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Isolation of Mouse Embryo Fibroblasts
小鼠胚胎成纤维细胞的分离   

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Abstract

Preparation of primary cultures of embryo fibroblasts from genetically engineered mouse strains can provide a valuable resource for analyzing the consequences of genetic alterations at the cellular level. Mouse embryo fibroblasts (MEFs) have been particularly useful in cancer research, as they have facilitated the identification of the genetic changes that allow cells to overcome senescence and proliferate indefinitely in culture. The immortalized MEFs can then acquire additional mutations that lead to anchorage-independent growth and the ability to form tumors in mice. Recently we developed an MEF model system for analysis of the role of the tumor suppressor gene DLC1 in cellular transformation (Qian et al., 2012). In this communication we describe a protocol for the isolation of MEFs from day 13.5-day 14.5 mouse embryos. The MEFs obtained by this procedure are suitable for use in biochemical assays and for further genetic manipulations.

Keywords: Mouse embryo fibroblasts(小鼠胚胎成纤维细胞), DLC1(DLC1), Primary culture(原代培养)

Materials and Reagents

  1. Mice 13-14 days pregnant
  2. Phosphate buffered saline (PBS), without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 10010 )
  3. Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L D-glucose (Life Technologies, Gibco®, catalog number: 11960044 or Mediatech, catalog number: 15-017-CV )
  4. Fetal bovine serum (Atlanta Biologicals, catalog number: S11550 )
  5. L-glutamine (Life Technologies, Gibco®, catalog number: 25030-149 )
  6. 1x penicillin-streptomycin solution (Gibco, catalog number: 15140-148 )
  7. 0.25% trypsin-EDTA (Life Technologies, Gibco®, catalog number: 25200 )
  8. 70% ethanol
    Note: Prepare in a sterile container with sterile distilled water.
  9. Dimethyl Sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
  10. MEF culture medium with 10% fetal bovine serum (see Recipes)

Equipment

  1. Plastic dissecting board or thick pad of blotting paper (such as Schleicher and Schuell GB004 paper)
  2. Masking tape or push pins
  3. Sterile Fine scissors (at least 3)
  4. Sterile Fine forceps (at least 3)
  5. Sterile 4 x 4 gauze pads (available from vendors of medical supplies)
  6. Sterile scalpel blades or single-edge razor blades
  7. Examination gloves
  8. Petri dishes (100 mm) (BD Biosciences, Falcon®, catalog number: 351029 , or equivalent)
  9. Sterile plastic serological pipettes, 5 and 10 ml
  10. Sterile disposable tubes (50 ml) (BD Biosciences, Falcon®, catalog number: 352070 , or equivalent)
  11. T75 tissue culture flasks (Corning, catalog number: 430641 , or equivalent)
  12. 37 °C 5% CO2 tissue culture incubator
  13. Tissue culture hood
  14. Inverted microscope
  15. Cryovials (such as Nunc®, catalog number: 375418 )

Procedure

  1. Harvest embryos from female mice 13-14 days after the appearance of the copulation plug. The female should be obviously pregnant at this stage.
    Note: Depending on the viability of the particular strain, 6-10 embryos can be expected from each pregnant female, and they should yield enough MEFs for several experiments.
  2. Euthanize the pregnant female by cervical dislocation (this can be done on the bench top, outside of the tissue culture hood.). Place the mouse on its back on a dissecting board or on a thick pad of blotting paper.
  3. Thoroughly soak the fur of mouse with 70% ethanol. Transfer the board or pad with the mouse to the tissue culture hood.
  4. Make a cut in the skin with scissors, then grab the skin with both hands and pull away to expose the abdominal wall. Use tape (or pins) to attach the feet of the mouse to the board (or pad).
  5. Put 10 ml of 0.25% trypsin-EDTA in one covered Petri dish and put approximately 20 ml PBS in another Petri dish.
  6. Set aside the scissors that were used to cut the fur, and put on a new pair of gloves. With new scissors, cut through the abdominal wall. Use forceps to lift up the uterine horns and cut away the uterus with scissors. Be careful not to let the uterus touch the fur. Place the uterus in the Petri dish with PBS.
    Note: Scissors and forceps can be placed in inverted Petri dish lids when not in use, and they can be cleaned by wiping with gauze pads wetted with 70% ethanol.
  7. Separate the embryos by slicing through the uterus in the regions between each embryo. The embryos may pop out spontaneously or they may come out after pressing gently with forceps. If they do not come out easily, carefully cut away the uterine tissue starting at the site where the dark red, disc-shaped placenta is located. If the embryo is still in the yolk sac, gently pull the sac off with forceps.
  8. Transfer the embryos to a new dish with PBS – use one dish of fresh PBS for every 3 embryos. Swirl the dish to remove blood from the embryos.
  9. With one hand, pick up an embryo with forceps and with the other hand, cut off the head above the eyes. Use another pair of forceps to tear out the red tissue (heart and liver). Place the rest of the embryo in the covered Petri dish with 0.25% trypsin-EDTA.
  10. After all the embryos have been isolated and placed in the dish with trypsin, chop up the embryos with scissors, and then use a scalpel blade or razor blade to mince the tissue into pieces of 1-2 mm. Pipet up and down several times with a 10 ml pipet, and then place the dish in the 37 °C tissue culture incubator for 10 min.
  11. Remove the dish from the incubator and pipet the embryo pieces up and down several times with a 5 ml pipet. Return the dish to the incubator for another 5-10 min.
  12. Transfer the cell suspension to a 50 ml tube. Add 20 ml MEF culture medium (see Recipe below) to inactivate the trypsin, and pipet up and down several times. Let the cell suspension sit for about 5 min to allow larger embryo fragments to sink to the bottom of the tube.
  13. Transfer the supernatant, consisting of single cells and cell clusters, to T75 flasks. Each flask should receive a volume of the cell suspension equivalent to 3 embryos. Add MEF culture medium to the flasks so that the total volume is 15-20 ml.
    Note: if some cell lysis has occurred, the cell suspension may be viscous, and it may not be possible to avoid transferring the larger fragments to the flasks. This is not a major problem, since some fibroblasts will migrate out from the tissue fragments and attach to the dish.
  14. The following morning, remove the old medium (with dead cells and debris) and replace with fresh medium. Check the cells later in the day; if the culture is very dense, split into 3 T75 flasks. Otherwise, split the cells on the following day or when they reach confluency. This is passage number 1.
  15. When cells are confluent, harvest by trypsinization, spin down, and resuspend the cell pellet in freezing medium (MEF culture medium with 10% fetal bovine serum and 10% DMSO). Aliquot the MEFs into cryovials and freeze the cells using standard methods for mammalian cell cryopreservation.
    Note: Some protocols call for 20% fetal bovine serum in the freezing medium, but 10% has worked for us.
  16. The MEFs will senesce faster if plated at low density. As the growth of the cells begins to slow down after several passages, the number of cells transferred per flask should be increased with each passage.
  17. After around 10 passages, the MEFs will reach the crisis phase when cell proliferation has greatly decreased. Immortalized lines can be derived in several weeks by following the protocols of Todaro and Green (Todaro and Green, 1963). We have found that immortalized MEF lines can be obtained after around 18 passages when all of the cells in a dish or flask are harvested and replated in fresh medium every 5 days.

Recipes

  1. MEF culture medium with 10% fetal bovine serum
    Mix 450 ml DMEM with 50 ml fetal bovine serum
    5 ml 200 mM L-glutamine
    5 ml 100x penicillin-streptomycin solution

Acknowledgments

This protocol was based on the method of Todaro and Green (1963) and subsequent modifications of the technique, as described in publications such as Coats et al. (1999). This work was supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.

References

  1. Coats, S., Whyte, P., Fero, M. L., Lacy, S., Chung, G., Randel, E., Firpo, E. and Roberts, J. M. (1999). A new pathway for mitogen-dependent cdk2 regulation uncovered in p27(Kip1)-deficient cells. Curr Biol 9(4): 163-173.
  2. Qian, X., Durkin, M. E., Wang, D., Tripathi, B. K., Olson, L., Yang, X. Y., Vass, W. C., Popescu, N. C. and Lowy, D. R. (2012). Inactivation of the Dlc1 gene cooperates with downregulation of p15INK4b and p16Ink4a, leading to neoplastic transformation and poor prognosis in human cancer. Cancer Res 72(22): 5900-5911.
  3. Todaro, G. J. and Green, H. (1963). Quantitative studies of the growth of mouse embryo cells in culture and their development into established lines. J Cell Biol 17: 299-313.

简介

来自基因工程小鼠品系的胚成纤维细胞的原代培养物的制备可以提供用于分析细胞水平的遗传改变的后果的有价值的资源。 小鼠胚胎成纤维细胞(MEF)已经在癌症研究中特别有用,因为它们促进了允许细胞克服衰老并且在培养中无限增殖的遗传变化的鉴定。 永生化MEF然后可获得导致锚定非依赖性生长和在小鼠中形成肿瘤的能力的其它突变。 最近我们开发了用于分析肿瘤抑制基因DLC1在细胞转化中的作用的MEF模型系统(Qian等人,2012)。 在这种沟通中,我们描述了从第13.5天14.5小鼠胚胎中分离MEF的方案。 通过该方法获得的MEF适用于生化测定和进一步的遗传操作。

关键字:小鼠胚胎成纤维细胞, DLC1, 原代培养

材料和试剂

  1. 小鼠13-14天怀孕
  2. 没有Ca 2+和Mg 2+的磷酸盐缓冲盐水(PBS)(Life Technologies,Gibco ,目录号:10010)
  3. 含有4.5g/L D-葡萄糖( Life Technologies,Gibco ,目录号:11960044或Mediatech,目录号:15-017-CV) />
  4. 胎牛血清(Atlanta Biologicals,目录号:S11550)
  5. L-谷氨酰胺(Life Technologies,Gibco ,目录号:25030-149)
  6. 1×青霉素 - 链霉素溶液(Gibco,目录号:15140-148)
  7. 0.25%胰蛋白酶-EDTA(Life Technologies,Gibco ,目录号:25200)
  8. 70%乙醇
    注意:在无菌容器中用无菌蒸馏水配制。
  9. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
  10. 具有10%胎牛血清的MEF培养基(参见Recipes)

设备

  1. 塑料解剖板或吸墨纸厚垫(如Schleicher和Schuell GB004纸)
  2. 遮蔽胶带或推针
  3. 无菌精剪刀(至少3件)
  4. 无菌精钳(至少3枚)
  5. 无菌4 x 4纱布垫(医疗用品供应商提供)
  6. 无菌手术刀刀片或单刃剃刀刀片
  7. 考试手套
  8. 培养皿(100mm)(BD Biosciences,Falcon ,目录号:351029或等同物)
  9. 无菌塑料血清移液管,5和10 ml
  10. 无菌一次性管(50ml)(BD Biosciences,Falcon ,目录号:352070或等同物)
  11. T75组织培养瓶(Corning,目录号:430641或等价物)
  12. 37℃,5%CO 2组织培养箱
  13. 组织培养罩
  14. 倒置显微镜
  15. 冷冻瓶(例如Nunc ®,目录号:375418)

程序

  1. 收获胚胎从雌性小鼠出现后13-14天的交配塞。 在这个阶段,女性应该明显怀孕。
    注意:根据特定菌株的生存力,可以预期每个怀孕女性有6-10个胚胎,并且对于几个实验应该产生足够的MEF。
  2. 安乐死怀孕女性颈椎脱位(这可以在台面上,组织文化罩外)。将鼠标放在解剖板上或厚垫的吸墨纸上
  3. 用70%乙醇彻底浸泡小鼠的毛皮。用鼠标将板或垫转移到组织培养罩
  4. 用剪刀剪切皮肤,然后用双手抓住皮肤,拉开露出腹壁。使用胶带(或针)将鼠标的脚连接到电路板(或焊盘)。
  5. 将10ml的0.25%胰蛋白酶-EDTA放入一个覆盖的培养皿中,并在另一个培养皿中放入约20ml PBS。
  6. 放置用来剪毛皮的剪刀,戴上一副新的手套。用新的剪刀,穿过腹壁。使用镊子提起子宫角,并用剪刀切掉子宫。小心不要让子宫接触毛皮。将子宫置于带有PBS的培养皿中。
    注意:不使用时,可以将剪刀和镊子放在倒置的培养皿盖中,然后用用70%乙醇润湿的纱布垫擦拭即可清洁。
  7. 通过切片通过子宫在每个胚胎之间的区域分离胚胎。胚胎可以自发弹出,或者用镊子轻轻按压后出来。如果他们不容易出来, 从暗红色,盘状胎盘所在的位置开始仔细切除子宫组织。如果胚胎仍在卵黄囊中,用镊子轻轻拉出囊。
  8. 转移胚胎到一个新的菜用PBS - 使用一个新鲜的PBS每3个胚胎。旋转菜,以从胚胎中移除血液。
  9. 用一只手,用镊子拿起一个胚胎,另一只手,切断眼睛上方的头。使用另一对镊子撕掉红色组织(心脏和肝脏)。将其余的胚胎在覆盖的培养皿中用0.25%胰蛋白酶-EDTA
  10. 在所有胚胎被分离并且用胰蛋白酶放置在培养皿中后,用剪刀剪下胚胎,然后使用解剖刀刀片或剃刀刀片将组织切碎成1-2mm的碎片。用10ml移液管上下吸移数次,然后将培养皿置于37℃组织培养箱中10分钟。
  11. 从培养箱中取出培养皿,用5ml移液管吸取胚胎片上下数次。将培养皿放回培养箱中5-10分钟。
  12. 转移细胞悬浮液到50ml管。加入20毫升MEF培养基(见下文配方)灭活胰蛋白酶,并用移液管上下数次。让细胞悬浮液静置约5分钟,使更大的胚胎碎片沉到管底部
  13. 将由单细胞和细胞簇组成的上清液转移到T75烧瓶中。每个烧瓶应该接受相当于3个胚胎的细胞悬浮液的体积。添加MEF培养基到烧瓶中,使总体积为15-20毫升。
    注意:如果发生了一些细胞裂解,细胞悬浮液可能是粘稠的,并且可能不能避免将较大的片段转移到烧瓶中。这不是一个主要问题,因为一些成纤维细胞将从组织碎片迁移出来并附着在培养皿上。
  14. 第二天早晨,取出旧的培养基(有死细胞和碎片),并更换新鲜培养基。检查细胞当天晚些时候;如果培养物非常致密,分成3个T75烧瓶。否则,在第二天或当它们达到汇合时拆分单元格。这是段落数1.
  15. 当细胞汇合时,通过胰蛋白酶消化收获,离心,并将细胞沉淀物重悬在冷冻培养基(具有10%胎牛血清和10%DMSO的MEF培养基)中。将MEFs等分到冷冻管中,并使用哺乳动物细胞冷冻保存的标准方法冷冻细胞。
    注意:一些方案需要在冷冻培养基中20%胎牛血清,但10%已经为我们工作。
  16. 如果以低密度电镀,MEFs将更快地衰减。由于细胞的生长在几次传代后开始减慢,每个传代每个烧瓶中转移的细胞数目应该增加。
  17. 大约10代后,当细胞增殖大大降低时,MEF将达到危机阶段。 永生化品系可以通过遵循Todaro和Green的方案(Todaro和Green,1963)在几周内得到。 我们已经发现,在收集盘或烧瓶中的所有细胞并且每5天重新在新鲜培养基中传代约18次后可以获得永生化MEF细胞系。

食谱

  1. 具有10%胎牛血清的MEF培养基 将450ml DMEM与50ml胎牛血清混合 5ml 200mM L-谷氨酰胺 5ml 100x青霉素 - 链霉素溶液

致谢

该方案基于Todaro和Green(1963)的方法和该技术的后续修改,如在例如Coats等人(1999)的出版物中所描述的。这项工作是由国家癌症研究所,国立卫生研究院癌症研究中心的校内研究计划支持。

参考文献

  1. Coats,S.,Whyte,P.,Fero,ML,Lacy,S.,Chung,G.,Randel,E.,Firpo,E.and Roberts,JM(1999)。在p27(Kip1)缺陷型细胞中揭示的促分裂原依赖性cdk2调节的新途径 Curr Biol em> 9(4):163-173。
  2. Qian,X.,Durkin,M.E.,Wang,D.,Tripathi,B.K.,Olson,L.,Yang,X.Y.,Vass,W.C.,Popescu,N.C。和Lowy,D.R。 dlc1基因的失活与 p15 基因的下调协同作用,/em> INK4b 和 p16 Ink4a ,导致肿瘤转化和预后不良在人癌症中。 Cancer Res 72(22):5900-5911。
  3. Todaro,G.J.and Green,H。(1963)。 对培养中小鼠胚胎细胞生长的定量研究及其发展成为已确立的 。 J Cell Biol 17:299-313。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Durkin, M. E., Qian, X., Popescu, N. C. and Lowy, D. R. (2013). Isolation of Mouse Embryo Fibroblasts. Bio-protocol 3(18): e908. DOI: 10.21769/BioProtoc.908.
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