DIFFERENTIATED HepaRG -NS CRYOPRESERVED Description and user guide For thawing, culture and use

Transcription

1 DIFFERENTIATED HepaRG -NS CRYOPRESERVED Descriptin and user guide Fr thawing, culture and use Catalg number: HPR116NS BACKGROUND HepaRG cells have the unique prperties f maintaining significant levels f hepatic cell functins, f being CYP450 inducible and supprting the cmplete replicative cycle f HBV. HPR116NS is a new frmat f the HPR116 with a freezing prcess that allws direct thawing and seeding f crypreserved differentiated cells withut the need fr pst-thaw washing, centrifugatin and cunting steps. This descriptin and use guide fr the thawing and culture f differentiated HepaRG -NS crypreserved includes three sectins: SECTION 1: MATERIALS, MEDIA AND CELLS... 2 SECTION 2: PROTOCOL FOR THE THAWING, SEEDING AND MAINTENANCE OF DIFFERENTIATED HEPARG -NS CRYOPRESERVED... 4 SECTION 3: CELL MORPHOLOGY LIMITED USE LICENSE HepaRG cells are patented and their use is strictly limited; cnsider the cells as a single-use, dispsable prduct that must be destryed upn cnclusin f a study r experiment. Prpagating, reprducing, clning, subclning r any ther use f the cells fllwing the cnclusin f a study is prhibited. Use f the cells t prduce r manufacture cmmercial prducts fr general sale r fr use in the manufacture f prducts intended fr general sale is prhibited. Transfer f the cells t anyne nt emplyed within the same rganizatin, whether fr financial benefit r nt, is prhibited. If yu are unwilling t accept the terms f this LIMITED USE LICENSE, d nt ORDER r use them, and immediately return the cells fr credit. Vilatrs f this Limited Use License will be prsecuted t the fullest extent f the law. Fr mre infrmatin and all publicatins n HepaRG, visit Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 1/11

2 SECTION 1: MATERIALS, MEDIA AND CELLS 1. Materials Water bath at +37 C Laminar flw hd Pipet-aid, pipettes and micrpipettes Multichannel pipettes 70% alchl prep/wipe/swab 40 ml Plystyrene rund-bttm tubes and 92 x 17 mm petri dishes r similar cntainers Incubatr at +37 C, 5% CO 2 and saturating humidity Phase-cntrast micrscpe Material fr cell cunt: cell cunting chamber, cverslips, 0.05% Trypan blue slutin 2. Cated cell culture supprts The fllwing are prvided by BIOPREDIC Internatinal with HepaRG, HPR116NS. Designatin Reference Shipping Cnditins f Strage 6-well plate cated with cllagen I* PLA C +4 C 12-well plate cated with cllagen I* PLA C +4 C 24-well plate cated with cllagen I* PLA C +4 C 48-well plate cated with cllagen I* PLA C +4 C 96-well plate cated with cllagen I* PLA C +4 C * BIOPREDIC Internatinal prprietary cating prcess t ensure prper seeding and culture f the HPR116NS Cllagen-cated plates will allw cells t attach faster, usually within three hurs; uncated plates generally require 4-6 hurs fr attachment t ccur. 3. Media Article Prvider Reference Strage Williams E Medium, n phenl red Invitrgen TM A * +4 C GlutaMAX TM I, 200 mm Invitrgen TM 35050* -20 C BIOPREDIC Internatinal recmmends Williams E Medium and GlutaMAX TM -I frm Invitrgen TM nly. *References in Nrth America The fllwing supplements are available frm BIOPREDIC Internatinal. Designatin HepaRG -NS Thawing and Plating Medium Supplement with crticids HepaRG -NS Maintenance/Metablism Medium Supplement with crticids HepaRG -NS Pre-inductin and Tx Medium Supplement with crticids HepaRG -NS Serum-free Inductin Medium Supplement with crticids Reference ADD411 ADD421 ADD431 ADD451 Cnditins f Shipping Strage -20 C r lwer -20 C r lwer -20 C r lwer -20 C r lwer Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 2/11

3 4. Cells HPR116NS 8M viable cells / 2 ml vial, shipped n dry ice r in LN dewar. If n dry ice, ensure shipment time did nt exceed 48 hurs. Immediately place the cryvial(s) in liquid nitrgen upn receipt. Keep the certificate f analysis handy fr calculating the ttal suspensin vlume, r recrd the ttal ppulatin f viable cells accrding t the certificate f analysis infrmatin fr use in sectin 2, 2.2. Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 3/11

4 SECTION 2: PROTOCOL FOR THE THAWING, SEEDING AND MAINTENANCE OF DIFFERENTIATED HEPARG -NS CRYOPRESERVED YOUR SAFETY OBSERVE UNIVERSAL PRECAUTIONS WHEN HANDLING HepaRG -NS CELLS AND TREAT ALL BIOLOGIC MATERIAL AS POTENTIALLY INFECTIOUS. THE FOLLOWING STEPS MUST BE PERFORMED UNDER A LAMINAR FLOW HOOD. 1 Media preparatin 1.1 Base medium preparatin Cmbine 99 ml f Invitrgen Williams E medium # A with 1 ml f Invitrgen GlutaMAX I # in a sterile cntainer. 1.2 Wrking Medium Preparatin Thaw the HepaRG -NS Supplement by placing the bttle in a +37 C water bath until cmpletely thawed. Prepare wrking HepaRG -NS Medium by adding the entire cntent f the bttle f HepaRG -NS Supplement t 100 ml f Base Medium. Wrking HepaRG -NS Medium shuld be stred at +4 C fr a maximum f ne mnth. See Sectin 2, 4 fr detailed instructins abut medium change. 2 Thawing and Seeding HepaRG cells 2.1 Thawing and suspending cells 1. Pre-warm wrking HepaRG -NS Thawing/Plating Medium 411 in a +37 C water bath. 2. Fr the thawing f ne vial, pipet 7.5 ml f pre-warmed, wrking HepaRG -NS Thawing/Plating Medium 411 int a sterile 40 ml plystyrene rund-bttm tube r similar cntainer. 3. Remve the cryvial frm the liquid nitrgen strage. Nte: Steps 4, 5, and 6 need t be accmplished within fur minutes t avid risk f any rm temperature thawing which will affect cell viability. 4. Under the laminar flw hd, briefly twist the cryvial cap a quarter t release internal pressure, and then clse it again a quarter turn. 5. Quickly transfer the cryvial int the +37 C water bath. D nt submerge the vial cmpletely, d nt allw water t penetrate int the cap. While hlding the tip f the cryvial, agitate the vial in a back and frth manner fr 90 secnds. Small ice crystals shuld remain when remved frm the water bath. 6. Under the laminar flw hd, wipe the utside f the cryvial with the 70% ethyl alchl. Then aseptically transfer with a pipette the semi -thawed HepaRG -NS cell suspensin int the 40 ml tube cntaining the pre-warmed wrking HepaRG -NS Thawing/Plating Medium. 7. Pipet apprximately 1 ml f the cell suspensin frm abve int the cryvial, swirl it, and return the resulting suspensin t the 40 ml tube, t ensure yu have recvered all cells. 8. Reduce cell clusters by using a pipette by gently drawing the suspensin int the pipette and then pipeting back ut. The cell suspensin shuld be hmgeneus t allw cells t plate evenly, but, under a micrscpe, it is nrmal t have sme clusters, as shwn belw. Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 4/11

5 Clusters Clusters X100 X Seeding the thawed HepaRG -NS Nte: HPR116NS is a new frmat that allws direct thawing and seeding f crypreserved differentiated cells withut the need fr pst-thaw washing, centrifugatin and cunting steps. But if a cunting is needed, fllw instructin in step Calculating the ttal vlume f suspensin/vial f cells The final vlume f suspended cells needed fr different well plate frmats is prvided n each lt s Certificate Of Analysis (Sec 3, Cnditins f Cell Seeding) based n the ttal ppulatin f viable cells/vial fr that specific lt. Once the cells are thawed prperly the initial vlume will be 8 ml, s the ttal vlume shwn includes that; in ther wrds, if the ttal vlume shwn n the COA is 10.4 ml, then 2.4 ml f wrking HepaRG -NS Thawing/Plating Medium shuld be added t the tube with the thawed cells. Fr general calculatin f vials and media needed, fllwing are the vlumes f suspensin and cells per well r per plate fr different multiwell plate cnfiguratins when the viable cell ppulatin in a vial is 8M cells: Well plate Ttal vlume f Media with Suspended Cells (ml) Required number f cells (10 6 ) Per Well Per Plate Per well Per Plate 6 well plate well plate well plate well plate well plate Seeding the plates Accrding t the cell culture supprt used, dispense in each well the vlume f suspended cells indicated in the table abve, with a micrpipette r a multichannel pipette. Except fr the 96 well plates, gently agitate the supprts in a back-and-frth and side-t-side manner, and visually cntrl the hmgeneity f the cell distributin. If 96 well plate(s) are seeded partially, fill the wells surrunding thse cntaining the cells with sterile water. Place the plate(s) in the incubatr at +37 C, 5% CO 2 and saturating humidity. Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 5/11

6 3 Cell viability and cunting Optinal and nt recmmended Nte: Steps 2.1 and 3 need t be accmplished within 30 minutes t avid damaging the cells. Cell viability measurement and cell cunting are determined by trypan blue (0.05% in D-PBS 1X) exclusin test. 1. Transfer 900 µl f trypan blue slutin (0.05% in D-PBS 1X) in a 5 ml plystyrene rund bttm tube. 2. Prepare a cell cunting chamber (e.g., Nagette chamber). T prepare the cunting chamber the mirrr-like plished surface is carefully cleaned with lens paper. The cverslip is als cleaned. The cverslip is placed ver the cunting surface befre prir t putting n the cell suspensin. 3. Gently hmgenize the cell suspensin by manual swirling. 4. Dilute 100 µl f the cell suspensin in the 900 µl f trypan blue slutin at 0.05%. 5. Gently hmgenize the btained cell suspensin. 6. Intrduce with a pipette arund 100 µl f cell suspensin between mirrr-like plished surface and cverslip. The area under the cverslip fills by capillary actin. Enugh liquid shuld be intrduced s that the mirrred surface is just cvered. 7. Prceed t bservatin under micrscpe. Cunt living and dead cells n at least fur rws distributed thrughut the cell cunting chamber. Living cells exclude the dye; dead cells take up the dye and appear blue. If the ttal number f cells is very different frm ne rw t anther, cunt ne r tw mre rws. Careful: Thawed differentiated HepaRG -NS cells can frm clusters. It s necessary t cunt all the cells including thse frming the clusters. 8. Determine the average number f viable cells and dead cells per rw. 9. Determine percentage f cell viability. Number f viable cells X 100 Number f viable cells + number f dead cells 10. Calculate the cell cncentratin in millin cells/ml. Sample calculatin with a Nagette chamber: Number f viable cells per rw X 10 (dilutin factr in trypan blue) X 800 (parameter relating t the Nagette cell)= M cell / ml 11. Calculate the ttal viable cell number: Cell cncentratin in millin cells / ml X Ttal vlume f cell suspensin = ttal number f cells 4 Medium change Suggestins abut remving/adding culture medium frm well plates with mnlayer cells. 4.1 Remving culture medium Slightly tilt the plate t reach the medium in the bttm f each well. Yu can use a vacuum aspiratin system r manual pipetr, but leave a small vlume f medium in the well, ding s helps t avid tuching the mnlayer. Take care t avid tuching the mnlayer with pipet tips. 4.2 Replenishing with new culture medium Pipet new medium dwn the walls f each well, let the fluid first tuch the plastic and then cllect n the mnlayer. Using a multichannel pipettes is recmmended, and avid high velcities in pipeting the medium int the wells. Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 6/11

7 5 Use f differentiated HepaRG -NS cells 5.1 METABOLISM Studies: Use f HepaRG in suspensin After thawing f differentiated HepaRG cells (Sectin 2, 2), cells can be used fr the metablism studies in suspensin accrding t yur standard prtcl with human hepatcytes. Incubate the cells with the test substrates accrding t yur prtcl fr metablism studies. SUSPENSION Day 1 - Thaw the cells using HepaRG -NS Thawing/Plating Medium Incubate the cells with the test substrates accrding t yur prtcl 5.2 METABOLISM studies: use f HepaRG in mnlayer Cell seeding See sectin 2, Cell maintenance fr metablism studies Yu have tw ptins: Either use the cells rapidly after thawing, r fllwing at least 3 days f culture. HepaRG keep a high level f CYP activities during the first 24 hurs fllwing thaw and plating, and these activities then decrease while the cells recnstitute the mnlayer, then the activities return during the furth day in culture, peaking at Day 7. At day 1, 4 hurs after plating Fur hurs after plating, bserve cell mrphlgy under phase-cntrast micrscpe, and when pssible, take phtmicrgraphs, fr yur wn recrds and als fr the best assistance frm yur vendr s experts. It is difficult fr the HepaRG experts t diagnse issues and prpse slutins withut pictures. Cells can be used fr the metablism studies accrding t yur standard prtcl with human hepatcytes. Incubate the cells with the test substrates accrding t yur prtcl fr metablism studies. MONOLAYER Day 1 4 hurs after plating - Thaw and seed the cells using HepaRG -NS Thawing/Plating Medium Fur hurs after plating, incubate the cells with the test substrates accrding t yur prtcl Day 5-Day 8 One day after thawing, bserve cell mrphlgy under phase-cntrast micrscpe, and when pssible, take phtmicrgraphs. Change frm the HepaRG -NS Thawing/lating Medium 411 t the HepaRG Maintenance/Metablism Medium 421. Maintain the HepaRG cells in HepaRG Maintenance/Metablism Medium 421 and use the cells: At Day 5 At day 5 after thawing and culture: a cell mnlayer can be bserved with a hepatcytelike cell rganizatin in clusters and metablic activities are slightly lwer than activities detected frm fresh cells. Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 7/11

8 MONOLAYER Use at Day 5 96 hrs Day 1 Thursday Thaw and seed the cells using HepaRG -NS Thawing/Plating Medium 411 Day 2 24 hrs Friday Remve HepaRG -NS Thawing/Plating Medium 411, and replace with the HepaRG Maintenance/Metablism Medium 421 Day 5 96 hrs Mnday Incubate the cells in mnlayer with the test substrates accrding t yur prtcl At Day 8 Fr ptimal activity levels, Maintenance/Metablism Medium 421 must have been renewed at Day 5 and Day 7. At day 8 after thawing and culture: cells are rganized in well-delineated trabeculae with many bright canaliculi-like structures and basal metablic activities similar t fresh cells. MONOLAYER Use at Day hrs Day 1 Thursday Thaw and seed the cells using HepaRG -NS Thawing/Plating Medium 411 Day 2 24 hrs Friday Remve HepaRG -NS Thawing/Medium 411, and replace with the HepaRG Maintenance/Metablism Medium 421 Day 5 96 hrs Day hrs Mnday Renew the HepaRG Maintenance/Metablism Medium 421 Wednesday Renew the HepaRG Maintenance/Metablism Medium 421 Day hrs Thursday Incubate the cells in mnlayer with the test substrates accrding t yur prtcl Nte: Cells can be used fr the metablism studies frm Day 5 t Day 8 accrding t yur standard prtcl with human hepatcytes. They can als be kept in HepaRG -NS Maintenance/Metablism Medium 421 fr 1 additinal week, prvided that renewal f the HepaRG -NS Maintenance/Metablism Medium 421 is perfrmed every 2-3 days. 5.3 INDUCTION Studies Cell seeding See sectin 2, Culture and maintenance fr inductin studies Six hurs after plating, bserve cell mrphlgy under phase cntrast micrscpe, and when pssible, take phtmicrgraphs. Remve HepaRG -NS Thawing/Plating Medium 411, and replace with the Pre-inductin/Tx Medium 431. Vlumes are specified in the tables in sectin 2, 2.2. At day 4, after 72 hurs f culture, bserve cell mrphlgy under phase-cntrast micrscpe, and when pssible, take phtmicrgraphs. Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 8/11

9 Change frm the HepaRG -NS Pre-inductin/Tx Medium 431 t HepaRG -NS Serum-free Inductin Medium 451 with the test articles. Incubate the cells with the test articles fr 48h. Renew the medium with the test articles daily with the HepaRG -NS Serum-free Inductin Medium 451. Nte: Maximal fld inductin f metablic activity may be achieved with 72 hurs treatment time, but vendr's data indicate that 48 hurs f treatment is sufficient t demnstrate significant inductin f CYP1A2, CYP2B6, and CYP3A4 metablic activity using prttypical inducers. Fr assessment f enzyme inductin by measuring mrna levels, 24 hurs treatment time is applied in mst cases, but 48 hurs incubatin is als retained by sme users Suggested timeline fr inductin studies Day 1 Friday mrning Thaw and seed the cells using HepaRG -NS Thawing/Plating Medium 411. Day 1 6 hrs Day 4 72 hurs Day 5 96 hurs Day hurs Friday end f afternn (6 h after plating) Mnday mrning Tuesday mrning Wednesday mrning Remve HepaRG -NS Thawing/Plating Medium 411, and replace with the Pre-inductin/Tx Medium 431. Remve the HepaRG -NS Pre-inductin/Tx Medium 431, and replace with the HepaRG -NS Serum-free Inductin Medium 451. Incubate the cells in mnlayer with the test articles accrding t yur study design. The renewal f the medium with the test articles shuld be perfrmed daily until Wednesday. Renew the HepaRG -NS Serum-free Inductin Medium 451 with the test articles. End f the incubatin with the test articles. Incubate the cells with the test substrates. 5.4 TOXICITY Studies Cell seeding See sectin 2, Culture and maintenance fr txicity studies One day after thawing, bserve cell mrphlgy under phase-cntrast micrscpe, and when pssible, take phtmicrgraphs. Remve HepaRG -NS Thawing/Plating Medium 411, and replace with the Pre-inductin/Tx Medium 431. Maintain the cells in HepaRG -NS Pre-inductin/Tx Medium 431 until the use f cells, at least 7 days, with a medium renewal every 2-3 days. Renew the HepaRG -NS Pre-inductin/Tx Medium 431, and incubate the cells in mnlayer with the test articles accrding t yur prtcl Suggested timeline fr txicity studies at day 7 Day 1 Thursday Thaw and seed the cells using HepaRG -NS Thawing/Plating Medium 411. Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 9/11

10 Day 2 24 hurs Day 5 96 hurs Day hurs Day hurs Friday Remve HepaRG -NS Thawing/Plating Medium 411, and replace with the Pre-inductin/Tx Medium 431. Mnday Renew the HepaRG -NS Pre-inductin/Tx Medium 431. Wednesday Renew the HepaRG -NS Pre-inductin/Tx Medium 431. Thursday Remve the HepaRG -NS Pre-inductin/Tx Medium 431 and incubate the cells in mnlayer with the test articles accrding t yur prtcl 5.5 UPTAKE AND TRANSPORT studies: use f HepaRG IN SUSPENSION - After thawing f differentiated HepaRG cells (Sectin 2, 2), cells can be used fr uptake and transprt studies in suspensin accrding t yur standard prtcl with human hepatcytes. - Incubate the cells with the test substrates accrding t yur prtcl fr uptake and transprt studies. SUSPENSION Day 1 - Thaw the cells using HepaRG -NS Thawing/Plating Medium Incubate the cells with the test substrates accrding t yur prtcl Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 10/11

11 SECTION 3: CELL MORPHOLOGY 6 hurs after plating, cells attach and spread t frm a mnlayer clse t the ne bserved with human hepatcytes (fig 1). After hurs f culture, a restructuring f cell mnlayer can be bserved with a hepatcyte-like cells rganizatin in clusters (fig 2). 120 hurs after plating, hepatcyte-like cells are rganized in well-delineated trabeculae with many bright canaliculi-like structures (fig 3). D1-6 hrs D4-72 hrs D6-120 hrs Fig 1 X100 Fig 2 X100 Fig 3 X100 Nte: Phtmicrgraphs taken n 96 well plates t be clser with reality, explaining a slight ptical distrtin. Differentiated HepaRG NS crypreserved Descriptin and user guide Thawing, culture and use 11/11