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Abstract
Introduction With the rise in obesity and insulin resistance, there is an increased incidence of liver diseases such as Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatotic Hepatitis (NASH), which can lead to the development of liver cancer. Liver biopsy is the gold standard for classification of liver steatosis (mild, moderate, or severe) to determine stage of NAFLD and NASH and the suitability of donor livers for transplantation. However, these observations are made visually and thus are prone to inter-observer variations.
Aim The aim of the project is to develop a simple quantitative test to assess fat content in isolated human hepatocytes.
Methods Nile Red was used to stain lipids in human hepatocyte batches (14 steatotic and 8 non-steatotic). 10,000 cells were placed per well in a black 96-well plate and diluted Nile Red solution was added. Plates were incubated (37°C, 10 min), then read using a fluorescence plate reader. Data were recorded and analysed. Cytospins of 10,000 cells per slide were allowed and the slides were then air-dried for 24h. They were stained with Nile Red (humidity chamber at 37°C, 10 min), cell nuclei were counterstained with DAPI, and slides were visualised under a fluorescence microscope. Images were taken. [In both techniques unstained cells were used as negative controls].
Results The t-test showed statistically significant difference in fluorescence values in steatotic vs non-steatotic hepatocytes (p<0.01) (figure 1). Pearson correlation coefficient showed positive linear correlation between fluorescence values and histological steatosis grading (r=0.4337; p<0.05). Greater fluorescence in steatotic hepatocytes was observed under fluorescence microscope due to high lipid content compared to non-steatotic cells (figure 2).
The t-test shows a statistically significant difference in the fluorescence values in normal and steatotic livers samples (p<0.01), t=3.55, df= 17
A) Steatotic hepatocytes stained with nile red and DAPI. B) Background fluorescence in unstained steatotic hepatocytes. C) Non-fatty hepatocytes stained with nile red and DAPI. D) Background fluorescence in unstained non-fatty hepatocytes.
Summary and Conclusion Our study shows that Nile Red staining was an easy, quick, and reliable technique for grading steatotic hepatocytes isolated from a liver biopsy of a patient with NAFLD or NASH, or from a donor liver to be used in transplantation. These results can be combined with a FibroScan to give a useful quantitative measure of steatosis and fibrosis in a sample of hepatocytes.