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Establishing Primary Malignant Pleural Mesothelioma (MPM) Cell Cultures
原代恶性胸膜间皮瘤(MPM)的细胞培养   

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Abstract

This is a general protocol for the isolation and maintenance of primary MPM cultures as a tool for the identification of Tumor Initiating Cells and early progenitor-targeting drugs (Cioce et al., 2010). Primary cultures can be propagated efficiently for 8-12 weeks and xenotransplanted in NOD/SCID mice while retaining the histofeatures of the originating tumor (Canino et al., 2012). The protocol is suitable for both MPM solid specimens and pleural effusion. For increased clarity, initially two separate sections addressing the isolation of MPM cells from solid tumors and pleural effusions are here provided.

Materials and Reagents

  1. Surgical specimen or pleural effusion
  2. Phosphate buffered saline (PBS)
  3. Collagenase type XI (300 U/ml) (Sigma-Aldrich, catalog number: C9407 )
  4. Hyaluronidase (100 U/ml) (Sigma-Aldrich, catalog number: H4272 )
  5. DMEM/F12 (1:1)+ GLUTAMAX (Life Technologies, Invitrogen™, catalog number: 10565-018 )
  6. FBS-non heat inactivated (Life Technologies, catalog number: 10437-028 )
  7. Human recombinant insulin (Sigma-Aldrich, catalog number: I-5500 )
  8. Bovine Serum Albumin-Fatty Acid Free (BSA-FAF) (Sigma-Aldrich, catalog number: A7030 )
  9. Non-heat inactivated FBS (Life Technologies, Gibco®, catalog number: 16000-044 )
  10. Ciprofloxacin (Sigma-Aldrich, catalog number: 17850 )
  11. Red blood lysis buffer (0.8% ammonium chloride) (Life Technologies, catalog number: A10492-01 )
  12. ACCUTASE (STEMCELL Technologies, catalog number: AT-104 )
  13. 0.4% trypan blu (Life Technologies, Gibco®, catalog number: 15250-061 )
  14. Digestion medium

Equipment

  1. Cell culture set up
  2. Scalpels (Becton, Dickinson and Company, catalog number: 371621 )
  3. Microdissecting forceps
  4. 5 ml Pasteur pipette (BD Biosciences, Falcon®)
  5. 15 ml centrifuge tubes
  6. 50 ml centrifuge tubes
  7. Centrifuge capable of running at ≥ 300 x g
  8. 70 μm nylon mesh (BD Biosciences, Falcon®, catalog number: 352350 )
  9. Ultralow attachment dishes (Corning Incorporated, catalog number: 3261 for 100 mm) (or alternatively, sterile Petri dishes non treated for cell culture)

Procedure

  1. Protocol 1A. Procedure for isolating MPM cells from surgical specimens
    Critical step: The time interval from tumor resection and digestion must be kept to a minimum, ideally ≤ 1 h.
    Critical step: To prevent undesired contamination, work under sterile condition at all steps and whenever possible.
    1. Wash the tumor specimen twice with PBS 1x supplemented with Ciprofloxacin (4 μg/ml) (2 x 40 ml in a 50 ml tube). To disaggregate the solid tumor follow three sequential steps (in a tissue-culture sterile hood):
      1. Manually cut the solid tumor into ≤ 1mm pieces with scalpels in a sterile Petri dish with 1 ml 1x PBS.
      2. Enzymatic disaggregation: Re-suspend tumor pieces in a 100 mm cell culture dish with 5 ml of digestion medium (DMEM-F12 + GLUTAMAX supplemented with 1% BSA-FAF and 5 μg/ml human insulin). After resuspension, add collagenase (final concentration 30 U/ml) and hyaluronidase (final concentration 10 U/ml) to the tumor suspension and leave cells in the incubator for 2 h at 37 °C, 5% CO2. Every 15 min re-suspend the semi-digested tumor with a 5 ml sterile Pasteur pipette by gently pipetting up and down to disperse tumor pieces.
    2. Filter the digested material through a 70 μm nylon mesh in a 50 ml tube.
    3. Transfer the filtered material to a 15 ml centrifuge tube.
    4. Spin at 300 x g for 10 min at room temperature (RT).
    5. Re-suspend the pellet in fresh medium DMEM F12 (1:1) + GLUTAMAX supplemented with 5% non-heat inactivated FBS, insulin (10 μg/ml) and ciprofloxacin (4 μg/ml). Count total live cell number with Trypan-Blue exclusion method.
    6. Seed cells in low-adhesion cell culture dishes at a cell density ≥ 1-1.5 x 106 cells/ml). Growth cells for 3 weeks and add 25% fresh medium once a week. After 3 weeks, a quite homogeneous population of mesothelioma cells can be observed in culture. These can be propagated (to a limited extent-see below) or harvested for further processing.

  2. Protocol 1B. Procedure for isolating MPM cells from pleural effusions
    1. Collect the pleural effusion in 15 ml FALCON tubes diluted 1:1 with 1x PBS supplemented with Ciprofloxacin (4 μg/ml).
    2. Harvest cells by centrifugation at 300 x g for 10 min at room temperature (RT). Keep the cell-free medium (supernatant - pleural effusion).
    3. Resuspend cells in Red Blood Lysis buffer (10 bed pellet volumes). Incubate for 5 min at room temperature (RT).
    4. Centrifuge (300 x g, 10 min) and discard supernatant.
    5. Re-suspend the pellet in fresh medium DMEM F12 (1:1) + GLUTAMAX supplemented with 5% non-heat inactivated FBS, insulin (10 μg/ml) and ciprofloxacin (4 μg/ml) + 30% cell free medium (as from step b). Count total live cell number with Trypan-Blue exclusion method.
    6. Seed cells in low-adhesion cell culture dishes at a cell density ≥ 1-1.5 x 106 live cells/ml. Size of the dish must be chosen according to the available number of cells in order to achieve the desired concentration.
    7. Grow cells for 3 weeks and add 25% fresh medium once a week. After an average of 3 weeks, a heterogeneous population of mesothelioma cells can be observed in culture. This is mainly composed of adherent and loosely attached cells (Figure 1), which can be propagated (to a limited extent-see below) or harvested for further processing.

  3. Protocol 2. Propagation of MPM cell cultures
    For the propagation of the primary cell cultures, always collect both floating (or loosely adherent) and adherent cell subpopulations.
    It is very important to keep conditioned medium during harvesting of the cells and add it back to cell culture during the establishment of the culture (Protocol 1, b) or during propagation of the cells (Protocol 2, a).
    All the volumes listed below refer to 100 mm dishes. Please vary volumes of solutions according the size of the cell culture dishes used.
    1. Collect cell culture medium in a centrifuge tube. Wash the adherent cells with 3-5 ml of 1x PBS and add it to the collected cell culture medium. This contains loosely adherent or floating cells (1x PBS should be ≤ 30% final volume in the collection tube).
    2. Wash cells again with 1x PBS, discard 1x PBS and add accutase (1.5 ml/dish).
    3. Incubate cells in the incubator for 5 min at 37 °C, 5% CO2.
    4. Harvest Accutase-treated (detached) cells with the collected cell culture medium/PBS 1x washing from step a. Count total live cell number with Trypan-Blue exclusion method.
    5. Seed cells in low-adhesion cell culture dishes at a density ≤ 0.5 x 106 live cells/ml). To achieve the required cell concentrations dilute the harvested cells with DMEM F12 (1:1) + GLUTAMAX supplemented with 5% non-heat inactivated FBS, insulin (20 μg/ml) and ciprofloxacin (4 μg/ml). The dilution medium must represent ≥ of the 50% of the final cell culture volume in the dish.


      Figure 1. Representative micrograph of a MPM cell culture (from a malignant pleural effusion) at 2 weeks after seeding.
      Arrows: adherent, fibroblast-like cells. Arrowheads: loosely adherent, rounded cells.

Notes

  1. Ciprofloxacin prevents contamination of the material from non-sterile handling of the tumor specimens during the harvesting of the sample.
  2. The obtained tumor digests consist initially of a heterogeneous population, comprising but not limited to Mesothelioma cells, macrophages, Immune infíltrate, stromal cells, adipocytes and remnant red blood cells. However, within 72-96 h from seeding most of the cells in culture consist of mesothelioma cells, since the mentioned accompanying cell subpopulations will not propagate in the experimental conditions used here, as revealed by morphological observations and clonogenic assays (Canino et al., 2012) The obtained populations have been shown to origínate MPM-like tumors when injected into NOD/SCID mice with very high resemblance to the originating tumor (Canino et al., 2012).
  3. A typical yield of 1 x 106 cells can be obtained from a 100 mg solid specimen. A typical yield of 10 x 106 cells can be obtained from 30 to 5 ml freshly collected pleural effusion (after red blood lysis removal).

Acknowledgments

This protocol was adapted from Canino et al. (2012). We acknowledge funding from INAIL (Italian Workers Compensation Authority) and AIRC-ROC and to GB.

References

  1. Canino, C., Mori, F., Cambria, A., Diamantini, A., Germoni, S., Alessandrini, G., Borsellino, G., Galati, R., Battistini, L., Blandino, R., Facciolo, F., Citro, G., Strano, S., Muti, P., Blandino, G. and Cioce, M. (2012). SASP mediates chemoresistance and tumor-initiating-activity of mesothelioma cells. Oncogene 31(26): 3148-3163.
  2. Cioce, M., Gherardi, S., Viglietto, G., Strano, S., Blandino, G., Muti, P. and Ciliberto, G. (2010). Mammosphere-forming cells from breast cancer cell lines as a tool for the identification of CSC-like- and early progenitor-targeting drugs. Cell Cycle 9(14): 2878-2887.

简介

这是用于分离和维持初级MPM培养物的一般方案,作为鉴定肿瘤启动细胞和早期祖细胞靶向药物的工具(Cioce等人,2010)。 原代培养物可以有效繁殖8-12周,并且在NOD/SCID小鼠中异种移植,同时保留起源肿瘤的组织特征(Canino等人,2012)。 该方案适用于MPM固体标本和胸腔积液。 为了增加清晰度,最初提供了两个分开的部分,其涉及从实体瘤和胸腔积液中分离MPM细胞。

材料和试剂

  1. 手术标本或胸腔积液
  2. 磷酸盐缓冲盐水(PBS)
  3. 胶原酶类型XI(300U/ml)(Sigma-Aldrich,目录号:C9407)
  4. 透明质酸酶(100U/ml)(Sigma-Aldrich,目录号:H4272)
  5. DMEM/F12(1:1)+ GLUTAMAX(Life Technologies,Invitrogen TM,目录号:10565-018)
  6. FBS-非热灭活(Life Technologies,目录号:10437-028)
  7. 人重组胰岛素(Sigma-Aldrich,目录号:I-5500)
  8. 牛血清白蛋白 - 无脂肪酸(BSA-FAF)(Sigma-Aldrich,目录号:A7030)
  9. 非热灭活的FBS(Life Technologies,Gibco ,目录号:16000-044)
  10. 环丙沙星(Sigma-Aldrich,目录号:17850)
  11. 红血液裂解缓冲液(0.8%氯化铵)(Life Technologies,目录号:A10492-01)
  12. ACCUTASE(STEMCELL Technologies,目录号:AT-104)
  13. 0.4%台盼蓝(Life Technologies,Gibco ,目录号:15250-061)
  14. 消化介质

设备

  1. 细胞培养设置
  2. 手术刀(Becton,Dickinson and Company,目录号:371621)
  3. 微切割钳
  4. 5ml的巴斯德吸管(BD Biosciences,Falcon )
  5. 15ml离心管
  6. 50ml离心管
  7. 离心机能够在≥300 em x/h下运行
  8. 70μm尼龙网(BD Biosciences,Falcon ,目录号:352350)
  9. 超低附件皿(Corning Incorporated,目录号:3261,100mm)(或者,未处理细胞培养物的无菌培养皿)

程序

  1. 协议1A。 从手术标本中分离MPM细胞的程序
    关键步骤:肿瘤切除和消化的时间间隔必须保持最小,理想情况下≤1小时。
    关键步骤:为了防止不必要的污染,在无菌条件下在所有步骤和任何可能的时候工作。
    1. 用补充有环丙沙星(4μg/ml)(2x40ml,在50ml试管中)的PBS 1x洗涤肿瘤样品两次。 按照三个连续步骤(在组织培养无菌罩中)分解实体瘤:
      1. 用无菌培养皿用1ml 1×PBS手术切除实体肿瘤,用手术刀切成≤1毫米
      2. 酶解:用5ml消化培养基(补充有1%BSA-FAF和5μg/ml人胰岛素的DMEM-F12 + GLUTAMAX)在100mm细胞培养皿中重悬细胞碎片。重悬后,向肿瘤悬浮液中加入胶原酶(最终浓度30U/ml)和透明质酸酶(终浓度10U/ml),并将细胞在37℃,5%CO 2中保持在培养箱中2小时, sub>。每15分钟用5ml无菌巴斯德吸管重新悬浮半消化的肿瘤,轻轻地上下吹打以分散肿瘤块。
    2. 在50ml管中通过70μm尼龙网过滤消化的材料
    3. 将过滤的材料转移到15ml离心管中
    4. 在室温(RT)下以300×g离心10分钟
    5. 将沉淀重新悬浮在补充有5%非热灭活的FBS,胰岛素(10μg/ml)和环丙沙星(4μg/ml)的新鲜培养基DMEM F12(1:1)+ GLUTAMAX中。使用台盼蓝排除法计算总活细胞数。
    6. 在低粘附细胞培养皿中以细胞密度≥1.1-5×10 6个细胞/ml接种细胞。 生长细胞3周,每周加入25%新鲜培养基。 3周后,在培养物中可观察到相当同质的间皮瘤细胞群。 这些可以繁殖(在有限程度上 - 见下文)或收获用于进一步处理

  2. 协议1B。 从胸腔积液中分离MPM细胞的步骤
    1. 收集胸腔积液在15毫升FALCON管中稀释1:1的1x PBS补充环丙沙星(4微克/毫升)。
    2. 通过在室温(RT)下以300xg离心10分钟收获细胞。 保持无细胞培养基(上清液 - 胸腔积液)。
    3. 重悬细胞在红血液裂解缓冲液(10床丸粒体积)。 在室温(RT)下孵育5分钟。
    4. 离心(300×g,10分钟)并弃去上清液
    5. 将沉淀重新悬浮在补充有5%非热灭活的FBS,胰岛素(10μg/ml)和环丙沙星(4μg/ml)+ 30%无细胞培养基(作为补充剂)的新鲜培养基DMEM F12(1:1)+ GLUTAMAX中。来自步骤b)。使用台盼蓝排除法计算总活细胞数。
    6. 种子细胞在低粘附细胞培养皿中,细胞密度≥1.1.5×10 6活细胞/ml。必须根据可用的细胞数量选择培养皿的大小,以达到所需的浓度
    7. 生长细胞3周,并添加25%新鲜培养基每周一次。平均3周后,可以在培养物中观察到间皮瘤细胞的异质群体。这主要由粘附和松散附着的细胞组成(图1),其可以繁殖(在有限程度上 - 见下文)或收获用于进一步加工。

  3. 方案2. MPM细胞培养物的繁殖
    对于原代细胞培养物的繁殖,总是收集浮动(或松散粘附)和贴壁细胞亚群。
    在培养物建立期间(方案1,b)或在细胞繁殖期间(方案2,a),在收获细胞期间保持条件培养基并将其添加回细胞培养物是非常重要的。 em>
    下面列出的所有卷均指100 mm盘。请根据所使用的细胞培养皿的大小,改变溶液的体积。
    1. 收集细胞培养基在离心管中。用3-5ml 1×PBS洗涤贴壁细胞,并将其加入收集的细胞培养基中。这包含松散粘附或浮动的细胞(1×PBS应≤收集管中的最终体积的30%)
    2. 用1×PBS再次洗涤细胞,弃去1×PBS,加入accutase(1.5ml /皿)
    3. 在37℃,5%CO 2孵育器中孵育细胞5分钟。
    4. 收获Accutase处理(分离)细胞与从步骤a收集的细胞培养基/PBS 1x洗涤。使用台盼蓝排除法计算总活细胞数。
    5. 低密度细胞培养皿中的种子细胞,密度≤0.5×10 6活细胞/ml)。为了达到所需的细胞浓度,用补充有5%非热灭活的FBS,胰岛素(20μg/ml)和环丙沙星(4μg/ml)的DMEM F12(1:1)+ GLUTAMAX稀释收获的细胞。稀释培养基必须占培养皿中最终细胞培养体积的50%

      图1.接种后2周的MPM细胞培养物(来自恶性胸腔积液)的代表性显微照片。箭头:粘附的成纤维细胞样细胞。箭头:松散粘附,圆形细胞

笔记

  1. 环丙沙星可防止在收集样品期间来自非无菌处理肿瘤样品的材料的污染。
  2. 所获得的肿瘤消化物最初包括异质群体,包括但不限于间皮瘤细胞,巨噬细胞,免疫抑制剂,基质细胞,脂肪细胞和残余红细胞。然而, 在72-96小时内从接种培养物中的大多数细胞由间皮瘤细胞组成,因为所提到的伴随的细胞亚群将不会在此处使用的实验条件下繁殖,如通过形态学观察和克隆形成测定所揭示的(Canino et al。 ,2012)当注射到与起始肿瘤非常相似的NOD/SCID小鼠中时,所获得的群体已经显示出起源于MPM样肿瘤(Canino等人,2012)。
  3. 可以从100mg固体样品获得1×10 6个细胞的典型产量。从30至5ml新鲜收集的胸腔积液(在除去红细胞溶解后)可以获得10×10 6个细胞的典型产量。

致谢

该协议改编自Canino等人(2012)。我们确认INAIL(意大利工人赔偿局)和AIRC-ROC和GB的资金。

参考文献

  1. Canino,C.,Mori,F.,Cambria,A.,Diamantini,A.,Germoni,S.,Alessandrini,G.,Borsellino,G.,Galati,R.,Battistini,L.,Blandino, Facciolo,F.,Citro,G.,Strano,S.,Muti,P.,Blandino,G.and Cioce,M。(2012)。 SASP介导间皮瘤细胞的化学抗性和肿瘤起始活性。癌基因 31(26):3148-3163。
  2. Cioce,M.,Gherardi,S.,Viglietto,G.,Strano,S.,Blandino,G.,Muti,P.and Ciliberto,G.(2010)。 乳腺癌细胞系的乳腺形成细胞作为识别CSC样和 早期祖细胞靶向药物。 细胞周期 9(14):2878-2887。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cioce, M., Canino, C., Strano, S. and Blandino, G. (2012). Establishing Primary Malignant Pleural Mesothelioma (MPM) Cell Cultures. Bio-protocol 2(21): e285. DOI: 10.21769/BioProtoc.285.
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