Summary of PMI's in vitro methods
Our toxicology studies involve in vitro methods to investigate the effects of smoke-free products on cultured cells (bacteria or mammalian cells) or tissues grown in the laboratory. Some of the most important types of in vitro toxicology tests we use for regulatory submissions are genotoxicity and cytotoxicity assays.
A genotoxicity assay is designed to detect genetic damage and gene mutations induced by a range of single chemicals and complex environmental and biological mixtures. The most common assay we use is the Ames Assay (or Bacterial Reverse Mutation Assay) which measures the rate of mutation in Salmonella strains. Other common genotoxicity assays employed are the Mouse Lymphoma Assay (MLA), which detects mutagenic events in the L5178Y mouse lymphoma tk+/- cell line, and the micronucleus assay. We have recently phased out the MLA in favor of the micronucleus assay.
A cytotoxicity assay is designed to determine the toxicity of a chemical on living cells. The most common assay we use is the Neutral Red Uptake Assay (a cell viability assay). This assay is based on the ability of viable mouse embryo cells (BALB/c 3T3 cells) to incorporate and bind a neutral red dye in the lysosomes of a cell after exposure to a range of chemicals.
For our in vitro research, we perform other studies to help us understand the effects of our products on different cell systems. Some of these include studies on lung cells, liver cells, and kidney cells. We usually start with 2D cell cultures before increasing in complexity with 3D cell cultures by using, for example, the Transwell system which enables the exposure of cells to air on one side and the cell culture liquid on the other side. This allows them to form an organotypic culture—one with 3D structure and behaviors more like what is found in the human airway. As such, we are using 3D cultures representing the different airway epithelia (nasal, buccal, gingival, bronchial, and small airway epithelium). With this method, our scientists can create cell cultures that better mimic tissues and organ substructures in the human body, as well as allowing whole aerosol exposures.