Meeting Abstract
A keystone species, the North Atlantic lobster, Homarus americanus, is a cold water invertebrate that is important fundamentally and commercially. Due to the lobster’s anatomical and physiological complexity, the transepithelial transport processes for any nutrient, ion, or heavy metal cannot be studied with ease. Therefore, dissociation of the organs, i.e. hepatopancreas, antennal gland, into cellular suspensions that can form functional confluent monolayers in vitro would provide a new method to study the physiological functions of nutrient and ion absorption and transepithelial transport. Cell culture would provide a technical means by which complex invertebrate organs can be studied in relation to the transcellular transport properties of the epithelial cells that comprise the organs. In this preliminary study, 3D culture techniques, cell dissociation techniques, cell viability, and cell density seeding were investigated to develop the best method for a successful crustacean cell culture with a functional confluent monolayer. It was found that enzymatic dissociation, rather than mechanical dissociation, yielded more viable cells that were likely undergoing mitosis, forming functional junctions with neighboring cells, and pseudopods on 3D collagen substrata. Cell density also plays an important role in the development of a confluent monolayer, in which cells must be seeded densely to form functional junctions. With these investigations, the functional analysis of nutrient absorption in cultured monolayers arranged on a nylon screen placed in an Ussing chamber that can separate the monolayer’s apical and basal membranes can tell us the nature of solute movement across the cell layer and what processes regulate those transport events.
