SC102A-1 Human iPS Cell Line User Manual
Contents
1. Transfer 1 mL of clumps in CRYO GOLD Human ESC iPSC Cryopreservation Medium into each labeled cryovial 6 Place vials into an isopropanol freezing container and place the container at 80 C overnight 7 Transfer to a liquid nitrogen tank the next day Page 6 ver 1 030713 www systembio com iPS Cell Lines Cat SC102A 1 C Validation of human iPS Cells Human iPSC Cell Line SC102A 1 Human iPS cell colonies were stained with antibodies for stem cell markers Nanog upper left Oct4 lower left SSEA3 lower right and TRA 1 60 upper right followed by fluorescent labeled Alexa Fluor secondary antibodies Cat No SAB KIT 1 ll References Rossant J 2007 Stem cells The magic brew Nature 448 260 262 Takahashi K and Yamanaka S 2006 Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 126 663 676 Takahashi K et al 2007 Induction of pluripotent stem cells from adult human fibroblasts by defined factors Cell 131 861 72 Park IH et al 2008 Reprogramming of human somatic cells to pluripotency with defined factors Nature 451 141 6 Baker Monya 2007 Adult cells reprogrammed to pluripotency without tumors Nature Reports Stem Cells 2007 12 11 Nakagawa M et al 2008 Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts Nature Biotechnology 26 101 106 Okita K et al 2007 Generation of germline2. SSBI System Biosciences Human iPS Cell Lines Cat SC102A 1 User Manual Store kit at 80 C on receipt A limited use label license covers this product By use of this product you accept the terms and conditions outlined in the Licensing and Warranty Statement contained in this user manual ver 1 030713 iPS Cell Lines Cat SC102A 1 Contents Le Human iPS Gells EE ES 3 Description AE EE RE N 3 B Growth Conditions for Human iPS Cells i iis see ee ee 4 C Validation of Human iPS Cells iese see ee ee ee 7 Il BEE RR RR EE EE 16 III Technical SUDPOF ese ese ee AA GR RA ee AA ee a 16 IV Licensing and Warranty StatemenNE sees ese ds see ee ee 17 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual List of Components Human iPS Cell Line Set SC102A 1 feeder free The product is shipped on dry ice and should be immediately stored in the gas phase of liquid nitrogen In general iPS cells are challenging to culture and should only be operated by researchers experienced in the intricacies of human embryonic stem hES cell and human induced pluripotent iPS cell culture The methods for iPS cell culture are nearly identical to hES cell culture although more careful maintenance will be required Page 2 ver 1 030713 www systembio com iPS Cell Lines Cat SC102A 1 L Human iPS Cells A Description Human induced pluripoten
3. competent induced pluripotent stem cells Nature 448 313 7 Yu J et al 2007 Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cells Science 318 1917 1920 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual lll Technical Support For more information about SBI products or to download manuals in PDF format please visit our website http www systembio com For additional information or technical assistance please call or email us at tech systembio com 650 968 2200 IV Licensing and Warranty Statement Limited Use License Use of the human iPS cells i e the Product is subject to the following terms and conditions If the terms and conditions are not acceptable return all components of the Product to System Biosciences SBI within 7 calendar days Purchase and use of any part of the Product constitutes acceptance of the above terms The purchaser of the Product is granted a limited license to use the Product under the following terms and conditions e The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use e The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI e This Product should be used in accordance with the NIH guidelines developed fo
4. er warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose SBI is committed to providing our customers with high quality products If you should have any questions or concerns about any SBI products please contact us at 888 266 5066 Page 8 ver 1 030713 www systembio com iPS Cell Lines Cat SC102A 1 2013 System Biosciences SBI 888 266 5066 Toll Free 650 968 2200 outside US Page 9
5. ll plate Then add 1 ml PSGro iPSC Growth Media containing 10 uM ROCK inhibitor Y 27632 5 Discard the supernatant from the human iPS cells and resuspend the cells with 1 ml fresh PSGro iPSC Growth Media containing 10 uM ROCK inhibitor Y 27632 Plate the cells on matrigel coated plate 6 After 24 hours remove the media and replace with fresh PSGro iPSC Growth Media without ROCK inhibitor 7 Incubate at 37 C with 5 CO until the cells reach 80 confluency The PSGro iPSC Growth Media must be changed every day and human iPS cells subcultured every 5 7 days 4 Maintenance and Passage of human iPSCs Grown under Feeder free condition In order to maintain pluripotency it is important to NOT keep human iPS cells in culture for long period of time without passaging 1 Look under microscope to identify regions of differentiation Mark the differentiated colonies using lens marker on the bottom of the plate 2 Remove regions of differentiation by scraping with a pipette tip or by aspiration 3 Aspirate medium from the hiPSC culture and rinse with DPBS 2 mL well 4 Add 0 5 mL per well of accutase Cat SCR005 Millipore diluted 1 1 with DPBS before use Let it stand at room temperature for 1 2 minutes 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual 5 Remove accutase and gently rinse each well 1 2 times with 2 mL of Knockout DMEM F 12 per well to dilute away remaining enzy
6. mes 6 Add 2 mL well PSGro Human ESC iPSC Growth Medium and scrape colonies off with a cell scraper 7 Transfer the detached cell aggregates to a 15 mL conical tube and rinse the well with an additional 2 mL PSGro Human ESC iPSC Growth Medium to collect any remaining aggregates Add the rinse to the 15 mL tube 8 Centrifuge the 15 mL tube containing the aggregates at 200 x g for 5 minutes at room temperature 9 Aspirate the supernatant Resuspend pellet in PSGro Human ESC iPSC Growth Medium containing 10 uM ROCK inhibitor by gently pipetting and ensure that cells are maintained as aggregates 10 Plate the hiPSC aggregates with PSGro Human ESC iPSC Growth Medium onto a new plate coated with matrigel Remove matrigel solution before plating If the colonies are at an optimal density the cells can be split every 5 7 days using 1 6 to 1 10 splits 11 Place the plate into the 37 C incubator and move the plate in quick side to side forward to back motions to evenly distribute the clumps within the wells Culture the cells at 37 C with 5 CO2 and 95 humidity 12 Change medium daily 5 Freezing Down iPSC Cells 1 Perform step 1 8 from Passaging hiPSCs Grown under Feeder free condition 3 Gently aspirate the supernatant taking care to keep the cell pellet intact 4 Gently resuspend the pellet in CRYO GOLD Human ESC iPSC Cryopreservation Medium taking care to leave the clumps larger than would normally be done for passaging 5
7. ore Matrigel BD Biosciences ver 1 030713 www systembio com iPS Cell Lines Cat SC102A 1 2 Coating plates with Matrigel Matrigel Cat 354277 BD should be aliquoted and stored at 80 C for long term use 1 Thaw matrigel on ice until liquid Dilute according to the manufacturer s instruction with pre chilled Knockout DMEM F12 2 Immediately use the diluted matrigel solution to coat tissue culture treated plates For a 6 well plate use 1 mL of diluted matrigel solution per well and swirl the plate to spread the matrigel solution evenly across the surface 3 Let the coated plate stand for at least 1 h at room temperature or overnight at 4 C If plate has been stored at 4 C allow the plate to incubate at room temperature for 1 h before removing the matrigel solution 3 Thawing human iPS cells To insure the highest level of viability be sure to warm medium to 37 C before using it on the cells Due to the low survival rate of cryopreserved human iPS cells the recovery is expected to take at least one week 1 Remove the vial from liquid nitrogen and thaw quickly in a 37 C water bath 2 Remove the vial from the water bath as soon as the cells are half thawed and sterilize by spraying with 70 ethanol 3 Transfer the cells with 10 ml of PSGro iPSC Growth Media to a 15 mL conical tube and pellet the cells by centrifugation at 200 g for 5 min 4 While centrifuging remove the matrigel solution from one well of 6 we
8. r stem cell research SBI has pending patent applications related to the Product For information concerning licenses for commercial use contact SBI Purchase of the product does not grant any rights or license for use other than those explicitly listed in this Licensing and Warranty Statement Use of the Product for any use other than described expressly herein may be covered by patents or subject to rights other than those mentioned SBI disclaims any and all responsibility for injury or damage which may be caused by the failure of the buyer or any other person to use the Product in accordance with the terms and conditions outlined herein Limited Warranty SBI warrants that the Product meets the specifications described in this manual If it is proven to the satisfaction of SBI that the Product fails to meet these specifications SBI will replace the Product or provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to SBI within 30 days of receipt of the Product SBI s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price SBls liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty SBI does not provide any oth
9. t stem cells iPSCs cat SC102A 1 were generated by transducing genetically unmodified human fibroblasts with viruses individually encoding the four human transcription factors Oct4 Sox2 KIf4 and c Myc that have been shown to induce the reprogramming of somatic cells to a pluripotent state The cells were derived using morphological selection criteria and without the use of fluorescent markers or drug selection When cultured under standard human ES cell culture conditions the morphology of SBI human iPSCs is identical to that of human ES cells The cells also express the pluripotency markers Nanog Oct4 SSEA3 and TRA 1 60 and demonstrate a strong endogenous AP activity Human iPS cells are adapted to feeder free conditions and should be grown on Matrigel Matrigel is available from BD Biosciences PSGro iPSC Growth Media SC500M 1 is strongly recommended as the optimal culture medium for those cells Human iPS cells from SBI are provided at approximately passage 10 and can be passaged 50 times before differentiation 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual B Growth Conditions for human iPS cells 1 Required media and reagents SC500M 1 SC150M 1 Information PSGro Human ESC iPSC Growth Medium for feeder free conditions CRYO GOLD Human ESC iPSC Cryopreservation Medium ZRD Y 01 05 25 ROCK Inhibitor Y 27632 SCR005 354277 Page 4 Accutase Millip
Download Pdf Manuals
Related Search