和培养方法，其特征在于：步骤2中，脐带使用I型胶原酶消化，I型胶原酶的质量体积比为0.2%-1%，每克脐带组织使用0.5-2毫升I型胶原酶消化，消化时间为1-3小时。 3. The extraction and culture method of human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: in step 2, the umbilical cord is digested with type I collagenase, and the mass to volume ratio of type I collagenase is 0.2%-1 %, each gram of umbilical cord tissue was digested with 0.5-2 ml type I collagenase, and the digestion time was 1-3 hours. 4.根据权利要求1所述的人脐带间充质干细胞的提取和培养方法，其特征在于：步骤4中使用的培养基成分包括胎牛血清，人成纤维生长因子，人上皮细胞生长因子，细胞转录因子，胆固醇。 4. The extraction and culture method of human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: the medium components used in step 4 include fetal bovine serum, human fibroblast growth factor, human epithelial growth factor, Cellular transcription factor, cholesterol. 5.根据权利要求4所述的人脐带间充质干细胞的提取和培养方法，其特征在于：培养基的成分配比为胎牛血清5%-10%，人成纤维生长因子5-10ng/ml，人上皮细胞生长因子5-10ng/ml，细胞转录因子1-5ng/ml，胆固醇10-20ng/ml。 5. the extraction and culture method of human umbilical cord mesenchymal stem cells according to claim 4, is characterized in that: the composition ratio of culture medium is fetal bovine serum 5%-10%, human fibroblast growth factor 5-10ng/ ml, human epithelial cell growth factor 5-10ng/ml, cell transcription factor 1-5ng/ml, cholesterol 10-20ng/ml. 6.根据权利要求1的人脐带间充质干细胞的提取和培养方法，其特征在于：培养基使用低糖DMEM培养基，并且加入了质量体积比0.5%-2%的青霉素和链霉素。 6. The method for extracting and culturing human umbilical cord mesenchymal stem cells according to claim 1, characterized in that: the medium uses low-sugar DMEM medium, and adds penicillin and streptomycin with a mass volume ratio of 0.5%-2%. the
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| CN103480040B (en) * | 2013-09-27 | 2014-12-17 | 中国人民解放军第三军医大学第一附属医院 | Bone matrix material containing various proteins secreted by umbilical cord mesenchymal stem cells and preparation method thereof |
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