This video features step-by-step instructions to help you prepare perfect hepatocytes.
Sekisui XenoTech’s cryopreserved hepatocytes are easy and convenient to use. Our patent pending, single-freeze process provides simple, customized pooling and long-term, consistent results with high cell yield and viability. However, this can only be achieved with proper thawing procedures.
We also strongly recommend using Sekisui XenoTech’s media, as it has been thoroughly tested for maximum performance with our hepatocytes.
First, prepare your work space. We recommend utilizing a biosafety containment hood to reduce the risk of contamination and minimize contact with potentially hazardous material.
Warm the OptiThaw media tube to 37° Celsius using a water bath, warmer, etc.
If you’re working with CryostaX pools, remove the cryotube from the liquid nitrogen storage unit and immediately dispense the frozen pellets into the pre-warmed OptiThaw media tube. Gently invert the tube until all of the pellets are melted.
For all non-CryostaX hepatocyte products, quickly transfer the cryotube into a 37° shaking water bath. The water level should be above the highest frozen point in the vial. Thaw the frozen cell pellet for about 80 seconds until it can move freely when the cryotube is inverted. Do not over-thaw!
Transfer the frozen pellet from the cryotube into the pre-warmed OptiThaw media tube. Rinse the cryotube with 1.5 mL of OptiThaw media, then pour this rinse into the OptiThaw tube, and gently invert until the pellet is melted.
Centrifuge the tube at 100 x g for 5 minutes at room temperature or 2-8°C. Do not vortex.
Carefully aspirate and discard the supernatant fluid without disturbing the cell pellet. Position the tip of the pipette at the top of the liquid to remove any dead cell debris from the suspension as you aspirate the liquid. Do not pour out the supernatant.
Resuspend the cell pellet(s) with the OptiPlate media for plating hepatocytes, or OptiIncubate media for suspension incubations, adding the media down the side of the tube, then mix gently until the cell pellet is distributed. Do not over-dilute.
Remove 50 μL of the homogenous cell suspension and dispense the 50 μL aliquot into the counting tube.
Mix gently. Cell viability can now be assessed by placing an aliquot from the counting tube on a hemacytometer and counting the dead (blue) cells and viable cell number. Two counts are recommended for verification.
Measure the volume of the cell suspension and q.s. to a final volume to bring the cells to the desired concentration.
This concludes our thawing demonstration video. Remember, cells are not a stable test system. Moving quickly yet gently can make all the difference in achieving healthier, more viable cells.
If you have any questions or concerns, please contact Sekisui XenoTech at 913-438-7450 (toll-free in N. America at 1-877-588-7530), www.xenotech.com or firstname.lastname@example.org.