A Simple Method to Obtain Low Density Marrow Cells for Human Marrow Transplantation

Abstract

Removal of more than 99% of the erythrocytes and 74% of the nucleated cells from marrow grafts was achieved by density floatation separation in Percoli gradients with a density of 1.070 g/ml in eight 250-ml tubes, containing up to 3 x 10^9 nucleated cells per gradient. More than 90% of the myeloid and erythroid progenitor cells were recovered in the low density fraction. It appeared mandatory to use a centrifuge with the possibility of a gradual acceleration and deceleration. Twenty-five patients received a marrow graft from a histocompatible sibling after additional lymphocyte depletion by counterflow centrifugation, and 5 patients with T lymphoblastic malignancies received an autograft after in vitro purging with immunotoxins. All evaluable patients engrafted within normal limits, except 1 patient with an autoimmune pancytopenia who responded to steroids and 1 patient with a CMV infection. Four patients died too early for complete evaluation. The described separation method is easy, cheap and requires only 2 h for the complete processing of a marrow graft.

Counterflow centrifugation allows addition of appropriate numbers of T cells to allogeneic marrow and blood stem cell grafts to prevent severe GVHD without substantial loss of mature and immature progenitor cells

“…After fat and plasma extraction by centrifugation, mononuclear cells (MNC) were isolated by density flotation separation in Percoll (Pharmacia, Uppsala, Sweden) gradients as described previously. 33 BM was mixed with 90% Percoll solution obtaining a density of 1.085 g/l. This suspension was pumped under a 1.073 g/l Percoll layer in 250 ml bottles.…”