ADME/Tox Support—Troubleshooting | Thermo Fisher Scientific - SE

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Hepatocytes

I’m getting low post-thaw cell viability with my hepatocytes. What should I do?

Please see the following causes and recommendations:

Possible cause	Recommendation
Improper thawing technique	Review thawing, plating and counting protocols Thaw cells <2 mins at 37 degrees C
Sub-optimal thawing medium	Use HTM Medium during thawing to remove cryoprotectant
Rough handling of hepatocytes during counting	Mix slowly, use wide-bore pipette tips Ensure a homogenous cell mixture prior to counting
Improper counting technique	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
Cells left out too long	Plate cells immediately after counting

I’m getting an unexpectedly low hepatocyte yield. What could be the cause of this?

Please see the following causes and recommendations:

Possible cause	Recommendation
Improper thawing technique	Review thawing, plating and counting protocols Thaw cells <2 mins at 37 degrees C
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	Mix slowly, use wide-bore pipette tips Ensure a homogenous cell mixture prior to counting
Improper counting technique	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

I’m getting low attachment efficiency with my hepatocytes. What should I do?

Please see the following causes and recommendations:

Possible cause	Recommendation
Not enough time for cells to attach	Wait before overlaying with Geltrex Matrix to see if attachment increases Compare cultures to pictures on the lot-specific characterization specification sheet (human cells)
Poor-quality substratum	Use Gibco Collagen I-Coated Plates
Hepatocyte lot not characterized as plateable	Check lot specifications to ensure it is qualified for plating Review thawing, plating, and counting protocols

I have a sub-optimal monolayer confluency for my hepatocytes. What should I do?

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.

I am seeing rounded up cell clumps or debris on top of the hepatocyte monolayer. What should I do?

Please see the following causes and recommendations:

Possible cause	Recommendation
Seeding density too high	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 Shake plate and wash cell monolayers prior to applying Geltrex Matrix overlay
Improper plating volume used for well format	Refer to literature or technical support for suggested plating volumes

I’m getting a loss of membrane integrity or cuboidal cell shape with my hepatocytes. What should I do?

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	Williams Medium E with Plating and Incubation Supplement Packs Refer to our plating protocol
Cells were cultured for too long	In general, plateable cryopreserved hepatocytes should not be cultured for more than five days

I’m seeing sub-optimal bile canalicular formation with my hepatocytes. What should I do?

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	Use Williams Medium E with Plating and Incubation Supplement Packs Refer to our plating protocol
Not enough time for bile canaliculi to form	In general, at least 4–5 days in culture is required for bile canalicular network formation

I’m getting unexpected induction results with my hepatocytes. What could be causing this?

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

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?

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	Use Williams Medium E with Plating and Incubation Supplement Packs Refer to our plating protocol
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

HepaRG Cells

My HepaRG cells have low post-thaw cell viability. Why is this?

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

I have an unexpected cell number for my HepaRG cells. Why is this?

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.

I have a sub-optimal monolayer confluency for my HepaRG cells. What should I do?

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.

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