Metformin, a biguanide class of chemical, was used as a treatment to reduce
blood sugar. It is the first choice of treatment for a wide range of diseases
including overweight and obese patients that have type 2 diabetes. It is also
recommended for patients with normal kidney function as well as gestational
diabetes. Polycystic ovary syndrome and insulin-resistant diseases are also
can be cured using Metformin (Smoak, 1999; Holland
et al., 2004). For the next two decades researchers focused on insulin
and other antidiabetic drugs in the expense of Metformin. New concerns on Metformin
were renewed in the late 1940s as several publications reported its efficacy
to reduce levels of blood sugar. According to Hirsch et
al. (2009), Jean Sterne was the first to publish work using metformin
to treat diabetes. The potential importance of metformin to reduce the possibility
of cancer risks is highlighted. The actual effect of metformin on cancer patient
has been still far away from complete understanding so far (Evans
et al., 2005).
The main systemic effect of metformin for the treatment of diabetes mellitus
is to increase insulin sensitivity and to reduce blood glucose concentrations
via lowering liver gluconeogenesis and increasing glucose uptake in peripheral
tissues, including skeletal muscles and adipose tissues (Bao
et al., 2012).
Fibroblast, the most common type of human cells in connective tissue, plays
a significant role in maintain the integrity of connective tissue. The major
components of extracellular matrix including the ground and fiber substances
are mainly produced by fibroblasts. Physical properties of connective tissues
are largely dependent on the nature of extracellular nature. In addition to
its pivot role in the structural support, fibroblast regulates wound healing,
self-tolerance, inflammation, fibrosis as well as organ development (Fries
et al., 1994). Placenta, skin, adipose tissue, thymus, muscle periosteum,
synovium, synovial fluid, fetal liver, blood and cord blood are considered to
be ideal tissues to isolate fibroblasts using tissue culture adherence technique
(Haniffa et al., 2009; Da
Silva Meirelles et al., 2006).
Oxygen tension is one of the most important conditions that should be controlled,
when planning to culture stem cells. According to Grimshaw
and Mason (2000), oxygen is considered as significant molecule with signaling
capabilities that are required for main issues in stem cell biology such as
self-renewal, proliferation, cell death, differentiation and migration (Grimshaw
and Mason, 2000).
It has been indicated that tissues have lower oxygen pressure compared with
atmospheric oxygen pressure commonly used in incubators (Csete,
2005). It has been shown by other studies that the oxygen pressure in bone
marrow is varied from site to site depending on how the tissue is far from the
blood capillaries. Generally oxygen tension ranges from 1-7%. For instant, oxygen
tension in articular cartilage ranges from 3-10% at the surface, whereas the
level is decreased to 1% in deeper tissues (Da Silva Meirelles
et al., 2006).
Production of cartilage during fetal development as well as formation of chondrocytes
in hyaline cartilage is an example of tissues that are synthesized under low
level of oxygen tension (Zscharnack et al., 2008).
So, the present investigation is carried out to study the effect of different
doses of antidiabetic drug-Metformin on proliferation of fibroblast derived
stem cells in the different concentrations of oxygen medium.
MATERIALS AND METHODS
Sample sourcing: Signed consent forms were taken from the patients to
participate in the study, human foreskin samples were taken by circumcision.
The ethics commission of Leipzig University approved the study which was performed
in accordance with the Declaration of Helsinki protocols. The study was carried
out in the Department of Cell Techniques and Applied Stem Cell Biology, Biotechnological-Biomedical
Center, University of Leipzig, Leipzig, Germany.
Cell isolation and culture:Fibroblasts were taken from human foreskin
samples. Immediately after circumcision foreskins were kept in sterile Phosphate
Buffered Saline (PBS) and stored at 4°C until use. They were immersed in
Betaisodona solution for 20 min and rinsed several times with PBS until they
were free of brown staining. After this disinfection the subcutaneous fatty
tissue were removed with scalpel and forceps. Foreskins were finally cut into
pieces of about 5x5 mm and digested with Dispose II solution (2.5 mg mL-1,
Dispose II in DMEM) for 3-4 h at 37°C or alternatively over night at 4°C.
After this enzymatic digestion the skin was washed with PBS and the epidermis
was removed from the dermis with forceps. The epidermis was discarded and the
dermis was cut in smaller pieces. After that, the dermis was incubated for 2
h with 1 mg mL-1 collagenase (in PBS with Ca2+ and Mg2+)
at 37°C with gently shaking. The pieces of dermis have to be dissolved.
A 70 μm cell strainer was used to filter the cell suspension and then centrifuged
for 5 min at 1800 RPM. The resulting cell pellet was suspended in Dulbeccos
Modified Eagle Medium (supplemented with 10% Fetal Bovine Serum (FCS), 1% penicillin/streptomycin
and Glutamax) and the fibroblasts were cultured in cell culture flasks. Cell
numbers were measured using trypan blue exclusion in an improved haemocytometer
after each passage. Fibroblasts were analysed for some mesenchymal stem cell
markers with flow cytometry analyses. Cell culture experiments were done in
6-well plates (30.000 cells well-1), 12-well plates (20.000 cells
well-1) and 24-well plates (10.000 cells well-1).
Measurement of cell viability: The MTT Cell Proliferation assay depends
on the observation that MTT dye is enzymatically reduced into formazan dye that
has a purple color. The activity of reductase responsible for this reaction
is directly proportional to changes in the color and as a result an indication
to measure cells viability. By using this method, both cell viability and cell
proliferation can be measured by cell counting and cell culture assay respectively.
Furthermore, this assay method can also be used to get an indication about variable
associated with medicinal agents' cytotoxicity since such materials stimulate
or inhibit cell viability and as a result growth.
All optical density values were measured at 570 nm, which correlates directly
with the number of metabolically active cells in the culture. After measurement
of absorbance by a Microtiter plate reader (Tecan) with 650 and 570 nm filters,
the mean values of approaches (duplicates) were allocated to mean values for
the respective control solution (control Metformin-medium without substance).
Description of experiments: Growth in 12-well plates with Metformin
in different dilutions, oxygen conditions (normoxia or hypoxia) and time intervals
for proliferation analyses (MTT). Dose of Metformin was 100 μg mL-1.
The Medium was contain DMEM, high Glucose, Natriumpyruvat, Glutamax with 10%
FCS and 1% penicillin/streptomycin, also 1 mL per well was used. Finally, the
number of the fibroblast cells was 20000 cells well-1.
At the first day cell seeding and stimulating with different concentration
of Metformin in the next day, then incubated to proceed MTT-test and measure
metabolic activity. In our experiment different concentration of oxygen in medium
of fibroblast stem cells are used. Two plates are used in our experiment, first
plate with 20% O2 and the second plate with 5% O2, MTT-Test
is carried at day 1 and 3.
MTT assay was used to study the impacts of metformin on cell proliferation.
The material of study in this experiment was skin samples derived from stem
cell. Metformin was found to significantly inhibit cell survival and that inhibition
rate was found to correlate with the dose concentration.
|| Influence of Metformin on the optical density of skin derived
|| Influence of Metformin on the percent of viability of skin
The three dose concentrations used in this experiment were: Metformin 10,
50 and Metformin 100 μg mL-1. Metformin decreased cell survival
in dose 100 μg mL-1 more than other two doses (Fig.
1) Comparable growth in all wells, differences in proliferation and percentage
of viability (Fig. 2).
Figure 3 Showed fibroblast stem cells in vitro cultured
after isolating with Percoll from skin and morphological changes of fibroblasts
by administration of different doses of metformin in day 1 and day 3. At day
3 I can see with increased metformin concentration the number of damaged cells
gets also increased, I can see the damaged cells as white clusters Fig.
3e, f. At day 3 the cells are denser than at day 1.
The rate of cell proliferation as well as the reduction in cell viability was
assessed using the MTT assay. The assay was carried out when cells undergo necrosis
and apoptosis conditions. MTT assay has another application in which the cytotoxicity
of potential medical agents and toxic materials can be evaluated since these
agents have a stimulatory or inhibitory effect on cell viability and growth.
The results of the MTT assay graphically represented indicate a definite negative
influence of Metformin on proliferation of skin derived stem cells during cell
culturing both on the first and the third day after administration. With a higher
concentration of Metformin 100 μg mL-1 it led to a correlative
decrease in stem cells activity, diagrammed as Fig. 1, in
the form of absolute measured values OD (optical density) or as Fig.
2 in the form of percentage of viability, the activity of fibroblast stem
cells increasing by low concentration of Metformin 50 and 10 μg mL-1
respectively. I find only low differences in respective data between the normoxia
and hypoxia conditions, for the last something better for absolute values of
day 1 but not for values of day 3, thus show no significant influence of oxidative
stress on cell proliferation in this part of experiment.
||Morphological changes of fibroblast stem cells, (a) 10 μg
mL-1 day 1, (b) 50 μg mL-1 day1 (c) 100 μg
mL-1 day 1, (d) 10 μg mL-1 day 3 (e) 50 μg
mL-1 day 3 and (F) 100 μg mL-1 day 3 (Original
magnification: a, b,c, d, e, f 100 x)
These researchers: Domm et al. (2002), Kurz
et al. (2004), Murphy and Polak (2004) and
Mizuno and Glowacki (2005). Studying the influence
of low oxygen (hypoxia) during the different type cells and fibroblast stem
cells proliferation in three dimensional systems. Studies have shown that there
are beneficial effects due to various precursors and articular stem cells. It
has also been pointed to the presence of partially contradicted results which
are thought to be caused by various cell types used and the application of different
cultural systems with different oxygen diffusion properties. I have put focus
on the impacts of low oxygen on fibroblast proliferation. Fibroblast expanded
at physiologically normxia 20% O2 was strongly accelerated and enhanced
compared to low oxygen tension 5% O2. This result was consistently
observed by cell viability in MTT test. Similar findings have been obtained
for cell proliferation in normaxia and hypoxia culture.
Our findings confirm the significance of appropriate fibroblast-culture conditions
during proliferation for potential application of such cells in regenerative
medicine. Our results agree with previous data (Sekiya
et al., 2002) in which it has been revealed that the initial monolayer
culture conditions for fibroblast proliferation, including seeding densities,
media, culture period and oxygen tension play an important role in defining
the fibroblast stem cells proliferation and differentiation.
In agreement with other works which used cells from other species (Grayson
et al., 2007; Zscharnack et al., 2008),
we show an advantage of using 20% oxygen culture for expansion and proliferate
stem cells in monolayer culture. Taken together, it has been shown that fibroblast
under the effects of 20% oxygen tension subsequently showed enhanced fibroblast
The results suggest that fibroblast stem cells proliferation is largely affected
by high dose (100 μg mL-1) of Metformin. Not only proliferation,
but also cell survival was found in this piece of work to be significantly inhibited
by metformin, also the experiment pointed to important effects of short-term
low oxygen (5% O2) treatment on fibroblast proliferation accompanied
with data that fibroblast which were under the effect of 20% oxygen tension
exhibited enhanced fibroblast proliferation and viability. There are no important
variations in the viability of fibroblast on the first and the third day after
This work was funded by the DFG programme and funded as consumables by department
of Cell Techniques and Applied Stem Cell Biology, Biotechnological- Biomedical
Centre at Leipzig University. I am grateful and thank Professor Augustinus Bader
for hosting me and Dr. Doris Stefanowa, Sabina and Katja for the management
of my work.