Shear effects on suspended marine sponge cells

Abstract

Fractions of viable cells, apoptotic and irreversibly damaged cells, dead whole cells and cell fragments were measured by flow cytometry during the production of freely suspended primary cells from explants of the marine sponge Axinella damicornis. The explants were disintegrated using the well-known Müller protocol [W.E.G. Müller, M. Wiens, R. Batel, R. Steffen, R. Borojevic, M.R. Custodio, Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula, Mar. Ecol. Progr. Ser. 178 (1999) 205–219]. Supplementation of the standard Ca 2+- and Mg 2+-free artificial seawater of the Müller protocol, with the shear protectant Pluronic F68 (0.1%, w/v) greatly reduced the cell damage and enhanced the recovery of viable cells at each of the four stages of the protocol. Agitation of cells on an orbital shaker at 75 rpm essentially killed all the viable cells within 2.5 h, but no loss of viability occurred at a higher agitation speed of 100 rpm for up to 6 h when the cells were supplemented with Pluronic F68. This time-dependent loss in viability could be significantly reduced by processing at 3 °C instead of the normal 17 °C. A four-step mechanistic model was shown to describe the kinetics of cell death and fragmentation within ±10% of the measured values. The damage to cells was modeled as a web of first-order processes that did not depend on cell–cell interactions. The forces in the agitated fluid killed the viable cells by impact, which was not accompanied by cell rupture (i.e. the cell was left dead, but intact).