Plateable hepatocytes are most commonly utilized in ADME research for CYP induction studies, but plated hepatocytes also see routine use in drug metabolism research, biochemistry, virology, host/pathogen interaction studies, and cell biology research.
Image Credit: BioIVT
This article explores a number of frequently asked questions around working with plateable hepatocytes. It also outlines common reasons that hepatocyte plating fails, providing a range of potential solutions to improving plated hepatocytes’ performance in in vitro assays.
What is considered an attaching lot?
Every hepatocyte lot is subject to rigorous testing to determine its specific attachment characteristics on behalf of BioIVT’s clients. The company also provides data on mRNA and enzymatic induction rates, CDFDA staining, MTT viability, and enzyme activity rates, as well as a micrograph of the monolayer.
Cryopreserved CryostaX hepatocytes benefit from a unique, patented cryopreservation format. Due to their distinctiveness, these hepatocytes come with a different characterization package featuring a micrograph of the monolayer, as well as mRNA and enzyme induction rates.
Cells must form a monolayer that is at least 70 % confluent to qualify as ‘attaching.’ This monolayer must be maintained for at least five days, although it is important to note that these cells will generally survive longer in culture.
When should a pooled hepatocyte lot be used?
Hepatocytes acquired from commonly available, small laboratory animals will typically exhibit similar activity between lots. Human donors differ significantly from one another, however. Pools are, therefore, utilized to minimize human donor-dependent variability in many experimental designs.
BioIVT offers different pooled hepatocyte formats—LIVERPOOL and CryostaX—with a range of donor pool sizes.
The design of the experiment may determine the most appropriate pool size; however, both LIVERPOOL and CryostaX pooled hepatocytes can be custom-pooled to meet specific research needs when an off-the-shelf product is not suitable.
How important is an accurate cell count?
Successful cell cultures are highly dependent on accurate and repeatable cell counts. Overseeding and underseeding can result in experimental variability and under-confluent cultures, so it is particularly important to employ a consistent technique to ensure that counts are as accurate as possible.
Hepatocytes settle quickly, so the cells should be gently but thoroughly resuspended regularly, ensuring they are fully mixed before sampling and counting. While resuspension is necessary throughout the process, it is important to avoid vortexing the hepatocytes.
Resuspension of Hepatocytes for Accurate Cell Count
Video Credit: BioIVT
Cell count samples should be taken from the center of the cell suspension to avoid selecting pockets of denser or settled cells that could skew the count. It is recommended to lower the pipette tip to the bottom of the tube, then raise it slightly to collect the sample from the center.
Hepatocyte Pipetting Tips
Video Credit: BioIVT
The pipette tip should barely touch the surface when adding the cell suspension to the counting solution. This ensures that cells on the outside of the pipette tip are not included in the cell count.
Adding Cell Suspension to Counting Solution
Video Credit: BioIVT
Pipetting up and down to mix a sample can shear hepatocytes and lower viability. It is, therefore, advisable to gently rock the sample back and forth prior to adding this to the hemocytometer.
Using automated cell counters to count hepatocytes typically leads to inaccurate viability and yield counts. This can also lead to issues with monolayer formation as too many or too few cells will not facilitate the formation of confluent monolayer.
The variability of automated cell counters means that these are not recommended for use with primary hepatocytes.
Mixing Cell Suspension with Counting Solution
Video Credit: BioIVT
How long can cells sit before plating?
In most cases, it is advisable to plate cells within 30 minutes of thawing. Plates and tubes should be labeled before removing the vial from cryostorage, and all media should be warmed to 37 °C prior to plating. However, it is not necessary to place the cells and media in a warmer during the counting process, as room temperature conditions are sufficient.
Why is it important to wait one day to overlay rat hepatocytes?
A variety of protocols exist for overlaying cryoplateable hepatocytes. While most species offer flexibility regarding when hepatocytes can be overlaid with an extracellular matrix, it is generally recommended to overlay them between 4 and 24 hours after plating, except for rat hepatocytes.
Rat hepatocytes should only be overlaid between 18 and 24 hours after plating due to their natural gapping pattern, which can cause cultures to appear under-confluent. Waiting one day before overlaying allows the cells more time to flatten and form a confluent monolayer.
Do cryoplateable hepatocytes require serum in culture?
Serum is used during specific steps of the plating protocol, but it is not needed once the hepatocyte monolayer has formed. In conventional ADME assays, serum is undesirable in the culture medium due to its high drug and protein binding capacity and the potential variability between lots.
BioIVT’s INVITROGRO HI, CryostaX Culture Medium, and INVITROGRO KHB are all serum-free, but certain non-standard assays may benefit from the use of serum in the culture medium. In these instances, it is possible to add serum to the hepatocyte culture mediums.
Can multichannel pipettes or repeater pipettes be used when seeding hepatocytes?
Manual multichannel pipettes are useful for efficiently seeding multiwell plates with more than 12 wells. Electronic multichannel pipettes can also be used, but their speed should be lowered, and only half a plate should be dispensed at a time to prevent cells from settling in the pipette tips, which can lead to uneven cell distribution.
Repeaters are best reserved for larger volume well formats and should also be operated at a low speed.
Acknowledgments
Produced from materials originally authored by Halee McElhaney, Dr. Chris Bohl, and Lucy Fahler from BioIVT.
About BioIVT
BioIVT, formerly BioreclamationIVT, is a leading global provider of high-quality biological specimens and value-added services. We specialize in control and disease state samples including human and animal tissues, cell products, blood and other biofluids. Our unmatched portfolio of clinical specimens directly supports precision medicine research and the effort to improve patient outcomes by coupling comprehensive clinical data with donor samples.
Our Research Services team works collaboratively with clients to provide in vitro hepatic modeling solutions. And as the world’s premier supplier of ADME-Tox model systems, including hepatocytes and subcellular fractions, BioIVT enables scientists to better understand the pharmacokinetics and drug metabolism of newly discovered compounds and the effects on disease processes. By combining our technical expertise, exceptional customer service, and unparalleled access to biological specimens, BioIVT serves the research community as a trusted partner in ELEVATING SCIENCE®.
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