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Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells
骨髓巨噬细胞和肿瘤细胞的跨室共培养   

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Abstract

Bone is a primary site of metastasis from prostate and breast cancers. Bone marrow macrophages (BMMs) are mediators of inflammatory processes and are thought to promote tumor growth in the skeletal sites. In order to elucidate how their interactions with tumor cells impact aggressiveness of metastatic tumors in bone in vitro methods are required. By employing a system in which BMMs and tumor cells are grown separately, yet share the media and exchange soluble factors, contribution of each cell type in the context of BMM-tumor cell relationship in the bone marrow can be investigated. Additional advantages of this system include the ability to study: 1) phenotypic changes in BMMs and tumor cells upon co-culture; 2) cell-specific modulation of protein and gene expression; and 3) effects on proliferation and cell survival. It is noteworthy, that this transwell co-culture system is not limited to BMMs and tumor cells and can be easily modified to include other components of bone marrow microenvironment (e.g., adipocytes, stromal cells, osteoblasts).

Materials and Reagents

  1. Mice (FvBN strain)
  2. PC3 cells (ATCC, catalog number: CRL 7934 )
  3. L929 cells (ATCC, catalog number: VR-1404 )
  4. DMEM (Sigma-Aldrich, catalog number: D2902 )
  5. MEM-alpha (Sigma-Aldrich, catalog number: M0644 )
  6. Fetal bovine serum (Thermo Fisher Scientific, HyClone catalog number: SH3008803 )
  7. Pencillin-Streptomycin (Life Technologies, Gibco®, catalog number: 15140-122 )
  8. 0.25% Trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25200-056 )
  9. 1x Sterile PBS (Sigma-Aldrich, catalog number: P3813 )
  10. 70% ethanol
  11. RNeasy Plus Mini Kit (QIAGEN, catalog number: 74134 )
  12. QIAshredder (QIAGEN, catalog number: 79654 )
  13. RLT buffer (part of RNeasy Plus Mini Kit, shown above), includes 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
  14. Sucrose (Sigma-Aldrich, catalog number: S9378 )
  15. MES (Sigma-Aldrich, catalog number: M8250 )
  16. Complete DMEM (see Recipes)
  17. L929 conditioned media (see Recipes)
  18. BMM media (see Recipes)
  19. SME lysis buffer (see Recipes)

Equipment

  1. 6-well plates (Corning, Costar®, catalog number: 3516 )
  2. Transwell inserts (Corning, catalog number: 3412 )
  3. Petri dishes (Thermo Fisher Scientific, catalog number: 08-757-13 )
  4. 100 mm tissue culture dishes (Corning, catalog number: 430293 )
  5. T75 flasks (Corning, catalog number: 430725 )
  6. Cell scrapers (SARSTEDT AG, catalog number: 83.1830 )
  7. Sterile fine-tip transfer pipets (Thermo Fisher Scientific, catalog number: 232-1S )
  8. 10 ml syringes (Luer-Lok) (BD, catalog number: 309604 )
  9. 5, 10, & 25 ml serological pipets
  10. Needles (26 Gauge & 20 Gauge) (BD, catalog numbers: 305111 & 305176 , respectively)
  11. Dissecting forceps and scissors
  12. Scalpel
  13. 1.5 ml tubes (Thermo Fisher Scientific, catalog number: 3464 )
  14. 15 ml centrifuge tubes (Thermo Fisher Scientific, Nunc®, catalog number: 339650 )
  15. 50 ml centrifuge tubes (Thermo Fisher Scientific, Nunc®, catalog number: 339652 )
  16. 5 K Millipore centrifugal concentrator (Amicon Ultra-4) (Millipore, model: UFC800596 )
  17. Centrifuge
  18. Incubator (5% CO2, 37 °C) (Napco Series 8000 DH CO2 Incubator)
  19. Biosafety cabinet (The Baker Company, SterilGARD®)

Procedure

  1. Obtaining BMMs from murine bone marrow
    1. Sacrifice donor mouse (male, 6-8 weeks of age, in-house bred) using CO2 inhalation and cervical dislocation.
    2. In sterile hood, rinse hind legs with 70% ethanol and remove, making sure to cut above head of femur. Place skinned legs in a petri dish with 1x sterile PBS.
    3. Remove muscle and connective tissue from bones (both femur and tibia), scraping them clean with scalpel and placing into fresh 1x sterile PBS.
    4. Place fresh scalpel and dissecting forceps into beaker of 70% ethanol. Remove one bone from PBS onto dry petri dish, and cut both ends of bone, exposing marrow.
    5. Using 26 gauge needle and 10 ml syringe, drill into bone marrow and slowly flush out marrow using BMM media into 50 ml centrifuge tube. Flush 2 ml from one side, flip bone, flush 2 ml from the other side - 4 ml total per bone.
    6. Repeat step A5 for all 4 bones into 1 x 50 ml centrifuge tube.
    7. Using 20 gauge needle and 10 ml syringe, gently mix the cell suspension 4x by aspiration into syringe to break up any clumps.
    8. Bring volume of BMM media and cells up to 36 ml. Plate into 3x petri dishes with 12 ml per 100 mm petri dish (~9-10 x 106 cells/dish). Allow cells to grow undisturbed for ~84 h in cell culture incubator (at 37 °C, 5% CO2).
      Note: Petri dishes are used instead of treated cell culture plates to allow easier passaging of BMMs. These cells tend to tightly adhere to tissue culture-treated cells and flasks and are difficult to lift for subsequent experiments.

  2. Transwell set up
    1. Both BMMs and PC3 cells should be at 70-80% confluency and ready for use the same day.
    2. BMMs:
      1. Remove media and lightly rinse BMM dish with 1x PBS. Add ~8 ml of fresh BMM media to dish and gently remove cells using a sterile fine-tip transfer pipet and cell scraper, collecting into a 15 ml centrifuge tube.
      2. Perform a cell count on a single-cell suspension, and plate 0.35 x 106 BMMs in 2 ml of BMM media directly in the 6-well plates.
    3. Using sterile forceps, gently place transwell inserts in each well above freshly plated cells. By their design inserts are secured in place by the walls of the well. They will remain suspended and touch the media, but not the bottom of the plate allowing for free exchange of soluble factors between the cells on the bottom and top of the insert.
    4. PC3s
      1. Allow PC3 cells to grow to ~70-80% confluency in T75 flask.  
      2. Rinse with 1x sterile PBS and dissociate the cells using 2 ml of 0.25% Trypsin-EDTA.
      3. Use 8 ml complete DMEM to stop the activity of the trypsin and collect all 10 ml in a 15 ml centrifuge tube.
      4. Centrifuge the cells at 130 x g for 5 min at RT to pellet, and re-suspend in an appropriate amount of complete DMEM (~8 ml) to perform a cell count.
      5. Plate 0.2 x 106 PC3s in 2 ml of complete DMEM on top of the transwell inserts.
    5. Allow the cells to grow for 48 h undisturbed in cell culture incubator.
    6. When plating for control conditions without the transwell, plate the same number of cells directly on the 6-well plate, in a mix of 2 ml complete DMEM and 2 ml BMM media.

  3. Changing co-culture to serum-free conditions (if necessary)
    1. For experiments requiring analysis of media conditioned by cells in co-culture and to avoid interference from serum in the media, transwell co-cultures may be changed to serum-free conditions 12-16 h prior to harvest.
    2. Briefly, after 48 h, gently wash the cells 3x with 1x sterile PBS. To do so, remove media from the top of the transwell, lift the transwell, remove media from the bottom of the well, add 2 ml of PBS to the bottom of the well, replace the transwell, add 2 ml of PBS to the transwell, and gently agitate. Repeat at least 2 times.
    3. Plate 2 ml of serum-free MEM-alpha to the bottom of the well, and 2 ml of serum-free DMEM to the transwell. Allow cells to sit for 12-16 h undisturbed in cell culture incubator.
    4. If change to serum-free conditions is not necessary, 3x PBS washes should be performed immediately prior to sample harvest for analyses.

  4. Harvesting experimental protein samples
    1. Media:
      1. Collect media into 15 ml centrifuge tube. Transfer any transwells to new, unused 6-well dishes.
      2. Remove any floating cells by centrifuging collected media at 130 x g (to keep the cells intact) for 5 min at 4 °C.
      3. Transfer media to new 15 ml centrifuge tube.
      4. Remove any additional cell debris by centrifuging at 830 x g for 10 min at 4 °C. It is necessary that the two spins are performed separately and intact cells are removed before 830 x g spin is performed.
      5. Freeze media as collected at -80 °C for future use or concentrate if needed using 5 K Millipore Centrifugal concentrator.
      6. Transfer media to the concentrator, and spin at 3,000 x g at 4 °C until desired volume is achieved.
      7. Store concentrated media at -80 °C for future use.
    2. Protein lysates - BMMs
      1. After media is collected, add required amount of SME (~200 μl) to cells and set plate on ice.
      2. Gently scrape the cells and collect into 1.5 ml tube.
      3. Freeze at -80 °C.
    3. Protein lysates - PC3s
      1. After media is collected, add 2 ml 0.25% Trypsin-EDTA to transwells, and 1 ml 0.25% Trypsin-EDTA to wells used as controls (i.e. directly on plastic).
      2. After ~5 min at 37 °C, cells should be dissociated. Stop the trysinization with 4 ml complete DMEM. This volume is sufficient for control wells.
      3. Using a sterile fine-tip transfer pipet, gently rinse and collect cells from the wells into a 15 ml centrifuge tube. For the transwells, add an additional 4 ml of complete DMEM to the transwell, and rinse again, adding the additional media to the original tube.
      4. Spin the cells at 130 x g for 5 min at RT.
      5. Remove the media, and gently wash the pellet by re-suspending in ~10 ml of 1x sterile PBS.
      6. Spin the cells at 130 x g for 5 min at RT, and remove all PBS.
      7. Re-suspend the pellet in SME (~200 μl) and transfer into 1.5 ml tube.
      8. Freeze at -80 °C.

  5. Harvesting samples for RNA analyses
    1. After 48 h, gently wash the cells 1x with PBS as described in step C1.  
    2. Collect the PC3 cells as indicated in steps D3a-d. Wash pellet an additional time with PBS (repeat steps D3e-f an additional time). Add RLT Buffer from RNeasy Plus Mini Kit to the pellet. Use ~250 μl per well collected. Transfer to fresh QIAshredder.
    3. Add ~250 μl of RLT Buffer per well directly to washed BMM cells. Scrape cells and collect into fresh QIAshredder.
    4. Proceed with RNA extraction according to directions supplied with RNeasy Plus Mini Kit.

Recipes

  1. Complete DMEM
    445 ml DMEM, sterile filtered
    50 ml heat-inactivated fetal bovine serum
    5 ml Pencillin-Streptomycin 10,000 U each/ml
  2. L929 conditioned media
    1. L929 cells grown to ~70-80% confluency in 10 ml using 100 mm tissue culture dish over 48 h in complete DMEM
    2. Media collected from cells into 15 ml centrifuge tube
    3. Cells removed from media by centrifuging at 130 x g for 5 min
    4. Media transferred to new 15 ml centrifuge tube
    5. Cell debris removed from media by centrifuging at 830 x g for 10 min
    6. Media transferred to new 15 ml centrifuge tube and used
  3. BMM media (30% L929 CM; 20% FBS)
    12 ml L929 conditioned media
    5.3 ml heat-inactivated fetal bovine serum
    22.7 ml MEM-alpha media
  4. SME lysis buffer
    250 mM sucrose
    25 mM MES
    1 mM EDTA
    In deionized water
    pH to 6.5, add 0.1% TritonX-100 before use

Acknowledgments

This protocol was adapted from our manuscript Herroon et al. (2013). Grant support was provided by DOD PC074031, DOD PC030325, Wayne State University start-up fund and MICR: P30 CA 22453.

References

  1. Herroon, M. K., Rajagurubandara, E., Rudy, D. L., Chalasani, A., Hardaway, A. L. and Podgorski, I. (2013). Macrophage cathepsin K promotes prostate tumor progression in bone. Oncogene 32(12): 1580-1593.

简介

骨是来自前列腺和乳腺癌的原发转移部位。骨髓巨噬细胞(BMM)是炎症过程的介质,并且被认为促进骨骼部位中的肿瘤生长。为了阐明它们与肿瘤细胞的相互作用如何影响骨髓中转移性肿瘤的侵袭性,需要体外方法。通过采用其中BMM和肿瘤细胞分开生长,但共享培养基和交换可溶性因子的系统,可以研究在骨髓中BMM-肿瘤细胞关系的上下文中每种细胞类型的贡献。该系统的另外的优点包括研究以下的能力:1)共培养时BMM和肿瘤细胞中的表型改变; 2)蛋白质和基因表达的细胞特异性调节;和3)对增殖和细胞存活的影响。值得注意的是,该transwell共培养系统不限于BMM和肿瘤细胞,并且可以容易地修饰以包括骨髓微环境的其他组分(例如,脂肪细胞,基质细胞,成骨细胞)。

材料和试剂

  1. 小鼠(FvBN株)
  2. PC3细胞(ATCC,目录号:CRL 7934)
  3. L929细胞(ATCC,目录号:VR-1404)
  4. DMEM(Sigma-Aldrich,目录号:D2902)
  5. MEM-α(Sigma-Aldrich,目录号:M0644)
  6. 胎牛血清(Thermo Fisher Scientific,HyClone目录号:SH3008803)
  7. 青霉素 - 链霉素(Life Technologies,Gibco ,目录号:15140-122)
  8. 0.25%胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:25200-056)
  9. 1x无菌PBS(Sigma-Aldrich,目录号:P3813)
  10. 70%乙醇
  11. RNeasy Plus迷你包(QIAGEN,目录号:74134)
  12. QIAshredder(QIAGEN,目录号:79654)
  13. RLT缓冲液(RNeasy Plus Mini Kit的一部分,如上所示)包括2-巯基乙醇(Sigma-Aldrich,目录号:M7522)
  14. 蔗糖(Sigma-Aldrich,目录号:S9378)
  15. MES(Sigma-Aldrich,目录号:M8250)
  16. 完成DMEM(参见配方)
  17. L929条件培养基(见配方)
  18. BMM媒体(参见配方)
  19. SME裂解缓冲液(参见配方)

设备

  1. 6孔板(Corning,Costar ,目录号:3516)
  2. Transwell插入物(Corning,目录号:3412)
  3. 培养皿(Thermo Fisher Scientific,目录号:08-757-13)
  4. 100mm组织培养皿(Corning,目录号:430293)
  5. T75烧瓶(Corning,目录号:430725)
  6. 细胞刮刀(SARSTEDT AG,目录号:83.1830)
  7. 无菌细尖移液管(Thermo Fisher Scientific,目录号:232-1S)
  8. 10ml注射器(Luer-Lok)(BD,目录号:309604)
  9. 5,10,& 25 ml血清移液器
  10. 针(26Gauge& 20Gauge)(BD,目录号分别为305111& 305176)
  11. 解剖钳和剪刀
  12. Scalpel
  13. 1.5ml管(Thermo Fisher Scientific,目录号:3464)
  14. 15ml离心管(Thermo Fisher Scientific,Nunc ,目录号:339650)
  15. 50ml离心管(Thermo Fisher Scientific,Nunc ,目录号:339652)
  16. 5 K Millipore离心浓缩器(Amicon Ultra-4)(Millipore,型号:UFC800596)
  17. 离心机
  18. 培养箱(5%CO 2,37℃)(Napco系列8000 DH CO 2培养箱)
  19. 生物安全柜(The Baker Company,SterilGARD ®

程序

  1. 从小鼠骨髓获得BMM
    1. 使用CO 2吸入和颈脱臼的牺牲供体小鼠(雄性,6-8周龄,内部繁殖)。
    2. 在无菌罩,用70%乙醇冲洗后腿和删除,使 肯定切开股骨头以上。 将皮肤的腿放在陪替氏培养皿中   1×无菌PBS
    3. 去除骨骼的肌肉和结缔组织   (股骨和胫骨),用手术刀和放置刮他们 到新鲜的1×无菌PBS中
    4. 放置新鲜的手术刀和解剖 镊子放入烧杯中的70%乙醇。 从PBS中取出一根骨头干燥 培养皿,切开骨头两端,暴露骨髓。
    5. 使用 26号针和10ml注射器,钻入骨髓并缓慢 使用BMM培养基冲洗出骨髓到50ml离心管中。 冲洗2 ml 从一侧,翻转骨,从另一侧冲洗2毫升 - 共4毫升 每个骨骼
    6. 对所有4个骨头重复步骤A5到1×50ml离心管中
    7. 使用20号针和10ml注射器,轻轻混合细胞 悬浮4x通过吸入注射器破碎任何团块
    8. 使BMM培养基和细胞的体积达到36ml。 板进入3x培养皿 每个100mm培养皿(〜9-10×10 6个细胞/培养皿)中培养。 允许 细胞在细胞培养箱中不受干扰地生长〜84小时(在37℃,   5%CO 2)。
      注意:使用培养皿代替处理的细胞 培养板以允许BMM的更容易的传代。 这些细胞倾向于 紧密粘附于组织培养处理的细胞和烧瓶 难以解除后续实验。

  2. Transwell设置
    1. BMM和PC3细胞应当在70-80%汇合,并且可以在同一天使用
    2. BMM:
      1. 取出介质,用1x PBS轻轻冲洗BMM培养皿。 加入〜8ml 新鲜的BMM培养基,并使用无菌细尖轻轻取出细胞   移液管和细胞刮刀,收集到15ml离心机中 管
      2. 对单细胞悬液进行细胞计数,并且 板中的0.35×10 6个BMM在2ml的BMM培养基中直接在6孔中 板。
    3. 使用无菌镊子,轻轻地放置transwell插入 在新铺板的细胞上方的每个孔中。 通过他们的设计插入 通过井的壁固定就位。 他们将保持暂停 并触摸媒体,但不是板的底部允许自由 在底部和顶部的细胞之间交换可溶性因子 插入。
    4. PC3s
      1. 使PC3细胞在T75烧瓶中生长至〜70-80%汇合。  
      2. 用1×无菌PBS冲洗,并使用2ml的0.25%胰蛋白酶-EDTA解离细胞
      3. 使用8毫升完整的DMEM停止胰蛋白酶的活动,收集所有10毫升在15毫升离心管。
      4. 在130×g离心细胞5分钟,在RT下沉淀,和 重悬于适量的完全DMEM(〜8ml)中以进行a   细胞计数。
      5. 板0.2×10 6 PC3s在2ml完全DMEM中的transwell插入物的顶部。
    5. 让细胞在细胞培养箱中不受干扰地生长48小时
    6. 当对没有transwell的对照条件进行电镀时, 相同数量的细胞直接在6孔板上,在2ml的混合物中 完全DMEM和2ml BMM培养基。

  3. 改变共培养至无血清条件(如有必要)
    1. 对于需要分析由细胞调节的培养基的实验 共培养和避免介质中的血清干扰,transwell 共培养可以在之前12-16小时改变为无血清条件 收获
    2. 简言之,48小时后,用1x轻轻地洗涤细胞3次 无菌PBS。 为此,请从transwell顶部取出介质,提起 transwell,从孔底部除去培养基,加入2ml PBS   到孔底部,更换transwell,加入2ml PBS transwell,轻轻搅拌。 重复至少2次。
    3. 板2毫升无血清MEM-α到底部的孔,和2毫升 的无血清DMEM至transwell。 让细胞静置12-16小时 在细胞培养箱中不受干扰
    4. 如果改为无血清 条件不是必需的,应进行3x PBS洗涤 紧接在样品收获之前用于分析。

  4. 收获实验蛋白样品
    1. 媒体:
      1. 将培养基收集到15ml离心管中。 将任何transwell转移到新的,未使用的6孔培养皿
      2. 通过在4℃下离心收集的培养基(130×g)(以保持细胞完整)5分钟,除去任何浮游细胞。
      3. 将培养基转移到新的15 ml离心管中
      4. 通过在830 x g离心10分钟,去除任何额外的细胞碎片 min。 有必要单独执行两个自旋   并且在进行830×g 旋转之前去除完整的细胞
      5. 冷冻介质在-80°C收集以备将来使用,或者如果需要,使用5 K Millipore离心浓缩器浓缩。
      6. 将培养基转移到浓缩器,并在4℃下以3,000×g转动,直到达到所需的体积。
      7. 将浓缩培养基储存于-80℃备用。
    2. 蛋白裂解物 - BMM
      1. 收集培养基后,向细胞中加入所需量的SME(〜200μl),置于冰上。
      2. 轻轻刮取细胞,收集到1.5 ml管中
      3. 在-80℃冷冻。
    3. 蛋白裂解物 - PC3s
      1. 收集培养基后,向转孔中加入2ml 0.25%胰蛋白酶-EDTA, 和1ml 0.25%胰蛋白酶-EDTA加到用作对照的孔中(即直接在孔上) 塑料)。
      2. 在37℃下〜5分钟后,细胞应解离。 停止用4ml完全DMEM的胰蛋白酶化。 这个卷是 足以控制井
      3. 使用无菌细尖转移   移液,轻轻冲洗和收集细胞从孔中进入15毫升 离心管。 对于transwells,添加另外4毫升完整 DMEM至transwell,并再次冲洗,添加额外的培养基 原管。
      4. 在室温下以130×g离心细胞5分钟。
      5. 取出培养基,轻轻地清洗沉淀,重悬在〜10毫升1×无菌PBS
      6. 在室温下将细胞在130×g下旋转5分钟,并除去所有PBS
      7. 将沉淀重悬在SME(约200μl)中,并转移到1.5ml管中
      8. 在-80℃下冷冻。

  5. 收集样品进行RNA分析
    1. 48小时后,如步骤C1中所述轻轻地用PBS洗涤细胞1次。  
    2. 收集PC3细胞,如步骤D3a-d所示。 洗涤丸 用PBS附加时间(重复步骤D3e-f另外的时间)。 加 从RNeasy Plus迷你试剂盒的RLT缓冲液到沉淀。 每孔使用〜250μl   集。 转移到新鲜QIAshredder。
    3. 每孔加入约250μlRLT缓冲液,直接冲洗BMM细胞。 刮细胞并收集到新鲜的QIAshredder
    4. 按照RNeasy Plus Mini Kit提供的说明进行RNA提取

食谱

  1. 完成DMEM
    445ml DMEM,无菌过滤
    50ml热灭活的胎牛血清 5ml青霉素 - 链霉素10,000U/ml/ml
  2. L929条件培养基
    1. L929细胞在完全DMEM中使用100mm组织培养皿在48小时内在10ml中生长至〜70-80%汇合。
    2. 将细胞收集到15ml离心管中
    3. 通过在130×g离心5分钟从培养基中除去细胞
    4. 将培养基转移到新的15ml离心管中
    5. 通过在830×g离心10分钟从培养基中除去细胞碎片
    6. 将培养基转移到新的15ml离心管中并使用
  3. BMM培养基(30%L929 CM; 20%FBS)
    12 ml L929条件培养基
    5.3ml热灭活的胎牛血清 22.7ml MEM-α培养基
  4. SME裂解缓冲液
    250mM蔗糖 25 mM MES
    1mM EDTA
    在去离子水中
    pH至6.5,使用前加入0.1%TritonX-100

致谢

这个协议改编自我们的手稿Herroon et al。(2013)。 Grant支持由DOD PC074031,DOD PC030325,Wayne State University启动基金和MICR:P30 CA 22453提供。

参考文献

  1. Herroon,M.K.,Rajagurubandara,E.,Rudy,D.L.,Chalasani,A.,Hardaway,A.L.and Podgorski,I.(2013)。 巨噬细胞组织蛋白酶K促进骨中的前列腺肿瘤进展 癌基因 32(12):1580-1593。
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引用:Herroon, M. K. and Podgorski, I. (2014). Transwell Co-culture of Bone Marrow Macrophages with Tumor Cells. Bio-protocol 4(9): e1121. DOI: 10.21769/BioProtoc.1121.
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