Abstract
BackgroundIn farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.ResultsBuffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3±0.66)×107 cells per gram of liver tissue with a viability of 82.3±3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.ConclusionWe developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene expression, regulation, hepatic genomics and proteomics in farm animals.
Highlights
Liver is the main organ for metabolism, biotransformation of drugs and xenobiotics and storage of different biomolecules
Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue
In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer
Summary
Liver is the main organ for metabolism, biotransformation of drugs and xenobiotics and storage of different biomolecules. Primary hepatocytes are well representative of in vivo hepatocytes and can be used for studying metabolic activities [2] as well as to explore pharmacological properties of drugs and xenobiotics [3, 4]. No ruminant specific immortal hepatic cell line is available to study the liver function in farm animals. Under such scenario, primary cell culture is the most viable material for studying metabolism, pharmacology of drugs and xenobiotics metabolism, gene expression and regulation to understand the liver function. Understanding of pharmacological and toxicological properties of various drugs and xenobiotics or even feed components may be exploited to manage animal health for improving milk and meat production in farm animals. There is no suitable cell line available to understand liver-specific functions. We provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes
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