Top Tips for Hepatocyte Handling

Top Tips for Hepatocyte Handling

Running a successful experiment using cryopreserved primary hepatocytes can be an effective and hassle-free experience when you follow all the correct hepatocyte handling procedures. The following are some key best practices in the handling of hepatocytes. Our optimized and standardized methods produce the highest quality hepatocytes in the industry.  To achieve the best results in your lab we have provided our top tips to guide you in your ADME-Tox research.

Storage

It is important to properly store your cryopreserved hepatocytes in order to guarantee they maintain their characteristics.

• Cells must be transferred from the cryoshipper upon delivery to a temperature-controlled cryogenic freezer
• Store cells at < -150°C in vapor phase of liquid nitrogen. Cells stored in liquid nitrogen may result in liquid nitrogen entering the vial and becoming a hazard upon thawing.
• If necessary you can transport vials under dry ice for less than 30 minutes, but this should be minimized as much as possible.

Thawing

When thawing a vial of cryopreserved hepatocytes, the goal is to thaw them just enough so that an ice pellet remains but is loose from the vial walls and can be decanted out into a conical tube. This prevents over-thawing and maintains good cell viabilities. Our thawing process sets us apart, as BioIVT’s cryoplateable hepatocytes do not require a percoll purification or wash step to achieve high viabilities. This not only saves you time and money but demonstrates the high quality of our cells.

• The appropriate INVITROGRO™ hepatocyte medium should be pre-warmed to 37°C prior to handling of the cells.
• Eliminate the need for a wash or percoll purification step by thawing cryoplateable hepatocytes directly into a small working volume of INVITROGRO CP medium. We recommend ~1mL for every million cells per vial being thawed.
• Thaw cells promptly upon removal from the storage unit
• After the cells have pulled away from the vial walls, but when a small ice pellet still remains, pour contents into pre-warmed medium to complete the thawing process.

Counting

Performing an accurate cell count is an important step when ensuring you have an accurate cell concentration in your thawed cell suspension. Errors in cell counts can cause errors in cell density or incubation concentrations which can impact your results.

• Trypan Blue exclusion counting using a Neubauer hemocytometer is the recommended method to determine cell viability and concentration
• A 1:5 dilution of Trypan Blue is recommended
• You can use our INVITROGRO KHB to dilute Trypan Blue
• (700µL KHB + 200µL trypan blue + 100µL cell suspension sample)
• Handle cells gently while in Trypan Blue to determine accurate cell viability
• Cell counting should be done within 5 minutes of cells being added to Trypan Blue
• Only count 2 of the four sides within the square

Resuspend cells before sampling or aliquoting, hepatocytes have a high sedimentation rate. Below is a time course of how quickly hepatocytes can settle.

Plating

Following proper plating techniques will allow you to seed and distribute the cells evenly to achieve proper confluency.

• Dilute the cells to 0.70 x 10⁶ viable cells/mL with recommended INVITROGRO hepatocyte medium for all species except mouse or when using 96 or 384-well plates. (Consult chart below)
• Mouse hepatocytes are larger than that of other species and require half the seeding density. (for example, 0.35 x 10⁶ viable cells/mL instead of 0.70 x 10⁶ viable cells/mL)
• Seed at the correct concentrations to avoid over or under seeding
• As a final step to plating, gently shake the plate in a north-south and then east-west direction to distribute the cells evenly
• Change the medium 3-4 hours after plating to remove any cells that did not attach

*Note that these seeding densities are for standard cryoplateable human and animal hepatocytes. TRANSPORTER CERTIFIED™ human hepatocyte lots have optimized seeding densities which can be referenced in the lot-specific COA.

Product Selection

Select the correct product for your application:

• Know your assay, does it require suspension or cryoplateable hepatocytes
• Our team can help you select the best products for your applications
• We have LIVERPOOL® Cryoplateable and Cryosuspension Human Hepatocyte lots of up to 200 donors to meet your diverse pooled donor needs
• Our extensive human donor liver inventory allows you to screen for specific characteristics including donor demographics, genotypes, pathology, HLA, transporter function, and CYP450 fold induction values

Visit our ADME-Toxicology page to learn more about all our liver-related products and services.