Germination of Clostridium oncolyticum in spheroid cell cultures as an in vitro tumor model

An aggregation technique is used for construction of multicellular spheroids from trypsinized cell cultures of several normal and cancerous cell lines. This technique is applicable also in the presence of 1 X 10(9)/ml spores of Clostridium oncolyticum, and spores and vegetative bacterial cells can be counted by a soft agar colonization technique after trypsinization of spheroids. 24 hours after aerobic cultivation of spore-containing spheroids, germination and multiplication of Clostridium is detected by microscopy, electron-microscopy, and by bacterial cultivation technique. In this response there was no considerable difference between spheroids from tumour or from fresh cell lines. By electron microscopy many spores and vegetatively multiplying Clostridia became visible in the central necrotic part of spheroids. In the peripheral aerobic rim of spheroids, however, also few vegetative Clostridia were detectable.