Initial observations 24 hours after cell culture preparation indicate no distinct differences in cell
growth or confirmation between wells that contain different media types and substratum
components. The same results for cell growth density were observed after 48 hours. The highest
concentration of cells occurs in the center of each well, with a clear decrease at the far edges of
the periphery. Cells in close contact with other cells, which appear to be initiating tissue
formation, are just as numerous as those that remained independent. Overall confluency is
approximately 50-60%. Cell shape varies as a result of proximity to other cells. Individual cells
exhibit a fan-shape appearance, while those in close contact have a cuboidal shape and are
arranged in a mosaic pattern. Nuclei are clearly visible in all cells.
After 72 hours, all wells were 100% confluent and the majority of cells exist in a single
layer tissue structure in a cuboidal confirmation. Cell density is equivalent in all media or
substratum conditions. With respect to cell confirmation, in growth and differential media, cells
exhibited a similar morphology. The vast majority of cells are cuboidal shaped arranged in a
mosaic pattern. The cells in basal media, specifically those grown on the plastic substratum,
appear slightly rounder at the edges and more three-dimensional shape. All cells in other basal
substratum conditions, as well as those in growth and differential media appeared flat and
formed uniform single layer tissues. The edges of the cells in growth media were slightly
rounder and brighter in comparison to those in differential media. In all media and substratum
types, cell nuclei were well defined with dark edges and two or three darker sports inside each.
The nucleus takes up the majority of internal cell space.
Prior to the addition of Apoptotic reagents (t = 7 days or 168 hours), all well conditions
appeared nearly identical with respect to all physical cell characteristics. Growth appears to have
slowed down overall as a result of the similar density of the single tissue layer as compared to
the last recorded observations (t = 72 hours) in addition to the appearance of minimal cellular
debris, prior to a change in media. Cell size and shape is consistent across all wells, and each
tissue appears indistinguishable from others grown in alternate conditions (i.e., media and
substratum). Distinctions appeared in the well with basal media on a substratum consisting only
of plastic. These cells were slightly rounder and thicker than their counterparts in other
wells(FIGURE1). Cells in growth media were brighter around the edges, but similar in all other
aspects to the tissues proliferating in basal or differential media(FIGURE2).
Observations five hours (t = A5) after the addition of Apoptotic reagents (Stauosporine to
cells previously in Differential Media, and Thapsigargin to those previously in Growth), few
changes were visible. Basal cells are indistinguishable from their appearance five hours ago.
Overall, all cells subject to apoptotic reagent addition appear slightly grainy, but the effect is
minimal. The only visible death is a few necrotic cells in all media types. These cells are
floating above the substratum, with internal components condensing and overall clearing of the
cytoplasm without any change in cell size.
All basal wells have a similar appearance, and the biggest change after 18 hours (t = A18)
is a slight overall rounding of the cells (edges and more three dimensionally, they are not as flat
as they were prior to addition of reagents). The most concentrated addition of Staurosporine,
5.0uM, has resulted in widespread cell death. The majority of cells have shrunk in size and
appear shriveled (FIGURE3). For the first time prior to reaching confluency, the substratum is
visible. Some cells have clumped together, while others are separate and equidistant from all
other cells. The lower concentration of 1.0uM Staurosporine has had no visible effects on tissue
growth as compared to the nature of the cells prior to the addition of the apoptotic reagent.
Subsequent decreased concentrations exhibit the same phenomenon.
Thapsigargin in its most concentrated dilution, 5.0uM, also had a much smaller effect on
this culture(FIGURE 4). Cells appear viable, but are no longer in a flat, uniform monolayer. All
cells are rounded and their morphology is similar to that of independent cells prior to reaching
100% confluency. In addition, there is also an increase in overall cell brightness.
FIGURE 1
FIGURE 2
FIGURE 3
FIGURE 4
Methods and Materials
Media for Cell Line Three media types were used to grow MCF-10-2A, non-tumorogenic,
transformed epithelial cells from a fibrocystic disease in a mammary gland(ATCC). Basal media
was comprised of a 2% serum of fetal origin, essential components to support growth (e.g.,
glucose, essential Amino Acids, vitamins, isosmotic H2O via small ions), and appropriate buffers.
In addition to Basal constituents, Growth media contained epidermal growth factor (EGF) and
insulin , whereas Differential Media added the hormones Insulin, Cortisol and Prolactin to Basal
Media.
Well Preparation The comparison of growth patterns in different media types was done using
four variable well conditions. Each of four types contained an extracellular matrix component,
either collagen IV, fibronectin, laminin or control blank (i.e., plastic) which was applied prior to
seeding each well.
Preparing Cell Cultures In order to seed the wells with an equal number of cells, the prepared
culture was split from a single 75 cm2 flask and quantified using a hemocytometer. The split
was performed in a sterile hood using pre-warmed media and reagents. Media was aspirated off
and 10 mL BSS (wash solution) was added and immediately removed. 4 mL BSS-trypsin was
added and incubated for 5-10 minutes or until cells are spherical and fully non-adherent. 2 mL
of F12-Growth medium was placed in a 15 mL Falcon tube to which the trypsinized cells were
also added. 6 mL of F12-Growth medium was added to the 75 cm2 flask to remove any
remaining cells and transferred to the 15 mL Falcon tube. Contents were centrifuged and media
was aspirated and pellet resuspended in 1mL F12-Basal medium and 3 mL regular Basal medium.
0.2 mL of resuspension were mixed with 0.2 mL of 0.4% Trypan Blue. The mixture was applied
to a hemocytometer in order to determine cell density and amount needed to achieve desired well
seeding density of 1.0 x 10^5 cells/well.
Feeding Cells All media types were aspirated and replaced with fresh media of same
composition after a period of 24 hours. Cells were subsequently fed in an identical manner every
48 hours, and growth patterns were observed and recorded via phase contrast microscopy.
Addition of Apoptotic Reagents After 7 days of incubation at 32°C, 5% CO2 media was
replaced with either basal media or four sequential dilutions (5.0 uM, 1.0 uM, 0.5 uM, 0.1 uM)
of Staurosporine (in what had been differential media) and Thapsigargin(in what had been
growth media). Observations were made after 5 and 18 hours.
Results
Cell Culture in Basal, Differential and Growth Medium
After 24 hours (t=24), the first media change was performed, and cells were observed.
There were no clear distinctions in growth patterns between media type. Cells were either fanshaped and independent from one another or cuboidal and arranged in a mosaic pattern. The
mosaic pattern appears to be the initiation of tissue formation, with the cells in this arrangement
appearing to be of similar size, shape and thickness. Neither cell morphology was more
prevalent in each field of vision; independent cells were evenly dispersed between clumps of
cells, which appear to be initiating tissue formation. Cells covered just over half the area of each
well, indicating that they were 50-60% confluent.
At 72 hours (t=72), cells were 90-100 % confluent in all well experimental types. There
are no distinctions in growth patterns among any of the given substratum components or various
media types: basal, growth, differential. Cells are actively growing and dividing as indicated
through the appearance and actions of now visible nuclei. Some cells appear to be multinucleated, suggesting cell division is about to occur. Nuclei are one of the only visible internal
cell components, and they take up the majority of the space. This phenomenon is apparent in all
cells, which regardless of media and substratum components in the well, appear homologous
with respect to size and shape. All cells exist in a single tissue layer with no visible overlaps or
rounding. However, at a certain point in the periphery, there is a clear demarcation between cells
that are part of the monolayer versus those that remained independent. These single cells still
exhibit a fan-like conformation.
Just prior to the addition of apoptotic reagents (t=7 days), cell density, uniform
throughout, is slightly greater than previously seen at 72 hours after initial seeding. The cells are
now approximately 100-110% confluent. The first signs of cell death due to necrosis are visible,
particularly in basal media wells. These cells are round and larger than those in the flat
epithelium-like tissue, which underlies this cell type. The internal components of these cells are
condensing into bright spots while the cytoplasm is much clearer in comparison to viable cells.
Tissue formation occurring in growth medium exhibits a brighter outline around cells. In
addition, the basal, plastic monolayer is comprised of cells that are visibly rounder than cells in
other wells although they appear to be just as numerous.
Addition of Apoptotic Reagents: Staurosporine and Thapsigargin
Observations five hours (t = A5) after the addition of Apoptotic reagents (Stauosporine to
cells previously in Differential Media, and Thapsigargin to those previously in Growth), few
changes were visible. Basal cells are indistinguishable from their appearance five hours ago.
Overall, all cells subject to apoptotic reagent addition appear slightly grainy, but the effect is
minimal. The only visible death is a few necrotic cells in all media types. These cells are
floating above the substratum, with internal components condensing and overall clearing of the
cytoplasm without any change in cell size.
All basal wells have a similar appearance, and the biggest change after 18 hours (t = A18)
is a slight overall rounding of the cells (edges and more three dimensionally, they are not as flat
as they were prior to addition of reagents). The most concentrated addition of Staurosporine,
5.0uM, has resulted in widespread cell death. Most cells have shrunk in size and appear
shriveled (FIGURE3). For the first time prior to reaching confluency, the substratum is visible.
Some cells have clumped together, while others are separate and equidistant from all other cells.
The lower concentration of 1.0uM Staurosporine has had no visible effects on tissue growth as
compared to the nature of the cells prior to the addition of the apoptotic reagent. Subsequent
decreased concentrations exhibit the same phenomenon.
Thapsigargin in its most concentrated dilution, 5.0uM, also had a much smaller effect on
this culture(FIGURE 4). Cells appear viable, but are no longer in a flat, uniform monolayer. All
cells are rounded and their morphology is similar to that of independent cells prior to reaching
100% confluency. In addition, there is also an increase in overall cell brightness.
Discussion
The hallmark of the observations made on the non-tumorogenic fibroblast cells was the
consistency of growth trends and cells morphologies given a variety of media and substratum
conditions. The similarity in the time it took for each test well to reach various levels of
confluency (TABLE) implies that components of the media and substratum had minimal effects
on cell proliferation. This was not the case, however, with Tumorgenic cell lines, which showed
reactions specific to single experimental conditions (other people’s data – cell lines that did
exhibit differences – which media type was best).
Literature review indicates that growth and differential media constituents should
decrease the time it takes for each well to become 100% confluent. The presence of insulin
increases the uptake of glucose and galactose, providing a larger amount of an essential source of
energy for metabolic activities, such as cell growth and division, in comparision to basal media,
which lacks the addition of this hormone (FIND REF). Differential media contains cortisol and
prolactin as well (FIND OUT WHAT THEY DO). The combination of these additional
supplements should provide for an even greater increase in density of the cell culture. This
hypthesis was not consistent with observations made on a transformed non-tumorogenic culture,
MCF-10-2A.
(OTHER DATA – where growth was best)
(why ours didn’t grow)
Cells exhibited one of two main morphologies, either a slightly rounded, fan shape or a
flat, seemingly adherent, cuboidal cell. These conformations were characteristic of independent
or confluent cells in contact with one another, respectively. The change in shape is indicative of
either an internal or external change in cellular activity, or possibly a combination of the two
factors. This might include (FIND OTHERS AND SOURCES – protein interactions,
cytoskeleton, and a change in receptor and ligands expressed). The novel interactions between
receptors and ligands that were not previously expressed in independent cells would generate
new intracellular events that could influence overall shape (EXAMPLES). Cell to cell
interactions, such as binding, signaling, or secretions, are now a likely explanation for this
phenomenon, as cuboidal cells exist in a mosaic pattern, which would enable cross talk between
cells.
Observations of tissue formation indicate that the cells that comprise this structure are
uniform in comparison to one another. All cells appear to be of approximately the same size,
shape and thickness in the same plane of growth. The only exception was seen in a single well
of control (basal) media, which ironically lacked any test substratum. These cells were slightly
rounder and appeared less adherent. The other substratum placed in the wells prior to seeding,
Collagen IV, Laminin, Fibronectin, indicated no distinction in either tissue characteristics or
adhesion in any instances of tissue formation. Previous(MAMMARY GLAND – secrtory tissue)
-probs with cell culture
-what we can’t observe, how substratum help mimick notmal
-previous lit on basement membrane
-us: no effect? Greatest importance in cell-cell interaction (END OF DISCUSSION)
(Apoptosis Discussion)
Senescense is an event that contributes to the formation of specific types of tissue. This
can occur by two morphologically and functionally distinct methods, Necrosis and Apoptosis.
The predominant form of cell death observed in a non-tumorogenic epithelial cell culture derived
from a fibrocystic disease appeared to be Necrotic in nature. Phase contrast microscopy
indicated that cells located above the cultured monolayer of non-tumorogenic epithelial cells
exhibited characteristics of necrosis, including cell swelling which is a result of membrane
disruption. Large bright spots inside the cell are indicative of the condensation of internal
components, another hallmark of necrosis. (GET NECROSIS PIC)(Los et al, 2002 ). Necrotic
cells were found sporatically throughout all wells in extremely low concentrations. The lack of
constency and the presence of programmed cell death, or apoptosis, indicates that this cell
culture is not actively participating in cell death at this stage. After seven days of observations,
though cell cultures were 100% confluent, cellular constituents of each monolayer appeared
smaller at each time interval, indicative of continued growth rather than death or elimination of
cells.
Lack of apoptosis in the first phase of cell culture may be attributed to either internal or
external factors. This cell line may not utilize this mechanism or it may not yet have reached a
stage in its life cycle that exhibits apoptosis. The MCF-10-2A cell line does have the ability to
undergo apoptosis, as effects were seen in a dose dependent manner using apoptotic reagents
Thapsigargin and Staurosporine. As expected, cultures treated with Staurosporine show signs
indicative of apoptosis, including reduced size and apparent chromatin condensation, indicated
by bright spots in each apoptotic cell (Figure S5). The effect was greatest at a concentration of
5.0uM, and concentrations less than 1.0uM indicated no signs of apoptosis. This is most likely a
result of decrease reagent concentration and not attributed to cell culture characteristics, as
growth was similar in all wells regardless of substratum components prior to addition of
Stauosporine, and control wells with just basal medium continued to indicate identical growth
patterns in all substratum types.
The reaction to the addition of Thapsigargin is not easily identified as apoptotic. The
only reaction was seen in a concentration of 5.0uM, and cells appear slightly smaller, but
rounded and bright along the edges (Figure T5), not commonly seen in apoptotic cells. The
physical changes in the cell line might be attributed to a reaction between Thapsigargin and the
substratum or media components (collagen IV and differential, respectively), which altered
certain pathways. A lack of previous experimentation on the effect of Thapsigargin on
mammary epithelial cells makes it difficult to attribute the effect to a specific causal agent.
-compare to BK+AF: maybe thapsigargin needs longer (they had bright edges)
most of results can be attributed to diff in starting conditions
Apoptotic sources
1. Los, M., Mozoluk, M., Ferrari,D., Stepczynska, A., Stroh,C., Renz, A., Herceg, Z., Wang,
Z., and Klaus Schulze-Osthoff. 2002. Activation and Caspase-mediated Inhibition of
PARP: A Molecular Switch between Fibroblast Necrosis and Apoptosis in Death
Receptor Signaling. Mol Biol Cell. 13(3): 978–988.
Figure S5 Staurosporine (5.0uM)
Figure T5 Thapsigargin (5.0uM)