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
LT142                      8-Mar-2011