A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants.

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