RETAINING CELL INTEGRITY DURING ORGANOTYPIC MODEL VIABILITY ASSAYS: ALTERNATIVES TO MTT.

This study by researchers at the State University of New York (SUNY) at Stony Brook documents the effort to the develop an alternative tissue viability endpoint analysis using a non-lytic agent (MTS, Alamar Blue or CalceinAM) in place of the industry-standard MTT for MatTek’s EpiAirway in vitro 3-D human tracheal/bronchial tissue equivalent and MatTek’s EpiDerm in vitro 3-D human skin tissue equivalent, thereby allowing users to preserve the tissues on which the viability assay was performed, permitting subsequent assays to be performed on those same tissues. Viability assays are important tools used for testing the cytotoxicity associated with synthetic reagents and environmental effectors. Our research frequently uses several of MatTek’s commercially produced 3-D, airlifted, organotypic models, EpiAirway™, EpiDerm™, EpiDermFT™ and MelanoDerm™. In each case MTT is the recommended end-point assay for cell viability. However, we often need to retain tissue integrity and MTT requires tissue lysis. Three non-lytic assays that our lab routinely uses on 2-D submerged cultures are MTS, Alamar Blue, and CalceinAM. MTS; a tetrazolium based assay similar to MTT, is reduced by metabolic activity to a soluble formazan. With Alamar Blue, resazurin is reduced to a fluorescent compound, resourufin. CalceinAM permeates the cell membrane and is hydrolyzed by intracellular esterases to green fluorescent calcein, which remains in the cytoplasm. Protocols for use of these non-lytic assays on air-lifted 3-D tissues have not been well described or systematically validated. We are endeavoring to adapt these assays to the organotypic models and clarify the best approaches to use. In our search for a reliable, reproducible, and robust tissue-preserving alternative to the current standard MTT viability assay, MTS is the most promising non-lytic assay. Alamar Blue and CalceinAM require further testing to determine if they can be suitably adapted for use on organotypic tissue models.

Alamar Blue, CalceinAM, EpiAirway, EpiDerm, LDH release, MTT assay

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