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Hepatocytes

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

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

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

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
  • 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

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

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

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
  • 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

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.