Article ; Online: Protocol for Isolation and Culture of Mouse Hepatocytes (HCs), Kupffer Cells (KCs), and Liver Sinusoidal Endothelial Cells (LSECs) in Analyses of Hepatic Drug Distribution.
Methods in molecular biology (Clifton, N.J.)
2022 Volume 2434, Page(s) 385–402
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. ... ...
| 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. |
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| MeSH term(s) | Animals ; Endothelial Cells/metabolism ; Hepatocytes/metabolism ; Kupffer Cells ; Liver/metabolism ; Mice ; Pharmaceutical Preparations/metabolism |
| Chemical Substances | Pharmaceutical Preparations |
| Language | English |
| Publishing date | 2022-02-25 |
| Publishing country | United States |
| Document type | Journal Article |
| ISSN | 1940-6029 |
| ISSN (online) | 1940-6029 |
| DOI | 10.1007/978-1-0716-2010-6_27 |
| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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