A miniaturized in vitro diffusion culture system

Abstract

A miniaturized diffusion culture apparatus holding 24 individual cultures and utilizing 1 10 the cells and volume of standard Marbrook-type cultures is described. This system was evaluated by studying the anti-sheep erythrocyte response of spleen cells from unprimed or primed and boosted mice. Compared to parallel Mishell-Dutton plate cultures memory responses in mini-diffusion cultures rise more slowly, peak 1–2 days later and thereafter decline less rapidly. Moreover, the diffusion cultures give a pronounced IgG response peaking at 6 days, while the abortive IgG response evident in plate cultures at 4 days rapidly disappears. While viability in the two systems is equivalent, cell recovery on days 7 and 8 is markedly higher in the diffusion cultures. Of 4 cell concentrations tested, 2 million cells are found to produce the optimum anti-sheep erythrocyte plaque-forming cell response. A 0.2 μ nuclepore membrane is preferable to a dialysis membrane and yields up to a 2-fold higher response. Replacement of the reservoir medium on day 4 impairs rather than improves the response. The culture system was also found to support and excellent primary response. This response is dependent on the presence of 2-mercapto-ethanol and does not require rocking.