Investigation and Standardization of the Conditions for Micro‐Lymphocyte Cultures

Abstract

Experiments were performed to establish optimal conditions for specific and non‐specific lymphocyte transformation in vitro, in cultures containing 1–3 × 105 human lymphocytes. The stimulation as measured by 14C‐thymidine incorporation was evaluated when using various methods of lymphocyte purification and different numbers of contaminating erythrocytes, different kinds of tubes, serum from different sources, and varying PHA concentrations. Furthermore, the dependence of the 14C‐thymidine incorporation on the thymidine concentration in the medium and on the total number of lymphocytes present per culture was determined. Two methods of harvesting the cells to measure the 14C‐thymidine incorporation were compared. On the basis of this analysis, conditions for maximal sensitivity and reproducibility could be defined in measuring lymphocyte reactivity to PHA, allogeneic lymphocytes and antigens in a miniaturized culture system.

Variable MLC stimulatory capacity of neoplastic B and non‐B/non‐T lymphocytes expressing HLA‐DR antigens

“…Mononuclear cells isolated from peripheral blood, spleen or lymph nodes from patients with NHL, CLL or ALL were used for this study*. Cells were isolated by Ficoll-Isopaque centrifugation of defibrinated blood (Du Bois et al 1973). Cells were freed from tissue fragments by teasing and mincing through tantalum gauze.…”

“…The capacity to stimulate allogenic cells in one-way mixed lymphocytic culture was determined as described previously (Du Bois et al, 1973). Tests were performed in microtest culture plates (round bottom), each well containing 4 x lo4 lymphocytes of both responding and stimulating cells in RPMI-I 640 medium (Flow Laboratories, Irvine, Ayrshire, Scotland), buffered with bicarbonate and supplemented with 20% heat-inactivated pooled human serum.…”

Further Miniaturization and Automation of in vitro Lymphocyte Cultures

“…Some of the currently available systems (Sengar & Terasaki 1971, Hartzmann et al 1971, Du Bois et al 1973) already represent a considerable development in this direction. In this context, we have made some efforts towards further reduction o,f cell numbers, media volumes and harvest-ing times.…”

The Use of Microtiter Plates in Mixed Lymphocyte Cultures

“…We have recently described our investigations on the optimalization of lymphocyte cultures; thus, a standardized technique was developed yielding optimal results with limited numbers of lymphocytes (1-3X 105) (Du Bois et al 1973).…”

“…I t was found that a maximal response in the presence of soluble antigen or allogeneic cells is critically dependent upon the number of lymphocytes sedimented per unit surface area, both greater and smaller cell numbers giving lower responses (Du Bois et al 1973, Schellekens & Eijsvoogel 1971) .This may be caused by two counteracting phenomena. Firstly, close membrane contact of the lymphocyte may be a prerequisite for optimal reactivity (Moorhead et al 1967, Leventhal & Oppenheim 1969.…”