Abstract
Development of the new generation of drugs (e.g., oligo- and polynucleotides administered intravascularly either as free compounds or as nano-formulations) frequently encounters major challenges such as lack of control of targeting and/or delivery. Uncontrolled or unwanted clearance by the liver is a well-known and particularly important hurdle in this respect. Hence, reliable techniques are needed to identify the type(s) of liver cells, receptors, and metabolic mechanisms that are responsible for unwanted clearance of these compounds.We describe here a method for the isolation and culture of the major cell types from mouse liver: hepatocytes (HCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs). The presently described protocol employs perfusion of the liver with a collagenase-based enzyme preparation to effectively transform the intact liver to a single cell suspension. From this initial cell suspension HCs are isolated by specified centrifugation schemes, yielding highly pure HC preparations, and KCs and LSECs are isolated by employing magnetic-activated cell sorting (MACS). The MACS protocol makes use of magnetic microbeads conjugated with specific antibodies that bind unique surface antigens on either KCs or LSECs. In this way the two cell types are specifically and separately pulled out of the initial liver cell suspension by applying a magnetic field, resulting in high purity, yield, and viability of the two cell types, allowing functional studies of the cells.If the drug compound in question is to be studied with respect to liver cell distribution of intravascularly administered drug compounds the isolated cells can be analyzed directly after isolation. Detailed studies of receptor-ligand interactions and/or dynamics of intracellular metabolism of the compound can be conducted in primary surface cultures of HCs, LSECs, and KCs established by seeding the isolated cells on specified growth substrates.
Highlights
Modern drug treatment modalities frequently include i.v. administration of large molecule compounds or nano-formulations
Apart from the one noteworthy example of i.v. administered oligonucleotide therapeutics patisiran that successfully targets the major type of liver cells, the hepatocytes (HCs) [2], the general rule is that large molecules/nanoformulations are rapidly cleared from the circulation by specialized scavenger cells lining the wall of the several hundred million capillary-like liver vessels called liver sinusoids
Exact studies designed to determine to what extent any i.v. administered compound accumulates in the different types of liver cells require reliable methods to isolate pure preparations of liver sinusoidal endothelial cells (LSECs), Kupffer cells (KCs), and HCs from a single liver
Summary
Modern drug treatment modalities frequently include i.v. administration of large molecule compounds or nano-formulations. Exact studies designed to determine to what extent any i.v. administered compound accumulates in the different types of liver cells require reliable methods to isolate pure preparations of LSECs, KCs, and HCs from a single liver. We pioneered the method of preparing LSECs, KCs, and HCs from a single rat liver, using collagenase perfusion to disperse the liver cells, followed by density separation on Percoll, and selective substrate adherence [9]. This method, which has been used with or without modifications by several laboratories, was later adapted by us for isolation and culture of mouse LSECs, KCs, and HCs [10]. This allows for both in vivo liver cell distribution of intravascularly administered compounds, as well as in vitro analysis of how compounds interact with isolated cultures of KCs, LSECs, and HCs
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