Abstract
The Bionas® 2500 system permits for the first time the long-term observation of cell lines, primary cells, tissue slices and biopsies in the presence of drug candidates under close-to-in-vivo conditions. The proprietary perfusion system allows the observation of regeneration effects.
Main
We present a sensor system that simultaneously measures three metabolically relevant parameters, such as oxygen consumption, the extracellular acidification rate and adhesion of test cells. The Bionas system is easy to use and allows the noninvasive and label-free monitoring of compound effects on cellular metabolism. The readout is continuous for up to several days.
The Bionas system is a valuable tool for optimizing target validation studies as it places the targets in near-physiological context. The proprietary perfusion system allows the monitoring of both acute (short-term) and chronic (long-term) effects of drug candidates in various cell types, and thus facilitates the selection of the most promising compounds for development.
Prediction of hepatotoxicity
The Bionas system provides for the first time a close-to-in-vivo environment that allows scientists to better predict hepatotoxicity and thus make more informed decisions earlier in the drug development process.
Primary hepatocytes grow on sensor chips
The Bionas system, in combination with the HEPAC2 cell cultivation method of primary hepatocytes (Primacyt GmbH), is a powerful tool for culturing primary hepatocytes and analyzing early signals of hepatotoxicity, a frequent side effect of pharmaceutical drugs.
To determine the most suitable cell type for predicting hepatotoxicity, we treated primary human and rat hepatocytes as well as human liver carcinoma cell line HepG2 cells with acetaminophen (AAP, paracetamol). AAP is metabolized to the toxic metaboliteN-acetyl-p-benzoquinone imine (NAPQI) by the hepatic cytochrome P450 system.
We seeded the cells directly on precoated sensor chips and cultured them as described1,2. Monitoring of the oxygen consumption rates, acidification rates and cellular adhesion with the Bionas system revealed distinct differences between these cells (Table 1).
Primary human hepatocytes are the best choice
The comparison of HepG2 cells and primary human and rat hepatocytes in response to AAP revealed the following:
-
Primary human hepatocytes were more similar to HepG2 cells than to primary rat hepatocytes.
-
Primary rat hepatocytes were more sensitive to AAP than the other cell types. Only rat hepatocytes showed strongly reduced cell adhesion and acidification rates in addition to a reduction of oxygen consumption.
-
Only primary human hepatocytes showed complete regeneration with 1.0 mg/ml AAP, whereas rat hepatocytes were irreversibly damaged under these conditions.
-
The comparison of primary human and rat hepatocytes demonstrates that rat cells behave differently in response to AAP. Rat hepatocytes, and probably other rodent cells, are therefore not suitable for predicting hepatotoxicity in humans even though they are widely used.
Toxic effects are independent of the donor
We isolated primary human hepatocytes from two different donors (P1 and P2) and cultured them on precoated sensor chips before assaying the metabolic parameters in the presence of AAP. The results show that in both preparations, respiration decreased in a dose-dependent manner (Fig. 1). This inhibition took effect very quickly and reached maximum values within the first 10 min of AAP exposure. The effects were independent of the donor, indicating that the response may be an intrinsic feature of the primary cells.
After removing AAP from the medium, the hepatocytes regenerated very quickly. Neither acidification rates nor cell adhesion were significantly influenced by AAP in both preparations (data not shown).
Dynamic IC 50 and RIC 50
Through the continuous respiration measurements (Fig. 1) taken by the Bionas system, it is possible to calculate the half-maximal inhibitory concentration (IC50) at each time point. Plotting IC50 versus the exposure time to the substance generates dynamic IC50 curves that illustrate the rapid inhibitory effect of AAP on primary human hepatocytes (Fig. 2).
Notably, the dynamic IC50 values of AAP in primary human hepatocytes are around 1,000 mg/l and hence are much lower than those described in the literature (IC50: 2,815 mg/l for a 4-h exposure, LS-L929 cells3) indicating greater than expected sensitivity. After removal of AAP, we observed an increase in the dynamic IC50 values reaching values around 2,000 mg/l. A residual inhibition of respiratory activity of 50% (residual IC50 or RIC50) was observed after exposure to 2,000 mg/l AAP.
Conclusion
Continuous measurements using the Bionas analyzing system reveal distinct effects on metabolism and therefore allow deeper insight into cellular pathways than is possible with endpoint-based methods. Not only does this allow the analysis of dose response in cells and tissues, but in addition recovery, regeneration and adaptation effects are easily detectable.
This article was submitted to Nature Methods by a commercial organization and has not been peer reviewed. Nature Methods takes no responsibility for the accuracy or otherwise of the information provided.
References
Runge, D. et al. Induction of cytochrome P450 (CYP)1A1, CYP1A2 and CYP3A4 but not of CYP2C9, CYP2C19, multidrug resistance (MDR-1) and multidrug resistance associated protein (MRP-1) by prototypical inducers in human hepatocytes. Biochem. Biophys. Res. Commun. 273, 333–341 (2000).
Thedinga, E. et al. In vitro system for prediction of hepatotoxic effects in primary human hepatocytes. ALTEX 24, 22–34 (2007).
Clemedson, C. et al. MEIC evaluation of acute systemic toxicity. Altern. Lab. Anim. 24, 273–311 (1996).
Acknowledgements
We thank D. Runge, Primacyt GmbH, for providing the primary hepatocytes and cultivating them on the sensor chips, and S. Duntze, b3c communications, for her support in scientific communications.
Author information
Authors and Affiliations
Corresponding author
Additional information
Disclaimer
This article was submitted to Nature Methods by a commercial organization and has not been peer reviewed. Nature Methods takes no responsibility for the accuracy or otherwise of the information provided.
Rights and permissions
About this article
Cite this article
Thedinga, E., Ehret, R. & Schulze, M. The Bionas® system bridges the gap between in vivo and in vitro. Nat Methods 4, iii–iv (2007). https://doi.org/10.1038/nmeth1071
Issue Date:
DOI: https://doi.org/10.1038/nmeth1071
This article is cited by
-
[N,N′-Bis(salicylidene)-1,2-phenylenediamine]metal complexes with cell death promoting properties
JBIC Journal of Biological Inorganic Chemistry (2009)