Neuro-Oncology Advance Access published December 12, 2013
Neuro-Oncology
Neuro-Oncology 2013; 0, 1 – 9, doi:10.1093/neuonc/not202
Brain tumor cell line authentication, an efficient alternative to capillary
electrophoresis by using a microfluidics-based system
Qian An, Helen L. Fillmore, Mikaella Vouri, and Geoffrey J. Pilkington
Corresponding Author: Dr. Qian An, MD, PhD, School of Pharmacy and Biomedical Sciences, University of Portsmouth, St Michael’s Building,
White Swan Road, Portsmouth PO1 2DT, UK ([email protected]).
Background. The current method for cell line authentication is genotyping based on short tandem repeat (STR) –PCR involving coamplification of a panel of STR loci by multiplex PCR and downstream fragment length analysis (FLA), usually performed by capillary electrophoresis. FLA by capillary electrophoresis is time-consuming and can be expensive, as the facilities are generally not accessible for many
research laboratories.
Methods. In the present study, a microfluidic electrophoresis system, the Agilent 2100 Bioanalyzer, was used to analyze the STR-PCR
fragments from 10 human genomic loci of a number of human cell lines, including 6 gliomas, 1 astrocyte, 1 primary lung cancer,
1 lung brain metastatic cancer, and 1 rhabdomyosarcoma; and this was compared with the standard method, that is, capillary electrophoresis, using the Applied Biosystems 3130xl Genetic Analyzer.
Results. The microfluidic electrophoresis method produced highly reproducible results with good sensitivity in sizing of multiple PCR fragments, and each cell line demonstrated a unique DNA profile. Furthermore, DNA fingerprinting of samples from 5 different passage
numbers of the same cell line showed excellent reproducibility when FLA was performed with the Bioanalyzer, indicating that no
cross-contamination had occurred during the culture period.
Conclusion. This novel application provides a straightforward and cost-effective alternative to STR-based cell line authentication. In addition, this application would be of great value for cell bank repositories to maintain and distribute precious cell lines.
Keywords: brain tumor cell authentication, DNA fingerprinting, microfluidics, STR profiling.
The importance of cell line authentication has been well recognized
by the research community, and recently a list of 360 crosscontaminated and misidentified cell lines has been published, to
be updated when necessary.1 At present the widely proposed technology for human cell line authentication is DNA genotyping by
short tandem repeat (STR) –PCR, and the standard method for analyzing STR fragments (ie, fragment length analysis [FLA]) is capillary
electrophoresis.2 – 5 There are a number of commercially available
STR kits commonly used that provide convenient multiplex PCR
amplification of the markers.4 The downstream FLA is, however,
normally done by capillary electrophoresis, which requires
state-of-the-art facilities and properly trained specialists to
analyze the data obtained, resulting in outsourcing of the cell authentication service for the majority of research laboratories. We
were interested in exploring the possibility of using other electrophoresis platforms for FLA, such as the Agilent 2100 Bioanalyzer,
which is a microfluidics-based Lab-on-Chip system for sizing and
quantification of DNA, RNA, proteins, and cells.6 – 9 It also provides
quality control of DNA, RNA, and protein samples in a broad
range of molecular assays.10 – 12 Recently the Bioanalyzer has
been employed to resolve STR fragments in forensic samples as
well as to identify fish species based on their restriction fragment
length polymorphism pattern.13,14
Our Cellular and Molecular Neuro-oncology Research Laboratories at the University of Portsmouth act as the brain tumor cell
culture repository for the South of England Brain Tumour Alliance
(SEBTA), a network formed in 2011 by 7 regional centers in the
south of England that are involved in diagnosis, treatment, and research in neuro-oncology (http://sebta.org). With its clearly identified strategic aims, SEBTA promotes and facilitates increased
collaboration among those centers, including sharing valuable
tissues and cell lines. Therefore, apart from our own group, we
are responsible for the authentication of cell lines that are distributed across the SEBTA centers by our brain tumor cell culture
Received 23 August 2013; accepted 2 October 2013
# The Author(s) 2013. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved.
For permissions, please e-mail: [email protected].
1 of 9
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
Cellular and Molecular Neuro-oncology Research Group, Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and
Biomedical Sciences, University of Portsmouth, Portsmouth, UK
An et al.: Brain tumor cell line authentication
repository, and it is critical for us to establish an efficient workflow
for the routine cell authentication tests. For that purpose, in the
present study we investigated the potential of this microfluidic
system in STR genotyping of human brain tumor cell lines, exploiting its capacity for DNA fragment separating and sizing. The
microfluidics-based electrophoresis proved to be an effective and
simple method for FLA in replacement of capillary electrophoresis.
In addition, it created accurate and reproducible DNA profiling data
for the cell lines studied, offering an efficient and reliable tool for
our routine cell line genotyping test.
Cell Lines
Fragment Length Analysis
All primary cell lines were derived from patient biopsy materials
under ethics permissions LREC 00-173, KCH 11-094, or 11/SC/
0048 in accordance with the National Research Ethics Service.
These cell lines in addition to 4 established commercially available
cell lines were used in the first part of the DNA profiling studies. The
primary glioma cell lines included a grade II astrocytoma UP-016/
Passage (P)2, a grade III astrocytoma UP-032 (P5), and 3 grade IV
glioblastoma multiforme (GBM) cell lines: IN699 (P17), UP-019
(P9), and UP-029 (P5). One primary lung brain metastatic cell
line, UP-024 (P4), was also included in this study. Commercially
available cell lines analyzed were: the GBM cell line SNB-19
(German Collection of Microorganisms and Cell Cultures [DSMZ];
P63), the human lung carcinoma cell line NCI-H1299 (American
Type Culture Collection [ATCC]; P25), the rhabdomyosarcoma cell
line TE-671 (ATCC; P22), and the normal human astrocyte
cell line CC2565 (Lonza Biologics; P10). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) with 10%
fetal calf serum (FCS; Sigma-Aldrich), with the exception of
NCI-H1299 cells that were cultured in Roswell Park Memorial Institute 1640 medium (Sigma-Aldrich).
To monitor potential changes (in particular, crosscontamination) in cell lines over long-term culture, UP-029 and
another biopsy-derived GBM cell line, SEBTA-003 (cultured in 10%
FCS/DMEM), were genotyped in the second part of these studies.
Cells were cultured for 5 continuous passages with a 5-passage
interval (ie, P10, P15, P20, P25, and P30 of UP-029; P8, P13, P18,
P23, and P28 of SEBTA-003).
Amplified alleles in the STR-PCR products were separated on the
Agilent 2100 Bioanalyzer with the Agilent DNA 1000 Kit according
to the standard protocol with slight modification.17 In essence, the
DNA chip was first primed with 9 mL of the gel-dye mixture, then
3 mL of the PCR product was added to each of the 12 sample
wells along with 3 mL of the internal marker (Agilent). One microliter of the Agilent DNA 1000 ladder, used as a sizing standard,
was loaded into the ladder well with 5 mL of the internal marker.
After electrophoresis, the amplified alleles from each sample
were analyzed using the manufacturer’s software, 2100 Expert,
provided with the instrument; the size of each fragment was determined based on the ladder and internal standards. In addition, the
STR markers were identified based on their size ranges.15 Fragment
length analysis was done with the Bioanalyzer for all samples from
3 independent STR-PCR experiments (1, 2, and 3). In comparison,
samples from the first experiment (Exp 1) were sent to Eurofins
MWG Operon (http://www.eurofinsgenomics.eu) and analyzed
using standard procedures of capillary electrophoresis on the
Applied Biosystems 3130xl Genetic Analyzer, as described in the
service information document (Eurofins MWG Operon).18 The relevant negative sample was also included in each of the FLA analyses
as a control.
Short Tandem Repeat– PCR
Genomic DNA was extracted from cultured cells using the QIAamp
DNA Mini Kit (Qiagen). STR-based multiplex PCR was carried out
with the StemElite ID System (Promega) according to the manufacturer’s protocol.15 DNA amplification was set up in a final
volume of 25 mL reaction, including 1 ×Enzyme Mix, 1 ×Primer
Pair Mix, and 2 ng of template DNA. PCR was performed on the
MyCycler Thermal Cycler (Bio-Rad) using the thermal cycling
program recommended for the Applied Biosystems GeneAmp
PCR System 9600 Thermal Cycler, according to the StemElite ID
System Manual (Promega).15 The step-by-step thermal cycling
protocol is as follows—step 1: 968C for 2 min; step 2: 948C for
30 s; step 3 (×10 cycles): ramp 60 s to 608C and hold for 30 s,
then ramp 50 s to 708C and hold for 45 s; step 4: 908C for 30 s;
step 5 (×22 cycles): ramp 60 s to 608C and hold for 30 s, then
ramp 50 s to 708C and hold for 45 s; step 6: 608C for 30 min. The
2 of 9
Results
DNA Profiling of Cell Lines by Microfluidics-based
Electrophoresis and Capillary Electrophoresis
To assess the application of the microfluidics-based electrophoresis method in STR-based DNA profiling of human cell lines, amplified STR fragments of the 10 cell lines from the first experiment (exp
1) were separated and analyzed by both microfluidic and capillary
electrophoresis. The former was performed in our laboratory using
the Agilent DNA 1000 Kit and the data were analyzed by the Agilent
2100 Expert software, the latter by Eurofins MWG’s FLA service
using the ABI 3130xl Genetic Analyzer as recommended for the
StemElite ID System.15 Amplified STR fragments from all 10 cell
lines were successfully separated by the Bioanalyzer, as demonstrated by the representative electropherograms (Fig. 1A and B). Although DNA fragment sizing was on average around 20 bp larger
than that obtained from capillary electrophoresis, likely due to
the dye-labeled PCR primers that had incorporated into the fragments and consequently reduced the migration speed of those
fragments, both sets of data were highly comparable in terms of
the DNA profiles of the cell lines studied (Table 1). There were
Neuro-Oncology
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
Materials and Methods
primers of the StemElite ID System Kit amplify 9 human STR loci, including D21S11, TH01, TPOX, vWA, CSF1PO, D16S539, D7S820,
D13S317, and D5S818, as well as amelogenin for gender identification. These markers are selected from the set of core STR loci that
has been approved by the human identity testing community.4,16
STR-PCR was performed in 3 independent experiments for all cell
lines in the first part of the studies (experiments 1, 2, and 3),
whereas 1 STR-PCR experiment was carried out for 5 continuous
passages each of UP-029 and SEBTA-003, in the crosscontamination test study. A negative PCR reaction was included
in each of the experiments where template DNA was replaced
with water.
An et al: Brain tumor cell line authentication
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
Fig. 1. Representative single (A and B) and comparison (C) electropherograms of DNA profiling by a microfluidic system, the Agilent 2100 Bioanalyzer. STR
profiles of the cell lines TE-671 (A) and UP-019 (B) are illustrated as DNA electrophoresis traces here. Fragments of the PCR-amplified STR markers are
separated based on their sizes, and the size of each peak is labeled above it in panels A and B. The overlaid traces of TE-671 and UP-019 in panel C,
obtained from the “Comparison” analysis (Agilent 2100 Expert software), show the unique STR profiles of these 2 cell lines.
differences in several of the STR markers when comparing these 2
methods. When the STR marker D21S11 was analyzed, 4 of the 10
cell lines using the microfluidic method failed to detect the heterozygous alleles compared with capillary electrophoresis (shaded
Neuro-Oncology
cells in Table 1). The STR markers D5S818, vWA, TPOX, and
CSF1PO showed homozygous alleles in 10% (1/10) of the cell
lines where capillary electrophoresis revealed heterozygosity of
the associated marker in those cell lines (shaded cells in Table 1).
3 of 9
An et al.: Brain tumor cell line authentication
Table 1. StemElite STR marker sizing by Agilent Bioanalyzer and ABI 3130xl Genetic Analyzer
Cell ID
D5S818 (bp)
Agilent
ABI
Agilent
ABI
Agilent
ABI
Agilent
ABI
Agilent
ABI
125;128
125;128
125;125
125;129
124;124
124;127
125;128
124;124
125;125
124;127
–a
103;109
103;109
103;103
104;109
103;103
103;109
103;109
103;103
104;104
103;109
–
139;139
148;148
143;143
155;158
150;154
137;150
159;163
159;159
146;146
155;163
–
111;124
132;132
128;128
128;132
124;128
124;136
132;136
132;132
132;132
132;136
–
151;151
159;159
155;165
165;173
173;173
163;167
177;177
168;168
156;169
168;181
–
150;158
158;158
146;154
146;154
154;154
146;150
158;158
150;150
138;150
138;150
–
170;177
178;188
174;190
190;190
189;189
187;187
204;204
188;188
175;190
194;194
–
172;175
164;172
160;175
175;175
175;175
175;175
160;160
175;175
160;175
168;168
–
186;203
206;211
214;214
206;211
219;219
208;221
214;214
205;214
206;214
213;213
–
178;190
182;186
190;190
182;186
194;194
187;198
190;190
182;190
182;190
190;190
–
D21S11 (bp)
CC2565
IN699
NCI-H1299
SNB-19
TE-671
UP-016
UP-024
UP-019
UP-029
UP-032
Negative
vWA (bp)
D7S820 (bp)
TH01 (bp)
TPOX (bp)
D13S317 (bp)
D16S539 (bp)
CSF1PO (bp)
Agilent
ABI
Agilent
ABI
Agilent
ABI
Agilent
ABI
Agilent
ABI
215;215
238;238
247;247
242;242
231;231
236;241
239;239
233;233
238;246
237;237
–
223;229
215;215
233;233
219;219
215;219
215;219
223;229
219;219
215;223
223;227
–
247;255
243;247
258;258
247;255
239;254
249;253
246;254
241;241
255;256
245;245
–
227;235
223;227
227;227
227;235
219;235
231;235
227;235
223;223
219;235
227;227
–
282;285
275;275
276;276
275;275
278;278
272;272
286;286
287;287
274;274
273;273
–
277;281
269;269
269;269
269;269
273;273
269;285
281;281
285;285
269;269
269;269
–
294;296
293;305
301;305
300;300
293;296
287;298
300;304
296;296
293;295
299;302
–
281;285
281;293
289;293
289;289
281;285
277;289
289;293
285;285
281;285
289;293
–
358;358
351;359
364;364
362;362
354;361
360;363
355;363
345;357
354;354
356;356
–
337;337
329;337
341;341
337;341
333;337
341;345
333;341
325;337
333;333
337;337
–
a
No DNA track detected.
On the contrary, the microfluidic system was highly consistent
in terms of analyzing homozygous STR markers and the homozygosity matched 100% between those 2 methods (Table 1). In
one cell line (UP-029), there was a discrepancy in the sizes of 2
alleles of D7S820 between the methods, with a 1-bp difference
in the microfluidic method, whereas there was 16 bp by capillary
electrophoresis (underlined cells in Table 1). In total, 92% (92/
100) of the analyzed markers showed excellent similarity
between the Bioanalyzer and capillary electrophoresis in terms of
fragment sizing. Importantly, both microfluidic and capillary electrophoresis revealed a unique DNA profile of each cell line, presented by fragment sizing for the former (Table 1) and by the
internationally recognized standard for the latter (Table 2).
The DNA profiles of NCI-H1299 and TE-671 in the present study
matched the ones published by ATCC and DSMZ respectively
(Table 3).19 Previous STR analysis at ATCC revealed that the
human GBM cell line SNB-19 had an identical profile to U373 MG,
another GBM cell line, which led to SNB-19 being discontinued by
ATCC.20 As mentioned, our laboratory obtained the SNB-19 from
the DSMZ, and this cell line was included in the DNA profiling
study to determine whether our SNB-19 cell line had been crosscontaminated. Our data confirmed that the SNB-19 cell line in
4 of 9
our cell bank was not cross-contaminated with U373 MG as
reported by ATCC, based on the comparison between STR profiles
of our SNB-19 cell line and the reported U373 MG (Table 3).21
According to the DSMZ data sheet, SNB-19 is a “subclone” of the
human GBM cell line U251 MG.22 This conclusion is supported by
previous studies indicating that those 2 cell lines carry 96% genotype similarity and identical genomic variants, that is, tumor
protein 53 (TP53), cyclin-dependent kinase inhibitor 2A (CDKN2A),
and phosphatase and tensin homolog (PTEN) mutations,16,23 although SNB-19 and U251 MG have diverged at the karyotypical
level.24 Interestingly, when STR matching analysis was performed
using DSMZ’s online tool for the SNB-19 cell line included in the
present study, the result showed that our SNB-19 had the identical
STR profile to those for U251 MG and SNB-19 in the DSMZ database
(Table 3). Hence the SNB-19 cell line cultured in our laboratories has
100% genotype similarity to both cell lines (U251 MG and SNB-19)
held at DSMZ. Our data also provide further evidence for the previous observation that SNB-19 is a derivative of U251 MG. In addition,
the STR profiles of 3 commercial cell lines, NCI-H1299, SNB-19, and
TE-671 obtained by capillary electrophoresis in our study were identical to those in published databases (Table 3), indicating good reproducibility of the conventional method.
Neuro-Oncology
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
CC2565
IN699
NCI-H1299
SNB-19
TE-671
UP-016
UP-024
UP-019
UP-029
UP-032
Negative
Amelogenin (bp)
An et al: Brain tumor cell line authentication
Table 2. STR genotypes of 10 cell lines
Amelogenin
D5S818
vWA
TH01
D13S317
D21S11
D7S820
TPOX
D16S539
CSF1PO
CC2565
IN699
NCI-H1299
SNB-19
TE-671
UP-016
UP-024
UP-019
UP-029
UP-032
Negative
X, Y
X, Y
X, X
X, Y
X, X
X, Y
X, Y
X, X
X, X
X, Y
–a
7, 10
12, 12
11, 11
11, 12
10, 11
10, 13
12, 13
12, 12
12, 12
12, 13
–
17, 19
19, 19
16, 18
16, 18
18, 18
16, 17
19, 19
17, 17
14, 17
14, 17
–
9, 9.3
7, 9
6, 9.3
9.3, 9.3
9.3, 9.3
9.3, 9.3
6, 6
9.3, 9.3
6, 9.3
8, 8
–
9, 12
10, 11
12, 12
10, 11
13, 13
11, 14
12, 12
10, 12
10, 12
12, 12
–
30, 31.2
28, 28
32.2, 32.2
29, 29
28, 29
28, 29
30, 31.2
29, 29
28, 30
30, 31
–
10, 12
9, 10
10, 10
10, 12
8, 12
11, 12
10, 12
9, 9
8, 12
10, 10
–
10, 11
8, 8
8, 8
8, 8
9, 9
8, 12
11, 11
12, 12
8, 8
8, 8
–
10, 11
10, 13
12, 13
12, 12
10, 11
9, 12
12, 13
11, 11
10, 11
12, 13
–
11, 11
9, 11
12, 12
11, 12
10, 11
12, 13
10, 12
8, 11
10, 10
11, 11
–
a
No DNA track detected.
Table 3. STR profiles of the NCI-H1299, SNB-19, and TE-671 cell lines by our data and published resources
Cell ID
Amelogenin
D5S818
vWA
TH01
D13S317
D21S11
D7S820
TPOX
D16S539
CSF1PO
NCI-H1299a
NCI-H1299b
SNB-19a
U-373 MGc
SNB-19d
U-251 MGd
TE-671a
TE-671d
X, X
X, X
X, Y
X, X
X, Y
X, Y
X, X
X, X
11, 11
11, 11
11, 12
12, 12
11, 12
11, 12
10, 11
10, 11
16, 18
16, 18
16, 18
17, 18
16, 18
16, 18
18, 18
18, 18
6, 9.3
6, 9.3
9.3, 9.3
7, 9.3
9.3, 9.3
9.3, 9.3
9.3, 9.3
9.3, 9.3
12, 12
12, 12
10, 11
8, 8
10, 11
10, 11
13, 13
13, 13
32.2, 32.2
N/A
29, 29
28, 29
29, 29
29, 29
28, 29
28, 29
10, 10
10, 10
10, 12
8.2, 12
10, 12
10, 12
8, 12
8, 12
8, 8
8, 8
8, 8
8, 8
8, 8
8, 8
9, 9
9, 9
12, 13
12, 13
12, 12
12, 13
12, 12
12, 12
10, 11
10, 11
12, 12
12, 12
11, 12
11, 12
11, 12
11, 12
10, 11
10, 11
a
Our data.
American Type Culture Collection.19
c
The Health Protection Agency Culture Collections.21
d
The Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures, online STR matching analysis, http://www.dsmz.de/services/
services-human-and-animal-cell-lines/online-str-analysis.html.
b
Reproducibility of FLA Using a Microfluidics-based
Electrophoresis System
In order to evaluate the reproducibility of this alternative electrophoresis platform in the application of STR FLA, 3 independent
experiments were carried out with multiplex PCR amplification
and fragment separation/sizing for the 10 cell lines in the DNA profiling study, using the same DNA sample of each cell line. Except for
D5S818 in UP-016 and D21S11 in IN699 (shaded cells in Table 4),
98% (294/300) of the STR markers analyzed amongst 3 experiments showed a difference of +1 –3 bp in their sizes, indicating
an excellent reproducibility of the Bioanalyzer in FLA (Table 4).
Interestingly, fragment sizes revealed in experiment 3 were slightly
larger in general for the majority of samples compared with those
in experiments 1 and 2 (Table 4), caused by slightly variable electrophoresis migration rates in those assays; however, due to the same
trend of sizing differences demonstrated by all STR markers
(instead of only 1 or 2 of them), the whole STR profile was maintained despite a slight shifting of the profile between different
experiments. Hence, all cell lines showed highly identical DNA
Neuro-Oncology
profiles from 3 independent experiments, as represented by
IN699 and SNB-19 (Fig. 2A and B).
Consistent DNA Profiling of Cell Line Over Multiple
Subcultures
Cross-contamination in cell culture invalidates research results and
compromises the comparison between laboratories. To further validate the application of the microfluidic system in cell line DNA profiling and to detect possible cross-contamination during long-term
cell culture, the same STR-based cell identity test was carried out
with 5 genomic DNA samples collected from each of the 2 brain
tumor cell lines, UP-029 and SEBTA-003, each with a 5-passage
interval. As the data indicated, each cell line showed consistent
STR profiles throughout 5 different passage numbers with +1 –
3 bp difference in sizing amongst 10 markers (Table 5A and B), suggesting that no cross-contamination occurred during the in vitro
culture period and each cell line had maintained its unique DNA fingerprint. The overlaid DNA profiles of 5 different passages further
5 of 9
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
Cell ID
Cell ID
Amelogenin (bp)
D5S818 (bp)
vWA (bp)
TH01 (bp)
D13S317 (bp)
Exp 1
Exp 2
Exp 3
Exp 1
Exp 2
Exp 3
Exp 1
Exp 2
Exp 3
Exp 1
Exp 2
Exp 3
Exp 1
Exp 2
Exp 3
CC2565
IN699
NCI-H1299
SNB-19
TE-671
UP-016
UP-024
UP-019
UP-029
UP-032
Negative
125;128
125;128
125;125
125;129
124;124
124;127
125;128
124;124
125;125
124;127
–a
124;127
125;128
124;124
125;128
124;124
124;126
124;127
124;124
125;125
124;127
–
125;129
126;129
125;125
126;130
125;125
124;128
125;128
125;125
125;125
124;128
–
139;139
148;148
143;143
155;158
150;154
137;150
159;163
159;159
146;146
155;163
–
138;138
148;148
143;143
155;159
150;154
138;150
158;162
159;159
147;147
155;162
–
139;139
148;148
144;144
155;160
151;155
145;151
160;163
160;160
148;148
155;164
–
151;151
159;159
155;165
165;173
173;173
163;167
177;177
168;168
156;169
168;181
–
150;150
159;159
154;165
165;174
173;173
163;167
176;176
168;168
156;169
168;182
–
151;151
160;160
155;166
166;174
174;174
164;168
178;178
169;169
156;169
169;182
–
170;177
178;188
174;190
190;190
189;189
187;187
204;204
188;188
175;190
194;194
–
169;177
178;188
173;190
191;191
190;190
189;189
203;203
189;189
176;190
204;204
–
170;178
178;189
174;191
191;191
191;191
189;189
206;206
190;190
176;190
205;205
–
186;203
206;211
214;214
206;211
219;219
208;221
214;214
205;214
206;214
213;213
–
188;202
206;210
213;213
206;211
218;218
208;221
213;213
205;214
205;214
213;213
–
188;203
207;210
215;215
207;212
220;220
210;222
215;215
206;215
206;215
214;214
–
Cell ID
D21S11 (bp)
CC2565
IN699
NCI-H1299
SNB-19
TE-671
UP-016
UP-024
UP-019
UP-029
UP-032
Negative
a
D7S820 (bp)
TPOX (bp)
D16S539 (bp)
CSF1PO (bp)
Exp 1
Exp 2
Exp 3
Exp 1
Exp 2
Exp 3
Exp 1
Exp 2
Exp 3
Exp 1
Exp 2
Exp 3
Exp 1
Exp 2
Exp 3
215;215
238;238
247;247
242;242
231;231
236;241
239;239
233;233
238;246
237;237
–
215;215
234;234
246;246
243;243
230;230
237;240
237;237
233;233
238;246
237;237
–
216;216
239;239
247;247
243;243
232;232
237;241
239;239
234;234
239;247
239;239
–
247;255
243;247
258;258
247;255
239;254
249;253
246;254
241;241
255;256
245;245
–
247;255
243;246
257;257
247;255
238;254
249;253
245;252
242;242
255;258
245;245
–
248;256
243;248
258;258
248;256
240;256
251;254
247;255
243;243
256;259
247;247
–
282;285
275;275
276;276
275;275
278;278
272;272
286;286
287;287
274;274
273;273
–
282;285
276;276
275;275
276;276
278;278
273;273
285;285
289;289
275;275
273;273
–
282;286
276;276
276;276
276;276
279;279
274;274
286;286
289;289
275;275
274;274
–
294;296
293;305
301;305
300;300
293;296
287;298
300;304
296;296
293;295
299;302
–
294;296
293;305
300;304
301;301
293;295
287;298
299;302
296;296
293;296
299;303
–
294;297
294;306
301;305
302;302
294;297
288;300
300;304
297;297
294;296
301;304
–
358;358
351;359
364;364
362;362
354;361
360;363
355;363
345;357
354;354
356;356
–
358;358
352;360
363;363
363;363
355;362
362;364
353;361
346;358
354;354
357;357
–
359;359
352;360
364;364
363;363
356;363
363;366
355;363
346;359
355;355
358;358
–
No DNA track detected.
An et al.: Brain tumor cell line authentication
6 of 9
Table 4. Comparison of StemElite STR marker sizing by Agilent BioAnalyzer from 3 independent experiments
Neuro-Oncology
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
An et al: Brain tumor cell line authentication
Fig. 3. Overlaid DNA profiles of UP-029 (A) and SEBTA-015 (B) from 5
continuous passage numbers, showing the identical profile throughout
different passages.
confirmed the identical STR fingerprints of the cells (Fig. 3A and B).
In accordance with the initial DNA profiling data, the sizing of all 10
markers in the cross-contamination test showed +1 –3 bp difference amongst different passages (Table 5A and B). Therefore, our
data indicate that DNA profiling by microfluidic electrophoresis
could be an effective approach to monitoring cross-contamination
over time and subcultures. Notably, UP-029 was also included in
the first part of this DNA fingerprinting study, where a different
Agilent DNA 1000 Kit was used, and the profile of UP-029 cells
maintained excellent similarity throughout different experiments/kits. These data provide further evidence for the reliability
of this method and the Agilent assay kits in the application of cell
line authentication.
In addition, the StemElite ID System used in this study incorporates a sensitive mouse marker that can be detected after multiplex PCR should the sample contain genomic DNA from mouse;15
Table 5. A. Consistent STR profiles of UP-029 cells from 5 continuous passages
Passage No. Amelogenin (bp) D5S818 (bp) vWA (bp) TH01 (bp) D13S317 (bp) D21S11 (bp) D7S820 (bp) TPOX (bp) D16S539 (bp) CSF1PO (bp)
10
126;126
148;148
159;171 174;190 208;216
15
125;125
147;147
157;170 175;190 206;215
20
125;125
146;146
156;169 174;189 206;215
25
126;126
147;147
157;170 175;189 206;215
30
126;126
148;148
157;170 175;190 207;216
B. Consistent STR profiles of SEBTA-003 cells from 5 continuous passages
8
126;129
157;157
171;171 176;180 212;221
13
127;129
157;157
171;171 177;180 212;221
18
126;129
157;157
170;170 176;179 212;220
23
126;129
157;157
171;171 176;180 212;220
28
125;128
156;156
170;170 176;179 211;219
Neuro-Oncology
241;248
239;247
238;246
239;246
240;248
257;257
256;256
255;255
255;255
256;256
276;276
275;275
274;274
275;275
276;276
296;298
294;296
293;296
294;296
295;297
355;355
354;354
353;353
354;354
355;355
237;241
237;241
236;241
236;241
235;240
248;256
248;256
248;255
248;256
247;255
276;287
276;287
276;287
276;287
275;286
290;307
291;307
290;307
290;306
289;306
356;359
356;359
356;359
357;359
355;358
7 of 9
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
Fig. 2. Overlaid DNA profiles of IN699 (A) and SNB-19 (B) from 3
independent analyses by the microfluidic system. All cell lines analyzed in
the 3 independent experiments showed reproducible STR profiles, in
particular between experiment (Exp) 1 and Exp 2, as represented by
IN699 and SNB-19.
An et al.: Brain tumor cell line authentication
and the data suggest that no mouse contamination was detected
in any cell lines analyzed in the present study (data not show).
Discussion
8 of 9
Neuro-Oncology
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
The present study demonstrated a new application of the
microfluidics-based electrophoresis method in STR-based DNA profiling of human cell lines, as shown here with brain tumor and brain
tissue–related cell lines. In this study, the Agilent 2100 Bioanalyzer
was used to analyze the STR fragments and was compared with the
standard capillary electrophoresis method. Since its launch in 1999,
the Bioanalyzer has been utilized for quality checks and quantification of DNA and RNA samples before downstream applications such
as sequencing and microarray analyses.11,25 – 28 Due to its capacity
to separate multiple DNA fragments within the same sample, it has
been utilized in STR-based DNA genotyping of forensic samples.13
The Agilent 2100 Bioanalyzer offers a broad range of prevalidated
analysis kits, including the DNA 1000 chip that was used in this
study. This DNA chip provides 5–25 bp sizing resolution for fragments of 100–500 bp.17 The size range of the STR markers in the
StemElite ID System is between 90 and 400 bp;15 therefore it had
been speculated that those amplified alleles would be separated
on the DNA 1000 chip. Notably, the StemElite ID System is designed
specifically for FLA by the Applied Biosystems capillary electrophoresis instruments, such as the ABI 3130 and 3130xl Genetic Analyzer.15 Here our data demonstrate that this cell identification kit is
valid for use with a microfluidic chip system, the Agilent 2100 Bioanalyzer. The STR profiles obtained from both FLA methods were
highly comparable in this study, despite some discrepancies in the
heterozygosity of several markers (Table 1; shaded cells). This
could have been caused by unbalanced PCR amplification of
those markers; that is, one allele was preferentially amplified over
the other, and therefore the Bioanalyzer failed to detect the other
allele due to low sample concentration, whereas the capillary electrophoresis system was able to capture the fluorescent signal and
detect the labeled fragment. The multiplex PCR in this study was
performed according to the manufacturer’s protocol for the StemElite ID System,15 but in future analyses, the amplification parameters could be modified in order to enhance balanced
amplification of the markers and subsequently improve the resolution of this novel method in detecting heterozygous alleles.
Notably, only 8% (8/100) of the total markers analyzed showed a
discrepancy in heterozygosity between the new and conventional
methods, and this had not affected the unique profile of each cell
line studied (Table 1).
In summary, the microfluidic system produced highly accurate
and reproducible DNA profiling data from 10 different cell lines
demonstrated in the present study, suggesting a new approach
to cell line authentication using this platform. Furthermore, the
comparison analysis featured by the Bioanalyzer 2100 Expert software allows a quick fingerprint check for different DNA samples
from the same cell line by overlaying DNA profiles from either independent or the same DNA chip assays, as demonstrated in Figs 2
and 3. This analysis can also be performed for any 2 cell lines in
order to compare and confirm their different profiles, as illustrated
in Fig. 1C for TE-671 and UP-019, showing the distinct profile of
each cell line. This could provide a reliable and cost-efficient downstream method for an STR-based cell line identity check in a cell
culture laboratory.
It has been fully recognized that routine identity check should
be conducted for human cell line cultures, and recently the importance of cell line verification prior to scientific publication has been
reiterated by Anja Torsvik and colleagues,29 as the authors discovered that the previously reported spontaneous transformation of
human mesenchymal stem cells was in fact caused by cell line
cross-contamination. Their report also sheds new light on the
urgent need fora cell validation test of primary cell cultures for therapeutic purposes.29 In the present study, the microfluidics-based
method was employed to monitor possible cross-contamination
over a long-term culture period, and excellent accuracy of this
novel method was illustrated by the identical DNA profiles obtained
from different passages of the same cell line (Table 5 and Fig. 3), indicating the potential of this method as a more effective alternative
for a cross-contamination check. However, further studies need to
be carried out to evaluate and compare the sensitivity and specificity
of both FLA methods in terms of detecting cross-contamination,
using carefully designed mixed cell cultures to represent the most
common contamination scenarios.
Recently new standards for cell line authentication using STR
fingerprinting have been proposed.1 – 5,30 It is critical that an internationally standardized STR panel be applied in the cell line identity
test. The StemElite ID System employed in this study comprises 9
STR markers, which are in accordance with the markers utilized
by ATCC and DSMZ, allowing comparison of the fingerprints of our
NCI-H1299, SNB-19, and TE-671 cells with those in the larger databases. Notably, a recent study by Pierre Bady and colleagues31 suggests that the commonly used similarity score of 0.8 is not
sufficiently stringent to differentiate the origin of a cell line based
on its DNA fingerprint consisting of only 9 STR loci, and it was proposed that either the number of STR markers measured should
be expanded or additional cell line characteristics be included,
such as specific mutations. The focus of this current study was to
evaluate an alternative method for the standard FLA by capillary
electrophoresis, which is time-consuming and costly for most laboratories. Both methods, however, are adaptable for expansion
of STR markers. The DNA fingerprints of 3 commercially available
cell lines recruited in the present study—NCI-H1299, SNB-19 and
TE-671—demonstrated 100% similarity in comparison with the
9-marker fingerprint database of ATCC and DSMZ, confirming the
origins of those cell lines held in our laboratories. The DNA profile
of CC2565, another commercially available cell line analyzed in
this study, was compared with the DSMZ database with no matching profile revealed (data not shown), indicating that the STR profile
of CC2565 is not included in this database. Each of the 6 “in-house”
cell lines presented in this study showed a unique DNA profile, suggesting that the 9-marker approach was sufficient to identify and
differentiate those biopsy-derived cultures.
It is likely that there will be a need in the future to expand the
panel of STR markers with an increasing number of “in-house”
cell lines to be tested, and this method will accommodate this
need. We have shown that this microfluidics-based electrophoresis
platform could provide a more straightforward and cost-effective
method for an STR-based cell line authentication test in our
hands, compared with the standard capillary electrophoresis for
DNA FLA. It is our hope that this novel application will promote
an increase in the routine cell line validation test not only by cell
bank repositories and distributors, but also by the research community. In addition, this method could be useful in a broader
range of applications, such as testing human cancer cells directly
An et al: Brain tumor cell line authentication
passaged and maintained in vivo using mouse xenograph models
and validating cells used in clinical cell therapies. It is also our intention to apply this method in our future studies to address several
key research questions, in particular with regard to monitoring
genetic instability caused by in vitro culture, assessing crosscontamination susceptibility levels in different cell lines, and developing personalized medicine.
14. Formosa R, Ravi H, Happe S, et al. DNA-based fish species identification
protocol. J Vis Exp. 2010;38:e1871.
TM
15. StemElite ID System, Promega Corporation, Technical Manual,
Publication Number TM307, 2008.
16. Lorenzi PL, Reinhold WC, Varma S, et al. DNA fingerprinting of the
NCI-60 cell line panel. Mol Cancer Ther. 2009;8:713 –724.
This work was supported by the brain tumor charities Brain Tumour Research and Headcase.
18. Fragment Length Analysis, Eurofins MWG Operson, Service Overview
Flyer, Version 1 06/2011.
Conflict of interest statement. None declared.
19. NCI-H1299 product information. American Type Culture Collection.
Available at http://www.atcc.org/Products/All/CRL-5803.aspx?Geo_
country=us#A7931A04156C4C7FA40828AEF707302F. Accessed
November 28, 2013.
References
1.
Capes-Davis A, Theodosopoulos G, Atkin I, et al. Check your cultures! A
list of cross-contaminated or misidentified cell lines. Int J Cancer. 2010;
127:1– 8.
2.
Barallon R, Bauer SR, Butler J, et al. Recommendation of short tandem
repeat profiling for authenticating human cell lines, stem cells, and
tissues. In vitro Cell Dev Biol Anim. 2010;46:727 –732.
3.
Nims RW, Sykes G, Cottrill K, Ikonomi P, Elmore E. Short tandem repeat
profiling: part of an overall strategy for reducing the frequency of cell
misidentification. In vitro Cell Dev Biol Anim. 2010;46:811 –819.
4.
Butler JM. Genetics and genomics of core short tandem repeat loci
used in human identity testing. J Forensic Sci. 2006;51:253– 265.
5.
Masters JR, Thomson JA, Daly-Burns B, et al. Short tandem repeat
profiling provides an international reference standard for human cell
lines. Proc Natl Acad Sci U S A. 2001;98:8012 –8017.
6.
Panaro NJ, Yuen PK, Sakazume T, Fortina P, Kricka LJ, Wilding P.
Evaluation of DNA fragment sizing and quantification by the Agilent
2100 Bioanalyzer. Clin Chem. 2000;46:1851 –1853.
7.
Isaksson HS, Nilsson TK. Preanalytical aspects of quantitative TaqMan
real-time RT-PCR: applications for TF and VEGF mRNA quantification.
Clin Biochem. 2006;39:373 –377.
8.
Kuschel M, Neumann T, Barthmaier P, Kratzmeier M. Use of Lab-ona-Chip technology for protein sizing and quantitation. J Biomol Tech.
2002;13:172 –178.
9.
Kataoka M, Fukura Y, Shinohara Y, Baba Y. Analysis of mitochondrial
membrane potential in the cells by microchip flow cytometry.
Electrophoresis. 2005;26:3025–3031.
10. Miller CL, Diglisic S, Leister F, Webster M, Yolken RH. Evaluating RNA
status for RT-PCR in extracts of postmortem human brain tissue.
Biotechniques. 2004;36:628– 633.
20. Misidentified Cell Lines, American Type Culture Collection. Available at
http://www.atcc.org/Products/Cells_and_Microorganisms/Cell_Lines/
Misidentified_Cell_Lines.aspx?Geo_country=us. Accessed November
28, 2013.
21. U-373 MG datasheet, the Health Protection Agency Culture Collections.
Available at http://www.phe-culturecollections.org.uk/products/
celllines/generalcell/detail.jsp?RefId=08061901&collection=ecacc_gc.
Accessed November 28, 2013.
22. SNB-19 datasheet, the DSMZ—German Collection of Microorganisms
and Cell Cultures. Available at http://www.dsmz.de/catalogues/
details/culture/ACC-325.html?Tx_dsmzresources_pi5%5BreturnPid%
5D=192. Accessed November 28, 2013.
23. Ikediobi ON, Davies H, Bignell G, et al. Mutation analysis of 24 known
cancer genes in the NCI-60 cell line set. Mol Cancer Ther. 2006;5:
2606 –2612.
24. Roschke AV, Tonon G, Gehlhaus KS, et al. Karyotypic complexity of the
NCI-60 drug-screening panel. Cancer Res. 2003;63:8634 –8647.
25. Thaitrong N, Kim H, Renzi RF, Bartsch MS, Meagher RJ, Patel KD.
Quality control of next-generation sequencing library through an
integrative digital microfluidic platform. Electrophoresis. 2012;33:
3506 –3513.
26. Wilkes TM, Devonshire AS, Ellison SL, Foy CA. Evaluation of a novel
approach for the measurement of RNA quality. BMC Res Notes.
2010;3:89.
27. Molas ML, Kiss JZ. The effect of column purification on cDNA indirect
labelling for microarrays. Plant Methods. 2007;3:9.
28. Colak T, Cine N, Bamac B, et al. Microarray-based gene expression
analysis of an animal model for closed head injury. Injury. 2012;43:
1264 –1270.
11. Grissom SF, Lobenhofer EK, Tucker CJ. A qualitative assessment of
direct-labeled cDNA products prior to microarray analysis. BMC
Genomics. 2005;6:36.
29. Torsvik A, Røsland GV, Svendsen A, et al. Spontaneous malignant
transformation of human mesenchymal stem cells reflects crosscontamination: putting the research field on track—letter. Cancer
Res. 2010;70:6393 –6396.
12. Becker C, Hammerle-Fickinger A, Riedmaier I, Pfaffl MW. mRNA and
microRNA quality control for RT-qPCR analysis. Methods. 2010;50:
237– 243.
30. American Type Culture Collection Standards Development
Organization, ASN-0002 Workshop. Cell line misidentification: the
beginning of the end. Nat Rev Cancer. 2010;10:441 – 448.
13. Aboud MJ, Gassmann M, McCord BR. The development of mini
pentameric STR loci for rapid analysis of forensic DNA samples on a
microfluidic system. Electrophoresis. 2010;31:2672 –2679.
31. Bady P, Diserens AC, Castella V, et al. DNA fingerprinting of glioma cell
lines and considerations on similarity measurements. Neuro Oncol.
2012;14:701 –711.
Neuro-Oncology
9 of 9
Downloaded from http://neuro-oncology.oxfordjournals.org/ at Pennsylvania State University on September 12, 2016
Funding
17. Agilent DNA 1000 Kit, Agilent Technologies, Inc. Manual, Publication
Number G2938–90014, edition 08/2006. Available at http://www.
chem.agilent.com/library/usermanuals/Public/G2938-90014_KitGuide
DNA1000Assay_ebook.pdf. Accessed November 28, 2013.