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2023 FDA Science Forum

Lessons learnt in establishing a reliable and low-cost assay for urea production in human primary hepatocytes cultured in a Liver-Chip for the study of drug hepatotoxicity

Authors:
Poster Author(s)
Shi, Qiang, FDA/NCTR; Ren, Lijun, FDA/NCTR; Papineau, Katy, FDA/NCTR; Sauld, John, Emulate Inc.; Kulkarni, Gauri, Emulate Inc.; Ewart, Lorna, Emulate Inc.; Avigan, Mark, FDA/CDER; Mendrick, Donna, FDA/NCTR; Hawes, Jessica, FDA/NCTR; Schnackenberg, Laura, FDA/NCTR
Center:
Contributing Office
National Center for Toxicological Research

Abstract

Poster Abstract

Background

Liver-on-a-chip (Liver-Chip) is a microphysiological system (MPS) designed to better maintain hepatic functions for liver cells cultured under in vitro conditions. Urea is synthesized exclusively by hepatocytes and is a proposed quality control marker for hepatocyte function maintained in MPS. Purpose: This study aimed to investigate the sensitivity and specificity of two urea assay methods for cells cultured in a Liver-Chip system for the study of acetaminophen-induced liver injury.

Methodology

Primary human hepatocytes (PHHs) were co-cultured with three types of non-parenchymal cells (NPCs) including liver sinusoid endothelial cells, Kupffer and stellate cells in Emulate S1-Chips®. The perfusing culture medium, also called effluent, was collected every 24 h to measure urea levels. Chemical and enzymatic methods, which have been reported for measuring urea in S1-Chips, were tested in parallel in the same samples.

Results

In the enzymatic method, culture medium alone led to a 6-fold increase of signal in urea measurements compared to blank buffer controls. Such background signals were directly proportional to levels of sodium pyruvate, a common component of many types of culture medium, including PHH cell cultures. This high background signal introduced large variations among replicates of the same sample, making it impossible to accurately determine the relatively low levels of urea, which were about 10 µg/mL, in the S1-Chips effluents. Effluents must be diluted by 5-fold to effectively reduce such interference, but this made it difficult to observe the percentage of reduction after drug treatment, as urea levels neared the limit of detection (1.2 µg/mL).

In contrast, the chemical method had negligible background signal for culture medium and a lower limit of detection (0.8 µg/mL). Additionally, the urea level could be accurately determined by chemical method at a fraction of the cost of the enzymatic method. As determined by the chemical method, the average urea production rate of hepatocytes from three human donors was 203 (± 29) µg/million cells/day, and acetaminophen treatment (0.1 to 10 mM) led to a concentration- and time-dependent reduction of 20% to 60%.

Conclusion

These data may guide selection of appropriate assays and aid optimization of procedures for urea detection in various liver cell culture platforms.


Poster Image
Lessons learnt in establishing a reliable and low-cost assay for urea production in human primary hepatocytes cultured in a Liver-Chip for the study of drug hepatotoxicity

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