piPS Cells Cat. #SC801A-1 & SC802A-1
Contents
1. 90 FBS plus 10 DMSO with 10 uM ROCK inhibitor Y 27632 NOTE This protocol is for growing piPS cells on MEF feeder cells These should already be growing before you plate your piPS cells Page 6 ver 1 020711 www systembio com piPS Cells Cats SC801A 1 SC802A 1 B Protocol for Human iPS Cell Culture Growth condition for mouse fibroblasts for feeder layers Gelatin treatment of plates 1 Add enough sterile autoclaved 0 1 gelatin to cover the bottom of the wells Approximate amounts 10cm 5ml 6 well 1 5ml well 24 well 0 5 ml well 96 well 200 pl well Incubate the gelatin coated dishes for at least 15 min at 37 C Aspirate excess gelatin solution before using Thawing MEF cells To insure the highest level of viability be sure to warm medium to 37 C before using it on the cells Cells should be plated at a minimum cell density of 1 x 10 cells cm 1 Remove the vial from liquid nitrogen and thaw quickly in 37 C water bath Remove the vial from the water bath as soon as the cells are half way thawed and sterilize by spraying with 70 ethanol Transfer the cells with 10 ml of MEF medium to a 15 cm conical tube and pellet the cells by centrifugation at 200x g for 5 min Discard the supernatant and resuspend the cells with 10 ml fresh MEF medium and plate the cells at seed density of 1 x 104 cells cm 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SB2. CK inhibitor to each well Page 10 ver 1 020711 www systembio com piPS Cells Cats SC801A 1 SC802A 1 4 When the edge of the human iPS cell colonies have fold up aspirate the Accutase solution and wash the cells three times with DMEM F12 medium 5 Add 1 ml per well of human ES medium and dislodge the cell colonies by using cell scraper 6 Transfer the contents into a 15 ml conical tube with 5 ml of pre warmed human ES medium Use another 1 ml of medium to wash the well one more time and combine it to the same tube Centrifuge at 200x g for 5 minutes at room temperature Carefully aspirate the overlaying medium then gently finger tap the tube bottom to dislodge the cell pellet 9 Gently add 3 to 6 ml of fresh human ES medium with 10 uM ROCK inhibitor and resuspend the cells by gently pipetting up and down 10 Add 1 ml of the human iPS cell suspension to each well of the 6 well plate Right after plating iPS cells gently swirl the plate back and forth and side to side to evenly distribute the cells and incubate at 37 C 11 The ES media must be changed every day and human iPS cells sub cultured every 5 7 days when the undifferentiated colonies are big enough Track passage number of iPS cells Note human iPS cells could also be grown on HFF feeders Freezing human iPS cells 1 Grow human iPS cells to the exponential phase in a 6 well plate and pre treat cells with 10 uM Y 27632 for one hour prior to free
3. I User Manual 5 6 Incubate at 37 C with 5 CO in air atmosphere until the cells reach 80 90 confluency Change medium twice a week or when pH decreases Passage of MEF cells Cells should be split when they reach confluency A split based on seed density of 0 5 x 10 cells cm is recommended 1 2 Discard the medium and wash the cells twice with PBS Aspirate PBS and add 1 ml per T75 flask of 0 25 trypsin EDTA and incubate for 1 min Add 5 ml of MEF medium and break up the cell clumps by gently pipetting up and down several times Transfer cells into a conical tube and centrifuge at 200 g for 5 min Discard the supernatant and resuspend the cell pellet in 10 ml MEF medium Count the number of cells plate cells at 0 5 x 10 cells cm and incubate at 37 C with 5 COs Freezing MEF cells 1 2 Follow steps 1 4 from the Passage of Cells above Discard the supernatant and resuspend the pellet in MEF medium Add approximately 1 ml for each T75 flask Count the number of cells and dilute the cell suspension to 1 x 10 cells ml Add an equal volume of cold 2X Freezing Media containing 20 DMSO and 80 FBS to the cell suspension Aliquot 1 ml of suspension into each cryovial 5 x 10 cells vial Page 8 ver 1 020711 www systembio com piPS Cells Cats SC801A 1 SC802A 1 6 Place the vials in a cell freezing container and keep it at 80 C overnight 7 Transfer the v
4. SSBI System Biosciences piPS Cells Cat SC801A 1 amp SC802A 1 User Manual Store cells in liquid nitrogen 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 ver 1 021111 contained in this user manual piPS Cells Cats SC801A 1 SC802A 1 Contents I Introduction and Background ececeeeeeeeeeteeeeeeetteeeeeeneeeeee 2 A Evolution of Reprogramming Technolog 0 ccseee 2 B Protein derived iPS Cell line 0 cceeeeeeeeeeeeeeeeeeeeneeeeeeeees 3 Il Protocols i2 s cicc need noel a a a tidied 5 A Materials nein e ane ee a eee A dee ENS 5 Gelatin treatment Of Plates eeceeccececeteeeeneeceeeeeseaeeesaeeseeeeeeaees 7 Thawing MEF celsesi eeni a E e 7 Thawing human iPS CellS eecceeeececeeeeeeeeeeeeeeeseeeeeseaeeeeaaeseeneeeaes 9 lil References niuno aes Mea Aes ae A ees 12 IV Technical Support ceeeeeeeeeeeeseeeeseceeeeeeseaeeesaeeteeeesaees 13 V Licensing and Warranty cccccceceeeeeseceeeeeeseeeeeseeeeeeeeeeees 13 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual I Introduction and Background A Evolution of Reprogramming Technology The concept of reprogramming was initially demonstrated by Gurdon et al in 1958 where they generated adult Xenopus from somatic cells by nuclear reprogramming After the dis
5. covery of induced pluripotent stem cells iPSC technique using defined transcription factors has been a basis and standard for generation of pluripotent stem cells In comparison to somatic cell nuclear transfer the iPSC technology offers an unprecedented technical simplicity and enables generation of patient specific pluripotent stem cells with less ethical concerns Although iPSC technology provides unprecedented opportunities in biomedical research and regenerative medicine there remains a great deal to learn about iPSC safety the reprogramming mechanisms the quality of iPSCs from different source cells and the variations using different reprogramming technology Since 2006 iPSC technology has evolved from integrated virus Retrovirus and lentivirus to non integrated virus Adenovirus viral methods to non viral methods plasmids genetic methods DNA or RNA vectors to non genetic methods proteins These technical advances provide safer iPSCs for more meaningful mechanistic studies iPSC based disease modeling and drug screening DO orcas 000 a Reprogramming factors Year 2006 Integrating Excisable Non integrating Small molecule Protein virus vector vector replacements Page 2 ver 1 020711 www systembio com piPS Cells Cats SC801A 1 SC802A 1 B Protein derived iPS cell line SBI offers the human protein iPS cell lines highlighted in Cell Stem Cell Generation of Human Induced Pluripotent Stem Cells by Direct D
6. ed for use in humans or for therapeutic or diagnostic use The Product may not be resold modified for resale or used to manufacture commercial products without prior written consent of SBI This Product should be used in accordance with the NIH guidelines developed for recombinant DNA and genetic research This Product shall be used by the purchaser for internal research purposes only and distribution is strictly prohibited without written permission by System Biosciences Limited Warranty SBI warrants that the Product meets the specifications described in the accompanying Product Analysis Certificate 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 SBI s 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 other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particu
7. elivery of Reprogramming Proteins Kim D et al 2009 4 472 476 The piPS cell lines were derived from the most commonly used source cells newborn human fibroblasts To date all methods to generate iPSCs require the use of genetic materials and or potentially mutagenic chemicals Using protein engineering technology stable piPSCs were induced from human fibroblasts by directly delivering four reprogramming proteins Oct4 Sox2 KIf4 and c Myc fused with a cell penetrating peptide These piPSCs exhibited similarities to human embryonic stem cells in morphology proliferation global gene expression DNA methylation patterns and expression of characteristic pluripotency markers PiPSC lines produced with recombinant proteins were successfully maintained for more than 35 passages and differentiated into derivatives of all three embryonic germ layers during the formation of embryoid body in vitro and teratomas in vivo the most stringent tests for the quality of human iPS cells This protein reprogramming system eliminates the potential risks associated with the viruses DNA transfection and potentially harmful chemicals Therefore it provides a promising safe source of patient specific cells for the future regenerative medicine Pluripotency of PiPSCs cat SC801A 1 SC802A 1 I oe Phase Contrast AP Staining SSEA4 Nanog Oct4 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual Mult
8. ials to a liquid nitrogen tank for long term storage Mitomycin C treatment of MEF At confluence MEF cells are treated with mitomycin C to halt the division of the cells when they are still able to condition the medium as the feeder layers for human iPS cells 1 Add 6 mL of fresh MEF medium containing 50 ul of mitomycin C solution 1mg ml to one T75 flask of confluent MEF cells and swirl it briefly Incubate at 37 C for at least 3 h After incubation aspirate the mitomycin C containing medium off the cells and wash the cells twice with 10 ml of PBS 4 Aspirate off PBS add 1 ml of 0 25 trypsin EDTA swirl to cover the entire surface and incubate for 1 min at room temperature 5 Add 5 ml of MEF medium and break up the cells to a single cell suspension by pipetting up and down Count the number of cells Seed the cells on gelatin coated dishes 1x 10 cells per 100 mm dish or 1 5 x 10 cells per well of 6 well plate 6 Cells should be ready to use by the next day Growth condition for human iPS cells 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 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI User Manual 1 Remove the vial from liquid nitrogen and thaw quickly in 37 C wate
9. iple Lineage Potential of PiPSCs Formation Teratoma Embryoid Body Formation Nestin Tuj Rosette HNF3beta Epithelium YE Desmine Cartilage Page 4 ver 1 020711 www systembio com piPS Cells Cats SC801A 1 SC802A 1 ll Protocols A Materials Human ESC medium tare Component Cat concentration Source Knockout serum 10828028 20 Invitrogen replacement Glutamax 1 35050061 2 mM Invitrogen A amino 11140050 1x 107M Invitrogen 2 mercaptoethanol M7522 1x10 M Sigma penicillin and 50 U and 50 streptomycin 15140122 ug ml Invitrogen 233 FB bFGF 025 10 ng ml R amp D KO DMEM F12 12660012 Invitrogen MEF medium Final Component Cat eareenirilon Source FBS 16000077 10 Invitrogen Glutamax 1 35050061 2mM Invitrogen penicillin and 50 U and 50 ug streptomycin 15140122 Iml Invitrogen DMEM 11995065 Invitrogen 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System Biosciences SBI User Manual Other Required Reagents Component Cat Final concentration Source Rock Inhibitor Y Y0503 10 uM ml Sigma 27632 Dissolve 0 5 g of gelatin from porcine 0 1 w v skin in 500 ml DPBS Gelatin G1890 and autoclave Stable Sigma for 1 yr at room temperature Aliquot in 10 ml and Accutase SCRO005 store in 20 C Dilute Millipore 1 1 with DPBS before use Human ES freezing medium
10. lar 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 2011 System Biosciences SBI All Rights Reserved Page 14 ver 1 020711 www systembio com
11. m Cell 2009 4 472 476 Fangjun Jia et al A nonviral minicircle vector for deriving human iPS cells Nature Methods 2010 7 3 197 9 Page 12 ver 1 020711 www systembio com piPS Cells Cats SC801A 1 SC802A 1 IV Technical Support For more information about SBI products and to download manuals in PDF format please visit our web site http www systembio com For additional information or technical assistance please call or email us at Phone 650 968 2200 888 266 5066 Toll Free Fax 650 968 2277 E mail General Information info systembio com Technical Support tech systembio com Ordering Information orders systembio com System Biosciences SBI 265 North Whisman Road Mountain View CA 94043 V Licensing and Warranty Use of the P iPS cell lines 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 The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual or warrant
12. r bath 2 Remove the vial from the water bath as soon as the cells are half way thawed and sterilize by spraying with 70 ethanol 3 Transfer the cells with 10 ml of human ES medium to a 15 cm conical tube and pellet the cells by centrifugation at 200x g for 5 min 4 While centrifuging remove MEF medium from the 6 well plate with MEF feeder cells wash the well twice with 1 ml of KO DMEM F 12 and add 1 ml of human ES medium supplemented with ROCK inhibitor Y 27632 10 uM 5 Discard the supernatant of the tube containing human iPS cells resuspend the cells with 1 ml of fresh human ES medium with ROCK inhibitor Y 27632 and plate the cells in the wells of 6 well plate with MEF feeder cells 6 Incubate at 37 C with 5 CO in air atmosphere until the cells reach 80 confluency 7 Change the medium everyday Note Y 27632 is not necessary for regular human iPS cell culture Maintenance of human iPS cells It is important to note that do NOT keep human iPS cells in culture for long periods in order to maintain the pluripotency 1 Aspirate the medium and wash the cells twice with 1 ml of PBS 2 Remove PBS completely add 0 5 ml of 1 1 Accutase to each well of a 6 well plate and incubate at room temperature for 1 min 3 While incubating remove a 6 well plate with MEF feeder cells from the incubator Aspirate MEF medium wash with 1 ml of KO DMEM F12 twice for each well and add 1 ml of human ES medium with 10 uM RO
13. ze 2 Remove PBS completely add 0 5 ml of 1 1 Accutase and incubate at room temperature for 1 min 888 266 5066 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual 3 While incubating remove a 6 well plate with MEF feeder cells from the incubator Aspirate MEF medium wash with 1 ml of KO DMEM F12 twice for each well and add 1 ml of human ES medium to each well 4 When the edge of the human iPS cell colonies fold up aspirate Accutase solution and wash the cells three times with KO DMEM F12 medium 5 Add 1 ml per well of human ES medium and dislodge the cell colonies using cell scraper 6 Transfer the contents into a 15 ml conical tube with 5 ml of pre warmed human ES medium Use another 1 ml of medium to wash the well one more time and combine it to the same tube Centrifuge at 200x g for 5 minutes at room temperature Carefully aspirate the overlaying supernatant then gently finger tap the tube bottom to dislodge the cell pellet 9 Gently add 2 ml of human ES freezing medium supplemented with 10 uM Y 27632 resuspend the cells by gently pipetting up and down and aliquot it at 1 ml per vial 10 Put the vials in a cell freezing container and store the vials at 80 C overnight 11 Transfer the vials to liquid nitrogen for long term storage lll References Kim D et al Generation of Human Induced Pluripotent Stem Cells by Direct Delivery of Reprogramming Proteins Cell Ste
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