RELEASE OF REPLICATION-DEFICIENT RETROVIRUSES FROM A PACKAGING CELL-LINE - INTERACTION WITH GLIOMA TUMOR SPHEROIDS IN-VITRO

The present study describes how various growth conditions affect gene expression and virus production from a retroviral packaging cell line (Lit 9), grown as monolayers and as multicellular spheroids. In additi on, to study the direct interaction between packaging cells and tumor tissue of glioma origin, Lit 9 spheroids were confronted with tumor sp heroids derived from a human glioma cell line, GaMg. The results show a progressive gene transfer into the tumor tissue, with 9% transfectio n efficacy after 5 days of co-culture. In comparison, no gene transfer was observed when the Lit 9 spheroids were confronted with normal bra in cell aggregates. The Lit 9 spheroids established from early-passage cultures (passages 7-14) showed limited growth during 28 days, wherea s those initiated from late-passage monolayer cultures (passages 39-49 ) showed extensive growth. Flow-cytometric DNA profiles of monolayers and of spheroids indicated no difference in cell-cycle distribution or ploidy between early and late passages. A cell-viability assay using scanning confocal microscopy revealed mostly viable cells in the Lit 9 spheroids, with only a few dead cells scattered within the structures . The lacZ-gene expression was maintained in early- and in late-passag e cultures. In comparison, in Lit 9 early-passage monolayers, the viru s titer was 3.1 x 10(4) +/- 0.4 x 10(4) CFU/ml, whereas no virus titer was found in late-passage cultures. The virus titer from the Lit 9 sp heroids was found to be between 10(3) and 10(4) CFU/ml. It is conclude d that the virus production from packaging cells may vary, depending o n passage number and tissue-culture conditions. In the present study, this is demonstrated by a complete loss in virus titer during prolonge d culture of packaging cells. In addition, the 3-dimensional confronta tion system described allows direct visualization of how packaging cel ls interact with tumor tissue. Thus, the co-culture system represents a model for studying the efficiency of packaging cells in transfecting heterogeneous tumor tissue in vitro. (C) 1997 Wiley-Liss, Inc.