Autologous Bone Marrow Transplantation - Blog Science Connections

80 Mafosfamide Purging in Leukemia
MATERIALS AND METHODS
In Vitro Studies
Progenitor Cell Culture in Semisolid Media
Blast cells were taken at initial diagnosis from the marrow of nine patients
with acte nonlymphoblastic leukemia (ANLL). "Normal" marrow hematopoietic
progenitors were derived from 37 patients ( 14 with acute lymphoblastic
leukemia [ALL], 23 with ANLL) in whom the disease was in complete remission
(CR). Leukemic cell progenitors from myeloblastic leukemia (CFCI-L, leukemic
colony-forming units) were cultured in methylcellulose according to the
technique of Chang et al. (3) and scored on day 7. Granulocyte-macrophage
progenitors (CFCI-GM) were assayed in semisolid agar by a modification of the
technique of Pike and Robinson (4). Erythroid progenitors or erythroid burstforming
units (BFÜ-E) were cultured in methylcellulose by a modification of the
technique of Iscove and Sieber (5). Results were expressed in terms of the
number of colonies per milliliter of marrow.
Continuous Liquid Cultures
Long-term liquid cultures were established using a modification of the
technique of Dexter et al. (6). Bone marrow buffy coat cells ( 10 6 ) from normal
donors were suspended in 1 ml of alpha medium (G1BCO Biocult) supplemented
with 20% fetal calf serum; 5 ml of the suspension was put into Falcon
25-cm 2
tissue culture flasks and incubated at 37°C in a 5% CO a
atmosphere.
Cultures were maintained by weekly demidepopulation with addition of fresh
medium. Cinder those conditions, a uniform adherent layer spread within 3 to 4
weeks. This primary culture was then depopulated of all suspension cells and
irradiated at 30 Gy. A new allogeneic inoculum of bone marrow cells was added
to the adherent layer. Cultures were maintained by replacing all of the medium
each week, without demipopulation. At the same time, suspension cells were
assayed for CFCI-GM as described above.
Cell Treatment
Bone marrow cells were incubated with increasing doses of mafosfamide
for 30 minutes at 37°C in a water bath with frequent agitation. Then they were
washed once in Hanks' balanced salt solution and cultured. Buffy coat cells
were obtained from bone marrow samples spun at 2,500 rpm for 10 minutes
and adjusted to 2 x 10 7
cells/ml in autologous plasma and TC 199 solution,
with a final hematocrit of 5%. The sensitivity of leukemic and normal progenitor
cells to mafosfamide was evaluated by the diminution of their number. For the
preincubation test (PIT), a 10-ml marrow aspirate was taken 15 days before
bone marrow collection. The sensitivity of remission CFCI-GM and BFCl-E to
increasing doses of mafosfamide was studied as described above, and a
dose-response curve was established for each patient. The optimal dose for