| 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
|