{"id":1006,"date":"2017-02-27T21:20:56","date_gmt":"2017-02-27T21:20:56","guid":{"rendered":"https:\/\/www.progeriaresearch.org\/?page_id=1006"},"modified":"2024-10-23T11:05:53","modified_gmt":"2024-10-23T15:05:53","slug":"fibroblast-cell-culture-protocols","status":"publish","type":"page","link":"https:\/\/www.progeriaresearch.org\/fr\/fibroblast-cell-culture-protocols\/","title":{"rendered":"Protocoles de culture de cellules fibroblastes"},"content":{"rendered":"

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Cellule fibroblaste<\/h1>\n

Protocoles de culture<\/h1>\n

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\n[\/et_pb_sidebar][\/et_pb_column][et_pb_column type=\u201d3_4\u2033 specialty_columns=\u201d3\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_row_inner _builder_version=\u201d4.16\u2033 background_color=\u201d#00b2e2\u2033 custom_margin=\u201d41px||\u201d custom_margin_tablet=\u201d0px||\u201d custom_margin_phone=\u201d\u201d custom_margin_last_edited=\u201don|phone\u201d custom_padding=\u201d39.4375px|20px|0|20px|false|true\u201d animation_style=\u201dslide\u201d animation_direction=\u201dtop\u201d animation_intensity_slide=\u201d25%\u201d box_shadow_style=\u201dpreset1\u2033 box_shadow_blur=\u201d38px\u201d box_shadow_spread=\u201d-12px\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_column_inner saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text admin_label=\u201dLe travail de la Progeria Research Foundation\u2026\u201d _builder_version=\u201d4.16\u2033 header_2_line_height=\u201d1.2em\u201d background_layout=\u201ddark\u201d custom_margin=\u201d||35px\u201d custom_padding=\u201d|||\u201d custom_padding_tablet=\u201d|50px||50px||true\u201d custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201dsur|bureau\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d\u201d header_font_size_last_edited=\u201dsur|bureau\u201d header_2_font_size_tablet=\u201d25px\u201d header_2_font_size_phone=\u201d\u201d header_2_font_size_last_edited=\u201dsur|tablette\u201d header_4_font_size_tablet=\u201d\u201d header_4_font_size_phone=\u201d19px\u201d header_4_font_size_last_edited=\u201dsur|t\u00e9l\u00e9phone\u201d header_4_line_height_tablet=\u201d\u201d header_4_line_height_phone=\u201d1.2em\u201d header_4_line_height_last_edited=\u201dsur|t\u00e9l\u00e9phone\u201d z_index_tablet=\u201d500\u2033 global_colors_info=\u201d{}\u201d]<\/p>\n

Protocole de sous-culture et de cong\u00e9lation des fibroblastes<\/strong><\/h2>\n

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Milieux de croissance<\/h4>\n

DMEM \u2013 ThermoFisher #11960-044 (glucose \u00e9lev\u00e9 sans L-glutamine)<\/p>\n

S\u00e9rum f\u0153tal bovin 15% FBS \u2013 ThermoFisher #10437-028<\/p>\n

1% (1X) P\u00e9nicilline-Streptomycine \u2013 ThermoFisher #15140-122<\/p>\n

1% (1X) GlutaMAX \u2013 ThermoFisher #35050-061<\/p>\n

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Trypsine<\/strong><\/h4>\n

Trypsine EDTA C 0,25% (ThermoFisher #25200-056)<\/p>\n

Solution saline \u00e9quilibr\u00e9e de Hank<\/strong><\/h4>\n

HBSS- (ThermoFisher #14170 (1X) (-) chlorure de calcium, (-) chlorure de magn\u00e9sium, (-) sulfate de magn\u00e9sium<\/p>\n

DMSO pour la cong\u00e9lation<\/strong><\/h4>\n

<\/u><\/strong>DMSO de qualit\u00e9 culture cellulaire \u2013 Sigma #D2438<\/p>\n

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D\u00e9cong\u00e9lation des lign\u00e9es cellulaires PRF<\/strong><\/h4>\n
    \n
  1. D\u00e9congeler rapidement les cellules dans un bain-marie \u00e0 37 \u00b0C.<\/li>\n
  2. Essuyez l\u2019ext\u00e9rieur du flacon avec de l\u2019\u00e9thanol 70%.<\/li>\n
  3. Transf\u00e9rer les cellules d\u00e9congel\u00e9es dans un flacon T25 contenant 5 ml de milieu de croissance.<\/li>\n
  4. Placer le flacon dans un incubateur \u00e0 37 \u00b0C pendant la nuit.<\/li>\n
  5. Le lendemain, observez au microscope pour vous assurer que les cellules se sont attach\u00e9es.<\/li>\n
  6. Retirez le support et remplacez-le par un nouveau support de croissance.<\/li>\n<\/ol>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n
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    Sous-culture de lign\u00e9es cellulaires PRF<\/strong><\/h4>\n
      \n
    1. Les cellules doivent \u00eatre divis\u00e9es lorsqu'elles sont confluentes.<\/li>\n
    2. Pour diviser les cellules (les volumes indiqu\u00e9s supposent que les cellules sont dans un T25)\u00a0:
      a) \u00c0 l'aide d'une technique st\u00e9rile, retirez le milieu et rincez le flacon avec 2 \u00e0 3 ml de HBSS st\u00e9rile. Retirez et jetez le HBSS.
      b) Ajoutez 1 ml de trypsine et laissez incuber pendant 2 \u00e0 3 minutes. Les cellules doivent \u00eatre examin\u00e9es au microscope invers\u00e9 pour d\u00e9terminer si elles ont commenc\u00e9 \u00e0 se redresser, \u00e0 se soulever et \u00e0 flotter. Si les cellules ne se d\u00e9tachent pas apr\u00e8s 3 minutes, vous pouvez laisser incuber 1 \u00e0 2 minutes suppl\u00e9mentaires.
      c) Serrez le bouchon et inclinez doucement le flacon d'un c\u00f4t\u00e9 \u00e0 l'autre pour d\u00e9loger toutes les cellules. Le flacon peut \u00eatre l\u00e9g\u00e8rement tapot\u00e9 sur le c\u00f4t\u00e9 pour d\u00e9tacher les cellules si n\u00e9cessaire.
      d) Ajoutez 4 ml de nouveau milieu au flacon d\u00e8s que les cellules flottent pour inactiver la trypsine. Rincez les parois du flacon plusieurs fois avec le nouveau milieu pour \u00e9liminer toutes les cellules du plastique et les placer dans la solution. Retirez tout le liquide contenant des cellules et transf\u00e9rez-le dans un r\u00e9cipient conique de 15 ml (ou de 50 ml si vous regroupez 3 flacons ou plus).
      e) \u00c0 l\u2019aide d\u2019une technique st\u00e9rile, pr\u00e9levez une aliquote pour la compter sur un h\u00e9mocytom\u00e8tre.
      f) Pendant que vous comptez les cellules, le reste de la solution doit \u00eatre centrifug\u00e9 dans la centrifugeuse clinique pendant 5 minutes \u00e0 1 000 tr\/min.<\/li>\n
    3. Apr\u00e8s avoir calcul\u00e9 votre nombre de cellules, placez les cellules sur une plaque \u00e0 2,5 x 105<\/sup>\u00a0<\/span>cellules par flacon filtrant T25.<\/li>\n
    4. Les cellules doivent \u00eatre nourries tous les 2 \u00e0 3 jours avec un milieu de croissance frais.<\/li>\n<\/ol>\n

      Gel:
      <\/u><\/strong><\/h4>\n

      Les cellules doivent \u00eatre congel\u00e9es \u00e0 au moins 5 x 105<\/sup>\u00a0<\/span>cellules\/ml\/cryotube dans un milieu de croissance contenant 10% DMSO et 30% FBS, puis plac\u00e9es dans une chambre de cong\u00e9lation \u00e0 l'isopropanol \u00e0 -80 \u00b0C pendant la nuit. Transf\u00e9rer dans l'azote liquide le lendemain.<\/p>\n

      Protocole de sous-culture et de cong\u00e9lation des fibroblastes<\/a><\/p>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n<\/div>\n

      [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner custom_padding_last_edited=\u201don|phone\u201d _builder_version=\u201d4.16\u2033 custom_padding=\u201d||0px|||\u201d custom_padding_tablet=\u201d|35px||35px||true\u201d custom_padding_phone=\u201d\u201d border_width_top=\u201d10px\u201d border_color_top=\u201d#8fd2ed\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_column_inner saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.16\u2033 global_colors_info=\u201d{}\u201d]<\/p>\n

      Pour plus d'informations, veuillez contacter :<\/strong><\/h4>\n

      [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][et_pb_row_inner column_structure=\u201d1_2,1_2\u2033 custom_padding_last_edited=\u201don|phone\u201d _builder_version=\u201d4.16\u2033 custom_padding=\u201d|35px|0|0px|false|false\u201d custom_padding_tablet=\u201d|35px||35px||true\u201d custom_padding_phone=\u201d\u201d animation_direction=\u201dtop\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_column_inner type=\u201d1_2\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|20px||\u201d custom_padding_tablet=\u201d||20px\u201d custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201don|desktop\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.19.5\u2033 global_colors_info=\u201d{}\u201d]<\/p>\n

      Dr Leslie B. Gordon, Ph. D.<\/h6>\n

      Professeur de recherche en p\u00e9diatrie, Warren Alpert Medical School de l'universit\u00e9 Brown et d\u00e9partement de p\u00e9diatrie, Hasbro Children's Hospital, Providence, RI D\u00e9partement d'anesth\u00e9sie, Children's Hospital Boston et Harvard Medical School, Boston, MA Directeur m\u00e9dical, The Progeria Research Foundation<\/p>\n

      T\u00e9l\u00e9phone : 978-535-2594
      T\u00e9l\u00e9copieur : 508-543-0377
      <\/span>      lgordon@progeriaresearch.org<\/a><\/p>\n

      [\/et_pb_text][\/et_pb_column_inner][et_pb_column_inner type=\u201d1_2\u2033 saved_specialty_column_type=\u201d3_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||20px\u201d custom_padding_tablet=\u201d20px|||0px\u201d custom_padding_phone=\u201d\u201d custom_padding_last_edited=\u201don|phone\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_text _builder_version=\u201d4.27.2\u2033 custom_padding=\u201d||0px|||\u201d custom_padding_last_edited=\u201dd\u00e9sactiv\u00e9|bureau\u201d hover_enabled=\u201d0\u2033 global_colors_info=\u201d{}\u201d sticky_enabled=\u201d0\u2033]<\/p>\n

      Wendy Norris<\/span><\/h6>\n
      Banque de cellules et de tissus PRF
      T\u00e9l\u00e9phone : 401-274-1122 x 48063
      wnorris@brownhealth.org<\/a>
      <\/span><\/div>\n

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      pour notre<\/h2>\n

      Bulletin!<\/h2>\n

      [\/et_pb_cta][\/et_pb_column][et_pb_column type=\u201d1_4\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_cta button_url=\u201dhttps:\/\/progeriaresearch.donorsupport.co\/-\/XZHJVWZR\u201d button_text=\u201dFaites un don maintenant\u201d admin_label=\u201dEnsemble, nous trouverons le rem\u00e8de !\u201d module_class=\u201dsign-btn\u201d _builder_version=\u201d4.16\u2033 header_font_size=\u201d25px\u201d background_color=\u201d#29327a\u201d animation_style=\u201dslide\u201d animation_direction=\u201dleft\u201d animation_intensity_slide=\u201d25%\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d30px\u201d header_font_size_last_edited=\u201don|desktop\u201d body_font_size_tablet=\u201d\u201d body_font_size_phone=\u201d\u201d body_font_size_last_edited=\u201don|desktop\u201d z_index_tablet=\u201d500\u2033 border_radii=\u201don|25px|25px|25px|25px\u201d global_colors_info=\u201d{}\u201d button_bg_color__hover_enabled=\u201don\u201d bouton_bg_color__hover=\u201d#8fd2ed\u201d bouton_border_color__hover_enabled=\u201don\u201d]<\/p>\n

      Ensemble, nous<\/h2>\n

      VOLONT\u00c9<\/em><\/h2>\n

      trouve le rem\u00e8de !<\/h2>\n

      [\/et_pb_cta][\/et_pb_column][et_pb_column type=\u201d1_2\u2033 _builder_version=\u201d4.16\u2033 custom_padding=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_image src=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2024\/08\/2024-strip-footer-strip-copy.png\u201d title_text=\u201dCopie de la bande de pied de page de la bande 2024\u201d _builder_version=\u201d4.27.0\u2033 _module_preset=\u201ddefault\u201d custom_margin=\u201d35px||||false|false\u201d global_colors_info=\u201d{}\u201d][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>","protected":false},"excerpt":{"rendered":"

      [et_pb_section fb_built=\u201d1\u2033 fullwidth=\u201don\u201d disabled_on=\u201doff|off|off\u201d _builder_version=\u201d4.16\u2033 border_width_bottom=\u201d55px\u201d border_color_bottom=\u201d#29327a\u201d locked=\u201doff\u201d global_colors_info=\u201d{}\u201d][et_pb_fullwidth_header _builder_version=\u201d4.16\u2033 title_font=\u201d||||||||\u201d title_font_size=\u201d55\u2033 background_color=\u201d#29327a\u201d background_image=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Aluisio.jpg\u201d background_position=\u201dcenter_left\u201d custom_padding=\u201d9vw||9vw||true\u201d custom_padding_tablet=\u201d\u201d custom_padding_phone=\u201d|56px||\u201d custom_padding_last_edited=\u201dsur|bureau\u201d title_font_size_tablet=\u201d45px\u201d title_font_size_phone=\u201d40px\u201d title_font_size_last_edited=\u201dsur|t\u00e9l\u00e9phone\u201d z_index_tablet=\u201d500\u2033 custom_css_main_element=\u201dbackground-position: center 18% !important;\u201d Protocoles de culture de cellules fibroblastes [\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 use_custom_gutter=\u201don\u201d gutter_width=\u201d1\u2033 specialty=\u201don\u201d padding_left_1=\u201d35px\u201d padding_left_2=\u201d35px\u201d padding_2_tablet=\u201d|||0px\u201d padding_2_phone=\u201d\u201d padding_2_last_edited=\u201don|desktop\u201d module_class_1=\u201dsidebar-secondary-nav\u201d module_class=\u201dhandprint-bg\u201d _builder_version=\u201d4.16\u2033 background_image=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png\u201d parallax=\u201don\u201d parallax_method=\u201doff\u201d inner_width=\u201d100%\u201d inner_max_width=\u201d100%\u201d custom_padding=\u201d0|0px|54px|0px|false|false\u201d z_index_tablet=\u201d500\u2033 border_width_top=\u201d10px\u201d border_color_top=\u201d#8fd2ed\u201d [\u2026]<\/p>","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"on","_et_pb_old_content":"\t\t\t\t[vc_row][vc_column][vc_custom_heading text=\"Fibroblast Cell Culture Protocols\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\n

      PROTOCOL FOR SUBCULTURING AND FREEZING FIBROBLASTS\u00a0<\/strong><\/p>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column width=\"1\/2\"][vc_column_text]Growth Media<\/u><\/strong>\r\n\r\nDMEM - ThermoFisher #11960-044 (high glucose without L-glutamine)\r\n\r\n15% FBS Fetal Bovine Serum \u2013 ThermoFisher #10437-028\r\n\r\n1% (1X) Penicillin-Streptomycin - ThermoFisher #15140-122\r\n\r\n1% (1X) GlutaMAX \u2013 ThermoFisher #35050-061[\/vc_column_text][\/vc_column][vc_column width=\"1\/2\"][vc_column_text]Trypsin<\/u><\/strong>\r\n\r\nTrypsin EDTA C 0.25% (ThermoFisher #25200-056)\r\n\r\nHank\u2019s Balanced Salt Solution\r\n<\/u><\/strong>HBSS- (ThermoFisher #14170 (1X) (-) calcium chloride, (-) magnesium chloride, (-) magnesium sulfate\r\n\r\nDMSO for Freezing\r\n<\/u><\/strong>Cell Culture Grade DMSO - Sigma #D2438[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Thawing PRF cell lines<\/u><\/strong>\r\n

        \r\n \t
      1. Thaw cells rapidly in a 37\u02daC water bath.<\/li>\r\n \t
      2. Wipe the outside of the vial with 70% ethanol.<\/li>\r\n \t
      3. Transfer the thawed cells to a T25 flask containing 5 ml of growth media.<\/li>\r\n \t
      4. Place flask in 37\u02daC incubator overnight.<\/li>\r\n \t
      5. The following day, observe under the microscope to ensure cells have attached.<\/li>\r\n \t
      6. Remove media and replace with fresh growth media.<\/li>\r\n<\/ol>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Subculturing PRF cell lines<\/u><\/strong>\r\n
          \r\n \t
        1. Cells should be split when confluent.<\/li>\r\n \t
        2. To split cells (volumes given are assuming cells are in a T25):\r\na)\u00a0 Using sterile technique, remove media and rinse flask with 2-3 ml of sterile HBSS. Remove and discard HBSS.\r\nb)\u00a0 Add 1 ml trypsin and incubate for 2-3 minutes. Cells should be checked under an inverted microscope to determine if they have begun to round up, lift and float.\u00a0 If cells are not detached after 3 minutes, you may incubate another 1-2 minutes.\r\nc)\u00a0 Tighten cap and gently tip flask from side to side to dislodge all cells. Flask may be tapped lightly on side to loosen cells if needed.\r\nd)\u00a0 Add 4 ml of new media to flask as soon as cells are floating to inactivate the trypsin. Rinse down sides of flask several times with the new media to wash all cells off the plastic and into the solution. Remove all liquid containing cells and transfer to a 15ml conical (or 50 ml if you are pooling 3 or more flasks).\r\ne)\u00a0 Using sterile technique, remove an aliquot for counting on a hemocytometer.\r\nf)\u00a0 While you are counting cells, the rest of the solution should be spun in the clinical centrifuge for 5 minutes at 1000 rpm.<\/li>\r\n \t
        3. After calculating your cell count, plate cells at 2.5 x 105<\/sup> cells per T25 filter top flask.<\/li>\r\n \t
        4. Cells should be fed every 2-3 days with fresh growth medium.<\/li>\r\n<\/ol>\r\n[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]Freezing:\r\n<\/u><\/strong>Cells should be frozen at no less than 5 x 105<\/sup> cells\/ml\/cryovial in growth media containing 10% DMSO and 30% FBS and subsequently placed in an isopropanol freezing chamber at -80\u02daC overnight.\u00a0 Transfer to the liquid nitrogen the next day.\r\n\r\n\"\"\u00a0Protocol for Subculturing and Freezing Fibroblasts<\/a>[\/vc_column_text][\/vc_column][\/vc_row][vc_row][vc_column][vc_column_text]For Further Information Please Contact:<\/strong>\r\n\r\nLeslie B. Gordon, MD, PhD\r\nProfessor of Pediatrics Research\u00a0Warren Alpert Medical School of Brown University and Department of Pediatrics, Hasbro Children's Hospital, Providence, RI\u00a0Department of Anesthesia, Children's Hospital Boston and Harvard Medical School, Boston, MA,\u00a0Medical Director, The Progeria Research Foundation\r\n\r\nPhone: 978-535-2594\r\nFax: 508-543-0377\r\nLeslie_Gordon@brown.edu\u00a0<\/a>\r\n
          Wendy Norris<\/span><\/div>\r\n
          PRF Cell and Tissue Bank\r\nPhone: 401-274-1122 x 48063\r\nwnorris@lifespan.org<\/a><\/span><\/div>\r\n[\/vc_column_text][\/vc_column][\/vc_row]\t\t","_et_gb_content_width":"","footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-1006","page","type-page","status-publish","hentry"],"yoast_head":"\nFibroblast | The Progeria Research Foundation<\/title>\n<meta name=\"description\" content=\"For info regarding Fibroblast Cell Culture Protocols, please contact Leslie Gordon at Leslie_Gordon@brown.edu, or Wendy Norris at wnorris@lifespan.org.\" \/>\n<meta name=\"robots\" content=\"index, follow, max-snippet:-1, max-image-preview:large, max-video-preview:-1\" \/>\n<link rel=\"canonical\" href=\"https:\/\/www.progeriaresearch.org\/fr\/fibroblast-cell-culture-protocols\/\" \/>\n<meta property=\"og:locale\" content=\"fr_FR\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Fibroblast | The Progeria Research Foundation\" 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