VI Autologous Bone Marrow Transplantation.pdf - Blog Science ...

Bielefeld, FRG) or control medium was performed by incubating the cells
(2x107/ml) for 30 min at 37°C in a water bath with frequent agitation. The cells
were subsequently incubated for 5 min on ice to stop the reaction.
Preparation of stromal layers. Stromal layers were prepared according to
the technique described by Gordon et al. (15). Normal bone marrow was obtained
from consenting donors by aspiration from the posterior iliac crest.
MNCs were isolated as above, washed and resuspended (5 x 105/ml) in alphamedium
supplemented with 15% FBS and 2 x 10 -6
MMethylprednisolone. One
ml aliquots were plated in 35 mm Petri dishes and incubated at 37°C in a humidified
atmosphere supplemented with 5% C02. The cultures were fed weekly
by complete replacement of medium and serum until confluent.
Clonogenic assay. The assay for pluripotent colony-forming units (CFU-
Mix), erythroid bursts (BFU-E), and granulocyte-macrophage colony-forming
units (CFU-GM) was carried out as described in detail elsewhere (16). Briefly,
5x104 were plated in 35-mm Petri dishes in 1-ml aliquots of Iscove's modified
Dulbecco's medium (IMDM, Miles Laboratories, Naperville, IL, U.S.A.) supplemented
with FBS (30%), 2-mercaptoethanol (5 x 10" 5
M), and methylcellulose
(1.1%, w/v). Cultures were stimulated with a mixture of human recombinant
colony-stimulating factors (CSFs) including: interleukin-3 (IL-3,10 ng/ml),
granulocyte-CSF (G-CSF, 10 ng/ml), granulocyte-macrophage-CSF (GM-CSF, 10
ng/ml) and erythropoietin (Epo, 1 U/ml). Recombinant IL-3, GM-CSF and Epo
were generously provided by Behringwerke AG (Marburg, FRG); G-CSF was
kindly provided by Amgen Inc. (Thousand Oaks, CA, U.S.A.). Progenitor cell
growth were scored according to previously described criteria (16) after incubation
of the dishes for 14 days. Four dishes were set up for each individual data
point per experiment.
Cytogenetic analysis. Cytogenetic analysis and standard GTG- or QFQbanding
techniques were performed according to standard methods (17). Single
colony karyotyping was performed according to Dube et al (l 8
).
Experimental design. Following incubation with mafosfamide (100 mg/ml)
or control medium, MNCs were seeded onto normal marrow-derived, allogeneic
stromal layers to allow attachment of stroma-adherent cells. After 3 hrs incubation
at 37°C, stroma non-adherent cells were harvested. Both adherent and
non-adherent cell fractions were then cultured in suspension (3 days, 37oC, 5%
CO 2
). At the end of the incubation period, stroma adherent cells were harvested
by repeatedly washing each dish. Stroma adherent and stroma non-adherent
cells were then cultured in methylcellulose to allow the growth of CFU-GM to
be analyzed by single colony karyotyping. Stromal layers were neither irradiated
nor fixed, and to exclude the possibility that Ph-negative metaphases were
derived from the stromal layers, the sex of the patient and the stroma donor
were mismatched.
RESULTS
Both the multilineage (CFU-Mix) as well as the granulocyte-macrophage
(CFU-GM) and erythroid (BFU-E) lineage-restricted progenitor cells were significantly
inhibited following exposure to 100 mg/ml of mafosfamide. The percentages
of surviving CFU-Mix, BFU-E, and CFU-GM were 0%, 3.3%, and 13%,
respectively. However, following stroma adherence and short-term suspension
SIXTH INTERNATIONAL SYMPOSIUM ON AUTOLOGOUS BONE MARROW TRANSPLANTATION 135