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Induced Pluripotent<\/h1>\n

Stem Cells<\/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

The Progeria Research Foundation Cell & Tissue Bank<\/strong> Human Induced Pluripotent Stem Cells (iPSC)<\/h4>\n
    \n
  1. Progeria iPSCs <\/strong>Background Information for the Non-scientist<\/strong><\/a>\u00a0<\/strong><\/li>\n
  2. Purpose of <\/strong>Induced Pluripotent Stem Cell Generation and Distribution by The Progeria Research Foundation\u00a0<\/strong>\u00a0<\/a><\/li>\n
  3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs)<\/strong>\u00a0<\/a><\/li>\n
  4. Quality Control: Validation and Characterization<\/strong>\u00a0<\/a><\/li>\n
  5. Original Starting Material from which iPSCs Were Derived<\/strong><\/a><\/li>\n
  6. Join our Email List for Future iPSC Updates and New Cell Lines<\/strong>\u00a0\u00a0<\/strong><\/a><\/li>\n
  7. Questions?\u00a0 Contact us.<\/strong><\/a><\/li>\n
  8. Ordering iPSC Lines<\/strong><\/a>\u00a0<\/strong><\/li>\n
  9. HGPS and Control iPSC Culture Media Preparation<\/strong>\u00a0<\/strong><\/a><\/li>\n
  10. Preparing Matrigel Plates\u00a0<\/strong><\/a><\/li>\n
  11. Thawing hESCs and iPSCs cells (per cryo-vial)<\/strong><\/a><\/li>\n
  12. Harvesting and Caring for hESC\/iPSC<\/strong>\u00a0<\/strong><\/a><\/li>\n
  13. Passaging <\/strong><\/a>hESC\/iPSC\u00a0<\/a><\/strong><\/li>\n
  14. Freezing hESC\/iPSC<\/a><\/strong><\/li>\n<\/ol>\n

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    1. iPSC Background information for the non-scientist<\/strong><\/h4>\n

    Stem 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>\u00a0. 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\u00a0We can now ask key\u00a0<\/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><\/p>\n

     <\/p>\n

    \n
      \n
    1. \n
        \n
      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.\u00a0Cell.\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. Induced pluripotent stem cell lines derived from human somatic cells.\u00a0Science.\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. Recapitulation of premature ageing with iPSCs from Hutchinson-Gilford progeria syndrome.\u00a0Nature.\u00a0<\/em>2011;472:221-225.<\/li>\n
      4. Misteli T. HGPS-derived iPSCs for the ages.\u00a0Cell Stem Cell.\u00a0<\/em>2011;8:4-6.<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n<\/div>\n

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        2.\u00a0Purpose of induced pluripotent stem cell (iPSC) generation and distribution by The Progeria Research Foundation<\/strong><\/h4>\n

        The 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\u2019s Sprott Centre for Stem Cell Research.<\/p>\n

        Our 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\u00a0<\/strong><\/p>\n

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        3. Generation of Hutchinson-Gilford Progeria Syndrome Induced-Pluripotent Stem Cells (iPSCs)<\/strong><\/h4>\n

        Induced-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).<\/p>\n

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        4. Quality Control: Validation and Characterization<\/strong><\/h4>\n

        The lines that are currently available have undergone several validation steps (see downloadable PDFs below):<\/p>\n

        \n
          \n
        1. \n
            \n
          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>\n
          2. Immunostaining for pluripotency markers Tra-1-60, Tra-1-81, and SSEA4.<\/li>\n
          3. Alkaline Phosphatase Staining as an indicator of pluripotency<\/li>\n
          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>\n
          5. Karyotype analysis.<\/li>\n
          6. Re-expression of lamin A in differentiated cells<\/li>\n
          7. Teratoma assays<\/li>\n<\/ol>\n<\/li>\n<\/ol>\n

            Additional validation in process:<\/strong>
            Some 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.<\/p>\n<\/div>\n

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            5.\u00a0\u00a0 Original starting material from which these iPS cells were derived<\/strong><\/h4>\n

            iPSCs were derived from PRF Cell & Tissue Bank non-transformed fibroblast cell lines.<\/p>\n

            The transduction method used for all iPS lines was Retrovirus MKOS.<\/p>\n

            <\/p>\n

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            \n\n\n\n\t\n\n\t\n\t\n\t\n\t\n\t\n\t\n\t\n\t\n\t
            iPSC Line ID<\/b><\/th>Mutation<\/b><\/th>Gender and DonationAge<\/b><\/th>Originating Cell Type Click here<\/a>.<\/b><\/th>Supporting Data<\/b><\/th>\n<\/tr>\n<\/thead>\n
            HGADFN003 iPS 1B<\/td> LMNAExon 11,
            \n1824 C>T<\/td>            		Male 2yr 0mo<\/td> Dermal Fibroblasts
            \nHGADFN003<\/td>            		003 iPS1B<\/a><\/td>\n<\/tr>\n
            HGADFN003 iPS 1C<\/td> LMNA Exon 11,
            \n1824 C>T<\/td>            		Male 2yr 0mo<\/td> Dermal Fibroblasts
            \nHGADFN003<\/td>            		003 iPS1C<\/a><\/td>\n<\/tr>\n
            HGDFN003
            \niPS 1D<\/td>            		LMNA Exon 11,
            \n1824 C>T<\/td>            		Male 2yr 0mo<\/td> Dermal Fibroblasts
            \nHGADFN003<\/td>            		003 iPS1D<\/a><\/td>\n<\/tr>\n
            HGADFN167 iPS 1J<\/td> LMNA Exon 11, 1824 C>T<\/td>Male 8yr 5mo<\/td>Dermal Fibroblasts HGADFN167<\/td>167 PS 1J<\/a><\/td>\n<\/tr>\n
            HGADFN167 iPS 1Q<\/td> LMNA Exon 11, 1824 C>T<\/td>Male 8yr 5mo<\/td>Dermal Fibroblasts HGADFN167<\/td>167 iPS1Q<\/a><\/td>\n<\/tr>\n
            HGMDFN090 iPS 1B<\/td> Mother of HGADFN167 (unaffected)<\/td>Female 37yr 10mo<\/td>Dermal Fibroblasts
            \nHGMDFN090<\/td>            		090 iPS1B<\/a><\/td>\n<\/tr>\n
            HGMDFN090 iPS 1C<\/td> Mother of HGADFN167 (unaffected)<\/td>Female 37yr 10mo<\/td>Dermal Fibroblasts
            \nHGMDFN090<\/td>            		090 iPS1C<\/a><\/td>\n<\/tr>\n
            HGFDFN168 iPS1 D2<\/td>Father of HGADFN167 (unaffected)<\/td>Male 40yr
            \n5mo<\/td>            		Dermal Fibroblasts HGFDFN168<\/td>168 iPS1 D2<\/a><\/td>\n<\/tr>\n
            HGFDFN168 iPS1P<\/td>Father of HGADFN167 (unaffected)<\/td>Male 40yr
            \n5mo<\/td>            		Dermal Fibroblasts
            \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. Join our email list for future iPSC updates and new cell lines<\/strong><\/h4>\n

            We 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\u00a0here<\/a><\/strong><\/p>\n

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            7.\u00a0Questions?<\/strong><\/h4>\n

            <\/a>Please contact Leslie Gordon, MD, PhD, Medical Director, with any questions or needs, at lgordon@progeriaresearch.org<\/a>\u00a0or 978-535-2594<\/p>\n

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            8.\u00a0 Ordering iPS cell lines<\/strong><\/h4>\n

            In 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.<\/p>\n

            For U.S. Federal Government Institutions or questions, please contact Wendy Norris at: wnorris@brownhealth.org<\/a><\/a>\u00a0or\u00a0401-274-1122 x 48063<\/a>.<\/p>\n

            Step 1: Complete an application and material transfer agreement
            <\/strong>
            <\/a>            Application and Agreement for Non-government Institutions<\/a><\/p>\n

            Material Transfer Agreement for Non-government Institutions<\/a><\/p>\n

            Step 2: Return the completed application and material transfer agreement to Wendy Norris at wnorris@brownhealth.org<\/a>.\u00a0 Once approved, you will receive an email confirming your order and anticipated shipping date.<\/b>\u00a0<\/strong><\/a><\/p>\n

            Step 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.<\/p>\n

            The Human Pluripotent Stem Cell Facility (directed by Dr. Stanford) at the Ottawa Hospital Research Institute offers virtual one-on-one training on basics of iPSC culture techniques specific to the Progeria iPSC cell lines. Training options and format are flexible depending on the scientists\u2019 level of experience. \u00a0For more information, please email hpscf@ohri.ca<\/a>.<\/u><\/strong><\/p>\n

            Step 4: The University of Ottawa will invoice you directly for each iPSC line plus courier costs, if any.<\/strong><\/p>\n

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            9.\u00a0 HGPS and Control iPS Cell Culture Media Preparation<\/strong><\/h4>\n

            iPSC and ESCs need to be fed with mTeSR Plus from Stem Cell Technologies (cat# 5825). Please follow the supplier\u2019s recommendations for storage.<\/p>\n

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            10. Preparing Matrigel Plates<\/strong><\/h4>\n

            Note<\/strong>: All steps involving matrigel should be done as quickly as possible and stay as cold as possible.<\/p>\n

              \n
            1. Thaw matrigel bottle at 4\u00b0<\/strong>C. Check the certificate of analysis on that lot to find its protein concentration.<\/li>\n
            2. Add enough cold DMEM\/F12 media to the thawed matrigel to reach a final concentration of 5mg\/mL.<\/li>\n
            3. Make 1mL aliquots of the prepared matrigel from step 2 in pre-chilled 15mL falcon tubes.<\/li>\n
            4. Freeze and store all aliquots at -20\u00b0<\/strong>C.<\/li>\n
            5. To make matrigel plates, remove one aliquot of matrigel (1mL) from -20\u00b0<\/strong>C and add 10mL of cold DMEM\/F12. Mix well until pellet thaws (without creating bubbles, and always keep solution cold).<\/li>\n
            6. Transfer to a 50mL tube, then add 20mL of cold DMEM\/F12 (1mL of matrigel aliquot is diluted in 30mL of DMEM\/F12), mix well.<\/li>\n
            7. Plate (1mL\/well for 6 well plate, 0.5mL\/well for 12 well plate, 0.25mL\/well for 24 well plate). Make sure that the solution is covering the entire surface area by shaking the plate gently. Do not re-freeze any leftover matrigel.<\/li>\n
            8. If using the plate immediately, let the plate sit at room temperature for 1 hour (or 30 minutes at 37\u00b0<\/strong>C), observe matrigel under the microscope. Matrigel should be well dispersed and not “clumpy”.<\/li>\n
            9. If using plates at another time, wrap the edge of the plate with parafilm and store at 4\u00b0<\/strong>C for up to 2 weeks.<\/li>\n<\/ol>\n

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

              DMEM\/F12 \u2013 Life Technologies, cat#11330-057<\/p>\n

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

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              11. <\/strong>Thawing ES or iPS cells (per cryo-vial)<\/strong><\/h4>\n
                \n
              1. Take a matrigel plate out of 4\u00b0C and warm at room temperature for one hour, or make a fresh matrigel plate (see preparing matrigel plate protocol).<\/li>\n
              2. Warm 4mL of mTeSR Plus in a 15mL falcon tube.<\/li>\n
              3. Remove cells from the liquid nitrogen tank and swirl in a 37\u00b0C bath until only a small ice chunk is left. Vial should thaw in 1-2 minutes. This step must be done quickly.<\/li>\n
              4. Ethanol cell tube and falcon media tube and place in the hood.<\/li>\n
              5. Use a 1mL wide mouth tip to slowly <\/u>add cells to 4mL of pre-warmed media (avoid mixing cell suspension).<\/li>\n
              6. Spin at 130 rcf for 5 minutes.<\/li>\n
              7. Remove supernatant.<\/li>\n
              8. Add 2mL of PSC media, and with a wide mouth tip break up clumps gently<\/u>. Transfer media to one well of a 6 well plate add 2uL of ROCK inhibitor (Y27632, final concentration of 10uM). Plate into one matrigel coated well (of a 6 well plate).<\/li>\n
              9. Rock cells gently to evenly distribute cells, and place in a hypoxic incubator (5%O2<\/sub>, 10% CO2<\/sub>). Avoid plate disturbance for 24 hours post seeding.NOTE: <\/strong>It is very important to avoid excessive breaking down of clumps or aggressive pipetting. This can significantly reduce survival rate. The cells should remain in chunks of 100-300 cells big at the time of seeding. While being gentle, try working quickly once cells are thawed to minimize time they are in contact with cryoprotectant.\u200b<\/li>\n
              10. 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 protocol.<\/li>\n<\/ol>\n

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

                mTeSR Plus from Stem Cell Technologies (cat# 5825). Please follow the supplier\u2019s recommendations for storage.<\/p>\n

                What is ROCK Inhibitor Y27632?<\/strong><\/p>\n

                ROCK Inhibitor Y27632 is a selective inhibitor of the Rho associated kinase p160 ROCK. Treatment with ROCK Inhibitor Y27632 prevents dissociation induced apoptosis of human embryonic stem cells (hESC) and human induced pluripotent stem cells (iPSC), 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. Note that Rock inhibitor aliquots are sensitive to light and repetitive freeze thaw cycles. Make sure to use these aliquots within the supplier\u2019s recommended shelf life.<\/p>\n

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

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                12. <\/strong>Harvesting and Caring for hESC\/ iPSC<\/strong><\/h4>\n
                  \n
                1. The day after cells were thawed take a look at them under the microscope to determine survival rate. NOTE: it is normal to observe a high number of unattached cells. As long as some cells are attached, colonies can arise from them within 3 – 7 days.<\/li>\n
                2. Remove media from the wells and pipette 2 mL (for a 6 well, 1mL for 12 well and 0.5mL for 24 wells plate) of fresh and warm PSC media per well. Return plate to incubator.<\/li>\n
                3. Cells are fed until 60-70% confluent (follow supplier recommendations).<\/li>\n
                4. As of day 2, cells should be observed and cleaned of any differentiated cells that might be growing.<\/li>\n
                5. To clean cells, use the picking hood and scrape off differentiated cells with a pipette tip.<\/li>\n
                6. Once cells are cleaned, change media as done in the above steps.<\/li>\n<\/ol>\n

                  (See Freezing hESC\/iPSC or Passing hESC\/iPSC)<\/p>\n

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

                  PSC media was determined to be more efficient in a hypoxic environment. We have also observed that less differentiation occurs when cells are grown in hypoxic incubators versus normoxic. Lastly, seeded cells have a better survival rate when using a hypoxic incubator.<\/p>\n

                  Normoxic: 37\u00b0<\/strong>C, 21% O2<\/sub>, 5% CO2<\/sub><\/p>\n

                  Hypoxic: 37\u00b0<\/strong>C, 5%O2<\/sub>, 10% CO2<\/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>Passing hESC\/iPSC<\/strong><\/h4>\n
                    \n
                  1. Add 2mL of PSC media to a 6 well matrigel coated plate and set aside.<\/li>\n
                  2. Take the plate to be passaged and remove the media from the well and wash once with 1mL of PBS(-\/-).<\/li>\n
                  3. Add 1mL of\u00a0 the EDTA solution to the well and leave for 3-4 minutes at room temperature.\u00a0 Don\u2019t move the plate around as cells can start detaching.<\/li>\n
                  4. Remove EDTA solution and add 1mL of PSC media. Do not leave EDTA on the cells for more than 4 minutes as this will cause the cells to lift off.<\/li>\n
                  5. Scrape cells using a cell scraper and divide cells amongst the 6 wells of your plate containing PSC media. Avoid excessive breaking up of the colony pieces, and try to be gentle with scraping. Try to keep cells in large chunks. Use a wide mouth pipette tip to break up clumps if needed. Excessive breaking up of cells may cause cell death or excessive spontaneous differentiation following passaging.<\/li>\n
                  6. Incubate at 37\u00b0<\/strong>C after evenly distributing cells in each well (8 figure or L shape shaking). Avoid plate disturbance for 24 hours post passaging.<\/li>\n<\/ol>\n

                    NOTE<\/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 5 minutes).<\/p>\n

                    If EDTA is left on the cells for more than 4 minutes, the cells can start to detach. If this happens, simply collect the cells in a 15mL falcon with 4mL of PSC media. Spin cells at 130 rcf for 5 minutes. Resuspend the pellet with 1mL of media and divide evenly among a 6 well matrigel coated plate (160uL per well).<\/p>\n

                    EDTA solution:\u00a0 Add 500uL of 0.5M EDTA (pH 8.0) into 500mL of DPBS (-\/-). Add 0.9g of NaCl. Filter the solution to sterilize and store it at 4\u00b0C for up to 6 months. <\/strong><\/p>\n

                    From the paper:<\/p>\n

                    Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions<\/strong><\/p>\n

                    Jeanette Beers<\/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> James A. Thomson<\/a>,2,3,6<\/sup> and 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>Freezing hESC\/iPSC <\/strong><\/h4>\n
                      \n
                    1. Turn on Bio-Cool (Controlled Rate Freezer) and adjust temperature to -7\u00b0C.<\/li>\n
                    2. Remove cells from the incubator and observe confluency and morphology under the microscope.<\/li>\n
                    3. If the wells are 70% confluent, remove old media and wash once with PBS(-\/-) then add 1 mL of EDTA solution (see passing with EDTA solution) per well.<\/li>\n
                    4. Incubate at room temperature for 3-4 minutes.<\/li>\n
                    5. Aspirate the EDTA solution and add 1 mL of cold mFreSR media (cat#05855, Stem Cell Technologies).<\/li>\n
                    6. Use a cell scraper to lift the cells gently. Keep cells in large chunks as much as possible and avoid pipetting up and down.<\/li>\n
                    7. Transfer cells\/mFreSR to a cryotube using a wide mouth tip. Keep vials on ice until ready for step 8.<\/li>\n
                    8. Place tubes in Bio-Cool and incubate for 10 minutes.<\/li>\n
                    9. Get liquid nitrogen.<\/li>\n
                    10. After 10 minutes, seed the cells by dipping a spatula into liquid nitrogen and touching the side of the cryo-vial for approximately 10-30 seconds or until you see a crystal form on the side of the cryo-vial.<\/li>\n
                    11. Start program 1 by pressing \u201cPROG\u201d button and going through the program by pressing the button again and you should see the rate of 0.5\u00b0C\/min. , then press \u201cRUN\u201d.<\/li>\n
                    12. Once temperature reaches -65\u00b0C the cryo-tubes can be transferred and stored in liquid nitrogen.<\/li>\n<\/ol>\n

                      Alternatives<\/p>\n

                      Another option is to place the cryo-tubes in a freezing container (Biocision-CoolCell) and store at -80\u00b0C overnight. Move the cryo-tubes to liquid nitrogen (liquid or vapor phase) on the following day.<\/p>\n

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

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                      [et_pb_section fb_built=”1″ fullwidth=”on” disabled_on=”off|off|off” _builder_version=”4.16″ border_width_bottom=”55px” border_color_bottom=”#29327a” locked=”off” global_colors_info=”{}”][et_pb_fullwidth_header _builder_version=”4.16″ title_font=”||||||||” title_font_size=”55″ background_color=”#29327a” background_image=”https:\/\/www.progeriaresearch.org\/wp-content\/uploads\/2020\/12\/Jes.jpg” background_position=”center_left” custom_padding=”9vw|0px|9vw||true|” custom_padding_tablet=”” custom_padding_phone=”|56px||” custom_padding_last_edited=”on|desktop” title_font_size_tablet=”45px” title_font_size_phone=”40px” title_font_size_last_edited=”on|phone” z_index_tablet=”500″ custom_css_main_element=”background-position: center 18% !important;” global_colors_info=”{}”] Induced Pluripotent Stem Cells   [\/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” […]<\/p>\n","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\/kk\/induced-pluripotent-stem-cells\/\" \/>\n<meta property=\"og:locale\" content=\"en_US\" \/>\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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