RESEARCH ARTICLE

Copyright 2011 American Scientific Publishers Advanced Science Letters

All rights reserved Vol. 4, 13, 2012
Printed in the United States of America

EFFECT OF DIMETHYL SULFOXIDE ON

PERIPHERAL BLOOD-ORIGINATING
MESENCHYMAL PROGENITOR CELLS
VIABILITY
Nadya Muthia Risky 1, Widya Wira Putri 1, Nungki Pramita Sari 1, Rufaida Mudrika 1, Rizka Kurnia Gemilang 1,Restu
Samsul Hadi 2, Yurika Sandra, 3, Indra Kusuma,4

1
Faculty of Medicine, Universitas Yarsi, Jakarta, Indonesia
2
Department of Anatomy, Faculty of Medicine, Universitas Yarsi, Jakarta, Indonesia
3
Department ofBiochemistry, Faculty of Medicine, Universitas Yarsi, Jakarta, Indonesia
4
Department ofPhysiology, Faculty of Medicine, Universitas Yarsi, Jakarta, Indonesia

(Abstract) Background: Mesenchymal Progenitor Cells Peripheral Blood (MPC-PB) are multipotent cells which acts as a
precursor of connective tissues and has the potential for the regeneration of joints cartilage. This study focuses on MPCs
originating from peripheral blood due to its less invasive nature for obtaining them compared to bone marrow aspiration.
Me2SO (dimethyl sulfoxide) were used in cryopreservation and culture processes for preventing cell damage from low
temparature. However, high concentrations of Me2SO may decrease cell viability. This study is aimed to determine the effect
of Me2SO towards peripheral blood-originating MPCs. Methods: MPC-PB were placed in a 96-well plate with a density of
2600 cells per well. The experiments were divided to 9 treatments; control, Me2SO 0,5%, 1%, 1,5%, 2%, 2,5%, 3%, 3,5%,
and 4%. The study was held for 15 days, and cell count were calculated using WST-1 reagent and microplate reader. Results:
We found a cell viability difference between each treatment. MPCs exposed to Me2SO for 9 days with a Me2SO
concentration of 0,5% dan 1% had a reversible change, while MPCs exposed to Me2SO above 1% had an irreversible
change.
Conclusion: The results of this study showed that certain Me2SO levels affect the viability of peripheral blood-originating in
vitro-cultured MPCs.

KEYWORDS: MPC-PB, Me2SO, viability

1 Adv. Sci. Lett. 4, 13, 2012 1936-6612/2012/4/3398/005 doi:10.1166/asl.2012.2049

Adv. Sci. Lett. 4, 13, 2012 RESEARCH ARTICLE

1. INTRODUCTION Me2SO 1% does not inhibits MPC-PB proliferation.

Stem cellis a cell equipped with self-renewal and plasticity

that enables differentiation and multiplication to various kind of
cells. Mesenchymal progenitor cells (MPC) is known for its
potential in joints cartilage regeneration.1To this day, the most
common source for Mesenchymal Stem Cells is bone marrow
and adipose tissue. Peripheral blood have also been proposed as
a less-invasive alternative to bone marrow.2 Peripheral blood
can induce cartilages cell migration, increase the number of
chondrocyte migration3 and stimulate chondrocyte hypertrophy
in endochondral ossification.4 This serves as the base for
several therapeutic modalities such as the one for osteoarthritic
joint,3 chondrodysplasia and some other bone pathologies.4
Dimethyl Sulfoxide(Me2SO) is a solvent commonly used
in toxicology, pharmaceutical investigation, and cells Figure 1. Me2SO effects on MPCs proliferation under control-
freezing.5 Me2SO is hydrophilic, stable, and has good treatment, Me2SO 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%
viscosity. That being said, Mes2SO harms cells viability at a on day 3rd, 6th, 13th (Me2SO OFF), 15th (Me2SO OFF)
concentration of > 15%,6 and therefore, we believe a study Aside from that, the highest absorbance difference was
about Me2SOs impact to MPC is of utmost importance in the observed on 3rd, 6th, 9th, 13th, and 15th day were seen in figure 2
development of clinical cell-based therapy. This study is which shows that highest viability on 3 rd and 6th day is observed
expected to be able to represent Indonesias contribution in in MPC with Me2SO 1%. Highest 9th-day-viability is seen in
medical therapy further advancement. MPC with Me2SO 0.5%. After Me2SO wash away on 9 th day
and 13th and 15th-day-check, the best viability is seen in MPC
2. METHODS with Me2SO 1%. On the 9th day of MPC incubation with
Me2SO 1%, there was an insignificant decrease of viability
This study used Me2SO of different concentrations for
(P>0.05) were seen in figure 3.
each of 9 groups: 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%
and a control. This study lasted for 5 months, starting from
April 2016 to August 2016 at YARSI Universitys laboratory.
Subjects were in-vitro Mesenchymal Progenitor Cell obtained
from YARSI Universitys biorepository.
MPC-PB was planted on plate 96 containing DMEM
(Dulbeccos Modified Eagles Medium) (Gibco), FBS (Fetal
Bovine Serum) (Gibco) 10%, penicillin 100 unit/mL, and
streptomycin (Sigma)100 g/mL, with a density of 2600 cells
per wells. Me2SO (Sigma) at the aforementioned
concentrations was then added to all but control group. They
were incubated for 15 days under temperature of 370 C and 5%
CO2. The medium were replaced every 2-3 times per week. On
the third, sixth, ninth, thirteenth (Me2SO OFF) and fifteenth
(Me2SO OFF), WST-1 (Roche) reagent was added and then the
cells were counted using microplate reader.
Data of absorbance at 450 nm wavelength were collected Figure 2. MPCs proliferation chart under control treatment,
and quantitatively analyzed using Microsoft Excel.7 The Me2SO 0.5%, 15, 1.5%, 2%, 2.5%, 3%, 3.5%, and 4% on 3 rd,
statistical test used was Student T-Test. 6th, 13th, (Me2SO OFF), 15th day (Me2SO OFF)

3. RESULT
Differences in viability and proliferation were observed
between MPCs exposed to various Me2SO concentrations.
From the microplate reader, after 15 days, were seen in figure
1which shows that Me2SO of more than 1% exposure inhibits
MPC-PB proliferation on 3rd, 6th, and 9th. Otherwise, Me2SO
0,5% and 1% still proliferates on 3 rd, 6th, and 9th.Similar result
was found when Me2SO was washed away on 13 th and 15thday ,
which shows that MPC after exposure Me2SO of>1% inhibits
MPC-PB proliferation, while MPC with Me2SO 0,5 % and
1%does not inhibits MPC-PB proliferation. This shows that

*Authors to whom correspondence should be addressed

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RESEARCH ARTICLE
Figure 3. Comparison of MPC proliferation in Me2SO 1% on
6th and 9th day (*P>0,05)
On the 9th day of Me2SO exposure, there is a significant
differences between all treatment except Me2SO 1.5% were
seen in figure 4.MPC with Me2SO 0.5 and 1% have higher and
significantly different absorbance compared with control
(P<0.05). MPC with Me2SO 1.5% has lower absorbance and is
not significantly different with control (P>0.05). MPC with
Me2SO 2% or more has significantly lower absorbance
compared with control (P<0.05)
Figure 4. Comparison of MPCs proliferation between control,
Me2SO 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% on 9 th day
(*P>0.05)
Microscopic pictures MPC-PB on 15th day(Me2SO OFF)
were seen in figure 5which shows that there were no
multinuclear cells seen.
(A) (B)
(C) (D)
Figure 5. MPC on 15th day as seen using Inverted Microscope.
MPC without Me2SO (A). MPC after Me2SO 1% exposure
(B). MPC after Me2SO 1.5% exposure (C). MPC after Me2SO
2% exposure (D)
4. DISCUSSION
Measurement of cell viability is one of the basic
examination in various form of cell cultures which tests healthy
cells within the samples.8In this study, Me2SO of more than
1% exposure inhibits MPC-PB proliferation on 3rd, 6th, and 9th.
This finding agrees with a study by Wang et al1which observed
cytotoxic property of Me2SO 2% in a cell culture.
3 Adv. Sci. Lett. Vol. 4, No. 2, 2011
Adv. Sci. Lett. 4, 13, 2012
Me2SO of 1% does not impact MPC-PBs viability on
3rd, 6th, and 9th day. Similar result was found when Me2SO was
washed away on 13th and 15th day (Figure 1), which shows that
the effect of Me2SO 0.5% and 1% is reversible, while MPC
with Me2SO of > 1% is irreversible. This finding is consistent
with Elisia et al9which stated that Me2SO induce cytotoxicity
on monocyte. There is a huge decline of viability on Me2SO
2%, mild hemolysis on Me2SO 5%, and dramatically
increasing cell death on Me2SO 10%. Me2SO has a narrow
therapeutic window, but at a concentration of 0.5% or lower, it
never seem to exhibit cytotoxic quality.
Highest viability on 3rd and 6th day is observed in MPC
with Me2SO 1%. Highest 9th-day-viability is seen in MPC with
Me2SO 0.5%. After Me2SO wash away on 9th day and 13th and
15th-day-check, the best viability is seen in MPC with Me2SO
1% (Figure 2). On the 9 th day of MPC incubation with Me2SO
1% (Figure 2), there was an insignificant decrease of viability
(P>0.05) (Figure 3).
On the ninth day, MPC with Me2SO 0.5 and 1% have
higher and significantly different absorbance compared with
control (P<0.05). MPC with Me2SO 1.5% has lower
absorbance and is not significantly different with control
(P>0.05). MPC with Me2SO 2% or more has significantly
lower absorbance compared with control (P<0.05) (figure 4).
According to Malinin& Perry,10an increase of Me2SO
concentration is linear with a decrease in absorbance, and thus a
decrease in cell numbers. HeLa cell with Me2SO 4% or more
after 72 hours of incubation has the same absorbance with a
cell-less well, and therefore, it can be concluded that at the
mentioned concentration either cells death occurs or metabolic
activity ceases.
Based on a study by Galvao et al, Me2SO 2% and 4% can
induce cell death because of increased expression of Bax in
mitochondria within 24 hour of exposure. The process involves
inhibition of mitochondria respiration followed by a decline in
oxygen consumption within minutes after exposure.
Cells movement velocity is seen to be slower under higher
concentration of Me2SO. Me2SO 5% inhibits cytokinesis but
cells growth is spared. It even tends to engorge 10 times
bigger. After Me2SO is washed away, giant multinuclear cells
undergo cytoplasmic division to normal-sized mononuclear
cells.11 On 15th day, in this study, a picture of the cell was taken
using inverted microscope, and there were no multinuclear cells
seen. (Figure 5)
Me2SO collapses the mitochondria membrane potential
and in turn causes cytochrome c release from mitochondria and
activation of caspase-9 and -3, but not caspase-8 (caspase
cascade from mitochondria apoptosis pathway, induced by
Me2SO). This leads to apoptotic changes in lymphoma murine
cells (concentration and time dependent effect) and down-
regulation of Bcl-2.12
This study concluded that MPC after 9-days-exposure to
Me2SO is reversible when the concentration is 0.5% and 1%,
and irreversible when it is more than 1%.
ACKNOWLEDGMENTS:
We thank dr. Insan Sosiawan A. Tunru, Ph.D as Dean
Faculty of Medicine Yarsi, dr. Lilian Batubara, M.Kes as Vice
Dean Faculty of Medicine Yarsi and dr. Rika Yuliwulandari as
Head of Yarsi Universitys laboratory
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