Katsuto Watanabe

A new compact and cell dense continuous culture system

A new compact and cell dense continuous culture system was developed. The system consisted of a culture bag, which had two compartments separated by a semipermeable membrane, and an external rotator. Cells were cultured in the inner compartment with medium containing serum or the necessary growth factors. The outer compartment was filled with medium without serum or growth factors. The concentration of cells in culture was normally in the order of 10(7). Due to the large number of cells, sufficient carbon dioxide was produced so that use of a CO2 incubator was unnecessary. The medium in the outer part was frequently changed, but no medium changes were necessary in the inner bag. Using this system, a concentrated yield of antibody as well as a large number of cells could be obtained.

Macrophage–T Cell Interaction Is Essential for the Induction of p75 Interleukin 2 (IL–2) Receptor and IL–2 Responsiveness in Human CD4+ T Cells

Fresh human CD8+ T cells showed a strong proliferative response to a high concentration of interleukin 2 (IL–2) in the absence of macrophages. In contrast, CD4+ T cells revealed no significant IL–2 responsiveness in the absence of macrophages. However, if CD4+ T cells were cocultured with macrophages, they showed higher proliferative response to IL–2 than CD8+ T cells. In accordance with the magnitude of IL–2 responsiveness, freshly isolated CD8+ T cells expressed significant amounts of p75 IL–2 receptor, while fresh CD4+ T cells did not express p75 IL–2 receptor. The expression of p75 IL–2 receptor on CD4+ T cells was induced by coculture with macrophages. The macrophage–induced p75 IL–2 receptor acquisition was blocked by monoclonal antibody (inAh) against class II antigen. Moreover, the addition of anti–CD4 mAb or anti–class II mAb to the culture caused a great inhibition of IL–2 responsiveness of CD4+ T cells. These results strongly suggest that macrophage–T cell interaction through CD4 and/or class II molecules is essential for the expression of p75 IL–2 receptor and IL–2 responsiveness in human CD4+, but not CD8+ T cells.

A new high-yield continuous cell-culture system for lymphokine-activated killer cells

SummaryWe performed basic studies on a new high-yield culture system (concentrate rotary tissue-culture system) for application to adoptive immunotherapy with lymphokine-activated killer (LAK) cells. Using this system, we demonstrated that up to 2 × 107 peripheral blood mononuclear cells/ml could be cultured in interleukin-2 with a sufficient recovery rate and cytotoxicity in short-term cultures (6 days). This system can also be used to proliferate LAK cells to four times the initial cell number with sufficient cytotoxicity for 14 days of culture. Thus, this system allows activation of sufficient numbers of cells to conduct clinical trials on humans.