The culture of lymph nodes in synthetic media

Abstract

Lymph nodes from young rats were cultured in fluid media. The cultures were supported at the surface of the medium on a grid of tantalum wire gauze. Each vessel contained 15–20 cultures in 5.5 ml of medium and the gas phase was 3 per cent CO 2 in oxygen. In most experiments the lymph nodes were cultured for 4 days. After culture, film preparations were made and the state of each culture was assessed on the following criteria:- (1) the percentage of dead lymphocytes, (2) the ‘quality’ of the lymphocytes—well nourished cells were bigger and more basophilic than starved ones, and (3) the amount of cell differentiation which had occurred. Synthetic culture media were prepared by adding various nutrient chemicals to a modified Tyrode solution. The best medium discovered contains inorganic salts, glucose, 19 amino acids, cocarboxylase, p-aminobenzoic and insulin, together with chloramphenicol as antibiotic and phenol red as indicator. This medium is designated TACPI. In it cultures survived reliably for 4 days and some survived fairly well for 8 days. After 4 days in TACPI the cultures contained about 2 per cent of dead lymphocytes, the lymphocytes looked bigger and better nourished than those in normal lymph nodes, and a certain amount of differentiation had occurred—many of the reticulum cells were turning into large lymphocytes and some of the small lymphocytes had turned into monocytes. By contrast, lymph nodes cultured for 4 days in rat serum contained about the same percentage of dead lymphocytes but the cells looked poorly nourished and there was no differentiation. Good quality lymphocytes and cell differentiation were only obtained when the medium contained both cocarboxylase and p-aminobenzoic acid, and the optimum concentrations of these substances were determined. Bicarbonate was also shown to be necessary. Many other substances, including most of the vitamins, purines and pyrimidines, CoA, DNA, peptones and proteins were added to the medium but none improved it. Occasional mitoses of medium and large lymphocytes were found in the cultures, but so far no mitotic stimulant has been discovered.