Studies on cell interactions using a micromethod for growing T-lymphocyte colonies in agar culture

Abstract

A micromethod is described for growing PHA-induced T-cell colonies from as few as 5 to 10×10³ peripheral blood mononuclear cells. When various numbers of cells were plated in agar at the same concentration under both standard and micromethod conditions, their plating efficiency remained constant. In contrast, the cell dose—response curves obtained in microculture supported previous observations that plating efficiency increases with cell concentration up to an optimum level. The mathematical analysis of these curves provided further indication that T-cell colony formation involved interactions between a colony progenitor cell (TCFC) and at least two populations of co-operating cells (CC). Pooled cells from primary colonies, which still contained TCFC but were usually depleted of CC, generated secondary colonies when transferred in subculture, provided medium conditioned by CC was added to the cultures. Under these conditions, the plating efficiency remained constant with increasing numbers of cells plated and thus, the number of colonies obtained was found to be strictly proportional to the number of cells plated. In addition, CC persisting occasionally in primary colonies could be detected by (a) their ability to generate secondary colonies in the absence of CC conditioned medium and (b) a modification of the dose—response curves obtained toward increasing plating efficiency with the number of cells seeded. With respect to the small numbers of cells and conditioned media required in this system, it was concluded that the micromethod presented should provide a useful tool for studies on cell interactions during T-cell colony formation.