| Popis: |
Counterflow centrifugal elutriation (CCE) has been used to separate nucleated cells from mammalian bone marrow on the basis of size with the resultant isolation of hematopoietic cells in varying stages of lineage development. We examined the feasibility of identifying and isolating such cells from feline bone marrow. CCE was performed with a Beckman J6MI centrifuge and a Sanderson chamber, using a fixed rotor speed of 3000 rpm and collection of cells at (1) 16-, (2) 21-, (3) 25-, (4) 32 ml/min, and (5) a rotor off fraction. Recovery of the total input cells in four replicate experiments averaged 86%, with the maximum number of recovered cells in fraction 4. Analysis by flow cytometry and monoclonal antibodies revealed mononuclear cells in fractions 1 and 2 and early and late differentiating myeloid/erythroid cells in fractions 2 through 5. T lymphocytes and alloreactivity in a mixed lymphocyte reaction (MLR) were restricted to fractions 1 and 2; removal of T cells and MLR activity was accomplished by immunomagnetic depletion. In vitro cultures for clonogenic cells revealed CFU-GM and BFU-E colonies in fractions 2 through 5, with fraction 4 containing the greatest absolute number of myeloid colonies and fractions 3 and 4 the majority of the erythroid colonies. More important, in examining the plating efficiency for clonogenic cells in the different fractions it was found that this increased significantly in fractions 2 and 3 when the culture time was extended from 7 to 14 days; in contrast, fractions 4 and 5 reached their maximum plating efficiency within 7 days with no further increase on day 14. We interpret these findings to indicate the presence of late differentiating progenitors in the large-cell size fractions 4 and 5, while the smaller mononuclear cells in fractions 2 and 3 represent an earlier, more primitive population of hematopoietic cells requiring an extended time in culture for full colony development. |
