DIFFERENTIATED HepaRG CELLS CRYOPRESERVED Descriptin and user guide Fr thawing, culture and use Catalg number: HPR116 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. This descriptin and use guide fr the thawing and culture f crypreserved differentiated HepaRG includes three sectins: SECTION 1: MATERIALS, MEDIA AND CELLS... 2 SECTION 2: PROTOCOL... 4 SECTION 3: CELL MORPHOLOGY... 11 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 www.heparg.cm Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 1/11
SECTION 1: MATERIALS, MEDIA AND CELLS 1. Materials Water bath at +37 C Laminar flw hd Pipet-aid, pipettes and micrpipettes Multichannel pipettes Plystyrene rund-bttm tubes (40 ml) and petri dishes (92 x 17 mm) r similar cntainers Incubatr at +37 C, 5% CO2 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, HPR116. Designatin Reference Cnditins f Shipping Strage 25 cm² flask cated with cllagen I* FLA125 +4 C +4 C 6-well plate cated with cllagen I* PLA135 +4 C +4 C 12-well plate cated with cllagen I* PLA138 +4 C +4 C 24-well plate cated with cllagen I* PLA137 +4 C +4 C 48-well plate cated with cllagen I* PLA139 +4 C +4 C 96-well plate cated with cllagen I* PLA136 +4 C +4 C *BPI prprietary cating prcess t ensure prper seeding and culture f the HPR116 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 Invitrgen TM 12551** +4 C GlutaMAX TM I, 200 mm Invitrgen TM 35050** -20 C Williams E Medium with GlutaMAX TM Invitrgen TM 32551* +4 C BPI recmmends using WEM and GlutaMAX TM -I frm Invitrgen TM. *Reference in Eurpe fr Base medium: crrespnds t 500mL f #32551. **References in Nrth America fr Base Medium: crrespnds t 495mL f #12551 cmbined with 5 ml f #35050. The fllwing supplements are available frm BIOPREDIC Internatinal. Designatin HepaRG Thawing/Plating/General Purpse Medium Supplement with antibitics HepaRG Maintenance/Metablism Medium Supplement with antibitics Reference ADD670 ADD620 Cnditins f Shipping Strage -20 C r lwer -20 C r lwer HepaRG Inductin Medium Supplement with antibitics ADD640-20 C r lwer HepaRG Serum-free Inductin Medium Supplement with antibitics ADD650-20 C r lwer Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 2/11
4. Cells (Immediately place the cryvial(s) in liquid nitrgen upn receipt) HepaRG cells Number f viable cells / vial (x10 6 ) Reference Cnditins f Shipping Strage Differentiated HepaRG cells crypreserved 8 HPR116-TA08 Dry ice r liquid nitrgen Liquid nitrgen Differentiated HepaRG cells crypreserved 12 HPR116-TA12* Dry ice r liquid nitrgen Liquid nitrgen *Fr Japan nly Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 3/11
SECTION 2: PROTOCOL YOUR SAFETY OBSERVE UNIVERSAL PRECAUTIONS WHEN HANDLING HepaRG 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 HepaRG THAWING/PLATING/GENERAL PURPOSE MEDIUM 670 - Thaw the HepaRG Thawing/Plating/General Purpse Medium Supplement (ADD670) by placing the bttle in a +37 C water bath until cmpletely thawed. - Recnstitute the HepaRG Thawing/Plating/General Purpse Medium 670 by adding the supplement (ADD670) t 100 ml f Base Medium: Williams E medium + GlutaMAX, r Williams E with GlutaMAX TM. - The recnstituted HepaRG Thawing/Plating/General Purpse Medium 670 is nw ready fr use. It shuld be stred at +4 C fr a maximum f ne mnth. 1.2 HepaRG MAINTENANCE/METABOLISM MEDIUM 620 - Thaw the HepaRG Maintenance/Metablism Medium Supplement (ADD620) by placing the bttle in a +37 C water bath until cmpletely thawed. - Recnstitute the HepaRG Maintenance/Metablism Medium 620 by adding the supplement (ADD620) t 100 ml f Base Medium: Williams E medium + GlutaMAX, r Williams E with GlutaMAX TM. - The recnstituted HepaRG Maintenance/ Metablism Medium 620 is nw ready fr use. It shuld be stred at +4 C fr a maximum f ne mnth. 1.3 HepaRG INDUCTION MEDIUM 640 - Thaw the HepaRG Inductin Medium Supplement (ADD640) by placing the bttle in a +37 C water bath until cmpletely thawed. - Recnstitute the HepaRG Inductin Medium by adding the supplement (ADD640) t 100 ml f Base Medium: Williams E medium + GlutaMAX, r Williams E with GlutaMAX TM. - The recnstituted HepaRG Inductin Medium 640 is nw ready fr use. It shuld be stred at +4 C fr a maximum f ne mnth. 1.4 HepaRG SERUM FREE INDUCTION MEDIUM 650 - Use ADD650 in place f ADD640 if yu prefer t use Serum-free medium; in such an instance, fllw all the steps in 1.3 fr use f ADD640 but use ADD650 instead. 2 Thawing and Seeding HepaRG cells 2.1 Thawing - Pre-warm the HepaRG Thawing/Plating/General Purpse Medium 670 (see 1.1) in the +37 C water bath. - Pipet 9 ml (per HepaRG cryvial t be used) f pre-warmed HepaRG Thawing/Plating/General Purpse Medium 670 int a sterile 40 ml plystyrene rund-bttm tube r similar cntainer. - Prepare an absrbent paper with 70% ethyl alchl. - Remve the cryvial frm the liquid nitrgen. - Under the laminar flw hd, briefly twist the cap a quarter turn (d nt pen the cryvial cmpletely) t release the internal pressure, and then clse it again. - Quickly transfer the cryvial t the water bath at +37 C. D nt submerge it cmpletely, being careful nt t allw water t penetrate int the cap. While hlding the tip f the cryvial, Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 4/11
gently agitate the vial fr abut 2 minutes. Small ice crystal shuld remain when remved frm the water bath. - Wipe the utside f the cryvial with 70% ethyl alchl n an absrbent paper, and place the cryvial under the laminar flw hd. - Aseptically transfer the semi -thawed HepaRG cell suspensin int the tube cntaining 9 ml f the pre-warmed HepaRG Thawing/Plating/General Purpse Medium 670 (resulting in a 1:10 rati f cell suspensin t ttal vlume). - Rinse ut the cryvial nce with apprximately 1 ml f the HepaRG Thawing/Plating/General Purpse Medium 670 and return the resulting suspensin t the 40 ml tube. - Centrifuge the differentiated HepaRG cell suspensin DURING 3 MIN AT 500G at rm temperature. D nt fllw hepatcyte prtcl fr centrifugatin, the HepaRG cells are smaller in size and need a lnger and faster centrifugatin. - Aspirate the supernatant. T avid aspiratin f cells, let a little vlume f medium n the pellet. - Resuspend gently the differentiated HepaRG cell pellet with HepaRG Thawing/Plating/General Purpse Medium 670 in 5 ml fr HPR116-TA08, r 7 ml fr HPR116-TA12. Dn t try t dissciate the bigger clusters. 2.2 Cell viability measurement and cunting Cell viability measurement and cell cunting are determined by trypan blue (0.05% in D-PBS 1X) exclusin test. - Transfer 900 µl f trypan blue slutin (0.05% in D-PBS 1X) in a 5 ml plystyrene rund bttm tube. - 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. - Gently hmgenize the cell suspensin by manual swirling. - Dilute 100 µl f the cell suspensin in the 900 µl f trypan blue slutin at 0.05%. - Gently hmgenize the btained cell suspensin. - 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. - 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 quite different frm ne rw t anther, cunt ne r tw mre rws. Careful: Thawed differentiated HepaRG cells can frm clusters. It s necessary t cunt all the cells including thse frming the clusters. - Determine the average number f viable cells and dead cells per rw. - Determine percentage f cell viability. Number f viable cells X 100 Number f viable cells + number f dead cells - 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 - Calculate the ttal viable cell number: Cell cncentratin in millin cells / ml X Ttal vlume f cell suspensin = ttal number f cells Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 5/11
3 Use f differentiated HepaRG cells 3.1 METABOLISM studies: use f HepaRG IN SUSPENSION - After thawing and cunting f differentiated HepaRG cells (Sec 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 - Thaw the cells in HepaRG Thawing/Plating/General Purpse Medium 670 - Incubate the cells with the test substrates accrding t yur prtcl 3.2 METABOLISM studies: use f HepaRG IN MONOLAYER 3.2.1 Cell seeding - After the thawing and the cunting f differentiated HepaRG cells (Sec 2), and using the Thawing/Plating/General Purpse Medium 670, seed the differentiated HepaRG cells int flatbttm multi-well plate(s) ) r flask(s) accrding t the table belw: Cell culture supprt Crypreserved differentiated HepaRG cells Number f viable cells per well (x10 6 ) Vlume per well/flask (ml) Cell cncentratin (x10 6 /ml) 25 cm² flask 5.2 5 1.04 6 well plate 2 2 1 12 well plate 0.8 1 0.8 24 well plate 0.48 0.5 0.96 48 well plate 0.16 0.2 0.8 96 well plate* 0.072 0.1 0.72 * If 96 well plate(s) are seeded partially, fill the wells surrunding thse cntaining the cells with sterile water. - 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. - Place the plate(s) r flask(s) in the incubatr at +37 C, 5% CO 2 and saturating humidity. 3.2.2 Cell maintenance fr metablism studies Yu have tw ptins: Either use the cells immediately 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 8. At day 1, 4 hurs after plating - Fur hurs after plating, bserve cell mrphlgy under phase-cntrast micrscpe, and when pssible, take phtmicrgraphs. - 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. Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 6/11
MONOLAYER 4 hurs after plating - Thaw and seed the cells using HepaRG Thawing/Plating/General Purpse Medium 670 - 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 Thawing/Plating/General Purpse Medium 670 t the HepaRG Maintenance/Metablism Medium 620. Fr 12-24-48-96 well plate(s): - Pre-warm the HepaRG Maintenance/Metablism Medium 620 in a sterile cntainer (12 ml/24 r 12 well plate, 9.6 ml/48 r 96 well plate, plus a little extra) at rm temperature. - Transfer the HepaRG Maintenance/Metablism Medium 620 pre-warmed int a 92 x 17 mm Petri dish r similar flat-bttm cntainer suitable fr use with multichannel pipette. - Remve the lid frm the multi-well plate. - Remve the existing medium frm the wells. - Gently add the pre-warmed HepaRG Maintenance/Metablism Medium 620 t the sides f each well with a multichannel pipette (fr vlume per well, see upper table in paragraph 3.2.1). D nt add the medium directly nt the cells. - Cntrl visually the medium level in the wells. - Put the lid back n the multi-well plate and place the plate(s) back in the +37 C incubatr. Fr 6 well plate(s): - Pre-warm the HepaRG Maintenance/Metablism Medium 620 in a sterile cntainer (12 ml/6 well plate) at rm temperature. - Remve the lid frm the multi-well plate. - Remve the existing medium frm the wells. - Gently add the pre-warmed HepaRG Maintenance/Metablism Medium 620 t the sides f each well with a pipette (2 ml per well). D nt add the medium directly nt the cells. - Cntrl visually the medium level in the wells. - Put the lid back n the multi-well plate and place the plate(s) back in the +37 C incubatr. Fr 25 cm² flask(s): - Pre-warm the HepaRG Maintenance/Metablism Medium 620 at rm temperature. - Remve the cap frm the flask(s). - Aspirate the existing medium frm the flask. - Transfer 5 ml f pre-warmed HepaRG Maintenance/Metablism Medium 620 int the 25 cm² flask. Take care nt t pipette dwn the medium directly n the cells. - Replace the cap n the flask and place the flask(s) back in the +37 C incubatr. - Maintain the HepaRG cells in HepaRG Maintenance/Metablism Medium 620 and use the cells: At Day 5 At day 5 after thawing and culture: a cell mnlayer can be bserved with a hepatcyte-like cell rganizatin in clusters and metablic activities are slightly lwer than activities detected frm fresh cells. Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 7/11
MONOLAYER Use at Day 5 96 hrs Day 2 24 hrs Day 5 96 hrs Thursday Friday Mnday Thaw and seed the cells using HepaRG Thawing/Plating/General Purpse Medium 670 Remve HepaRG Thawing/Plating/General Purpse Medium 670, and replace with the HepaRG Maintenance/Metablism Medium 620 Incubate the cells in mnlayer with the test substrates accrding t yur prtcl At Day 8 Fr ptimal activity levels, Maintenance/Metablism Medium 620 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 8 168 hrs Day 2 24 hrs Day 5 96 hrs Day 7 144 hrs Day 8 168 hrs Thursday Friday Thaw and seed the cells using HepaRG Thawing/Plating/General Purpse Medium 670 Remve HepaRG Thawing/Plating/General Purpse Medium 670, and replace with HepaRG Maintenance/Metablism Medium 620 Mnday Renew the HepaRG Maintenance/Metablism Medium 620 Wednesday Renew the HepaRG Maintenance/Metablism Medium 620 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 Maintenance/Metablism Medium 620 fr 1 additinal week, prvided that renewal f the HepaRG Maintenance/Metablism Medium 620 is perfrmed every 2-3 days. 3.3 Inductin studies: use f HepaRG in mnlayer 3.3.1 Cell seeding See 3.2.1 3.3.2 Culture and maintenance fr inductin study - Six hurs after plating (see the suggested timeline), bserve cell mrphlgy under phase-cntrast micrscpe, and when pssible, take phtmicrgraphs. - Renew the HepaRG Thawing/Plating/General Purpse Medium 670 (fr methd, see 3.2.2). - At day 4, after 72 hurs f culture, bserve cell mrphlgy under phase-cntrast micrscpe, and when pssible, take phtmicrgraphs. - Cells can be used fr inductin studies: chse between tw media with: N Serum Lw level f Serum HepaRG Serum-free Inductin Medium 650 HepaRG Inductin Medium 640 Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 8/11
- Change frm the HepaRG Thawing/Plating/General Purpse Medium 670 t either the HepaRG Inductin Medium 640 r HepaRG Serum-free Inductin Medium 650 with the test articles. - Incubate the cells with the test articles fr 48h. - Renew the medium with the test articles daily and always with the medium chsen at the beginning f the study (either HepaRG Inductin Medium 640 r HepaRG Serum-free Inductin Medium 650). 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. 3.3.3 Suggested timeline fr inductin studies 6 hrs Friday mrning Friday end f afternn (6 h after plating) Thaw and seed the cells using HepaRG Thawing/Plating/General Purpse Medium 670 Renew the HepaRG Thawing/Plating/General Purpse Medium 670 Day 4 72 hurs Day 5 96 hurs Day 6 120 hurs Mnday mrning Tuesday mrning Wednesday mrning Remve the HepaRG Thawing/Plating/General Purpse Medium 670, and replace with the HepaRG Inductin Medium 640 r HepaRG Serum-free Inductin Medium 650 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 Inductin Medium 640 r HepaRG Serumfree Inductin Medium 650 with the test articles End f the incubatin with the test articles Incubate the cells with the test substrates 3.4 UPTAKE AND TRANSPORT studies: use f HepaRG IN SUSPENSION - After thawing and cunting f differentiated HepaRG cells (Sec 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 - Thaw the cells in HepaRG Thawing/Plating/General Purpse medium 670 - Incubate the cells with the test substrates accrding t yur prtcl Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 9/11
3.5 TOXICITY studies: use f HepaRG IN MONOLAYER 3.5.1 Cell seeding See 3.2.1 3.5.2 Culture and maintenance fr txicity study - One day after thawing, bserve cell mrphlgy under phase-cntrast micrscpe, and when pssible, take phtmicrgraphs. - Renew the HepaRG Thawing/Plating/General Purpse Medium 670 (fr methd, see 3.2.2). - Maintain the HepaRG cells in HepaRG Thawing/Plating/General Purpse Medium 670 until the use f cells at day 8. - Renew the HepaRG Thawing/Plating/General Purpse Medium 670, and incubate the cells in mnlayer with the test articles accrding t yur prtcl. 3.5.3 Suggested timeline fr txicity studies Thursday Thaw and seed the cells using HepaRG Thawing/Plating/General Purpse Medium 670 Day 2 24 hurs Day 5 96 hurs Day 7 144 hurs Friday Renew the HepaRG Thawing/Plating/General Purpse Medium 670 Mnday Renew the HepaRG Thawing/Plating/General Purpse Medium 670 Wednesday Renew the HepaRG Thawing/Plating/General Purpse Medium 670 Day 8 168 hurs Thursday Remve the Thawing/Plating/General Purpse Medium 670 and incubate the cells in mnlayer with the test articles accrding t yur prtcl See the next page fr phtmicrgraphs f HepaRG cells in culture at different time pints. Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 10/11
SECTION 3: CELL MORPHOLOGY - After 24 hurs f culture, hepatcyte-like cells appear in small, differentiated clnies, individualized (fig 1). - After 72-96 hurs f culture, a restructuring f cell mnlayer can be bserved with a hepatcyte-like cells rganizatin in clusters (fig 2). - 120-144 hurs after plating, hepatcyte-like cells are rganized in well-delineated trabeculae with many bright canaliculi-like structures (fig 3). D1 D3 D6 Fig 1 Fig 2 Fig 3 Differentiated HepaRG cells crypreserved Descriptin and user guide Thawing, culture and use 11/11