Cellartis® Enhanced hiPS
Contents
1. 1 C Thawing Medium and add to the cells in Thawing Medium 5 Mix by carefully inverting the tube with cell suspension approximately 10 times Do not mix by pipetting 6 Incubate the cell suspension in Thawing Medium at RT for 15 20 minutes Longer incubation may negatively impact cell viability 7 Optional Count the viable cells using a hemocytometer Take 50 ul cell suspension add 5 pl trypan blue count viable cells and multiply the number of viable cells by 1 1 to obtain the concentration of viable cells The cell suspension contains both single cells and small cell clusters Make sure to count the viable cells in the clusters as well 8 Centrifuge at 100 x g at RT for 2 min with the slowest deceleration possible 9 Remove the Thawing Medium with a pipette without disturbing the cell pellet Loosen the cell pellet by flicking the tube and resuspend the cell pellet very carefully by slowly adding 15 ml of 37 C 1 C Plating Medium It is not recommended to use more than 15 ml Plating Medium per vial 10 Remove the Cellartis HEP Coat from the cell culture wells just prior seeding the cells 11 Carefully seed the cells into the coated cell culture units using 150 ul well for 96 well plates or 1 000 ul well for 24 well plates 12 Place the culture vessels in an incubator at 37 C 1 C 5 CO 29096 humidity Ideally plates should be spread out in the incubator avoid stacking of several plates to allow them to warm quickly and2. A Takara Bio Company Cellartis Enhanced hiPS HEP User Manual Notice to Purchaser 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 is a trademark 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 100615 www clontech com Page 10 of 10 Takara Bio Europe AB A Takara Bio Company
3. should not be moved the next day at the earliest 100615 www clontech com Page 6 of 10 Takara Bio Europe AB A Takara Bio Company Cellartis Enhanced hiPS HEP User Manual C Thawing Multiple Vials Several vials can be thawed at the same time using the protocol in section VIII B Consider the following points when thawing multiple vials e Scale up the volume of Thawing Medium according to the number of vials e g 80 ml for four vials e Pour the thawed cell suspensions from all vials into the total volume of Thawing Medium and distribute the cell suspension in 50 ml centrifuge tubes adding no more than 40 ml per tube e Add Plating Medium according to the number of vials e g 30 ml for two vials equally distributed to the centrifugation tubes e Cell suspension in different tubes can be pooled before seeding by combining into one bottle or tube IX Maintenance of Cellartis Enhanced hiPS HEP It is recommended that manual pipetting is used for medium changes instead of a vacuum pump use a multichannel pipette for 96 well plates To make sure that the cells are not left without medium for longer than a few seconds change medium only in four to eight wells at a time NOTE Do not remove the entire Cellartis Enhanced hiPS HEP Maintenance Medium volume Leave approximately 10 of the medium during each medium change making sure that the cells are covered by a thin layer of medium at all times Do not let the cells dry com
4. EP Maintenance Medium to 37 C x 1 C as described in section VILE 2 Medium Change 1 Very gently remove approximately 90 of the Cellartis Enhanced hiPS HEP Maintenance Medium from the cell culture plate and discard i e a maximum of 150 ul per 96 well or 0 94 ml per 24 well Do not use a vacuum pump pipette manually use a multichannel pipette for 96 well plates 2 Very carefully add warm Cellartis Enhanced hiPS HEP Maintenance Medium to the cell culture plate using 0 47 ml cm i e 150 ul per 96 well and 0 94 ml per 24 well 3 Place the Cellartis Enhanced hiPS HEP in an incubator at 37 C 1 C 5 CO 29096 humidity 4 Discard any leftover warm Cellartis Enhanced hiPS HEP Maintenance Medium X Images of Thawed Cellartis Enhanced hiPS HEP Bs z re r 4 j 100615 Page 8 of 10 Takara Bio Europe AB A Takara Bio Company Cellartis Enhanced hiPS HEP User Manual Appendix A CYP Activity Assay After 6 days in culture Cellartis Enhanced hiPS HEP cells can be used for Cytochrome P450 CYP activity assays Samples can be analysed using LC MS to measure the formation of the following specific metabolites acetaminophen CYP1A 4 OH Diclofenac CYP2C9 4 OH Mephenytoin CYP2C19 OH Bufuralol CYP2D6 and 1 OH Midazolam CYP3A A Additional Material Required e HEPES Solution 1 M e L Glutamine Solution 200 mM e CYP Substrate Cocktail Phenacetin Sigma Aldrich Cat No 77440 Bupropion Sigma A
5. Takara Bio Europe AB Cellartis amp Enhanced hiPS HEP User Manual Cat Nos Y10050 Y10056 Y10058 100615 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 Page 1 of 10 800 662 2566 1 650 919 7300 amp 46 0 31 758 0900 81 0 77 543 6116 Cellartis Enhanced hiPS HEP User Manual Table of Contents L Httoductionm uo acie tite redet etin Oen It Lee uem tte Rel cubed totem E Det E iced Pt E tn deu debaters 3 H ListofCompOoLnents oie Er e inei e E ei d cet ia e En ODER vnd abe OD vefte n eri oh eens 3 III Additional Material Required iore eer deter erret nest tad lareo svaedsas sbabasin adea E seri PE ERE OISE uua eoa 3 IV General Considerations iniecto i ERR et ete epe Etui edo ee LED pea EETR Ren LEE cba e be Pepe Les E Pep eee Pag Eg 4 egeo run AsEurilinp ET 4 V Culture of Cellartis Enhanced hiPS HEP sees eene nennen nennen rene en rennen tese trennen nenne 4 VI Coating of Cell Culture Vessels rni there eode ede Eee Co HE ETR REGE BARRE HE TER LEER LRL p eR FEDERE de TUBE Eun 5 VIL Medium Preparation iioi neo iieri d ete ie e n bei RR E He UVP e EE OH dte ic dte aon a Puit ea Ee 5 A X 27632 Stock SOLO deser inenen eth rige ei EEEE E EERE OTEO estu vrbe ep adr a e RRE A ERE ERE 5 B DIhawimg Medium r
6. able of Figures Figure 1 Cellartis Enhanced hiPS HEP cells in culture 00 cele ceeeceseceseceseeeeeeeeeeeeneeeaeeeaeecsaecaaecsaecaeceaeenaeeseeeneeseneeees 8 Table of Tables Table I Recommended Culture Schedule for Cellartis Enhanced hiPS HEP sssssseseeeeeeeeene 4 Table ll CYP Substrate Cocktail iiec c cr er horrea epe EN aee ee EET ERE CERE UNSERE Ce RR NEUE NN Pe ERA CEUEE 9 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 9 takara clontech eu e mail tech cellartis takara clontech eu 100615 www clontech com Page 2 of 10 Takara Bio Europe AB A Takara Bio Company Cellartis Enhanced hiPS HEP User Manual Introduction Cellartis Enhanced hiPS HEP are human hepatocyte like cells derived from human induced pluripotent stem iPS cell lines The cells have been differentiated to hepatocyte like cells in vitro dissociated into cell suspensions and frozen in vials Cellartis Enhanced hiPS HEP cells are provided with coating substrate and maintenance medium supplement 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 reag
7. at ente Ere Re Ree PR PER BOL EE rra LE EE PRU MEER RH La EUR RU BEER a e Eo ERR e EE epe Pe EOD Dep 5 C Plating M dium eene rne ttr tei REEL bik sari os de cused Hbi CoL eg CERE NEL Fe ken Led EEA EE Do taper eo Fee doux To Eden 5 D Base Mamtenance Medt mi 1 rte tetigisse polo tpe exce dx ge sal bep cp ed ape ca ER ees Ra dene ol Ue pue deb puse Dee Lao vp x eden e unu 5 E Cellartis Enhanced hiPS HEP Maintenance Medium eese eene ener enne enne 5 VIII Thawing of Cellartis Enhanced hiPS HEP sees nent tenente tnnt nete enr enn enne 6 PX ucc E 6 B Thawn Cells nee reote e d n e qd tellu pee EIE onsets HR e eH TAIEEN 6 C Thawing Multiple Vials uenire eerte E nete EHE eH eve etr kae IE rue LEER E a SD Ha pe EO a aa SE 7 IX Maintenance of Cellartis Enhanced hiPS HEP essere entrent enr enne 7 Dry LEE 7 VEO np ec 8 X Images of Thawed Cellartis Enhanced hiPS HEP esses enne enne enne nesn tenente enne 8 Appendix As CYP Activily ASSAY uicit pter EE ER essc e ge Uer E u arae dre En Up e PE E Lope PUN aee Ee uS AER 9 A Additional Material Required oinnia rrt sul Epub aget Pr eel dene sh Ue pace tob puso Ld eta erp EE ESEESE 9 I EN Pr pat ath OW p P 9 Co NGUVICJ ASSAY i oe eter e pere eer e Re pe eo ERE AE Pene eo io evo p este AAEREN En t insu ee ea RE Pe Yd exe e Eee Reset 9 T
8. ents according to this manual for optimal performance of the cells Takara Bio Europe AB cannot guarantee correct technical feedback on customer cultures unless the below culture instructions have been followed List of Components e Cellartis Enhanced hiPS HEP from ChiPSC18 Kit Cat No Y10050 o Cellartis Enhanced hiPS HEP from ChiPSC18 Cat No Y10051 not sold separately o 2 tubes Cellartis HEP Coat Cat No Y10052 3 ml o 2 tubes Cellartis HEP Supplement Cat No Y10053 1 ml e Cellartis Enhanced hiPS HEP from ChiPSC22 Kit Cat No Y10056 o Cellartis Enhanced hiPS HEP from ChiPSC22 Cat No Y10057 not sold separately o 2 tubes Cellartis HEP Coat Cat No Y10052 3 ml o 2tubes Cellartis HEP Supplement Cat No Y10053 1 ml e Cellartis Enhanced hiPS HEP from ChiPSC12 Kit Cat No Y10058 o Cellartis Enhanced hiPS HEP from ChiPSC12 Cat No Y10059 not sold separately o 2tubes Cellartis HEP Coat Cat No Y10052 3 ml o 2tubes Cellartis HEP Supplement Cat No Y10053 1 ml Additional Material Required The following materials are required but not supplied e DMSO e InVitroGRO CP medium BioreclamationIVT Cat No Z99029 e InVitroGRO HT medium BioreclamationIVT Cat No Z99019 e Penicillin Streptomycin PEST 10 000 units ml of penicillin and 10 000 ug ml of streptomycin e Williams Medium E WME Life Technologies Cat No 32551 e Y 27632 e Cell culture vessels tissue culture treated polyst
9. g Medium and plated at a density of 4 0 x 10 viable cells cm 150 ul well of a 96 well plate or 1 000 ul well of a 24 well plate To count the number of viable cells refer to step 7 in section VII B However it is not recommended to use more than 15 ml Plating Medium per vial NOTE Newly thawed Cellartis Enhanced hiPS HEP cells are very fragile Do not use a pipette for mixing the cell suspension Only use pipette when seeding the cells A Preparation e Coat the appropriate number of cell culture vessels with Cellartis HEP Coat according to section VI e Prepare and warm the appropriate volume of Thawing Medium and Plating Medium to 37 C 1 C B Thawing Cells NOTE FOR YOUR PROTECTION Wear a protective face mask and protective gloves Use forceps when handling frozen vials Never hold the vial in your hand as the cryovial may explode due to rapid temperature changes 1 Transfer the vial directly from liquid nitrogen to a 37 C 1 C water bath using forceps 2 The cell suspension should be thawed just until it is possible to pour it from the vial including any frozen parts 3 After approximately 1 minute check if the cell suspension is sufficiently thawed by carefully turning the tube upside down Decontaminate the external surface with an appropriate disinfectant and place into the biological safety cabinet 4 Once thawed pour the cell suspension into 19 ml of 37 C 1 C Thawing Medium Wash the vial with 1 ml of 37 C
10. ldrich Cat No B102 Mephenyotin Santa Cruz Cat No sc 200975A Diclofenac Sigma Aldrich Cat No D6899 Bufuralol Becton Dickinson Cat No 451034 o Midazolam Loradan Cat No MID 111 HC e Pierce BCA Protein Assay Kit Life Technologies Cat No 23225 O Q Q OQ Preparation e Prepare a stock solution for the CYP substrate cocktail In the assay use the final assay concentrations listed in Table II e Warm the appropriate volume of Base Maintenance Medium to 37 C 1 C e Prepare and warm CYP activity assay medium Basal Maintenance Medium 25 mM HEPES 2 mM L Glutamine Add the CYP substrate cocktail just prior to use Table II CYP Substrate Cocktail CYP Substrate Final Assay Concentration 1A Phenacetin 10 uM 2B6 Bupropion 10 uM 2C19 Mephenytoin 50 uM 2C9 Diclofenac 10 uM 2D6 Bufuralol 10 uM 3A Midazolam 5 uM Activity Assay 1 Wash Cellartis Enhanced hiPS HEP cells twice very gently with 0 5 ml cm warm Base Maintenance Medium Add 110 ul cm warm CYP activity assay medium to the cells Incubate for 2 hours at 37 C 1 C 5 CO 29096 humidity Collect 100 ul supernatant and store at 80 C until LC MS analysis Determine the amount of protein per well using the Pierce BCA Protein Assay Kit QUARUM Normalize the metabolite concentrations measured by LC MS to the amount of protein per well and the assay duration 120 min 100615 www clontech com Page 9 of 10 Takara Bio Europe AB
11. ml cm Make sure the entire surface is covered The recommended volume for coating is 50 ul well for 96 well plates or 300 ul well for 24 well plates 3 Incubate at room temperature RT 15 25 C for 30 60 min 4 Remove excess Cellartis HEP Coat from the wells just before seeding Vil Medium Preparation A Y 27632 Stock Solution Prepare a 5 mM stock solution of Y 27632 by dissolving 10 mg Y 27632 in 5 92 ml sterile distilled water Aliquot in appropriate volumes and store at 20 C for up to 12 months Thawed aliquots can be stored at 4 C and should be used within one week B Thawing Medium 1 Decontaminate the external surfaces of all bottles with an appropriate disinfectant and place into the biological safety cabinet 2 Add PEST final concentration 0 596 to the whole bottle InVitroGRO HT 250 ml This medium can be used for up to one month when stored at 4 C 3 Add Y 27632 final concentration 5 uM to the required amount of medium to prepare complete Thawing Medium This medium should be freshly prepared immediately before use C Plating Medium 1 Decontaminate the external surfaces of all bottles with an appropriate disinfectant and place into the biological safety cabinet 2 Add PEST final concentration 0 596 to the whole bottle InVitroGRO CP 250 ml This medium can be used for up to one month when stored at 4 C 3 Add Y 27632 final concentration 5 uM to the required amount of medium to prepare complete Plating Medium Thi
12. pletely A Day 1 Wash the hepatocytes twice Base Maintenance Medium and change medium Cellartis Enhanced hiPS HEP Maintenance Medium 0 5 ml cm 1 Preparation e Prepare and warm an appropriate volume of Base Maintenance Medium to 37 C 1 C as described in section VILD e Prepare and warm complete Cellartis Enhanced hiPS HEP Maintenance Medium to 37 C x 1 C as described in section VILE 2 Medium Change 1 Wash the cells twice very gently with warm Base Maintenance Medium 0 5 ml cm to remove unattached cells Do not use a vacuum pump pipette manually use a multichannel pipette for 96 well plates 2 After the second wash step carefully add warm Cellartis Enhanced hiPS HEP Maintenance Medium to the cell culture plate 0 5 ml cm 3 Place the Cellartis Enhanced hiPS HEP in an incubator at 37 C 1 C 5 CO 29096 humidity 4 Discard any leftover warm Cellartis Enhanced hiPS HEP Maintenance Medium 100615 www clontech com Page 7 of 10 Takara Bio Europe AB A Takara Bio Company Cellartis Enhanced hiPS HEP User Manual B Day 3 Onward The medium should be changed every second to third day If leaving the Cellartis Enhanced hiPS HEP without a medium change over the weekend add 50 more medium on Friday i e 1 5 ml well of a 24 well plate and 230 ul well of a 96 well plate It is best to change the medium late on Friday and early on Monday 1 Preparation e Thaw and warm complete Cellartis Enhanced hiPS H
13. s medium should be freshly prepared immediately before use D Base Maintenance Medium 1 Decontaminate the external surfaces of all bottles with an appropriate disinfectant and place into the biological safety cabinet 2 To prepare 500 ml Base Maintenance Medium add 0 1 PEST to 500 ml WME Base Maintenance Medium can be stored at 4 C for up to one month E Cellartis Enhanced hiPS HEP Maintenance Medium NOTE Cellartis Enhanced hiPS HEP Maintenance Medium contains DMSO Therefore use nitrile gloves when preparing and changing medium and discard old medium in a closed container as hazardous waste 1 Decontaminate the external surface of all bottles with an appropriate disinfectant and place into the biological safety cabinet 2 To prepare Cellartis Enhanced hiPS HEP Maintenance Medium add 250 ul DMSO and 1 ml Cellartis HEP Supplement per 49 ml of Base Maintenance Medium 3 Mix the complete medium carefully aliquot appropriate amounts for medium changes day 1 3 5 etc and store the aliquots at 20 C 4 The complete medium can be stored at 20 C for up to one month Once thawed use the same day and discard any remaining media 100615 www clontech com Page 5 of 10 Takara Bio Europe AB A Takara Bio Company Vill Cellartis Enhanced hiPS HEP User Manual Thawing of Cellartis Enhanced hiPS HEP One vial of Cellartis Enhanced hiPS HEP contains 71 23 x 10 viable cells It is recommended that cells are resuspended in 15 ml Platin
14. seeded in 24 or 96 well plates The day after thawing the cultures are washed to remove non attached cells and medium is replaced with Cellartis Enhanced hiPS HEP Maintenance Medium Subsequently medium is changed every second to third day The workflow is depicted in Table I The hepatocytes are ready to use after five days in culture The cells can be maintained at least until day 11 after thawing if handled according to the provided protocol For applications that require high CYP activity use cells between days 6 and day 11 after thawing For optimal results in CYP activity assays change the medium the day before starting the assay Table I Recommended Culture Schedule for Cellartis Enhanced hiPS HEP Corresponding sections of this user manual are referenced in parentheses Day Coating Thawing amp Wash Maintenance Usage High amp Stable VI Plating IX A Medium Window CYP Activity VIII IX A amp IX B 0 0 15 4 0 x 105 ml cm cells cm 0 5 ml cm 1 2 x 0 5 ml cm 0 5 ml cm 2 3 0 47 ml cm 4 5 0 47 ml cm 6 7 0 47 ml cm 8 9 0 47 ml cm 10 11 NOTE Always work under aseptic conditions 100615 www clontech com Page 4 of 10 Takara Bio Europe AB A Takara Bio Company Cellartis Enhanced hiPS HEP User Manual VI Coating of Cell Culture Vessels 1 Thaw the frozen Cellartis HEP Coat overnight at 4 C Keep thawed Cellartis HEP Coat cold until use 2 Add Cellartis HEP Coat to the cell culture vessels 0 15
15. yrene surface e General cell culture equipment used in cell culture laboratory 100615 www clontech com Page 3 of 10 Takara Bio Europe AB A Takara Bio Company Cellartis Enhanced hiPS HEP User Manual IV General Considerations A Storage and Handling Cellartis Enhanced hiPS HEP cells should be stored at lt 150 C Under the recommended storage conditions the cells can be stored for up to one year from the date of receipt Cellartis Enhanced hiPS HEP cells should be maintained in an incubator at 37 C 1 C 5 CO and 29096 humidity Cellartis HEP Supplement should be stored at 20 C and expires as indicated on the label Use Cellartis HEP Supplement to prepare Cellartis Enhanced hiPS HEP Maintenance Medium directly when thawed Cellartis Enhanced hiPS HEP Maintenance Medium should be used fresh or aliquoted and frozen the same day Always discard warmed leftover Cellartis Enhanced hiPS HEP Maintenance Medium Both Cellartis HEP Supplement and Enhanced hiPS HEP Maintenance Medium are light sensitive therefore avoid unnecessary exposure to light Cellartis HEP Coat should be stored at 20 C and expires as indicated on the label Thawed Cellartis HEP Coat should be kept cold until handling and should be used directly V Culture of Cellartis Enhanced hiPS HEP Cellartis Enhanced hiPS HEP cells are thawed in Thawing Medium and plated in coated culture vessels using Plating Medium It is recommended that the cells are
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