Five Reasons Why Your Hepatocyte Plating Failed

#1:  You left them in the cryopreservation medium after thawing.  

Hepatocytes are very fragile during thawing procedures and if left casually floating in the cryopreservation medium for too long then poor viability may be the result.  Here are some tips for thawing hepatocytes for optimum viability:

1. Carefully, thaw in a clean waterbath set no higher than 37 degrees.
2. Watch closely for ice crystals in the vial to thin and then remove from waterbath when small spindle of ice still remains.
3. Quickly transfer cells into pre-warmed thawing medium 

#2: You didn't properly disperse the cells when plating them.

Hepatocyte cell health is best when in a tight 100% confluent monolayer.  To accomplish this, use a shaking method where plate is setting on a flat surface and pushed in a N-S and then E-W directions several times. 

Repeat this shaking every 15 minutes during the first hour of plating. With exception is the 96 or 384 wells plate where you should pre-fill wells with ½ of the total media without cells.  When a 2x concentration of cells are added, the plate should be kept as still as possible. 

#3:  You plated too few or too many cells.

Hepatocyte cell health is best when in a tight 100% confluent monolayer.   Cell-cell interactions are important to maintaining metabolic activity in hepatocytes, long-term. If cells are too sparse, de-differentiation occurs quickly.  If cells are too dense, you can have poor attachment.  Double check you cell density calculations and carefully monitor the morphology of the monolayer using microscopy.  Healthy hepatocytes have bright cell borders, a very distinct nucleus, and cobblestone shapes with low cytoplasm to nucleus area ratios. 

#4: You didn’t use the right media or plates.

So you can’t replicate the beautiful morphology promised from the manufacturer. There are a few possible causes and solutions:

1. Make sure you are using plates coated with type I collagen. We have success with Corning BioCoat^® plates that come in all the major configurations for plating hepatocytes.
2. Your cell density may be incorrect. Check your calculations to make sure that your cell density matches the recommended values.
3. Your plating or maintenance medium may not have the right ingredients to promote attachment. Lonza provides plating and maintenance media optimized for superior hepatocyte health.

# 5: You incorrectly measured the post-thaw viability. 

 Your Trypan blue concentration may be too high. Unlike many other cell types, hepatocyte membranes are full of highly active transporters that can make cells appear bluish color in high concentrations of Trypan Blue when then are not dead. Underestimating your viability will lead to over-plating and ugly monolayers.  Ensure that the final Trypan blue concentration in counting medium is no more than 0.04%. Many automated counters use higher concentrations of Trypan blue.  Using the appropriate Trypan blue concentration and manually counting by eye with a hemacytometer is the best way to ensure you know the accurate number of viable hepatocytes in your culture.