Modification of the alkaline comet assay with humanmesenchymal stem cellsRobert Fuchs1,2*, Ingeborg Stelzer1*,{, Christoph M. P. Drees*,{, Christian Rehnolt*,{, Elisabeth Schraml{, AntonSadjak* and Wolfgang Schwinger1

* Institute of Pathophysiology and Immunology, Center of Molecular Medicine, Medical University of Graz, Heinrichstrasse 31A, 8010 Graz, Austria{ Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, Auenbruggerplatz 15, 8036 Graz, Austria{ Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences Vienna, Muthgasse 18, 1190 Vienna, Austria1

Division of Pediatric Hematology/Oncology, Department of Pediatrics and Adolescent Medicine, Medical University of Graz, Auenbruggerplatz 30,8036 Graz, Austria

AbstractMSCs (mesenchymal stem cells) are planned foruse in regenerative medicine to offset age-dependent alterations.

However, MSCs are affected by replicative senescence associated with decreasing proliferation potential, telomere

shortening and DNA damage during in vitro propagation. To monitor in vitro senescence, we have assessed the integrity of

DNA by the alkaline comet assay. For optimization of the comet assay we have enhanced the stability of comet slides in

liquid and minimized the background noise of the method by improving adhesion of agarose gels on the comet slides and

concentrating cells on a defined small area on the slides. The modifications of the slide preparation increase the overall

efficiency and reproducibility of the comet assay and minimize the image capture and storage. DNA damage of human

MSCs during in vitro cultivation increased with time, as assessed by the comet assay, which therefore offers a fast and easy

screening tool in future efforts to minimize replicative senescence of MSCs in vitro.

Keywords: comet assay; DNA damage; human mesenchymal stem cell (MSC); replicative senescence

1. Introduction

The comet assay, originally established by Ostling and Johanson

(1984), is a frequently used assay to detect single- and double-

strand breaks of DNA at the level of single cells (Singh et al.,

1988; Tice et al., 2000). It is suitable for a wide range of applications,

including the screening of genotoxity (Nousis et al., 2005; Mughal

et al., 2010) and DNA damage due to ROS (reactive oxygen species;

Schraml et al., 2009; Cao et al., 2010). Although highly sophisticated

techniques as breakpoint mapping or array-comparative geno-

mic hybridization (Baptista et al., 2008; Pfragner et al., 2009) are

currently available, the comet assay holds its position, at least for

screening purposes, because it is simple, fast and cost efficient.

An important field of application of the comet assay is in aging

research, as the cellular aging process is accompanied by

enhanced generation of ROS (Passos and Von Zglinicki, 2006;

Schraml et al., 2007), genomic instability (Burhans and Weinberger,

2007) and altered regulation of cell death (Zheng et al., 2005; Hinkal

et al., 2009). To overcome aging accompanying pathological

alterations like neurodegenerative diseases, a great deal of

expectation is placed on MSCs (mesenchymal stem cells; Sadan

et al., 2009; Whone and Scolding, 2009). To use MSCs in

regenerative medicine, it is necessary to expand MSCs in vitro

before application, as the numbers of MSCs available from the cord

blood or BM (bone marrow) are limited (Schallmoser et al., 2008;

Reinisch and Strunk, 2009). However, MSCs are subjected to

senescence-associated alterations when expanded in vitro (Fehrer

and Lepperdinger, 2005; Bonab et al., 2006; Wagner et al., 2008,

2010; Schallmoser et al., 2010), which include decreasing prolifera-

tion potential, accumulation of SA-b-gal (senescence-associated b-

galactosidase), telomere shortening, DNA damage and continuous

changes in gene expression (Fehrer and Lepperdinger, 2005; Bonab

et al., 2006; Wagner et al., 2008, 2010; Galderisi et al., 2009;

Schallmoser et al., 2010). To screen for the occurrence of DNA

damage during in vitro cultivation of human MSC, we used the

alkaline comet assay. Since the stability of comet slides and edge

effects are weak points of the comet assay, some simple

modifications were introduced to minimize these sources of error.

2. Materials and methods

2.1. Isolation and cultivation of human MSCs

MSCs were isolated from BM taken from patients with haemo-

poietic disorders or malignancies (Figure 2B, Table I), aspirations

being performed according to treatment protocols and used only

after informed consent of the donor. The study protocol was

approved by the local ethics committee (decision number 21-197

ex 09/10). For enrichment of MSCs, MNCs (mononuclear cells)

were isolated from BM by Ficoll density centrifugation using Ficoll-

Paque PLUS (GE Healthcare Bio-Sciences AB) according to the

manufacturer’s instructions. MNCs were incubated at 37uC in a

humidified atmosphere in MEM-a (Invitrogen/Gibco) supplemented

1 These authors contributed equally to this study.2 To whom correspondence should be addressed (email robert.fuchs@medunigraz.at).Abbreviations: BM, bone marrow; DAPI, 49,6-diamidino-2-phenylindol; FBS, foetal bovine serum; MSC, mesenchymal stem cells; MNC, mononuclear cells; ROS,reactive oxygen species.

Cell Biol. Int. (2012) 36, 113–117 (Printed in Great Britain)

Short Communication

E The Author(s) Journal compilation E 2012 Portland Press Limited Volume 36 (1) N pages 113–117 N doi:10.1042/CBI20110251 N www.cellbiolint.org 113

with 10% FBS (foetal bovine serum) at 56104 cells/cm2 in tissue

culture flasks. Adherent cells were maintained in culture and

passaged weekly. After three passages, the purity of MSC

cultures was examined using a panel of mononuclear antibodies

by means of multi-parameter flowcytometry with a FACS-

Caliburj flow cytometer (Becton Dickinson). Antibodies were

purchased from BD Biosciences, Immunotech or Dako. MSCs

were lineage negative for CD2, 4, 7, 8, 13, 14, 15, 19, 22, 30, 45,

56 and HLA-DR. Starting with the third passage, a sample of

cells was taken for the comet assay during the passaging

process. MSCs were cultivated as long as at least 56106

cells could be harvested for continuation of the culture. The

osteosarcoma cell line U2-OS (A.T.C.C. Number: HTB-96TM)

was used to optimize the comet assay protocol. U2-OS cells

were maintained in DMEM (Dulbecco’s modified Eagle’s

medium; Invitrogen/Gibco) supplemented with 10% FBS,

penicillin/streptomycin (100 units/ml respectively 100 mg/ml)

and glutamine (2 mM) at standard cell culture conditions. Cell

numbers were assessed by a haemacytometer or an automatic

CASYj cell counter (Innovatis).

2.2. Alkaline comet assay

Alkaline comet assay was done as by Schraml et al. (2009), based

on a protocol developed by Singh et al. (1988) with 2 modifications.

To improve adhesion of agarose gels on the comet slides, the

pretreatment procedures of slides as described for the first time by

Klaude et al. (1996) was changed. Stable slides are the essential

prerequisite for successful comet assay scoring (Tice et al.,

2000). Standard microscopy slides were dipped into melted 1%

normal agarose – the agarose on the underside of the slides

was wiped off – and placed immediately on a 100uC hot plate until

the first agarose layer on the surface of the slides was fully

dehydrated (Figure 1A). After pretreatment, slides were covered

with 3 more layers of agarose (Klaude et al., 1996; Tice et al., 2000).

The second modification allowed concentration of cells on a

defined small area on the slides; to do this, 500 ml of 1% normal

melting agarose (5second layer) was poured on pretreated

slides and covered by a combination of a 24660 mm and a

18618 mm coverslip (Figure 1B) that had been stuck together

with a drop of melted agarose. After setting in the refrigerator for

5 min, a 18618 mm chamber was formed on the slides by

detaching both coverslips (Figure 1B). Then 80 ml of a cell

suspension (10 ml cell suspension containing 56104 cells mixed

with 70 ml 1% low melting agarose (5third layer) was put into the

chambers and covered with 18618 mm coverslips (Figure 1C).

After setting for 10 min in the refrigerator and covering the slides

with a layer of 1% normal melting agarose (5fourth layer,

Figure 1D), cells were lysed in the dark for 1 h in lysis buffer

(pH 10) containing 2.5 M NaCl, 100 mM EDTA, 10 mM Tris Base

and 1% Triton X-100. After 20 min incubation in electrophore-

sis buffer (300 mM NaOH and 1 mM EDTA, pH.13) for DNA

unwinding, the voltage was set at 20 V for 30 min. For elec-

trophoresis a special, light protected electrophoresis chamber

optimized for the comet assay (Cleaver Scientific) was used. For

neutralization, slides were incubated in neutralization buffer

containing 0.4 M Tris, pH 7.4, for 10 min and stained with the

DNA-dyes, ethidium bromide (Sigma) or DAPI (49,6-diamidino-

2-phenylindol; Sigma). After washing in distilled water, slides

were covered with coverslips and analysed by a fluorescence

microscope. For data evaluation, visual scoring of ethidium

bromide-stained comets was performed (Dusinska and Collins,

2008). Comets were graded into 4 classes (C05cell without

comet tail, and C1–C4 depending on tail intensity; see Figure 4A).

One hundred comets of each cell sample were selected ran-

domly, and the score was calculated according to the following

formula: score [arbitrary units]5nC1+nC262+nC363+nC464,

resulting in values between 0 and 400. A minimum of two comet

slides for each sample were prepared. The scoring procedure was

repeated twice; in total, therefore, 300 cells per passage on two

different slides of each sample were included in the statistical

analysis.

Figure 1 Modified preparation of comet slidesSlides are tipped into melted agarose and placed on a heating plate until the agarose is fully dehydrated (A). On a pretreated comet slide, melted agaroseis filled with a syringe and covered with a combination of two coverslips (B). After setting of the agarose, a chamber is formed on the slide (B). The cellsuspension mixed with low melting agarose is added into this chamber and capped with a coverslip (C). After setting, a final coat of low melting agarose isfilled on the slide and covered with a coverslip again (D).

Comet assay with human mesenchymal stem cells

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2.3. Statistics

For statistical analysis, SigmaPlot 11.0 was used. Differences

between two data records were evaluated by Student’s t test.

P#0.05 were considered as significant.

3. Results and discussion

3.1. The proliferative potential of human MSCs isdecreasing with duration of in vitro cultivation

After three passages, no haemopoietic cells, as assessed by

flow cytometric examination, were detectable in the cultures,

confirming enrichment of MSCs (data not shown). MSC cultures

could be maintained at a median of 10 weeks (n55, range: 8–10

weeks) until cells reached the Hayflick limit (Schallmoser et al.,

2010) and ceased proliferation (Figure 2). The proliferation

potential of MSCs decreased in a time-dependent manner, with

typical sigmoidal progression (Figure 2C). MSC cultures

decreased in density, were enlarged and lost their typical

spindle-shaped morphology the longer they were cultured

(Figure 2A), as previously described (Fehrer and Lepperdinger,

2005; Bonab et al., 2006; Wagner et al., 2008, 2010). Perez-Simon

et al. (2009) showed that MSCs from patients with IPT (immune

thrombocytopenic purpura) are functionally abnormal. This dys-

function is reflected in impaired proliferative capacity and weaker

inhibition of T-cell proliferation. We did not find similar changes in

Figure 2 Proliferative potential of human MSCs decreases with duration of in vitro cultivation(A) Representative culture of MSCs at the time of passage 3 and passage 8 is shown. Original magnification 6200. (B) Table I: MSCs obtained frompatients suffering from haemopoietic disorders or malignancies could be maintained in culture for a limited time until reaching a state of replicativesenescence. BM, bone marrow; ITP, idiopathic thrombocytopenic purpura; SSA/MDS, severe aplastic anaemia and myelodysplasia. (C) The median cellnumber¡the mean deviation from the median of 5 cultures of MSCs during cultivation up to passage 9 is shown. The data represent the mathematicallycalculated total cell mass of MSCs in culture at the time of passaging. The numbers above the line indicate the fold increase in proliferation of MSCsrelated to the last passage of the culture.

Figure 3 How to stop comets from falling into water(A) In preliminary experiments comet slides without (w/o) pre-coating were used. The numbers of slides with detaching gels (5failure) and stable slides ofthese experiments are shown in comparison with experiments using pretreated slides. (B) The introduction of a cell chamber on comet slides protects cellsagainst the induction of additional DNA damage during processing of the cells. Untreated U2-OS cells were investigated by means of comet assay eitherusing slides with or without cell chamber. The mean comet score values of at minimum 2 slides for each condition were obtained by optical scoring (n57).

Cell Biol. Int. (2012) 36, 113–117

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MSCs derived from patients with haemopoietic disorders or

different malignancies.

3.2. Cellular senescence of human MSCs isaccompanied by a time-dependent increase ofthe comet score

After each passage of the MSCs, a sample of cells was taken and

analysed using the comet assay. Using the described modifica-

tions of the comet protocol, the performance of the test was

significantly improved. It was previously necessary to prepare a

couple of slides of a sample under investigation, because gels

had a tendency to detach from slides in liquid, making it both a

common and a serious problem in comet assay (Thomas et al.,

1998; Olive and Banath, 2006). In 180 slide preparations for

preliminary experiments, only 64 (35%) could be examined

microscopically (Figure 3). Pre-coating slides enhanced stability

and prevented detachment of the gels. In experimental work,

100% of a total of 256 slides could be examined (Figure 3A).

Thus pre-coating protocol minimizes the number of slides

needed per experiment, thus accelerating the overall work flow

and saving precious samples. The small ‘cell chamber’ within the

agarose gel on the comet slides improved the efficiency as well

as the resolution of the method. Using ‘standard comet slides’,

edge effects are a common problem (Olive, 2002; Collins, 2004;

Azqueta et al., 2009). Therefore, comets lying at the edges of

the slides were usually ignored (Olive, 2002; Collins, 2004). The

same applies for air bubbles, which are an interfering factor in

comet slides (Collins, 2004). The introduction of a ‘cell chamber’

on the slide resolved this problem and provided standardized

conditions for all cells under investigation. This conclusion is

supported by experiments assessing the scores of untreated

U2-OS cells processed either on comet slides with or without

cell chamber; cells processed on slides without a chamber

had significantly higher scores than those within a cham-

ber (P50.002; Figure 3B). Therefore, the cell chamber is useful

in the approach to reduce background noise and to get homo-

geneous slides (Tice et al., 2000). Furthermore, comets could

be found on a small and defined area of the slide, which

accelerated the optical scoring procedure. The chamber also

minimizes the need of microscopic photos which are the base

for analysing comets by means of automatic comet scoring

software (Chaubey, 2005). The number and size of the chambers

can also be adapted by using different size coverslips.

The comet score of MSCs during in vitro propagation

increased with the duration of cultivation (Figure 4), being

significantly elevated pronounced at the plateau phase of MSC

growth (i.e. senescence).

In summary, the comet assay is an optimal screening tool to

detect DNA damage as an indicator of senescence in human

MSCs grown in vitro. Our improved pretreatment procedure

ensures stability of comet slides throughout the whole comet

assay protocol. The introduction of the cell chamber avoids edge

effects. These modifications contribute to better overall efficiency

and reproducibility of the comet assay.

Author contribution

Robert Fuchs contributed to the experimental setup, analysed

data and wrote the paper. Ingeborg Stelzer performed the experi-

ments, analysed data and designed the study. Christoph Drees

and Christian Rehnolt performed the comet assay experiments.

Elisabeth Schram contributed to the experimental setup. Anton

Sadjak supervised the study and discussed the results. Wolfgang

Schwinger designed, planned and supervised the study and

contributed to the preparation of the paper.

Figure 4 In vitro cultivation of human MSCs is accompanied by a time-dependent increase in comet score(A) MSCs processed by comet assay and stained with DAPI, viewed by fluorescence microscopy. Original magnification: 6200. Comets were classifiedinto 5 categories by microscopically inspection ranging from C0 (Class0, no DNA damage, no detectable comet tail) up to C4 (high level of DNA damage,almost all DNA in the tail). (B) Statistical analysis of the comet assay with MSCs during cultivation. (I) The comet score value, shown in arbitrary units, of invitro cultured MSCs increases with duration of cultivation. n55. (II) Increase of DNA damage is strongly and significantly (P,0.001) correlated withduration of culture. The medians of the results of 5 independent experiments are shown.

Comet assay with human mesenchymal stem cells

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Acknowledgements

We thank Elvira Kloibhofer, Anita Puregger, Nicole Albrecher and

Nathalie Allard for excellent technical assistance.

Funding

This research received no specific grant from any funding agency

in the public, commercial or not-for-profit sectors.

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Received 2 May 2011/ 20 July 2011; accepted 16 September 2011

Published as Immediate Publication 16 September 2011, doi 10.1042/CBI20110251

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