User Manual - System Biosciences
Contents
1. SBI System Biosciences Disease Specific iPS Cells Cat SC60XA Series User Manual Store in Gas Phase of 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 031011 contained in this user manual Disease Specific iPS Cell Lines Cat SC60xA xxx Contents Human Disease Specific iPS Cell eeeeeseeeeeeeees 3 A Description 2 2 e cece cece eeeeeeceeeeeeeaeeeeaaeseeeeeseaeeeseaeeeaaeeeeeneeeaas 3 B Type Diabetes iPS cell line Cat SC602A DT 3 C Metachromatic Leukodystrophy MLD iPS cell line Cat SGG60UASMLD zrase E aE aaa 4 D Culture conditions for MEF feeder Cells ccssceeeeees 5 Culture Conditions for HFF feeder cells 9 E F Growth Conditions for Human Disease Specific iPS cells 11 G Validation of human iPS Cells 0 cceeeeceeeeesteeeeeeees 15 Ile FROTEKONCOS ace soeeetesec dines dances sedans aa iaaa aaa 17 Ill Technical Support ce eesceceeeceeeeeeeseceeeeeeseaeeesaeeseneeeeaees 18 IV Licensing and Warranty Statement 18 888 266 5066 Toll Free 650 968 2200 outside US Page 1 System Biosciences SBI User Manual List of Components Each disease specific iPS cell line set comes as one vial with 2 x 10 cells The product is shipped on dry ice and should be immediately stored in the gas phase of liquid ni2. outside US Page 17 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 Disease Specific 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 for 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
3. Biosciences SBI User Manual 2 3 Incubate the gelatin coated dishes for at least 15 min at 37 Aspirate excess gelatin solution before use 3 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 104 cells cm 1 2 6 Remove the vial from liquid nitrogen and thaw quickly in a 37 C water bath Remove the vial from the water bath as soon as the cells are half thawed and sterilize the tube 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 200 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 10 cells cm Incubate at 37 C with 5 COs until the cells reach 80 90 confluency Change medium twice a week or when pH decreases 4 Passaging MEF cells Cells should be split when they reach confluency We recommend splitting the cells based on 0 5x10 cells cm 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 Page 6 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx 3 Add 5 ml of MEF medium and break up the cell clumps by gently pipetting up and down several time
4. and SSEA4 top right were determined by immunocytochemistry using antibodies for SSEA4 Abcam and Nanog Abcam followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Phase contrast bottom left shows the morphology of the MLD iPS cell line Positive AP staining Cat AP100R 1 is shown on the bottom right Page 16 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx 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 germline 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
5. markers or drug selection When cultured under standard human ES cell culture conditions the morphology of SBI human disease specific iPSCs is identical to that of human ES cells The cells also express the pluripotency markers SSEA 4 and Nanog and demonstrate a strong endogenous AP activity All disease specific iPS cell lines have also been karyotyped Human iPS cells must be grown either on a feeder cell layer Appropriate feeder cells for human iPS cells are mouse embryonic fibroblasts MEFs available from Applied Stemcell Inc htto Awww appliedstemcell com or human foreskin fibroblasts HFFs available from System Biosciences Cat PC502B HFF B Type I Diabetes iPS cell line Cat SC602A DTI Type Diabetes is an autoimmune condition where the pancreatic beta cells are destroyed Patients display diabetes related autoantibodies The cause of type diabetes is polygenic and chronic insulin therapy is required for survival 888 266 5066 Toll Free 650 968 2200 outside US Page 3 System Biosciences SBI User Manual The type diabetes human iPS cells were derived from human patient fibroblasts from cultured skin explants from a single donor The patient was a 29 year old female of Hispanic Caucasian descent who was diagnosed with Type Diabetes at age 12 C Metachromatic Leukodystrophy MLD iPS cell line Cat SC601A MLD Metachromatic leukodystrophy MLD is a group of disorders marked by storage buildup in the wh
6. to the exponential phase in a 6 well plate Aspirate the medium and wash the cells twice with 2 ml PBS Add 0 5 ml of 1x accutase and incubate 2 min at 37 C Aspirate the accutase Add 1 ml of Knockout DMEM F12 to the plate and carefully wash the feeder cells Add 1 ml of human ES medium to the plate Scrape the colonies off using a cell scraper Transfer the cell suspension to a 15 ml conical tube and spin the cells at 200 g for 5 min Discard the supernatant and resuspend the cells with 0 5 ml FBS Add an equal volume of 2x freezing medium and aliquot it at 1 ml per vial Put the vials in a cell freezing container and store the vials at 80 C overnight 10 Transfer the vials to liquid nitrogen for long term storage Page 14 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx G Validation of human iPS Cells Type Diabetes iPS cell line Stem cell markers for Nanog top left and SSEA4 top right were determined by immunocytochemistry using antibodies for SSEA4 Abcam and Nanog Abcam followed by fluorescent labeled Alexa Fluor secondary antibodies Invitrogen Phase contrast bottom left shows the morphology of the Type Diabetes iPS cell line Positive AP staining Cat AP100R 1 is shown on the bottom right 888 266 5066 Toll Free 650 968 2200 outside US Page 15 System Biosciences SBI User Manual MLD iPS cell line Stem cell markers for Nanog top left
7. 66 Toll Free 650 968 2200 outside US Page 11 System Biosciences SBI User Manual 2 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 2 Remove the vial from liquid nitrogen and thaw quickly in a 37 C water bath Remove the vial from the water bath as soon as the cells are half thawed and sterilize by spraying with 70 ethanol Transfer the cells with 10 ml of human ES medium to a 15 ml conical tube and pellet the cells by centrifugation at 200g for 5 min While centrifuging remove MEF HFF medium from the feeder cell plates and wash the wells twice with Knockout DMEM F12 Then add 1 ml of human ES Medium with 10 uM ROCK inhibitor Y 27632 Discard the supernatant from the human iPS cells and resuspend the cells with 1 ml fresh human ES medium containing 10 uM ROCK inhibitor Y 27632 Plate the cells on MEF or HFF feeder cells After 24 hours remove the media and replace with human ES media without ROCK inhibitor Incubate at 37 C with 5 CO until the cells reach 80 confluency The ES media must be changed every day and human iPS cells subcultured every 5 7 days 3 Maintenance and Passage of human iPS cells In order to maintain pluripotency it is important not to keep human iPS cells in culture for long peri
8. ite matter of the central nervous system in the peripheral nerves and to some extent in the kidneys Similar to Krabb disease MLD affects the myelin that covers and protects the nerves This autosomal recessive disorder is caused by a deficiency of the enzyme arylsulfatase A Both males and females are affected by this disorder and death generally occurs within 6 to 14 years after onset of symptoms MLD has a juvenile onset that is marked by proximal and distal weakness hypotonia decreased or absent tendon reflexes and mild distal sensory loss The MLD iPS cell line were drived from human patient fibroblasts cultured from skin explants from a single They can be used as a model to study other polyneuropathies as well Page 4 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx D Culture conditions for MEF feeder cells 1 Required media and reagents Reagent Information MEF Medium DMEM containing 10 FBS 2 mM glutamine 1x10 M nonessential amino acids and 50 U and 50 ug ml penicillin and streptomycin 2x Cold 20 DMSO and 80 FBS Freezing Media Mitomycin C 1 mg ml solution 2 Gelatin treatment of plates for MEF feeder cells 1 Add enough sterile autoclaved 0 1 gelatin to cover the bottom of the wells Approximate amounts 10cm plate 5 ml 6 well 1 5 ml well 24 well 0 5 ml well 96 well 200 ul well 888 266 5066 Toll Free 650 968 2200 outside US Page 5 System
9. ods of time Page 12 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx 1 2 Aspirate the medium and wash the cells twice with 1 ml of PBS Remove PBS completely and add 0 5 ml of 1x Accutase and incubate for 2 min at room temperature Tap the bottom of the plate to dislodge the cells from the bottom of the plate Then aspirate the accutase Add 1 ml of DMEM F 12 to the plate and carefully wash the feeder cells and aspirate the medium Repeat Add 1 ml of human ES medium containing 10 uM ROCK inhibitor to the plate and suspend the cell colonies by pipetting up and down It is important not to break up the colonies into single cells Remove a plate of MEF or HFF feeder cells from the incubator Aspirate the MEF medium Wash once with KO DMEM F12 medium Distribute 0 2 0 3 ml of the human iPS cell suspension to each well of a 6 well plate Add hES medium with ROCK inhibitor to a final volume of 2 ml per well Right after plating the iPS cells gently swirl the plate back and forth and side to side and incubate at 37 C After 24 hours remove the media and replace with human ES media without ROCK inhibitor The ES media must be changed every day and human iPS cells subcultured every 5 7 days Track the passage number of the iPS cells 888 266 5066 Toll Free 650 968 2200 outside US Page 13 System Biosciences SBI User Manual 4 Freezing human iPS Cells 9 Grow cells
10. 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 2011 System Biosciences SBI 888 266 5066 Toll Free 650 968 2200 outside US Page 19
11. rights or license for use other than those explicitly listed in this Licensing and Warranty Page 18 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx 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 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
12. s 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 5x10 cells cm and incubate at 37 C with 5 COs 5 Freezing MEF cells 1 2 Follow steps 1 4 from the Passaging MEF cells protocol 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 to the cell suspension Aliquot 1 ml of suspension into each cryovial 5 x 10 cells vial Place the vials in a cell freezing container and keep it at 80 C overnight Transfer the vials to a liquid nitrogen take for long term storage 888 266 5066 Toll Free 650 968 2200 outside US Page 7 System Biosciences SBI User Manual 6 Mitomycin C treatment of MEF Mitomycin C acts to halt the division of MEF cells so that they can be used to condition the medium for human iPS cells MEF cells should be at confluence when treated with mitomycin C 1 Add 6 ml of fresh MEF medium contain 50 ul of mitomycin C solution 1 mg ml to one T75 flask of confluent MEF cells and swirl it briefly The final concentration of mitomycin C is 8 yg ml Incubate at 37 C for at least 3 hrs Aspirate the mitomycin C containing medium off the cells and wa
13. ser Manual 3 Thawing HFF 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 104 cells cm 1 2 Remove the vial from liquid nitrogen and thaw quickly in 37 C Remove the vial from the water bath as soon as the cells are half thawed and sterilize by spraying with 70 ethanol Transfer the cells with 10 ml of HFF medium to a 15 cm conical tube and pellet the cells by centrifugation at 200 g for 5 min Discard the supernatant and resuspend the cells with 10 ml fresh HFF medium and plate the cells at seed density of 1 x 10 cells cm Incubate at 37 C with 5 COs until the cells reach 80 90 confluency Change medium twice a week or when pH decreases Page 10 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx F Growth Conditions for Human Disease Specific iPS cells 1 Required media and reagents Reagent Information Human ES DMEM F12 containing 20 knockout Knockout serum replacement 2mM glutamine 0 1 M Medium nonessential amino acids 0 1 M 2 mercaptoethanol 10 ng ml bFGF and 50 U and 50 ug ml penicillin and streptomycin StemPure SBI Cat 5C050 1 May be substituted Medium for Human ES medium 2x Cold Freezing 20 DMSO and 80 FBS Media Knockout Invitrogen DMEM F12 ROCK Inhibitor Sigma Y 27632 Accutase Millipore 888 266 50
14. sh the cells twice with 10 ml PBS Aspirate PBS and add 1 ml of 0 25 trypsin EDTA swirl to cover the entire surface and incubate for 1 min at room temperature Add 5 ml 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 2 x 10 cells per 100 mm dish or 3 x 10 cells per well of a 6 well plate Cells should be ready to use by the next day Page 8 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx E Culture Conditions for HFF feeder cells HFF cells for feeder cells can be obtained from SBI Cat PC502B HFF The HFF cells provided in the iPSC kits are not suitable for use as feeder cells 1 Required media and reagents Reagent Information HFF Medium DMEM containing 10 FBS 2mM glutamine 0 1 M nonessential amino acids and 50 U and 50 ug ml penicillin and streptomycin 2x Cold 20 DMSO and 80 FBS Freezing Media 2 Gelatin treatment of plates for HFF feeder cells optional 1 Add enough sterile autoclaved 0 1 gelatin to cover the bottom of the wells Approximate amounts 10cm plate 5 ml 6 well 1 5 ml well 24 well 0 5 ml well 96 ell 200 ul well 2 Incubate the gelatin coated dishes for at least 15 min at 377 3 Aspirate excess gelatin solution before using 888 266 5066 Toll Free 650 968 2200 outside US Page 9 System Biosciences SBI U
15. trogen 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 culture The methods for culture are nearly identical to HES cell culture although more care and increased maintenance will be required Disease specific iPS cells must be grown on feeder cells for culture MEF cells for feeder cells can be obtained from Applied Stemcell Inc http Avww appliedstemcell com HFF cells for feeder cells can be obtained from SBI Cat PC502B HFF Page 2 ver 1 030811 www systembio com Disease Specific iPS Cell Lines Cat SC60xA xxx I Human Disease Specific iPS Cells A Description SBI and DV Biologics have partnered to develop novel human iPS cell lines from patient derived sources Utilizing iPS cell lines from these disorders represents an oopportunity to recapitulate both normal and pathological tissue formation in vitro for the ideal drug development screening cell line to facilitate new therapeutic discovery and disease modeling Human disease specific induced pluripotent stem cells iPSCs cat were generated by transducing source cells with retroviruses individually encoding the four human transcription factors Oct4 Sox2 Klf4 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
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