Problem Possible cause Recommendation
Low cell viability, post-thaw
  • Improper thawing technique
  • Sub-optimal thawing medium
  • Rough handling of hepatocytes during counting
  • Improper counting technique
  • Cells left out too long
  • Review Life Technologies thawing, plating, and counting protocols
  • Thaw cells <2 min at 37°C
  • Use CHRM® Medium during thawing to remove cryoprotectant
  • Mix slowly; use wide-bore pipette tips
  • Ensure a homogenous cell mixture prior to counting
  • Count cells on 2 of the 4 grid lines
  • Do not let cells sit in trypan blue mixture for more than 1 min prior to loading
  • Plate cells immediately after counting
Unexpected cell yield
  • Improper thawing technique
  • Sub-optimal thawing medium
  • Incorrect centrifugation speed
  • Rough handling of hepatocytes during counting
  • Improper counting technique
  • Review Life Technologies thawing, plating, and counting protocols
  • Thaw cells <2 min at 37°C
  • Use CHRM® Medium during thawing to remove cryoprotectant
  • Check thawing protocol for proper centrifugation speed and time (varies by species; human is 100 x g for 10 min at RT)
  • Mix slowly; use wide-bore pipette tips
  • Ensure a homogenous cell mixture prior to counting
  • Count cells on 2 of the 4 grid lines
  • Do not let cells sit in trypan blue mixture for more than 1 min prior to loading
Low attachment efficiency
  • Not enough time for cells to attach
  • Poor-quality substratum
  • Hepatocyte lot not characterized as plateable
  • Compare cultures to pictures on Life Technologies lot-specific characterization specification sheet (human cells)
  • Wait before overlaying with Geltrex™ matrix to see if attachment increases
  • Use GIBCO® Collagen I Coated Plates
  • Review Life Technologies thawing, plating, and counting protocols (see above section for additional protocol suggestions)
  • Check lot specifications to ensure it is qualified for plating
Sub-optimal monolayer confluency
  • Seeding density too low
  • Insufficient dispersion of hepatocytes during plating
  • Insufficient plating volume used for well format
  • Low attachment efficiency (see above)
  • Some animal lots are not >80% confluent
  • Check Life Technologies lot-specific characterization specification sheet for appropriate seeding density (human cells)
  • Observe cells under microscope for appropriate seeding prior to incubation
  • Disperse cells evenly by moving plate slowly in a figure-eight and back & forth pattern in incubator
  • Refer to Life Technologies literature or technical support for suggested plating volumes
Dirty monolayers
(rounded up cell clumps or debris on top of monolayer)
  • Seeding density too high
  • Insufficient dispersion of cells during plating
  • Improper plating volume used for well format
  • Check Life Technologies lot-specific characterization specification sheet for appropriate seeding density (human cells)
  • Observe cells under microscope for appropriate seeding prior to incubation
  • Disperse cells evenly by moving plate slowly in a figure-eight and back & forth pattern in incubator
  • Shake plate and wash cell monolayers prior to applying Geltrex™ overlay
  • Refer to Life Technologies literature or technical support for suggested plating volumes
Loss of membrane integrity or cuboidal cell shape
  • Hepatocyte lot not characterized as plateable
  • Sub-optimal culture medium
  • Cells were cultured for too long
  • Check lot specifications to ensure it is qualified for plating
  • Use GIBCO® Williams Medium E with GIBCO® Plating and Incubation Supplement Packs
  • Refer to Life Technologies plating protocol
  • In general, plateable cryopreserved hepatocytes should not be cultured for more than five days
Sub-optimal bile canalicular formation
  • Hepatocyte lot not transporter-qualified
  • Sub-optimal culture medium
  • Not enough time for bile canaliculi to form
  • Check lot specifications to ensure it is transporter-qualified
  • Use GIBCO® Williams Medium E with GIBCO® Plating and Incubation Supplement Packs
  • Refer to Life Technologies plating protocol
  • In general, at least 4–5 days in culture is required for bile canalicular network formation
Unexpected induction results
  • Sub-optimal monolayer confluency (see above)
  • Poor monolayer integrity (see above)
  • Inappropriate positive control
  • Incorrect concentration of positive control
  • Compare results to those reported on Life Technologies  lot-specific characterization specification sheet (human cells)
  • Refer to Life Technologies enzyme induction protocol
  • Check positive control to ensure suitability
Rounding up of cells, cellular debris, and/or holes in monolayer indicative of dying cells
  • Toxicity of test compound
  • Sub-optimal culture medium
  • Hepatocyte lot not characterized as plateable
  • Cells were cultured for too long
  • Compare cell morphology of treated and non-treated cells
  • Refer to the Life Technologies plating protocol
  • Check lot specifications to ensure it is qualified for plating
  • In general, plateable cryopreserved hepatocytes should not be cultured for more than five days

Ordering Information

Catalog # Name Size List Price (USD) Qty
HMCPIS Human Plateable Hepatocytes, Induction Qualified 1.5 mL 1,314.00
RTCS10 Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Male 1.5 mL 191.00
RTCS20 Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Female 1.5 mL 191.00
RTCP10 Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Plateable Male 1.5 mL 387.00
RTCP20 Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Plateable Female 1.5 mL 387.00
MKCS10 Monkey (cynomolgus) Cryopreserved Hepatocytes, Male 1.5 mL 363.00
MKCS20 Monkey (cynomolgus) Cryopreserved Hepatocytes, Female 1.5 mL 363.00
MKCP10 Monkey (cynomolgus) Cryopreserved Hepatocytes, Plateable Male 1.5 mL 720.00
MKCP20 Monkey (cynomolgus) Cryopreserved Hepatocytes, Plateable Female 1.5 mL 720.00
MSCS10 Mouse (CD-1) Cryopreserved Hepatocytes, Male 1.5 mL 520.00
MSCS20 Mouse (CD-1) Cryopreserved Hepatocytes, Female 1.5 mL 520.00
MSCP10 Mouse (CD-1) Cryopreserved Hepatocytes, Plateable Male 1.5 mL 980.00
MSCP20 Mouse (CD-1) Cryopreserved Hepatocytes, Plateable Female 1.5 mL 980.00
DGCS10 Dog (Beagle) Cryopreserved Hepatocytes, Male 1.5 mL 273.00
DGCS20 Dog (Beagle) Cryopreserved Hepatocytes, Female 1.5 mL 273.00
DGCP10 Dog (Beagle) Cryopreserved Hepatocytes, plateable Male 1.5 mL 564.00
DGCP20 Dog (Beagle) Cryopreserved Hepatocytes, plateable Female 1.5 mL 564.00
HMCPMS Human Plateable Hepatocytes, Metabolism Qualified 1.5 mL 1,026.00
HMCPTS Human Plateable Hepatocytes, Transporter Qualified 1.5 mL 1,520.00
HMCSPS Human Suspension Hepatocytes, Polymorphic Donors, (4-8 x 106 cells) 1 vial 788.00
HMCSPL Human Suspension Hepatocytes, Polymorphic Donors, (9-12 x 106 cells) 1 vial 994.00
HMCPUS Human Plateable Hepatocytes, Uptake Qualified 1.5 mL 1,026.00
HMCS1S Human Suspension Hepatocytes, Metabolism Qualified, Male, (4-8 x 106 cells) 1 vial 479.00
HMCS1L Human Suspension Hepatocytes, Metabolism Qualified, Male, (9-12 x 106 cells) 1 vial 640.00
HMCS2S Human Suspension Hepatocytes, Metabolism Qualified, Female, (4-8 x 106 cells) 1 vial 479.00
HMCS2L Human Suspension Hepatocytes, Metabolism Qualified, Female, (9-12 x 106 cells) 1 vial 640.00
HMCSTS Human Suspension Hepatocytes, Transporter Qualified, (4-8 x 106 cells) 1 vial 670.00
HMCSTL Human Suspension Hepatocytes, Transporter Qualified, (9-12 x 106 cells) 1 vial 876.00
LT142                      8-Mar-2011