{"id":1010,"date":"2017-02-27T21:26:38","date_gmt":"2017-02-27T21:26:38","guid":{"rendered":"https:\/\/www.progeriaresearch.org\/?page_id=1010"},"modified":"2026-03-17T10:50:58","modified_gmt":"2026-03-17T14:50:58","slug":"induced-pluripotent-stem-cells","status":"publish","type":"page","link":"https:\/\/www.progeriaresearch.org\/id\/induced-pluripotent-stem-cells\/","title":{"rendered":"Sel Punca Pluripoten yang Diinduksi"},"content":{"rendered":"

Bahasa Indonesia: [et_pb_section fb_built=\u201d1\u2033 lebar_penuh=\u201daktif\u201d dinonaktifkan_aktif=\u201dnonaktif|nonaktif|nonaktif\u201d _builder_version=\u201d4.16\u2033 lebar_batas_bawah=\u201d55px\u201d warna_batas_bawah=\u201d#29327a\u201d terkunci=\u201dnonaktif\u201d info_warna_global=\u201d{}\u201d][et_pb_fullwidth_header _builder_version=\u201d4.16\u2033 judul_font=\u201d|||||||||\u201d ukuran_font_judul=\u201d55\u2033 warna_latar_belakang=\u201d#29327a\u201d gambar_latar_belakang=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Jes.jpg\u201d posisi_latar_belakang=\u201dtengah_kiri\u201d bantalan_khusus=\u201d9vw|0px|9vw||benar|\u201d custom_padding_tablet=\u201d\u201d custom_padding_phone=\u201d|56px||\u201d custom_padding_last_edited=\u201ddi|desktop\u201d ukuran_font_judul_tablet=\u201d45px\u201d ukuran_font_judul_ponsel=\u201d40px\u201d ukuran_font_judul_terakhir_edited=\u201ddi|ponsel\u201d z_index_tablet=\u201d500\u2033 custom_css_main_element=\u201dposisi_latar belakang: tengah 18% !penting;\u201d info_warna_global=\u201d{}\u201d]<\/p>\n

Pluripoten yang Diinduksi<\/h1>\n

Sel Punca<\/h1>\n

 <\/p>\n

[\/et_pb_fullwidth_header][\/et_pb_section][et_pb_section fb_built=”1″ use_custom_gutter=”on” gutter_width=”1″ specialty=”on” padding_left_1=”35px” padding_left_2=”35px” padding_2_tablet=”|||0px” padding_2_phone=”” padding_2_last_edited=”on|desktop” module_class_1=”sidebar-secondary-nav” module_class=”handprint-bg” _builder_version=”4.16″ background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2019\/04\/blue-handprint-only.png” parallax=”on” parallax_method=”off” inner_width=”100%” inner_max_width=”100%” custom_padding=”0|0px|54px|0px|false|false” z_index_tablet=”500″ border_width_top=”10px” border_color_top=”#8fd2ed” use_custom_width=”on” width_unit=”off” custom_width_percent=”100%” global_colors_info=”{}”][et_pb_column type=”1_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_sidebar area=”et_pb_widget_area_14″ disabled_on=”on|on|off” module_class=”subpage-sidebars” _builder_version=”4.16″ animation_style=”fade” z_index_tablet=”500″ border_width_right=”5px” locked=”off” global_colors_info=”{}”][\/et_pb_sidebar][\/et_pb_column][et_pb_column type=”3_4″ specialty_columns=”3″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_row_inner custom_padding_last_edited=”on|phone” _builder_version=”4.27.5″ custom_padding=”|35px|0|0px|false|false” custom_padding_tablet=”|35px||35px||true” custom_padding_phone=”” animation_direction=”top” global_colors_info=”{}”][et_pb_column_inner saved_specialty_column_type=”3_4″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_text _builder_version=”4.27.5″ background_size=”initial” background_position=”top_left” background_repeat=”repeat” custom_padding=”||0px||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

Bank Sel & Jaringan Yayasan Penelitian Progeria<\/strong> Sel Punca Pluripoten yang Diinduksi Manusia (iPSC)<\/h4>\n
    \n
  1. iPSC Progeria <\/strong>Informasi Latar Belakang untuk Non-Ilmuwan<\/strong><\/a>\u00a0<\/strong><\/li>\n
  2. Tujuan dari <\/strong>Generasi dan Distribusi Sel Punca Pluripoten yang Diinduksi oleh Yayasan Penelitian Progeria\u00a0<\/strong>\u00a0<\/a><\/li>\n
  3. Pembuatan Sel Punca Pluripoten yang Diinduksi Sindrom Progeria Hutchinson-Gilford (iPSC)<\/strong>\u00a0<\/a><\/li>\n
  4. Kontrol Kualitas: Validasi dan Karakterisasi<\/strong>\u00a0<\/a><\/li>\n
  5. Bahan Awal Asli dari mana iPSC Dihasilkan<\/strong><\/a><\/li>\n
  6. Bergabunglah dengan Daftar Email kami untuk Pembaruan iPSC Masa Depan dan Lini Sel Baru<\/strong>\u00a0\u00a0<\/strong><\/a><\/li>\n
  7. Ada pertanyaan? Hubungi kami.<\/strong><\/a><\/li>\n
  8. Pemesanan Jalur iPSC<\/strong><\/a>\u00a0<\/strong><\/li>\n
  9. Persiapan Media Kultur iPSC HGPS dan Kontrol<\/strong>\u00a0<\/strong><\/a><\/li>\n
  10. Mempersiapkan Pelat Matrigel\u00a0<\/strong><\/a><\/li>\n
  11. Mencairkan sel hESC dan iPSC (per cryo-vial)<\/strong><\/a><\/li>\n
  12. Pemanenan dan Perawatan hESC\/iPSC<\/strong>\u00a0<\/strong><\/a><\/li>\n
  13. Melewati <\/strong><\/a>hESC dan iPSC\u00a0<\/a><\/strong><\/li>\n
  14. Membekukan hESC\/iPSC<\/a><\/strong><\/li>\n<\/ol>\n

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    1. Informasi latar belakang iPSC untuk non-ilmuwan<\/strong><\/h4>\n

    Sel punca adalah sel yang "belum matang" dan belum berkomitmen untuk menjadi satu jenis sel. Sel punca bersifat lentur karena memiliki potensi untuk berkembang menjadi berbagai jenis sel matang di dalam tubuh, seperti sel yang membentuk jantung atau pembuluh darah, serta jaringan dan organ lainnya. Pada tahun 2007, para peneliti menemukan strategi untuk menciptakan sel punca di laboratorium dengan memprogram ulang sel dewasa matang yang biasa kita kembangkan untuk tujuan penelitian.1, 2<\/sup><\/strong>\u00a0Sel punca yang dibuat secara artifisial ini disebut Sel Punca Pluripoten Terinduksi (\u201ciPSC\u201d). Bagi bidang Progeria, ini merupakan terobosan besar. Untuk pertama kalinya, para ilmuwan kini dapat membuat sel punca Progeria dan mengajukan pertanyaan tentang bagaimana sel punca berfungsi dan berkembang dalam Progeria. Sebelumnya tidak ada sumber sel punca Progeria manusia, dan oleh karena itu terdapat kekosongan informasi tentang bagaimana sel punca Progeria berfungsi dibandingkan dengan sel punca dari orang tanpa Progeria. Selain itu, para ilmuwan dapat memprogram ulang sel punca Progeria untuk menciptakan, untuk pertama kalinya, pembuluh darah Progeria yang matang, sel jantung, dan jenis sel lainnya. Hingga saat ini, tidak ada sumber sel jantung atau pembuluh darah Progeria manusia.\u00a0\u00a0Sekarang kita bisa bertanya kunci\u00a0<\/strong>pertanyaan tentang penyakit jantung yang menyebabkan kematian dini pada Progeria akibat serangan jantung dan stroke. Kita dapat membandingkan penemuan ini dengan penyakit jantung dan penuaan pada populasi umum dan menemukan lebih banyak tentang apa yang memengaruhi penuaan pada kita semua. Sudah ada beberapa penelitian luar biasa yang diterbitkan menggunakan sel induk Progeria.3-5<\/sup> \u00a0Tujuan kami di The Progeria Research Foundation adalah memfasilitasi lebih banyak penemuan dengan menggunakan alat yang sangat berharga ini. Untuk informasi dasar tentang sel punca, silakan lihat situs web pemerintah AS ini:\u00a0https:\/\/stemcells.nih.gov\/info\/basics.htm<\/a><\/p>\n

     <\/p>\n

    \n
      \n
    1. \n
        \n
      1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induksi sel punca pluripoten dari fibroblas manusia dewasa dengan faktor-faktor yang ditentukan.\u00a0Sel.\u00a0<\/em>2007;131:861-872.<\/li>\n
      2. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin, II, Thomson JA. Garis sel induk berpotensi majemuk yang diinduksi berasal dari sel somatik manusia.\u00a0Sains.\u00a0<\/em>2007;318:1917-1920.<\/li>\n
      3. Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J, 3rd, Izpisua Belmonte JC. Rekapitulasi penuaan dini dengan iPSC dari sindrom progeria Hutchinson-Gilford.\u00a0Alam.\u00a0<\/em>2011;472:221-225.<\/li>\n
      4. Misteli T. iPSC turunan HGPS untuk segala usia.\u00a0Sel Sel Induk.\u00a0<\/em>2011;8:4-6.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<\/div>\n

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        2. Tujuan dari generasi dan distribusi sel induk pluripoten terinduksi (iPSC) oleh The Progeria Research Foundation<\/strong><\/h4>\n

        Misi dari The Progeria Research Foundation adalah menemukan pengobatan dan penyembuhan untuk Sindrom Progeria Hutchinson-Gilford dan gangguan terkait penuaan. Pada tahun 2009, PRF menjalin kerja sama dengan tim ilmuwan ahli di University of Toronto, Kanada, di bawah arahan William Stanford, PhD, untuk menghasilkan iPSC Progeria berkualitas tinggi. Dr. Stanford adalah Ketua Riset Kanada dalam Biologi Sel Punca Integratif. Hingga tahun 2011, PRF terus bekerja sama dengan Dr. Stanford di University of Ottawa, Kanada, tempat ia menjabat sebagai Profesor Kedokteran Seluler dan Molekuler, Fakultas Kedokteran, dan Ilmuwan Senior di Pusat Riset Sel Punca Sprott di Ottawa Hospital Research Institute.<\/p>\n

        Tujuan kami adalah menyediakan alat yang sangat berharga ini bagi para peneliti di seluruh dunia. Alat penelitian baru ini akan digunakan untuk menghasilkan penelitian baru dan inovatif dalam Progeria, serta hubungannya dengan penyakit jantung dan penuaan.\u00a0\u00a0<\/strong><\/p>\n

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        3. Pembuatan Sel Punca Pluripoten yang Diinduksi Sindrom Progeria Hutchinson-Gilford (iPSC)<\/strong><\/h4>\n

        Sel Punca Pluripoten Terinduksi (iPSC) diperoleh dengan menggunakan transduksi retrovirus pseudotyped VSVG dari empat faktor manusia, Oct4, Sox2, Klf4, dan c-Myc ke dalam fibroblas. Koloni iPSC diperoleh dari fibroblas embrionik tikus (MEF). Prosedur yang digunakan pada dasarnya sama seperti yang dijelaskan sebelumnya tetapi tanpa menggunakan reporter EOS (Nature Protocols 4: 1828-1844, 2009).<\/p>\n

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        4. Kontrol Kualitas: Validasi dan Karakterisasi<\/strong><\/h4>\n

        Baris yang tersedia saat ini telah menjalani beberapa langkah validasi (lihat PDF yang dapat diunduh di bawah):<\/p>\n

        \n
          \n
        1. \n
            \n
          1. Pengujian Mikoplasma untuk setiap lini: Laboratorium Dr. Stanford telah melakukan analisis mikoplasma dengan PCR untuk setiap lini sel. Selain itu, setelah ekspansi dan sebelum pengiriman sel, lini sel akan diuji ulang untuk mikoplasma.<\/li>\n
          2. Pewarnaan imun untuk penanda pluripotensi Tra-1-60, Tra-1-81, dan SSEA4.<\/li>\n
          3. Pewarnaan Alkaline Phosphatase sebagai indikator pluripotensi<\/li>\n
          4. Pembentukan badan embrioid dan imunopewarnaan selanjutnya untuk penanda tiga lapisan germinal. Penanda yang diuji adalah \u03b2III-Tubulin (Ektoderm), Aktin Otot Halus (Mesoderm), dan Gata4 atau AFP (Endoderm)<\/li>\n
          5. Analisis kariotipe.<\/li>\n
          6. Pengekspresian kembali lamin A pada sel-sel yang berdiferensiasi<\/li>\n
          7. Pengujian teratoma<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n

            Validasi tambahan sedang dalam proses:<\/strong>
            Beberapa lini telah menyelesaikan uji teratoma seperti yang ditunjukkan dalam data pendukung. Untuk semua lini lainnya, uji teratoma sedang dalam proses dan statusnya akan diperbarui saat uji ini selesai.<\/p>\n<\/div>\n

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            5. Bahan awal asli dari mana sel iPS ini diturunkan<\/strong><\/h4>\n

            iPSC berasal dari garis sel fibroblas nontransformasi PRF Cell & Tissue Bank.<\/p>\n

            Metode transduksi yang digunakan untuk semua lini iPS adalah Retrovirus MKOS.<\/p>\n

            <\/p>\n

            [\/et_pb_text][et_pb_text admin_label=”Table” module_id=”original” _builder_version=”4.27.5″ custom_padding=”||0px|||” hover_enabled=”0″ z_index_tablet=”500″ global_colors_info=”{}” sticky_enabled=”0″]<\/p>\n

            \n\n\n\n\t\n\n\t\n\t\n\t\n\t\n\t\n\t\n\t\n\t\n\t
            ID Baris iPSC<\/b><\/th>Mutasi<\/b><\/th>Jenis Kelamin dan Usia Donasi<\/b><\/th>Jenis Sel Asal klik disini<\/a>.<\/b><\/th>Data pendukung<\/b><\/th>\n<\/tr>\n<\/thead>\n
            HGADFN003 iPS 1B<\/td> LMNAEkson 11,
            \ntahun 1824 C>T<\/td>            		Laki-laki 2 thn 0 bln<\/td> Fibroblas Dermal
            \nHGADFN003<\/td>            		003 iPS1B<\/a><\/td>\n<\/tr>\n
            HGADFN003iPS 1C<\/td> LMNA Ekson 11,
            \ntahun 1824 C>T<\/td>            		Laki-laki 2 thn 0 bln<\/td> Fibroblas Dermal
            \nHGADFN003<\/td>            		003 iPS1C<\/a><\/td>\n<\/tr>\n
            HGDFN003
            \niPS 1D<\/td>            		LMNA Ekson 11,
            \ntahun 1824 C>T<\/td>            		Laki-laki 2 thn 0 bln<\/td> Fibroblas Dermal
            \nHGADFN003<\/td>            		003 PS1D<\/a><\/td>\n<\/tr>\n
            HGADFN167iPS 1J<\/td> LMNA Ekson 11, 1824 C>T<\/td>Pria 8 thn 5 bln<\/td>Fibroblas Dermal HGADFN167<\/td>167 PS 1J<\/a><\/td>\n<\/tr>\n
            HGADFN167iPS 1Q<\/td> LMNA Ekson 11, 1824 C>T<\/td>Pria 8 thn 5 bln<\/td>Fibroblas Dermal HGADFN167<\/td>167 iPS1Q<\/a><\/td>\n<\/tr>\n
            HGMDFN090iPS 1B<\/td> Induk HGADFN167 (tidak terpengaruh)<\/td>Perempuan 37 thn 10 bln<\/td>Fibroblas Dermal
            \nHGMDFN090<\/td>            		090 iPS1B<\/a><\/td>\n<\/tr>\n
            HGMDFN090iPS 1C<\/td> Induk HGADFN167 (tidak terpengaruh)<\/td>Perempuan 37 thn 10 bln<\/td>Fibroblas Dermal
            \nHGMDFN090<\/td>            		090 iPS1C<\/a><\/td>\n<\/tr>\n
            HGFDFN168iPS1D2<\/td>Ayah HGADFN167 (tidak terpengaruh)<\/td>Pria 40 tahun
            \n5 bulan<\/td>            		Fibroblas Dermal HGFDFN168<\/td>168 PS1 D2<\/a><\/td>\n<\/tr>\n
            HGFDFN168iPS1P<\/td>Ayah HGADFN167 (tidak terpengaruh)<\/td>Pria 40 tahun
            \n5 bulan<\/td>            		Fibroblas Dermal
            \nHGFDFN168<\/td>            		168 iPS1P<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/h4>\n

            [\/et_pb_text][et_pb_button button_url=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2023\/05\/PRF-AVAILABLE-CELL-LINES-5-24-23.pdf” button_text=”PRF Available Cell Lines” button_alignment=”left” admin_label=”Cell lines pdf” _builder_version=”4.27.5″ background_layout=”dark” custom_margin=”||25px||false|false” hover_enabled=”0″ z_index_tablet=”500″ global_colors_info=”{}” sticky_enabled=”0″][\/et_pb_button][et_pb_text admin_label=”join” module_id=”join” _builder_version=”4.27.5″ custom_padding=”||||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

            6. Bergabunglah dengan milis kami untuk mendapatkan pembaruan iPSC dan lini sel baru di masa mendatang<\/strong><\/h4>\n

            Kami terus menghasilkan galur iPSC. Jika Anda menginginkan informasi terkini secara berkala tentang iPSC yang disimpan di PRF Cell & Tissue Bank, silakan bergabung dengan milis kami dengan mengklik\u00a0Di Sini<\/a><\/strong><\/p>\n

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            7. Ada pertanyaan?<\/strong><\/h4>\n

            <\/a>Silakan hubungi Leslie Gordon, MD, PhD, Direktur Medis, jika Anda memiliki pertanyaan atau membutuhkan sesuatu, di lgordon@progeriaresearch.org<\/a>\u00a0atau 978-535-2594<\/p>\n

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            8. Memesan lini sel iPS<\/strong><\/h4>\n

            Pada tahun 2014, PRF memberlakukan kebijakan tidak ada perubahan pada MTA kami. Ini merupakan hasil dari pengaturan kontrak selama 12 tahun dengan 70 tim peneliti yang bekerja di berbagai lembaga di 14 negara. PRF dan penasihat hukumnya telah mempertimbangkan berbagai masalah yang muncul dalam kurun waktu tersebut dan mengedit perjanjian tersebut sesuai dengan itu, sehingga menghasilkan apa yang kami rasa sebagai ketentuan yang adil dan wajar.<\/p>\n

            Untuk Lembaga Pemerintah Federal AS atau pertanyaan, silakan hubungi Wendy Norris di: alamat email: wnorris@brownhealth.org<\/a><\/a>\u00a0atau\u00a0401-274-1122x48063 cm<\/a>.<\/p>\n

            Langkah 1: Lengkapi aplikasi dan perjanjian transfer material
            <\/strong>
            <\/a>            Aplikasi dan Perjanjian untuk Lembaga Non-Pemerintah<\/a><\/p>\n

            Perjanjian Pengalihan Material untuk Lembaga Non-Pemerintah<\/a><\/p>\n

            Langkah 2: Kembalikan aplikasi yang sudah lengkap dan perjanjian transfer material ke Wendy Norris di alamat email: wnorris@brownhealth.org<\/a>Setelah disetujui, Anda akan menerima email yang mengonfirmasi pesanan Anda dan perkiraan tanggal pengiriman.<\/b>\u00a0<\/strong><\/a><\/p>\n

            Langkah 3: <\/strong>Laboratorium Dr. Stanford saat ini sedang mendistribusikan lini dalam kriovial beku. Laboratoriumnya akan mengirimkan email kepada Anda saat kultur telah dikirim, dengan informasi pengiriman dan pelacakan. Peneliti yang belum berpengalaman diarahkan untuk mendapatkan pelatihan di kursus khusus yang penting untuk pekerjaan sel induk embrionik manusia\/iPSC.<\/p>\n

            Fasilitas Sel Punca Pluripoten Manusia (dipimpin oleh Dr. Stanford) di Institut Penelitian Rumah Sakit Ottawa menawarkan pelatihan tatap muka virtual tentang dasar-dasar teknik kultur iPSC yang khusus untuk lini sel Progeria iPSC. Pilihan dan format pelatihan fleksibel tergantung pada tingkat pengalaman para ilmuwan. \u00a0Untuk informasi lebih lanjut, silakan email hpscf@ohri.ca<\/a>.<\/u><\/strong><\/p>\n

            Langkah 4: Universitas Ottawa akan menagih Anda secara langsung untuk setiap jalur iPSC ditambah biaya kurir, jika ada.<\/strong><\/p>\n

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            9. Persiapan Media Kultur Sel HGPS dan Kontrol iPS<\/strong><\/h4>\n

            iPSC dan ESC perlu diberi makan dengan mTeSR Plus dari Stem Cell Technologies (cat# 5825). Harap ikuti rekomendasi pemasok untuk penyimpanan.<\/p>\n

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            10. Mempersiapkan Pelat Matrigel<\/strong><\/h4>\n

            Catatan<\/strong>: Semua langkah yang melibatkan matrigel harus dilakukan secepat mungkin dan tetap sedingin mungkin.<\/p>\n

              \n
            1. Cairkan botol matrigel pada suhu 4\u00b0<\/strong>C. Periksa sertifikat analisis pada lot tersebut untuk mengetahui konsentrasi proteinnya.<\/li>\n
            2. Tambahkan media DMEM\/F12 dingin secukupnya ke matrigel yang telah dicairkan hingga mencapai konsentrasi akhir 5 mg\/mL.<\/li>\n
            3. Buat alikuot 1 mL dari matrigel yang telah disiapkan dari langkah 2 dalam tabung falcon 15 mL yang telah didinginkan sebelumnya.<\/li>\n
            4. Bekukan dan simpan semua alikuot pada suhu -20\u00b0<\/strong>C.<\/li>\n
            5. Untuk membuat pelat matrigel, keluarkan satu bagian matrigel (1mL) dari -20\u00b0<\/strong>C dan tambahkan 10 mL DMEM\/F12 dingin. Aduk rata hingga pelet mencair (tanpa menimbulkan gelembung, dan selalu jaga agar larutan tetap dingin).<\/li>\n
            6. Pindahkan ke tabung 50mL, lalu tambahkan 20mL DMEM\/F12 dingin (1mL alikuot matrigel diencerkan dalam 30mL DMEM\/F12), aduk rata.<\/li>\n
            7. Plat (1 mL\/sumur untuk plat 6 sumur, 0,5 mL\/sumur untuk plat 12 sumur, 0,25 mL\/sumur untuk plat 24 sumur). Pastikan larutan menutupi seluruh permukaan dengan mengocok plat secara perlahan. Jangan bekukan kembali matrigel yang tersisa.<\/li>\n
            8. Jika akan menggunakan plat segera, biarkan plat pada suhu ruangan selama 1 jam (atau 30 menit pada suhu 37\u00b0<\/strong>C), amati matrigel di bawah mikroskop. Matrigel harus tersebar dengan baik dan tidak "menggumpal".<\/li>\n
            9. Jika menggunakan piring di lain waktu, bungkus tepi piring dengan parafilm dan simpan pada suhu 4\u00b0<\/strong>C hingga 2 minggu.<\/li>\n<\/ol>\n

              Matrigel \u2013 BD\/Fisher, cat#CB-40230<\/p>\n

              DMEM\/F12 \u2013 Teknologi Kehidupan, cat#11330-057<\/p>\n

              Dr. William Stanford-2022<\/p>\n

              [\/et_pb_text][et_pb_text admin_label=”inactivating” module_id=”inactivating” _builder_version=”4.27.5″ custom_padding=”||||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

              11. <\/strong>Mencairkan sel ES atau iPS (per cryo-vial)<\/strong><\/h4>\n
                \n
              1. Keluarkan pelat matrigel dari suhu 4\u00b0C dan hangatkan pada suhu ruangan selama satu jam, atau buat pelat matrigel baru (lihat protokol persiapan pelat matrigel).<\/li>\n
              2. Hangatkan 4mL mTeSR Plus dalam tabung falcon 15mL.<\/li>\n
              3. Keluarkan sel dari tangki nitrogen cair dan aduk dalam air bersuhu 37\u00b0C hingga hanya tersisa bongkahan es kecil. Botol akan mencair dalam 1-2 menit. Langkah ini harus dilakukan dengan cepat.<\/li>\n
              4. Tabung sel etanol dan tabung media falcon lalu tempatkan dalam sungkup.<\/li>\n
              5. Gunakan 1mL ujung mulut lebar perlahan <\/u>tambahkan sel ke 4mL media yang telah dihangatkan sebelumnya (hindari pencampuran suspensi sel).<\/li>\n
              6. Putar pada 130 rcf selama 5 menit.<\/li>\n
              7. Buang cairan supernatan.<\/li>\n
              8. Tambahkan 2mL media PSC, dan dengan ujung mulut lebar hancurkan gumpalan dengan lembut<\/u>. Pindahkan media ke satu sumur dari pelat 6 sumur, tambahkan 2uL inhibitor ROCK (Y27632, konsentrasi akhir 10uM). Masukkan pelat ke dalam satu sumur berlapis matrigel (dari pelat 6 sumur).<\/li>\n
              9. Goyangkan sel dengan lembut untuk mendistribusikan sel secara merata, dan tempatkan dalam inkubator hipoksia (5%O2<\/sub>, 10% Perusahaan2<\/sub>). Hindari gangguan pada pelat selama 24 jam setelah penyemaian.CATATAN: <\/strong>Sangat penting untuk menghindari pemecahan gumpalan yang berlebihan atau pemipetan yang agresif. Hal ini dapat mengurangi tingkat kelangsungan hidup secara signifikan. Sel-sel harus tetap dalam potongan-potongan yang besarnya 100-300 sel pada saat penyemaian. Meskipun berhati-hati, cobalah bekerja dengan cepat setelah sel-sel dicairkan untuk meminimalkan waktu kontak dengan krioprotektan.<\/li>\n
              10. Buang media setelah 24 jam dan tambahkan 2 mL media PSC (untuk 6 sumur, 1 mL untuk 12 sumur, dan 0,5 mL untuk pelat 24 sumur). Lihat Pemanenan dan Perawatan untuk protokol hESC\/iPSC.<\/li>\n<\/ol>\n

                media PSC<\/strong><\/p>\n

                mTeSR Plus dari Stem Cell Technologies (cat# 5825). Harap ikuti rekomendasi dari pemasok untuk penyimpanan.<\/p>\n

                Apa itu ROCK Inhibitor Y27632?<\/strong><\/p>\n

                Inhibitor ROCK Y27632 merupakan inhibitor selektif dari Rho associated kinase p160 ROCK. Pengobatan dengan Inhibitor ROCK Y27632 mencegah apoptosis yang diinduksi oleh disosiasi pada sel punca embrionik manusia (hESC) dan sel punca pluripoten yang diinduksi manusia (iPSC), meningkatkan tingkat kelangsungan hidup dan mempertahankan pluripotensi selama subkultur dan pencairan hESC dan hiPSC. Inhibitor ROCK Y27632 juga telah terbukti meningkatkan tingkat kelangsungan hidup sel punca selama kriopreservasi. Perhatikan bahwa alikuot inhibitor Rock sensitif terhadap cahaya dan siklus pembekuan-pencairan yang berulang. Pastikan untuk menggunakan alikuot ini dalam masa simpan yang direkomendasikan oleh pemasok.<\/p>\n

                Dr. William Stanford-2022<\/p>\n

                [\/et_pb_text][et_pb_text admin_label=”thawing” module_id=”thawing” _builder_version=”4.27.5″ custom_padding=”||||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

                12. <\/strong>Pemanenan dan Perawatan hESC\/iPSC<\/strong><\/h4>\n
                  \n
                1. Sehari setelah sel dicairkan, amati sel-sel tersebut di bawah mikroskop untuk menentukan tingkat kelangsungan hidup. CATATAN: adalah normal untuk mengamati sejumlah besar sel yang tidak menempel. Selama beberapa sel menempel, koloni dapat muncul dari sel-sel tersebut dalam waktu 3 \u2013 7 hari.<\/li>\n
                2. Keluarkan media dari sumur dan pipet 2 mL (untuk 6 sumur, 1 mL untuk 12 sumur dan 0,5 mL untuk pelat 24 sumur) media PSC segar dan hangat per sumur. Kembalikan pelat ke inkubator.<\/li>\n
                3. Sel diberi makan hingga konfluen 60-70% (ikuti rekomendasi pemasok).<\/li>\n
                4. Pada hari ke-2, sel harus diamati dan dibersihkan dari sel-sel berdiferensiasi yang mungkin tumbuh.<\/li>\n
                5. Untuk membersihkan sel, gunakan penutup pengambil dan kikis sel-sel yang telah berdiferensiasi dengan ujung pipet.<\/li>\n
                6. Setelah sel dibersihkan, ganti media seperti yang dilakukan pada langkah di atas.<\/li>\n<\/ol>\n

                  (Lihat Pembekuan hESC\/iPSC atau Melewatkan hESC\/iPSC)<\/p>\n

                  CATATAN:<\/strong><\/p>\n

                  Media PSC terbukti lebih efisien dalam lingkungan hipoksia. Kami juga mengamati bahwa diferensiasi yang terjadi lebih sedikit ketika sel ditumbuhkan dalam inkubator hipoksia dibandingkan dengan inkubator normoksik. Terakhir, sel yang disemai memiliki tingkat kelangsungan hidup yang lebih baik saat menggunakan inkubator hipoksia.<\/p>\n

                  Normoksik: 37\u00b0<\/strong>C, 21% O2<\/sub>, 5% Perusahaan2<\/sub><\/p>\n

                  Hipoksia: 37\u00b0<\/strong>C, 5%O2<\/sub>, 10% Perusahaan2<\/sub><\/p>\n

                  Dr. William Stanford-2022<\/p>\n

                  [\/et_pb_text][et_pb_text admin_label=”routine” module_id=”routine” _builder_version=”4.27.5″ custom_padding=”||||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

                  13. <\/strong>Lulus hESC\/iPSC<\/strong><\/h4>\n
                    \n
                  1. Tambahkan 2 mL media PSC ke dalam pelat berlapis matrigel 6 sumur dan sisihkan.<\/li>\n
                  2. Ambil pelat yang akan disalurkan dan keluarkan media dari sumur lalu cuci sekali dengan 1 mL PBS(-\/-).<\/li>\n
                  3. Tambahkan 1 mL larutan EDTA ke dalam sumur dan biarkan selama 3-4 menit pada suhu ruangan. Jangan menggerakkan pelat karena sel-sel dapat mulai terlepas.<\/li>\n
                  4. Buang larutan EDTA dan tambahkan 1 mL media PSC. Jangan biarkan EDTA pada sel selama lebih dari 4 menit karena ini akan menyebabkan sel terangkat.<\/li>\n
                  5. Kikis sel menggunakan pengikis sel dan bagi sel di antara 6 sumuran pelat yang berisi media PSC. Hindari pemecahan berlebihan pada potongan koloni, dan cobalah untuk berhati-hati saat mengikis. Cobalah untuk menjaga sel dalam potongan besar. Gunakan ujung pipet bermulut lebar untuk memecah gumpalan jika perlu. Pemecahan sel yang berlebihan dapat menyebabkan kematian sel atau diferensiasi spontan yang berlebihan setelah melewati lintasan.<\/li>\n
                  6. Inkubasi pada suhu 37\u00b0<\/strong>C setelah mendistribusikan sel secara merata di setiap sumur (pengocokan berbentuk 8 angka atau L). Hindari gangguan pada pelat selama 24 jam setelah pengoperan.<\/li>\n<\/ol>\n

                    CATATAN<\/strong>: Setelah sel dikikis, Anda ingin memindahkannya ke pelat baru sesegera mungkin karena sel akan menempel kembali dengan cepat (dalam waktu 5 menit).<\/p>\n

                    Jika EDTA dibiarkan pada sel selama lebih dari 4 menit, sel dapat mulai terlepas. Jika ini terjadi, cukup kumpulkan sel dalam falcon 15mL dengan 4mL media PSC. Putar sel pada 130 rcf selama 5 menit. Suspensikan kembali pelet dengan 1mL media dan bagi secara merata di antara pelat berlapis matrigel 6 sumur (160uL per sumur).<\/p>\n

                    Larutan EDTA: Tambahkan 500uL EDTA 0,5M (pH 8,0) ke dalam 500mL DPBS (-\/-). Tambahkan 0,9g NaCl. Saring larutan untuk mensterilkannya dan simpan pada suhu 4\u00b0C hingga 6 bulan. <\/strong><\/p>\n

                    Dari makalah:<\/p>\n

                    Pengalihan dan perluasan koloni sel induk pluripoten manusia melalui disosiasi bebas enzim dalam kondisi kultur yang ditentukan secara kimia<\/strong><\/p>\n

                    Bir Jeanette<\/a>,1<\/sup> Daniel R. Gulbranson<\/a>,2,3<\/sup> Nicole George<\/a>,4<\/sup>Lauren I. Siniscalchi<\/a>,1<\/sup> Jeffrey Jones<\/a>,4,5<\/sup> Penulis: James A. Thomson<\/a>,2,3,6<\/sup> Dan Guokai Chen<\/a>1,2<\/sup><\/p>\n

                    [\/et_pb_text][et_pb_text admin_label=”culturing” module_id=”culturing” _builder_version=”4.27.5″ custom_padding=”||||false|false” z_index_tablet=”500″ global_colors_info=”{}”]<\/p>\n

                    14. <\/strong>Membekukan hESC\/iPSC <\/strong><\/h4>\n
                      \n
                    1. Nyalakan Bio-Cool (Controlled Rate Freezer) dan atur suhu ke -7\u00b0C.<\/li>\n
                    2. Keluarkan sel dari inkubator dan amati konfluensi dan morfologi di bawah mikroskop.<\/li>\n
                    3. Bila sumurnya konfluen 70%, buang media lama dan cuci sekali dengan PBS(-\/-), kemudian tambahkan 1 mL larutan EDTA (lihat melewatkan dengan larutan EDTA) per sumur.<\/li>\n
                    4. Inkubasi pada suhu ruangan selama 3-4 menit.<\/li>\n
                    5. Aspirasikan larutan EDTA dan tambahkan 1 mL media mFreSR dingin (cat#05855, Stem Cell Technologies).<\/li>\n
                    6. Gunakan pengikis sel untuk mengangkat sel dengan hati-hati. Sebisa mungkin, buat sel dalam potongan besar dan hindari pemipetan ke atas dan ke bawah.<\/li>\n
                    7. Pindahkan sel\/mFreSR ke tabung krio menggunakan ujung bermulut lebar. Simpan botol dalam es hingga siap untuk langkah 8.<\/li>\n
                    8. Tempatkan tabung dalam Bio-Cool dan inkubasi selama 10 menit.<\/li>\n
                    9. Dapatkan nitrogen cair.<\/li>\n
                    10. Setelah 10 menit, taburkan sel dengan mencelupkan spatula ke dalam nitrogen cair dan menyentuh sisi botol kriogenik selama kurang lebih 10-30 detik atau sampai Anda melihat kristal terbentuk pada sisi botol kriogenik.<\/li>\n
                    11. Mulailah program 1 dengan menekan tombol \u201cPROG\u201d dan jalankan program dengan menekan tombol tersebut lagi dan Anda akan melihat laju 0,5\u00b0C\/menit. , lalu tekan \u201cRUN\u201d.<\/li>\n
                    12. Setelah suhu mencapai -65\u00b0C, tabung kriogenik dapat dipindahkan dan disimpan dalam nitrogen cair.<\/li>\n<\/ol>\n

                      Alternatif<\/p>\n

                      Pilihan lainnya adalah dengan menempatkan tabung krio dalam wadah pembeku (Biocision-CoolCell) dan menyimpannya pada suhu -80\u00b0C semalaman. Pindahkan tabung krio ke nitrogen cair (fase cair atau uap) pada hari berikutnya.<\/p>\n

                      Dr. William Stanford-2022<\/p>\n

                      Bahasa Indonesia: [\/et_pb_text][\/et_pb_column_inner][\/et_pb_row_inner][\/et_pb_column][\/et_pb_section][et_pb_section fb_built=\u201d1\u2033 kelas_modul=\u201dfooter\u201d _builder_version=\u201d4.21.0\u2033 warna_latar_belakang=\u201d#29327a\u201d margin_khusus=\u201d-2px|||||\u201d custom_padding=\u201d0|0px|0|0px|false|false\u201d z_index_tablet=\u201d500\u2033 border_width_top=\u201d12px\u201d border_color_top=\u201d#00b2e2\u2033 global_module=\u201d133\u2033 terkunci=\u201dmati\u201d global_colors_info=\u201d{}\u201d][et_pb_row struktur_kolom=\u201d1_4,1_4,1_2\u2033 buat_sama=\u201daktif\u201d kelas_modul=\u201d et_pb_row_lebar_penuh\u201d _builder_version=\u201d4.16\u2033 lebar=\u201d89%\u201d lebar_tablet=\u201d80%\u201d lebar_ponsel=\u201d\u201d lebar_terakhir_diedit=\u201daktif|desktop\u201d lebar_maks=\u201d89%\u201d lebar_tablet=\u201d80%\u201d lebar_ponsel_maks=\u201d\u201d max_width_last_edited=\u201dpada|desktop\u201d z_index_tablet=\u201d500\u2033 buat_lebar_penuh=\u201daktif\u201d unit_lebar=\u201dnonaktif\u201d persentase_lebar_khusus=\u201d100%\u201d info_warna_global=\u201d{}\u201d][et_pb_column jenis=\u201d1_4\u2033 _builder_version=\u201d4.16\u2033 bantalan_khusus=\u201d|||\u201d global_colors_info=\u201d{}\u201d custom_padding__hover=\u201d|||\u201d][et_pb_cta button_url=\u201dhttps:\/\/lp.constantcontactpages.com\/sl\/88gWWwz\u201d button_text=\u201dDaftar Sekarang\u201d admin_label=\u201dDaftar untuk Pembaruan\u201d module_class=\u201dsign-btn\u201d _builder_version=\u201d4.27.4\u2033 header_font_size=\u201d25px\u201d background_color=\u201d#29327a\u201d animation_style=\u201dslide\u201d animation_direction=\u201dleft\u201d animation_intensity_slide=\u201d25%\u201d link_option_url=\u201dhttps:\/\/lp.constantcontactpages.com\/sl\/88gWWwz\u201d header_font_size_tablet=\u201d\u201d header_font_size_phone=\u201d30px\u201d header_font_size_last_edited=\u201don|desktop\u201d z_index_tablet=\u201d500\u2033 border_radii=\u201daktif|25px|25px|25px|25px\u201d global_colors_info=\u201d{}\u201d button_bg_color__hover_enabled=\u201daktif\u201d button_bg_color__hover=\u201d#8fd2ed\u201d button_border_color__hover_enabled=\u201daktif\u201d]<\/p>\n

                      Mendaftar<\/h2>\n

                      untuk Kami<\/h2>\n

                      Pembaruan!<\/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=\u201dDonasi Sekarang\u201d admin_label=\u201dBersama-sama, kita akan menemukan obatnya!\u201d modul_kelas=\u201dtanda-btn\u201d _builder_version=\u201d4.16\u2033 ukuran_font_header=\u201d25px\u201d warna_latar belakang=\u201d#29327a\u201d gaya_animasi=\u201dslide\u201d arah_animasi=\u201dkiri\u201d intensitas_animasi_slide=\u201d25%\u201d ukuran_font_header_tablet=\u201d\u201d ukuran_font_header_phone=\u201d30px\u201d ukuran_font_header_last_edited=\u201dpada|desktop\u201d ukuran_font_body_tablet=\u201d\u201d ukuran_font_body_phone=\u201d\u201d ukuran_font_body_last_edited=\u201dpada|desktop\u201d z_index_tablet=\u201d500\u2033 radius_batas=\u201dpada|25px|25px|25px|25px\u201d info_warna_global=\u201d{}\u201d warna_latar_belakang_tombol__hover_enabled=\u201dpada\u201d tombol_warna_latar_belakang_hover=\u201d#8fd2ed\u201d tombol_warna_batas_belakang_hover_diaktifkan=\u201daktif\u201d]<\/p>\n

                      Bersama-sama, kita<\/h2>\n

                      AKAN<\/em><\/h2>\n

                      temukan obatnya!<\/h2>\n

                      [\/et_pb_cta][\/et_pb_column][et_pb_column type=”1_2″ _builder_version=”4.16″ custom_padding=”|||” global_colors_info=”{}” custom_padding__hover=”|||”][et_pb_image src=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2026\/03\/2026-footer-image-copy.png” title_text=”2026 footer image copy” _builder_version=”4.27.5″ _module_preset=”default” custom_margin=”35px||||false|false” global_colors_info=”{}”][\/et_pb_image][\/et_pb_column][\/et_pb_row][\/et_pb_section]<\/p>","protected":false},"excerpt":{"rendered":"

                      Bahasa Indonesia: [et_pb_section fb_built=\u201d1\u2033 lebar_penuh=\u201daktif\u201d dinonaktifkan_aktif=\u201dnonaktif|nonaktif|nonaktif\u201d _builder_version=\u201d4.16\u2033 lebar_batas_bawah=\u201d55px\u201d warna_batas_bawah=\u201d#29327a\u201d terkunci=\u201dnonaktif\u201d info_warna_global=\u201d{}\u201d][et_pb_fullwidth_header _builder_version=\u201d4.16\u2033 judul_font=\u201d|||||||||\u201d ukuran_font_judul=\u201d55\u2033 warna_latar_belakang=\u201d#29327a\u201d gambar_latar_belakang=\u201dhttps:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Jes.jpg\u201d posisi_latar_belakang=\u201dtengah_kiri\u201d bantalan_khusus=\u201d9vw|0px|9vw||benar|\u201d custom_padding_tablet=\u201d\u201d custom_padding_phone=\u201d|56px||\u201d custom_padding_last_edited=\u201ddi|desktop\u201d title_font_size_tablet=\u201d45px\u201d title_font_size_phone=\u201d40px\u201d title_font_size_last_edited=\u201ddi|ponsel\u201d z_index_tablet=\u201d500\u2033 custom_css_main_element=\u201dposisi-latar belakang: tengah 18% !penting;\u201d global_colors_info=\u201d{}\u201d] Sel Punca Pluripoten yang Diinduksi [\/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 metode_paralaks=\u201dmati\u201d lebar_dalam=\u201d100%\u201d lebar_maks_dalam=\u201d100%\u201d bantalan_khusus=\u201d0|0px|54px|0px|salah|salah\u201d z_index_tablet=\u201d500\u2033 lebar_batas_atas=\u201d10px\u201d warna_batas_atas=\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=\"Induced Pluripotent Stem Cells\" font_container=\"tag:h1|text_align:center\" use_theme_fonts=\"yes\"][vc_column_text]\r\n\r\n\r\n\r\n
                      \"\"<\/td>\r\n<\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\nThe Progeria Research Foundation Cell & Tissue Bank\r\n<\/strong>Human Induced Pluripotent Stem Cells (iPSC)\r\n
                        \r\n \t
                      1. Progeria iPSCs <\/strong>Background Information for the Non-scientist<\/strong><\/a>\u00a0<\/strong><\/li>\r\n \t
                      2. Purpose of <\/strong>Induced Pluripotent Stem Cell Generation and Distribution by The Progeria Research Foundation\u00a0<\/strong>\u00a0<\/a><\/li>\r\n \t
                      3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs)<\/strong>\u00a0<\/a><\/li>\r\n \t
                      4. Quality Control: Validation and Characterization<\/strong>\u00a0<\/a><\/li>\r\n \t
                      5. Original Starting Material from which iPSCs Were Derived<\/strong><\/a><\/li>\r\n \t
                      6. Join our Email List for Future iPSC Updates and New Cell Lines<\/strong>\u00a0\u00a0<\/strong><\/a><\/li>\r\n \t
                      7. Questions?\u00a0 Contact us.<\/strong><\/a><\/li>\r\n \t
                      8. Ordering iPSC Lines<\/strong><\/a>\u00a0<\/strong><\/li>\r\n \t
                      9. HGPS and Control iPSC Culture Media Preparation<\/strong>\u00a0<\/strong><\/a><\/li>\r\n \t
                      10. Preparing Madrigal Plates\u00a0<\/strong><\/a><\/li>\r\n \t
                      11. Thawing hESCs and iPSCs<\/strong><\/a><\/li>\r\n \t
                      12. Harvesting and Caring for hESCs and iPSCs<\/strong>\u00a0<\/strong><\/a><\/li>\r\n \t
                      13. Passaging with EDTA solution<\/strong><\/a><\/li>\r\n \t
                      14. Freezing hESCs and iPSCs<\/a><\/strong><\/li>\r\n<\/ol>\r\n\r\n
                        \r\n\r\n                        <\/a>1. iPSC Background information for the non-scientist<\/strong>\r\nStem cells are \u201cimmature\u201d cells that have not yet committed to becoming any one cell type.\u00a0 They are pliable because they have the potential to develop into many different types of mature cells in the body, such as cells that make up the heart or blood vessels, and other tissues and organs.\u00a0 In 2007, researchers discovered a strategy for creating stem cells in the laboratory by reprogramming mature adult cells that we commonly grow for research purposes.1, 2<\/sup><\/strong> . These artificially created stem cells are called Induced Pluripotent Stem Cells (\u201ciPSCs\u201d). For the field of Progeria, this is a huge breakthrough.\u00a0 For the first time, scientists can now make Progeria stem cells and ask questions about how stem cells function and develop in Progeria.\u00a0 Previously there was no source of human Progeria stem cells, and there was therefore a void of information about how Progeria stem cells function compared with stem cells from people without Progeria.\u00a0 In addition, scientists can re-program the Progeria stem cells to create, for the first time, mature Progeria blood vessels, heart cells, and other cell types.\u00a0 Until now, there was no source of human Progeria heart or blood vessel cells.\u00a0 We can now ask key <\/strong>questions about the heart disease that leads to early death in Progeria from heart attacks and strokes. We can compare these discoveries with the heart disease and aging in the general population and discover more about what influences aging in all of us.\u00a0 Already there have been several excellent studies published using Progeria stem cells.3-5<\/sup> \u00a0Our goal at The Progeria Research Foundation is to facilitate many more discoveries using this invaluable tool.\u00a0 For a primer on stem cells, please see this US government website:\u00a0https:\/\/stemcells.nih.gov\/info\/basics.htm<\/a>\r\n
                        \r\n
                          \r\n \t
                        1. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. <\/em>2007;131:861-872.<\/li>\r\n \t
                        2. Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin, II, Thomson JA. Induced pluripotent stem cell lines derived from human somatic cells. Science. <\/em>2007;318:1917-1920.<\/li>\r\n \t
                        3. Liu GH, Barkho BZ, Ruiz S, Diep D, Qu J, Yang SL, Panopoulos AD, Suzuki K, Kurian L, Walsh C, Thompson J, Boue S, Fung HL, Sancho-Martinez I, Zhang K, Yates J, 3rd, Izpisua Belmonte JC. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome. Nature. <\/em>2011;472:221-225.<\/li>\r\n \t
                        4. Misteli T. HGPS-derived iPSCs for the ages. Cell Stem Cell. <\/em>2011;8:4-6.<\/li>\r\n<\/ol>\r\n<\/div>\r\n<\/a>2.\u00a0Purpose of induced pluripotent stem cell (iPSC) generation and distribution by The Progeria Research Foundation<\/strong>\r\nThe mission of The Progeria Research Foundation is to discover treatments and the cure for Hutchinson-Gilford Progeria Syndrome and its aging-related disorders. In 2009, PRF entered into a collaboration with an expert team of scientists at the University of Toronto, Canada, under the direction of William Stanford, PhD, to generate high quality Progeria iPSCs. Dr. Stanford is the Canada Research Chair in Integrative Stem Cell Biology. As of 2011, PRF continues to collaborate with Dr. Stanford at the University of Ottawa, Canada where he is Professor of Cellular and Molecular Medicine, Faculty of Medicine, and Senior Scientist at Ottawa Hospital Research Institute's Sprott Centre for Stem Cell Research.<\/a>\r\n\r\nOur goal is to provide this invaluable tool to researchers throughout the world.\u00a0 This new research tool will be used to generate new and innovative research in Progeria, as well as its relationship to heart disease and aging. \u00a0<\/strong>\r\n\r\n3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs)<\/strong><\/a>\r\nInduced-Pluripotent Stem Cells (iPSCs) were derived using VSVG-pseudotyped retroviral transduction of four human factors, Oct4, Sox2, Klf4, and c-Myc into fibroblasts. \u00a0iPSC colonies were derived on mouse-embryonic fibroblasts (MEFs). The procedure used was essentially as previously described but without the use of the EOS reporter (Nature Protocols 4: 1828-1844, 2009). \u00a0<\/strong>\r\n\r\n4. Quality Control: Validation and Characterization<\/strong>\r\nThe lines that are currently available have undergone several validation steps (see downloadable PDFs below):\r\n
                          \r\n
                            \r\n \t
                          1. Mycoplasma Testing for each line: Dr. Stanford\u2019s lab has performed mycoplasmaanalysis by PCR for each cell line.\u00a0 In addition, after expansion and prior to shipping cells, the lines will be retested for mycoplasma.<\/li>\r\n \t
                          2. Immunostaining for pluripotency markers Tra-1-60, Tra-1-81, and SSEA4.<\/li>\r\n \t
                          3. Alkaline Phosphatase Staining as an indicator of pluripotency<\/li>\r\n \t
                          4. Embryoid body formation and subsequent immunostaining for markers of the three germ-layers. Markers tested were \u03b2III-Tubulin (Ectoderm), Smooth-Muscle Actin (Mesoderm), and Gata4 or AFP (Endoderm)<\/li>\r\n \t
                          5. Karyotype analysis.<\/li>\r\n \t
                          6. Re-expression of lamin A in differentiated cells<\/li>\r\n \t
                          7. Teratoma assays<\/li>\r\n<\/ol>\r\n<\/div>\r\n<\/a>\r\nAdditional validation in process:<\/strong>\r\nSome lines have completed teratoma assays as shown in supporting data. For all other lines, teratoma assays are in process and status will be updated as these assays are completed.\r\n\r\n5.\u00a0\u00a0 Original starting material from which these iPS cells were derived<\/strong>\r\niPSCs were derived from PRF Cell & Tissue Bank non-transformed fibroblast cell lines.\r\n\r\nThe transduction method used for all iPS lines was Retrovirus MKOS.\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n
                            \r\n
                            iPSC Line<\/strong> ID<\/strong>\r\n<\/strong><\/div><\/td>\r\n
                            		\r\n
                            Mutation<\/strong>\r\n<\/strong><\/div><\/td>\r\n
                            		\r\n
                            Gender<\/strong> and Donation<\/strong> Age\r\n<\/strong><\/div><\/td>\r\n
                            		\r\n\r\n
                            Supporting Data\u00a0\"\"<\/strong><\/div><\/td>\r\n<\/tr>\r\n
                            \r\n
                            HGADFN003 iPS 1B<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA<\/em>Exon 11,\r\n1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 2yr 0mo<\/div><\/td>\r\n
                            		\r\n
                            \u00a0<\/strong>Dermal Fibroblasts\r\nHGADFN003<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGADFN003 iPS 1C<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA<\/em> Exon 11,\r\n1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 2yr 0mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGADFN003<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGDFN003\r\niPS 1D<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA <\/em>Exon 11,\r\n1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 2yr 0mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGADFN003<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGADFN167 iPS 1J<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA <\/em>Exon 11, 1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 8yr 5mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts HGADFN167<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGADFN167 iPS 1Q<\/div><\/td>\r\n
                            		\r\n
                            \u00a0LMNA<\/em> Exon 11, 1824 C>T<\/div><\/td>\r\n
                            		\r\n
                            Male 8yr 5mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts HGADFN167<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGMDFN090 iPS 1B<\/div><\/td>\r\n
                            		\r\n
                            \u00a0<\/em>Mother of HGADFN167 (unaffected)<\/div><\/td>\r\n
                            		\r\n
                            Female 37yr 10mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGMDFN090<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGMDFN090 iPS 1C<\/div><\/td>\r\n
                            		\r\n
                            \u00a0<\/em>Mother of HGADFN167 (unaffected)<\/div><\/td>\r\n
                            		\r\n
                            Female 37yr 10mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGMDFN090<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGFDFN168 iPS1 D2<\/div><\/td>\r\n
                            		\r\n
                            \u00a0Father of HGADFN167 (unaffected)<\/div><\/td>\r\n
                            		\r\n
                            Male 40yr\r\n5mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts HGFDFN168<\/div><\/td>\r\n
                            		\r\n
                            \r\n
                            HGFDFN168 iPS1P<\/div><\/td>\r\n
                            		\r\n
                            Father of HGADFN167 (unaffected)<\/div><\/td>\r\n
                            		\r\n
                            Male 40yr<\/div>\r\n
                            5mo<\/div><\/td>\r\n
                            		\r\n
                            Dermal Fibroblasts\r\nHGFDFN168<\/div><\/td>\r\n
                            		<\/a>\r\n
                            168 iPS1P<\/a><\/div><\/td>\r\n<\/tr>\r\n<\/tbody>\r\n<\/table>\r\n
                            <\/div>\r\n\"\"<\/a>\u00a0PRF AVAILABLE CELL LINES<\/a><\/strong>\u00a0<\/strong>\r\n\r\n6.\u00a0Join our email list for future iPSC updates and new cell lines<\/strong>\r\nWe are continuing to generate iPSC lines.\u00a0 If you would like periodic updates on iPSCs held in the PRF Cell & Tissue Bank,\u00a0please join our emailing list by clicking here<\/a><\/strong>\r\n\r\n7.\u00a0Questions?<\/strong><\/a>\r\nPlease contact Leslie Gordon, MD, PhD, Medical Director, with any questions or needs, at lgordon@progeriaresearch.org<\/a> or 978-535-2594\r\n<\/strong>\r\n\r\n8.\u00a0 Ordering iPS cell lines<\/strong>\r\n\r\nIn 2014, PRF instituted a policy of no changes to our MTA. This is the result of 12 years of contractual arrangements with 70 research teams working at institutions in 14 countries. PRF and its counsel have taken into consideration the issues that have arisen in that time period and edited the agreement accordingly, resulting in what we feel are fair and reasonable terms.\r\n\r\nFor U.S. Federal Government Institutions or questions, please contact<\/span>\u00a0<\/span>Wendy Norris at:\u00a0<\/span>wnorris@lifespan.org<\/a><\/span>\u00a0or\u00a0401-274-1122 x 48063<\/a>.\r\n\r\nStep 1: Complete an application and material transfer agreement\r\n<\/strong>\r\n<\/a>\u00a0 Application and Agreement for Non-government Institutions<\/a>\r\n\r\n\u00a0Material Transfer Agreement for Non-government Institutions*<\/a>\r\n\r\nStep 2: Return the completed application and material transfer agreement to PRF at info@progeriaresearch.org<\/a>.\u00a0 Once approved, you will receive an email confirming your order and anticipated shipping date.\u00a0<\/strong><\/a>\r\n\r\nStep 3: <\/strong>Dr. Stanford\u2019s laboratory is currently distributing lines\u00a0in frozen cryovials.\u00a0 His laboratory will email you when the culture has been shipped, with shipping and tracking information. Inexperienced researchers are directed to obtain training at specialized courses essential to human embryonic stem cell\/iPSCs work.<\/a>\r\n\r\nStep 4: The University of Ottawa will charge $84.00 per iPSC line plus courier costs, if any, and will send you a bill directly.<\/strong>\r\n\r\n9.\u00a0 HGPS and Control iPS Cell Culture Media Preparation<\/strong>\r\niPSC and ESCs need to be fed with either TeSR-E8 from Stem Cell\u00a0Technologies (cat# 05990) or Essential 8 medium from ThermoFisher Scientific (cat#A1517001). Please follow the supplier's recommendations for storage.\r\n\r\n10.Preparing Madrigal Plates<\/strong>\r\n\r\nNote<\/strong>: All steps involving matrigel should be done as quickly as possible and stay as cold as possible.\r\n
                              \r\n \t
                            1. Thaw matrigel bottle (10ml) at 4C.<\/li>\r\n \t
                            2. Take matrigel bottle and add 10ml of cold DMEM\/F12 media.<\/li>\r\n \t
                            3. Aliquot 1ml of matrigel in pre-chilled 15ml falcon tubes.<\/li>\r\n \t
                            4. Freeze all aliquots at -20C<\/li>\r\n \t
                            5. To make matrigel plate, remove one aliquot of matrigel (1ml) from -20C and add 18ml of cold DMEM\/F12. Let pellet thaw and once thawed mix well (without creating bubbles) and plate (1ml\/well for 6 well plate, 0.5ml\/well for 12 well plate, 0.25ml\/well for 24 well plate). These plates are labelled as MG1.<\/li>\r\n \t
                            6. If using plate immediately, let plate sit at room temperature for 1 hour, observe matrigel under the microscope. Matrigel should be well dispersed and not \u201cclumpy\u201d.<\/li>\r\n \t
                            7. Once 1 hour is passed transfer matrigel to a second plate (this plate is labelled MG2). If using right away, incubate at room temperature for 1 hour.<\/li>\r\n \t
                            8. If using plates at another time, wrap edge of plate with paraffin and store at 4 degrees.<\/li>\r\n<\/ol>\r\n<\/a>\r\nMatrigel \u2013 BD\/Fisher, cat#CB-40230\r\nDMEM\/F12 \u2013 Life Technologies, cat#11330-057\r\n\r\nDr. William Stanford-2018\r\n\r\n11. Thawing hESCs and iPSCs<\/strong>\r\n\r\nThawing ES or iPS cells (per cryo-vial)<\/u><\/strong>\r\n
                                \r\n \t
                              1. Take matrigel plate out of 4C and warm at room temperature for one hour or make fresh matrigel plate (see preparing matrigel plate protocol<\/strong>).<\/li>\r\n \t
                              2. Warm 4ml of PSC media (pluripotent stem cell media) in a 15ml falcon tube.<\/li>\r\n \t
                              3. Remove cells from freezer and swirl in 37 degree water bath until only a small ice chunk is left. This step must be done quickly.<\/li>\r\n \t
                              4. Ethanol cell tube and falcon media tube and place in the hood.<\/li>\r\n \t
                              5. Use a 1ml wide mouth tip to slowly add cells to 4ml of prewarmed media<\/li>\r\n \t
                              6. Spin at 850rpm for 5min<\/li>\r\n \t
                              7. Remove supernatant<\/li>\r\n \t
                              8. Add 2ml of PSC media and with a\u00a0wide mouth tip break up clumps. Transfer media to one well of a 6 well plate add\u00a02ul of\u00a0ROCK inhibitor (Y27632, final concentration of 10uM)\u200b.<\/li>\r\n \t
                              9. Remove media after 24 hours and add 2ml of PSC media (for a 6 well, 1ml for 12 well and 0.5ml for 24 well plate). See Harvesting and Caring for hESC\/iPSC <\/strong>protocol<\/li>\r\n<\/ol>\r\nPSC media\r\n<\/u><\/strong>TeSR-E8 from Stem Cell Technologies, catalogue # 05990 or Essential 8 Medium from ThermoFisher Scientific, catalogue # A1517001\r\n\r\nWhat is ROCK Inhibitor Y27632?<\/a>\r\n<\/u><\/strong>ROCK Inhibitor Y27632 is a selective inhibitor of the Rhoassociated kinase p160ROCK. Treatment with ROCK Inhibitor Y27632 prevents dissociationinduced apoptosis of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC), increasing the survival rate and maintaining pluripotency during subcultivation and thawing of hESCs and hiPSCs. ROCK Inhibitor Y27632 also has been shown to enhance the survival rate of stem cells during cryopreservation.\r\n\r\nDr. William Stanford-2018\r\n\r\n12.Harvesting and Caring for hESCs and iPSCs<\/strong>\r\n
                                  \r\n \t
                                1. Remove media from 4C and place in 37C water bath.<\/li>\r\n \t
                                2. The day after cells were thawed take a look at them under the microscope to determine survival rate. (If cells were frozen only with mFreSR the survival rate is appr. 20%, if cells were frozen using the Bio-Cool survival rate is appr. 60%).<\/li>\r\n \t
                                3. Remove media from the wells and pipette 2ml (for a 6 well, 1ml for 12 well and 0.5ml for 24 wells plate) of fresh PSC media per well.<\/li>\r\n \t
                                4. Place plate in 37C incubator (hypoxic condition: 10% CO2 and 5% O2).<\/li>\r\n \t
                                5. Cells are fed every day for the next 3-5 days until 80% confluent.<\/li>\r\n \t
                                6. Cells should be observed and cleaned of any differentiated cells that might be growing.<\/li>\r\n \t
                                7. To clean cells use the picking hood and \u201cscrap off\u201d differentiated cells with a pipette tip.<\/li>\r\n \t
                                8. Once cells are cleaned changed media as done in above steps.<\/li>\r\n<\/ol>\r\n(See Freezing hESC\/iPSC or Passing hESC\/iPSC<\/strong>)\r\n\r\nNOTE:\r\n<\/u><\/strong>PSC media was determine to be more efficient\u00a0 in a hypoxic environment. We have also observed that less differentiation occurs when cells are grown in hypoxic incubators versus normoxic. Also when seeding cells there\u2019s a better survival rate when using a hypoxic incubator.<\/a>\r\n\r\nNormoxic: 5% CO2\r\nHypoxic: 5%O2, 10% CO2\r\n\r\nDr. William Stanford-2018\r\n\r\n13.Passaging with EDTA solution<\/strong>\r\n
                                    \r\n \t
                                  1. Add 2ml of PSC media to a 6 well matrigel coated plate and set aside.<\/li>\r\n \t
                                  2. Take the plate to be passaged and remove the media from the well and wash once with 1ml of PBS(-\/-).<\/li>\r\n \t
                                  3. Add 1ml of\u00a0 the EDTA solution to the well and leave for 3-4min at room temperature. \u00a0Don\u2019t move the plate around as cells can start detaching.<\/li>\r\n \t
                                  4. Once 4 min. is up remove EDTA solution and add 1ml of PSC media. Do not leave EDTA on the cells for more than 4 min as this will cause the cells to lift off.<\/li>\r\n \t
                                  5. Scrape cells (with cell scrapper) and divide cells amongst the 6 wells of your plate containing PSC media (I\u2019ve been taking 160ul into each well). Avoid breaking up the pieces in very small pieces. Try to keep large chunks. Preferably use a wide mouth pipette tip.<\/li>\r\n<\/ol>\r\nNOTE<\/u><\/strong>: Once the cells have been scraped you want to transfer them to the new plate as soon as possible because the cells will reattach quickly (within 5min).\r\n\r\nIf EDTA is left on the cells for more than 4mins. the cells can start to detach. If this happen simply scrap cells and collect in a 15ml falcon with 4ml of PSC media. Spin cells at 850rpm for 5 min. Resuspend the pellet with 1ml of media and divide evenly among a 6 well matrigel coated plate (160ul per well).\r\n\r\nEDTA solution<\/u><\/strong>:\u00a0 Add 500ul of 0.5M EDTA (pH 8.0) into 500ml of DPBS (-\/-). Add 0.9g of NaCl and adjust the osmolarity to 340 mOsm. Filter the solution to sterilize and store it at 4C for up to 6 months. We want the least amount of disturbance for the cells during dissociation therefore the EDTA solution is at the same osmolarity as the PSC media. <\/u><\/strong>\r\n\r\nFrom the paper:<\/a>\r\nPassaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions\r\n<\/strong>Jeanette Beers<\/a>,1<\/sup>\u00a0Daniel R. Gulbranson<\/a>,2,3<\/sup>\u00a0Nicole George<\/a>,4<\/sup>Lauren I. Siniscalchi<\/a>,1<\/sup>\u00a0Jeffrey Jones<\/a>,4,5<\/sup>\u00a0James A. Thomson<\/a>,2,3,6<\/sup>\u00a0and\u00a0Guokai Chen<\/a>1,2<\/sup>\r\n\r\n14.Freezing hESCs and iPSCs<\/strong>\r\n
                                      \r\n \t
                                    1. Turn on Bio-Cool (Controlled Rate Freezer) and adjust SP temperature to -7C.<\/li>\r\n \t
                                    2. Remove cells from incubator and observe confluency and morphology under the microscope.\r\nNOTE:<\/strong> Looking for ~80% confluency<\/li>\r\n \t
                                    3. If confluent, remove old media and wash once with PBS(-\/-) then add 1 ml of EDTA solution (see passaging with EDTA solution) per well.<\/li>\r\n \t
                                    4. Incubate at room temperature for 3-4 minutes.<\/li>\r\n \t
                                    5. Aspirate the EDTA solution and add 1 ml of mFreSR media (cat#05855, Stem Cell Technologies)<\/li>\r\n \t
                                    6. Use cell scraper to detach the cells from the well (gently).<\/li>\r\n \t
                                    7. Transfer cells\/mFreSR to a cryotube using a wide mouth tip.<\/li>\r\n \t
                                    8. Place tubes in Bio-Cool and incubate for 10min.<\/li>\r\n \t
                                    9. Get liquid nitrogen.<\/li>\r\n \t
                                    10. After 10min. seed the cells by dipping a spatula into liquid nitrogen and touching the side of the cryo-vial for approximately 10 seconds or until you see a crystal form on the side of the cryo-vial.<\/li>\r\n \t
                                    11. Start program 1 by pushing \u201cPROG\u201d button and going through the program by pushing the button again and you should see the rate of 0.5C\/min. , then press \u201cRUN\u201d.<\/li>\r\n \t
                                    12. Once temperature reaches -65C the cryo-tubes can be transferred and stored in liquid nitrogen.<\/li>\r\n<\/ol>\r\nAlternatives\r\n<\/u><\/strong>1-Another option is to place the cryo-tubes in a freezing container (Biocision-CoolCell) and store at -80C overnight. The next day store cryo-tubes in liquid nitrogen.\r\n\r\nDr. William Stanford-2018[\/vc_column_text][\/vc_column][\/vc_row]\t\t","_et_gb_content_width":"","footnotes":"","_links_to":"","_links_to_target":""},"class_list":["post-1010","page","type-page","status-publish","hentry"],"yoast_head":"\nInduced | The Progeria Research Foundation<\/title>\n<meta name=\"description\" content=\"To learn more about Induced Pluripotent Stem Cells, please contact Leslie Gordon, MD, PhD, Medical Director at lgordon@progeriaresearch.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\/id\/induced-pluripotent-stem-cells\/\" \/>\n<meta property=\"og:locale\" content=\"id_ID\" \/>\n<meta property=\"og:type\" content=\"article\" \/>\n<meta property=\"og:title\" content=\"Induced | The Progeria Research Foundation\" \/>\n<meta property=\"og:description\" content=\"To 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