Tips to Work with Primary Hepatocytes

Tips to Work with Primary Hepatocytes

Following proper primary liver cell culture handling guidelines is essential to grant success in your in vitro experiments.

To obtain the best results, BeCytes Biotechnologies has outlined essential practices for working with these cells, from thawing to keeping them in culture.

Step 1 – Thawing your cells

  • If you don’t want to lose viability, do not leave hepatocytes floating in the cryopreservation medium for too long. After removing the cryopreserved hepatocytes from the liquid nitrogen, immediately place them at 37
  • Do not remove a small aliquot from the vial, because you are exposing primary hepatocytes to repeated freezing and thawing cycles and this procedure causes additional stress and damage on the cells and their subcellular components

Step 2 – Seeding your cells

  • Seed the correct cell density to obtain a confluent monolayer of hepatocytes. Check the certificate of analysis of each cell lot to know the right seeding density. Distribute the cells by manually shaking the plate in a T-shape and checking it under a microscope.

An accurate cell count is vital to the success of your experiment because both under-seeding and over-seeding influence the quality of the sample. BeCytes always includes the number of cells and the viability with the respective standard deviation for each parameter at the certificate of analysis so you can compare your cell count to theirs.

  • When seeding non-parenchymal cells (NPCs), please be careful! Identifying cell debris and hepatocytes (bigger cells) is probable during cell counting. Do not consider these cells as part of the cell counting. NPCs should be identified as the smallest and brightest cells.
  • If you work with plateable primary hepatocytes, 2D configurations are based on hepatocytes cultured on collagen-coated tissue culture wells or flasks to maintain the hepatocytes in culture for extended periods. The sandwich culture, between two collagen hydrogel layers, has been described to maintain a high level of hepatic functions, such as albumin secretion and cytochrome P450 activities, reaching culture period times superior to 4 weeks.
  • To better mimic the liver microenvironment and avoid hepatocyte dedifferentiation in long-term culture, 3D configurations are recommended. Hepatocyte spheroids that help to maintain cell polarity can be generated using low-attachment plates, hanging drop cultures, microwells, etc…

BeCytes hepatocytes are 3D spheroid-qualified and can self-assemble into a 3D spheroid using their protocol, ultra-low attachment plates and their 3D plating and maintenance medium.

Step 3 – Keeping your cells in culture

  • Do not split primary hepatocytes since these cells are not proliferative and are very sensitive.


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

Originally posted by BeCytes Biotechnologies on:

Caltag Medsystems is the distributor of BeCytes Biotechnologies products in the UK and Ireland. If you have any questions about these products, please contact us.

Tips to Work with Primary Hepatocytes

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