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Please see the following causes and recommendations:
Possible cause | Recommendation |
Improper thawing technique |
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| Sub-optimal thawing medium | Use HTM Medium during thawing to remove cryoprotectant |
| Rough handling of hepatocytes during counting |
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Improper counting technique |
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Cells left out too long | Plate cells immediately after counting |
Please see the following causes and recommendations:
Possible cause | Recommendation |
Improper thawing technique |
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| Sub-optimal thawing medium | Use HTM Medium during thawing to remove cryoprotectant |
| Incorrect centrifugation speed | Check thawing protocol for proper centrifugation speed and time (varies by species; human is 100 x g for 10 min at RT) |
Rough handling of hepatocytes during counting |
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Improper counting technique |
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Please see the following causes and recommendations:
Possible cause | Recommendation |
| Not enough time for cells to attach |
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| Poor-quality substratum | Use Gibco Collagen I-Coated Plates |
| Hepatocyte lot not characterized as plateable |
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Please see our recommendations above for:
Possible cause | Recommendation |
Seeding density too low | Check lot-specific characterization specification sheet for appropriate seeding density (human cells) Observe cells under microscope for appropriate seeding prior to incubation |
Insufficient dispersion of hepatocytes during plating | Disperse cells evenly by moving plate slowly in a figure-eight and back and forth pattern in incubator |
Insufficient plating volume used for well format | Refer to literature or technical support for suggested plating volumes |
Low attachment efficiency | Please see our recommendations above for: “I’m getting low attachment efficiency with my hepatocytes. What should I do?” |
Some animal lots are not >80% confluent | Check lot-specific characterization specification sheet for appropriate seeding density. Note: Some animal species create chains or islands of cells rather than being 100% confluent. |
Please see the following causes and recommendations:
Possible cause | Recommendation |
| Seeding density too high |
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| Insufficient dispersion of hepatocytes during plating |
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| Improper plating volume used for well format | Refer to literature or technical support for suggested plating volumes |
Please see the following causes and recommendations:
Possible cause | Recommendation |
| Hepatocyte lot not characterized as plateable | Check lot specifications to ensure it is qualified for plating |
Sub-optimal culture medium |
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| Cells were cultured for too long | In general, plateable cryopreserved hepatocytes should not be cultured for more than five days |
Please see the following causes and recommendations:
Possible cause | Recommendation |
| Hepatocyte lot not transporter-qualified | Check lot specifications to ensure it is transporter-qualified |
Sub-optimal culture medium |
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| Not enough time for bile canaliculi to form | In general, at least 4–5 days in culture is required for bile canalicular network formation |
First of all, we recommend comparing results to those reported on our lot-specific characterization specification sheet (human cells) and also referring to our enzyme induction protocol. Here are other potential causes and recommendations:
Possible Cause | Recommendation |
Sub-optimal monolayer confluency
| Please see our recommendations above for ‘I have a sub-optimal monolayer confluency for my hepatocytes. What should I do?’ |
Poor monolayer integrity
| Please see our recommendations below for ‘With my hepatocytes, I’m seeing rounding up of the cells, cellular debris, and/or holes in the monolayer, indicating dying cells. What should I do?’ |
| Inappropriate positive control | Check positive control to ensure suitability |
| Incorrect concentration of positive control | Use the correct concentration of positive control |
This could be due to the toxicity of the test compound. Here are other potential causes and recommendations:
Possible cause | Recommendation |
| Sub-optimal culture medium |
|
| Hepatocyte lot not characterized as plateable | Check lot specifications to ensure it is qualified for plating |
Cells were cultured for too long | In general, plateable cryopreserved hepatocytes should not be cultured for more than five days |
Here are possible reasons:
- Storage temperature not maintained below -80 degrees C or repeated transient increase in temperature to RT
- Cells were thawed incorrectly
- Thawing medium is not correct or was at the incorrect temperature
- Cells were not handled gently
- Counting difficulty: Perform a repeat count with a new sample; avoid vortexing the cells to mix them
- Shipment temperature not maintained below -70 degrees C
Here are possible reasons:
- Improper thawing technique: Review Section 2.1: Protocol for thawing of HepaRG cells.
- Thawing medium is not correct: Use the recommended medium - Review Section 1: Recommended materials, media, and cells.
- Centrifugation speed is not correct: Follow the recommended speed for centrifugation. Review Section 2.1: Protocol for thawing of HepaRG cells. Make sure that the centrifuge is calibrated. If the viability is very high but the cell number is low, then the centrifugation speed is too low - increase the speed and repeat centrifugation (this may require some optimization). By contrast, if the viability is lower than expected and the number of cells is very high, then the centrifugation speed is too high and is pelleting dead cells as well as viable cells.
- Loss of cells: After centrifugation, aspirate the supernatant while leaving a small volume on the pellet. Be careful not to aspirate the pellet.
- Improper counting technique: Review Section 2.2: Protocol for counting of HepaRG cells. Ensure a homogeneous cell mixture prior to counting. Due to the normal presence of aggregates in the cell suspension, we recommend counting the cells with a hemocytometer.
Here are possible causes and suggestions:
- Low seeding density: Follow recommended procedure for seeding the cells depending upon your application in the HepaRG Cell User Guide.
- Low attachment efficiency; inadequate time allowed for attachment:
- Low attachment efficiency may be due to:
- Poor-quality matrix: Use only Collagen I coated plates from recognized manufacturers.
- Cells not handled gently: Freezing and thawing procedures are stressful for cells and cause them to be fragile. Handle the cells gently.
- Low attachment efficiency may be due to:
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