Abstract
Counterflow centrifugal elutriation (CCE) has been a highly efficient physical method for separating T cells from bone marrow (BM) without impairing cell function and yield. To investigate the usefulness of CCE, the hematopoietic potential as well as the level of T cell contamination in rotor-off (R/O) fraction of BM was studied using a murine bone marrow transplantation (BMT) model [C3H/He (H-2 k) →BALB/C (H-2 d)]. The total recovery of cells after CCE procedure was 71.4%. Morphologically, R/O fraction contained abundant mononuclear cells and a few lymphocytes. The numbers of colony forming unit for granulocyte/monocyte (CFU-GM), Sca-1 + cells, and T cells were compared among four fractions of CCE (fractions at flow rate of 17, 25, 28 mL/min, and R/O fraction). The number of CFU-GM per 10 5 nucleated cells in each fraction were significantly higher in R/O fraction (331.3 ± 34.4) compared to unfractionated marrow (UM) (21.1 ± 1.3) and fraction of 17 mL/min (FR 17) (23.7 ± 2.2 ) ( χ 2 = 0.0044 ). Neither fraction of 25 mL/min (FR 25) nor fraction of 28 mL/min (FR 28) contained CFU-GM colonies. The concentration of Sca-1 + cells in R/O fraction was significantly higher (1.96-fold) than UM ( p < 0.05), and 80.0 ± 10.1 % of Sca-1 + cells in UM were recovered in R/O fraction; 88.1% of Thy-1.2 + T cells were eliminated in R/O fraction ( p < 0.05). Mice receiving UM after lethal irradiation (875cGy) suffered from severe graft-versus-host disease (GVHD) and all five died within 7 days after BMT procedure (Group A). Of interest, mice receiving mixture of R/O fraction with lymphocyte-rich fraction (FR 25 plus FR 28) to equalize T cell number as UM, developed severe GVHD and four out of five died (probability of survival; 20%) (Group B). Mice receiving R/O fraction had mild GVHD and four out of five survived for at least 90 days (probability of survival; 80%) (Group C). In group C, probability of survival ( p = 0.0006 ) was higher, and severity of GVHD ( p = 0.0043 ) and progression rate of GVHD ( p = 0.02 ) was lower. In conclusion, the elutriated R/O fraction cells of BM have the advantages of stable engraftment and tolerable GVHD in murine allogeneic BMT with complete major histocompatibility disparity. This could be directly applicable to patients with high risk of GVHD and graft failure in upcoming clinical trials.
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