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 (Liz 9), grown as monolayers and as multicellular spheroids. In addition, to study the direct interaction between packaging cells and tumor tissue of glioma origin, Liz 9 spheroids were confronted with tumor spheroids derived from a human glioma cell line, GaMg. The results show a progressive gene transfer into the tumor tissue, with 9% transfection efficacy after 5 days of co-culture. In comparison, no gene transfer was observed when the Liz 9 spheroids were confronted with normal brain-cell aggregates. The Liz 9 spheroids established from early-passage cultures (passages 7-14) showed limited growth during 28 days, whereas 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 Liz 9 spheroids, with only a few dead cells scattered within the structures. The lacZ-gene expression was maintained in early- and in late-passage cultures. In comparison, in Liz 9 early-passage monolayers, the virus 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 Liz 9 spheroids was found to be between 10(3) and 10(4) CFU/ml. It is concluded that the virus production from packaging cells may vary, depending on passage number and tissue-culture conditions. In the present study, this is demonstrated by a complete loss in virus titer during prolonged culture of packaging cells. In addition, the 3-dimensional confrontation system described allows direct visualization of how packaging cells 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.
Thorsen
F
F
Department of Anatomy and Cell Biology, University of Bergen, Norway. frits.thorsen@pki.uib.no
Visted
T
T
Lehtolainen
P
P
Ylä-Herttuala
S
S
Bjerkvig
R
R
D000818:Animals; D001921:Brain / Q000166:cytology; D002449:Cell Aggregation; D002460:Cell Line; D002470:Cell Survival; D018920:Coculture Techniques; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D005910:Glioma / Q000378:metabolism*; D006801:Humans; D051379:Mice; D011817:Rabbits; D051381:Rats; D012190:Retroviridae / Q000502:physiology*; D018874:Spheroids, Cellular / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication; D001616:beta-Galactosidase / Q000235:genetics
D004247:DNA; D001616:beta-Galactosidase