autologous blood and marrow transplantation - Blog Science ...

Henschler et al. 553
MATERIALS AND METHODS
CD34 +
cells were isolated from patients with a diagnosis of solid tumors or
lymphoma, after informed consent, by leukaphereses and subsequent CD34 +
purification
(CellPro) as previously described. 21
CD34 +
cells were inoculated at a density
of 3 to 5X 10 4
/mL into serum-free medium (CellGro; CellGenix GmbH, Freiburg,
Germany) in the presence of recombinant hematopoietic growth factors in standard
2 1
tissue culture flasks at 37°C and 5% C0 2. Colony-forming assays and determination
of LTC-IC was performed as previously described. 11
NOD/SCID mice were
maintained and transplanted according to a previously described protocol for SCID
mice. 22
' 23
Human engraftment was assessed by flow cytometry using humanspecific
antibodies against CD45 and HLA class I surface antigens.
Tumor cells were isolated from ascites or pleural effusions of patients with
metastatic breast cancer and cultured in RPMI, 10% fetal calf serum (FCS) at a
starting cell density of 5Xl0 4
/mL. 9,24
Tumor cells were measured by immunostaining
with anticytokeratin antibodies as described. 924
Recombinant immunotoxins
recognizing the erbB2 and EGF receptor antigens have also been described. 9
RESULTS
Ex vivo expansion of total cell numbers followed a rapid time course with a
more than 10-fold amplification within 1 week of culture (Fig. 1). Also, numbers
of colony-forming cells increased exponentially in the presence of the cytokine
combination, flt3 ligand, SCF, and IL-3. In contrast, numbers of LTC-IC increased
to a relatively lower degree, by a factor of up to three within this time period (Fig.
1). When equal aliquots of approximately 0.5X10 5
CD34 +
cells were cultured for
various time periods under the same conditions and injected into preirradiated (300
cGy) NOD-SCID mice, their ability to reconstitute human hematopoiesis in vivo
was maintained for a period of only 4 days (Table 1). In contrast, on day 5 or day
7 of ex vivo expansion culture, no or nearly no human engraftment could be
recorded in the mice (Table 1). These data show that primitive cell populations as
assayed in vitro follow kinetics during serum-free ex vivo expansion of CD34 +
progenitor cells that are different from those of the in vivo repopulating cells.
To study the behavior of solid tumor cells during ex vivo expansion, primary
breast carcinoma cells were used. These cells were isolated from malignant
effusions, as numbers of contaminating tumor cells are usually very low within
bone marrow. The tumor cells were identified using anticytokeratin immunostains.
These stains were complemented by further immunohistochemical analyses to
distinguish tumor cells from mesothelial cells, which might also grow from the
effusion. The tumor cell origin of the analyzed cells could be verified by a positive
BerEP4 stain and negative staining for calretinin. When primary tumor cells were