Ultrastructural study on the in vitro interaction between haemopoietic cells and stromal cells. A new method using...

The ultrastructural study of the interaction between stroma and haemopoiesis is not an easy task because the loose attachment may be damaged during manipulation. This paper describes a technique by which the loose connection between preestablished stromal layer and attached haemopoietic cells (derived from blast colony forming cells) can be preserved and studied ultrastructurally. Stromal cultures were obtained from human bone marrow cells. Blast colony forming cells were studied by co-incubating the stroma with fetal calf serum supplemented McCoy's medium containing bovine plasma, thrombin and calcium to form a gel ('plasma clot'). Colony formers attached to the stroma formed myeloid colonies within 6 days. The semisolid plasma clot which solidifiers rapidly on the addition of glutaraldehyde or formaldehyde entraps the blastic colonies and haemopoietic cells in their position. Even the non-attached or mobile cells can be entrapped by this technique. The immature cells were found to be attached to the stromal surface and/or to the extracellular matrix, while the more mature cells migrated either to the surface of the colony or attached to the non-covered areas of the plastic surface. This method may offer a special technique to study dynamic interactions in other situations (e.g. chemotaxis etc.), too.