Cellartis® Human iPS Cell Lines User Manual
Contents
1. Cellartis DEF CS Additives GF 1 GF 2 and GF 3 should be stored at 20 C shelf life specified on product label At first use thaw provided vials mix gently and aliquot into appropriate volumes Store at 220 C according to expiry date on original vial Thawed vials may be stored at 4 C for up to one week Do not re freeze aliquots after thawing NOTE All three Cellartis DEF CS Additives GF 1 GF 2 and GF 3 are used when thawing and passaging human iPS cells Only Additives GF 1 and GF 2 are needed when changing medium on human iPS cells V Culturing Cellartis Human iPS Cell Lines Cellartis human iPS cell lines are cultured in DEF CS Medium which is sold as part of the Cellartis DEF CS Culture System and subsequently frozen as a single cell suspension with approximately 3 x 10 cells per vial After thawing cells need to be passaged at least once in Cellartis DEF CS Culture System for recovery After this period the cells can be transferred to other media although we recommend to continue culturing them in Cellartis DEF CS Culture System We recommend up to 10 passage expansions in Cellartis DEF CS Culture System from each vial in order to ensure consistent phenotype and genotype A schematic picture of thawing maintenance medium changes and passage and cryopreservation of hiPS cell lines in Cellartis DEF CS Culture System is shown in Figure 1 061215 www clontech com Page 4 of 12 Takara Bio Europe AB A Takara Bio Compa2. 100 ml DEF CS COAT 1 for 100 ml 800 ul DEF CS GF 1 for 100 ml 300 ul DEF CS GF 2 for 100 ml 100 ul DEF CS GF 3 for 100 ml 40 ul O d Qoo Additional Materials Required The following materials are required but not supplied e PBS Dulbecco s with Ca amp Mg D PBS e PBS Dulbecco s without Ca or Mg D PBS www clontech com Page 3 of 12 Takara Bio Europe AB A Takara Bio Company Cellartis Human iPS Cell Lines User Manual e TrypLE Select Enzyme 1X no phenol red e Cell culture vessels Tissue culture treated polystyrene surface e General cell culture equipment used in cell culture laboratory IV General Considerations A Storage and Handling 1 Cellartis Human iPS Cell Lines Cellartis human iPS cell lines should be stored at 150 C The cells can be stored for one year from date of receipt under proper storage conditions Cellartis human iPS cell lines should be maintained in an incubator at 37 C 1 C 5 CO and gt 90 humidity NOTE When transferring the cells from the transport vessel to long term storage immediate transfer is essential since variations in temperature may have an adverse effect on cell survival and quality Cellartis DEF CS 100 Culture System Cellartis DEF CS Basal Medium and Cellartis DEF CS COAT 1 should be stored at 4 C shelf life specified on product label The Cellartis DEF CS Basal Medium formulation contains penicillin and streptomycin
3. CS Culture System eese 9 Figure 3 ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF CS Culture System eese 10 Figure 4 ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF CS Culture System esee 11 Table of Tables Table I Weekly schedule for medium changes and passaging eescesscesecssecsseceeeeeseeesneeeneeeaeeeaeessaecsaecaeceaeesseeeaeeees 6 061215 www clontech com Page 2 of 12 Takara Bio Europe AB A Takara Bio Company 061215 Cellartis amp Human iPS Cell Lines User Manual Introduction Cellartis human induced pluripotent stem iPS cell lines are available from several donors and are supplied from fully characterised cell banks Please refer to the certificate of analysis for cell line specific information such as donor information and karyotype Cellartis human iPS cell lines are delivered with the Cellartis DEF CS 100 Culture System not sold separately which is a complete system for efficient expansion and scale up manufacturing of human iPS cells in a feeder free and defined environment The Cellartis DEF CS Culture System is sold in a 500 ml size as Cat No Y30010 This product should only be handled by persons who have been trained in laboratory techniques and should only be used in accordance with the principles of good cell culture practice Takara Bio Europe AB recommends the use of media and reagents according to this ma
4. EF CS medium RT 6 Rinse the vial with 1 ml supplemented Cellartis DEF CS medium warmed to RT Add to the cell suspension 7 Centrifuge at 300 x g for 1 minute 8 After centrifugation aspirate the supernatant and gently resuspend the pellet in 4 ml supplemented Cellartis DEF CS medium 37 C 1 C It is not necessary to count the cells at this time 9 Pipet the cell suspension into the cell culture unit 10 Ensure that the cells and medium are evenly distributed across the surface of the cell culture unit and place the cell culture unit in the incubator Passaging Cellartis Human iPS Cell Lines As a general rule cells should be seeded at a density of 4 0 5 0 x 10 cells cm use 4 0 x 10 cells cm if leaving the cells four days between passages and 5 0 x 10 cells cm if leaving three days between passages When passaging the cells we strongly recommend growing them to a confluence of 1 5 3 0 x 10 cells cm see Figures 2 4 for images of a variety of Cellartis human iPS cell lines in culture If cultures should appear suboptimal after a few passages we recommend to vary seeding density and passage interval A Preparations Coat cell culture flasks as described above Section VI Prepare the appropriate volume of supplemented Cellartis DEF CS maintenance medium as described above Section VII B and warm it to 37 C 1 C before use Warm all other reagents to RT before use www clontech com Page 7 of 12 Takar
5. Takara Bio Europe AB Cellartis amp Human iPS Cell Lines User Manual Cat Nos Y00185 Y00225 Y00265 Y00275 Y00285 Y00305 Y00315 Y00325 061215 Takara Bio Europe AB A Takara Bio Company Arvid Wallgrens backe 20 SE 413 46 G teborg Sweden Europe Technical Support tech cellartis takara clontech eu United States Canada Asia Pacific Europe Japan 800 662 2566 1 650 919 7300 46 0 31 758 0900 81 0 77 543 6116 Page 1 of 12 Cellartis amp Human iPS Cell Lines User Manual Table of Contents I seleiUiM pH tm 3 M Listo Components cereo pec o eri ost IEEE E ed b itae ih n Pre nan RM er OUR Ce RE nee 3 Cellartis Human iPS Cell Lines aee erit erede nene ere ter eta tei eo Rene rta epo EXER seas KEVER E ETERNE ra vn ea ede 3 B Cellartis DEF CS 100 Cult re System niii ere e a e o eee SERRE Eee pe Ld EA 3 III Additional Materials Required eee tiere tete net Ee eet dare Ferte iet E denne a nba EERTE 3 IV General Considerations cete e e t see e oie PER te Pe ien Lo hasbedabsusouescoueangbUdenaloesdaeselasoareseedoceay 4 As Storage and Handling oet nced eite a eeieea Erie eene FER Hb E EC Rue HAUS caveebdh LEER LEA RES ka ASE EEEE 4 V Culturing Cellartis Human iPS Cell Lines eese eren nennen nennen nne tnt E EA EOE enne 4 VI Coating Cell Culture Vessels tte eremo esteri sede saaphoensetinds cauene tuna sd eases isi eben pe esee vaste epa EES 6 VIL Preparing Cellarti
6. a Bio Europe AB A Takara Bio Company XI XII 061215 Cellartis amp Human iPS Cell Lines User Manual B Passaging 1 Check cells under microscope photo document as necessary 2 Aspirate medium from cell culture flasks and wash the cell layer once with D PBS 3 Add20 ul cm of TrypLE Select to the cell culture flasks and incubate for 5 minutes or until the cell layer has detached Detachment can be aided by swirling the cell culture flask or by tapping the side of the cell culture flask firmly but gently 4 Resuspend the cells in the supplemented Cellartis DEF CS medium and pipet up and down several times to ensure a single cell suspension The cells will aggregate if left too long in TrypLE Select 5 OPTIONAL To remove TrypLE Select if present at greater than 1 10 ratio Centrifuge the cells at 200 x g for 2 5 minutes There is no need to centrifuge the cell suspension after dissociation if the TrypLE Select was diluted at least 1 10 6 Count the cells in a haemocytometer or in a cell counter optimized for the cell type 7 Add the appropriate volume of cell suspension and medium to the newly coated cell culture flasks to obtain the selected density The seeding volume of supplemented Cellartis DEF CS medium should be 0 15 0 25 ml cm 8 Tilt the flask backwards and forwards gently to ensure that the cell suspension is dispersed evenly over the surface then place in the incubator Changing Medium for Cellar
7. age 6 of 12 Takara Bio Europe AB A Takara Bio Company Cellartis amp Human iPS Cell Lines User Manual Vill Thawing Cellartis Human iPS Cell Lines Thaw one vial of your Cellartis human iPS cell line in one 12 5 cm cell culture flask in 4 ml of supplemented Cellartis DEF CS medium 061215 A Preparations Coat cell culture units as described above Section VI Prepare supplemented Cellartis DEF CS medium as described above Section VII A and warm it to the appropriate temperature See below for recommended volumes Thawing Cells NOTE FOR YOUR PROTECTION Wear a protective face mask and protective gloves Use forceps when handling a frozen vial Never hold the vial in your hand as the cryovial may explode due to rapid temperature changes 1 Transfer 4 ml of supplemented Cellartis DEF CS medium to a sterile centrifuge tube and warm to RT 2 Using forceps transfer the vial directly from liquid nitrogen into a container of 37 C 1 C water Thaw the vial by gently pushing it under the surface of the water Do not submerge the cap of the vial in the water bath as this could contaminate the cells 3 Allow the vial to thaw until the cell suspension can be poured out of the vial It is okay if the suspension has a slushy consistency as long as it can be poured out 4 Decontaminate the vial in an appropriate disinfectant 5 Pour the entire contents of the vial into the sterile tube containing 4 ml supplemented Cellartis D
8. chaser This product is for research use only It is not intended for use in therapeutic or diagnostic procedures for humans or animals Also do not use this product as food cosmetic or household item etc This product may not be resold or transferred modified for resale or transfer or used to manufacture commercial products without written approval from Takara Bio Europe AB If you require licenses for other use please contact us by phone at 46 31 758 0900 Your use of this product is also subject to compliance with any applicable licensing requirements as detailed in our catalogues on our website at http www clontech com on the label or other documentation accompanying the goods It is your responsibility to review understand and adhere to any restrictions imposed by such statements Takara and the Takara logo are trademarks of TAKARA HOLDINGS INC Kyoto Japan Cellartis and DEF CS are a trademarks of Takara Bio Europe AB All other trademarks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Takara Bio Europe AB is a Takara Bio company 2015 Takara Bio Europe AB This document has been reviewed and approved by the Takara Bio Europe AB Quality Assurance Department 061215 www clontech com Page 12 of 12 Takara Bio Europe AB A Takara Bio Company
9. nd down 3 Add the appropriate volume of diluted Cellartis DEF CS COAT 1 solution to the cell culture flasks use 0 1 ml cm make sure the entire surface is covered 4 Place the cell culture flasks for a minimum of 20 minutes in the incubator at 37 C 1 C or 0 5 3 hr at room temperature RT 15 25 C 5 Aspirate Cellartis DEF CS COAT 1 solution from cell culture flasks just before use Vil Preparing Cellartis DEF CS Medium A Medium for Thawing or Passaging Human iPS Cells 1 Decontaminate the external surface of all additives and the medium bottle with an appropriate disinfectant and place in the biological safety cabinet 2 Prepare the appropriate volume of supplemented Cellartis DEF CS medium by adding DEF CS GF 1 dilute 1 333 GF 2 dilute 1 1000 and GF 3 dilute 1 1000 to Cellartis DEF CS Basal Medium 3 Prepare fresh medium on the day of intended use Discard any left over warm medium B Medium for Maintenance of Human iPS Cells 1 Decontaminate the external surface of all additives and the medium bottle with an appropriate disinfectant and place into the biological safety cabinet 2 Prepare the appropriate volume of supplemented Cellartis DEF CS medium by adding DEF CS GF 1 dilute 1 333 and GF 2 dilute 1 1000 to Cellartis DEF CS Basal Medium Do not add Cellartis DEF CS GF 3 to maintenance medium 3 Prepare fresh medium on the day of intended use Discard any left over warm medium 061215 www clontech com P
10. nual Takara Bio Europe AB cannot guarantee correct technical feedback on customer cultures unless the below culture instructions have been followed List of Components A Cellartis Human iPS Cell Lines e Cellartis human iPS cell line P11025 Cat No Y00180 not sold separately sold as a part of Cat No Y00185 e Cellartis human iPS cell line P11032 Cat No Y00220 not sold separately sold as a part of Cat No Y00225 e Cellartis human iPS cell line 4 ChiPSC4 Cat No Y00260 not sold separately sold as a part of Cat No Y00265 e Cellartis human iPS cell line 7 ChiPSC7 Cat No Y00270 not sold separately sold as a part of Cat No Y00275 e Cellartis human iPS cell line 12 ChiPSC12 Cat No Y00280 not sold separately sold as a part of Cat No Y00285 e Cellartis human iPS cell line 18 ChiPSC18 Cat No Y00300 not sold separately sold as a part of Cat No Y00305 e Cellartis human iPS cell line 21 ChiPSC21 Cat No Y00310 not sold separately sold as a part of Cat No Y00315 e Cellartis human iPS cell line 22 ChiPSC22 Cat No Y00320 not sold separately sold as a part of Cat No Y00325 B Cellartis DEF CS 100 Culture System e Cellartis DEF CS 100 Culture System Cat No Y30020 not sold separately sold as a part of Cat Nos Y00185 Y00225 Y00265 Y00275 Y00285 Y00305 Y00305 and Y00315 A larger 500 ml version of this product is sold as Cat No Y00310 o DEF CS Basal Medium
11. ny Cellartis Human iPS Cell Lines User Manual Thawing 0 Medium Changes X Cryopreservation Coating VI Medium Preparation XD Medium Preparation VILB VILA Passage IX Coating VI Medium Preparation VILA Figure 1 Schematic presentation of the Cellartis human iPS cell line work flow Corresponding sections of this user manual are referenced in brackets 061215 www clontech com Page 5 of 12 Takara Bio Europe AB A Takara Bio Company Cellartis amp Human iPS Cell Lines User Manual Human iPS cell lines that are maintained in Cellartis DEF CS should be passaged every three to four days with daily medium changes When the cell density is sparse you can change the medium every other day however it is important to change medium the day after passage or thawing and the day before passage or freezing It is recommended that the cells are grown to a maximum confluence of 1 5 3 0 x 10 cells em A suggested weekly schedule is depicted in Table I Table I Weekly schedule for medium changes and passaging Monday Tuesday Wednesday Thursday Friday Saturday Sunday Passage Change Change Passage Change Change medium medium medium medium NOTE Always work under aseptic conditions VI Coating Cell Culture Vessels 1 Dilute the required volume of Cellartis DEF CS COAT 1 in D PBS before use Make a 1 20 dilution 2 Mixthe diluted Cellartis DEF CS COAT 1 solution gently and thoroughly by pipetting up a
12. pe AB A Takara Bio Company Cellartis amp Human iPS Cell Lines User Manual XIII Images of Cellartis Human iPS Cell Lines Maintained in the Cellartis DEF CS Culture System Cell line ChiPSC4 Cell line ChiPSC18 Flattened homogenous layer Flattened homogenous layer EX ds c Pee 4X magnification xx C y mis 10X magnification 20X magnification 061215 www clontech com Page 9 of 12 Takara Bio Europe AB A Takara Bio Company 061215 Cellartis amp Human iPS Cell Lines User Manual Cell line ChiPSC4 Cell line ChiPSC18 Flattened homogenous layer Flattened homogenous layer 4X magnification 10X magnification 20X magnification Culture www clontech com Page 10 of 12 Takara Bio Europe AB A Takara Bio Company Cellartis amp Human iPS Cell Lines User Manual Cell line ChiPSC4 Cell line ChiPSC18 Flattened homogenous layer Flattened homogenous layer 4X magnification 10X magnification 20X magnification 061215 www clontech com Page 11 of 12 Takara Bio Europe AB A Takara Bio Company Cellartis Human iPS Cell Lines User Manual Contact Us Customer Service Ordering Technical Support tel 46 0 31 758 0900 tel 46 0 31 758 0900 fax 46 0 31 758 0910 fax 46 0 31 758 0910 web www clontech com web www clontech com e mail info cellartis takara clontech eu e mail tech cellartis takara clontech eu Notice to Pur
13. s DEF CS Medium leitete dene e eenae a Ph se Rabe oppo e UE aia 6 A Medium for Thawing or Passaging Human iPS Cells eseseseeseeeseeeeeeeeeeen nennen rennen rennen 6 B Medium for Maintenance of Human iPS Cells eese eene rennen eren 6 VIN Thawing Cellartis Human iPS Cell Lines essent irinin ener enne nenne 7 Piz APLC PAaratlOMs M 7 B rvannaedicm 7 IX Passaging Cellartis Human iPS Cell Lines iersinii neii eik aie oreinen is Esia tnn 7 Preparations iuis e te EUR EA EE De E ET EAA A A RE A E EA E RA 7 ME e cic iea aE E T EAEE EEA ERE E EE 8 X Changing Medium for Cellartis Human iPS Cell Lines eese enne nennen nennen 8 lucc E 8 B Medium Change one Ep EIER CELERE Pe Ee ECT blip E eu EE eae eue dives LEER ED dies Le DER AE ET pud ERA 8 XI Cryopreserving Cellartis Human iPS Cell Lines eeeeesesseeeeeeee eene eene emen en rennen nennen 8 XII Transferring to Other Culture Media esses enrennren eren eese neen tenerent nennen 8 XIII Images of Cellartis Human iPS Cell Lines Maintained in the Cellartis DEF CS Culture System 9 Table of Figures Figure 1 Schematic presentation of the Cellartis human iPS cell line work flow 5 Figure 2 ChiPSC4 and ChiPSC18 cells cultured in the Cellartis DEF
14. tis Human iPS Cell Lines Medium change is recommended daily except day of passage Use 0 25 0 4 ml cm of medium If the medium turns yellow due to high metabolic activity increase the medium volume A Preparation Prepare the appropriate volume of supplemented Cellartis DEF CS medium as described above Section VII B and warm it to 37 C 1 C before use Do not add Cellartis DEF CS GF 3 at medium change Discard any leftover warm medium B Medium Change 1 Check cells under microscope photo document as necessary 2 Carefully aspirate the medium and pipet newly warmed medium into the cell culture flask Avoid flushing medium directly onto the cell layer 3 Place the cell culture flask in the incubator Cryopreserving Cellartis Human iPS Cell Lines Cellartis human iPS cells cultured in Cellartis DEF CS Culture System can be cryopreserved using common slow freezing protocols for cell suspensions with STEM CELLBANKER Takara Clontech Cat No CB041 or DMSO and FBS As a general guide 2 5 3 5 x 10fcells in 1 ml freezing medium should be frozen in a 2 ml cryovial Transferring to Other Culture Media We recommend thawing and maintaining Cellartis human iPS cell lines in the Cellartis DEF CS Culture System After thawing and recovery at least one regular passage the iPS cells can be transferred to other culture systems if desired following the instructions of the preferred culture system www clontech com Page 8 of 12 Takara Bio Euro
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