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Generation of Mouse Lung Epithelial Cells
制备小鼠肺上皮细胞   

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Abstract

Although in vivo models are excellent for assessing various facets of whole organism physiology, pathology, and overall response to treatments, evaluating basic cellular functions, and molecular events in mammalian model systems is challenging. It is therefore advantageous to perform these studies in a refined and less costly setting. One approach involves utilizing cells derived from the model under evaluation. The approach to generate such cells varies based on the cell of origin and often the genetics of the cell. Here we describe the steps involved in generating epithelial cells from the lungs of KrasLSL-G12D/+;p53LSL-R172/+ mice (Kasinski and Slack, 2012). These mice develop aggressive lung adenocarcinoma following cre-recombinase dependent removal of a stop cassette in the transgenes and subsequent expression of Kra-G12D and p53R172. While this protocol may be useful for the generation of epithelial lines from other genetic backgrounds, it should be noted that the Kras; p53 cell line generated here is capable of proliferating in culture without any additional genetic manipulation that is often needed for less aggressive backgrounds.

Keywords: Mouse cells(鼠标电池), Kras(KRAS), P53(p53), Cancer(癌症), Lung(肺)

Materials and Reagents

  1. Collagenase/Dispase (F. Hoffmann-La Roche, catalog number: 10269638001 )
  2. PureCol Collagen I (Bovine-Fisher, catalog number: 50-360-230)
  3. Fibronectin (Life Technologies, Gibco®, catalog number: PHE-0023 )
  4. Dulbecco's Phosphate-buffered saline (D-PBS)
  5. RPMI-1640 with L-Glutamine (Life Technologies, Gibco®, catalog number: 11875-093 )
  6. Fetal bovine serum (FBS) (from multiple vendors)
  7. 0.25% Trypsin-EDTA (1x) (Life Technologies, Gibco®, catalog number: 25200-056 )
  8. Collagen Coating Mix (see Recipes)
  9. Fibronectin Coating Mix (see Recipes)
  10. 1 mg/ml Collagenase (see Recipes)

Equipment

  1. 10 ml syringes
  2. 25 gauge needles
  3. 37 °C 5% CO2 cell culture incubator
  4. Refrigerated centrifuge
  5. Inverted microscope
  6. Tissue culture hood equipped with UV light source
  7. Vacuum aspirator

Procedure

  1. Coating tissue culture plates
    Plates are coated with collagen and fibronectin to facilitate cell adhesion acting as a cellular matrix. Fibronectin specifically aids in anchoring the cells to the collagen.
    1. Prepare collagen coating mix under sterile conditions.
    2. Add adequate volume of collagen coating mix to cover the bottom of the plate being coated (e.x. 5 ml/10 cm plate).
    3. Leave plates covered overnight in the tissue culture hood under the UV light to prevent contamination.
    4. The following day aspirate the coating mix.
    5. Air-dry the plates in the tissue culture hood.
    6. Rinse the plate 2x with D-PBS (2.5 ml/10 cm plate).
    7. Air-dry plates in the tissue culture hood.
    8. Cover the plates, seal with parafilm, and store at 4 °C until ready to coat with fibronectin (duration of storage has not been tested extensively; however, plates left at 4 °C for one week were successfully used).
    9. Prepare fibronectin coating mix.
    10. Add adequate volume of fibronectin mixture to cover the bottom of the plate being coated (e.x. 5 ml/10 cm plate).
    11. Incubate coated plates at 37 °C overnight in 5% CO2 cell culture incubator.
    12. The following day rinse the plates 2x with D-PBS (leave D-PBS in plates if not using immediately -do not allow plates to dry).

  2. Extracting cells from Tumor tissue
    Individual tumors, small areas of lung tissue, or entire lungs can be used to generate epithelial cells.
    1. Sacrifice animals per the university/institute established animal care and use protocol.
    2. Open up the thoracic cavity immediately after sacrifice.
    3. Perfuse the lungs by slowly injecting 7-10 ml of D-PBS into the left ventricle of the heart using a 25 gauge needle and syringe. Successful perfusion will result in the lungs changing color from pink to white, representing displacing the RBCs.
    4. Carefully remove the lungs and place them into D-PBS.
    5. If large tumors are evident dissect them out and proceed; otherwise continue the procedure using the entire lung (see Figure 1 for lung tumors that can easily be harvested from the lung).


      Figure 1. A KrasLSL-G12D/+; p53flx/flx mutant mouse was intratracheally infected with adenoviral particles expressing cre-recombinase to induce transgene recombination. Ten weeks following infection the mouse was sacrificed, lungs were perfused and harvested, and imaged. Multiple large tumor nodules are present on the surface.

    6. Wash the tumors/lungs by rinsing the exterior 4x with D-PBS.
    7. Mince the tissue in and equal volume ice-cold D-PBS with a sterile blade in tissue culture hood until the mass represents a slurry and few if any larger solid pieces are evident; however care should be taken to perform this step in a timely fashion to avoid cell death. The mincing step allows for easier retrieval of individual cells for propagating.
    8. Add the minced tissue to an equal volume of RPMI-1640 supplemented with 1 mg/ml collagenase and incubate for 1 h at 37 °C in 5% CO2 cell culture incubator.
    9. Remove cells intermittently from supernatant. Do not centrifuge. Let the larger pieces settle and remove the top-half of the supernatant containing individual cells every 10-15 min. Replenish RPMI/collagenase solution as needed and repeat 4-5 times.
    10. Pool and spin the collected supernatant at 1,000 x g at 4 °C for 5 min.
    11. Remove the supernatant and add 10 ml of RPMI-1640 supplemented with 10% FBS.
    12. Remove the D-PBS from the collagen/fibronectin coated plates.
    13. Immediately transfer the cell suspension to the coated plates being sure to supplement with additional RPMI-1640/10% FBS to cover the surface of the plate if necessary (10 ml/10 cm plate).
    14. Incubate cells at 37 °C in 5% CO2 cell culture incubator.
    15. Continue to passage on coated plates for three passages.

  3. Selecting epithelial cells
    1. Over the course of culturing select epithelial looking colonies. There are a few mechanisms to help in the selection process. Firstly, the fibroblasts are often more sensitive to trypsin and can therefore be removed from the plate while the epithelial cells will adhere for a longer time. This is done by treating the cells with trypsin and removing the first cells begin to slough off the plate as visualized under a microscope. This step helps to increase the epithelial cell population. Secondly, epithelial clones will become visible and are easily discernible from the fibroblast population.
    2. Once epithelial clones are evident, the clones are treated directly with a small amount of trypsin (50 μl dispensed directly on the clone) and visualized under a microscope until they begin to round up. The cells are then abducted using a sterile transfer pipette. The bulb of the pipette is squeezed in and held in that position while the tip of the pipette is placed over the individual clone. Once in place the pressure on the bulb is released slowly to encourage the cells to enter into the pipette. The entire contents are then transferred to individual wells in 12-well standard tissue-culture treated plates to further propagate (to enhance visualization of the clones, allow clones to be followed, and increase the abduction step, once identified, clones can be circled on the bottom of the plate with a sharpie).
    3. To confirm that cells are of epithelial origin stain with standard markers such as keratins. For the cells described here, keratin 14 was used.
    4. Thus far, cells generated by this procedure have been able to propagate beyond 50 passages.
      Note: It is inherently difficult to generate cultures of normal cells. All the cells that formed clones and were isolated by this procedure (irrespective of being generated from tumors or the whole lung) were confirmed to be mutant for Kras and p53. This procedure will specifically allow one to "select" for epithelial cells that can propagate outside of the organ.

Recipes

  1. Collagen coating mix (10 ml)
    Item
    Final
    Stock
    Volume
    Collagen
    0.4 mg/ml
    3 mg/ml in water
    1.33 ml
    D-PBS


    8.67 ml
  2. Fibronectin coating mix (10 ml)
    Item
    Final
    Stock
    Volume
    Fibronectin
    5 μg/ml
    500 μg/ml in water
    100 μl
    Ice-cold-D-PBS


    9.9 ml
  3. 1 mg/ml collagenase (2 ml)
    Dilute collagenase stock in RPMI-1640
    Add 20 μl of stock to 1980 μl of RPMI-1640.

Acknowledgments

The protocol presented herein was adapted from Kasinski and Slack (2012). This work was supported by an NIH grant to FJS and Joanne Weidhaas (NCI R01 CA131301). AK was supported by a US National Institutes of Health (NIH) grant (1F32CA153885-01) and an American Cancer Society Postdoctoral Fellowship (120,766-PF-11-244-01-TBG).

References

  1. Kasinski, A. L. and Slack, F. J. (2012). MiRNA-34 prevents cancer initiation and progression in a therapeutically resistant K-ras and p53-induced mouse model of lung adenocarcinoma. Cancer Res 72(21): 5576-5587.

简介

尽管体内模型对于评估整个生物体生理学,病理学和对治疗的总体反应的各个方面是优异的,但是在哺乳动物模型系统中评估基本细胞功能和分子事件是具有挑战性的。因此,有利的是在精制和较便宜的设置中进行这些研究。一种方法涉及利用来自所评估的模型的细胞。产生这样的细胞的方法基于起始细胞和通常细胞的遗传学而变化。在这里,我们描述了从肺部产生上皮细胞的步骤 LSL-G12D/+ ; LSL-R172/+ 小鼠(Kasinski和Slack,2012)。这些小鼠在重组酶依赖性去除转基因中的终止盒并随后表达Kra -G12D 和后产生侵袭性肺腺癌> p53 R172 。虽然该方案可用于从其他遗传背景产生上皮细胞系,但应注意的是,Kras ;此处产生的p53 细胞系能够在培养物中增殖,而没有任何额外的遗传操作,其对于侵略性较低的背景通常是需要的。

关键字:鼠标电池, KRAS, p53, 癌症, 肺

材料和试剂

  1. 胶原酶/分散酶(F.Hoffmann-La Roche,目录号:10269638001)
  2. PureCol Collagen I(Bovine-Fisher,目录号:50-360-230)
  3. 纤连蛋白(Life Technologies,Gibco ,目录号:PHE-0023)
  4. Dulbecco磷酸盐缓冲盐水(D-PBS)
  5. 具有L-谷氨酰胺的RPMI-1640(Life Technologies,Gibco ,目录号:11875-093)
  6. 胎牛血清(FBS)(来自多个供应商)
  7. 0.25%胰蛋白酶-EDTA(1x)(Life Technologies,Gibco ,目录号:25200-056)
  8. 胶原涂层混合(见配方)
  9. 纤连蛋白涂层混合物(参见配方)
  10. 1 mg/ml胶原酶(见配方)

设备

  1. 10ml注射器
  2. 25号针
  3. 37℃5%CO 2细胞培养孵育器
  4. 冷冻离心机
  5. 倒置显微镜
  6. 装有紫外线光源的组织培养罩
  7. 真空吸气器

程序

  1. 涂层组织培养板
    板用胶原和纤连蛋白包被以促进作为细胞基质的细胞粘附。 纤连蛋白特异性地帮助将细胞锚定到胶原。
    1. 在无菌条件下制备胶原涂层混合物
    2. 添加足够体积的胶原涂层混合物以覆盖正在涂覆的板的底部( e 5 ml/10cm板)。
    3. 将组织培养罩中的板在UV光下覆盖过夜以防止污染
    4. 第二天吸入涂料混合物。
    5. 在组织培养罩中对板进行空气干燥
    6. 用D-PBS(2.5ml/10cm平板)冲洗平板2次
    7. 组织培养罩中的空气干燥板
    8. 覆盖板,用石蜡膜密封,并在4℃下储存,直到准备用纤连蛋白包被(储存的持续时间没有广泛测试;然而,板在4℃下保持一周成功使用)。
    9. 准备纤连蛋白涂层混合物
    10. 加入足够体积的纤连蛋白混合物以覆盖被包被的平板的底部(例如5毫升/10厘米平板)。
    11. 将包被的平板在37℃下在5%CO 2细胞培养箱中孵育过夜。
    12. 第二天用D-PBS冲洗板2次(如果不立即使用,则在板中留下D-PBS,不允许板干燥)。

  2. 从肿瘤组织中提取细胞
    个体肿瘤,肺组织的小区域或整个肺可用于产生上皮细胞。
    1. 每所大学/学院的牺牲动物建立动物护理和使用协议
    2. 在牺牲后立即打开胸腔。
    3. 通过使用25号针和注射器将7-10ml D-PBS缓慢注射入心脏的左心室来灌注肺。成功的灌注将导致肺将颜色从粉红色变为白色,代表置换RBC。
    4. 小心取出肺,将其放入D-PBS中。
    5. 如果大肿瘤是明显的,将它们分开并继续进行;否则继续使用整个肺的程序(参见图1肺容易从肺收获)

      图1. A Kras LSL-G12D/+ 使用表达cre重组酶的腺病毒颗粒气管内感染大鼠 以诱导转基因重组。 在感染后10周,处死小鼠,灌注和收获肺,并成像。表面存在多个大的肿瘤结节。

    6. 通过用D-PBS冲洗外部4x来洗涤肿瘤/肺
    7. 用组织培养罩中的无菌刀片将组织和等体积的冰冷D-PBS剁碎,直到质量表示浆液,并且很少(如果有任何较大的固体块)是明显的;但应注意及时执行此步骤以避免细胞死亡。切碎步骤允许更容易地检索用于繁殖的单个细胞。
    8. 将切碎的组织加入等体积的补充有1mg/ml胶原酶的RPMI-1640中,并在37℃下在5%CO 2细胞培养箱中孵育1小时。
    9. 从上清液中间歇地除去细胞。不要离心。让更大的块沉降并且每10-15分钟去除含有单个细胞的上清液的上半部分。根据需要补充RPMI /胶原酶溶液,重复4-5次。
    10. 合并并在4℃下以1,000×g离心收集的上清液5分钟。
    11. 取出上清液,加入10ml补充有10%FBS的RPMI-1640
    12. 从胶原/纤连蛋白包被的板上除去D-PBS
    13. 立即将细胞悬液转移到涂覆的板上,必要时补充RPMI-1640/10%FBS以覆盖板的表面(10ml/10cm板)。
    14. 在37℃下在5%CO 2细胞培养箱中孵育细胞
    15. 继续在涂布板上通过三次。

  3. 选择上皮细胞
    1. 在培养选择上皮寻找菌落的过程中。有几种机制可以帮助选择过程。首先,成纤维细胞通常对胰蛋白酶更敏感,因此可以从板上除去,而上皮细胞将粘附更长的时间。这通过用胰蛋白酶处理细胞来进行,并且除去第一细胞开始从板上脱落,如在显微镜下可视化的。此步骤有助于增加上皮细胞群体。其次,上皮克隆将变得可见,并且容易从成纤维细胞群中辨别出来
    2. 一旦上皮克隆是明显的,直接用少量胰蛋白酶(50μl直接分配在克隆上)处理克隆并在显微镜下观察直到它们开始圆形。然后使用无菌转移移液管将细胞包裹。移液管的球管被挤入并保持在该位置,而移液管的尖端放置在个体克隆上。一旦就位,球泡上的压力缓慢释放以促进细胞进入移液管。然后将全部内容物转移到12孔标准组织培养物处理的平板中的单个孔中,以进一步繁殖(以增强克隆的可视化,允许克隆被跟踪,并且增加外展步骤,一旦鉴定,克隆可以在板的底部有一个锐利)。
    3. 以确认细胞是具有标准标记例如角蛋白的上皮起源染色。对于本文所述的细胞,使用角蛋白14。
    4. 到目前为止,通过该程序产生的细胞能够增殖超过50代。
      注意:产生正常细胞的培养物本质上是困难的。通过该方法分离的所有形成的克隆(不管是从肿瘤还是从整个肺产生的)都被证实是 突变体为Kras和p53。 该过程将特异性地允许人们"选择"可以在器官外部繁殖的上皮细胞。

食谱

  1. 胶原涂层混合物(10ml)
    项目
    最后
    库存

    胶原蛋白
    0.4 mg/ml
    3mg/ml的水溶液 1.33 ml
    D-PBS


    8.67 ml
  2. 纤连蛋白涂层混合物(10ml)
    项目
    最后
    库存

    纤连蛋白
    5μg/ml
    500μg/ml的水中 100微升
    冰冷的D-PBS


    9.9毫升
  3. 1mg/ml胶原酶(2ml) 稀释RPMI-1640中的胶原酶原料
    加入20微升的股票到1980微升的RPMI-1640

致谢

本文提出的协议改编自Kasinski和Slack(2012)。 这项工作得到NIH授予FJS和Joanne Weidhaas(NCI R01 CA131301)的支持。 AK由美国国家卫生研究院(NIH)资助(1F32CA153885-01)和美国癌症协会博士后研究金(120,766-PF-11-244-01-TBG)支持。

参考文献

  1. Kasinski,A.L。和Slack,F.J。(2012)。 MiRNA -34在肺腺癌的治疗抗性K-ras和p53诱导的小鼠模型中防止癌症的起始和进展。癌症研究72(21):5576-5587。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kasinski, A. L. and Slack, F. J. (2013). Generation of Mouse Lung Epithelial Cells. Bio-protocol 3(15): e837. DOI: 10.21769/BioProtoc.837.
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