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Preparation of Primary Astrocyte Culture Derived from Human Glioblastoma Multiforme Specimen
源自人多形性胶质母细胞瘤标本的原发性星形细胞培养物的制备   

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Molecular Neurobiology
Oct 2016

Abstract

Glioblastoma multiforme (GBM) is a grade 4 astrocytoma tumor in central nervous system. Astrocytes can be isolated from human GBM. Study of astrocytes can provide insights about the formation, progression and recurrence of glioblastoma. For using isolated astrocytes, new studies can be designed in the fields of pharmacology, neuroscience and neurosurgery for glioblastoma treatment. This protocol describes the details for preparing high purity primary astrocytes from human GBM. Tumor tissue is disrupted using mechanical dissociation and chemical digestion in this protocol. 2 weeks after plating the cell suspension in culture, primary astrocytes are available for further subculturing and immunocytochemistry of S100-beta antigen.

Keywords: Glioblastoma multiforme (多形性胶质母细胞瘤), Astrocyte (星形胶质细胞), S100-beta antigen (S100-β抗原), Primary cell culture (原代细胞培养), Malignant brain tumor (恶性脑肿瘤), Astrocytoma tumor (星形细胞瘤)

Background

Astrocytes are glial cells that provide structural and nutritional support for brain neurons. The cell cycle of astrocytes seems to be disrupted in astrocytoma brain tumors. The World Health Organisation (WHO) has classified astrocytomas into four grades according to their malignancy. Glioblastoma multiforme (GBM, grade IV) is the most malignant form of astrocytoma. Glioblastoma is characterized by the invasive cells with the rapid proliferation rate and angiogenesis. Prognosis is poor for patients with glioblastoma. Current therapeutic approaches including surgery, chemo-therapy and radiation don’t have good effects on the treatment of suffering patients. Median survival time for patients is about one year after treatment (Stuup et al., 2005; Wen and Kesari, 2008). So many researchers focus on the study of evaluating the physiological function and apoptosis of glioblastoma cells in order to detect more effective treatment methods. Here we present a method for isolation of high purity primary astrocyte from human glioblastoma specimen without fibroblast contamination (Hashemi et al., 2016).

Materials and Reagents

  1. 15 ml centrifuge tubes (Corning, Falcon®, catalog number: 352096 )
  2. Petri dish culture (Nest Biotechnology, catalog number: 704001 )
  3. No. 10 scalpel blade surgical tool (BD, catalog number: 371610 )
  4. Cell strainer sieve (Corning, Falcon®, catalog number: 352340 )
  5. 50 ml centrifuge tubes (Corning, Falcon®, catalog number: 352070 )
  6. T25 flask culture (Nest Biotechnology, catalog number: 707003 )
  7. 1 ml microtube (Nest Biotechnology, catalog number: 613111 )
  8. 24-well plates of polystyrene with high clarity (Nest Biotechnology, catalog number: 702001 )
  9. 5 ml pipette (Nest Biotechnology, catalog number: 326001 )
  10. Glioblastoma multiforme sample (Human)
  11. Hanks’ balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 24020117 )
  12. Antibiotic-antimycotic (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  13. 0.25% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  14. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  15. Trypan blue solution 0.4% (Sigma-Aldrich, catalog number: T8154 )
  16. Primary antibody of rabbit anti-S100-beta (Sigma-Aldrich, catalog number: S2644 )
  17. Primary antibody of rabbit anti-fibronectin (Abcam, catalog number: ab23751 )
  18. FITC-conjugated goat anti-rabbit (Abcam, catalog number: ab6717 )
  19. Dulbecco’s modified Eagle medium/F12 (DMEM/F12) (Thermo Fisher Scientific, GibcoTM, catalog number: 31331028 )
  20. Phosphate buffer saline (PBS) (tablet) (Thermo Fisher Scientific, GibcoTM, catalog number: 18912014 )
  21. Paraformaldehyde (powder) (Sigma-Aldrich, catalog number: P6148 )
  22. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  23. Bovine serum albumin (powder) (Sigma-Aldrich, catalog number: A2058 )
  24. Culture medium (see Recipes)
  25. Phosphate buffer saline (PBS) (see Recipes)
  26. 4% paraformaldehyde (see Recipes)
  27. 0.2% Triton X-100 (see Recipes)
  28. 10% bovine serum albumin (see Recipes)

Equipment

  1. Ventilation hood (VISION SCIENTIFIC, model: VS-7120LV )
  2. 37 °C water bath (Memmert, model: WNB14 )
  3. Centrifuge machine (Hettich Lab Technology, model: Universal 320R )
  4. Hemocytometer (Sigma-Aldrich, catalog number: Z359629 )
  5. CO2 cell culture incubator (Memmert, model: INC108 T2T3 )
  6. Inverted fluorescence microscope (Optika, model: XDS-2FL )
  7. Small forceps surgical tools (Fine Science Tools, catalog number: 11050-10 )

Procedure

  1. Collection of primary glioblastoma multiforme (GBM) tissue
    1. A glioblastoma multiforme sample was obtained from a patient with glioblastoma who underwent surgical treatment.
    2. This study was approved by the Ethical Commission of the Tehran University of Medical Sciences. Before operation, a signed written consent was given by the patient.
    3. After coordination with the neurosurgery team, the neurosurgeon placed the resected glioblastoma sample in a 15 ml Falcon tube containing 5 ml cold HBSS with 10% antibiotics (antibiotic-antimycotic).
    4. The glioblastoma sample was delivered to the lab on dry ice.

  2. Dissociation of GBM sample into single primary astrocytes
    1. Under biology’s hood, remove excess of the HBSS in the Falcon tube containing glioblastoma tissue. Wash the tissue 3 times with 5 ml cold PBS to remove the blood.
    2. Place the glioblastoma tissue in a Petri dish filled with 1 ml cold PBS.
    3. Shred and mince the tissue with a scalpel blade for 5 min until the sample is converted to a milky suspension. Mincing can help to speed up the trypsinization process through increasing the efficacy of tissue dissociation.
    4. Add 3 ml of warm (37 °C) 0.05% trypsin-EDTA to minced tissue. Then, transfer the suspension with trypsin into a new 15 ml Falcon tube.
    5. Incubate the produced suspension for 10 min at a 37 °C water bath for digestion of the minced tissue.
    6. Add 3 ml of DMEM/F12 medium containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic to inhibit the activity of trypsin.
    7. Pipette the suspension 10 times with a 5 ml pipette and centrifuge at 180 x g for 10 min at room temperature.
    8. Remove the upper liquid and resuspend the resulting pellet slowly into 5 ml DMEM/F12 containing 2% FBS and 1% antibiotic-antimycotic by pipetting 10 times with a 5 ml pipette.
    9. Filter the cell suspension through a 40 micron cell strainer into a 50 ml tube to remove un-dissociated pieces and debris.
    10. Centrifuge the filtered suspension at 180 x g for 5 min at room temperature.
    11. Resuspend the pelleted cells into 1 ml DMEM/F12 containing 2% FBS and 1% antibiotic-antimycotic (Figure 1).


      Figure 1. Procedure to isolate primary astrocyte for culture from human glioblastoma sample. A. A human glioblastoma sample after surgery; B. Glioblastoma sample was washed using cold PBS; C. Glioblastoma sample was transferred to Petri dish; D. Glioblastoma sample was shred and minced with scalpel blade; E. After enzymatic digestion, cell suspension was plated in a T25 flask.

  3. Cell counting and plating
    1. Add 10 μl of the cell suspension to a 1 ml tube containing 10 μl of 0.4% trypan blue in PBS.
    2. Transfer 10 μl of the cell-trypan blue mixture to a hemocytometer chamber. Count the number of the cells in each individual square (total, 4 squares) under an inverted microscope. Determine the average number of the cells per square and calculate the total cell number by multiplying the cell quantity per square by 2 x 104.
    3. Seed 1 x 105 cells in 5 ml DMEM/F12 containing 2% FBS and 1% antibiotic-antimycotic into T25 flasks.
    4. Place the flasks into an incubator at 37 °C, 5% CO2 and 95% humidity.
    5. Astrocytes usually attach to the bottom of flask after the first 24 h. Astrocytes neurites extend gradually after 3-4 days. Astrocytes start to grow and proliferate after the complete extension of their neurites at day 7 of culture.
    6. Replace half of the cell medium with fresh medium every 2 days. FBS concentration is gradually increased after each change of the culture medium, from 2% to 10% in 2 weeks (Table 1). 10% FBS amount in culture medium is preserved in the following primary astrocyte culture.

      Table 1. FBS concentration in culture medium at different days after seeding
      Days after seeding to change medium
      FBS concentration
      Seeding (0)
      2%
      4
      3%
      6
      4%
      8
      5%
      10
      6.5%
      12
      8%
      14
      10%

    7. Gradual increase of FBS amount might avoid fibroblast contamination of the cell culture (Figure 2).


      Figure 2. Phase contrast images of primary astrocytes of glioblastoma multiforme in the different days of cell culture. A. 2 days after cell culture; B. 4 days after cell culture; C. 9 days after cell culture; D. 14 days after cell culture. Scale bars = 100 µm.

  4. Sub-culture of primary astrocytes of GBM
    1. Check the level of cell confluency using an inverted microscope. Subculture the cells when they reach 80% of confluence 3 weeks after seeding.
    2. Remove the medium from each T25 flask under a biological hood with a 5 ml sterile pipette.
    3. Wash the cells with 3 ml of warm (37 °C) PBS and then remove PBS.
    4. Detach the cells with 1 ml of warm (37 °C) 0.05% trypsin-EDTA for 1 min in an incubator.
    5. Inhibit trypsin-EDTA reaction by adding 4 ml of DMEM/F12 containing 10% FBS and 1% antibiotic-antimycotic into each T25 flask and then transfer the cell suspension from each flask to a 15 ml Falcon tube.
    6. Centrifuge the cell suspensions at 250 x g for 5 min at room temperature.
    7. Remove the medium from each tube with a 5 ml sterile pipette.
    8. Resuspend the pellet in each tube with 1 ml of fresh medium containing DMEM/F12, 10% FBS and 1% antibiotic-antimycotic.
    9. After re-suspend and counting, plate the cells in 24-well dishes (at a density of 5 x 103 cells per well) for immunocytochemistry analysis and also T25 flasks for sub-culture.

  5. S100-beta staining
    1. Seed 5 x 103 cells into each well of a 24-well culture dish.
    2. Wash each well three times with phosphate-buffered saline (PBS) after 24 h at room temperature.
    3. Fix cells with 4% paraformaldehyde in PBS for 20 min.
    4. Wash cells three times in PBS at room temperature for 5 min each.
    5. Permeate cell membranes with 0.2% Triton X-100 for 15 min.
    6. Wash cells three times in PBS at room temperature for 5 min each.
    7. Block cells with 10% bovine serum albumin for 1 h.
    8. Dilute the primary antibody of rabbit anti-S100-beta 1:100 with PBS containing 1% BSA.
    9. Incubate cells with primary antibody of rabbit anti-S100-beta or primary antibody of rabbit anti-fibronectin overnight at 4 °C.
    10. Wash cells three times in PBS at room temperature for 5 min each.
    11. Dilute the secondary antibody of FITC-conjugated goat anti-rabbit 1:500 with PBS containing 1% BSA.
    12. Incubate cells with secondary antibody of FITC-conjugated goat anti-rabbit for 2 h at room temperature in the dark.
    13. Wash cells three times in PBS at room temperature for 5 min each.
    14. Image cells by an inverted fluorescence microscopy (Figures 3 and 4).
      Note: Only the secondary antibody was added as a negative control.


      Figure 3. Expression of S100-beta antigen in purified primary astrocytes after 3 weeks of culture. A. Primary astrocytes were expressed astrocytic antigen of S100-beta (negative control); Primary astrocytes were expressed astrocytic antigen of S100-beta observed under an inverted fluorescence microscope with 10x objective lens (B) and 40x objective lens (C), scale bars = 50 µm (B), 100 µm (C).


      Figure 4. Assessment of fibronectin antigen in purified primary astrocyte culture in order to observe fibroblast cell contamination after 3 weeks of culture. A. Fibroblastic antigen of fibronectin was not observed in the negative control. B. Primary astrocytes were observed with phase contrast microscopy, scale bar = 100 µm. C. Fibroblastic antigen of fibronectin was not observed in primary astrocyte culture.

Data analysis

Confirmation of cultured primary astrocytes was done by detection of marker S100-beta via fluorescence immunocytochemical analysis. This analysis demonstrated that isolated cells from glioblastoma expressed S100-beta antigen. As a result, this protocol is efficient to isolate a purified population of astrocytes. Test results are obtained from three independent experiments.

Notes

  1. Pathological result should confirm that patient tumor specimen is glioblastoma multiforme.
  2. 15 ml Falcon tube containing 5 ml cold HBSS and 10% antibiotic-antimycotic was preserved in refrigerator and glioblastoma sample was transferred into the tube after surgery. Then, the tube containing HBSS, 10% antibiotic-antimycotic and glioblastoma sample was transferred to lab on dry ice.
  3. Freezed glioblastoma sample can’t be used to isolate primary cell culture. We have examined several freezed samples and we could not derive primary astrocyte from these freezed samples. So, sample must be fresh. Whereas, primary astrocytes derived from fresh sample have freeze-thaw tolerance.
  4. Most steps should be performed in a sterile ventilation hood.
  5. All reagents used in Procedures B-E should be pre-warmed at 37 °C before use. Exception is for washing and mincing tissue with cold PBS.
  6. The amount of trypsin directly depends on the sample size of glioblastoma. Increase the Trypsin volume for large tumor sample according to Table 2.

    Table 2. Relation between sample diameter and trypsin volume
    Diameter (millimeter) 
    Trypsin volume (milliliter)
    5
    1
    10
    2
    15
    3
    20
    4

  7. Long time and vigorous mechanical dissociation decrease cell viability (survival rate and growth). Primary cells are sensitive.
  8. We don’t use any growth factor in this method and FBS is nutrition substance in culture medium.
  9. Glioblastoma tumors have abundant blood vessels. Blood vessels can transfer fibroblast cells to culture. According to this protocol, if add fetal bovine serum gradually to culture, fibroblast cells can’t attach to the bottom of culture flask. Finally, the purity of isolated astrocytes from glioblastoma can be increased. 

Recipes

  1. Culture medium (200 ml)
    178 ml DMEM/F12
    20 ml FBS
    2 ml Pen/Strep
    Store at 4 °C
  2. Phosphate buffer saline (PBS) (500 ml)
    Dissolve1 tablet PBS in 500 ml MiliQ water
    Filter-sterilize and store at 4 °C
  3. 4% paraformaldehyde
    Dissolve 4 g paraformaldehyde in 100 ml PBS
  4. 0.2% Triton X-100
    Add 10 µl Triton X-100 in 5 ml PBS
  5. 10% bovine serum albumin
    Dissolve 0.5 g bovine serum albumin in 5 ml PBS

Acknowledgments

This study was supported by grant 20035 from School of Advanced Technologies in Medicine and grant 19925 from Brain and Spinal Cord Injury Research Center, Tehran University of Medical Sciences. The protocol was adopted from Hashemi et al. (2016).

References

  1. Hashemi, M., Fallah, A., Aghayan, H. R., Arjmand, B., Yazdani, N., Verdi, J., Ghodsi, S. M., Miri, S. M. and Hadjighassem, M. R. (2016). A new approach in gene therapy of glioblastoma multiforme: human olfactory ensheathing cells as a novel carrier for suicide gene delivery. Mol Neurobiol 53(8): 5118-5128.
  2. Stupp, R., Mason, W. P., van den Bent, M. J., Weller, M., Fisher, B., Taphoorn, M. J., Belanger, K., Brandes, A. A., Marosi, C., Bogdahn, U., Curschmann, J., Janzer, R. C., Ludwin, S. K., Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, J. G., Eisenhauer, E., Mirimanoff, R. O., European Organisation for, R., Treatment of Cancer Brain, T., Radiotherapy, G. and National Cancer Institute of Canada Clinical Trials, G. (2005). Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352(10): 987-996.
  3. Wen, P. Y. and Kesari, S. (2008). Malignant gliomas in adults. N Engl J Med 359(5): 492-507.

简介

多形性葡萄球菌(GBM)是中枢神经系统中的4级星形细胞瘤。星形胶质细胞可以从人类GBM中分离出来。研究星形胶质细胞可以提供关于胶质母细胞瘤的形成,进展和复发的见解。对于使用孤立的星形胶质细胞,可以在药理学,神经科学和神经外科学领域设计新的研究用于成胶质细胞瘤治疗。该方案描述了从人类GBM制备高纯度原发性星形胶质细胞的细节。在本方案中使用机械解离和化学消化破坏肿瘤组织。培养细胞悬液2周后,初级星形胶质细胞可用于S100-β抗原的进一步传代培养和免疫细胞化学。

星形胶质细胞是提供脑神经元结构和营养支持的神经胶质细胞。星形胶质细胞的细胞周期似乎在星形细胞瘤脑肿瘤中被破坏。世界卫生组织根据恶性肿瘤将星形细胞分为四级。多形性成胶质细胞瘤(GBM,IV级)是星形细胞瘤最恶性的形式。成胶质细胞瘤的特征在于具有快速增殖速率和血管生成的侵袭性细胞。胶质母细胞瘤患者预后差。目前的治疗方法,包括手术,化学治疗和放射治疗对患者的治疗没有很好的疗效。患者的中位生存时间大约在治疗后一年(Stuup等人,2005; Wen和Kesari,2008)。许多研究人员专注于评估胶质母细胞瘤细胞的生理功能和凋亡的研究,以便检测更有效的治疗方法。在这里,我们提出了一种从没有成纤维细胞污染的人胶质母细胞瘤标本中分离高纯度原代星形胶质细胞的方法(Hashemi等人,2016)。

关键字:多形性胶质母细胞瘤, 星形胶质细胞, S100-β抗原, 原代细胞培养, 恶性脑肿瘤, 星形细胞瘤

材料和试剂

  1. 15ml离心管(Corning,Falcon ®,目录号:352096)
  2. 培养皿培养(Nest Biotechnology,目录号:704001)
  3. 10号手刀刀片外科手术刀(BD,目录号:371610)
  4. 细胞过滤筛(Corning,Falcon ®,目录号:352340)
  5. 50ml离心管(Corning,Falcon ®,目录号:352070)
  6. T25烧瓶培养(Nest Biotechnology,目录号:707003)
  7. 1 ml微管(Nest Biotechnology,目录号:613111)
  8. 具有高透明度的聚苯乙烯的24孔板(Nest Biotechnology,目录号:702001)
  9. 5ml移液管(Nest Biotechnology,目录号:326001)
  10. 成胶质细胞瘤多形性样品(人)
  11. Hanks的平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:24020117)
  12. 抗生素 - 抗真菌药(Thermo Fisher Scientific,Gibco TM,目录号:15240062)
  13. 0.25%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  14. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  15. 台盼蓝溶液0.4%(Sigma-Aldrich,目录号:T8154)
  16. 兔抗S100-β的一级抗体(Sigma-Aldrich,目录号:S2644)
  17. 兔抗纤连蛋白的抗体(Abcam,目录号:ab23751)
  18. FITC缀合的山羊抗兔(Abcam,目录号:ab6717)
  19. Dulbecco改良的Eagle培养基/F12(DMEM/F12)(Thermo Fisher Scientific,Gibco TM,目录号:31331028)
  20. 磷酸盐缓冲盐水(PBS)(片剂)(Thermo Fisher Scientific,Gibco TM,目录号:18912014)
  21. 多聚甲醛(粉末)(Sigma-Aldrich,目录号:P6148)
  22. Triton X-100(Sigma-Aldrich,目录号:T8787)
  23. 牛血清白蛋白(粉末)(Sigma-Aldrich,目录号:A2058)
  24. 培养基(见食谱)
  25. 磷酸盐缓冲盐水(PBS)(见食谱)
  26. 4%多聚甲醛(见食谱)
  27. 0.2%Triton X-100(见配方)
  28. 10%牛血清白蛋白(见食谱)

设备

  1. 通风罩(VISION SCIENTIFIC,型号:VS-7120LV)
  2. 37°C水浴(Memmert,型号:WNB14)
  3. 离心机(Hettich Lab Technology,型号:Universal 320R)
  4. 血细胞计数器(Sigma-Aldrich,目录号:Z359629)
  5. CO 2细胞培养箱(Memmert,型号:INC108 T2T3)
  6. 倒置荧光显微镜(Optika,型号:XDS-2FL)
  7. 小镊子手术工具(精细科学工具,目录号:11050-10)

程序

  1. 原始胶质瘤多形性(GBM)组织的收集
    1. 从进行手术治疗的胶质母细胞瘤患者获得多形性成胶质细胞瘤样品。
    2. 本研究由德黑兰医科大学伦理委员会批准。手术前,患者签署书面同意书。
    3. 与神经外科组协调后,神经外科医生将切除的胶质母细胞瘤样品置于含有10%抗生素(抗生素 - 抗真菌药)的5ml冷HBSS的15ml Falcon管中。
    4. 胶质母细胞瘤样品在干冰上输送到实验室。

  2. GBM样品分离成单原发性星形胶质细胞
    1. 在生物学的罩下,去除含有胶质母细胞瘤组织的Falcon管中多余的HBSS。用5ml冷PBS洗涤组织3次以除去血液。
    2. 将胶质母细胞瘤组织置于装有1ml冷PBS的培养皿中。
    3. 用解剖刀刀片切碎并切碎组织5分钟,直到样品转化成乳状悬浮液。粉碎可以通过增加组织解离的功效来帮助加速胰蛋白酶消化过程。
    4. 向切碎的组织中加入3ml温(37℃)0.05%胰蛋白酶-EDTA。然后,用胰蛋白酶将悬浮液转移到新的15 ml Falcon管中
    5. 在37℃水浴下孵育生产的悬浮液10分钟,以消化切碎的组织。
    6. 加入3ml含有10%胎牛血清(FBS)和1%抗生素抗真菌剂的DMEM/F12培养基,以抑制胰蛋白酶的活性。
    7. 用5ml移液管吸取悬浮液10次,并在室温下以180 x g离心10分钟。
    8. 取出上层液体,并用5ml移液管吸取10次,将得到的颗粒缓慢地悬浮于含有2%FBS和1%抗生素抗真菌剂的5ml DMEM/F12中。
    9. 将细胞悬浮液通过40微米的细胞过滤器过滤到50ml管中以除去未解离的碎片和碎屑
    10. 将过滤的悬浮液在室温下离心180分钟5分钟
    11. 将沉淀的细胞重悬于含有2%FBS和1%抗生素抗真菌剂的1ml DMEM/F12中(图1)。


      图1.从人胶质母细胞瘤样品中分离初级星形胶质细胞进行培养的步骤A.手术后人类成胶质细胞瘤样品; B.使用冷PBS洗涤成胶质细胞瘤样品;将胶质母细胞瘤样品转移到培养皿中; D.胶质母细胞瘤样品用手术刀刀片切碎并切碎; E.酶消化后,将细胞悬浮液接种在T25烧瓶中
  3. 细胞计数和电镀
    1. 将10μl细胞悬浮液加入含有10μl0.4%台盼蓝在PBS中的1ml管中。
    2. 将10μl细胞 - 台盼蓝混合物转移到血细胞计数器室。在倒置显微镜下,计算每个单个平方(总共4个正方形)中的细胞数。确定每平方厘米的平均细胞数,并通过将每平方厘米的细胞数乘以2×10 4。
    3. 将含有2%FBS和1%抗生素抗真菌剂的5ml DMEM/F12中的种子1×10 5个细胞置于T25烧瓶中。
    4. 将烧瓶放入37℃,5%CO 2和95%湿度的培养箱中。
    5. 前24小时,星形胶质细胞通常附着在烧瓶的底部。星形胶质细胞神经突在3-4天后逐渐延长。星形胶质细胞在培养第7天完全延伸其神经突后开始生长和增殖。
    6. 每2天更换新鲜培养基的一半细胞培养基。培养基每次变化后,FBS浓度逐渐增加,2周内为2%〜10%(表1)。培养基中的10%FBS量保留在以下初级星形胶质细胞培养物中。

      表1.播种后不同天培养基中的FBS浓度

      播种后更改媒体天数
      FBS浓度
      播种(0)
      2%
      4
      3%
      6
      4%
      8
      5%
      10
      6.5%
      12
      8%
      14
      10%

    7. FBS量的逐渐增加可能会避免细胞培养物的成纤维细胞污染(图2)

      图2.细胞培养不同日期,多形性成胶质细胞瘤原代星形胶质细胞的相位图。。细胞培养2天后。 B.细胞培养4天后C.细胞培养9天; D.细胞培养14天。比例尺=100μm
  4. GBM原发性星形胶质细胞的亚文化
    1. 使用倒置显微镜检查细胞融合水平。在播种后3周达到融合的80%时对细胞进行细菌培养。
    2. 使用5ml无菌移液管在生物罩下从每个T25烧瓶中取出培养基
    3. 用3ml温热(37℃)PBS洗涤细胞,然后除去PBS。
    4. 在孵育器中用1ml温(37℃)0.05%胰蛋白酶-EDA分离细胞1分钟。
    5. 通过向每个T25烧瓶中加入含有10%FBS和1%抗生素抗真菌剂的4ml DMEM/F12,然后将细胞悬浮液从每个烧瓶转移到15ml Falcon管中,从而抑制胰蛋白酶-EDTA的反应。
    6. 将细胞悬浮液在室温下以250×g离心5分钟
    7. 用5ml无菌移液管从每个管中取出培养基。
    8. 在每个管中用1ml含有DMEM/F12,10%FBS和1%抗生素 - 抗霉菌剂的新鲜培养基重悬。
    9. 重新悬浮和计数后,将细胞在24孔培养皿(每孔5×10 3个细胞密度)中平板化,进行免疫细胞化学分析,并用T25烧瓶进行亚培养。 >
  5. S100-β染色
    1. 将种子5×10 3个细胞注入24孔培养皿的每个孔中
    2. 在室温下24小时后用磷酸盐缓冲盐水(PBS)将每孔洗涤三次
    3. 用PBS中的4%多聚甲醛固定细胞20分钟
    4. 在PBS中,室温洗涤细胞三次,每次5分钟
    5. 用0.2%Triton X-100渗透细胞膜15分钟
    6. 在PBS中,室温洗涤细胞三次,每次5分钟
    7. 用10%牛血清白蛋白封闭细胞1 h
    8. 用含有1%BSA的PBS稀释兔抗S100-β1:100的一抗
    9. 在4℃下将细胞与兔抗S100-β的一级抗体或兔抗纤维连接蛋白的一级抗体孵育过夜。
    10. 在PBS中,室温洗涤细胞三次,每次5分钟
    11. 用含有1%BSA的PBS稀释FITC-缀合的山羊抗兔1:500的二抗。
    12. 在室温下在黑暗中孵育细胞与FITC-缀合的山羊抗兔二抗2小时。
    13. 在PBS中,室温洗涤细胞三次,每次5分钟
    14. 通过倒置荧光显微镜观察图像细胞(图3和图4) 注意:只有第二抗体作为阴性对照添加。


      图3.培养3周后,纯化的原代星形胶质细胞中S100-β抗原的表达。A.原代星形胶质细胞表达S100-β的星形胶质细胞抗原(阴性对照);在具有10倍物镜(B)和40倍物镜(C),比例尺=50μm(B),100μm(C)的倒置荧光显微镜下观察到原代星形胶质细胞的S100-β的星形胶质细胞抗原。

      图4.纯化的原代星形细胞培养物中纤连蛋白抗原的评估,以便在培养3周后观察成纤维细胞污染。 A.在阴性对照中未观察到纤连蛋白的成纤维细胞抗原。用相差显微镜观察原发性星形胶质细胞,比例尺= 100μm。原发性星形细胞培养中未观察到纤连蛋白的成纤维细胞抗原

数据分析

通过荧光免疫细胞化学分析检测标记S100-β,进行培养的原代星形胶质细胞的确认。该分析表明,胶质母细胞瘤分离的细胞表达S100-β抗原。因此,该方案对于分离纯化的星形胶质细胞群是有效的。测试结果从三个独立实验中获得。

笔记

  1. 病理结果应确认患者肿瘤标本为多形性胶质母细胞瘤。
  2. 将含有5ml冷HBSS和10%抗生素抗真菌剂的15ml Falcon管保存在冰箱中,并将成胶质细胞瘤样品在手术后转移到管中。然后,将含有HBSS,10%抗生素 - 抗真菌和成胶质细胞瘤样品的管转移到干冰上的实验室。
  3. 冷冻胶质母细胞瘤样品不能用于分离原代细胞培养。我们检查了几个冻结的样品,我们不能从这些冷冻样品中获得原始星形胶质细胞。所以样品必须是新鲜的。而来自新鲜样品的原发性星形胶质细胞具有抗冻融耐受性
  4. 大多数步骤应在无菌通风罩中进行。
  5. 程序B-E中使用的所有试剂应在37℃预热,然后再使用。异常是用冷PBS洗涤和切碎组织。
  6. 胰蛋白酶的量直接取决于成胶质细胞瘤的样品量。根据表2增加大肿瘤样本的胰蛋白酶体积。

    表2.样品直径与胰蛋白酶体积之间的关系
    直径(毫米) 
    胰蛋白酶体积(毫升)
    5
    1
    10
    2
    15
    3
    20
    4

  7. 长时间和剧烈的机械解离降低细胞活力(存活率和生长)。原代细胞是敏感的。
  8. 我们在这种方法中不使用任何生长因子,FBS是培养基中的营养物质。
  9. 胶质母细胞瘤具有丰富的血管。血管可以将成纤维细胞转移到培养物中。根据该方案,如果将胎牛血清逐渐培养,成纤维细胞不能附着在培养瓶的底部。最后,可以增加成胶质细胞瘤分离的星形胶质细胞的纯度。

食谱

  1. 培养基(200 ml)
    178 ml DMEM/F12
    20毫升FBS
    2 ml Pen/Strep
    储存于4°C
  2. 磷酸盐缓冲盐水(PBS)(500 ml)
    将500ml MiliQ水溶解1片PBS 过滤灭菌并在4℃下储存
  3. 4%多聚甲醛
    将4g多聚甲醛溶解于100ml PBS中
  4. 0.2%Triton X-100
    在5ml PBS中加入10μlTriton X-100
  5. 10%牛血清白蛋白 将0.5 g牛血清白蛋白溶解在5ml PBS中

致谢

这项研究得到了医学先进技术学院授予的20035年资助,并授予了德黑兰医科大学脑脊髓损伤研究中心19925年。该协议由Hashemi等人采用。 (2016)。

参考文献

  1. Hashemi,M.,Fallah,A.,Aghayan,HR,Arjmand,B.,Yazdani,N.,Verdi,J.,Ghodsi,SM,Miri,SM and Hadjighassem,MR(2016)。  多形态成胶质细胞瘤基因治疗的新方法:人类嗅鞘细胞作为新载体用于自杀基因递送。 Mol Neurobiol 53(8):5118-5128。
  2. Stupp,R.,Mason,WP,van den Bent,MJ,Weller,M.,Fisher,B.,Taphoorn,MJ,Belanger,K.,Brandes,AA,Marosi,C.,Bogdahn,U.,Curschmann, J.,Janzer,RC,Ludwin,SK,Gorlia,T.,Allgeier,A.,Lacombe,D.,Cairncross,JG,Eisenhauer,E.,Mirimanoff,RO,European Organization for,R.,Treatment of Cancer Brain ,T.,Radiotherapy,G. and National Cancer Institute of Canada Clinical Trials,G.(2005)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/15758009"target ="_ blank">放射治疗加用于胶质母细胞瘤的辅助性佐剂替莫唑胺。 N Engl J Med 352(10):987-996。
  3. Wen,PY和Kesari,S.(2008)。成人恶性神经胶质瘤。 N Engl J Med 359(5):492-507。
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引用:Hashemi, M. and Hadjighassem, M. (2017). Preparation of Primary Astrocyte Culture Derived from Human Glioblastoma Multiforme Specimen. Bio-protocol 7(8): e2241. DOI: 10.21769/BioProtoc.2241.
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