Common Mistakes When Handling Hepatocytes and How to Avoid Them

Hepatocyte cell culture is a key technique in absorption, distribution, metabolism, and excretion (ADME) and toxicology research, requiring careful handling to maintain cell viability and achieve reliable experimental results.

After years of experience, we have gathered common mistakes and best practices for hepatocyte handling for successful experiments.

.

Common Errors in Culture and Handling of Cryopreserved Primary Hepatocytes

Thawing

The first step to start a primary hepatocyte culture using cryopreserved hepatocytes is thawing. 

One of the prevalent mistakes in hepatocyte culture is thawing cryopreserved hepatocytes too quickly or at incorrect temperatures, leading to decreased viability. After removing the cryovial from liquid nitrogen, place it into a 37°C water bath until the cell suspension is partially thawed, usually within 1-2 minutes. 

Tip:  Ensure partial thawing by leaving a small ice pellet in the cryovial to prevent over-thawing.

.

.

Another common hepatocyte culture error is not pre-warming the thawing medium at 37°C. The cryovial content, including the ice pellet, should be poured into a 50 mL tube with a pre-heated thawing medium to ensure optimal recovery. It is essential to gently move the Falcon tube to create a homogeneous suspension.

Tip: Add 1 mL of medium to the cryovial, to recover the cells that have remained inside. We recommend using a 1000 µL pipette and rinsing any remaining content in the cryovial with thawing medium to maximize recovery.

.

Counting 

Accurate cell counting is essential for experimental success, as over-seeding can lead to nutrient depletion and waste accumulation, while under-seeding can result in poor cell-cell interactions. BeCytes includes a detailed certificate of analysis (CoA) providing cell number, cryopreserved hepatocyte viability, and their standard deviations. Strictly follow the protocol and CoA to optimize your time by seeding the correct concentration on the first attempt. 

Incorrect homogenization of the cell suspension after thawing is a common mistake when handling cryopreserved hepatocytes. Carefully homogenize and resuspend the cell suspension, but avoid vortexing, before taking an aliquote for cell counting using a hemocytometer. 

Tip: Primary human hepatocytes are very sensitive. Avoid pipetting up and down for homogenization. Instead, resuspend by gently tapping. 

Take 100 μL of the cell suspension and mix it in a 1.5 mL tube with 100 μL of Trypan Blue solution and 800 μL of Phosphate Buffered Saline (PBS). 

.

.

Avoid counting the 4 sides within the square in the hemocytometer, only count 2 of them.  Apply the following formula to obtain cell density: 

Viability (%) = (Live cell count/Total cell count) x 100

Total Cell number = Viable cell count/Quadrants counted x Dilution factor x 104* x Current volume (mL)

.

Seeding

Another common mistake in hepatocyte culture is using suboptimal hepatocyte culture media. The selection of appropriate media is vital for maintaining hepatocyte function and viability. 

BeCytes media has been designed to successfully culture primary hepatocytes following specific formulations to support the thawing, plating, and maintenance of primary cells. Most of the products are ready-to-use and are available in a variety of formats.

Once cells are counted, adjust cell suspension to the desired density. The recommended seeding density for each lot is stated on the accompanying data sheet. Indicative seeding densities for BeCytes primary human plateable hepatocytes are shown in Table 1. 

Table 1. Recommended seeding densities for BeCytes human primary plateable hepatocytes in diverse well plates. 

.

Tip: Once the plates have been seeded shake vigorously in T form, except the 96-well plates that should NOT be shaken. During the first hour after the seeding, move the plate in T form every 15 minutes.

.

Extra Tips for Working with Cryopreserved Hepatocytes Successfully

If you need extra help, here are some additional tips to optimize thawing, counting, and seeding procedures, helping you achieve the best possible results:

.

Thawing

• Do not refreeze hepatocytes after thawing. Refreezing hepatocytes after thawing, even if you haven’t cultured them yet, involves repeated freezing and thawing cycles. These cycles can cause additional stress and damage to hepatocytes and their subcellular components. 
• Hepatocytes are non-proliferative and very sensitive cells, any assays designed with them should be endpoint-based, avoiding splitting. 
• Primary hepatocytes from different animal species require adapted centrifugation conditions. Check Table 2 for centrifugation details. 

Table 2. Centrifugation details for different species.

.

Seeding

• Collagen I-coated tissue culture wells are recommended for primary human hepatocytes 2D configurations to maintain the hepatocytes in culture for extended periods. The sandwich configuration also helps mimic the in vivo phenotype. Collagen I-coated plates are used, where hepatocytes are seeded first, followed by the addition of Matrigel, creating the sandwich conformation or 2.5D culture.

We provide top-tier hepatocyte products along with a detailed protocol to guide you through every step, as well as a specific Certificate of Analysis (CoA) for each lot. Whether you need cryopreserved hepatocytes, optimized culture media, or technical guidance, our team is here to help you achieve the best possible results.

Do you need more info? Do you require personalized assistance? 

Don’t hesitate to contact us! You will obtain personalized advice from BeCytes scientific and sales experts.

.

References

Kaur I, Vasudevan A, Rawal P, et al. Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements. Bioengineering. 2023;10(2):131. doi:10.3390/bioengineering10020131

Weiskirchen S, Schröder SK, Buhl EM, Weiskirchen R. A Beginner’s Guide to Cell Culture: Practical Advice for Preventing Needless Problems. Cells. 2023 Feb 21;12(5):682. doi: 10.3390/cells12050682.