Leukemia Research 27 (2003) 455–464
In vitro culture of human acute lymphoblastic leukemia (ALL) cells in
serum-free media; a comparison of native ALL blasts, ALL cell
lines and virus-transformed B cell lines
Øystein Bruserud a,∗ , Nils Glenjen a , Anita Ryningen a , Elling Ulvestad b
b
a Medical Department, Division of Hematology, Institute of Medicine, Haukeland University Hospital, N-5021 Bergen, Norway
Department of Microbiology and Immunology, Section for Immunology, The Gade Institute, The University of Bergen, Bergen, Norway
Received 31 May 2002; accepted 28 September 2002
Abstract
The aim of this study was to standardize in vitro culture conditions for human acute lymphoblastic leukemia (ALL) cells. The cells were
cultured in medium containing 10% fetal calf serum (FCS) and in the four serum-free media X-vivo 10® , X-vivo 15® , X-vivo 20® and
Stem SpanTM . Native ALL blasts could proliferate in all four serum-free media, but the strongest responses were usually observed with
Stem SpanTM . Native leukemia blasts were also cultured in the presence of various single cytokines or cytokine combinations. The highest
proliferation was usually observed in the presence of Flt3-Ligand (Flt3-L) when single cytokines were examined, and these responses
could be further increased especially by combining Flt3-L with interleukin 3 (IL3), IL7 or stem cell factor (SCF). Proliferation could also
be increased when ALL blasts were cultured in the presence of two commercially available fibroblast cell lines (Hs27 and HFL1). Based on
these results we suggest that in vitro culture conditions for native human ALL blasts can be standardized by using serum-free culture media
supplemented with exogenous Flt3-L + IL3 + SCF, and the use of accessory cells can also be standardized by using well-characterized
fibroblast cell lines. Detectable ALL blast proliferation can then be observed for most patients. Our experimental model can thereby be
used for in vitro evaluation of possible antileukemic treatment strategies, and it will then allow comparison of experimental results between
different studies.
© 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Acute lymphoblastic leukemia; In vitro culture; Serum-free media
1. Introduction
Acute lymphoblastic leukemia (ALL) is characterized by
clonal proliferation and accumulation of immature lymphoid
cells [1]. Clinical data together with analysis of membrane
molecule expression and cytogenetic abnormalities are commonly used for classification and prognostic evaluation of
these patients [1–3], and functional studies of in vitro cultured ALL cells can be used for further characterization of
the disease. Several studies have used culture media supplemented with inactivated fetal calf serum (FCS) [4,5], but
serum-free culture conditions can also be used [6–9]. One
of these serum-free media is very similar to media used for
culture of normal hematopoietic progenitors [8] and human
acute myelogenous leukemia (AML) cells [10]. However, in
vitro blast proliferation is low or undetectable for a substantial number of patients even when media are supplemented
∗
Corresponding author. Tel.: +47-55-97-50-00; fax: +47-55-97-29-50.
E-mail address: [email protected] (Ø. Bruserud).
with exogenous growth factors [5,8,11–16], and for this reason experimental models based on coculture of ALL blasts
with various irradiated or non-irradiated accessory cells have
been used [11,15,17–20]. The accessory cells can then be
cultured either in direct contact with the leukemia cells or
separated by a semipermeable membrane [11], and a wide
range of different accessory cells have been used including
murine fibroblasts [17] as well as human monocytes [15],
bone marrow stromal cells [18–20], normal fibroblasts and
fibroblast cell lines [9,15]. This variation in experimental
models makes it difficult to compare results from different
studies.
The aim of the present study was to characterize growth
requirements of ALL cells and thereby suggest standardized in vitro models. For this reason we focused on: (i) the
use of commercially available and serum-free culture media that also can be used for culture of AML blasts and
normal hematopoietic stem cells; this allows comparison of
ALL blasts with normal progenitors and myeloid leukemic
cells; (ii) the identification of a standardized growth factor
0145-2126/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 5 - 2 1 2 6 ( 0 2 ) 0 0 2 2 7 - 8
456
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
combination suitable for stimulation of ALL blast proliferation; and (iii) the use of commercially available cell lines
as accessory cells for in vitro cultured ALL cells.
2. Materials and methods
Recombinant human cytokines were used at the following
concentrations: Flt3-Ligand (Flt3-L, Peprotech; Rocky Hill,
NJ, USA) 20 ng/ml; Interleukin 2 (IL2, Peprotech) 50 ng/ml;
IL3 (Peprotech) 20 ng/ml, IL4 (Peprotech) 50 ng/ml, IL5
(Peprotech) 50 ng/ml, IL7 (Peprotech) 20 ng/ml, and stem
cell factor (SCF; Peprotech) 50 ng/ml.
2.1. Patients
2.3. Cell preparation
ALL blasts were derived from 12 consecutive patients
with high peripheral blood blast counts. Clinical and biological characteristics of the patients together with the
ALL-subclassification [21] are presented in Table 1.
2.3.1. Native ALL blasts
Leukemic peripheral blood mononuclear cells (PBMC)
were isolated by density gradient separation (Ficoll-Hypaque;
NyCoMed, Oslo, Norway; specific density 1.077) from the
peripheral blood of ALL patients with a high percentage
of leukemia blasts among blood leukocytes (see Table 1).
Cells were stored frozen in liquid nitrogen until used in the
experiments [23]. The percentage of blasts among leukemic
PBMC exceeded 95% for all patients judged by light microscopy of May-Grünwald-Giemsa stained cytospin smears
[24–26].
2.2. Reagents
Serum-containing culture medium was RPMI 1640 with
hepes plus glutamine (BioWhitacker, Walkersville, MA,
USA) and supplemented with 10% heat-inactivated FCS
(BioWhitacker). The four serum-free media X-vivo 10® ,
X-vivo 15® , X-vivo 20® (BioWhitacker) and Stem Span
H2000TM (referred to as Stem SpanTM or SS; Stem Cell
Technologies, Vancouver, BC, Canada) were also studied.
The X-vivo media are commonly used for culture of normal lymphocytes, whereas the Stem SpanTM medium is
recommended for culture of normal hematopoietic stem
cells (manufacturer’s information) and AML blasts [10,22].
Dulbecco’s Modified Eagle’s Medium (DMEM, American Type Culture Collection ATCC, Manassas, VA, USA)
and F12K medium (ATCC) were used for fibroblast culture. All media were supplemented with 100 ␮g/ml of
gentamicin.
2.3.2. Malignant cell lines
Six lymphoid cell lines were included in the experiments
(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ, Braunschweig, Germany). The biological characteristics of each cell line are included in Table 2.
2.3.3. Virus-transformed B-lymphoblastoid cell lines
Five Epstein-Barr virus (EBV)-transformed cell lines
were studied. These cell lines were originally prepared
by long-term in vitro expansion in RPMI-medium supplemented with 10% inactivated FCS.
Table 1
Clinical and biological characteristics of ALL patients
Patient
Sex
Age
Previous disease or chemotherapy
ALL subclassificationa
Cytogenetic analysisb
WBC countsc
1
2
3
4
5
6
7
8
9
10
11
12
M
M
F
F
F
M
M
F
M
F
F
F
18
82
23
58
32
24
21
28
74
54
22
14
Post-transplant relapse
Pro-B-ALL
B-ALL
Pro-B-ALL
Pre-B-ALL
Pre-B-ALL
Pro-B-ALL
B-ALL
T-ALL
Common-B-ALL
Common-B-ALL
Common-B-ALL
Common-B-ALL
nt
nt
nt (bcr/abl+ )
nt
nt
t(9;22) (bcr/abl+ )
dic(7;9) (p11;p11)
nt (bcr/abl− )
nt
t(9;22) (bcr/abl+ )
nt (bcr/abl− )
nt
87
125
48.7
34
35
89
18.8
68
78
560
3.6
25
a
Previous chemotherapy
ALL relapse
Previous chemotherapy for testicular carcinoma
ALL relapse
ALL blasts were regarded as positive for membrane molecules when >20% of the cells stained positive judged by flow-cytometric analysis. The
classification was based on the guidelines given by the European Group for the Immunological Classification of Acute Leukemias [21]. According to
this classification B lineage ALL blasts are positive for at least two of the three markers CD19, CD22 and CD79a. Patients classified as pro-B-ALL
(also referred to as B-I or null ALL) express no other B-cell differentiation antigens, common ALL (also referred to as c-ALL, pre-pre-B-ALL or B-II)
express CD10, pre-B-ALL (B-III) express cytoplasmic Ig and mature-B-ALL (also referred to as B-ALL or B-IV) express surface membrane Ig [21].
b Routine screening for chromosomal abnormalities was done by analysis of cells in mitosis. The abbreviation nt (not tested) means that cells were
not available for testing or did not proliferate in vitro, for three of these patients the presence of the bcr/abl translocation (Philadelphia chromosome)
was analyzed by Fish technique.
c White blood cell (WBC) counts in peripheral blood are expressed as ×109 l−1 (normal range 3.5–10.5 × 109 l−1 ). The WBC included at least 80%
leukemia blasts.
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
Table 2
In vitro culture of human cell lines derived from patients with lymphoid
malignancies; a comparison of proliferative responses in various culture
media
Cell line
Medium
Nalm 6 (B cell precursor ALL)
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
5.5
5.5
4.3
4.4
3.8
Tanoue (B-ALL, FAB L2)
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
8.7
10.0
5.1
10.0
10.2
SD1 (B-lymphoblastoid cell line
derived from pre-B ALL)
X-vivo 10®
5.2
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
5.3
1.6
5.3
3.5
697 (B cell precursor ALL)
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
6.4
7.0
6.4
7.3
6.2
Jurkat (T-ALL)
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
3.5
3.3
1.0
4.3
3.8
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
5.6
4.7
3.8
4.0
5.1
Daudi (Burkitt’s lymphoma)
Proliferation
(population
doublings)
All cell lines were cultured in parallel in the various media, and the
results are expressed as the number of population doublings during a 10
days culture period. All cultures were prepared in 24-well tissue culture
plates with 2 ml medium per well, half of the medium was changed
three times weekly, and cultures were divided when the cells formed a
monolayer. The results in bold represent a proliferation equal to (within
±0.2 population doubling) or exceeding the response in the recommended
FCS-containing growth medium.
2.4. Assays for cell proliferation
2.4.1. Native ALL blasts
As described previously for AML blasts [24,25], 5 × 104
leukemia cells per well were cultured in 150 ␮l medium in
flat-bottomed microtiter plates (Costar 3796; Cambridge,
MA, USA). Cultures were incubated at 37 ◦ C in a humidified atmosphere of 5% CO2 . After 6 days 3 H-thymidine
(37 kBq per well; TRA 310, Amersham International,
Amersham, UK) was added in 20 ␮l saline and nuclear
radioactivity assayed 18 h later by liquid scintillation
counting.
457
2.4.2. Lymphoid cell lines
All cell lines were cultured in 24-well tissue culture plates
(Costar 3524) with 2 ml medium per well. The cultures were
initiated with 2 × 105 cells per well. All cultures were thereafter microscoped regularly, and divided when the cells were
confluent. Light microscopy without cell counting was used
because several cultures could then be compared and handled in parallel in the same culture plate, and the time needed
for handling of cultures outside the incubator could thereby
be kept to a minimum. Control experiments verified that the
cell concentrations then were <1 × 106 ml−1 throughout the
culture period. Half of the medium was renewed three times
weekly, 1 ml fresh medium was also added if division of cultures was necessary between the regular medium renewals.
All cell lines were cultured for 10 days before the cells were
counted and the number of population doublings calculated
for the whole culture period.
2.5. Coculture of native ALL cells with fibroblasts
The two human fibroblast cell lines Hs27 (ATTC number
CRL-1634) and HFL1 (ATTC number CCL-153) were used
as accessory cells for the ALL blasts. Both cell lines are
commercially available (ATCC). The optimal medium for
expansion of Hs27 is DMEM + 10% FCS, and for HFL1
F12K + 10% FCS (information from ATCC).
2.5.1. Coculture of fibroblast cell lines and ALL blasts
The fibroblasts were cultured in their optimal medium for
4 days (Costar 3524 24-well culture plates; 2 ml medium
per well). At the end of this period the cells were regularly distributed and covered <50% of the bottom, and the
ALL cells were then added. For certain experiments 2 × 106
ALL blasts were added per well by changing 1 ml of the
medium per well, in these cases the cells were thus cultured in the original FCS-containing medium for additional
6 days before 3 H-thymidine incorporation was determined
(see later). Alternatively, after 4 days the FCS-containing
medium was removed and 2 × 106 ALL blasts added in 2 ml
Stem SpanTM . 3 H-thymidine incorporation was then determined 6 days later (see later).
2.5.2. Culture of fibroblasts and ALL cells separated
by a semipermeable membrane
The fibroblasts were initially cultured alone as described
above in the lower chamber of 12-well tissue culture plates
(Costar Transwell, 12 wells per plate, pore size 0.4 ␮m). After 4 days 2.5 × 106 ALL blasts were added by changing
0.5 ml in the upper chamber, and 3 H-thymidine incorporation was assayed 6 days later. The FCS-containing medium
optimal for each of the fibroblast cell lines was then used
throughout the culture period.
2.5.3. 3 H-thymidine incorporation in cocultured cells
Fibroblasts and ALL cells were cocultured for 6 days before 278 kBq of 3 H-thymidine was added in 150 ␮l saline
458
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
and cultures incubated for additional 18 h. The nonadherent
cells were then resuspended (either the whole well or only
for the upper chamber of transwell cultures), and nuclear radioactivity of 100 ␮l cell suspension then determined. The
adherent cells were thereafter extensively washed in saline,
incubated for 15 min with 300 ␮l trypsin solution (Stem
Cells), and nuclear radioactivity then determined for 70 ␮l
of this cell suspension.
2.6. Flow-cytometric analysis
Analysis of membrane molecule expression was performed as described earlier [27]. Flow-cytometric analysis
was also used to determine the percentage of apoptotic
and/or dead cells by the use of annexin-V and propidiumiodide, see also reference [28].
2.7. Presentation of the data
3 H-thymidine incorporation was assayed in triplicates and
the mean counts per minute (cpm) used for all calculations.
The incremental response was defined as the cpm for cultures with ALL blasts minus cpm for negative controls, and
significant blast proliferation was defined as an incremental
response exceeding 1000 cpm. Incremental responses were
used for all calculations. A significant alteration of a proliferative response was defined as a conversion to/from nonresponsiveness; or a difference: (i) exceeding 2000 cpm, and
(ii) the difference being >20% of the control response. For
statistical analysis the Wilcoxon’s test for paired samples
was used, and differences were regarded as significant when
P < 0.05.
3. Results
3.1. Culture of native ALL blasts in serum-free media
Cells from eight ALL patients (Table 1, patients 1–8)
were cultured in X-vivo 10® , X-vivo 15® , Stem SpanTM
and FCS-containing medium for 7 days before 3 H-thymidine
incorporation was determined. The cells were cultured in
each medium alone and in media supplemented with Flt3-L,
Flt3-L + IL3 and Flt3-L + SCF. The overall results are
presented in Fig. 1. Detectable proliferation corresponding
to >1000 cpm was only observed for 10 of the 32 combinations (eight patients times four cytokine alternatives)
when using FCS-containing medium, whereas higher fractions were observed both for X-vivo 10® (14/32), X-vivo
15® (12/32) and especially Stem SpanTM (17/32). The median response for those combinations with detectable proliferation was also highest for Stem SpanTM (Fig. 1). Thus,
several serum-free media seem to be equal or superior to the
FCS-containing medium for in vitro analysis of ALL blast
proliferation.
Fig. 1. Spontaneous and cytokine-dependent ALL blast proliferation in
FCS containing and serum-free media. The ALL blasts were cultured in
RPMI 1640 + 10% inactivated FCS (RPMI), X-vivo 10® (X10), X-vivo
15® (X15) and Stem SpanTM (SS). Proliferation was determined for
cells cultured in medium alone (䊉) and medium supplemented with
Flt3-L (䊐), Flt3-L + IL3 (䉲) and Flt3-L + SCF (䊏). Open symbols indicate that proliferation was undetectable. The median value for those
patients showing detectable levels of 3 H-thymidine incorporation is indicated in the figure. All results are presented as the mean of triplicate
determinations.
3.2. Culture of native ALL blasts in the presence of
single cytokines
When ALL blasts from nine patients (Table 1, patients
1–9) were cultured in Stem SpanTM and FCS-containing
medium supplemented with various exogenous cytokines
(IL2, IL3, IL4, IL5, IL7, SCF or Flt3-L), the maximal proliferation was always observed with Stem SpanTM medium
and usually in the presence of Flt3-L (Table 3). ALL blast
proliferation was generally lower with the FCS-containing
medium, but for most patients the highest responses were
Patient
Proliferation in
medium alone
Proliferation in medium supplemented with single exogenous cytokines
IL2
1
2
3
4
5
6
7
8
9
552
712
754
310
370
281
1115
58639
1902
±
±
±
±
±
±
±
±
±
154
131
404
91
256
96
431
4266
296
3018
865
980
596
210
308
982
37536
1873
IL3
±
±
±
±
±
±
±
±
±
104
104
278
127
75
15
93
3919
384
8425
723
2567
446
361
325
3876
56974
2738
IL4
±
±
±
±
±
±
±
±
±
1002
55
315
210
54
53
141
7782
449
126
448
1046
275
190
109
360
65549
4030
IL5
±
±
±
±
±
±
±
±
±
895
63
32
105
90
17
83
5875
374
835
1243
1023
247
197
215
459
75281
2445
IL7
±
±
±
±
±
±
±
±
±
681
276
520
51
73
44
76
3296
463
691
756
3170
994
148
1025
1981
139885
2711
SCF
±
±
±
±
±
±
±
±
±
441
120
573
334
48
78
254
6096
223
2092
776
1821
752
341
263
1119
63.425
2706
Flt3-L
±
±
±
±
±
±
±
±
±
697
181
79
198
35
68
254
4210
454
31769
1375
3564
280
1625
2884
1458
78189
8709
±
±
±
±
±
±
±
±
±
1964
151
664
17
350
78
106
2194
948
Proliferation was assayed as 3 H-thymidine incorporation after 7 days of in vitro culture in Stem SpanTM . The results are presented as the mean ± S.D. of triplicate determinations. The proliferative responses
presented in bold represent either (i) the highest response for the patient; or (ii) responses that exceeded the cytokine-free control by >10.000 cpm. Negative controls generally corresponded to <300 cpm.
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
Table 3
Cytokine-dependent proliferation of native human ALL blasts cultured in serum-free Stem SpanTM medium
459
460
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
Table 4
Proliferative responses of native ALL blasts cultured in Stem SpanTM medium and FCS-containing medium: comparison of maximal cytokine-dependent
proliferation for FCS containing and Stem SpanTM medium when ALL blasts were cultured in the presence of various single cytokines or cytokine
combinations
Patient
1
2
3
4
5
6
7
9
Exogenous cytokine(s) giving the
highest ALL blast proliferationa
ALL blast proliferationb
Flt3-L + IL3
Flt3-L + IL3
Flt3-L + IL3
Flt3-L + SCF
Flt3 + SCF
Flt3-L + IL7
Flt3-L + IL3
Flt3-L + IL3
28458
352
202
354
78
104
313
218
RPMI + 10% FCS
±
±
±
±
±
±
±
±
Stem SpanTM
5542
74
52
72
15
82
18
81
52985
1393
11033
1280
2152
5847
5541
9023
±
±
±
±
±
±
±
±
4682
209
928
490
274
369
48
944
a ALL blasts were cultured in the presence of single cytokines or combinations of two different cytokines, the media then containing either IL7,
SCF or Flt3-L alone or these three cytokines combined with each other or with IL2, IL3, IL4 and IL5. The table presents the cytokine or cytokine
combination that resulted in the highest ALL blast proliferation.
b Cells from all patients were cultured with Stem SpanTM and RPMI + 10% FCS, and proliferation was assayed as the 3 H-thymidine incorporation
after 7 days of in vitro culture. All results are presented as the mean ± S.D. of triplicate determinations, and the responses presented in bold represent
the maximal response. Negative controls generally corresponded to <300 cpm.
Flt3-L + SCF and Flt3-L + IL7. However, for one patient
(Table 1, patient 7) we detected an IL7-induced inhibition
in repeated experiments, whereas combining Flt3-L with
SCF and IL3 did not inhibit proliferation for any patient.
Combinations of Flt3-L plus IL2, IL4 or IL5 had divergent
effects on ALL blast proliferation (Table 5).
detected in the presence of Flt3-L also when using this
medium (Table 1, patients 1–8, 10–12; data not shown).
3.3. Culture of native ALL blasts in the presence of
cytokine combinations
ALL blasts were cultured in the presence of IL7, SCF or
Flt3-L alone or in combination with one additional cytokine.
These three cytokines were then combined either with each
other or with IL2, IL3, IL4 and IL5. Cultures were prepared
in Stem SpanTM and in FCS-containing medium (patients
1–7, 9). The highest proliferation was then observed with
Stem SpanTM (Table 4), and the maximal response was
always detected when using Flt3-L alone or Flt3-L in combination with another cytokine. The overall results for various Flt3-L combinations in Stem SpanTM are presented in
Table 5 (patients 1–9). It can be seen that maximal proliferation was usually detected in the presence of Flt3-L + IL3,
3.4. Constitutive secretion of IL10 and IL15 by
native ALL blasts
ALL blasts derived from eight patients (Table 1, patients
1–3, 6–10) were cultured in the five different media (1×106
cells per ml), and concentrations of IL10 and IL15 were determined in the supernatants after 48 h. Cells were cultured
both in medium alone and in medium supplemented with
IL3+SCF +Flt3-L. For seven of these patients neither IL10
nor IL15 could be detected for any medium, and the results
were similar for cells cultured in media alone and media
Table 5
Cytokine-dependent proliferation of native human ALL blasts cultured in Stem SpanTM medium: the effect of combining Flt3-L with other exogenous
cytokines
Patient
1
2
3
4
5
6
7
8
9
Proliferation with
Flt3-L alone
32374
1074
3564
383
1940
2884
1327
78196
6452
±
±
±
±
±
±
±
±
±
1587
198
664
114
363
78
282
6845
1211
ALL blast proliferation in cultures containing Flt3-L together with another exogenous cytokine
IL2
50345
1307
3831
714
667
2104
1728
42675
5109
IL3
±
±
±
±
±
±
±
±
±
4630
152
543
227
379
199
93
4917
626
52985
1393
11033
845
1504
2744
5541
73173
9023
IL4
±
±
±
±
±
±
±
±
±
4682
209
928
75
359
316
48
4187
944
27735
439
6542
462
171
662
518
78801
8585
IL5
±
±
±
±
±
±
±
±
±
935
69
1552
128
99
33
109
3196
160
40805
1377
4504
275
950
1739
738
81118
8680
IL7
±
±
±
±
±
±
±
±
±
2826
200
1453
31
72
128
738
3708
853
35022
1221
5705
930
207
5847
2172
133425
8026
SCF
±
±
±
±
±
±
±
±
±
4968
190
716
132
31
369
54
6172
587
39965
1429
9002
1280
2152
2673
1670
78304
8281
±
±
±
±
±
±
±
±
±
4039
400
1432
490
274
233
226
5704
1099
Proliferation was assayed as the 3 H-thymidine incorporation after 7 days of in vitro culture in Stem SpanTM . The results are presented as the mean ± S.D.
of triplicate determinations. The proliferative responses marked in bold represent 3 H-thymidine incorporation that was significantly higher than for cultures
containing Flt3-L alone. Negative controls generally corresponded to <300 cpm.
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
with exogenous cytokines. Detectable levels of IL10 (but
nor IL15) were observed only for patient 7, and the highest levels were then observed in FCS-containing medium
supplemented with exogenous cytokines (18.4 pg/ml in
medium alone and 37.2 pg/ml in the presence of exogenous
cytokines).
3.5. Coculture of fibroblasts and native ALL blasts
in medium alone
Native ALL blasts derived from six patients (Table 1, patients 2, 6–10) were cultured with the two fibroblast cell lines
Hs27 and HFL1, and cultures were prepared both with ALL
cells and fibroblasts in direct contact and with the cells separated by a semipermeable membrane (referred to as transwell
cultures). The culture medium was DMEM + 10% FCS for
cultures containing Hs27 and F12K + 10% FCS for cultures
including HFL1, and these two media were used throughout
the whole culture period (also after addition of ALL blasts).
Significant proliferation (>1000 cpm) was observed only for
one patient (Table 1, patient 8) both when cells were incubated in medium alone and with fibroblasts. For this patient
increased proliferation was observed for ALL cells cultured
with HFL1 in transwell cultures (6729±342) compared with
control cultures without fibroblasts (3959 ± 296), whereas
ALL blast proliferation was not altered when cells were cultured in direct contact.
3.6. Coculture of native ALL cells with fibroblasts
and exogenous cytokines
Cells from 10 ALL patients (Table 1, patients 1–10)
were cultured either in medium alone, medium with exogenous cytokines (Flt3-L + IL3 + SCF), fibroblasts alone
461
(Hs27 or HFL1) or HFL1 fibroblasts plus the three exogenous cytokines. The medium during the period of coculture
was Stem SpanTM as described in Section 2. The highest
proliferation of the non-adherent ALL blast fraction was
usually observed when cells were cultured with HFL1 fibroblasts plus exogenous cytokines (Table 6). We regard
this non-adherent cell fraction to contain mainly ALL blasts
and only a negligible contamination of proliferating fibroblasts because: (i) altered 3 H-thymidine incorporation of
adherent and non-adherent cells showed no correlation (data
not shown); (ii) light microscopy verified that resuspension removed the non-adherent cells whereas adherent cells
were present after washing and efficiently removed only by
15–20 min of trypsination (data not shown). Furthermore,
the two fibroblast cell lines were also cultured in 10%
FCS-containing media (RPMI 1640, F12K and McCoy’s
medium) and the four serum-free media for 1 week, and
both cell lines showed no detectable 3 H-thymidine incorporation after 7 days of culture in serum-free media not even
when bFGF was added. These observations further suggest
that the 3 H-thymidine incorporation by the nonadherent cell
fraction is caused by the ALL blast population.
ALL blasts (Table 1, patients 1–3, 6–10) were also cultured with the two fibroblast cell lines for 7 days (Stem
SpanTM medium without cytokines) before the membrane
molecule phenotype of the nonadherent cells was analyzed.
Analysis of forward and side scatter showed only a single
population of viable cells. For six of these patients >90%
of these cells were judged to be positive for at least one
leukocyte-associated membrane molecule (CD19, CD20,
CD45), and for the last two patients the percentage of positive cells exceeded 60% for at least one of these molecules.
Furthermore, the cell distribution curves for all patients
and membrane molecules were consistent with a single
Table 6
ALL blast proliferation in the presence of fibroblast accessory cells; proliferation of non-adherent ALL blasts cultured with exogenous cytokines and in
direct contact with fibroblasts
ALL blasts
ALL blasts alone
None
1
2
3
4
5
6
7
8
9
10
319
2293
219
5435
885
975
327
441
14911
5671
29571
Fraction of patients with
detectable proliferation
Highest ALL blast
proliferation
±
±
±
±
±
±
±
±
±
±
±
220
25
59
99
46
56
103
39
360
365
922
ALL blasts + Hs27
908
22784
2217
28867
2799
6767
1743
834
41136
9317
40318
±
±
±
±
±
±
±
±
±
±
±
150
433
23
776
328
776
113
23
898
558
932
ALL blasts + HFL1
359
38739
7693
21412
4679
7861
5488
1481
37872
5373
49538
±
±
±
±
±
±
±
±
±
±
±
37
909
555
508
134
281
62
152
1714
151
392
ALL blasts + HFL1
+ cytokines
197
28754
5325
23374
14774
14406
25385
7547
53496
12159
32906
±
±
±
±
±
±
±
±
±
±
±
20
609
74
184
595
372
866
303
1471
1098
213
ALL blasts
+ cytokines
128 ±
9039 ±
231 ±
26647 ±
nt
4554 ±
2277 ±
3578 ±
20465 ±
15162 ±
30642 ±
4/10
7/10
10/10
10/10
8/9
0/10
1/10
3/10
5/10
1/9
36
490
27
651
253
143
161
534
206
390
Proliferative responses (3 H-thymidine incorporation) are presented as the mean ± S.D. cpm of triplicate determinations. The cytokines used in the cultures
were Flt3-L 20 ng/ml + IL3 20 ng/ml + SCF 20 ng/ml. The highest proliferative response for each patient is marked in bold. The fraction of patients with
highest ALL blast proliferation is indicated at the bottom of the table for each of the experimental models.
462
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
population, and light microscopy of the wells after removal
of nonadherent cells showed a layer of cells with the morphological characteristics of adherent fibroblasts.
3.7. Culture of ALL cell lines and EBV-transformed B
cell lines
Four B-lineage ALL cell lines (Nalm-6, SD1, Tanoue and
697), one T leukemia cell line (Jurkat) and one B-lymphoma
cell line (Daudi) were cultured in the four serum-free media
and in RPMI with 10% FCS. All cell lines were cultured for
10 days, and the results are expressed as the number of population doublings during this period (Table 2). Serum-free
media could be used for expansion of all these cell lines,
and for each cell line proliferation in at least one of the
serum-free media was equal to or exceeded proliferation in
the recommended FCS-containing medium.
Four EBV-transformed B cell lines were cultured for 10
days in the four serum-free media and in RPMI-medium
with 10% FCS. All the EBV-transformed cell lines could
be expanded in serum-free media, but the optimal medium
differed between the lines (Table 7). However, for three
of the four cell lines the proliferation in at least one
serum-free medium was equal to or exceeded proliferation
in FCS-containing medium.
Table 7
In vitro culture of EBV-transformed B cell lines; a comparison of proliferative responses in various culture media
Cell line
Medium
Proliferation
(population doublings)
AL10
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
4.3
2.3
2.3
2.6
2.7
ALK
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
1.1
2.8
0.5
1.4
2.5
DOC
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
4.0
3.6
2.3
4.7
3.3
EPA
X-vivo 10®
X-vivo 15®
X-vivo 20®
Stem SpanTM
RPMI + 10% FCS
1.6
0
0
0
3.8
All cell lines were cultured in parallel in the various media, and the
results are expressed as the number of population doublings during a
10 days culture period. All cultures were prepared in 24-well tissue
culture plates with 2 ml medium per well, half of the medium was
changed three times weekly, and cultures were divided when the cells
formed a monolayer. The results in bold represent a proliferation equal
to (within ±0.2 population doubling) or exceeding the response in the
FCS-containing growth medium.
4. Discussion
The overall long-term ALL-free survival after intensive
chemotherapy is only 40–50%, and the prognosis is even
worse for patients with unfavorable prognostic parameters and for elderly patients that cannot receive the most
intensive chemotherapy due to an unacceptable risk of
treatment-related mortality [1–3]. Thus, there is a need for
new treatment strategies in ALL, and several of these approaches require an initial experimental evaluation before
they can be further investigated in clinical trials. It will then
be important to use standardized in vitro models for the
biological characterization of native human ALL blasts.
Our present study included consecutive patients with high
peripheral blood blast counts, and highly enriched ALL blast
populations could then be prepared by gradient separation
alone. More extensive cell separation procedures can induce
functional alterations in immature myeloid leukemia cells
[26,29,30], and our use of a simple separation procedure
would possibly reduce the risk of inducing similar effects
in the ALL blasts. However, due to this patient selection
our results should be interpreted with caution and may be
representative only for patients with high blood blast counts.
Culture media supplemented with inactivated FCS are
widely used for experimental studies of hematopoietic cells
[26]. The concentrations of FCS used by different investigators show a wide variation, some investigators use 10%
FCS [6] but in certain colony formation assays concentrations up to 30% have been used [31]. Furthermore, different media (e.g. RPMI 1640, ␣-Modified Eagle’s Medium,
Iscove’s Modified Dulbecco’s Medium) have been combined with FCS in these previous studies [4,6,18]. The
use of RPMI + 10% FCS in our experiments was based
on the recent study by Srivannaboon et al. [6] who used
this medium when they investigated apoptosis regulation of
ALL blasts cultured in a stroma-based experimental model.
However, the use of FCS represents nonstandardized experimental conditions because: (i) the characteristics of FCS
will differ between batches; and (ii) the serum represents
a source of unidentified soluble mediators that may affect
functional in vitro characteristics of immature leukemia
cells [32]. To improve the standardization of experimental
models it will therefore be important to include serum-free
experimental conditions in future in vitro studies.
In a previous study we demonstrated that a serum-free
medium originally developed for culture of normal
hematopoietic progenitors, could be used for in vitro culture
of native human AML cells [10]. A very similar medium
has also been used for culture of ALL blasts [8]. Taken
together these results suggest that the growth requirements
of normal progenitors, native AML blasts and native ALL
blasts are very similar. In our present studies of in vitro cultured ALL blasts we therefore included the serum-free Stem
SpanTM medium that can be used for culture of normal stem
cells (manufacturer’s information) as well as native AML
blasts and AML cell lines [21]. Our results demonstrated
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
that all four serum-free media (including Stem SpanTM )
could be used for in vitro culture of native ALL blasts and
ALL cell lines, and the use of such standardized in vitro
conditions will thereby allow comparative studies of normal
immature hematopoietic cells and immature leukemia cells
of myeloid as well as lymphoid origin.
Previous studies have demonstrated that ALL blast proliferation can be increased by several cytokines, including
Flt3-L, IL3 and IL7 [5,8,11–16]. Our present results confirmed that ALL blast proliferation was increased by these
three cytokines, especially Flt3-L. Furthermore, although a
previous study concluded that SCF does not affect ALL
blast proliferation [12], our present results demonstrated that
SCF could increase ALL blast proliferation for a subset of
patients when being present together with Flt3-L. We also
tested a wide range of cytokine combinations both in FCS
containing and in the optimal serum-free medium (Stem
SpanTM ), and based on these experiments we suggest that
the triple combination of Flt3-L +IL3+SCF should be used
for experimental studies when a standardized growth factor
combination is required, because: (i) the highest proliferative
responses to a single cytokine were usually observed in the
presence of Flt3-L; (ii) Flt3-L-initiated proliferation was often increased by combining Flt3-L with IL3 and/or SCF; (iii)
although high responses were often detected with IL7 alone
or IL7 + Flt3-L, for one patient a reproducible IL7-induced
inhibition was observed; (iv) in those cases when IL7 or
IL7 + Flt3-L induced the highest proliferative response, the
responses to Flt3-L plus IL3 or SCF were also high.
Constitutive cytokine secretion is an important functional
characteristic of several immature leukemic cells, and a recent study suggested that secretion of IL10 and IL15 is
common in ALL [33]. We therefore examined the supernatant levels of these two cytokines after culture of native
ALL blasts in various media. Although the previous study
reported that IL10 and IL15 producing ALL blasts could
be easily detected, the supernatant levels of these cytokines
were either low or undetectable for all culture media investigated in our study. Thus, the constitutive cytokine release
seems to be less dependent on variations in culture conditions than ALL blast proliferation.
Several experimental studies have used coculture of human ALL cells with various irradiated or non-irradiated accessory cells, including murine fibroblasts as well as human
fibroblasts, monocytes, endothelial cells and bone marrow
stromal cells [11,15–20]. In this context we investigated two
well-characterized fibroblast cell lines (Hs27, HFL1) as accessory cells. Although a previous study concluded that it
is not optimal to use fibroblast cell lines as accessory cells
[9], our present results demonstrated that the cell lines can
be used in combination with an optimal serum-free culture
medium. The highest ALL blast proliferation was then usually detected with HFL1 fibroblasts, and the responses could
often be further increased by adding exogenous Flt3-L +
IL3 + SCF. By using these standardized culture conditions
detectable ALL blast proliferation was observed for all our
463
patients, and control experiments verified that this was a true
proliferation of non-adherent ALL cells and not contaminating fibroblasts.
We also investigated the proliferation of ALL cell
lines and EBV-transformed B cells that were cultured in
serum-free media and the recommended FCS-containing
medium. These results demonstrated that serum-free culture
conditions can also be used for ALL cell lines. Although
EBV-transformed cell lines were heterogeneous with regard to growth requirements, serum-free conditions could
be used for several of these cell lines too.
The present results demonstrate that both native human
ALL blasts, ALL cell lines and EBV-transformed B cell
lines can be studied in vitro under standardized serum-free
conditions. Our observations may thereby form a basis for
a standardized experimental evaluation of new antileukemic
treatment strategies in ALL.
Acknowledgements
The work was supported by the Norwegian Cancer Society, Olaf Ruunshaugens Foundation and The Rakel and
Otto-Kristian Brun Foundation. O. Bruserud is responsible
for the conception and design, drafting the article, provision
of study materials or patients, statistical and expertise obtaining funding; all authors were responsible for analysis and
interpretation of data, critical revision of the article for important intellectual content, final approval of the article and
collection or assembly of data; O. Bruserud, E. Ulvestad, L.
Menzoni for administrative, technical and logical support.
References
[1] Hoelzer D, Gokbuget N. Recent approaches in acute lymphoblastic
leukemia in adults. Crit Rev Oncol Hematol 2000;36:49–58.
[2] Berman E. Recent advances in the treatment of acute leukemia: 1999.
Curr Opin Hematol 2000;7:205–11.
[3] Cortes JE, Kantarjian H, Freireich EJ. Acute lymphoblastic leukemia:
a comprehensive review with emphasis on biology and therapy.
Cancer Treat Res 1996;84:291–323.
[4] Shao L-E, Dialynas DP, Yu AL, Mackintosh E, Yu J. Human cord
blood conditioned medium enhances leukemia colony formation in
vitro. Leuk Res 2000;24:1041–8.
[5] Pontvert-Delucq S, Hibner U, Vilmer E, Baillou C, Rohrlich P,
Heymann D, et al. Heterogeneity of B lineage acute lymphoblastic
leukemias (B-ALL) with regard to their in vitro spontaneous
proliferation, growth factor response and BCL-2 expression. Leuk
Lymphoma 1996;21:267–80.
[6] Srivannaboon K, Shanafelt AB, Todisco E, Forte CP, Behm FG,
Raimondi SC, et al. Interleukin 4 variant (BAY 36-1677) selectively
induces apoptosis in acute lymphoblastic leukemia cells. Blood
2001;97:752–8.
[7] Köhler T, Plettig R, Wetzstein W, Schmitz M, Ritter M, Mohr B, et
al. Cytokine-driven differentiation of blasts from patients with acute
myelogenous and lymphoblastic leukemia into dendritic cells. Stem
Cells 2000;18:139–47.
[8] Duyn AEJ, Kaspers GJL, Pieters R, van Zantwijk CH, Broekema
GJ, Hählen K, et al. Effects of interleukin 3, interleukin 7,
464
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
Ø. Bruserud et al. / Leukemia Research 27 (2003) 455–464
and B-cell growth factor on proliferation and drug resistance in
vitro in childhood acute lymphoblastic leukemia. Ann Haematol
1999;78:163–71.
Manabe A, Coustan-Smith E, Behm FG, Raimondi SC, Campara D.
Bone marrow derived stromal cells prevent apoptotic cell death in
B-lineage acute lymphoblastic leukemia. Blood 1992;79:2370–7.
Bruserud Ø, Frostad S, Foss B. In vitro culture of acute myelogenous
leukemia blasts: a comparison of four different culture media. J
Hematother 1999;8:63–73.
Bradstock KF, Makrynikola V, Bianchi A, Shen W, Hewson J,
Gottlieb DJ. Effects of the chemokine stromal cell-derived factor-1
on the migration and localization of precursor-B acute lymphoblastic
leukemia ells within the bone marrow. Leukemia 2000;14:882–8.
Petrucci MT, de Felice L, Riccardi MR, Ariola C, Mascolo MG,
Fenu S, et al. Stem cell factor and PIXY-321 is acute lymphoblastic
leukemia: in vitro study on proliferation effects and apoptosis.
Cytokines Mol Ther 1996;2:225–30.
Eder M, Hemmati P, Kalina U, Ottman OG, Höltzer D, Lyman SD,
et al. Effects of Flt3-L and interleukin 2 on in vitro growth of acute
lymphoblastic leukemia cells. Exp Hematol 1996;24:371–7.
Campana D, Coustan-Smith E, Kumagai MA, Manabe A. Growth
requirements of normal and leukemic human B cell progenitors.
Leuk Lymphoma 1994;13:359–71.
Eder M, Ottmann OG, Hansen-Hagge TE, Bartram CR, Falk S, Gillis
S, et al. In vitro culture of common acute lymphoblastic leukemia
blasts: effects of interleukin 3, interleukin 7 and accessory cells.
Blood 1992;79:3274–84.
Makrynikola V, Kabral A, Bradstock KF. Effects of recombinant
human cytokines on precursor-B acute lymphoblastic leukemia cells.
Exp Hematol 1991;19:674–9.
Buske C, Hiddemann W, Wormann B. Proliferation of human
leukemic pre-B cells induced by human bone marrow stromal cells
and murine fibroblasts. Cell Biol Int 1994;18:1049–58.
Fortney JE, Zhao W, Wenger SL, Gibson LF. Bone marrow
stromal cells regulate caspase 3 activity in leukemic cells during
chemotherapy. Leuk Res 2001;25:901–7.
Bradstock K, Bianchi A, Makrynikola V, Filshie R, Gottlieb
D. Long-term survival and proliferation of precursor-B acute
lymphoblastic leukemia cells on human bone marrow stroma.
Leukemia 1996;10:813–20.
Ashley DM, Bol SJ, Kannourakis G. Viable bone marrow stromal
cells are required for the in vitro survival of B-cell precursors acute
lymphoblastic leukemic cells. Leuk Res 1995;19:113–20.
Bene MC, Castoldi G, Knapp W, Ludwig WD, Matutes E, Orfao
A, et al. Proposals for the immunological classification of acute
leukemias. Leukemia 1995;9:1783–6.
[22] Bruserud Ø, Gjertsen BT, von Volkman HL. In vitro culture of acute
myelogenous leukemia (AML) cells in serum-free media: studies of
native AML blasts and AML cell lines. J Hematother Stem Cell Res
2000;9:923–32.
[23] Bruserud Ø. Effects of dipyridamole, theophyllamine and verapamil
on spontaneous proliferation of myelogenous leukemia cells. Acta
Oncol 1992;31:53–8.
[24] Bruserud Ø, Gjertsen BT, Brustugun OT, Bassoe CF, Akselsen PE,
Bühring HJ, et al. Effect of interleukin 10 on blast cells derived from
patients with acute myelogenous leukemia. Leukemia 1995;9:1910–
20.
[25] Bruserud Ø. Effects of interleukin 13 on cytokine secretion by human
acute myelogenous leukemia blasts. Leukemia 1996;10:1497–503.
[26] Bruserud Ø, Gjertsen BT, Foss B, Huang TS. New strategies in the
treatment of acute myelogenous leukemia (AML): in vitro culture of
AML cells—the present use in experimental studies and the possible
importance for future therapeutic strategies. Stem Cells 2001;19:1–
11.
[27] Bruserud Ø, Ulvestad E. Acute myelogenous leukemia blasts as
accessory cells during in vitro T lymphocyte activation. Cell Immunol
2000;206:36–50.
[28] Abrahamsen JF, Bakken AM, Bruserud Ø, Gjertsen BT. Flow
cytometric measurement of apoptosis and necrosis in cryopreserved
PBPC concentrates from patients with malignant diseases. Bone
Marrow Transplant 2002;29:165–71.
[29] Sissolak G, Hoffbrand AV, Mehta AB, Ganeshaguru K. Effects
of interferon (IFN) alpha on the expression of interleukin 1
beta (IL1), interleukin 6, granulocyte-macrophage colony-stimulating
factor (GM-CSF) and tumor necrosis factor alpha (TNF) in acute
myeloid leukemia blasts. Leukemia 1992;6:1155–60.
[30] Fiedler W, Suciu E, Wittlief C, Ostertag W, Hossfeld DK.
Mechanisms of growth factor expression in acute myeloid leukemia
(AML). Leukemia 1990;4:459–61.
[31] Espinoza-Hernandez L, Cruz-Rico J, Benitez-Aranda H, MartinezJaramillo G, Rodriguez-Zepeda MC, Velez-Ruelas MA, et al. In vitro
characterization of the hematopoietic system in pediatric patients
with acute lymphoblastic leukemia. Leukemia Res 2001;25:295–303.
[32] Bloch A. Dynamics of interaction between DNA-specific antitumor
agents and serum-contained cytokines in the initiation of
ML-1 human myeloblastic leukemia cell differentiation. Leukemia
1993;7:1219–24.
[33] Kebelmann-Betzing C, Korner G, Badiali L, Buchwald D, Moricke A,
Korte A, et al. Characterization of cytokine, growth factor receptor,
costimulatory and adhesion molecule expression patterns of bone
marrow blasts in relapsed childhood B cell precursor ALL. Cytokine
2001;13:39–50.