Preparation of equine isolated hepatocytes.

This research presents a detailed process for isolating liver cells, or hepatocytes, from horses. The researchers obtained livers from slaughtered horses, then cleans them and protected them from injury. They warmed and chelated the livers, then digested them with collagenase, consistently yielding large batches of viable cells.

Study Overview

• The researchers collected livers from horses at a local slaughterhouse to isolate hepatocytes. Hepatocytes are the main functional cells of the liver, responsible for protein synthesis, metabolism of proteins, carbohydrates and fats, and detoxification of metabolic products.
• The first step in the protocol involved perfusion with an oxygenated HBSS (Hank’s Balanced Salt Solution) at 0-2 degrees Celsius, with a continuous flow rate of 500-800 ml/min for 3-6 minutes. This was to remove blood from the liver and preserve it.
• Next, they protected the liver from ischemia (restricted blood supply) injuries by flushing it with an ice-cold University of Wisconsin (UW) Solution, with a continuous flow rate of 500-800 ml/min. The liver lobe was then immersed in UW solution at 2 degrees Celsius for transport to the laboratory. Ischemia injury could otherwise cause cell death and damage the tissue.
• In the laboratory, the researchers performed a three-step perfusion procedure: rewarming, chelating, and collagenase perfusion. These steps served to prepare the hepatocytes for isolation.

Optimal Conditions for Hepatocyte Isolation

• For rewarming, the liver was perfused with UW solution at 38 degrees Celsius for 8-14 minutes. This raised the tissue’s temperature after its cold transport.
• During the chelating step, the liver tissue was perfused with a calcium-free HBSS solution at 38 degrees Celsius, supplemented with a chelator (1 mM ethylene glycol-bis[beta-aminoethyl ester]-N,N,N’,N’-tetracetic acid) at a flow rate of 450 ml/min for 6 minutes. Chelating agents are used to sequester metal ions in solution, which in this case helps to soften the tissue, facilitating cell isolation.
• Finally, the researchers digested the liver tissue by perfusing with a HBSS solution supplemented with 0.1% collagenase, at 38 degrees Celsius and with a flow rate of 450 ml/min for 8-27 minutes. Collagenase is an enzyme that breaks down collagen, a structural protein present in the extracellular matrix and connective tissue. The breakdown of collagen aids in the isolation of the hepatocytes.
• Consistently carrying out this specified process resulted in approximately 21 million viable hepatocytes isolated per gram of perfused liver tissue, with an average cell viability rate of 82.7%.