Methods and compositions for separating cells

#The present invention relates generally to compositions and methods for separating cells, and more particularly, to compositions and methods for specifically removing erythrocytes from blood-cell containing samples to facilitate enhanced recovery of leukocytes, thrombocytes, stem cells, and other non-erythrocytic components of blood.

#Isolation of cells for in vitro studies or for applications in cellular therapies usually requires an initial separation of blood cell components mainly based on the bulk depletion of erythrocytes, which comprise >99% of the cellular mass of blood. These techniques for erythrocyte removal are based on hypotonic lysis of erythrocytes, density gradient separation, or enhanced centrifugal sedimentation using heta starch. Hypotonic lysis, while useful in low volume in vitro studies, is inefficient and impractical for the large volumes of blood tissues processed for cellular therapies.

#Density-gradient separation relies on small differences in the density of different cell types causing them to segregate at different levels in a fluid medium of variable density. Differences in density between the cell types can be small, and individual cells types can be heterogeneous in size and density. Consequently, particular cell types can become distributed throughout a density-gradient medium rather than precisely segregating at a discrete area in the density medium, resulting in poor recovery of desired cells and/or contamination with undesired cell types. In procedures that enrich for rare blood cell types such as hematopoietic progenitor cells, density-gradient sedimentation generally results in poor yields. For example, using conventional density-gradient methods to isolate progenitor cells (e.g., CD34+ hematopoietic stem cells) from umbilical cord blood reportedly results in a significant loss of the desired stem cells. See e.g., Wagner, J. E., Am J Ped Hematol Oncol 15:169 (1993). As another example, using conventional density-gradient methods to isolate lymphocytes reportedly results in selective loss of particular lymphocyte subsets. See e.g., Collins, D. P., J Immunol Methods 243:125 (2000).

#The invention features compositions and methods for separating cells. As described herein, the compositions specifically aggregate erythrocytic cells via surface antigen recognition and stimulate the enhanced sedimentation of erythrocytes at 1×g. Non-erythrocytic cells, including, for example, leukocytes, thrombocytes, and stem cells can be recovered from the supernatant phase of the fractionated blood sample.

#Cell Separation Compositions

#A cell separation composition in accord with the invention can contain dextran, heparin, a calcium chelator, and anti-glycophorin A antibodies. Dextran is a polysaccharide consisting of glucose units linked predominantly in alpha (1 to 6) mode. Dextran can cause stacking of erythrocytes (i.e., rouleau formation) and thereby facilitate the removal of erythroid cells from solution. Typically, soluble dextran having a molecular weight of 500,000 (e.g., from 400,000 to 550,000, Sigma Chemical Co., St. Louis) is used in compositions of the invention.