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Glioma Associated Stem Cells (GASCs) Isolation and Culture
胶质瘤相关干细胞(GASC)的分离和培养   

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Abstract

Glioma Associated Stem Cells (GASCs) represent a population of non-tumorigenic multipotent stem cells hosted in the microenvironment of human gliomas. In vitro, these cells are able, through the release of exosomes, to increase the biological aggressiveness of glioma-initiating cells. The clinical importance of this finding is supported by the strong prognostic value associated with the GASCs surface immunophenotype thus suggesting that this patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma (Bourkoula et al., 2014).

Keywords: Human glioma(人脑胶质瘤), Stem cells(干细胞), Microenvironment(微环境), Tumor-supporting function(肿瘤支持功能)

Materials and Reagents

  1. Hank’s balanced salt solution (HBSS) without calcium and magnesium (Sigma-Aldrich, catalog number: H2387 )
  2. Collagenase type II (Sigma-Aldrich, catalog number: C6885-500MG )
  3. Fetal bovine serum (FBS) (EuroClone, catalog number: ECS0180L )
  4. Trypan blue (Sigma-Aldrich, catalog number T6146-5G )
  5. Dulbecco’s phosphate buffered saline (D-PBS) (Life Technologies, catalog number: 14190-250 )
  6. Fibronectin from human plasma 0.1% solution (cell culture tested) (Sigma-Aldrich, catalog number: F0895 )
  7. Modified Eagle’s medium (MEM) Joklik without NaHCO3 (Sigma-Aldrich, catalog number: M0518 )
  8. Hepes (powder) (Sigma-Aldrich, catalog number: H3375 )
  9. L-glutamine (Sigma-Aldrich, catalog number: G7513 )
  10. Taurine (Sigma-Aldrich, catalog number: T0625 )
  11. Penicillin-streptomycin (100x solution stabilized, sterile-filtered, suitable for cell culture) (Sigma-Aldrich, catalog number: P4333-100ML )
  12. Insulin (Sigma-Aldrich, catalog number: I5523-50MG )
  13. Dulbecco’s modified Eagle’s medium (DMEM) low glucose (Life Technologies, Gibco®, catalog number: 31600-091 )
  14. MCDB-201 (Sigma-Aldrich, catalog number: M6770 )
  15. Linoleic acid-bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: L9530 )
  16. Dexamethasone (powder) (Sigma-Aldrich, catalog number: D2915 )
  17. Ascorbic acid-2 phosphate (powder) (Sigma-Aldrich, catalog number: A8960 )
  18. 100x insulin-transferrin-selenium (ITS) (Life Technologies, Gibco®, catalog number: 41400-045 )
  19. Stem cell tested fetal bovine serum (Stem Cells Technologies, catalog number: 10M37180 )
  20. Human platelet derived growth factor-BB (hPDGF-BB) (Pepro Tech, catalog number: 100-14B )
  21. Human epidermal growth factor (hEGF) (Pepro Tech, catalog number: AF-100-15 )
  22. TrypLE-express dissociation reagent (Life Technologies, Gibco®, catalog number: 12605 )
  23. Basic buffer (BB) (see Recipes)
  24. Incubation buffer (IB) (see Recipes)
  25. 0.025% Collagenase type II (see Recipes)
  26. 0.4% Trypan blue solution (see Recipes)
  27. Fibronectin-coated 100 mm petri dish (see Recipes)
  28. Multipotent adult stem cell (MASC) medium (see Recipes)

Equipment

  1. Sterile tissue culture dishes 100 x 20 mm style (BD Biosciences, Falcon®, catalog number: 353003 )
  2. Scalpel blades (Sigma-Aldrich, catalog number 27046 )
  3. Sterile serological transfer pipettes
  4. Serological pipettes
  5. Sterile Falcon tubes
  6. Mesh filter (70 µm) (BD Biosciences, Falcon®, catalog number 352350 )
  7. Biological hood (Faster, model: Safe FAST Elite )
  8. Agitation rotor (Celbio, model: MINI OVEN )
  9. Burker counting chamber (Sigma-Aldrich, catalog number: Z359629-1EA )
  10. 37 °C, 5% CO2, 5% O2 force air incubator (New Brunswick, model: Galaxy 170 R )
  11. Centrifuge (Heraeus Instruments, model: Megafuge 1.0 R )
  12. Light microscope (Leica Microsystems, model: DMIL )

Procedure

  1. Isolation and primary culture of primitive cells from human glioma (see Video 1)
    1. Under biological hood, place the glioma sample into sterile 100 mm Petri dish and cut the fragments into ~1 mm pieces using scalpels.
    2. Re-suspend glioma fragments into 3-5 ml of BB and transfer them into a 15 ml Falcon tube.
    3. Let the fragments deposit to the bottom of the 15 ml Falcon tube by gravity at room temperature. If fragments will not deposit into the bottom, centrifuge the suspension at 300 x g for 1 min.
    4. Remove the supernatant and re-suspend the volume of the pellet in an equal volume of collagenase solution (0.025%).
    5. Incubate the suspension for 5 min in an agitation rotor at 37 °C.
    6. At the end of the incubation period, gently shake the bottom of the Falcon tube and add 5 ml of IB and gently pipette it with a 10 ml serological pipette for 4-5 min.
    7. Pre-wet a 70 μm mesh filter by filtering 5 ml of sterile, fresh HBSS using a 5 ml sterile serological pipette into a 50 ml Falcon tube.
    8. Filter the cell suspension through the pre-wet 70 μm mesh filter.
    9. Centrifuge the filtered suspension at 500 x g for 5 min at room temperature and re-suspend it into 1 ml of MASC medium.
    10. Count the cells using the Burker Counting chamber: Remove 10 μl of cell suspension, mix it with an equal volume of 0.4% Trypan blue in D-PBS and apply 10 μl of diluted cells to the Burker chamber. Count at least four squares and calculate the number of cells by multiplying the medium value obtained for 2 x 104. You will obtain the total amount of cells/ml of cell suspension.
    11. Seed 5,000 cells/cm2 into fibronectin-coated dishes and culture them in MASC medium at 37 °C in a 5% CO2, 5% O2 force air incubator.

  2. Sub-culture of glioma associated stem cells (GASCs)
    1. Take out from the incubator (5% CO2, 5% O2, at 37 °C) the dish containing the cells that has to be split. Check at the microscope that cells have reached 80% of confluence (7-10 days after seeding).
    2. Transfer the dish below the biological hood, open the dish and discard the supernatant with a 10 ml sterile serological pipette.
    3. Add 5 ml of HBSS or D-PBS to the dish, gently move the plate in order to cover the entire surface, and then remove the HBSS or D-PBS. Repeat the washing procedure. Remove carefully the HBSS or D-PBS.
    4. Add 2 ml of TrypLE Express solution for each dish. Since cells will detach very fast, check continuously at the microscope the process in order to avoid over-digestion.
    5. When cells are finally detached, add 5 ml of HBSS or D-PBS to dilute the TrypLE Express solution.
    6. Re-suspend the cells by gently pipetting them through a 10 ml sterile serological pipette. To recover all cells from the dishes, wash the Petri dish one more time, by adding 3 ml of fresh sterile HBSS or D-PBS and put the solution in the Falcon Tube containing the previous obtained cell suspension.
    7. Centrifuge the cell suspension at 500 x g for 5 mins at room temperature, discard the supernatant and re-suspend the cell pellet in 1 ml of MASC medium.
    8. Seed the cells onto fibronectin-coated dishes at the concentration of 3,500 cells/cm2.
    9. Change the medium every three days. Split the cells when they will reach 80% confluence.

Representative data


Video 1. GASC isolation procedure. Video 1 is representing the steps from 1 to 11 of the GASCs isolation protocol.


Figure 1. Phase contrast image of GASCs at the third passage in culture

Recipes

  1. Basic buffer (BB)
    11 g MEM Joklik without NaHCO3 (powder)
    4.7 g Hepes (powder)
    0.3 g L-glutamine
    0.25 g taurine
    5 ml penicillin-streptomycin
    Insulin 20 U/L
    Add non-pyrogenic dH2O up to 900 ml and adjust the pH to 7.3
    Add non-pyrogenic dH2O up to 1,000 ml and filter sterilize (0.2 µm)
    Stored at 4 °C
  2. Incubation buffer (IB)
    Add 10% of fetal bovine serum to HBSS
    Stored at 4 °C
  3. 0.025% Collagenase type II
    0.025 g Collagenase type II
    Add 10 ml of BB and carefully pipette for 2-3 min
    Add BB up to 100 ml, filter sterilize and stored at -20 °C
  4. 0.4% Trypan blue solution
    0.1 g Trypan blue
    Add 25 ml sterile 1x D-PBS
    Vortex and stored at room temperature
  5. Fibronectin-coated 100 mm petri dish
    For each 100 mm petri dish, add 5 µl 0.1% fibronectin in 1 ml of HBSS
    Carefully cover the bottom of the dish with the fibronectin solution and keep for at least 20 min at room temperature
    Remove the exceeding solution from the dish
  6. Multipotent adult stem cell (MASC) medium
    Base medium (60% DMEM low glucose + 40% MCDB-201)
    2% stem cell tested fetal bovine serum
    10 ng/ml hPDGF-BB
    10 ng/ml hEGF
    1 mg/ml linoleic acid-BSA
    10-9 M dexamethasone
    1x ITS 100x
    10-4 M ascorbic acid-2 phosphate
    1x penicillin-streptomycin 100x
    Adjust the pH to 7.3
    Filter sterilize (0.2 µm) and stored at 4 °C

Acknowledgments

FIRB Accordi di programma 2011 Pr. RBAP11Z4Z9. FIRB accordi di programma 2011 Pr. RBAP11ETKA_007. Programma per la Cooperazione Transfrontaliera Italia-Slovenia 2007-2013. Title: “Identificazione di nuovi marcatori di cellule staminali tumorali a scopo diagnostico e terapeutico”; AIRC 5 per mille Special program 2011, Pr. 12214. Project ERC- 7FP SP 2 IDEAS QUIDPROQUO G.A. n. 269051. The protocol was adopted from Bourkoula et al. (2014).

References

  1. Andolfi, L., Bourkoula, E., Migliorini, E., Palma, A., Pucer, A., Skrap, M., Scoles, G., Beltrami, A. P., Cesselli, D. and Lazzarino, M. (2014). Investigation of adhesion and mechanical properties of human glioma cells by single cell force spectroscopy and atomic force microscopy. PLoS One 9(11): e112582.
  2. Beltrami, A. P., Cesselli, D., Bergamin, N., Marcon, P., Rigo, S., Puppato, E., D'Aurizio, F., Verardo, R., Piazza, S. and Pignatelli, A. (2007). Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow). Blood 110(9): 3438-3446.
  3. Bourkoula, E., Mangoni, D., Ius, T., Pucer, A., Isola, M., Musiello, D., Marzinotto, S., Toffoletto, B., Sorrentino, M., Palma, A., Caponnetto, F., Gregoraci, G., Vindigni, M., Pizzolitto, S., Falconieri, G., De Maglio, G., Pecile, V., Ruaro, M. E., Gri, G., Parisse, P., Casalis, L., Scoles, G., Skrap, M., Beltrami, C. A., Beltrami, A. P. and Cesselli, D. (2014). Glioma-associated stem cells: a novel class of tumor-supporting cells able to predict prognosis of human low-grade gliomas. Stem Cells 32(5): 1239-1253.

简介

胶质瘤相关干细胞(GASC)代表在人类神经胶质瘤的微环境中存在的非致瘤性多能干细胞群体。 在体外,这些细胞能够通过外来体的释放增加胶质瘤起始细胞的生物攻击性。 该发现的临床重要性得到与GASCs表面免疫表型相关的强预后价值的支持,因此表明这种基于患者的方法可以提供开创性的方法来预测预后并开发靶向肿瘤基质的新策略(Bourkoula等人, et al。,2014)。

关键字:人脑胶质瘤, 干细胞, 微环境, 肿瘤支持功能

材料和试剂

  1. 不含钙和镁的Hank's平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H2387)
  2. 胶原酶II型(Sigma-Aldrich,目录号:C6885-500MG)
  3. 胎牛血清(FBS)(EuroClone,目录号:ECS0180L)
  4. 台盼蓝(Sigma-Aldrich,目录号T6146-5G)
  5. Dulbecco's磷酸盐缓冲盐水(D-PBS)(Life Technologies,目录号:14190-250)
  6. 来自人血浆0.1%溶液(测试的细胞培养物)(Sigma-Aldrich,目录号:F0895)的纤连蛋白
  7. 不含NaHCO 3的改良的Eagle培养基(MEM)Joklik(Sigma-Aldrich,目录号:M0518)
  8. Hepes(粉末)(Sigma-Aldrich,目录号:H3375)
  9. L-谷氨酰胺(Sigma-Aldrich,目录号:G7513)
  10. 牛磺酸(Sigma-Aldrich,目录号:T0625)
  11. 青霉素 - 链霉素(100x溶液稳定,无菌过滤,适用于细胞培养)(Sigma-Aldrich,目录号:P4333-100ML)
  12. 胰岛素(Sigma-Aldrich,目录号:I5523-50MG)
  13. Dulbecco改良的Eagle培养基(DMEM)低葡萄糖(Life Technologies,Gibco ,目录号:31600-091)
  14. MCDB-201(Sigma-Aldrich,目录号:M6770)
  15. 亚油酸 - 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:L9530)
  16. 地塞米松(粉末)(Sigma-Aldrich,目录号:D2915)
  17. 抗坏血酸-2磷酸盐(粉末)(Sigma-Aldrich,目录号:A8960)
  18. 100x胰岛素 - 转铁蛋白 - 硒(ITS)(Life Technologies,Gibco ,目录号:41400-045)
  19. 干细胞测试的胎牛血清(Stem Cells Technologies,目录号:10M37180)
  20. 人血小板衍生生长因子-BB(hPDGF-BB)(Pepro Tech,目录号:100-14B)
  21. 人表皮生长因子(hEGF)(Pepro Tech,目录号:AF-100-15)
  22. TrypLE-表达解离试剂(Life Technologies,Gibco ,目录号:12605)
  23. 基本缓冲区(BB)(参见配方)
  24. 孵育缓冲液(IB)(参见配方)
  25. 0.025%II型胶原酶(见配方)
  26. 0.4%台盼蓝溶液(见配方)
  27. 纤连蛋白包被的100mm培养皿(参见配方)
  28. 多能成体干细胞(MASC)培养基(参见配方)

设备

  1. 将100×20mm样式的无菌组织培养皿(BD Biosciences,Falcon ,目录号:353003)
  2. 手术刀(Sigma-Aldrich,目录号27046)
  3. 无菌血清学转移移液器
  4. 血清移液器
  5. 无菌猎管
  6. 网孔过滤器(70μm)(BD Biosciences,Falcon ,目录号352350)
  7. 生物罩(更快,型号:Safe FAST Elite)
  8. 搅拌转子(Celbio,型号:MINI OVEN)
  9. Burker计数室(Sigma-Aldrich,目录号:Z359629-1EA)
  10. 37℃,5%CO 2,5%O 2强制空气培养箱(New Brunswick,型号:Galaxy 170 R)
  11. 离心机(Heraeus Instruments,型号:Megafuge 1.0R)
  12. 光学显微镜(Leica Microsystems,型号:DMIL)

程序

  1. 人神经胶质瘤原始细胞的分离和原代培养(见视频1)
    1. 在生物罩下,将胶质瘤样品置于无菌的100mm培养皿中   并使用手术刀将碎片切成〜1毫米的块
    2. 重新悬浮胶质瘤片段到3-5毫升BB,并将他们转移到15毫升Falcon管。
    3. 让片段沉积到15毫升Falcon管的底部 重力在室温。 如果碎片不会沉积到 底部,将悬浮液在300×g离心1分钟
    4. 取出上清液,并在等体积的胶原酶溶液(0.025%)中重悬浮沉淀的体积。
    5. 在37℃的搅拌转子中孵育悬浮液5分钟
    6. 在孵化期结束时,轻轻摇动底部 Falcon管并加入5毫升IB,轻轻地用10毫升吸管 血清移液器4-5分钟
    7. 通过预湿润70微米的网过滤器 使用5ml无菌血清学过滤5ml无菌,新鲜HBSS 移液至50ml Falcon管中
    8. 通过预湿的70μm网过滤器过滤细胞悬液
    9. 在室温下将过滤的悬浮液在500×g离心5分钟,并将其重悬浮在1ml MASC培养基中。
    10. 计数细胞使用Burker计数室:删除10μl的 细胞悬浮液,将其与等体积的0.4%台盼蓝混合 D-PBS并将10μl稀释的细胞应用于Burker室。 计数 至少四个正方形并通过乘以计算细胞的数量 对于2×10 4个中值获得。 您将获得总金额 细胞/ml细胞悬浮液
    11. 种子5,000个细胞/cm 2 纤维连接蛋白包被的培养皿,并在37℃下在MASC培养基中培养 5%CO 2 2,5%O 2强制空气培养箱。

  2. 胶质瘤相关干细胞(GASCs)的亚培养物
    1. 从培养箱(5%CO 2,5%O 2,在37℃)取出培养皿 含有必须分裂的细胞。 检查显微镜说 细胞已达到80%的汇合(接种后7-10天)
    2. 转移在生物罩下面的菜,打开菜和丢弃 上清液用10ml无菌血清移液管
    3. 添加5 ml的HBSS或D-PBS加入培养皿中,轻轻地移动板以覆盖   整个表面,然后去除HBSS或D-PBS。 重复 洗涤程序。 小心取出HBSS或D-PBS。
    4. 加入2 ml 的TrypLE Express溶液。 因为细胞会非常分离 快速,在显微镜连续检查过程,以避免 过度消化。
    5. 当细胞最终分离时,加入5ml HBSS或D-PBS稀释TrypLE Express溶液。
    6. 重新悬浮细胞,轻轻地吸取他们通过10毫升 无菌血清移液管。 要从菜中回收所有细胞,洗涤 培养皿再次通过加入3ml新鲜无菌HBSS或 D-PBS,并将溶液放在含有上述的Falcon管中 获得细胞悬液
    7. 在500×下离心细胞悬浮液   g,在室温下放置5分钟,弃去上清液 将细胞沉淀重悬于1ml MASC培养基中
    8. 将细胞以3,500个细胞/cm 2的浓度接种在涂有纤连蛋白的培养皿上。
    9. 每三天更换一次培养基。 当细胞达到80%汇合时分裂细胞。

代表数据


视频1. GASC隔离过程。 视频1表示从1到11的GASCs隔离协议的步骤。
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图1.培养物第三代的GASCs的相差图像


食谱

  1. 基本缓冲区(BB)
    11g MEM Joklik,无NaHCO 3(粉末)
    4.7克Hepes(粉末)
    0.3g L-谷氨酰胺 0.25克牛磺酸 5ml青霉素 - 链霉素 胰岛素20 U/L
    加入无热原的dH 2 O至高达900ml,并将pH调节至7.3。
    加入无热原的dH 2 O至1000ml并过滤除菌(0.2μm)
    储存在4°C
  2. 孵育缓冲液(IB)
    将10%胎牛血清加入HBSS中
    储存在4°C
  3. 0.025%II型胶原酶
    0.025g II型胶原酶
    加入10 ml BB,小心移液2-3分钟
    加入BB至100毫升,过滤除菌并储存在-20℃下
  4. 0.4%台盼蓝溶液
    0.1克台盼蓝
    加入25ml无菌1x D-PBS
    涡旋并在室温下贮存
  5. 纤连蛋白包被的100mm培养皿
    对于每个100mm培养皿,在1ml HBSS中加入5μl0.1%纤连蛋白 小心地用纤连蛋白溶液覆盖盘的底部,并在室温下保持至少20分钟
    从培养皿中除去过量的溶液
  6. 多潜能成体干细胞(MASC)培养基
    基础培养基(60%DMEM低葡萄糖+ 40%MCDB-201)
    2%干细胞测试胎牛血清
    10 ng/ml hPDGF-BB
    10 ng/ml hEGF
    1mg/ml亚油酸BSA 10 -9 M地塞米松
    1x ITS 100x
    10 -4 M抗坏血酸-2磷酸盐 1×青霉素 - 链霉素100×
    将pH调节至7.3
    过滤灭菌(0.2μm)并在4℃下保存

致谢

FIRB Accordi di programma 2011 Pr。 RBAP11Z4Z9。 FIRB计划2011年。 RBAP11ETKA_007。 Programma per la Cooperazione Transfrontaliera Italia-Slovenia 2007-2013。 标题:"Identificazione di nuovi marcatori di cellule staminali tumorali a scopo diagnico e terapeutico"; AIRC 5 per mille 2011年特别计划, 12214.项目ERC- 7FP SP 2 IDEAS QUIDPROQUO G.A. n。 该方案获自Bourkoula等人(2014)。

参考文献

  1. Andolfi,L.,Bourkoula,E.,Migliorini,E.,Palma,A.,Pucer,A.,Skrap,M.,Scoles,G.,Beltrami,AP,Cesselli,D。和Lazzarino, )。 通过单细胞力谱和原子力显微镜检查人神经胶质瘤细胞的粘附和力学性质。/a> PLoS One 9(11):e112582。
  2. Beltrami,AP,Cesselli,D。,Bergamin,N.,Marcon,P.,Rigo,S.,Puppato,E.,D'Aurizio,F.,Verardo,R.,Piazza,S.and Pignatelli, (2007)。 多能细胞可以在体外从几个成人器官(心脏)产生,肝和骨髓)。 血液 110(9):3438-3446。
  3. Bourkoula,E.,Mangoni,D.,Ius,T.,Pucer,A.,Isola,M.,Musiello,D.,Marzinotto,S.,Toffoletto,B.,Sorrentino,M.,Palma, Caponnetto,F.,Gregoraci,G.,Vindigni,M.,Pizzolitto,S.,Falconieri,G.,De Maglio,G.,Pecile,V.,Ruaro,ME,Gri,G.,Parisse, Casalis,L.,Scoles,G.,Skrap,M.,Beltrami,CA,Beltrami,AP和Cesselli,D。(2014)。 胶质瘤相关干细胞:一种新型的能够预测人类低度预后的肿瘤支持细胞 - 等级神经胶质瘤。干细胞 32(5):1239-1253。
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引用:Bourkoula, E., Mangoni, D., Caponnetto, F., Ius, T., Skrap, M., Beltrami, A. P. and Cesselli, D. (2015). Glioma Associated Stem Cells (GASCs) Isolation and Culture. Bio-protocol 5(9): e1457. DOI: 10.21769/BioProtoc.1457.
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