Cell based antiviral assays for screening and profiling of Anti dengue agents
Mammalian cell culture growth response to animal serum
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Experiment1– MammalianCell Cultures
Matt Dower- 1021492
Mammalian cell culture: the growth response of cells to varying animal serum
concentrations
Introduction
For the study of mammalian cells to take place in vitro cell cultures needs to be created. In
the laboratory the most commonly used mammalian cell is the HeLa cell, followed closely by
Baby Hamster Kidney cells (BHK), mostly used to study transfections and the growth of
viruses.
These cells require a medium to grow on; Dulbecco’s Modified Eagle Medium (DMEM) and
Glasgow Minimum Essential Medium (GMEM) are both frequently used. To aid further in the
growth on these mediums some essential compounds are needed, these are usually
provided in the form of foetal calf serum (FCS) also known as foetal bovine serum.
HeLa cells originated in 1951 from the cervical cancer patient Henrietta Lacks (hence the
name HeLa). Studies of these cells revealed them to be extremely robust and reproduce
quickly, even faster than cancer cells, and were later identified to be in an immortal cell line.
This means, due to a mutation, that they can grow over very long periods in vitro because
of their over active telomerase during cell division. These telomerase prevent the shortening
of telomerase which is responsible for aging and cell death, making them ideal for laboratory
work.
Baby Hamster Kidney Cells (BHK) came to light in 1961 from 5 Syrian hamsters at 1 day old.
They are fibroblasts meaning they play an important role in the structure of animal tissues.
They synthesise collagen, a protein abundant in the connective tissue and flesh of
mammals, reticular, glycosaminoglycans, glycoproteins and elastic fibres.
DMEM basic components are glucose, non-essential amino acids, sodium pyruvate, vitamins,
potassium and magnesium.
GMEM is a modified version of DMEM as it contains 10% more tryptose phosphate and twice
the concentration of amino acids and vitamins, the standard form of this medium contains
800 mg/L glycine, 110 mg/L sodium pyruvate 2750 mg/L sodium bicarbonate, without ferric
nitrate and L-glutamine. It was initially created for the study of the polyoma virus on BHK
cells.
Both of these mediums contain phenol Red, a red pigment that changes colour depending
on its pH, making it a useful tool when estimating the size of cell cultures.
Foetal calf serum (FCS) is the plasma of coagulated blood after the plasma protein
fibrinogen is converted into fibrin during the healing of wounds. As its name suggests, FCS
is extracted from a bovine foetus and contains relatively no antibodies and a high level of
growth factors making it the most widely used serum-subsidiary when dealing with in vitro
eukaryotic cell cultures.
Phosphate buffered saline (PBS) is a water-based salt solution containing sodium chloride,
potassium phosphate , sodium phosphate, and potassium chloride. It acts as a buffer to
help maintain a constant pH and simulates human osmolality.
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The purpose of this practical is to understand the growth response and protein
concentration of Hela Cells on DMEM with 2% and 10% concentration of FCS, and BHK cells
on GMEM with 2% and 10% concentration of FCS.
Method
The method of this experiment can be seen in close detail on page 8-16 in the lab manual.
This section will go into detail on the purpose of these methods and how they aid the
experiment.
The cells were observed under a microscope every day for 4 days (Appendix A-B). A cell
suspension was obtained using aspiration and a mixture of trypsin and versene.
Trypsin causes lysis in cells, useful for obtaining cells from a petri dish and versene provides
protection against metal-catalysed reactions that can cause harmful reactions and adverse
results. After a cell concentration was calculated using a Neubauer counting chamber.
Next the cell underwent centrifugation, it was necessary at this step to wash the cell pellets
in media +2% FCS before re-plating them in medium, whether it was 2% or 10% serum.
This is so the cells would still be supplied with the necessary growth factors and not dry out
whilst being replated, as well as becoming accustom to the stresses of the laboratory.
If they had been washed in 10% serum it would have made the results of the 2% serum
inaccurate as they are exposed to a higher level of growth factors.
The 10% FCS cell cultures on the other hand was unaffected as they are exposed to less
serum than they were during the experiment.
The solution was then diluted to 5x104
cells/ml with their respected serums and plated out
on 4 dishes each, distributed evenly.
Standard pellets were obtained by adding 0.4 ml of the suspension to a microfuge tube and
centrifuged, aspirated and centrifuged again.
These standard cells pellets were then washed with PBS before being frozen for study on
the final day.
This is necessary as the cells metabolic rate slow down when frozen, therefore changing its
pH. The PBS maintained this pH and kept the cells alive for longer in the cold unfavourable
conditions. It also wipes the cells of any irrelevant extracellular proteins; water cannot be
used as it causes the cells to burst due to the osmolarity. It also formed a thin film of water
around the cell pellets to prevent denaturation or conformational changes.
This helps make the results of this experiment more accurate as the standard pellets will
stay preserved from day 1 and a true comparison can be made to the other cultures on day
5.
A number of aseptic methods were put in place to prevent contamination such as the
flaming of glass pipettes and use of sterile plastic pipettes which are changed after every
use. The petri dish lids were taken off for the shortest time possible when aspirating or
adding a solution, and a number of processes were done near a Bunsen burner as the hot
air rises and pushes away any contaminants.
Day 2, one of the 4 cell cultures were harvested through a series of aspirations and washed
with PBS, marking the first time point at 24 hours, then were put into microfuges and stored
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on dry ice (-70°C). The same process was repeated for the next 3 days, marking the
respected time periods, 48, 72 and 96.
The cells were then lysed with cell lysis buffer and an assay was done on the standard
pellets to obtain a standard curve (Appendix E), the same process was repeated on the
samples obtained throughout the week and a protein concentration was determined using
the standard curve. (Appendix D)
The total cell number was also calculated using the standard pellets and absorbance
readings at 590nm, the doubling time is calculated from this (appendix G, Figure 5)
Equipment list
For a detailed list of the equipment used in this experiment refer to pages 9 and 10 of the
lab manual. This list consists of the essentials used throughout the experiment.
DMEM/2% FCS
DMEM/10% FCS
GMEM-BHK/2% FCS
GMEM-BHK/10% FCS
Versene
Trypsin Solution
FCS
Neubauer counting chamber
Aspirator
PBS
Bio-Rad protein assay reagent
Microfuge tubes
Results
Appendix E
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Figure 1 shows the readings of a series of dilutions from 50µl of standard protein in water
from 0mg/ml to 1 mg/ml. The readings were taken 590nm and were used to calculate the
protein concentration in the HeLa and BHK cultures (figure 3).
Appendix D
Figure 2 shows the readings of the 4 day samples from HeLa and BHK cell cultures at
590nm.
0.000
0.100
0.200
0.300
0.400
0.500
0.600
Day 1 Day 2 Day 3 Day 4
590nm
Age of cell culture
590 nm of Cell cultures
HeLa DMEM/2% FCS
HeLa DMEM/10% FCS
BHK GMEM-BHK/2%
FCS
BHK GMEM-BHK/10%
FCS
Figure 2
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Figure 3
Appendix D + E
Figure 3a and 3b used the standard curve to estimate the protein concentration present in
each of the cell cultures over 4 days.
Figure 4 shows the total cell number present in each cell culture. This was calculated using
the standard curve (Figure 1) as the total cell number and absorbance of the standard cell
pellet was known (Appendix F).
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Appendix G
Figure 5 shows the doubling time of each cell type in a bar chart for simple visual
comparison making it easier to draw a conclusion from.
Discussion
From the observational data (Appendix A-B) it can be seen that the cell coverage of the petri
dish increases over the 4 days in all cases, meaning that the cells were growing. Both the
GMEM and DMEM possess pH indicator in the form of its colour. In all cases it starts as red
and pH 7.0-7.4, but over time it changes colour to pink, orange and peach depending on the
pH level.
The DMEM in the 2% FCS HeLa culture had change to pink by day 3, then purple by day 4
with 70% coverage meaning its pH has become more alkaline.
The DMEM in the 10% FCS HeLa culture changes to orange in day 3, then to light pink by
day 4, again meaning its pH is alkaline, with an estimated 85% coverage.
The GMEM in the BHK/2% FCS culture was orange/pink by day 3, however the petri dish of
the 4th
day sample had been turned upside down, resulting in the culture falling into the lid
and evaporating whilst in incubation, meaning many of the cells had died, resulting in a drop
from 30% coverage to 20% coverage.
The GMEM in the BHK/10% FCS culture was a peach colour by day 4 with 100% coverage,
meaning the pH has become more acidic.
From the observational data there seems to be a correlation in the increase of cell numbers
and pH, in the HeLa culture, more cells result in a lower alkaline pH.
In the BHK cultures the increase in cell numbers results in a peachy colour with a more
acidic pH.
A reason for this change in pH may be due to the uptake of amino acids from the medium
as well as products of metabolic reactions.
The cell culture coverage of the petri dish may be slightly inaccurate as the process of
0
10
20
30
40
50
60
70
80
90
BHK 2%FCS BHK 10%
FCS
HeLa 2%
FCS
HeLa 10%
FCS
Time(Hours)
Cell type and FCS %
Doubling Time (Hours)
Doubling times Time
(Hours)
Figure 5
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Matt Dower- 1021492
estimation comes with experience, which may be lacking.
Figure 1 is a standard curve of the BSA/Water that has been used to determine the total
protein concentration released from each culture using the absorbance readings from Figure
2, these results can be seen in figure 3a and 3b.
There is a positive correlation between the age of the cell cultures and the estimated
number of cells as they had more time to undergo mitosis. The more cells present the
higher protein concentration mg/ml as the cells produce the protein (appendix F + Figure
3a-3b). This is true of both the HeLa and BHK 10% FCS cultures as they had a higher
protein concentration than their 2% counterparts.
This might be because of the more epidermal growth factors (EGF) and fibroblast growth
factors (FGF) present in the 10% FCS than the 2% FCS. EGF regulate cell growth,
differentiation and proliferation making them extremely useful for the cells growth, they bind
using three intermolecular disulphide bonds making it stable in various conditions such as
the 37 ℃ incubator.
FGF are heparin-binding proteins and involved in embryo development, proliferation, wound
healing and angiogenesis; the growth of blood cells from other blood cells. As it is a mitogen
it plays a major role in triggering cell division giving rise to cell numbers.
The cell doubling time was calculated using the standard cell samples and reading from the
graph the time it takes for the cells to double in population (Figure 5, appendix G).
The BHK 10% FCS had the fastest doubling rate at 30 hours followed by HeLa 10% FCS
with 44 hours then HeLa 2% with a doubling rate of 48 hours.
The BHK 2% FCS doubling time was obtained using the first 3 samples, 24, 48 and 72 hours
due to the evaporated of the medium, as the cell number was not a true representation of
the growth rate, yielding 80 hours, the slowest doubling time.
The reason for the BHK 10% FCS having the fastest doubling time may be due to the GMEM
as it contained 10% more tryptose phosphate and twice the concentration of amino acids
and vitamins than DMEM. GMEM was also specifically designed for the growth of BHK
whereas DMEM was created for a wide range of cell types including monkeys, humans, fish
and rats, so therefore is not specific for the growth requirements of HeLa cells.
HeLa 10% FCS was second with a doubling rate of 44 hours. This could be due to the HeLa
cell line being immortal, meaning they can divide unlimited amounts of time in a laboratory
when presented with sustainable conditions, which the DMEM provided.
On the other hand the HeLa 2% FCS had a doubling time of 48 hours, suggesting that the
difference of 4 hours in doubling time was, again, due to the 10% and 2% FCS that
provided the cultures with EGF and FGF (see above).
The difference in doubling time between the BHK 2% FCS and BHK 10% FCS was 50 hours
suggesting that the doubling time was highly reliant on the FCS and not the GMEM, whereas
the different in HeLa was not so extreme suggesting it has a faster natural growth rate not
as dependent on provided compounds, supporting the immortal cell line hypothesis.
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Conclusion
To conclude, the data suggest that as cell numbers increase so does the protein
concentration. It also puts forward that BHK 10% FCS has a faster doubling time than HeLa
10%, whereas HeLa cells may have a faster natural growth rate when mediums are
excluded. However this may be false as the 2% BHK lacks accuracy as only 3 of the 4 data
points where used.
Although the standard curve shows that the assay ceases to be linear at higher absorbance
values and so protein inputs determined from the upper part of the standard curve are less
accurate than those from the lower part, as well as the absorbance readings as very low
amounts of light are transmitted through the sample. Both BHK 10% FCS and HeLa 10%
FCS results are taken from the higher end of the standard curve making their results and
the data discussion less accurate.
Overall for a more accurate conclusion the BHK and HeLa should be grown in both the
GMEM and DMEM mediums with the FCS to eliminate any differences in growth factors,
therefore a result based solely on the cell type can be obtained.
By Matt Dower
Appendix
Appendix A
HeLa Cells – Observational data
DMEM with FCS concentraion Day 1 Day 2 Day 3 Day 4
DMEM/2% FCS
40% Coverage,
cellsshow
signsof
dividingand
growing,no
contaminates.
50%
Coverage,
cellsmostly
inmiddle
60%
Coverage,
Pink
70%
Coverage,
pink/purple
DMEM/10% FCS
60% coverage,
mild
contaminents,
cellscompact
65%
coverage,
cells
clustered
80%
Coverage,
Orange/pink
85%
Coverage,
lightpink
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Appendix B
BHK Cells –Observational Data
GMEM-BHKwith FCS concentraion Day 1 Day 2 Day 3 96 hours
GMEM-BHK/2% FCS
20%
coverage,
cellsspaced
out
25%
Coverage,
cellsspaced
out
30%
Coverage,
orange pink
20%
coverage,
cellsdying,
as medium
evaporated,
lidupside
down
DMEM-BHK/10% FCS
25%
coverage,
cellsevenly
spread,some
bunched
together.
40%
Coverage,
clusters
75%
Coverage,
Peach
colour
100%
Coverage,
Peachcolour
Appendix C
Quantity
Cell
type FCS concentration
24
hours
48
hours
72
hours
96
hours
HeLa
DMEM/2% FCS
990
µl
975
µl
975
µl
1000
µl
DMEM/10% FCS
1080
µl
980
µl
970
µl
1000
µl
BHK
GMEM-BHK/2% FCS
985
µl
985
µl
965
µl
1000
µl
DMEM-BHK/10%
FCS
985
µl
970
µl
975
µl
1000
µl
Appendix D
Absorbance at 590nm
Cell
type FCS concentration
Day
1
Day
2
Day
3
Day
4
HeLa
DMEM/2% FCS 0.104 0.140 0.220 0.235
DMEM/10% FCS 0.111 0.260 0.229 0.445
BHK
GMEM-BHK/2% FCS 0.130 0.156 0.213 0.127
GMEM-BHK/10% FCS 0.246 0.244 0.449 0.511
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Appendix E
Standard Curve
BSAµl
Water
µl
BSA µl / Water
µl
590nm
Reading
0 50 0 0.000
5 45 0.1 0.083
10 40 0.2 0.137
15 35 0.3 0.219
20 30 0.4 0.261
25 25 0.5 0.300
30 20 0.6 0.352
35 15 0.7 0.492
40 10 0.8 0.522
45 5 0.9 0.576
50 0 1 0.723
Appendix F
Cell number
A590
nm
HeLA 1.02x106
0.171
BHK 1.26x107
0.443
Total cell numbers.
Cell culture age (hours)
Cell
type
FCS concentration 24 48 72 96
HeLa
DMEM/2% FCS 6203508.772 8350877.193 13122807.02 14017543.86
DMEM/10% FCS 8350877.193 15508771.93 13659649.12 26543859.65
BHK
GMEM-BHK/2% FCS 3668171.558 4401805.869 6010158.014 3583521.445
GMEM-BHK/10% FCS 4119638.826 6884875.847 12669300.23 14418735.89
Example of calculation for HeLa DMEM/2% FCS 24 hours:
0.104/0.171 x 1.02x106
= 6203508.772
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Appendix G
Cell doubling times:
HeLa – DMEM/2% FCS = 78 – 30= 48 hours
HeLa – DMEM/10% FCS = 34 - 78 =44 hours
BHK – GMEM/2% FCS (based on 24, 48 and 72 samples) = 54 – 34 = 20 x 4 = 80 hours
BHK – GMEM/10% FCS = 64 – 34 = 30 Hours
The 20 x 4 because 20 hours is time to increase by 10000000, we want to know how long it
takes to increase by 40000000.
