Studies on Cell Aggregation: Demonstration of Materials with Selective Cell-binding Activity.

The mechanisms involved in the mutual attachments of cells and in selectivity of cell contacts are of major interest in studies on growth, developmental interactions, and differentiation in multicellular systems (recent reviews'). With the advent of procedures for dissociating tissues into cells and for controlled reaggregation of single cells into tissues, various aspects of these problems became accessible to critical analysis. Work on aggregation of dissociated embryonic cells directed attention to the possibility that specific cell products, functioning at the cell surface or between cells, might be involved in developmental attachment and interactions of cells.2 This raised the question, considered previously by others,3 , 4whether selective binding, grouping and "affinities" of cells, and related morphogenetic phenomena might not depend on the molecular features of such products.5 Suggestive in this context was the role of cell surface materials in selective contact of mating types of microorganisms,6 in virus attachment to cells,7 in antigenic specificity of blood cells,8 in fertilization,9 etc. The pursuit of these propositions hinged on validating the postulated existence of experimentally accessible cell-binding materials. Bioassays were therefore developed which would enable their detection,1" and tests were conducted for evidence of cell-binding activity in various preparations from embryonic cells,5 and from sponge cells. This report deals with work on marine sponge cells; the experimental advantages of these cells are that, unlike vertebrate cells, sponge cells function normally in sea water (or in a similarly defined salt solution) and can be dissociated without treatment with enzymes. It was found that (1) materials with cell-binding activity can be obtained cell-free from living sponge cells; (2) their removal results in loss of mutual adhesiveness of the cells; (3) their replacement reendows the cells with selective adhesiveness. Materials and Methods.-Microciona prolifera (referred to as 11), a bright red sponge, and Haliclona occulata (H), a light purple-brown sponge, (both from Cape Cod waters) were used. The difference in pigments served to identify cells in interspecific mixtures. Only freshly collected material was used. Preparation of cell suspensions: Two critically different dissociation procedures were used: (a) mechanical dissociation by pressing sponge fragments through fine cloth into sea water;" the expressate contained cells and numerous cell clusters; and (b) chemical dissociation' whereby suspensions of single cells were produced by removing divalent cations. To do this, mechanically dissociated cells were washed in calciumand magnesium-free artificial sea water (CMF-SW) at 4°C by swirling them for 6 hr on a gyratory shaker at 80 rpm. The cells collected by low speed centrifugation were resuspended by pipetting in cold CMF-SW to make a stock suspension of single cells. Cell aggregation: The procedure of cell aggregation by rotation was used;10 3 ml aliquots of cell suspension in sea water, or in a test medium, were rotated in 25 ml