Abstract

Publisher Summary This chapter presents a method for cloning anchorage-dependent cells in agarose. The method allows single cells to be isolated and cloned while retaining the conditioning effects of a high cell concentration. It is an extension of the method of Metcalf, Bradley, and Robinson, in which feeder cells were incorporated in an agar base layer, and bone marrow cells in an agar top layer and conditioning factors stimulated the growth of colonies of erythropoietic stem cells. The technique is restricted to cells which can grow in suspension in agar and however, most cells require anchorage to a solid substrate in order to grow. The method incorporates cells and small pieces of solid substrate into an agarose gel. Some of the cells attach to the substrata and are able to grow. Cells to be cloned are trypsinized to yield a single-cell suspension, and resuspended in an upper layer of 0.25% agarose, together with a number of glass platelets, over a preset base layer of 0.5% agarose containing feeder cells. Two days later, platelets with only one cell attached are marked, and, after incubation, those with a confluent layer of cells are removed, and placed in tissue culture wells, for further growth in liquid medium.

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