A miniaturized in vitro diffusion culture system.
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 micron nucleopore 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 an excellent primary response. This response is dependent on the presence of 2-mercaptoethanol and does not require rocking.
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004306:Dose-Response Relationship, Immunologic; D004912:Erythrocytes / Q000276:immunology; D005260:Female; D007158:Immunologic Techniques; D008214:Lymphocytes / Q000276:immunology*; D008566:Membranes; D008623:Mercaptoethanol / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D012756:Sheep
D008623:Mercaptoethanol