Liver hepatocytes in cryopreservation contain major drug-metabolizing enzymes. For growing liver hepatocyte cell culture, a quick-thaw protocol is performed in the case of cryopreserved primary cells. However, if the thawing procedure is incorrect, the viability and functionality of the primary cells might be impaired.
Inhibit Cryoinjury effects
Before going for liver hepatocyte thawing, cryoinjury phenomena should be kept in mind. Cryoinjury causes intra- and extra-cellular ice formation, besides having osmotic effects on the primary cells, thus altering native protein conformation. The major cryoinjury on primary liver hepatocytes deals with cell membrane disruption, DNA breaks, and reduced cell attachment.
A simple tip to reduce cryoinjury effects is to quickly transfer primary liver hepatocyte vial from liquid nitrogen to 37°C water bath. The least time should be taken to transfer the vial of liver hepatocytes from liquid nitrogen to the cell culture lab at room temperature.
Time is Critical
Over-thawing of cryopreserved liver hepatocytes will result in low viability and yield. A time of 60-80 sec is generally enough. After thawing, it is also crucial to do the washing and centrifugation step quickly to limit the toxic effect of the cryoprotectant. Re-suspension should also be with gentle rocking motion rather than vortex or vigorously shaking.
Washing and Debris Removal
A single washing step is generally enough to ensure clean hepatocyte culture seeding after thawing. It is suggested to aspirate the supernatant rather than pouring off the supernatant after centrifugation. The pouring poses a risk of losing viable cell pellets. When aspirating, the tip of the aspirator should be at the highest media level to ensure that cell debris is removed before the viable cell pellet is reached.
Primary liver hepatocytes in culture will be represented by monolayer after plating with cuboidal hepatocytes having and one or two centrally located nuclei. Thawing of these primary cells often requires a simple understanding of the process to avoid errors and restrict low yield or bad viability. If your lab is currently looking for primary liver hepatocytes for research or industrial purposes, you can contact KOSHEEKA and we will provide you the best quality of cells along with expert technical assistance & support for hassle-free primary cell culture practice. Contact info@kosheeka.com for further inquiries.
