Cryopreserved HepaRG Cells and Media Supplements

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1 Cryopreserved HepaRG Cells and Media Supplements Catalog No. MMHPR116 Catalog No. MMADD621 Catalog No. MMADD631 Catalog No. MMADD641 Catalog No. MMADD651 Catalog No. MMADD671 FOR RESEARCH USE ONLY Not for use in diagnostic procedures. USA & Canada Phone: +1(800) Fax: +1 (951) Australia

2 Limited Use License HepaRG cells are patented and their use is strictly limited; consider the cells as a single-use, disposable product that must be destroyed upon conclusion of a study or experiment. Propagating, reproducing, cloning, sub-cloning or any other use of the cells following the conclusion of a study is prohibited. Use of the cells to produce or manufacture commercial products for general sale or for use in the manufacture of products intended for general sale is prohibited. Transfer of the cells to anyone not employed within the same organization, whether for financial benefit or not, is prohibited. If you are unwilling to accept the terms of this Limited Use License, do not use them, and immediately return the cells for credit. Violators of this Limited Use License will be prosecuted to the fullest extent of the law. Media and Cells Cryopreserved HepaRG Cells (Cat. No. MMHPR116, Store in liquid nitrogen) Medium Supplements: HepaRG Thawing/Plating Medium Supplement (Cat. No. MMADD671, 12.5 ml, Store at -20 C) HepaRG Culture Medium Supplement (Cat. No. MMADD621, 14 ml, Store at -20 C) HepaRG Induction Medium Supplement (Cat. No. MMADD641, 2.6 ml, Store at -20 C) HepaRG Serum Free Induction Medium Supplement (Cat. No. MMADD651, 0.6 ml, Store at -20 C) HepaRG Tox Medium Supplement (Cat. No. MMADD631, 12.5 ml, Store at -20 C) Base Medium: Williams Medium E (WEM) (LONZA EU Cat. No.: BE12-761F; or Invitrogen US Cat. No.: ) Ultraglutamine (200 mm) (LONZA EU Cat. No.: BE17-605E/U1) or GlutaMAX I (200 mm) (Invitrogen US Cat. No.: ) Materials Not Provided: +37 C water bath, laminar flow hood, pipet-aid, pipettes and micropipettes, multichannel pipettes, polystyrene round-bottom tubes (40 ml), glass bottles (capacity 125mL) with screw-tops, +37 C, 5% CO 2 and saturating humidity incubator, phase-contrast microscope, cell counting chamber, coverslips, 0.05% Trypan blue solution, 96-well, 48-well or 24-well collagen I coated plate. Protocols Note: Observe universal precautions when handling HepaRG cells and treat all biological materials as potentially infectious. The following protocol should be performed under a laminar flow hood. It is recommended that photomicrographs are taken at regular intervals during the protocol. Medium Preparation 1. Thaw the appropriate HepaRG Medium Supplement (be sure it is completely thawed). 2. Depending on your experiment, prepare the medium according to Table 1.

3 Table 1. Medium Base Medium HepaRG Thawing/Plating Medium HepaRG Culture Medium HepaRG Induction Medium HepaRG Serum Free Induction Medium HepaRG Tox Medium Prepare by Adding Column A to Column B A B 1 ml GlutaMAX I (200 mm) 99 ml Williams Medium E 12.5 ml HepaRG Thawing/Plating Medium Supplement 14 ml HepaRG Culture Medium Supplement 2.6 ml HepaRG Induction Medium Supplement 0.6 ml HepaRG Serum Free Induction Medium Supplement 12.5 ml HepaRG Tox Medium Supplement Supplemented HepaRG media may be stored at +4 C for a maximum of one month. The working medium should be kept in a sterile glass bottle with a screw-top cap, and the bottle should be dated with the date of preparation. (Note: The supplements do not contain Pen/Strep; some laboratories prohibit it from their premises. If your laboratory uses Pen/Strep, we recommend the addition of 1.1mL of Pen/Strep to the Medium.) Thawing of Cryopreserved HepaRG Cells 1. Warm HepaRG Thawing/Plating Medium in a water bath. 2. Pipette 9 ml of HepaRG Thawing/Plating Medium (+37 C) into a sterile 40 ml polystyrene round-bottom tube. 3. Prepare an absorbent paper with 70 % ethyl alcohol. 4. Remove one Cryopreserved HepaRG Cells (MMHPR116) cryovial from liquid nitrogen storage. 5. In the laminar flow hood, turn the cryovial cap a quarter turn (do not open the cryovial completely) to release internal pressure, and then close it again. 6. Immediately transfer the cryovial into a +37 C water bath (do not submerge the cryovial or allow water to penetrate into the cap; we recommend holding it by the cap). 7. Gently agitate the vial for 1 to 2 minutes (small ice crystals should remain when removed from the water bath). 8. Wipe the outside of the cryovial with the 70% ethyl alcohol absorbent paper, and place the cryovial in the laminar flow hood. 9. Aseptically transfer the semi -thawed HepaRG cell suspension into the tube containing 9 ml of the HepaRG Thawing/Plating Medium (resulting in a 1:10 ratio of cell suspension to total volume). 10. Rinse the cryovial with ~1mL of HepaRG Thawing/Plating Medium and pipet the resulting suspension into the 40 ml tube. 11. Centrifuge the cell suspension for 3 minutes at 500 G at room temperature (do not follow typical cryopreserved hepatocyte protocol for centrifugation, the HepaRG cells require the centrifugation detailed above).

4 12. Aspirate the supernatant from the 40 ml tube. To avoid aspiration of cells, leave a small volume of medium on the cell pellet. 13. Gently resuspend the differentiated HepaRG cell pellet with 5 ml of HepaRG Thawing/Plating Medium (do not try to dissociate the bigger clusters, at this point you should have ~8M viable cells in ~5.1mL of suspension). Viability Assessment and Counting of HepaRG Cells (optional) 1. Pipette 900 µl of a 0.05% Trypan blue solution into a 5 ml polystyrene round-bottom tube. 2. Homogenize the HepaRG cell suspension with gentle manual swirling. 3. Pipette 100 µl of the cell suspension into the tube containing the Trypan blue solution. 4. Gently homogenize the resulting cell suspension by manual swirling. 5. Introduce an aliquot of this cell suspension into a cell counting chamber (e.g. Nageotte chamber). 6. Perform cell observation and count under the microscope. 7. Living cells exclude the dye while dead cells take it up and appear blue. 8. Count the living and dead cells to determine the cell viability and concentration. Use of HepaRG in Suspension for Metabolism or Uptake and Transport Studies After thawing of differentiated HepaRG cells, cells can be incubated with test substrate as for standard metabolism or uptake and transport studies in suspension according to your standard protocol with human hepatocytes. Cell Seeding for Use of HepaRG Cells in Monolayer After thawing (and optionally counting) the HepaRG cells, seed them into flat bottom multi-well plates as follows: For 24 well plates: 1. You will need two vials of thawed HepaRG cells; 11.5 M cells are required to fully seed a 24 well plate, and each vial will yield ~8.3 M viable cells (consult COA), so this process should be followed with each vial. 2. Aliquot ~7.1 ml of Thawing/Plating Medium into a 40 ml polystyrene tube and allow to equilibrate to room temperature. 3. Add the Thawing/Plating Medium to the 40 ml tube containing the suspended HepaRG, and gently mix to achieve homogeneous solution. 4. Dispense 0.5 ml of the HepaRG suspended in the Thawing/Plating Medium into each well, and gently rock (back-and-forth and side-side) the plate; levels in each well should be similar. Place the plate in the incubator at 37 C, 5% CO 2, and saturating humidity. 5. This achieves a seeding density of ~0.48 M cells/well at a concentration of 0.96 M cells/ml. For 48 well plates: 1. One vial of HepaRG cells is sufficient to fully seed a 48 well plate. 2. Aliquot 5.1 ml of Thawing/Plating Medium into a 40 ml polystyrene tube and allow to equilibrate to room temperature.

5 3. Add the Thawing/Plating Medium to the 40 ml tube containing the suspended HepaRG, and gently mix to achieve homogeneous solution. 4. Dispense 0.2 ml of the HepaRG suspended in the Thawing/Plating Medium into each well, and gently rock (back-and-forth and side-side) the plate; levels in each well should be similar. Place the plate in the incubator at 37 C, 5% CO 2, and saturating humidity. 5. This achieves a seeding density of ~0.16 M cells/well at a concentration of 0.8 M cells/ml. For non-imaging studies on 96 well plates: 1. One vial of HepaRG cells is sufficient to fully seed a 96 well plate. 2. Aliquot 6.2 ml of HepaRG Thawing/Plating Medium into a 40 ml polystyrene tube and allow to equilibrate to room temperature. 3. Add the HepaRG Thawing/Plating Medium to the 40 ml tube containing the suspended HepaRG, and gently mix to achieve homogeneous solution. 4. Dispense 0.1 ml of the HepaRG suspended in the HepaRG Thawing/Plating Medium into each well, and gently rock (back-and-forth and side-side) the plate; levels in each well should be similar. Place the plate in the incubator at 37 C, 5% CO 2, and saturating humidity. 5. This achieves a seeding density of ~0.072 M cells/well at a concentration of 0.72 M cells/ml. For imaging studies on 96 well plates: 1. One vial of HepaRG cells is sufficient to fully seed a 96 well plate. 2. Aliquot 8.9 ml of HepaRG Thawing/Plating Medium into a 40mL polystyrene tube and allow to equilibrate to room temperature. 3. Add the HepaRG Thawing/Plating Medium to the 40mL tube containing the suspended HepaRG, and gently mix to achieve homogeneous solution. 4. Dispense 0.1 ml of the HepaRG suspended in the HepaRG Thawing/Plating Medium into each well, and gently rock (back-and-forth and side-side) the plate; levels in each well should be similar. Place the plate in the incubator at 37 C, 5% CO 2, and saturating humidity. 5. This achieves a seeding density of ~0.050 M cells/well at a concentration of 0.57 M cells/ml. Protocol for Changing/Replacement of Media 1. Allow the new HepaRG Medium (12 ml/24 well plate; 9.6 ml/48 or 96 well plate) to warm to room temperature. 2. Remove the existing medium from the wells of the multi-well plate. 3. Gently add the HepaRG Medium applicable to your analysis to each well using a multichannel pipette (100 µl/well for a 96 multi-well plate, 200 µl/well for a 48 multi-well plate and 500 µl/well for a 24 multi-well plate) (Do not add the medium directly onto the cells). 4. Replace the lid onto the multi-well plate and return the plate to the +37 C incubator.

6 Cell Culture and Maintenance for Metabolism Studies Plated cells may be used either after 4 hours but within 24 hours of plating, or following at least 4 days of culture. HepaRG cells keep a high level of Cytochrome P450 (CYP) activity during the first 24 hours after plating, these activities then decrease while the cells reconstitute the monolayer, activity returns during the fourth day in culture, peaking at day 7. To use HepaRG cells for metabolism studies after 4 hours but within 24 hours of plating, incubate the cells with test substrates according to your standard protocol for metabolism studies with human hepatocytes. Use of HepaRG for Metabolism Studies After 4 Days of Culture 1. One day after plating (day 1), observe cell morphology under phase-contrast microscope, and take photomicrographs. 2. Replace the HepaRG Thawing/Plating Medium with HepaRG Culture Medium days after plating and culturing a cell monolayer can be observed with a hepatocyte-like cell organization in clusters (metabolic activity may be slightly lower than that detected in fresh cells). 4. Renew the HepaRG Culture Medium on day 4 and day 6 (if the study duration dictates) after plating to attain optimal activity levels days after plating cells are organized in well-delineated trabeculae with many bright canaliculi-like structures and basal metabolic activities similar to fresh cells. 6. HepaRG cells can be used for the metabolism studies from day 4 to day 7 according to your standard protocol with human hepatocytes. 7. HepaRG cells can also be kept in HepaRG Culture Medium for 7 additional days, provided that renewal of the HepaRG Culture Medium is performed every 2-3 days. Cell Culture and Maintenance for Induction Studies 1. Six hours after plating (day 0), observe cell morphology under phase-contrast microscope, and take photomicrographs. 2. Renew the HepaRG Thawing/Plating Medium 3. On day 3, replace the HepaRG Thawing/Plating Medium with either HepaRG Induction Medium with test compound or HepaRG Serum Free Induction Medium with test compound by the same procedure. 4. Incubate the cells with the test articles for 24h (for assessment of mrna induction) or 48h (maximal induction of metabolic activity may be achieved with 72h treatment time, but data indicate that 48h of treatment is sufficient to demonstrate significant induction of CYP1A2, CYP2B6, and CYP3A4 metabolic activity using prototypical inducers). 5. Renew the chosen induction medium with the test compound daily. Cell Culture and Maintenance for Toxicity Studies 1. One day after thawing (day 1), observe cell morphology under phase-contrast microscope and take photomicrographs. Replace the HepaRG Thawing/Plating Medium with HepaRG Tox Medium. 2. On day 4, renew the HepaRG Tox Medium; after doing so you may treat the cells with test compounds at this time.

7 3. If your design is intended to study toxicity following extended exposure time, you may continue to treat the cells with test article through day 8, provided that the HepaRG Tox Medium is renewed every two days. Cell Morphology After 1 day of culture, hepatocyte-like cells appear in small, differentiated colonies, individualized (Figure 1.). After 3-4 days of culture, a restructuring of cell monolayer can be observed with a hepatocyte-like cells organization in clusters (Figure 2.). 6-7 days after plating, hepatocyte-like cells are organized in well-delineated trabeculae with many bright canaliculi-like structures (Figure 3.). Day 1 Day 3 Day 6 Fig 1 Fig 2 Fig 3 For Research Use Only; Not for use in diagnostic procedures.

8 Warranty Millipore Corporation ( Millipore ) warrants its products will meet their applicable published specifications when used in accordance with their applicable instructions for a period of one year from shipment of the products. MILLIPORE MAKES NO OTHER WARRANTY, EXPRESSED OR IMPLIED. THERE IS NO WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. The warranty provided herein and the data, specifications and descriptions of Millipore products appearing in Millipore s published catalogues and product literature may not be altered except by express written agreement signed by an officer of Millipore. Representations, oral or written, which are inconsistent with this warranty or such publications are not authorized and if given, should not be relied upon. In the event of a breach of the foregoing warranty, Millipore s sole obligation shall be to repair or replace, at its option, the applicable product or part thereof, provided the customer notifies Millipore promptly of any such breach. If after exercising reasonable efforts, Millipore is unable to repair or replace the product or part, then Millipore shall refund to the Company all monies paid for such applicable Product. MILLIPORE SHALL NOT BE LIABLE FOR CONSEQUENTIAL, INCIDENTAL, SPECIAL OR ANY OTHER DAMAGES RESULTING FROM ECONOMIC LOSS OR PROPERTY DAMAGE SUSTAINED BY ANY COMPANY CUSTOMER FROM THE USE OF ITS PRODUCTS. Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals. (c) 2009: Millipore Corporation. All rights reserved. No part of these works may be reproduced in any form without permission in writing

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