Vivek Patel1, Khalid Amin2, David Allen3, Lindsey Ukishima1, Adam Wahab1, Chad Grodi1 and Holger Behrsing1
1Respiratory Toxicology, Institute for In Vitro Sciences, Inc., Gaithersburg, MD, USA
2Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA
3Integrated Laboratory Systems, Inc., Morrisville, NC, USA
This report details the maintenance of hPCLS in culture for 28 days, comparing three media and three commonly used culture methods. Viability and functionality were assessed by measuring several markers. The ALI culture method permits hPCLS to be directly exposed in nebulisers like the Vitrocell® Cloud. This device is designed to help evaluate products such as aerosolised cleaning agents or electronic cigarettes.
As non-animal alternatives gain acceptance, a need for harmonised testing strategies has emerged. Arguably the most physiologically-relevant model for assessing potential respiratory toxicants, that based on human precision-cut lung slices (hPCLS) has been utilised in many laboratories, but a variety of culture methodologies are employed. In this pilot study, combinations of three different hPCLS culture methods (dynamic organ roller culture (DOC), air–liquid interface (ALI) and submersion) and various media (based on E-199, DMEM/F12 and RPMI-1640) were compared. The hPCLS were assessed in terms of their viability and responsiveness to challenge. The endpoints selected to compare the medium–method (M–M) combinations, which included histological features and viability, were evaluated at day 14 (D14) and day 28 (D28); protein and adenylate kinase (AK) content, and cytokine response to immunostimulants (lipopolysaccharide (LPS) at 5 μg/ml; polyinosinic:polycytidylic acid (Poly I:C) at 15 μg/ml) were evaluated at D28 only. Based on the set of endpoints assessed at D28, it was clear that certain culture conditions significantly affected the hPCLS, with the tissue retaining more of its native features and functionality (in terms of cytokine response) in some of the M–M combinations tested more than others. This pilot study indicates that the use of appropriate M–M combinations can help maintain the health and functional responses of hPCLS, and highlights the need for the standardisation of culture conditions in order to facilitate effective inter-laboratory comparisons and encourage greater acceptance by the regulatory community.