Molecular adhesion development in a neural cell monolayer forming in an ultrasound trap

A 2-dimensional aggregate of C6 neural cells was formed rapidly ( within 30 s) in suspension in a recently developed 1.5 MHz ultrasound standing wave trap. A typical 1 mm diameter aggregate contained about 3,500 cells. Spreading of membrane occurred between the aggregated cells. The rate of spreading of the tangentially developing intercellular contact area was 0.19 mu m/min. The form of the suspended aggregate changed from one of a hexagonal arrangement of cells to one of a cell-monolayer-like continuous sheet of mostly quadrilateral and pentagonal cells as in a cell monolayer on a solid substratum. A range of fluorescent indicators showed that the > 99% viability of the cells did not change during 1 h exposures; therefore cell viability was not compromised during the monolayer development. The average integral intensities from stained actin filaments at the spreading cell-cell interfaces after 1, 8 and 30 min were 14, 25 and 46 mm 2 respectively. The cells in this work progressed from physical aggregation, through molecular adhesion, to displaying the intracellular consequences of receptor interactions. The ability to form mechanically strong confluent monolayer structures that can be monitored in situ or harvested from the trap provides a technique with general potential for monitoring the synchronous development of cell responses to receptor-triggered adhesion.