Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap.

Chick wing bud mesenchymal cell micromass culture allows the study of a variety of developmental mechanisms, ranging from cell adhesion to pattern formation. However, many cells remain in contact with an artificial substratum, which can influence cytoskeletal organisation and differentiation. An ultrasound standing wave trap facilitates the rapid formation of 2-D monolayer cell aggregates with a defined zero time-point, independent from contact with a surface. Aggregates formed rapidly (within 2 min) and intercellular membrane spreading occurred at points of contact. This was associated with an increase in peripheral F-actin within 10 min of cell-cell contact and aggregates had obtained physical integrity by 30 min. The chondrogenic transcription factor Sox9 could be detected in cells in the ultrasound trap within 3 h (ultrasound exposure alone was not responsible for this effect). This approach facilitates the study of the initial cell-cell interactions that occur during condensation formation and demonstrates that a combination of cell shape and cytoskeletal organisation is required for the initiation and maintenance of a differentiated phenotype, which is lost when these phenomena are influenced by contact with an artificial substrate.

A quantitative inverse relationship between connexin32 expression and cell proliferation in a rat hepatoma cell line.

Gap junctions comprised of connexin proteins are involved in direct intercellular communication and the regulation of cell behaviour and homeostasis. Reduced connexin expression and loss of gap junction function is a characteristic of many cancer cells and of the effect of many non-genotoxic carcinogens that induce cell proliferation. Moreover, when certain cancer cell lines are transfected with specific connexin genes, cells can regain control over proliferation. We have employed RNA interference and dexamethasone to modulate connexin32 expression in MH(1)C(1) cells to a range of concentrations. This allowed the determination of the quantitative relationship between connexin32 protein expression and cell proliferation. The magnitude of cell proliferation, measured by bromodeoxyuridine incorporation, was inversely proportional to the level of connexin32 expression. Q-PCR indicated a lack of change of expression of a range of cell cycle-related genes at 24h. The inverse relationship between Cx32 expression and proliferation was continuous, and a threshold level of reduction of connexin32 was not observable for an influence on proliferation.

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates.

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.