HepaRG™ Cells, Cryopreserved

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 (http://www.thermofisher.com/us/en/home/references/protocols/drug-discovery/adme-tox-protocols/heparg-cell-user-guide.html).
-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.

Here are possible reasons:

-Improper thawing technique: Review Section 2.1: Protocol for thawing of HepaRG cells (https://www.thermofisher.com/us/en/home/references/protocols/drug-discovery/adme-tox-protocols/heparg-cell-user-guide.html#3).
-Thawing medium is not correct: Use the recommended medium - Review Section 1: Recommended materials, media, and cells (https://www.thermofisher.com/us/en/home/references/protocols/drug-discovery/adme-tox-protocols/heparg-cell-user-guide.html#2).
-Centrifugation speed is not correct: Follow the recommended speed for centrifugation. Review Section 2.1: Protocol for thawing of HepaRG cells (https://www.thermofisher.com/us/en/home/references/protocols/drug-discovery/adme-tox-protocols/heparg-cell-user-guide.html#3). 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 (https://www.thermofisher.com/us/en/home/references/protocols/drug-discovery/adme-tox-protocols/heparg-cell-user-guide.html#3). 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 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

The cells are licensed for single-use applications.

Upon receipt, cryovials must be stored in liquid or vapor phase nitrogen.