Corning Life Sciences – Discovery Labware For Research Use Only

Corning

Cryop reserved HepatoCells

Instructions for Use

Corning

cryopreserved HepatoCells are derived from single-donor human hepatocy tes using proprietary technology

for immortalizing primary cells. HepatoCells retain the physiological properties of their parental hepatocytes, show

mature hepatocyte-like morphology, and express the metabolic enzymes, nuclear receptors, and drug transporters

common to mature hepatocytes. These cells have been characterized for CYP3A4, CYP 1A2, and CYP 2B6 inductio n

response to prototypical inducers, as well as cytotoxicity applications.

Cryopreserved HepatoCells were developed to overcome so me of the shortcomings of primary human hepatocytes

such as large lot-to-lot variability, short life span, tendency to dedifferentiate in culture, and limited supply of high

quality materials. With virtually unlimited supply and consistent performance, cryopreserved HepatoCells can work as

a reliable alternative in vitro hepatic model for ADME/Tox screening by providing the convenience of being readily

available and supporting long-term studies with large quantities of cells.

Process Overview

This instruction for use describes the procedure for thawing and plating cryopreserved HepatoCells to form a

monolayer culture and the procedure for performing CYP450 induction assay with plated cultures. One vial contains >8

million viable cells, enough for one 96-well plate or one 24-well plate when following the procedure.

Note: Corning cryopreserved HepatoCells have been validated for CYP induction application; although we are in the

process of developing other applications, users are welcome to test the samples for any applications of their interest.

Corning cryopreserved HepatoCells show typical mature hepatocyte morphology after 3 to 4 days in culture.

Caution

When working with cryopreserved HepatoCells, follow the same safety recommendations as working with primary

human hepatocytes (i.e., treat the cells as potential biohazards). Always wear suitable personal protective equipment

such as latex gloves, safety glasses and/or a face shield, and a polyester lab coat while working with these cells. The

steps involved with the cell culture should be carried out in a sterile biological safety cabinet and standard practices for

biohazard materials apply.

Material s S upplied

Dry shipper

Corning cryopreserved HepatoCells (Corning Cat. No. 3548 81): >80% viability, >8 million cells/vial. Store cells in vapor

phase of liquid nitrogen tank immediately upon receipt.

Material s and Equipmen t to be Ordered or Supplied by the User

 Corning culture medium for HepatoCells (CMH) (Corning Cat. No. 354882)

 Corning Matrigel

basement membrane matrix, phenol red-free (Corning Cat. No. 356237)

 Antibiotics, such as Penicillin-Streptomycin solution (100x) (Corning Cat. No. 30-002-CI)

 Fetal Bovine Serum, heat inactivated (Corning Cat. No. 35-016-CV)

 Corning

BioCoat™ Collagen I-coated 96-well plates (Corning Cat. No. 354407)

 Certified tissue culture hood

 Phase-contrast microscope

 Cell culture incubator (5% CO

2, 37°C, 95% humidity)

 Water bath (37°C)

 Liquid nitrogen storage unit

Discovery Labware, Inc ., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)

CLSTechServ@Corning.com www.corning.com/lifesciences

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

For a listing of trademarks, visit www.corning.com/lifesciences/trademarks

Corning Life Sciences – Discovery Labware For Research Use Only

 Refrigerator or equivalent storage chamber (4°C)

 Centrifuge capable of achieving 150 x g

 Aspirating device (i.e., pipettor, vacuum aspirator)

 Falcon 50 ml Polypropylene Conical Tubes (Cat. No. 352070)

 Multi-channel pipets and reagent reservoirs

 Materials or equipment for cell count

Preparation B efo re Use

1. All the media used in the protocol is recommended to be supplemented with Penicillin-Streptomycin solution

(1:100 dilut ion) .

2. Prepare plating medium by adding FBS (final concentration of 10%) to CMH. Note that serum is on l y needed

for the plating step, therefore, DO NOT add FBS to the entire bottle of CMH.

3. Pre-warm plating medium in a 37°C water bath.

Protocol for Thawing Corning Cryopreserved HepatoCells

1. For each vial of cells, aliquot 10 to15 mL of warm plating media into a 50 mL conical centrifuge tube.

2. Remove a cryovial of Corning cryopreserved HepatoCells from liquid nitrogen storage unit and put in dry ice.

3. Transfer the cryovial into 37°C water bath immediately.

4. Thaw the cryovial for 1 minute (no more than 2 minutes) by gently moving the vial back and forth in the water

bath to facilitate heat transfer, until there is a small ball of frozen cells remaining in the vial.

5. Remove the cryovial from the water bath and quickly transfer the cryovial to a tissue culture hood.

6. Wipe the cryovial with a 70% alcohol pad before removing lid.

7. Pour the vial content to the 10 to15 mL pre-aliquoted plating medium in the 50 mL tube.

8. Rinse the cryovial with 1 mL warm plating medium and transfer the rinse medium back to the above 50 mL

tube.

9. Use a 10 mL serological pipet to break any large cell clumps by quickly pipetting up and down. It is fine to

have some small cell clumps at this step.

10. Add plating medium to the centrifuge tube until the solution volume is equal to 50 mL.

11. Centrifuge at 150 x g for 10 minutes at room temperature.

12. Aspirate supernatant carefully without disturbing the pellet and leave a small amount of medium (~0.5 mL) in

the tube.

13. Add about 5 mL of plating medium to resuspend the pellet.

14. Use a 10 mL serological pipet to break any cell clumps by quickly pipetting up and down 10 to 20 times to

make a homogeneous cell suspension. It is important to have homogeneous cell suspension at this step for

accurate cell counting.

15. Count cell number to determine viability and cell concentration.

16. Adjust cell suspension volume with plating medium to achieve desired cell concentration (see Table 1 for

seeding density).

Discovery Labware, Inc ., Two Oak Park, Bedford, MA 01730, USA. Tel: +1.978.442.2200 (U.S.)

CLSTechServ@Corning.com www.corning.com/lifesciences

For Research Use Only. Not for use in diagnostic or therapeutic procedures.

For a listing of trademarks, visit www.corning.com/lifesciences/trademarks