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Developing cancer therapeutics requires the ability to investigate their effects using in vitro models of a specific type of tumor. This protocol provides a method for the isolation and adoption to growth in culture of cells from primary tumors. This is particularly valuable for studying mouse models where original tumor cells can be evaluated, for example for gene modifications, and subsequently injected back to the same background mice to create more tumors for in vivo efficacy studies.

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Isolation of Mammary Epithelial Cells and Fibroblasts From Mouse Tumor
小鼠肿瘤乳腺上皮细胞与成纤维细胞的分离

癌症生物学 > 通用技术 > 动物模型 > 细胞分离和培养
作者: Shiva Kazerounian
Shiva KazerounianAffiliation: Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, USA
For correspondence: shiva.kazerounian@gmail.com
Bio-protocol author page: a559
Vol 4, Iss 4, 2/20/2014, 6412 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1050

[Abstract] Developing cancer therapeutics requires the ability to investigate their effects using in vitro models of a specific type of tumor. This protocol provides a method for the isolation and adoption to growth in culture of cells from primary tumors. This is particularly valuable for studying mouse models where original tumor cells can be evaluated, for example for gene modifications, and subsequently injected back to the same background mice to create more tumors for in vivo efficacy studies.
Keywords: Cell isolation(细胞分离), Tumor cell(肿瘤细胞), Fibroblast(成纤维细胞), Primary cells(原代细胞)

[Abstract]

Materials and Reagents

  1. Mouse mammary tumor
  2. DMEM (Life Technologies, Gibco®)
  3. 2.5 mg/ml trypsin (0.06 g/25 ml) (Sigma-Aldrich, catalog number: T4799-10G )
  4. 5 mg/ml albumin (125 mg/25 ml) (Thermo Fisher Scientific, catalog number: BP1600-100 )
  5. 850 units/ml of collagenase type II (0.064 g/25 ml) (Worthington Biochemical, catalog number: 46J8959-A or 4176)
  6. EGF (Pepro Tech, Catalog number: 100-15 )
  7. Penicillin/Streptomycin (Pen/Strep) (Life Technologies, Gibco®)
  8. F12 (Life Technologies, Gibco®)
  9. Gentamycin (50 μg/ml)
  10. Fetal Bovine Serum (Life Technologies, Gibco®)
  11. Insulin (Sigma-Aldrich, catalog number: I1882 )
  12. Hydrocortisone (Sigma-Aldrich, catalog number: H0135 )
  13. Gentamycin (Sigma-Aldrich, catalog number: G1397 )
  14. Digestion buffer (Collagenase solution) (see Recipes)
  15. Wash buffer (see Recipes)
  16. Growth Media (see Recipes)

Equipment

  1. 100 mm or 60 mm cell culture plate
  2. Sterile single edged razor blade
  3. Sterile scissors and tweezers
  4. 10 ml beaker/flask with a sterile small stirrer bar
  5. Cell strainers (BD, FalconTM Cell Strainers for 50 ml Conical Tubes, 100 μm)
  6. Centrifuge 250 x g
  7. 15 ml centrifuge tube
  8. Tissue culture freezer
  9. 37 °C with 5% CO2 incubator

Procedure

  1. Euthanize mouse when tumor reaches around 1 cm3, sterilize the area around the tumor.
  2. Remove the tumor using autoclaved instruments and place in 100 mm sterile dish.
  3. Rinse the tumor several times with 5 ml wash buffer until media is clear of blood.
  4. Finely mince the tumor with sterile single edged razor blade and sterile scissors.
  5. Again rinse the tumor pieces several times with wash buffer until media is clear of blood.
    Note: Be careful not to aspirate the tissue.
  6. Isolate the cells as described previously (Yee et al., 2009). Transfer the tumor pieces into a small autoclaved 10 ml beaker with a small sterile stir bar. Add 5 ml of collagenase solution made fresh at room temperature. Cover the beaker with sterile foil. Incubate for 20 min at 37 °C while stirring. Do not stir too fast.
  7. Remove the 1st 20 min collagenase solution (label as first collagenase). The best way to do this is pour through a cell strainer fitted on the top of a 50 ml conical tube. Once all the liquid with single cells passed through, put the tissue pieces back in the same beaker. If you want fibroblast, keep this solution and continue with step 8. However, if you only want to culture mammary tumor epithelial cells, then discard this solution and only keep the tissue pieces and go to step 9.
  8. For fibroblast cells only, dilute the 5 ml (first collagenase) solution up to 20 ml with wash buffer. Spin the cells down at 250 x g for 10 min. Remove the supernatant and discard. Re-suspend the pellet in 5 ml growth media, plate it in a 60 mm dishes and incubate at 37 °C in 5% CO2. Note that it is important that cells are not too spaced after plating. If the number of cells are less than 5 x 106, re-suspend them in a smaller volume of media and use a smaller cell culture dish because many cells might not survive after plating. 6-well plates can also be used in case of fewer cell numbers.
  9. Add another 5 ml of collagense solution to the remaining tissue pieces in the beaker and cover with sterile foil (label as second collagenase). Incubate for 20 min at 37 °C with stirring again. Do not stir too fast.
  10. Stop the second collagenase by adding 10 ml of wash buffer. The total volume will be 15 ml now. Transfer the solution and tissue pieces from the beaker into a fresh 15 ml centrifuge tube.
  11. Spin at 250 x g for 10 min. Transfer only 10 ml of the solution (label as second collagenase) into a fresh tube. Again, if you want fibroblasts, keep the 10 ml solution and repeat step 8. However, if you only want to culture mammary tumor epithelial cells, then discard this solution and only keep the 5 ml pellet containing tissue pieces and go to step 12.
  12. To the pellet (remaining 5 ml) add 10 ml of wash buffer and resuspend (this is considered first wash) and incubate at room temperature in laminar flow hood for 20 min. Epithelial cells and fibroblasts were separated by a modification of the sedimentation technique as described previously (Lanari et al., 2001). Therefore, allow the epithelial cells to settle down for 20 min at this step.
  13. Remove 10 ml of the first wash, transfer to a fresh tube and keep at room temperature (label as “first wash”). Add 10 ml of fresh wash buffer to the remaining 5 ml with pellet and allow the cells to settle down by gravity by incubating at room temperature for another 20 min.
  14. Repeat step 13 10 times, i.e. do a further 10 washes. Each time collect 10 ml of the wash into a fresh tube and label it with the wash number and add 10 ml fresh wash buffer to the remaining.
  15. After the last wash, spin the pellet down in 10 ml wash buffer at 250 x g for 10 min. Remove the supernatant and discard. Add 10 ml growth media to the pellet and transfer the cells into a 100 mm cell culture plate with all the pieces of tissue. This is the final epithelial pellet.
  16. At the end, spin all the tubes of all the washes down separately at 250 x g for 10 min. Remove the supernatant and discard. Add 5 ml of growth media to the pellet and transfer the cells to separate 60 mm cell culture plates. If any of the pellets are too small, use less growth media and smaller cell culture plate such as 6-well plates. Alternately, you can combine some of the washes that have few cells and transfer them all in one 60 mm cell culture plate. After plating, cells need to be about 70-80% confluent. Confluence is important at this step. If the cells are too far space out, they will not survive. Using separate plates for each wash gives you the possibility of having more homogenous culture.
  17. Leave the cells at 37 °C with 5% CO2 incubator for couple of days before changing the media. The plate that has pellet and pieces of tissue will reach confluence sooner. Cells will grow away from tissue pieces once they are on a plate. This plate can be trypsenized and the extra cells and tissues can be frozen for future use. This is considered “Passage Zero” cells.
  18. Each wash will be a non-homogenous cell mix with different numbers of fibroblast and epithelial cells. You can identify them using a light microscope. At this step, according to your preference you can keep the cultures with mainly epithelial cells or mainly fibroblast cells. In general there is high percentage of fibroblast cells in earlier digestion and washes and more epithelial cells in last washes and final pellet.


    Figure 1. Tumor epithelial cells isolated from mouse mammary tumor (20x, Images was acquired using Nikon TE300 inverted microscope equipped with differential interference contrast microscope/phase/fluorescence optics, connected to a Leica DC200 digital camera and analyzed using DCViewer software).


    Figure 2. Tumor fibrolast cells isolated from mouse mammary tumor (20x, Images was acquired using Nikon TE300 inverted microscope equipped with differential interference contrast microscope/phase/fluorescence optics, connected to a Leica DC200 digital camera and analyzed using DCViewer software).


    Figure 3. Mixed culture of tumor fibrolast and epithelial cells. Fibroblasts are surrounding tumor epithelial cells. Cells were isolated from mouse mammary tumor (20x, Images was acquired using Nikon TE300 inverted microscope equipped with differential interference contrast microscope/phase/fluorescence optics, connected to a Leica DC200 digital camera and analyzed using DCViewer software).

Recipes

  1. Digestion buffer (25 ml)
    2.5 mg/ml trypsin
    5 mg/ml albumin
    850 units/ml of collagenase type II
    25 ml of PBS
  2. Wash Buffer
    1,000 ml F12
    5 ml Gentamycin (50 μg/ml)
    50 ml FBS (5%)
  3. Growth Media
    Bring up to volume in F12: DMEM
    Filter Sterilize, stored at 4 °C
    Insulin
    Final 5 μg/ml
    (stock 1 mg/ml)
    500 μl/100 ml
    5 ml/1,000 ml
    Hydrocortisone
    1 μg/ml
    Stock 1 mg/ml
    100 μl/100 ml
    1 ml/1,000 ml
    EGF
    5 ng/ml
    Stock 10 μg/ml
    50 μl/100 ml
    500 μl/1,000 ml
    Gentamycin
    50 μg/ml
    Stock 10 mg/ml
    500 μl/100 ml
    5 ml/1,000 ml
    Pen/Strep
    10 U/ml
    Stock 10,000 U/ml
    1 ml/100 ml
    10 ml/1,000 ml
    FBS
    5%


    50 ml

Acknowledgments

This protocol was adapted from previous work (Yee et al., 2009; Lanari et al., 2001).

References

  1. Yee, K. O., Connolly, C. M., Duquette, M., Kazerounian, S., Washington, R. and Lawler, J. (2009). The effect of thrombospondin-1 on breast cancer metastasis. Breast Cancer Res Treat 114(1): 85-96.
  2. Lanari, C., Luthy, I., Lamb, C. A., Fabris, V., Pagano, E., Helguero, L. A., Sanjuan, N., Merani, S. and Molinolo, A. A. (2001). Five novel hormone-responsive cell lines derived from murine mammary ductal carcinomas: in vivo and in vitro effects of estrogens and progestins. Cancer Res 61(1): 293-302.

材料和试剂

  1. 小鼠乳腺肿瘤
  2. DMEM(Life Technologies,Gibco )
  3. 2.5mg/ml胰蛋白酶(0.06g/25ml)(Sigma-Aldrich,目录号:T4799-10G)
  4. 5mg/ml白蛋白(125mg/25ml)(Thermo Fisher Scientific,目录号:BP1600-100)
  5. 850单位/ml II型胶原酶(0.064g/25ml)(Worthington Biochemical,目录号:46J8959-A或4176)
  6. EGF(Pepro Tech,目录号:100-15)
  7. 青霉素/链霉素(Pen/Strep)(Life Technologies,Gibco )
  8. F12(Life Technologies,Gibco )
  9. 庆大霉素(50μg/ml)
  10. 胎牛血清(Life Technologies,Gibco )
  11. 胰岛素(Sigma-Aldrich,目录号:I1882)
  12. 氢化可的松(Sigma-Aldrich,目录号:H0135)
  13. 庆大霉素(Sigma-Aldrich,目录号:G1397)
  14. 消化缓冲液(胶原酶溶液)(参见配方)
  15. 洗涤缓冲液(见配方)
  16. 生长介质(参见配方)

设备

  1. 100 mm或60 mm细胞培养板
  2. 无菌单刃剃须刀
  3. 无菌剪刀和镊子
  4. 10ml烧杯/烧瓶用无菌小搅拌棒
  5. 细胞过滤器(BD,用于50ml锥形管的Falcon TM细胞过滤器,100μm)
  6. 离心机250
  7. 15ml离心管
  8. 组织培养箱
  9. 37℃,5%CO 2培养箱

程序

  1. 当肿瘤达到约1cm 3时,安乐死小鼠,消毒肿瘤周围的区域。
  2. 使用高压灭菌器械取出肿瘤,置于100mm无菌培养皿中
  3. 用5ml洗涤缓冲液冲洗肿瘤几次,直到培养基清除血液。
  4. 用无菌单刃刀片和无菌剪刀细致地切碎肿瘤
  5. 再次用洗涤缓冲液冲洗肿瘤块几次,直到培养基清除血液。
    注意:小心不要吸入组织。
  6. 如前所述分离细胞(Yee等人,2009)。将肿瘤块转移到小型灭菌搅拌棒的高压灭菌的小型烧杯中。加入5ml室温下新鲜制备的胶原酶溶液。用无菌箔覆盖烧杯。在37℃下搅拌20分钟。不要太快搅拌。
  7. 去除第1 20分钟胶原酶溶液(标签作为第一胶原酶)。最好的方法是通过安装在50ml锥形管顶部的细胞过滤器倒入。一旦所有液体与单个细胞通过,将组织片放回到同一个烧杯。如果你想要成纤维细胞,保持这个解决方案,并继续第8步。但是,如果你只想培养乳腺肿瘤上皮细胞,然后丢弃这个解决方案,只保留组织片段,走到第9步。
  8. 对于成纤维细胞,用洗涤缓冲液稀释5毫升(第一胶原酶)溶液到20毫升。将细胞以250×g离心10分钟。取出上清液并弃去。将沉淀重悬在5ml生长培养基中,将其平板在60mm培养皿中并在37℃在5%CO 2中孵育。注意,重要的是细胞在电镀后不会太间隔。如果细胞数小于5×10 6个,将它们重悬在较小体积的培养基中,并使用较小的细胞培养皿,因为许多细胞可能在铺板后不能存活。 6孔板也可用于较少细胞数的情况。
  9. 添加另外5毫升胶原溶液到烧杯中的剩余组织片,并用无菌箔(标记为第二胶原酶)覆盖。在37℃下再次搅拌孵育20分钟。不要太快搅拌。
  10. 通过加入10毫升洗涤缓冲液停止第二个胶原酶。总体积现在是15毫升。将溶液和组织片从烧杯转移到新鲜的15ml离心管中。
  11. 在250xg下旋转10分钟。只转移10ml的溶液(标签作为第二胶原酶)到一个新的管。再次,如果你想要成纤维细胞,保持10毫升的解决方案,重复第8步。但是,如果你只想培养乳腺肿瘤上皮细胞,然后丢弃这个解决方案,只保留5毫升含有组织碎片的球团,并转到步骤12。
  12. 向沉淀(剩余5ml)中加入10ml洗涤缓冲液并重悬(这被认为是第一次洗涤),并在室温下在层流通风橱中孵育20分钟。通过如先前所述的沉淀技术的修饰来分离上皮细胞和成纤维细胞(Lanari等人,2001)。因此,允许上皮细胞在此步骤沉降20分钟。
  13. 取出10ml的第一次洗涤,转移到一个新的管,并保持在室温(标记为"第一次洗涤")。加入10ml新鲜的洗涤缓冲液到剩余的5毫升与丸,并允许细胞通过重力沉降在室温下再温育20分钟。
  14. 重复步骤13 10次,即进行另外10次洗涤。每次将10ml洗涤液收集到新管中,并用洗涤次数标记,并向剩余的洗涤缓冲液中加入10ml新鲜洗涤缓冲液。
  15. 在最后一次洗涤后,将所述沉淀物在10ml洗涤缓冲液中以250×g离心10分钟。取出上清液并弃去。向沉淀中加入10ml生长培养基,并将细胞转移到具有所有组织片的100mm细胞培养板中。这是最终的上皮沉淀。
  16. 最后,将所有洗涤物的所有管分别在250×g下旋转10分钟。取出上清液并弃去。向沉淀中加入5ml生长培养基,并将细胞转移到分开的60mm细胞培养板上。如果任何颗粒太小,使用较少的生长培养基和较小的细胞培养板如6孔板。或者,您可以组合一些具有少量细胞的洗涤物,并将其转移到一个60mm细胞培养板中。铺板后,细胞需要约70-80%汇合。融合在这一步很重要。如果细胞太远的空间,他们将无法生存。每次洗涤使用单独的板给你有更多的同质文化的可能性。
  17. 在更换培养基之前,将细胞在37℃下用5%CO 2培养箱保持几天。具有丸和组织片的板将更快地到达汇合。一旦它们在板上,细胞将从组织片生长。该板可以被冷冻并且额外的细胞和组织可以冷冻以备将来使用。这被认为是"通道零"单元。
  18. 每次洗涤将是具有不同数目的成纤维细胞和上皮细胞的非均质细胞混合物。您可以使用光学显微镜识别它们。在这一步,根据你的喜好,你可以保持文化与主要上皮细胞或主要成纤维细胞。通常,在较早消化和洗涤中存在高百分比的成纤维细胞,在最后一次洗涤和最终沉淀中存在更多上皮细胞。


    图1.从小鼠乳腺肿瘤分离的肿瘤上皮细胞(20x,使用配备有微分干涉对比显微镜/相/荧光光学器件的Nikon TE300倒置显微镜获得图像,连接到Leica DC200数字照相机并分析使用DCViewer软件)。


    图2.从小鼠乳腺肿瘤分离的肿瘤纤维母细胞(20x,使用配备有微分干涉对比显微镜/相位/荧光光学器件的Nikon TE300倒置显微镜获得图像,连接到Leica DC200数字照相机并分析使用DCViewer软件)。


    图3.肿瘤fibrolast和上皮细胞的混合培养物。成纤维细胞围绕肿瘤上皮细胞。细胞从小鼠乳腺肿瘤(20x,使用配备有微分干涉对比显微镜/相位/荧光光学,连接到Leica DC200数码相机和使用DCViewer软件分析的Nikon TE300倒置显微镜获得的图像)获取。

食谱

  1. 消化缓冲液(25ml)
    2.5mg/ml胰蛋白酶
    5mg/ml白蛋白 850单位/ml II型胶原酶
    25 ml PBS
  2. 洗涤缓冲液
    1,000 ml F12
    5ml庆大霉素(50μg/ml) 50ml FBS(5%)
  3. 成长媒体
    在F12:DMEM
    中调出音量 过滤灭菌,储存于4°C
    胰岛素
    最终5μg/ml
    (原液1mg/ml)
    500μl/100 ml
    5 ml/1,000 ml
    氢化可的松
    1μg/ml
    库存1 mg/ml
    100μl/100 ml
    1 ml/1,000 ml
    EGF
    5 ng/ml
    库存10μg/ml
    50μl/100 ml
    500μl/1000 ml
    庆大霉素
    50μg/ml
    库存10mg/ml
    500μl/100 ml
    5 ml/1,000 ml
    Pen/Strep
    10 U/ml
    库存10,000 U/ml
    1 ml/100 ml
    10ml/1000ml
    FBS
    5%


    50毫升

致谢

该协议改编自以前的工作(Yee等人,2009; Lanari等人,2001)。

参考文献

  1. Yee,K.O.,Connolly,C.M.,Duquette,M.,Kazerounian,S.,Washington,R.and Lawler,J.(2009)。 血小板反应蛋白-1对乳腺癌转移的影响乳腺癌研究 处理 114(1):85-96。
  2. Lanari,C.,Luthy,I.,Lamb,C.A.,Fabris,V.,Pagano,E.,Helguero,L.A.,Sanjuan,N.,Merani,S。和Molinolo,A.A。(2001)。 五种来自鼠乳腺导管癌的新型激素反应性细胞系: em>和 in vitro effect of estrogens and progestins。 Cancer Res 61(1):293-302。
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How to cite this protocol: Kazerounian, S. (2014). Isolation of Mammary Epithelial Cells and Fibroblasts From Mouse Tumor. Bio-protocol 4(4): e1050. DOI: 10.21769/BioProtoc.1050; Full Text



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