Viable interspecies cytoplasmic-nuclear hybrid cells were constructed by fusion of karyoplasts prepared from the highly tumorigenic A9 mouse fibroblast cell line and cytoplasts prepared from the Detroit 532 normal human diploid cell strain. The identity of the hybrid cells was ascertained using a variety of morphological, immunological, and genetic criteria, including: nuclear pattern of staining with the fluorochrome Hoechst 33258, appearance of the actin-myosin containing cytoskeleton, presence of fibronectin, and resistance to azaguanine and diphtheria toxin. About 90% of the hybrid cells were viable, that is, capable of division. Changes in the morphology of the hybrid cells, apparently nuclear directed, were observed before cell division occurred. Using the techniques described here, large numbers of interspecies hybrid cells suitable for many types of biochemical analyses can be routinely produced.

A general procedure for identifying viable hybrid cells was developed. One cell type was labeled by a brief incubation in the Kodak laser dye rhodamine 123, which accumulates in the mitochondria; a second cell type was labeled by a brief incubation in the Hoechst fluorochrome 33258, which binds to chromatin. The substances which are eventually lost from the organelles, appeared to be nontoxic; the plating efficiencies of numerous cell lines tested was unaffected. Either whole cells or cytoplasts labeled with rhodomine 123 were fused, using inactivated Sendai virus, to whole cells or karyoplasts labeled with Hoechst 33258. When living cells were illuminated with ultraviolet light, individual whole cell hybrids, cybrids or cytoplasmic- nuclear hybrid cells could be rapidly identified by the appropriate staining pattern.

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