Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions
Aim. To optimize the cultivation of Wharton jelly-derived mesenchyma stem cells (WJ-MSCs) using physiological oxygen concentrations, and to compare the effect of “hypoxic” gas mixtures, based on nitrogen and argon, on their proliferative activity. Methods. From the first passage, WJ-MSCs were cultiv...
Gespeichert in:
| Datum: | 2015 |
|---|---|
| Hauptverfasser: | , |
| Format: | Artikel |
| Sprache: | English |
| Veröffentlicht: |
Інститут молекулярної біології і генетики НАН України
2015
|
| Schriftenreihe: | Вiopolymers and Cell |
| Schlagworte: | |
| Online Zugang: | http://dspace.nbuv.gov.ua/handle/123456789/152476 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions / N.S. Shuvalova, V.A. Kordium // Вiopolymers and Cell. — 2015. — Т. 31, № 3. — С. 233-239. — Бібліогр.: 37 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
irk-123456789-152476 |
|---|---|
| record_format |
dspace |
| spelling |
irk-123456789-1524762019-06-12T01:26:36Z Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions Shuvalova, N.S. Kordium, V.A. Methods Aim. To optimize the cultivation of Wharton jelly-derived mesenchyma stem cells (WJ-MSCs) using physiological oxygen concentrations, and to compare the effect of “hypoxic” gas mixtures, based on nitrogen and argon, on their proliferative activity. Methods. From the first passage, WJ-MSCs were cultivated during five passages in the nitrogen-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % nitrogen) and argon-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % argon), 7 days before replating. At each passage the final cell number was estimated and the number of population doublings was calculated. Results. The proliferation level of WJ-MSCs, cultured in both gas mixtures with 3 % of O2, was significantly higher compared to that under the regular CO2-incubator conditions. In argon-based mixture, the WJ-MSCs proliferation was higher than in the control but lower than in nitrogen-based mixture. Conclusion. Cultivation of human WJ-MSCs under 3 % O2 had a stimulating effect on the cell proliferation potential. The highest intensity of the cell multiplication was observed in the nitrogen-based mixtures. Мета. Оптимізувати культивування і порівняти вплив газових сумішей зі зниженим вмістом кисню, на основі азоту та аргону, на процеси проліферації в культурах мезенхімальних стовбурових клітин Вартонового студню (МСК-ВС). Методи. Протягом 5 пасажів. МСК-ВС культивували в газових сумішах на основі азоту (кисень – 3 %, вуглекислий газ – 4 %, азот – 93 %) та аргону (кисень – 3 %, вуглекислий газ – 4 %, аргон – 93 %). На кожному пасажі після 7 днів культивування підраховували кількість клітин, і визначали число подвоєнь культури. Результати. Чисельність клітин МСК-ВС, культивованих в сумішах, що містять 3 % кисню, була вищою, ніж в загальноприйнятих умовах СО2-інкубатора. Рівень проліферації в суміші на основі аргону був нижчим, ніж в суміші на основі азоту, при цьому будучи вищим за такий для контрольних груп. Висновки. Культивування в газових сумішах, що містять 3 % кисню, мало стимулюючий вплив на процеси проліферації МСК-ВС. Найбільш високий рівень мультиплікації спостерігали в сумішах на основі азоту. Цель. Оптимизировать культивирование и сравнить влияния газовых смесей с пониженным содержанием кислорода на основе азота и аргона на процессы пролиферации в культурах мезенхимальных стволовых клеток Вартонова студня (МСК-ВС). Методы. На протяжении 5 пассажей. МСК-ВС культивировали в газовых смесях на основе азота (кислород – 3 %, углекислый газ – 4 %, азот – 93 %) и аргона (кислород – 3 %, углекислый газ – 4 %, аргон – 93 %). На каждом пассаже после 7 дней культивирования подсчитывали количество клеток, и определяли число удвоений культуры. Результаты. Численность клеток МСК-ВС, культивированных в смесях, содержащих 3 % кислорода, была выше, чем в общепринятых условиях СО2-инкубатора. Уровень пролиферации в смеси на основе аргона был несколько ниже, чем в смеси на основе азота, при этом будучи выше такого показателя для контрольных групп. Выводы. Культивирование в газовых смесях, содержащих 3 % кислорода, оказывало стимулирующее влияние на процессы пролиферации МСК-ВС. Наиболее высокий уровень мультипликации наблюдали в смесях на основе азота. 2015 Article Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions / N.S. Shuvalova, V.A. Kordium // Вiopolymers and Cell. — 2015. — Т. 31, № 3. — С. 233-239. — Бібліогр.: 37 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.0008E5 http://dspace.nbuv.gov.ua/handle/123456789/152476 576.533 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Methods Methods |
| spellingShingle |
Methods Methods Shuvalova, N.S. Kordium, V.A. Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions Вiopolymers and Cell |
| description |
Aim. To optimize the cultivation of Wharton jelly-derived mesenchyma stem cells (WJ-MSCs) using physiological oxygen concentrations, and to compare the effect of “hypoxic” gas mixtures, based on nitrogen and argon, on their proliferative activity. Methods. From the first passage, WJ-MSCs were cultivated during five passages in the nitrogen-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % nitrogen) and argon-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % argon), 7 days before replating. At each passage the final cell number was estimated and the number of population doublings was calculated. Results. The proliferation level of WJ-MSCs, cultured in both gas mixtures with 3 % of O2, was significantly higher compared to that under the regular CO2-incubator conditions. In argon-based mixture, the WJ-MSCs proliferation was higher than in the control but lower than in nitrogen-based mixture. Conclusion. Cultivation of human WJ-MSCs under 3 % O2 had a stimulating effect on the cell proliferation potential. The highest intensity of the cell multiplication was observed in the nitrogen-based mixtures. |
| format |
Article |
| author |
Shuvalova, N.S. Kordium, V.A. |
| author_facet |
Shuvalova, N.S. Kordium, V.A. |
| author_sort |
Shuvalova, N.S. |
| title |
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions |
| title_short |
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions |
| title_full |
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions |
| title_fullStr |
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions |
| title_full_unstemmed |
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions |
| title_sort |
comparison of proliferative activity of wharton jelly mesenchymal stem cells in cultures under various gas conditions |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2015 |
| topic_facet |
Methods |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/152476 |
| citation_txt |
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions / N.S. Shuvalova, V.A. Kordium // Вiopolymers and Cell. — 2015. — Т. 31, № 3. — С. 233-239. — Бібліогр.: 37 назв. — англ. |
| series |
Вiopolymers and Cell |
| work_keys_str_mv |
AT shuvalovans comparisonofproliferativeactivityofwhartonjellymesenchymalstemcellsinculturesundervariousgasconditions AT kordiumva comparisonofproliferativeactivityofwhartonjellymesenchymalstemcellsinculturesundervariousgasconditions |
| first_indexed |
2025-07-13T03:17:01Z |
| last_indexed |
2025-07-13T03:17:01Z |
| _version_ |
1837500167727611904 |
| fulltext |
233
ISSN 0233-7657
Biopolymers and Cell. 2015. Vol. 31. N 3. P. 233–239
doi: http://dx.doi.org/10.7124/bc.0008E5
Methods
UDC 576.533
Comparison of proliferative activity of Wharton jelly
mesenchymal stem cells in cultures under various gas conditions
N. S. Shuvalova1, V. A. Kordium1, 2
1 State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine
67, Vyshhorodska Str., Kyiv, Ukraine, 04114
2 Institute of Molecular Biology and Genetics, NAS of Ukraine
150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
riyena@yandex.ua
Aim. To optimize the cultivation of Wharton jelly-derived mesenchymal stem cells (WJ-MSCs) using
physiological oxygen concentrations, and to compare the effect of “hypoxic” gas mixtures, based on nitrogen
and argon, on their proliferative activity. Methods. From the fi rst passage, WJ-MSCs were cultivated
during fi ve passages in the nitrogen-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % nitrogen)
and argon-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % argon), 7 days before replating. At
each passage the fi nal cell number was estimated and the number of population doublings was calculated.
Results. The proliferation level of WJ-MSCs, cultured in both gas mixtures with 3 % of O2, was signifi cantly
higher compared to that under the regular CO2-incubator conditions. In argon-based mixture, the WJ-MSCs
proliferation was higher than in the control but lower than in nitrogen-based mixture. Conclusion. Cultivation
of human WJ-MSCs under 3 % O2 had a stimulating effect on the cell proliferation potential. The highest
intensity of the cell multiplication was observed in the nitrogen-based mixtures.
K e y w o r d s: mesenchymal stem cells, Wharton jelly, hypoxia, physiological oxygen concentration,
proliferation.
© 2015 N.S. Shuvalova et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited
Introduction
Numerous experimental works and theoretical anal-
ysis, focused on mesenchymal stem cells (MSCs),
allow considering them not only as an interesting
object of studies on fundamental processes of fetal
and adult life, but also as a key element in various
methods of regenerative therapy. A promising posi-
tion of MSCs in the cell-based therapeutic strategies
results from their high proliferative and differential
potential, unique paracrine effects and immune prop-
erties [1, 2, 3].
MSCs were fi rst identifi ed in bone marrow and
described as a population of non- hematopoietic
mul tipotent cells [4, 5]. Further studies showed that
the cells with similar properties can be found in both
tissues of adult organism and birth-associated tis-
sues: amnion, placenta and umbilical cord [6]. Ac-
cor ding to the current literature, the latter are often
defi ned as the «perinatal» stem cells, possessing the
properties of both adult and embryonic stem cells [7].
Among them, MSCs from umbilical cord matrix –
Wharton jelly (WJ-MSCs), are thought to be espe-
cially attractive. The formation of WJ-MSCs popu-
lation at the early stages of embryogenesis [8], per-
mits them to preserve the features of the embryonic
stem cells [7, 9, 10], prominent differentiation [11],
immune [12] and paracrine properties [13]. At the
same time, they possess the characteristics of adult
somatic mesenchymal multipotent stromal cells [14],
determined by the International Society for Cellular
Therapy [15].
One of the most important characteristics of MSCs,
particularly for the clinical usage, is their prolifera-
234
N. S. Shuvalova, V. A. Kordium
tive activity. For instance, according to the literature,
during the treatment of acute «graft-versus-host»
disease, patients received 2 × 106–8×106 MSCs/kg
body weight [16]. The percent of MSCs in their clas-
sical source – bone marrow for the newborns is
0.01 %, and decreases to 0.001–0.005 % with aging
[17]. Therefore, it is vitally important to develop the
cultivation technologies, which would allow maxi-
mal cell multiplication with preservation of the
MSCs therapeutically relevant properties. The com-
plexity of this task is related to the process of culti-
vation itself. Some works report that a long-term
cultivation increases the risks of genetic abnormali-
ties [18], thus a reasonable approach to provide the
most effective MSCs cultivation should grant ob-
taining the maximal number of cells while minimiz-
ing the duration of culturing.
In the organism, a crucial role in the regulation of
MSCs behavior and preservation of their properties
belongs to their natural site of localization – «stem
cell niche». Its components include the extracellular
matrix, surrounding cells and signal molecules, pro-
duced by them. One of the key factors of niche regula-
tion is the oxygen concentration, which is generally
lower comparing not only to ambient atmospheric
concentration , but also to that in other regions of the
tissue. For instance, the oxygen concentration in bone
marrow ranges from 2 % to 7 %, depending on the
distance from capillary, and MSCs locate in the areas
remote from vessels , which are, the most «hypoxic»
zones [19]. Thus, the generally accepted environmen-
tal conditions of CO2-incubator, where the oxygen
concentration is similar to atmospheric, are actually
«hyperoxic» for MSCs, which inevitably leads to the
oxidative damages [20, 21]. Taking this into account,
the MSCs cultivation at physiological oxygen con-
centrations, often referred to as «hypoxic», is consid-
ered to be a perspective approach [22–24].
The studies focused on the infl uence of hypoxic
conditions on the MSCs cultivation reported their
benefi cial effect on multiplication, reducing oxida-
tive stress, and engraftment in the transplantation
[25–28]. However, it is hard to compare the results
obtained in different works. MSCs, used in various
studies, originate from different sources, the design
of experiments varies from short-term precondition-
ing to a long-term cultivation, and the O2 concentra-
tions used range from 1.5 % to 8 %. The works on
the infl uence of hypoxic conditions on WJ-MSCs,
still remain rare cases.
Generally, the gas mixtures used for cultivation
include nitrogen as a major «fi lling» component
[29–31]. However, recent works have shown the cy-
toprotective effect of noble (or inert) gases (argon
and xenon) on the cell cultures [32]. Taking this into
account, we hypothesized, that using the «hypoxic»
gas mixture based on the noble gas would enhance
the benefi cial effect of physiological oxygen con-
centration on the MSCs culture.
Thus, the aim of the present work was to optimize
the MSCs cultivation using physiological oxygen
concentrations, and to compare the effects of «hy-
poxic» nitrogen- and argon-based gas mixtures on
the human WJ-MSCs proliferation.
Materials and Methods
MSCs were obtained from WJ of umbilical cord
(UC) from three healthy donors (39–40 weeks of
gestation, normal delivery), after obtaining the in-
formed written consent, in Kyiv maternity clinic
N 5. The cells were isolated using the explant meth-
od [33]. The UC fragment (5–10 cm) was washed
with PBS, the vessels were mechanically removed.
WJ was mechanically sliced, the fragments were
placed in the cultural fl acks, 75 cm2, containing com-
plete growth medium (DMEM with low glucose
(PAA Austria) supplemented with 10 % fetal bovine
serum (PAA, Austria), glutamine 2 mM (PAA,
Austria), penicillin 100 U/ml (Arterium, Ukraine),
streptomycin 100 μg/ml (Arterium, Ukraine). The
fi rst adherent cells were visible on 7–10 day. After
14 days the clones reached 70–80 % confl uence, and
the cells were passed using trypsin-EDTA (0.1 %
trypsin and 0.02 % EDTA) solution. At the fi rst pas-
sage the cells were characterized for the surface
marker proteins CD90, CD73, CD105 expression
(over 85 % positive), using fl ow cytometry ( BD
FACS Aria) with fl uorescein- and rhodamine-conju-
gated antibodies (UsBiological, USA). For micros-
copy, inverted microscope Leica DMIL was used.
235
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions
From the fi rst passage, MSCs were seeded on plas-
tic fl acks (25 cm2) at a density of 75,000 per fl ack and
cultivated during 5 passages in the nitrogen-based gas
mixture (3 % oxygen, 4 % carbon dioxide, 93 % ni-
trogen) and argon-based gas mixture (3 % oxygen,
4 % carbon dioxide, 93 % argon), 7 days before re-
plating. The control group was maintained under
standard CO2 incubator conditions. For creating the
hypoxic conditions, the cultural fl acks with cells we-
re placed in polyethylene bags with hermetical clasp
«ZipLock». The bags were washed twice with the
oxygen-free gas mixture, containing 4 % CO2 and
96 % nitrogen or argon (depending on the group),
and after that were fi lled with the cultivation gas
mixture (see above). The bags were placed in the
vacuum containers (Scarlet). The percentage «liquid
media/gas» for normal gas exchange must be 1:100
[34], the volume of gas mixture must be no less than
0.7 l. The volume of bags used was 1.5 l.
At each passage, after 7 days of culturing, the cells
were replated using trypsin-EDTA (0.1 % trypsin and
0.02 % EDTA) solution and counted in hemocytom-
eter (Goryaev chamber). Population doubling was cal-
culated as: PD = log (Nf/Ni)/log 2, where Nf – fi nal
cell number; Ni – initial cell number [22].
The data on cell numbers are represented as me-
an ± standard deviation for 3 samples. Statistical sig-
nifi cance was determined using Mann-Whitney U-test
at P < 0.05.
Results and Discussion
WJ-MSCs were expanded for fi ve consecutive pas-
sages under 3 % O2, in the gas mixtures based on
nitrogen, argon, and ambient O2 concentration (near-
ly 20 %) in CO2-incubator. In order to assess the pro-
liferation activity, the cells were counted at each pas-
sage. The results, summarized in Table 1 an Figure
1, show a fi nal number of cells after 7 days of culti-
vation.
The proliferation level of WJ-MSCs, cultured at 3 %
O2, in both gas mixtures was signifi cantly higher com-
pared to that of WJ-MSCs under the CO2-incubator
conditions. This was observed at each passage.
Interestingly, a comparison of the culture growth
rates at different passages within each group allows
distinguishing two separate «phases» during the cul-
tivation. The fi rst «phase» – at the fi rst and second
passages the cultures had practically similar prolif-
eration levels. The second «phase» – beginning from
the third passage, a progressive decrease in prolifera-
tion can be observed with each new passing. For WJ-
0
100
200
300
400
500
600
1 2 3 4 5
Ce
ll
nu
m
be
r,
t
ho
th
an
d
passage
WJ-MSC proliferation
nitrogen
argon
athmospheric
Fig. 1. Numbers of cells in WJ-MSCs cultures at 1–5 passages,
after 7 days of cultivation. «Nitrogen» – nitrogen-based gas mix ture
(3 % oxygen, 4 % carbon dioxide, 93 % nitrogen), «argon» –
argon-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 %
argon), «atmospheric» – CO2 incubator conditions. * – P <
0.05)
Table 1. Numbers of cells in WJ-MSCs cultures at 1–5 passage, after 7 days of cultivation.
«Nitrogen» – nitrogen-based gas mixture (3 % oxygen, 4 % carbon dioxide, 93 % nitrogen), «argon» – argon-based gas
mixture (3 % oxygen, 4 % carbon dioxide, 93 % argon), «atmospheric» – CO2 incubator conditions (P < 0.05)
N of passage Nitrogen Argon Atmospheric
1 513.68 ± 9.58 488.3 ± 20.91 471.72 ± 9.7
2 525.47 ± 7.32 508.6 ± 25.69 444.58 ± 19.07
3 465.6 ± 2.91 460.67 ± 21.06 317.53 ± 25.23
4 332.39 ± 15.16 315.27 ± 16.22 233.62 ± 5.1
5 288.93 ± 11.39 258 ± 26.17 149.77 ± 36.93
* *
* *
* *
* * * *
236
N. S. Shuvalova, V. A. Kordium
MSCs, cultured in the gas mixtures, this tendency
was less pronounced.
At the fi rst passage of culture under 3 % O2 in the
nitrogen-based and argon-based gas mixtures, the fi -
nal number of expanded WJ-MSCs was respectively
6.8 and 6.4 times higher, was higher comparing to
cells from control group (6.1-fold increase). The re-
sults for the second passage were 6.9, 6.4 and 6 re-
spectively. The data for the entire culture duration
are summarized in Table 2.
The PD estimation showed that, despite a general
decrease in the number of PD up to the 5th passage, the
MSCs cultured under the physiological oxygen tension,
had generally higher intensity of division (Table 3).
The proliferative activity is an important criterion
for an estimation of the state of culture, and a suffi -
cient activity is necessary for practical application. It
strongly depends on the conditions of cultivation,
each factor of environment having its own mecha-
nisms of infl uence. In this context, proliferation itself
can be considered a subject of research. In the present
work, the proliferation rates of MSCs cultures, ex-
panded under physiological oxygen tensions – 3 %, in
the mixtures based on nitrogen and argon, were vali-
dated. The estimation of cells number at 1–5 passages
showed signifi cantly higher numbers in the gas mix-
tures at each passage, comparing to the groups from
control CO2-incubator. The results also revealed a
higher level of PD under hypoxic conditions up to the
5th passage. The effect of mild hypoxia appeared to
be stimulating in both mixtures, at the same time, the
differences between these conditions were detected.
Although a high variability in the object and meth-
ods of studies on hypoxia complicates the comparison
of results, we can assume that the tendencies, observed
in the present work, are generally in line with those
described in literature. Nevertheless, it is important to
point out the differences. The work by Basciano et al.
revealed the slowing of culture growth at early pas-
sages under hypoxic conditions [22]. We have not ob-
served any similar effect. Ren et al detected the mor-
phological signs of accelerated cellular senescence in
hypoxic conditions, probably associated with a higher
population doublings number [35]. In the present
study there was no evidence of degenerative changes
of morphology in the cultures expanded under hypox-
ic conditions (data not shown).
The current literature distinguishes several poten-
tial mechanisms of physiological oxygen concentra-
tions impact. First, the reactive oxygen species are
one of the major sources of DNA damage [36].
Hypoxic conditions were shown to prevent the ac-
cumulation of damages of genetic apparatus, which
in control groups can be detected since the second
passage [17]. This can play an important role during
long-term cultivation. Next, the researches show that
hypoxic conditions increase the level of cytokine re-
ceptors expression, as well as the production of
growth factors. In this case, hypoxia « sensibilizes «
the cells to serum growth factor, and to that produced
by the cells themselves [27]. Next, there are data
Table 2. Multiplication in WJ- MSCs cultures
at 1–5 passages, n-fold increase in number after 7 days
of cultivation
Passage Nitrogen Argon Atmospheric
1 6.8 6.4 6.1
2 6.9 6.8 6
3 6 6 4.1
4 4.4 4.2 3.1
5 3.8 3.4 1.9
N o t e. «Nitrogen» – nitrogen-based gas mixture (3 % oxygen, 4 %
carbon dioxide, 93 % nitrogen), «argon» – argon-based gas mix-
ture (3 % oxygen, 4 % carbon dioxide, 93 % argon), « atmo-
spheric» – CO2 incubator conditions (P < 0.05)
Table 3. Population doublings in WJ-MSCs cultures
at 1–5 passages, after 7 days of cultivation
N of passage Nitrogen Argon Atmosperic
1 2.76 2.68 2.61
2 2.79 2.76 2.59
3 2.59 2.59 2.04
4 2.15 2.07 1.64
5 1.95 1.78 1
N o t e. «Nitrogen» – nitrogen-based gas mixture (3 % oxygen, 4 %
carbon dioxide, 93 % nitrogen), «argon» – argon-based gas mix-
ture (3 % oxygen, 4 % carbon dioxide, 93 % argon), « atmo-
spheric» – CO2 incubator conditions (P < 0.05)
237
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions
showing that hypoxia activates the signal cascades
of cell survival. The cultures in hypoxic conditions
are shown to have lower levels of necrosis comparing
to those cultured under the ambient oxygen concen-
tration [24]. Each of these mechanisms can contribute
to the general effect of physiological oxygen concen-
tration, and determination of their roles is still consid-
ered the question of interest.
To date, the data about the effect of noble gases
on MSCs culture are still lacking. There are only
few works conducted to determine the infl uence of
inert gases (xenon and argon), which demonstrated
a cytoprotective effect on the cell cultures of neural
origin. For example, the modeling of ischemic and
traumatic damages of hippocampal slice culture in
gas mixtures, containing various concentrations of
argon (25 %, 50 % and 74 %) and atmospheric oxy-
gen concentration (21 %), showed a decreased level
of cellular death compared to the control samples.
The underlying mechanisms are unknown [32].
Though, Fahlenkamp et al. demonstrated the acti-
vation of ERK 1/2, kinase that plays an important
role in the processes of cells proliferation and sur-
vival, in the primary cultures of mouse embryonic
astrocytes and neurons, cultured under 20 % O2 and
50 % argon [37]. It is possible that MSCs possess
the similar mechanism.
The present work showed that the level of prolif-
eration of WJ-MSCs, cultured in argon-based gas
mixtures was higher comparing to WJ-MSCs from
CO2-incubator conditions, but lower than that for the
cultures from nitrogen-based mixtures. To explain
these results, further research is required. Besides,
taking into account the variety of existing cultivation
protocols, changing the cultural strategy could prob-
ably lead to a different effect.
Conclusions
Cultivation of human WJ-MSCs under 3 % O2 in gas
mixtures, based on nitrogen and argon, had a benefi -
cial effect on the cells proliferative activity and pres-
ervation of multiplication potential. The hypoxic ar-
gon- and nitrogen-based gas mixtures had different
effects. The highest intensity of cell proliferation was
observed in the nitrogen-based mixtures.
REFERENCES
1. Caplan AI. Why are MSCs therapeutic? New data: new in-
sight. J Pathol. 2009;217(2):318–24.
2. Xin H, Li Y, Chopp M. Exosomes/miRNAs as mediating cell-
based therapy of stroke. Front Cell Neurosci. 2014;8: 377.
3. Khubutiya MS, Vagabov AV, Temnov AA, Sklifas AN. Pa rac-
rine mechanisms of proliferative, anti-apoptotic and anti-
infl ammatory effects of mesenchymal stromal cells in mod-
els of acute organ injury. Cytotherapy. 2014;16(5):579–85.
4. Friedenstein AJ, Piatetzky-Shapiro II, Petrakova KV. Osteo-
ge nesis in transplants of bone marrow cells. J Embryol Exp
M orphol. 1966;16(3):381–90.
5. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibroblast pre-
cursors in normal and irradiated mouse hematopoietic or-
gans. Exp Hematol. 1976;4(5):267–74.
6. Shachpazyan NK, Astrelina TA, Yakovleva MV. Mesenchymal
stem cells from various human tissues: biological proper-
ties, assessment of quality and safety for clinical use. CTTE
2011;7(1): 23–33.
7. Taghizadeh RR, Cetrulo KJ, Cetrulo CL. Wharton’s Jelly
stem cells: future clinical applications. Placenta. 2011;32
(Suppl 4):S311–5.
8. Wang XY, Lan Y, He WY, Zhang L, Yao HY, Hou CM, Tong Y,
Liu YL, Yang G, Liu XD, Yang X, Liu B, Mao N. Identifi cation
of mesenchymal stem cells in aorta-gonad-mesonephros and
yolk sac of human embryos. Blood. 2008;111(4):2436–43.
9. Fong CY, Richards M, Manasi N, Biswas A, Bongso A.
Comparative growth behaviour and characterization of stem
cells from human Wharton’s jelly. Reprod Biomed Online.
2007;15(6):708–18.
10. Zhang H, Zhang B, Tao Y, Cheng M, Hu J, Xu M, Chen H.
Isolation and characterization of mesenchymal stem cells
from whole human umbilical cord applying a single enzyme
approach. Cell Biochem Funct. 2012;30(8):643–9.
11. Lindenmair A, Hatlapatka T, Kollwig G, Hennerbichler S,
Gabriel C, Wolbank S, Redl H, Kasper C. Mesenchymal
stem or stromal cells from amnion and umbilical cord tissue
and their potential for clinical applications. Cells. 2012;1
(4):1061–88.
12. Le Blanc K. Immunomodulatory effects of fetal and adult
mesenchymal stem cells. Cytotherapy. 2003;5(6):485–9.
13. Hsieh JY, Wang HW, Chang SJ, Liao KH, Lee IH, Lin WS,
Wu CH, Lin WY, Cheng SM. Mesenchymal stem cells from
human umbilical cord express preferentially secreted fac-
tors related to neuroprotection, neurogenesis, and angiogen-
esis. PLoS One. 2013;8(8):e72604.
14. Troyer DL, Weiss ML. Wharton’s jelly-derived cells are a
primitive stromal cell population. Stem Cells. 2008;26(3):
591–9.
15. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I,
Marini F, Krause D, Deans R, Keating A, Prockop Dj, Hor-
witz E. Minimal criteria for defi ning multipotent mesenchy-
238
N. S. Shuvalova, V. A. Kordium
mal stromal cells. The International Society for Cellular The-
rapy position statement. Cytotherapy. 2006;8(4):315–7.
16. Kebriaei P, Isola L, Bahceci E, Holland K, Rowley S, Mc Guirk
J, Devetten M, Jansen J, Herzig R, Schuster M, Mon roy R,
Uberti J. Adult human mesenchymal stem cells added to corti-
costeroid therapy for the treatment of acute graft-ver sus-host
disease. Biol Blood Marrow Transplant. 2009;15 (7):804–11.
17. Lund TC, Kobs A, Blazar BR, Tolar J. Mesenchymal strom-
al cells from donors varying widely in age are of equal cel-
lular fi tness after in vitro expansion under hypoxic condi-
tions. Cytotherapy. 2010;12(8):971–81.
18. Rubio D, Garcia-Castro J, Martín MC, de la Fuente R,
Cigudosa JC, Lloyd AC, Bernad A. Spontaneous human adult
stem cell transformation. Cancer Res. 2005;65(8): 3035–9.
19. Watt FM, Hogan BL. Out of Eden: stem cells and their nich-
es. Science. 2000;287(5457):1427–30.
20. Choo KB, Tai L, Hymavathee KS, Wong CY, Nguyen PN,
Huang CJ, Cheong SK, Kamarul T. Oxidative stress-in-
duced premature senescence in Wharton’s jelly-derived me-
sen chymal stem cells. Int J Med Sci. 2014;11(11): 1201–7.
21. Kim M, Kim C, Choi YS, Kim M, Park C, Suh Y. Age-related
alterations in mesenchymal stem cells related to shift in dif-
ferentiation from osteogenic to adipogenic potential: impli-
cation to age-associated bone diseases and defects. Mech
Ageing Dev. 2012;133(5):215–25.
22. Basciano L, Nemos C, Foliguet B, de Isla N, de Carvalho
M, Tran N, Dalloul A. Long term culture of mesenchymal
stem cells in hypoxia promotes a genetic program maintain-
ing their undifferentiated and multipotent status. BMC Cell
Biol. 2011;12:12.
23. Dos Santos F, Andrade PZ, Boura JS, Abecasis MM, da
Silva CL, Cabral JM. Ex vivo expansion of human mesen-
chymal stem cells: a more effective cell proliferation kinet-
ics and metabolism under hypoxia. J Cell Physiol. 2010;
223(1):27–35.
24. Lavrentieva A, Majore I, Kasper C, Hass R. Effects of hy-
poxic culture conditions on umbilical cord-derived human
mesenchymal stem cells. Cell Commun Signal. 2010;8:18.
25. Krinner A, Zscharnack M, Bader A, Drasdo D, Galle J. Im-
pact of oxygen environment on mesenchymal stem cell ex-
pansion and chondrogenic differentiation. Cell Prolif. 2009;
42(4):471–84.
26. Fehrer C, Brunauer R, Laschober G, Unterluggauer H, Rei-
tinger S, Kloss F, Gülly C, Gassner R, Lepperdinger G. Re-
du ced oxygen tension attenuates differentiation capacity of
human mesenchymal stem cells and prolongs their lifespan.
Aging Cell. 2007;6(6):745–57.
27. Hung SC, Pochampally RR, Hsu SC, Sanchez C, Chen SC,
Spees J, Prockop DJ. Short-term exposure of multipotent
stro mal cells to low oxygen increases their expression of
CX3CR1 and CXCR4 and their engraftment in vivo. PLoS
One. 2007; 2(5):e416.
28. Annabi B, Lee YT, Turcotte S, Naud E, Desrosiers RR, Cha-
m pa gne M, Eliopoulos N, Galipeau J, Béliveau R. Hypoxia
promotes murine bone-marrow-derived stromal cell migra-
tion and tube formation. Stem Cells. 2003;21(3):337–47.
29. D’Ippolito G, Diabira S, Howard GA, Roos BA, Schiller PC.
Low oxygen tension inhibits osteogenic differentiation and
enhances stemness of human MIAMI cells. Bone. 2006;39
(3):513–22.
30. Carrancio S, López-Holgado N, Sánchez-Guijo FM, Villa-
rón E, Barbado V, Tabera S, Díez-Campelo M, Blanco J,
San Miguel JF, Del Cañizo MC. Optimization of mesenchy-
mal stem cell expansion procedures by cell separation and
culture conditions modifi cation. Exp Hematol. 2008;36(8):
1014–21.
31. Ivanovic Z, Hermitte F, Brunet de la Grange P, Dazey B,
Belloc F, Lacombe F, Vezon G, Praloran V. Simultaneous
maintenance of human cord blood SCID-repopulating cells
and expansion of committed progenitors at low O2 concen-
tration (3 %). Stem Cells. 2004;22(5):716–24.
32. Loetscher PD, Rossaint J, Rossaint R, Weis J, Fries M,
Fahlenkamp A, Ryang YM, Grottke O, Coburn M. Argon:
neuroprotection in in vitro models of cerebral ischemia and
traumatic brain injury. Crit Care. 2009;13(6):R206.
33. Maslova OO, Shuvalova NS, Sukhorada OM, Zhukova SM,
Deryabina OG, Makarenko MV, Govseiev DO, Kordium VA.
Heterogeneity of umbilical cords as a source for mesenchy-
mal stem cells. Dataset Pap Biol. 2013;2013:1–4.
34. Freshney RI. Animal cell culture a practical approach.
Oxford University Press; 2nd edition 1986; 264 p.
35. Ren H, Cao Y, Zhao Q, Li J, Zhou C, Liao L, Jia M, Zhao Q,
Cai H, Han ZC, Yang R, Chen G, Zhao RC. Proliferation
and differentiation of bone marrow stromal cells under hy-
poxic conditions. Biochem Biophys Res Commun. 2006;347
(1):12–21.
36. Wiseman H, Halliwell B. Damage to DNA by reactive oxy-
gen and nitrogen species: role in infl ammatory disease and
progression to cancer. Biochem J. 1996;313 (Pt 1):17–29.
37. Fahlenkamp AV, Rossaint R, Haase H, Al Kassam H,
Ryang YM, Beyer C, Coburn M. The noble gas argon mod-
ifi es extracellular signal-regulated kinase 1/2 signaling in
neurons and glial cells. Eur J Pharmacol. 2012;674(2–3):
104–11.
Порівняльна оцінка ростових показників
мезенхімальних стовбурових клітин Вартонового
студня в різних газових сумішах
Н. С. Шувалова, В. А. Кордюм
Мета. Оптимізувати культивування і порівняти вплив газо-
вих сумішей зі зниженим вмістом кисню, на основі азоту та
аргону, на процеси проліферації в культурах мезенхімаль-
них стовбурових клітин Вартонового студню (МСК-ВС). Ме-
тоди. Протягом 5 пасажів. МСК-ВС культивували в газових
сумішах на основі азоту (кисень – 3 %, вуглекислий газ –
4 %, азот – 93 %) та аргону (кисень – 3 %, вуглекислий газ –
239
Comparison of proliferative activity of Wharton jelly mesenchymal stem cells in cultures under various gas conditions
4 %, аргон – 93 %). На кожному пасажі після 7 днів культи-
вування підраховували кількість клітин, і визначали число
подвоєнь культури. Результати. Чисельність клітин МСК-
ВС, культивованих в сумішах, що містять 3 % кисню, була
вищою, ніж в загальноприйнятих умовах СО2-інкубатора.
Рівень проліферації в суміші на основі аргону був нижчим,
ніж в суміші на основі азоту, при цьому будучи вищим за
такий для контрольних груп. Висновки. Культивування в
газових сумішах, що містять 3 % кисню, мало стимулюючий
вплив на процеси проліферації МСК-ВС. Найбільш високий
рівень мультиплікації спостерігали в сумішах на основі азоту.
Ключов і слова: мезенхімальні стовбурові клітини, Вар-
тонів студень, гіпоксія, фізіологічні концентрації кисню, про-
ліферація
Сравнительная оценка ростовых показателей
мезенхимальных стволовых клеток Вартонова студня в
различных газовых смесях
Н. С. Шувалова, В. А. Кордюм
Цель. Оптимизация культивирования и сравнение влияния
газовых смесей с пониженным содержанием кислорода на
основе азота и аргона на процессы пролиферации в культу-
рах мезенхимальных стволовых клеток Вартонова студня
(МСК-ВС). Методы. На протяжении 5 пассажей. МСК-ВС ку-
льтивировали в газовых смесях на основе азота (кислород –
3 %, углекислый газ – 4 %, азот – 93 %) и аргона (кислород –
3 %, углекислый газ – 4 %, аргон – 93 %). На каждом пасса-
же после 7 дней культивирования подсчитывали количество
клеток, и определяли число удвоений культуры. Результаты.
Численность клеток МСК-ВС, культивированных в смесях,
содержащих 3 % кислорода, была выше, чем в общеприня-
тых условиях СО2-инкубатора. Уровень пролиферации в сме-
си на основе аргона был несколько ниже, чем в смеси на
основе азота, при этом будучи выше такого показателя для
контрольных групп. Выводы. Культивирование в газовых
смесях, содержащих 3 % кислорода, оказывало стимулиру-
ющее влияние на процессы пролиферации МСК-ВС. Наи-
более высокий уровень мультипликации наблюдали в сме-
сях на основе азота.
Ключевые слова: мезенхимальные стволовые клетки,
Вартонов студень, гипоксия, физиологические концентра-
ции кислорода, пролиферация.
Received 16.02.2015
|