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View additional product information for Rat (Sprague-Dawley) Cryopreserved Hepatocytes, Plateable Male - FAQs (RTCP10)
13 product FAQs found
Thermo Fisher Scientific has a dedicated team to answer questions regarding hepatocytes. You can send a question by email (hepaticproducts@thermofisher.com) or contact us by phone: US toll-free: (866) 952 3559
This could be due to the toxicity of the test compound. Here are other potential causes and recommendations:
-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
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:
-Sub-optimal monolayer confluency: Please see our recommendations for 'I have a sub-optimal monolayer confluency for my hepatocytes. What should I do?'
-Poor monolayer integrity: Please see our recommendations 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
Please see the following causes and recommendations:
-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
Please see the following causes and recommendations:
-Hepatocyte lot not characterized as plateable: Check lot specifications to ensure it is qualified for plating
-Sub-optimal culture medium: Use 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:
-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:
-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 for: 'I'm getting low attachment efficiency with my hepatocytes. What should I do?'
-Some animal lots are not greater than 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:
-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 the following causes and recommendations:
-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:
-Improper thawing technigue: Review thawing, plating and counting protocols; thaw cells less than 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
Unlike immortalized cell lines, hepatocytes are primary cells that cannot be cultured indefinitely. The use of thawed suspension hepatocytes should be limited to short-term experiments with a maximum of 4-6 hour incubations. Plateable hepatocytes, which attach to collagen-coated plasticware in culture media, are generally metabolically active for anywhere from 2-7 days depending on which assay they are qualified for use with.
If properly stored, cryopreserved hepatocytes can be maintained in the vapor phase of liquid nitrogen (-135 degrees C or below) for several years. This makes them ideal for a series of experiments conducted over many months.
Cryopreserved hepatocytes are shipped in the vapor phase of liquid nitrogen contained in a non-hazardous container called a dry liquid nitrogen (LN2) vapor shipper or dewar. The internal temperature of the dewar is maintained between -140 degrees C and -160 degrees C, generally for up to 7-10 days if unopened. Once hepatocytes are received and transferred to a long term LN2 dewar in the laboratory, dewers are returned to the address listed on the pre-paid return shipping label for reuse. Upon receipt of a shipment of cryopreserved hepatocytes, carefully and quickly transfer the vials to the vapor phase of liquid nitrogen and keep at -135 degrees C or below until use. Any increase in the temperature of the cryovials before an experiment threatens the viability, functionality, and activity of the hepatocytes.