Journal of Biomaterials and Nanobiotechnology, 2011, 2, 378-389
doi:10.4236/jbnb.2011.24047 Published Online October 2011 (http://www.SciRP.org/journal/jbnb)
Assessment of Biological Properties of Mouse
Embryonic Stem Cells Characteristics Prior to
Differentiation
Gary Adams1,2*, Lee Buttery3, Snow Stolnik3, Stephen E. Harding2, Nan Wang1
1Insulin and Diabetes-Related Experimental Research Group, Faculty of Medicine and Health Sciences, University of Nottingham,
Nottingham, United Kingdom; 2National Centre Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham,
Sutton, United Kindom; 3Drug Delivery and Tissue Engineering Division, School of Pharmacy, University of Nottingham, Notting-
ham, United Kingdom.
Received October 30th, 2010; revised February 11th, 2011; accepted July 20th, 2011.
ABSTRACT
Mouse embryonic stem (ES) cells are continuous cell lines derived directly from the fetal founder tissue of the pre-im-
plantation embryo and can be expanded in vitro and give rise to cells from ectodermal, mesodermal and endodermal
layers. Mouse ES cells can be maintained and their numbers expanded by culture on feeder layer cells with LIF present
in the culture medium. This study shows that changes in seeding density can significantly influence cell number expan-
sion rates. Culturing ES cells in the absence of feeder layer cells and LIF stimulates EB formation when cultured in
non-adherent culture plates. Formation of EBs particularly numbers, size of EBs formed, rates of cell proliferation
within EBs and viability of cells can also be controlled based on seeding density. All these factors are important for
optimizing approaches to co-ordinate differentiation towards a specific cell type. A key goal of ES cell research is to
develop specific functional cell types which can be potentially used to study mechanisms of tissue development and as a
therapy to repair or replace damaged or diseased tissues.
Keywords: Assessment, Biological Properties, Mouse, Embryonic, Stem Cells
1. Introduction
Mouse embryonic stem (ES) cells are continuous cell
lines derived directly from the fetal founder tissue of the
pre-implantation embryo [1]. They can be expanded in
vitro and give rise to cells from all three germ layers:
ectoderm, mesoderm and endoderm [1]. A key goal of
ES cell research is to develop specific functional cell
types which can be potentially used to study mechanisms
of tissue development and as a therapy to repair or re-
place damaged or diseased tissues. Fundamental to achiev-
ing these goals is a good understanding of the factors
influencing ES cell proliferation and differentiation and
application to efficient in vitro expansion and controlled
differentiation.
Under appropriate culture conditions, ES cells can be
maintained as pluripotent undifferentiated cells and their
numbers expanded almost indefinitely. In general, mouse
ES cells are maintained in a proliferating undifferentiated
state by co-culture on feeder layers of immortalized
mouse embryonic fibroblast cell lines. Leukaemia inhibi-
tory factor (LIF) is also required to help maintain pluri-
potency and is added to the culture medium although
some feeder cell lines are genetically engineered to over-
express LIF helping to reduce the amounts of exogenous
LIF required. Some mouse ES cell lines can be grown in
the absence of feeder layers, but usually require higher
concentrations of LIF and may also require conditioned
media from feeder cells to be added to the culture me-
dium [2].
Differentiation of mouse ES cells can be induced in a
variety of ways, but the most common technique is via
formation of cell aggregates referred to as embryoid
bodies (EBs) where ES cells in the absence of feeder
cells and LIF spontaneously differentiate as tissue-like
spheroids in suspension culture [3,4]. The differentiation
of EBs has been shown as aspects of early embryogene-
sis, including the formation of a complex three-dimen-
sional architecture wherein cell-cell and cell-matrix in-
teractions are thought to support the development of the

Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation379
three embryonic germ layers and their derivatives [5].
Standard methods of EB formation include hanging drop
method, liquid suspension method, and methylcellulose
culture. These culture systems maintain a balance be-
tween allowing ES cell aggregation necessary for EB
formation and preventing EB over-agglomeration for
efficient cell growth and differentiation [6]. There are
also methods for scalable production of EBs using spin-
ner flask, slow turning lateral vessel (STLV), and high
aspect rotating vessel (HARV) bioreactor techniques
[7-9].
Here, we assess mouse embryonic stem cells characte-
ristics using various techniques to gain fundamental
knowledge of their biological properties prior to investi-
gating directed differentiation. ES cell proliferation con-
ditions were optimized for different cell culture systems,
which included the optimization of cell culture surface,
cell seeding density and presence or absence of serum in
the culture medium. Moreover, EB formation was stu-
died in order to evaluate the ability of ES cells to differ-
rentiate. Assessments were performed at different time
points (after 1 day and 4 days culture) and included EB
morphology, EB diameter assessments, total cell counts
within each EB and monitoring cell viability. Although
much research is now carried out on human stem cells,
the use of animal stem cells still plays a vital role in
characterising biological structure and function.
2. Materials and Methods
2.1. Cells Culture
Mouse ES cells (cell line CEE 14) were maintained by
culturing them on the mouse embryonic fibroblast feeder
layer cells with Dulbecco’s Modified Eagles Medium
(DMEM) supplemented with 10% fetal bovine serum
(FBS) L- glutamine (2 mM), 1% Penicillin /Streptomycin
(100 units/ml/0.1 mg/ml) and 0.1% β-mercaptoethanol
and leukaemia inhibitory factor (LIF) (1000 U/ml) at
37˚C with 5% CO2.
Fibroblast feeder layer cells were mitotically arrested
by treatment with mitomycin C (0.02 mg/ml) and re-
plated at a density of ~80,000 cells per ml on gelatin-
coated tissue culture T25 flasks. ES cells were then
seeded on the feeders after being allowed to attach for
approximately 12 hours. Culture medium was changed
every day.
2.2. Cell Counting
In order to monitor ES cell proliferation under different
culture conditions, total cell number counts were per-
formed by haemocytometer. ES cell colonies were first
detached from the feeder cells by brief trypsinization
(~30 - 60 seconds) and gentle tapping of the plate dis-
lodged the colonies. Colonies were collected by aspira-
tion and trypsinized for a further 2 - 3 minutes with gen-
tle pipetting to break up the colonies and achieved an
even single cell suspension. Total cell count and cell vi-
ability was assessed by the trypin blue exclusion method.
2.3. Trypin Blue Assay
Trypin Blue is one of several stains recommended for use
in dye exclusion procedures for viable cell counting. This
method is based on the principle that live (viable) cells
do not take up certain dyes, whereas dead (non-viable)
cells do. Staining also facilitates the visualization of cell
morphology. In this study, trypin blue was widely used to
assess ES cells viability under varied culture conditions
in both two dimensional and three dimensional culture
systems.
Practically, cell pellets were harvested and re-sus-
pended to gain a homogenous suspension. Aliquot 50 µl
of the cell suspension was mixed with an equal volume
of the 0.4% (v/v) trypin blue solution and allowed to
stand for 5 - 15 min. The cell suspension and trypin blue
mixture was transferred to a haemocytometer and viable
(non-stained) and non-viable (blue) cells were counted
separately.
2.4. Alamar Blue Assay
The Alamar Blue assay incorporates a fluorometric/calo-
rimetric growth indicator based on the detection of meta-
bolic activity. The system incorporates an oxidation-
reduction (REDOX) indicator that both fluoresces and
changes colour in response to a chemical reduction of
growth medium resulting from cell growth. As cells grow
in culture, innate metabolic activity results in a chemical
reduction of the immediate surrounding environment.
Continued growth maintains a reduced environment
while inhibition of growth maintains an oxidized envi-
ronment. Reduction related to growth causes the REDOX
indicator to change from oxidised (non-fluorescent, blue)
to a reduced form (fluorescent, red). In this study, Ala-
mar blue assay was mainly used to monitor cells pro-
liferation in the continuous culture systems and further
explore the proliferation trend within specific time pe-
riod.
In general, Alamar blue stock solution was diluted into
Hanks Balanced Salts Solution (HBSS) without Phenol
Red (Sigma; Cat.No. H1387) to get 10% working solu-
tion. This working solution was mixed and sterilised with
0.2 µm filters and then kept in the dark or wrapped in foil
until ready to use.
To assess cell viability over a period of time, triplicate
samples were required for each measurement. Cells were
allowed to grow on appropriate surfaces until confluent.
Harvest and count cells and dilute to gain a cell concen-

Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation 380
tration of 1 × 105 cells/mL in complete cell culture media.
1 ml cell suspension added to each assay well of the 24
well plate and incubated at 37˚C, 5% CO2, to allow cells
to attach. The media was aspirated from the wells and
washed with 3 x warm, sterile PBS. 1 ml Alamar Blue
working solution was transferred into each well and in-
cubate at 37˚C, 5% CO2 for 90 min. 100 µl of the Alamar
Blue working solution (post-incubation with cells) was
transferred to a 96 microtitre well plate and then wrapped
in foil until ready to measure fluorescence (Ex 560 nm/
Em 590 nm). Empty wells with no cells attached were
used as blank.
2.5. Live: DeadTM Staining
The LIVE/DEAD Viability/Cytotoxicity Kit for animal
cells is a fluorescence-based method for determining
viability of adherent or nonadherent cells and for a-
ssaying cytotoxicity. The kit comprises two probes: cal-
cein AM and ethidium homodimer-1. Calcein AM is a
fluorogenic esterase substrate that is hydrolyzed to a
greenfluorescent product (calcein); thus, green fluores-
cence is an indicator of cells that have esterase activity as
well as an intact membrane to retain the esterase products.
Ethidium homodimer-1is a high-affinity, red-fluorescent
nucleic acid stain that is only able to pass through the
compromised membranes of dead cells. In this study,
live/dead staining was applied to show the viable cells
distribution with three dimensional ES cells aggregates
(EBs) after different incubation time.
In general, studied cells were allowed to growing till
confluent. After thoroughly washes, live/dead working
solution was applied to cover the cells and incubated for
30 - 40 minutes at room temperature. Remove the live/
dead working solution and wash with PBS. Fluorescent
staining can be visualized using confocal microscopy
(ex/em 495/515 nm Calcein: ex/em 495/635 nm EB).
3. Results
3.1. Maintaining Mouse ES Cells in an
Undifferentiated State and Expaneding Cell
Numbers
Mouse ES cells used in this study were maintained in an
undifferentiated state in an attempt to obtain large num-
bers of cells before initiating directed differentiation. In
order to achieve this goal LIF and mouse embryonic fi-
broblast feeder layer cells were used.
In order to study the feeder layer cells’ ability to main-
tain undifferentiated ES cells, 2 × 105 mouse ES cells
were plated on mitotically arrested mouse embryonic
fibroblast feeder layer cells in tissue culture T25 flasks
with and without LIF present in the culture medium.
Cells were incubated at 37˚C with 5% CO2 for 4 days.
During 4 days culture, ES cells morphology was ob-
served by light microscopy and total cell counts were
performed by trypin blue exclusion.
Results clearly demonstrated that mouse fibroblast
feeder layers offered a good culture environment for ES
cells in terms of the attachment and expansion. ES cells
could attach to the feeder layer cells and distinct colonies
were formed after two days of culture. Proliferation on
feeders was robust over the four days of study particu-
larly in the presence of LIF. Total cell counts revealed an
increase of up to 20 times of the original starting num-
bers (Table 1) and morphologically the ES cells formed
well defined colonies (Figure 1(a)). By comparison, ES
cell in the presence of fibroblasts and without LIF re-
vealed a lower expansion of cell numbers. Morphologi-
cally, ES cell colonies were seen to be less well defined
with colonies often touching and merging into irregular
colonies (Figure 1(b)).
The fibroblast-free culture of ES cells in the presence
of LIF showed a much lower increase in cell numbers
compared to culture on feeders and cell attachment to the
culture surface was also poor (data not shown).
3.2. Optimizing ES Cells Seeding Density
To assess for optimum cell seeding density, feeder layer
free culture was established on gelatin-coated tissue cul-
ture surface. These cells were transferred onto gelatine
coated (0.1%) tissue culture surface and cultured using
serum containing medium with LIF present in the me-
dium. Cell growth was assessed by microscopy observa-
tions and the Alamar blue assay at different time points
(on each culture day from day 1 to day10). Four different
original plating densities were used in the study; 5 × 104
cells/ml, 105 cells/ml, 2 × 105 cells/ml and 5 × 105
cells/ml.
Light microscopy observations showed that ES cells
attached to the culture plates and formed defined colo-
nies, which at the lower plating densities of 5 × 104 and
105 cells/ml, were small isolated clusters as compared to
the higher plating densities of 2 × 105 or 5 × 105 cells/ml,
where the cells formed large defined clusters comprising
Table 1. ES cell total counts cultured on mouse fibroblast
feeder layer +/-LIF in the culture medium. The original
cells plating density on each flask was 200,000 cells/ml.
Culture conditions Cells original density Cells final density
Feeder layer cellsLIF(Cells/ml) (Cells/ml)
Y Y 200,000 4,000,000
Y N 200,000 2,950,000
N Y 200,000 800,000
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Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation 381
(a)
(b)
Figure 1. (a) Morphological appearance of mouse embryonic stem cells maintained on mouse fibroblast feeder layers plus
LIF (Magnification × 10); (b) Mouse embryonic stem cells maintained on mouse fibroblast feeder layers in the absence of LIF
(Magnificat i o n × 10).

Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation 382
Figure 2. Mouse ES cell growth curves at different seeding densities with ES cell morphology after four days culture.
many more cells (Figure 2).
Alamar Blue assay showed that from initial cell plat-
ing densities of 5 × 104 and 1 × 105 cells/ml, cell num-
bers increased slowly and after 8 days in culture with
fluorescent intensity readings shifting from 2240.9 ±
24.3 units to 2212.6 ± 6.9 units and from 2961.5 ± 96.8
units to 4768.4 ± 62.4 units, respectively. From an initial
plating density of 2 × 105 cells/ml, ES cells numbers
continued to increase and after 10 days in culture, fluo-
rescence intensity was 15561.4 ± 131.8 units, re- pre-
senting an approximately 4 times increase. At the highest
investigated seeding density of 5 × 105 cells/ml fluores-
cence intensity peaked on day four at 28684.4 ± 396.1
units, roughly 3 times of the original density but then
fluorescence intensity readings showed a continual de-
creased over the remaining 5 days of the study (Figure
2). Based on these results a plating density 2 × 105
cells/ml indicated that cells remained proliferating for at
least 10 days and yielded the biggest fold increase in cell
numbers. Hence this plating density was using in the
following characterization and differentiation studies.
3.3. Investigating Cell Culture Surfaces for ES
Cell Culture
ES cells were cultured on feeder layer cells in the pre-
sence of LIF until confluent. Followed by that, the ES
cells cultured in the absence of feeders but with LIF pre-
sent in the medium on various tissue culture surfaces,
including non-treated tissue culture 6 well plates, tissue
culture treated 6 well plates, plastic petri dishes and gela-
tin-coated tissue culture-treated 6 well plates. Cells were
plated at an initial density of 2 × 105 cells/ml and adher-
ence and proliferation recorded by microscopy observa-
tions and Alamar blue assay. Microscopy images showed
that ES cells do not readily attach to the non- treated tis-
sue culture surface or to the Petri dish surface, however,
on gelatin-coated tissue culture plates; ES cells were
confluent after four days culture (Figure 3).
Alamar blue data (Figure 3) revealed that on gelatin-
coated plates, fluorescence intensity increased to 15561.4
± 131.8 units by day 7, representing an approximate in-
crease of 4 times that recorded on day 1. Alamar blue
data recorded for the non-treated surfaces where cells did
not readily adherent revealed decreases in cell number,
which makes it difficult to have maximum cells before
the commence of ES cells differentiation.
3.4. Serum and Serum Free Culture Medium
Serum (fetal bovine/calf serum, FBS/FCS) is a very com-
mon supplement for cell culture, but it should be noted
that FBS/FCS is a complex natural product and the qua-
lity and concentration of specific proteins and other mo-
lecules may vary from batch to batch and can hold a
marked effect on cell growth. Here, the expansion of
mouse ES cells in the presence of serum or serum-free
medium was evaluated (Figure 4).
Confluent mouse ES cells were plated on tissue cul-
ture T25 flasks at a density of 2 × 105 cells/ml in 10%
FBS supplemented DMEM or FBS free DMEM (sup-
plemented by L-glutamine, Penicillin /Streptomycin and
β-mercaptoethanol). Images of cells growing in the
separate culture media were taken every 24 hours in or-
der to monitor cell morphology and ability to form de-
fined clusters. After 72 hours incubation, total cell num-
ber counts and cell viability were assessed by trypin blue
extrusion methods using hemocytometer. In both
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Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation383
Figure 3. Mouse ES cell growth curves on different surfaces and ES cell morphology after four days culture.
Figure 4. ES cells expansion and proliferation wi thin serum/serum free medium. (a) Total cells count on culture day one and
day three using serum/serum free culture medium; (b) Cells colony area on culture day one, day three and day five using
serum/serum free culture medium.
FBS and FBS-free medium cells maintained similar high
levels of viability of 98.7% ± 0.4% and 99.2% ± 0.8%
re- spectively. However, cell counts revealed a marked
difference with a 17.7 ± 0.7 fold increase in cell numbers
grown in FBS supplemented culture medium compared
to an increase of 7.2 ± 0.8 fold in FBS-free medium (P =
0.0007). It should also be mentioned that during 72 hours
incubation, the size of the cell clusters within both FBS
supplemented and FBS free medium increased signi-
ficantly. The cell clusters were monitored every 24 hours
and the size was recorded. In general, 6 cell clusters were
randomly picked up from culture plate and their diameter
was determined by microscopy analysis software. The
data on colony size measurements over the 72 hours of
culture showed that in FBS medium, average cluster size
increased from 775.5 ± 82.5 µm2 to 4779.4 ± 525.4 µm2
and finally reached 11772.4 ± 1602.9 µm2. Within FBS
free culture medium, the size of cell cluster changed
from 815.1 ± 51.2 µm2 and then increased to 4554.2 ±
505.1 µm2 and finally reached 11089.2 ± 1649.3 µm2. No
significant difference between cells cluster size within
the different culture media were found. Consequently,
FBS supplemented culture medium was chosen as opti-
mum condition for ES cells expansion.

Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation 384
However, according to the special requirements for
cells differentiation, serum-free culture medium is ap-
plied to the designed ES cells differentiation culture
systems.
3.5. Induction of ES Cell Differentiation
3.5.1. ES Cell Differentiation via EB Formation on
Different Cell Culture Surfaces
ES cells were cultured on gelatin-coated tissue culture 6-
well plates in LIF supplemented culture medium at the
original density as 2 × 105 cells/ml to gain large numbers
of cells for subsequent differentiation studies. Confluent
ES cells were collected and dispersed into single cell
suspension by brief treatment with trypsin and gently
pipetting. Cells were then allowed to grow in suspension
culture without the presence of both feeder layer cells
and LIF to induce differentiation via embryoid body for-
mation. Various tissue culture surfaces were investigated
in order to study their effects on cell aggregation and EB
formation, including 10 cm Petri dish, treated and non-
treated tissue culture 6 well plates. Cell morphology was
observed after four days incubation on the different sur-
faces and culture plates.
EBs formed readily when cultured in petri dishes,
however the shape and size of EBs varied markedly.
Conversely formation of EBs was more challenging when
ES cells were plated on treated and non-treated tissue
culture surfaces, with many cells adhering to the surfaces
and few cells forming EBs. This was also confirmed
from day 1 cell culture images which showed that cells
can attach and expand on these two kinds of surfaces
(Figure 5).
3.5.2 Effect of Cell Seeding Density on EB Formation
Suspension cultures were initiated at various seeding densi-
ties of ES cells (8 × 104, 2 × 105, 6 × 105 and 106 cells/ml)
and EB formation assessed by general morphological ob-
servation and analysis of EB diameter (Figure 5 ).
The morphological observations showed that at the
lowest seeding density investigated of 8 × 104 cells/ml,
the ES cells had formed small aggregates by day 1 and
by day 4 had formed EBs over a wide range of different
sizes. At a plating density of 2 × 105 cells/ml many small
spherical EBs with fairly uniform size and shape were
formed by day 1. By day 4 the EBs had increased mar-
kedly in size and still retained a fairly uniform size range.
At a density of 6 × 105 cells/ml many EBs were formed
after day one, although EB size was quite variable. After
four days of culture EBs had grown much larger and it
was evidence then many EBs were coalescing to form
larger irregular shaped aggregates. At the highest cell
density investigated of 106 cells/ml, many EBs were
formed but with a wide range of sizes after day 1 and by
day 4 the EBs were much larger, generally more irregu-
lar in shape and many EBs had coalesced to form larger
aggregates (Figure 5).
3.5.3. Char acterization of EB Growt h
The growth of EBs was assessed by measuring average
EBs diameters and total cell counts of EBs dispersed to
single cell suspensions by trypsinization. At various time
points, an aliquot sample of EBs (The whole EBs were
evenly suspended into 2 ml culture medium and 100 µl
of aliquot was taken for EBs observations) was taken and
numbers and average diameter of EBs were recorded by
microscopy. 12 EBs were randomly picked up from 12
different observation areas and their diameters were mea-
sured by image analysis software and recorded. These
EBs were then trypsinized into single cell suspension and
total cell counts performed by haemocytometer to deter-
mine the average number of cells per EB.
At a seeding density of 80,000cells/ml EB diameters
increased in the first three days culture before level out
to reach an average diameter 140.1 ± 5.9µm by day 5. A
similar trend of an increase in EB diameter during the
first 3 days of culture before levelling out by day 5 was
seen at the other seeding densities of 200,000 cells/ml,
600,000 cells/ml and 1,000,000 cells/ml. At each of these
densities there was also an increase in average EB di-
ameter increasing from 194.2 ± 15.2 µm to 300.9 ± 15.5
µm and 328.7 ± 25.0 µm, respectively (data not shown).
3.5.4. Assessment of EB Viability by Live: DeadTM
Staining
EBs were formed in suspension in non-adherent petri
dish by plating 2 × 105 cells/ml ES cells in absence of
both feeder layer and LIF in the culture medium. Live
Dead staining results suggested that during the first day
of EB formation most cells within the EBs were viable
(Figure 6(a)) but by day 5 (Figure 6(b)) some dead cells
were observed within the EBs. After a further 10 days
culture more dead cells were seen (Figure 7).
Further analyses were performed using a Leica Con-
focal microscope to optically section the EBs and deter-
mine the numbers of Live dead cell present within the
EBs. Each EB was optically sectioned into 8 slices (the
thickness of each slices is approximately 7 µm) and the
average ratio of live to cells determined by counting vi-
able (green) and dead (red) cells. Based on this analysis,
EBs comprised 84.12% ± 8.78% live cells after 5 days
culture and after 10 days, the ratio of live cells is 85.26%
± 4.30%. The percentage of live cells in EBs remained in
the same level, which indicates EBs could be maintained
stably in vitro for relatively long term.
4. Discussion
Mouse ES cells can be maintained and their numbers
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Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation 385
Figure 5. Growth of EBs based on average diameters at different seeding densities and their morphological appearance.
Dotted arrow represents increasing EB density.
Figure 6. Growth of EBs over time based on total numbers of cells per of EB. Confocal microscopy images showing living and
dead/dying cells within EBs based on differential uptake of the Live/Dead stain at different time points. (a) EB formation day
one; (b) EBs formation day five; (c) EBs formation day ten.
Figure 7. Confocal microscopy images showing living and and dead/dying cells within EBs based on differential uptake of the
Live/Dead stain. Optical section of a day 10 EB.

Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation 386
expanded by culture on feeder layer cells with LIF pre-
sent in the culture medium. This study also showed that
changes in seeding density can significantly influence
cell number expansion rates. Culturing ES cells in the
absence of feeder layer cells and LIF stimulates EB for-
mation when cultured in non-adherent culture plates such
as Petri dishes. Formation of EBs particularly numbers,
size of EBs formed, rates of cell proliferation within EBs
and viability of cells can be controlled based on seeding
density. All these factors are important for optimizing
approaches to co-ordinate differentiation towards a spe-
cific cell type.
Stem cells can be defined as cells that must choose to
transit through alternative “gates” of self-renewal and
differentiation at each division [10]. It has been proven
that when effectively maintained under particular culture
conditions to prevent their differentiation, these cells can
self-renew continuously for a long period of time [10]. It
is particular important to maintain self-renewal capacity
by culturing ES cells on layers of mitotically-arrested
embryonic fibroblast cells and inclusion of the cytokine
LIF in the culture medium. In this study, to gain maxi-
mum amount of cells, ES cells were cultivated on gela-
tine-coated tissue culture flasks with LIF present in the
culture medium. Results indicated that cells could ex-
pand on the surface and formed well defined clusters by
applying optimized original seeding density. LIF exerts
its effects by binding to a two-part receptor com- plex
that consists of the LIF receptor and the gp130 receptor.
The binding of LIF triggers the activation of the latent
transcription factor STAT3, a necessary event in vitro for
the continued proliferation of mouse ES cells [11]. It was
also reported that two transcription factors, STAT3 and
Oct-4, may interact and affect the function of a common
set of target genes [12].
If ES cells are to be used effectively in the clinic to
treat, for example, regenerative diseases affecting tissues
like the pancreas, an important step is to be able to con-
sistently supply a large number of pluripotent cells that
can be then guided towards the desired differentiated cell
type, such as pancreatic islet cells. The purpose of this
study was to investigate the basic fundamental effects of
cell seeding densities, culture conditions on ES cell
growth and initial differentiation via EB formation with
the aim of optimizing conditions from which to investi-
gate differentiation of ES cells into pancreatic islet cells
capable of synthesizing and secreting insulin in response
to specific stimuli. Investigation of several seeding den-
sities and analysis of growth by Alamar blue assay over
several days suggested that a seeding density of 2 × 105
cells/ml produced the growth curves consistent with
rapid and sustained proliferation. At lower seeding den-
sities, cells grew much more slowly, whereas at higher
densities while cells initially grew rapidly within 4 - 5
days they apparently stopped growing and cell numbers
actually declined. The changing of cells seeding density
also produced changes in morphology of the ES cell
colonies and particularly at higher seeding densities with
the well defined colonies that characterize ES cell
growth quickly merging into and overgrowing each other
and is known to stimulate differentiation.
Serum, usually from fetal calves, is an extremely
complex mixture of many small and large bio-molecules
with different, physiologically balanced growth pro-
moting and growing inhibiting activities. A concentration
5% - 20% v/v serum is usually needed for optimum cell
growth. Due to the endocrinal differentiation require-
ments, the induction of insulin-producing cells from
mouse ES cells was carried out in serum-free conditions
in the later sections. In the context of developing culture
conditions to promote application of stem cells in the
clinic, use of FCS is a significant issue as it may contain
potentially harmful xenogeneic compounds. Bovine se-
rum proteins may be internalized in stem cells stimu-
lating immunogenicity [13,14], consequently a host of
potential problems can arise including viral trans- mis-
sion and immunological reactions due to the bovine pro-
tein attachment to cells in culture that act as antigenic
substrates once transplanted [15].
Another key goal was to evaluate serum-supplement
and serum-free culture medium and optimize the culture
medium for ES cells’ expansion. The data from these
studies demonstrated that the mouse ES cells can be ex-
panded in both serum-supplemented and serum-free cul-
ture medium. The cells remained viable over several
days of culture and formed characteristic well defined
colonies. Not surprisingly, the rate of expansion of cell
numbers was significantly lower compared to cells cul-
tured in FCS-containing medium although culture in se-
rum-free conditions still produced numbers of cells for
subsequent differentiation experiments and may have
had the added advantage that the factors to which the
cells had been exposed could be defined.
Formation of EBs has been shown to be an important
step in the process of ES cell in vitro differentiation.
Induction of EB formation in the suspension culture has
been applied to initiate the differentiation of ES cells into
a variety of differentiated cell types. For mouse ES cells,
neural progenitors [16], vascular cells [17,18], cardio-
myocytes [19], chondrocytes [20], hepatic cells [21],
insulin-producing cells [22,23] and germ cells [24] are
induced from EBs formation in suspension culture. For
human ES cells, neural cells [5], hematopoietic cells [5],
cardiomyocytes [25], insulin-producing cells [26], and
endothelial cells [27] are induced from EBs and as dis-
cussed above in the culture of ES cells induction of EB
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Assessment of Biological Properties of Mouse Embryonic Stem Cells Characteristics Prior to Differentiation387
formation can be variable and can have marked effects
on the subsequent differentiation. Suspension culture is
the most basic method that is used for EB formation
from both mouse and human ES cells and while there is a
certain amount of variability associated with this ap-
proach it generally produces the highest yields of differ-
entiating cells. Although this method can be used to get
large amount of EBs, it can only offer limited control
over the size of EBs. To improve the homogeneity of
EBs formed in suspension culture, rotating suspension
culture was introduced [28]. The use of this rotation cul-
ture system improved oxygen supply and enabled high
density culture. In this study, approaches to optimize EB
formation in simple suspension culture have been inves-
tigated focussing on investigating fundamental bio-
physical stimuli such as seeding densities, and adhesion
to cell culture plates.
Methylcellulose culture was originally employed to
form cell aggregates of a cloned origin [29]. When ES
cells are plated in semi-solid methylcellulose media, they
tend to remain as single cells and these single cells can
develop into cells aggregations (EBs). Therefore, me-
thylcellulose culture allows reproducible formation of
EBs from single ES cells and it has been used to induce
haematopoietic differentiation [30] and endothelial cells
differentiation [31].
Hanging drop method provides good control over EBs
size and shape since the number of ES cells aggregated
in a hanging drop can be controlled by varying the num-
ber of cells in the initial cells suspension. The hanging
drop method, in which cells are dispersed in 15 - 20 μl
drops suspended from the lid of a Petri dish, has been
used to more precisely control the microenvironment for
EB formation. This method is normally used to get fairly
homogeneous EBs since individual EBs are not able to
agglomerate each physically separated drops. It has been
reported that hanging drop method can be used to gener-
ate broad range of cell types including neuronal cells
[32], lymphoid [33], cardiomyocytes [34], smooth mus-
cle cells [35], chondrocytes [36], renal cells [37], adipo-
cytes [38], hepatocytes [39], insulin-producing cells [40],
and gametes [41]. However it is not ideal for large scale
applications since the liquid volume of a drop is limited
due to maintaining hanging drops on the lid by surface
tension. Medium exchange for a drop is not practically
possible and it is difficult for a direct microscopic ob-
servation during cultivation.
In this study suspension culture was used to form EBs
since there is no special requirement for the size distribu-
tion of EBs and this method holds the benefit of practi-
cally easy operation in the cells culture process. At this
experiment stage, there is no need to produce large
amount of EBs, hence large scale EBs culture system
was not introduced, such as rotary suspension culture
system.
Results also indicated that EBs are formed only on
non/low-adherent surface in the suspension culture.
Moreover, EBs should be induced only after one day in
the suspension culture with all the explored original
plating densities. It has been demonstrated [6] that indi-
vidual mouse ES cells could form EBs with high effi-
ciency (at least 42%). This finding implied the use of
methylcellulose differentiation cultures that induce single
cell colony formation. Along with the increase of ES
cells original plating density, EBs size increase within
same incubation time as well. However, after 5 days in-
cubation, the size of EBs started from 1 × 106cells/ml is
in the same level with the culture from 6 × 105 cells/ml.
Cell culture images and EBs size growth indicate that
EBs agglomerations between two or more EBs appeared
during the incubation process. In this culture system, the
control of EBs’ aggregation and agglomeration is very
important since the centre of very large aggregates may
experience cell death due to limitations in nutrients and
oxygen delivery [42]. However in this study, we de-
monstrated that with optimized culture conditions, with
plating cell density of 2 × 105cells/ml on non-adherent
Petri dish, ES cells’ viability can be maintained at high
level for at least 10 days. A two-step mechanism for
mouse EB agglomeration has been reported [43]. First,
cell-cell adhesion molecule E-cadherin was determined
to mediate attachment between neighbouring EBs. Fo-
llowing attachment, cells actively migrated and re- mod-
elled, assimilating cells into a single spheroid. Based on
this finding, it has also been demonstrated that EB ag-
glomeration can be controlled by ES cells mass encapsu-
lation [44].
5. Conclusions
An optimized cell culture condition has been determined
which can maintain ES cells in an undifferentiated stage
and holding high self renew ability. After large amounts
of ES cells were achieved, EBs were formed in suspen-
sion culture and the size and agglomeration of EBs can
be controlled by optimizing starting ES cells densities.
ES cells viability remained at a high level within EBs
structures during in vitro incubation. High standard ES
cells culture can strongly secure the reproducible ES
cells based differentiation strategy.
Considerable research is now carried out on human stem
cells, but the use of animal stem cells still plays a crucial
role in characterising biological structure and function.
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