建立从小学成人成纤维细胞培养鼠害

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Summary

本文介绍了一种从野生啮齿类动物的皮肤和肺组织的初级成纤维细胞培养的隔离和维护协议。

Cite this Article

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Seluanov, A., Vaidya, A., Gorbunova, V. Establishing Primary Adult Fibroblast Cultures From Rodents. J. Vis. Exp. (44), e2033, doi:10.3791/2033 (2010).

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Abstract

在生物学研究中使用的主要细胞,而不是肿瘤细胞株,的重要性是越来越广泛的认可。原代细胞在细胞周期调控,细胞凋亡的研究中的首选,DNA修复,癌细胞进行,在这些过程中所涉及的基因突变。原电池不能无限期由于复制衰老或aneuploidization开始培养。因此,新的文化需要建立定期。啮齿类动物胚胎成纤维细胞的分离过程是公认的,但隔离成年成纤维细胞培养,往往提出了严峻挑战。成人啮齿类动物从人类疾病小鼠模型中分离出的成纤维细胞,可能是来自人类患者的成纤维细胞进行比较时,他们首选的控制。此外,成人的成纤维细胞与怀孕的女性不能轻易获得的野生啮齿类动物工作时,唯一可用的材料。在这里,我们提供了一个从啮齿类动物的皮肤和肺部成年成纤维细胞的分离和文化协议。我们用这个程序成功地隔离开来超过20个啮齿类动物从实验室小鼠和大鼠的成纤维细胞,如河狸,豪猪,松鼠等野生啮齿类动物。

Protocol

1。开始之前

  1. 用70%乙醇消毒的剪刀和镊子。
  2. 置于30 mL烧杯内的小型磁力搅拌器,盖上两层铝箔和高压灭菌。
  3. 无菌水,准备一个28 Wunsch算法Liberase Blendzyme 3单位/毫升原液。在-20 ° C,0.5毫升分装和冻结解冻之前,每次使用新的等分。解冻后的解决方案可能会出现阴天。旋涡的解决方案,直到它变得清晰。
  4. 热身细胞培养基。

2。动物样品制备

注意 :野生动物可能含有病原体,如狂犬病毒。始终意识到开放锐器。

  1. 安乐死的动物,胴体放置在4 ° C。这是最好立即使用的胴体,但是,如果这是不实际的的,例如,如果动物是在外地收集,尸体可能会在24小时内使用。 24小时后的细胞产量下降。
  2. 动物解剖手术套件或化学罩(而不是在组织培养罩)。
  3. 腋下区是一个便捷的网站收集皮肤样本,腋下的皮肤较薄,含脂肪少,和皮毛密度较小。用70%乙醇的清洁切口部位。确保毛皮是用乙醇浸泡。
  4. 剃须用锋利的手术刀切开现场周围的皮毛。剃须比预期的切口部位的面积较大。尽量减少对皮肤的削减。喷雾用70%乙醇的面积,并让它干。
  5. 为了收集皮肤样本消费约1厘米2的一个片段。捏皮肤组织钳,用剪刀剪开。尽量不减少与皮肤的脂肪层,脂肪干扰胶原酶消化。要收集的肺样本,用70%乙醇洗胸部,胸部用剪刀打开一个T形切口,除了皮肤的皮瓣拉的皮肤。用70%乙醇洗开的肌肉面积,并让干燥。切肋骨,用无菌镊子和剪刀(用于较大的动物骨铣刀)。使用无菌技术,以避免污染五脏六腑。请勿触摸内部器官与仪器接触动物皮毛。切约1厘米,用无菌镊子和剪刀 2肺片段。
  6. 广场组织碎片,用无菌PBS 50 mL管,以避免干燥。
  7. 50 mL管外组织碎片用乙醇清洗,并把他们组织培养罩。
  8. 妥善地处理动物尸体。

3。提取细胞

  1. 转移到一个10厘米的组织培养盘用无菌手术刀的组织碎片。不要太多PBS样品转移。
  2. 〜1毫米,使用两个手术刀片切成组织。使用两个叶片,用剪刀的动作,从中心出发,除了拉。保持组织揉成团,不切一块一块。当切割是足够的皮肤像腻子,它不会单独成片,但会拉伸薄。肺组织是比较容易切割,将分成小块。
  3. 使用手术刀,转移到无菌的30 mL烧杯中,用无菌搅拌棒切割组织。用于切割组织与10毫升的DMEM/F12媒体与文施0.14单位/毫升Liberase Blendzyme 3,1X抗生素/ antimycotic板洗净,和解决方案添加到30毫升的烧杯组织碎片。
  4. 用无菌铝箔盖住烧杯中,并在37 ° C孵育,慢慢地搅拌,30至90分钟。的潜伏期的长短取决于品种及组织类型。小心不要过度消化组织。组织碎片时,仍处于消化结束,获得最佳产量。皮肤需要更长的时间消化比肺癌。大型动物的皮肤需要更长的时间来消化。检查消化后30分钟,然后每10分钟。当皮肤消化完成后,媒体变得混浊和皮肤相互分离的片段,并件的边缘变得“模糊”。肺片段改变颜色由红色变为白色,并开始形成粘纤维时,应停止龙消化。
  5. 吸取的解决方案,含有组织碎片向上和向下突破的团块。传输的解决方案,以50毫升无菌试管。如果碎片移动10毫升吸管切割和消化容易做得很好。冲洗烧杯3次,与10毫升与15%FBS,抗生素/ antimycotic 1X温暖的DMEM/F12媒体和媒体组织碎片添加50毫升管。关闭50毫升管和颠倒几次混合。 FBS在媒体将停止Liberase消化。
  6. 摆动桶组织文化离心5分钟旋转524克。去除上清。在温暖的DMEM/F12媒体10毫升与15%FBS,抗生素1X / antimycotic的重悬沉淀。吸管悬挂最大的力量来打破组织块。
  7. 添加15%胎牛血清,1X抗生素/ antimycotic摆动桶组织文化离心5分钟在524克,混合和离心机的DMEM/F12媒体的另一个30毫升。重复一个更多的时间,以消除Liberase痕迹。
  8. 重悬在10毫升的DMEM/F12媒体颗粒,含15%FBS 1X抗生素/ antimycotic和3%转移到10 cm组织培养皿中并放置在一个组织文化的孵化器在37 ° C,5%CO 2,O 2
  9. 检查板每天都为成纤维细胞和媒体的色彩。如果隔离是成功的,成纤维细胞爬出来的组织碎片和附加板(图1)。成纤维细胞开始退出在2-5天内组织碎片。
  10. 如果媒体改变颜色为黄色,这表明潜在污染或细胞的挤迫情况。在高倍显微镜下检查板。如果存在的细菌,真菌,或蠕虫丢弃板(这是很少与实验动物的问题,但是可能会发生时从野外收集的样本)。如果没有污染的存在,但媒体变了颜色,这是造成要么太多的细胞或组织放置在同一盘菜太多片段。如果超过60%的板是由附加的成纤维细胞覆盖,改变盘上的媒体,并转移到一个新的板块与新媒体的组织碎片。如果没有很多的成纤维细胞附着板,改变媒体和分裂组织块2-4板块。
  11. 7天之后,如果媒体没有改变早些时候,改变媒体和组织碎片转移到与新媒体的新盘。
  12. 孵育一个额外的7天的细胞和组织碎片。第14天,已经退出所有可行​​的成纤维细胞的组织碎片。
  13. 从细胞分离开始的14天之后,摒弃旧的媒体和组织碎片,收获15%胎牛血清,1X青霉素/链霉素,非必需氨基酸的细胞和一个新的板块板他们在5 × 10 5细胞/板EMEM ,和丙酮酸钠。 EMEM媒体将支持只有和其他类型的细胞会死亡或停止增殖的成纤维细胞的生长。
  14. 细胞达到80%-90%汇合后,冻结等分细胞以供将来使用。
  15. 当细胞达到80%-90%汇合,继续培养细胞分裂他们在5 × 10 5细胞/板。

4。代表性的成果

正常成纤维细胞具有显着突起(lamellipodia)(图2)的大细胞。成纤维细胞生长在一个单层。健康成长的文化包含1-10%M期细胞,四舍五入超过板面升高的细胞,但不能脱离板(图3)确认。通常情况下,与5X10 5细胞接种10厘米的菜在3-4天相连。倍增时间物种之间的差别很大,可能是一些长寿命的啮齿类动物 1 。当细胞填补他们逮捕在G1期的扩散板。典型的成纤维细胞融合板包含一个紧凑层细胞(图4)。当细胞达到90%汇合,他们准备分裂。如果需要的话,细胞可以保持长时间与常规媒体的变化(每周一次或两次)被逮捕的合流板。

图1
图1。被改变成纤维细胞从老鼠的皮肤(A)和肺癌(二)隔离。媒体之前,到第7天的图片删除独立的细胞和碎片。

图2
图2。小鼠肺成纤维细胞。

图3
图3。一个领域的小鼠成纤维细胞含有细胞在M级,10倍的放大倍率拍摄。

图4
图4。一个小鼠成纤维细胞的融合板,10倍的放大倍率拍摄

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Discussion

普通小学的成纤维细胞生物学研究癌症细胞株的建立提供了一个绝佳的替代方案。成纤维细胞的一个重要的优势是,他们不进行癌基因和肿瘤抑制基因的突变,并保持完好的细胞周期检查点。这使得一个首选系统研究了细胞周期调控,DNA修复,和凋亡的正常成纤维细胞。这里所描述的协议提供了一个简单的初级成纤维细胞的分离和维护配方。该协议被用来成功地分离出超过20种啮齿类动物细胞。

重要的是时刻保持无菌细胞培养工作,以避免污染。如果实验室文化的侵略性癌症,如HeLa细胞的细胞系,成纤维细胞,应另案处理癌细胞。这是首选指定的引擎盖和小学文化的孵化器。

最好是保持在3%的生理氧气浓度的初级细胞培养。大气(20%)的氧气缩短寿命的文化,并增加氧化应激。小鼠成纤维细胞是敏感的氧化应激和衰老或进入内保持在20%(大气)的氧气时〜14人口倍增危机。小鼠成纤维细胞可以无限期地保持在3% 氧气 2 。

始终保持人口数量翻一番的文化的纪录。大型动物(8,000克以上的身体质量)可能出现复制衰老,和文化的寿命有限,甚至在3%的氧气 ,1 ,。从较小的品种,如小鼠和大鼠,细胞无​​限增殖在3%的氧气,但最终可能会成为非整倍体。如果可能的话,开始实验使用细胞在低人口倍增。

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Disclosures

没有利益冲突的声明。

Acknowledgements

我们感谢为我们提供与本议定书​​的第一个版本博士史蒂芬Austad。这项工作是由从美国国立卫生研究院和埃利森医学基金会VG的赠款和支持

Materials

Name Company Catalog Number Comments
DMEM/F12 media Invitrogen 11330-032
Fetal Bovine Serum (FBS), Qualified Invitrogen 01437-036 The Qualified serum had been pre-tested to provide good growth support for primary fibroblasts.
Antibiotic/Antimycotic Invitrogen 15420-096
Penicillin/Streptomycin Invitrogen 15140-122
Liberase TM Research Grade Roche 05401127001 Replacement enzyme.
A note from the authors: Since Roche discontinued Liberase Blendzyme 3 (11814184001), they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 05401127001 - 100mg) instead. We have switched to this enzyme successfully with no issues.
EMEM media ATCC 30-203 The EMEM media from ATCC already contains nonessential amino acids and sodium pyruvate.
Feathered #21 disposable, sterile scalpel Multiple suppliers
Three Gas Control incubator Forma or Heraeus

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References

  1. Seluanov, A. Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan. Aging . Cell. 7, 813-823 (2008).
  2. Parrinello, S. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nat Cell Biol. 5, 741-747 (2003).

Comments

29 Comments

  1. Very helpful protocol. Got it working at the very first try, Would highly recommend the protocol.

    Reply
    Posted by: Anonymous
    February 14, 2012 - 5:10 PM
  2. I have recently isolated fibroblasts from fresh fibrotic human lung. I used collagenase and plated the cells via your protocol. the cells seem to be of ² populations: one is long (²0% of plate) typical of fibroblast but the other cells are round. They look like they just started to adhere to the plate. What cells could these be? Is this happen often? COuld they be a subset of fibroblasts?

    Reply
    Posted by: Barbara S.
    April 4, 2012 - 9:33 AM
  3. Hi
    The round cells are epithelial cells that always accompany lung fibroblast isolation. These cells will just float. After you switch the media to EMEM (mentioned in Step 13) and start splitting the fibroblasts, these cells will go away.

    Reply
    Posted by: Anonymous
    April 5, 2012 - 12:11 PM
  4. I have used your protocol successfully before on human lung tissue. However this time, the majority of our cells were epithelial- look very much like keritanocytes. Is there something we could do to only keep the fibroblasts growing- change to MEM media?
    Also, the next time we try this protocol, is there anything we can do to not get so many epithelial cells?WIll be happy for any suggestions. Thanks.

    Reply
    Posted by: Barbara S.
    April 5, 2012 - 8:14 AM
  5. Hi Barbara,
    We always get the round epithelial cells during our lung fibroblast isolation too. When you switch to EMEM media and start splitting the fibroblasts, they will be selected for and the epithelial cells will be lost.
    Next time you try the protocol, you can use EMEM media for the whole isolation procedure instead of DMEM/F1² to minimize the epithelial cells you get. But the trade-off is that the efficiency of isolation might decrease. Good luck.

    Reply
    Posted by: Anonymous
    April 5, 2012 - 12:15 PM
  6. A note from the authors:
    Since Roche discontinued Liberase Blendzyme 3, they recommend using Liberase TM Research Grade medium Thermolysin (Cat. no. 05401119001 - 10 mg, Cat. no. 054011²7001 - 100mg) instead. We have switched to this enzyme successfully with no issues.

    Reply
    Posted by: Anonymous
    April 5, 2012 - 12:31 PM
  7. Thank you so much for your reply. I split them yesterday and changed the media to MEM.

    Reply
    Posted by: Barbara S.
    April 6, 2012 - 7:35 AM
  8. What is the best way to remove tissue peices on day 7? Especially with the larger tissue peices, ones that you can clearly see, produce more epithelial cells, where as the small peices (as in your pictures) produce mostly pure fibroblasts. Also, do you transplant the tissues on day 7 regardless of the confluency of your plate?

    Reply
    Posted by: Anonymous
    April 6, 2012 - 11:59 AM
  9. On day 7, we gently aspirate the supernatant (with the tissue pieces) with a pipette, transfer it to a new plate and provide the original plate with fresh media. In this way, the small pieces attached to the plate are not disturbed and the other tissue debris with the potential to give out more fibroblasts can do so on a new plate.
    Also, yes, it is a good idea to transfer the tissues on a new plate on day 7.

    Reply
    Posted by: Anonymous
    April 6, 2012 - 3:43 PM
  10. When you trypsinize initially, do you do this aggressively and try to remove all the cells from the plate (fibroblast and epithelial)? I have found that the fibroblasts seem to trypsinize first and can thus help to purify the culture, but my cell yield is significantly smaller (too small). Thank you for you help.

    Reply
    Posted by: Anonymous
    April 6, 2012 - 4:05 PM
  11. The first trypsinization dŒs not have to be aggressive. As long as all the fibroblasts are off the plate and floating, we dont worry about the epithelial cells. They usually disappear after the first couple of passages with EMEM media. The fibroblasts are fast growing and will be selected for with passaging.
    To improve the yield, I would suggest ² things in the isolation procedure : Mincing the tissue very well and incubating the tissue pieces for a longer time in the Collagenase enzyme (while taking care to avoid over-digestion).

    Reply
    Posted by: Anonymous
    April 6, 2012 - 4:18 PM
  12. You recommend storing 0.5 mL aliquots of Blendzyme at ²8 units/ml (i.e. 14 wunsch units per aliquot) but you recommend using 0.14 units/ml in 10 mls of media, or 1.4 wunsch units per digestion) This implies each aliquot is for 10 digestions. Is this correct?

    Reply
    Posted by: Anonymous
    April 16, 2012 - 10:59 AM
  13. Yes

    Reply
    Posted by: Anonymous
    April 16, 2012 - 11:04 AM
  14. After 14 days I achieve confluence and then split into fibroblast specific growth media and these cells have no trouble reaching 80-90%. When I subsequently split this population the cells senesce. Is there any obvious reason for this? It would think that 14 days of growth would cause a primary cell to senesce but your protocol suggests that I should be able to continue splitting my cells and further purfy in the process. I appreciate your help!

    Reply
    Posted by: Justin K.
    May 4, 2012 - 10:49 AM
  15. Hi Justin
    Are these mouse fibroblasts? Are you growing them in ²0% oxygen? If you are, then they will senesce. We grow all our cells in 3% oxygen and 5% CO² conditions.

    Reply
    Posted by: Amita V.
    May 9, 2012 - 3:06 PM
  16. Hi, one of the first steps recommends to euthanize the animal and store it at 4oC. Do we complete this step even if we plan on using the animal right away? If so, for how long do we store the animal at 4oC? Is putting it on ice also acceptable?

    Reply
    Posted by: J A.
    May 13, 2012 - 7:49 PM
  17. No..It is best to use the animal right away. The 4C step is only if circumstances make it impossible to process it right away. In this case, we store the corpse at 4C(better) or on ice(in case of wild caught animals) for not more than ²4 hours.

    Reply
    Posted by: Amita V.
    May 13, 2012 - 7:57 PM
  18. Also, another question I have is about the tissue fragments in the media. When would be the best time to remove them from the cells and replace them with media?

    Reply
    Posted by: Anonymous
    May 14, 2012 - 12:42 AM
  19. It depends. Usually, if the media turns yellow a few days after isolation and if there aren't enough fibroblasts on the plate, we collect the fragments, suspend them in fresh media and plate them back. As the protocol says, day 14 is when we usually get rid of the tissue fragments.

    Reply
    Posted by: Amita V.
    May 14, 2012 - 2:51 PM
  20. Hi,

    I a couple of questions about the reagents used. My first question is about the liberase enzyme used. I noticed that the liberase enzyme 3 from Roche has been discontinued. Would you recommend replacing it with the Liberase TM Research Grade medium enzyme with Thermolysin? If so, would you recommend keeping the concentration the same (.14 U/mL)? My second question is about the Fetal Bovine Serum used in this experiment. I understand that you recommend using the Qualified Fetal Bovine Serum because it has been shown to support the growth of primary fibroblast cultures. In my lab we use heat-inactivated FBS. Would you recommend me switching from heat-inactivated to qualified FBS?

    Thank you in advance for your help :)

    Reply
    Posted by: J A.
    May 21, 2012 - 11:25 AM
  21. Yes. We now use TM instead of Liberase 3 with the same concentration.
    I understand that heat-inactivation of FBS is usually done to get rid of complement factors. I don't know how critical it is for your experiments to do this but we never use heat-inactivated serum.

    Reply
    Posted by: Amita V.
    May 24, 2012 - 3:06 PM
  22. I also have a question about plating the tissue into the media. ²4- hours after I plated my tissue pieces into the media, I looked at my cultures under the microscope and saw little round cells that adhered to the plate. I understand that the protocol mentions that fibroblasts do not typically exit the tissue for at least 48 hours, and the round epithelial cells that assist the growth of fibroblasts usually float through the media. I was just wondering if seeing these cells after ²4 hours is normal.

    Reply
    Posted by: J A.
    May 24, 2012 - 12:59 PM
  23. Yes it is normal, more for lung fibroblast isolation. Once the fibroblasts start growing, the round cells will be selected against.

    Reply
    Posted by: Amita V.
    May 24, 2012 - 2:57 PM
  24. Hi,

    I was wondering what concentration of trypsin EDTA you used? I use trypsin 0.²5% trypsin EDTA to remove my cells during splitting (after an incubation time of 4 minutes). After trypsinizing the cells for 4 minutes, I have noticed that less than half of them are detached from the plate. I am afraid to leave the cells in trypsin for longer than 4 minutes because I do not want to damage them. Any suggestions?

    Reply
    Posted by: J A.
    July 18, 2012 - 1:05 AM
  25. We use Red trypsin with 0.²5% EDTA..Its very strong and we only treat cells with it for 1-² minutes. So you wash/rinse the plate with PBS after aspirating media, before adding trypsin? Because the FBS in the media inhibits trypsin activity and if the plates are not washed with PBS first, trypsin cannot act properly.

    Reply
    Posted by: Amita V.
    July 18, 2012 - 11:28 AM
  26. Thank you for such detailed procedure. I am curious is it possible that the keratinocytes also exist in the fibroblast culture? Thank you.

    Reply
    Posted by: Hua-Ling C.
    September 19, 2012 - 3:16 AM
  27. Dear all, my adult fibroblasts (GFP positive C67Bl/6 mice) are not growing very well (DMEM, high glucose, Na-pyrovate, Pen/Strp, 15%FBS). Do you have any suggestions about what I could add to the media to increase proliferation? Thanks a lot. Gunnar

    Reply
    Posted by: Gunnar P.
    March 1, 2013 - 2:03 PM
  28. Can this protocol be adapted to isolate cancer-associated fibroblasts?

    Reply
    Posted by: Wei L.
    July 9, 2013 - 2:59 PM
  29. Do you know what is the best marker to check the purity of mouse lung fibroblasts?

    Reply
    Posted by: Jenny t.
    July 15, 2014 - 5:02 PM

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