搜索

Isolation and Primary Culture of Adult Mouse Cardiac Fibroblasts
成年小鼠心脏成纤维细胞的分离和原代培养   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

Fibroblasts are often used as a feeder layer for progenitor or stem cells in co-culture systems. In the heart fibroblasts are important for cardiac development, homeostasis, and remodelling. They provide cardiomyocytes and progenitor cells not only with nutrition but also secrete extracellular matrix that forms the microenvironment that ensures cell survival and function. Although different kinds of mouse fibroblasts have been used in co-cultures (embryonic, skin and cardiac fibroblasts) adult mouse cardiac fibroblasts (AMCFs) create the closest microenvironment to the adult murine heart for culturing adult mouse cardiac progenitor cells. This protocol describes the isolation of cardiac fibroblasts from adult mouse hearts as well as their maintenance in culture.

Materials and Reagents

  1. Falcon tubes (50 ml) (Sarsdtedt)
  2. Sterile pipets (Sarsdtedt)
  3. 100 mm cell culture dishes (Sarsdtedt)
  4. Aluminum foil
  5. Adult mouse hearts from freshly euthanized mice (C57Bl6/J)
  6. NaCl (Carl Roth, catalog number: 3957.2 )
  7. KCl (Carl Roth, catalog number: HNO2.3 )
  8. Na2HPO4 (Carl Roth, catalog number: 4984.1 )
  9. KH2PO4 (AppliChem, catalog number: A2946 )
  10. CaCl2 (AppliChem, catalog number: A3652 )
  11. MgSO4.7H2O (AppliChem, catalog number: A1037 )
  12. NaHCO3 (AppliChem, catalog number: A1940 )
  13. Collagenase II (Worthington, catalog number: LS004177 )
  14. 2.5% Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 15090-046 )
  15. DMEM/F12 (Thermo Fisher Scientific, GibcoTM, catalog number: 31331-028 )
  16. PBS (without CaCl2, MgCl2) (Thermo Fisher Scientific, GibcoTM, catalog number: 14190-94 )
  17. FBS (Thermo Fisher Scientific, GibcoTM, catalog number: 10270-106 )
  18. PenStrep (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  19. L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 25030-024 )
  20. Ascorbic acid (AppliChem, catalog number: A1052 )
  21. PBS (see Recipes)
  22. Hanks balanced salt solution (HBSS) (see Recipes)
  23. Collagenase II stock (see Recipes)
  24. Digestion buffer (see Recipes)
  25. Fibroblast medium (see Recipes)

Equipment

  1. Scalpel, forceps and scissors (Fine Science Tools)
  2. Autoclaved glass beaker (100 ml) with magnetic stirrer
  3. Sterile cell culture hood
  4. Cell culture incubator (Labotect Labor-Technik Göttingen)
  5. Magnetic stirrer with thermostat (IKA®-Werke GmbH & CO. KG)
  6. Cell culture centrifuge (Eppendorf, model: 5417R )
  7. Autoclave

Procedure

  1. Remove 3 adult mouse hearts from freshly euthanized mice and place them on a Petri dish containing ice cold PBS.
  2. Place the hearts under a sterile cell culture hood and perform the following steps under sterile conditions.
  3. Pump the blood out of the hearts by the use of forceps.
  4. Mince the 3 hearts on a Petri dish on ice. By the use of scissors cut the heart into 10 pieces and by the use of a scalpel reduce them into a size of 1 mm.
  5. Transfer the minced tissue (using the scalpel as a spatula) in an autoclaved glass beaker with a magnetic stirrer and cover the beaker with aluminum foil.
  6. Add 25 ml digestion buffer and digest tissue under constant stirring at 37 °C for 5 min.
    Note: The stirring speed should be adjusted so that all the pieces of tissue just flow and do not sit at the bottom but should not be too strong to destroy the cells.
  7. Leave the mixture for 1 min for the tissue to sit down and discard the first supernatant that contains debris and blood cells.
  8. Add 25 ml digestion buffer and digest tissue under constant stirring at 37 °C for 10 min.
  9. Leave the mixture 1 min for the tissue to sit down and collect the supernatant with a 25 ml pipette (around 22 ml). Do not collect the tissue pieces that tend to float.
  10. Place the supernatants in plastic 50 ml falcon tubes on ice containing 2 ml fibroblast medium. If possible supernatants can be combined in one falcon but for each supernatant 2 ml of fibroblast medium is required.
  11. Repeat steps 6-10 until all the tissue is dissolved (typically 7-10 times).
  12. Spin the cells down for 5 min at 300 x g at 4 °C.
  13. Resuspend cells in 20 ml fibroblast medium.
  14. Plate the cell suspension into two 100 mm cell culture dishes (10 ml cell suspension/dish) and incubate them at 37 °C in a cell culture incubator with 5% CO2 for 2 h (“Pre-plating”).
  15. 2 h upon plating alive and healthy fibroblasts should have adhered to the dish. Check the cell confluence under the microscope (it should be around 50%). At this point fibroblasts resemble little round dots.
  16. Collect and discard supernatant. Wash 3x with 2.5 ml warm PBS and replenish with 10 ml fresh fibroblast medium. Cultivate at 37 °C and 5% CO2 in a cell culture incubator.
  17. Primary adult mouse fibroblasts divide less than embryonic ones and under our culture conditions have a doubling time of 36-48 h. Typically, 2-3 days upon plating the fibroblasts reach 90% confluence.
    Note: Caution! Do not split the fibroblasts until they reached a minimum confluence of 80% because the fibroblasts growth rate will decrease and they will differentiate.
  18. Once confluence is reached fibroblasts are trypsinized as follows.
  19. Wash two times with 2 ml pre-warmed PBS and then add 2 ml warm trypsin. Incubate for 5 min at 37 °C, in a cell culture incubator. Control if fibroblasts detached from plate. If not prolong incubation until the cells detach.
  20. Neutralize trypsin by adding 7 ml pre-warmed fibroblast medium.
  21. To achieve a re-plating confluence of 30%, split 1:3 into 3 new 100 mm plates. Add 3 ml of fibroblast suspension in a plate containing 7 ml fresh and warm fibroblast medium (final volume/plate 10 ml)

Representative data

For representative photos and results please refer to Zafiriou et al. (2014).

Notes

Note that primary isolated cells as fibroblasts are differentiated when cultured for a long time period (more than 20 days) cease to divide (Bayreuther et al.,1988). Moreover, using cells from the same passage leads to better reproducibility. Therefore, we recommend using passage 4 fibroblasts for all co-culture experiments.

Recipes

  1. PBS, 1 L
    Dissolve the following in 800 ml distilled H2O (dH2O)
    8 g of NaCl
    0.2 g of KCl
    1.44 g of Na2HPO4
    0.24 g of KH2PO4
    Adjust pH to 7.4 with HCl, add dH2O to 1 L, sterilized by autoclaving
  2. Hanks balanced salt solution (HBSS) (100 ml)
    1. Stock solution 1: 8.0 g NaCl, 0.4 g KCl in 100 ml dH2O
    2. Stock solution 2: 0.358 g Na2HPO4, 0.6 g KH2PO4 in 100 ml dH2O
    3. Stock solution 3: 0.72 g CaCl2 in 50 ml dH2O
    4. Stock solution 4: 1.23 g MgSO4.7H2O in 50 ml dH2O
    5. Stock solution 5: 0.35 g NaHCO3 in 10 ml dH2O
    6. HBSS buffer: 10 ml Solution 1, 1 ml Solution 2, 1 ml Solution 3, 86 ml dH2O, 1 ml Solution 4, 1 ml Solution 5 (0.137 M NaCl, 5.4 mM KCl, 0.25 mM Na2HPO4, 0.44 KH2PO4, 1.3 mM CaCl2, 1 mM MgSO4, 4.2 mM NaHCO3)
  3. Collagenase II stock (1 ml)
    Weight the appropriate amount of collagenase (varies from lot to lot) to obtain 10,000 U and dissolve in 1 ml sterile water
  4. Digestion buffer (100 ml)
    95 ml HBSS buffer
    1 ml collagenase II (final conc. 100 U/ml)
    4 ml 2.5% Trypsin (final conc. 0.1%)
    Filter sterilize
    For optimal enzymatic activity always prepare fresh.
  5. Fibroblast medium
    DMEM/F12
    10% FBS
    100 U/ml PenStrep
    1x L-glutamine
    100 µM ascorbic acid
    Filter sterilize the whole medium and keep at 4 °C

Acknowledgments

This protocol was adapted by methods described by Lafontant et al. (2006) and Ohkubo et al. (1997).
The HBSS recipe was retrieved from the internet site labrat.com (link: http://www.thelabrat.com/protocols/Hanks.shtml).
This work was supported by the Deutsche Forschungsgemeinschaft (ZE900/1-1; ZI708/7-1, 8-1, 10-1; SFB1002; exFLIII) and the German Center for Cardiovascular Research (DZHK).

References

  1. Bayreuther, K., Rodemann, H. P., Hommel, R., Dittmann, K., Albiez, M. and Francz, P. I. (1988). Human skin fibroblasts in vitro differentiate along a terminal cell lineage. Proc Natl Acad Sci U S A 85(14): 5112-5116.
  2. Lafontant, P. J., Burns, A. R., Donnachie, E., Haudek, S. B., Smith, C. W. and Entman, M. L. (2006). Oncostatin M differentially regulates CXC chemokines in mouse cardiac fibroblasts. Am J Physiol Cell Physiol 291(1): C18-26.
  3. Ohkubo, N., Matsubara, H., Nozawa, Y., Mori, Y., Murasawa, S., Kijima, K., Maruyama, K., Masaki, H., Tsutumi, Y., Shibazaki, Y., Iwasaka, T. and Inada, M. (1997). Angiotensin type 2 receptors are reexpressed by cardiac fibroblasts from failing myopathic hamster hearts and inhibit cell growth and fibrillar collagen metabolism. Circulation 96(11): 3954-3962.
  4. Zafiriou, M. P., Noack, C., Unsold, B., Didie, M., Pavlova, E., Fischer, H. J., Reichardt, H. M., Bergmann, M. W., El-Armouche, A., Zimmermann, W. H. and Zelarayan, L. C. (2014). Erythropoietin responsive cardiomyogenic cells contribute to heart repair post myocardial infarction. Stem Cells 32(9): 2480-2491.

简介

成纤维细胞通常用作共培养系统中的祖细胞或干细胞的饲养层。 在心脏成纤维细胞对于心脏发育,体内平衡和重塑是重要的。 他们提供心肌细胞和祖细胞不仅与营养,而且分泌细胞外基质,形成微环境,确保细胞的生存和功能。 尽管不同种类的小鼠成纤维细胞已用于共培养(胚胎,皮肤和心脏成纤维细胞),但是成年小鼠心脏成纤维细胞(AMCFs)为培养成年小鼠心脏祖细胞产生了与成年鼠心脏最接近的微环境。 该协议描述了从成年小鼠心脏分离心脏成纤维细胞以及它们在培养物中的维持。

材料和试剂

  1. 猎鹰管(50ml)(Sarsdtedt)
  2. 无菌移液器(Sarsdtedt)
  3. 100 mm细胞培养皿(Sarsdtedt)
  4. 铝箔
  5. 来自新鲜安乐死的小鼠(C57B16/J)的成年小鼠心脏
  6. NaCl(Carl Roth,目录号:3957.2)
  7. KCl(Carl Roth,目录号:HNO2.3)

  8. (Carl Roth,目录号:4984.1)。

  9. (AppliChem,目录号:A2946)
  10. CaCl 2(AppliChem,目录号:A3652)
  11. (AppliChem,目录号:A1037)。
  12. NaHCO 3(AppliChem,目录号:A1940)
  13. 胶原酶II(Worthington,目录号:LS004177)
  14. 2.5%胰蛋白酶(Thermo Fisher Scientific,Gibco TM ,目录号:15090-046)
  15. DMEM/F12(Thermo Fisher Scientific,Gibco TM ,目录号:31331-028)
  16. PBS(无CaCl 2,MgCl 2)(Thermo Fisher Scientific,Gibco< sup>,目录号:14190-94)
  17. FBS(Thermo Fisher Scientific,Gibco TM ,目录号:10270-106)
  18. PenStrep(Thermo Fisher Scientific,Gibco TM ,目录号:15140-122)
  19. L-谷氨酰胺(Thermo Fisher Scientific,Gibco TM ,目录号:25030-024)
  20. 抗坏血酸(AppliChem,目录号:A1052)
  21. PBS(请参阅配方)
  22. Hanks平衡盐溶液(HBSS)(参见配方)
  23. 胶原酶II原液(见配方)
  24. 消化缓冲液(参见配方)
  25. 成纤维细胞培养基(参见配方)

设备

  1. 手术刀,镊子和剪刀(Fine Science Tools)
  2. 具有磁力搅拌器的高压灭菌的玻璃烧杯(100ml)
  3. 无菌细胞培养罩
  4. 细胞培养孵化器(Labotect Labor-TechnikG?ttingen)
  5. 具有恒温器的磁力搅拌器(IKA -Werke GmbH& CO.KG)
  6. 细胞培养离心机(Eppendorf,型号:5417R)
  7. 高压灭菌器

程序

  1. 从新鲜安乐死的小鼠中取出3只成年老鼠心脏,并将它们放在含有冰冷PBS的培养皿中
  2. 将心脏放置在无菌细胞培养罩下,并在无菌条件下进行以下步骤
  3. 通过使用镊子将血液泵出心脏。
  4. 剁碎在一个培养皿的3心脏在冰。通过使用剪刀将心脏切成10片,使用手术刀将它们切成1毫米的大小。
  5. 在带有磁力搅拌器的高压灭菌玻璃烧杯中转移切碎的组织(使用手术刀作为刮刀),并用铝箔覆盖烧杯。
  6. 加入25ml消化缓冲液,消化组织在37℃恒定搅拌5分钟。
    注意:搅拌速度应该调整,使所有的组织只是流动,不要坐在底部,但不应太强,以破坏细胞。
  7. 离开混合物1分钟以使组织坐下,并丢弃含有碎片和血细胞的第一上清液
  8. 加入25毫升消化缓冲液,消化组织,在37℃持续搅拌10分钟
  9. 离开混合物1分钟使组织坐下,并用25ml移液管(约22ml)收集上清液。不要收集易于浮起的纸巾片。
  10. 将上清液放置在含有2ml成纤维细胞培养基的冰上的塑料50ml falcon管中。如果可能的上清液可以在一只猎鹰中组合,但是每个上清液需要2ml成纤维细胞培养基。
  11. 重复步骤6-10,直到所有组织溶解(通常7-10次)。
  12. 在4℃下以300×g离心细胞5分钟。
  13. 将细胞重悬在20ml成纤维细胞培养基中
  14. 将细胞悬浮液置于两个100mm细胞培养皿(10ml细胞悬浮液/皿)中,并在37℃下在具有5%CO 2的细胞培养箱中孵育2小时("电镀")。
  15. 2小时电镀活的和健康的成纤维细胞应该已经粘附到菜。检查细胞汇合在显微镜下(它应该在50%左右)。此时成纤维细胞类似小圆点。
  16. 收集并弃去上清液。用2.5ml温热PBS洗涤3次,并补充10ml新鲜成纤维细胞培养基。在37℃和5%CO 2下在细胞培养箱中培养
  17. 原代成年小鼠成纤维细胞分裂少于胚胎的,在我们的培养条件下具有36-48小时的倍增时间。通常,在铺板后2-3天,成纤维细胞达到90%汇合。
    注意:注意!不要分裂成纤维细胞,直到它们达到80%的最小汇合,因为成纤维细胞生长速率会降低,并且它们会分化。
  18. 一旦达到融合,成纤维细胞如下胰蛋白酶化。
  19. 用2ml预热的PBS洗涤两次,然后加入2ml温热胰蛋白酶。在37℃下,在细胞培养箱中孵育5分钟。控制成纤维细胞是否从板上分离。如果不延长孵育直到细胞分离。
  20. 通过加入7ml预热的成纤维细胞培养基中和胰蛋白酶。
  21. 为了实现30%的重铺镀层汇合,1:3分裂成3个新的100mm板。在含有7ml新鲜和温热成纤维细胞培养基(终体积/板10ml)的平板中加入3ml成纤维细胞悬浮液。

代表数据

有关代表性照片和结果,请参阅Zafiriou等人(2014)。

笔记

注意,作为成纤维细胞的初级分离细胞在长时间(超过20天)停止分裂时分化(Bayreuther等人,1988)。此外,使用来自相同通道的细胞导致更好的再现性。因此,我们建议使用通道4成纤维细胞进行所有共培养实验。

食谱

  1. PBS,1 L
    将以下物质溶于800ml蒸馏的H 2 O(dH 2 O)中。
    8克NaCl
    0.2克KCl
    1.44g的Na 2 HPO 4
    0.24g的KH 2 PO 4 sub/
    用HCl调节pH至7.4,加入dH 2 O至1L,通过高压灭菌灭菌
  2. Hanks平衡盐溶液(HBSS)(100ml)
    1. 储备溶液1:8.0g NaCl,0.4g KCl在100ml dH 2 O中
    2. 储备溶液2:在100ml dH中的0.358g Na 2 HPO 4,0.6g KH 2 PO 4, sub> 2 O
    3. 储备溶液3:0.72g CaCl 2在50ml dH 2 O中
    4. 储备溶液4:1.23g MgSO 4。在50ml dH 2 O中的7H 2 O溶液
    5. 储备溶液5:在10ml dH 2 O中的0.35g NaHCO 3
    6. HBSS缓冲液:10ml溶液1,1ml溶液2,1ml溶液3,86ml dH 2 O,1ml溶液4,1ml溶液5(0.137M NaCl,5.4mM KCl,0.25 1mM Na 2 HPO 4,0.44KH 2 PO 4,1.3mM CaCl 2,3mM CaCl 2 ,1mM MgSO 4,4.2mM NaHCO 3)
  3. 胶原酶II原液(1ml) 称量适当量的胶原酶(根据批次不同),以获得10,000U,并溶解于1ml无菌水中
  4. 消化缓冲液(100ml)
    95 ml HBSS缓冲液
    1ml胶原酶II(终浓度100U/ml) 4ml 2.5%胰蛋白酶(最终浓度0.1%) 过滤灭菌
    为了最佳酶活性总是准备新鲜。
  5. 成纤维细胞培养基
    DMEM/F12
    10%FBS
    100 U/ml PenStrep
    1x L-谷氨酰胺
    100μM抗坏血酸
    过滤灭菌整个培养基,并保持在4℃

致谢

该方案通过Lafontant等人(2006)和Ohkubo等人(1997)描述的方法进行修改。
HBSS食谱是从互联网网站labrat.com检索的(链接: http ://www.thelabrat.com/protocols/Hanks.shtml )。
这项工作得到了德国心脏病研究中心(ZE900/1-1; ZI708/7-1,8-1,10-1; SFB1002; exFLIII)和德国心血管研究中心(DZHK)的支持。

参考文献

  1. Bayreuther,K.,Rodemann,HP,Hommel,R.,Dittmann,K.,Albiez,M.and Francz,PI(1988)。  制瘤素M差异调节小鼠心脏成纤维细胞中的CXC趋化因子。 Am J Physiol Cell Physiol 291(1):C18- 26.
  2. Ohkubo,N.,Matsubara,H.,Nozawa,Y.,Mori,Y.,Murasawa,S.,Kijima,K.,Maruyama,K.,Masaki,H.,Tsutumi,Y.,Shibazaki, Iwasaka,T。和Inada,M。(1997)。  血管紧张素2型受体由来自衰竭的肌病仓鼠心脏的心脏成纤维细胞再表达,并抑制细胞生长和纤维胶原蛋白代谢。 循环 96(11):3954-3962。
  3. Zafiriou,MP,Noack,C.,Unsold,B.,Didie,M.,Pavlova,E.,Fischer,HJ,Reichardt,HM,Bergmann,MW,El-Armouche,A.,Zimmermann,WHand Zelarayan,LC (2014)。  红细胞生成素反应性心肌形成细胞有助于心脏修复post myocardial infarction。 Stem Cells 32(9):2480-2491。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zafeiriou, M. P., Noack, C. and Zelarayan, L. C. (2016). Isolation and Primary Culture of Adult Mouse Cardiac Fibroblasts. Bio-protocol 6(13): e1860. DOI: 10.21769/BioProtoc.1860.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。