Tissue-derived fractions commonly used in hepatocytes, S9, and in vitro tests

2018/12/212019/5/11

Here is a memo about the characteristics and differences of liver tissue-derived components that are widely used in in-vitro studies of pharmacokinetics and drug metabolism research.

table of contents

1. hepatocyte
2. S9 fraction
3. Liver microsomes
4. Cytosol

hepatocyte

Hepatocytes isolated from a suspension (liver homogenate, liver free cells) obtained by crushing and homogenizing the liver.

Since hepatocytes are retained as they are, they are relatively close to in-vivo. The reproducibility of the conjugation reaction, which is difficult to proceed with the S9 and microsome fractions, is also good.It can be used for uptake via transporters, drug-drug interactions, and evaluation of CYP enzyme induction.On the other hand, it is necessary to pay attention to the survival rate during cryopreservation and thawing.

S9 fraction

As an image, a fraction containing metabolic enzymes is extracted from hepatocytes.

The supernatant (supernatant) obtained by centrifuging the liver homogenate removes cell membranes, nuclei, and mitochondria.It contains many enzymes involved in drug metabolism such as the following.

Cytochrome P450 (CYP), flavin-containing monooxygenase (FMO), carboxyesterase (CES), epoxide hydrolase (EH), UDP glucuronosyltransferase (UGT), sulfate transferase (SULT), glutathione-S-transferase (GST) , Methyltransferase, acetyltransferase, etc.

Liver microsomes

Precipitation when S9 is further centrifuged.Includes endoplasmic reticulum fragments (torn during centrifugation) and lysosomes.

Endoplasmic reticulum cells
One of the organelles.It has a structure with a reticulated film.
It has important functions in sugar / lipid metabolism and steroid / protein synthesis.
Cytochrome P450 and oxygenase are localized in a film.

Lysosome
A vesicle consisting of a single layer of membrane.
Contains hydrolases such as glycosidases and peptidases.
Important for foreign substances in cells and autolysis.

Since many drugs and foreign substances are metabolized and detoxified by CYP, "microsome fraction, which is a simple system including CYP" is widely used in experiments (although metabolism other than CYP is also important in recent years).It is easy to use for reasons such as stability against cryopreservation, reproducibility, and abundant commercial products.

CYP, UGT, CES, etc.