SEPARATION OF FELINE BONE-MARROW CELLS BY COUNTERFLOW CENTRIFUGAL ELUTRIATION - IDENTIFICATION AND ISOLATION OF PRESUMPTIVE EARLY AND LATE MYELOID ERYTHROID PROGENITORS/

Counterflow centrifugal elutriation (CCE) has been used to separate nu cleated cells from mammalian bone marrow on the basis of size with the resultant isolation of hematopoietic cells in varying stages of Linea ge development. We examined the feasibility of identifying and isolati ng such cells from feline bone marrow, CCE was performed with a Beekma n J6MI centrifuge and a Sanderson chamber, using a fixed rotor speed o f 3000 rpm and collection of cells at (1) 16-, (2) 21-, (3) 25-, (4) 3 2 ml/min, and (5) a rotor off fraction. Recovery of the total input ce lls in four replicate experiments averaged 88%, with the maximum numbe r of recovered cells in fraction 4. Analysis by flow cytometry and mon oclonal 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 rea ction (MLR) were restricted to fractions 1 and 2; removal of T cells a nd MLR activity was accomplished by immunomagnetic depletion. In vitro cultures for clonogenic cells revealed CFU-GM and BFU-E colonies in f ractions 2 through 5, with fraction 4 containing the greatest absolute number of myeloid colonies and ii actions 3 and 4 the majority of the erythroid colonies. More important, in examining the plating efficien cy for clonogenic cells in the different fractions it was found that t his increased significantly in fractions 2 and 3 when the culture time was extended from 7 to 14 days; in contrast, fractions 4 and 5 reache d their maximum plating efficiency within 7 days with no further incre ase on day 14, We interpret these findings to indicate the presence of late differentiating progenitors in the large-cell size fractions 4 a nd 5, while the smaller mononuclear cells in fractions 2 and 3 represe nt an earlier, more primitive population of hematopoietic cells requir ing an extended time in culture for full colony development.