Mammalian cell culture growth response to animal serum

1Experiment 1 – Mammalian Cell Cultures

Mammalian cell culture: the growth response of cells to varying animal serum concentrations

IntroductionFor 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

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acts as a buffer to help maintain a constant pH and simulates human osmolality.

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.

MethodThe 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 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

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).

Day 1 Day 2 Day 3 Day 40.000

0.100

0.200

0.300

0.400

0.500

0.600

590 nm of Cell cultures

HeLa DMEM/2% FCSHeLa DMEM/10% FCSBHK GMEM-BHK/2% FCSBHK GMEM-BHK/10% FCS

Age of cell culture

590

nm

Figure 2

Appendix D

Figure 2 shows the readings of the 4 day samples from HeLa and BHK cell cultures at 590nm.



Figure 3Appendix 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).

BHK 2%FCS BHK 10% FCS

HeLa 2% FCS

HeLa 10% FCS

0102030405060708090

Doubling Time (Hours)

Doubling times Time (Hours)

Cell type and FCS %

Tim

e (H

ours

)

Figure 5

Appendix GFigure 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 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.

ConclusionTo 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 dataDMEM with FCS concentraion Day 1 Day 2 Day 3 Day 4

DMEM/2% FCS

40% Coverage, cells show signs of dividing and growing, no contaminates.

50% Coverage, cells mostly in middle

60% Coverage, Pink

70% Coverage, pink/purple

DMEM/10% FCS

60% coverage, mild contaminents, cells compact

65% coverage, cells clustered

80% Coverage, Orange/pink

85% Coverage, light pink

Appendix B

BHK Cells –Observational DataGMEM-BHK with FCS concentraion Day 1 Day 2 Day 3 96 hours

GMEM-BHK/2% FCS

20% coverage, cells spaced out

25% Coverage, cells spaced out

30% Coverage, orange pink

20% coverage, cells dying, as medium evaporated, lid upside down

DMEM-BHK/10% FCS

25% coverage, cells evenly spread, some bunched together.

40% Coverage, clusters

75% Coverage, Peach colour

100% Coverage, Peach colour

Appendix C

Quantity

Cell type FCS concentration

24 hours

48 hours

72 hours

96 hours



HeLaDMEM/2% FCS 990

µl975 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 590nmCell type FCS concentration

Day 1

Day 2

Day 3

Day 4

HeLaDMEM/2% FCS 0.10

40.14

00.22

00.23

5

DMEM/10% FCS 0.111

0.260

0.229

0.445

BHKGMEM-BHK/2% FCS 0.13

00.15

60.21

30.12

7GMEM-BHK/10% FCS

0.246

0.244

0.449

0.511

Appendix E

Standard CurveBSAµl

Water µl

BSA µl / Water µl

590nm Reading

0 50 0 0.0005 45 0.1 0.083

10 40 0.2 0.13715 35 0.3 0.21920 30 0.4 0.26125 25 0.5 0.30030 20 0.6 0.35235 15 0.7 0.49240 10 0.8 0.52245 5 0.9 0.57650 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

HeLaDMEM/2% FCS 6203508.772 8350877.193 13122807.02 14017543.86DMEM/10% FCS 8350877.193 15508771.93 13659649.12 26543859.65

BHKGMEM-BHK/2% FCS 3668171.558 4401805.869 6010158.014 3583521.445GMEM-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

Appendix G



Cell doubling times:HeLa – DMEM/2% FCS = 78 – 30= 48 hoursHeLa – 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.

Doubling times

Cell type Time (Hours)

BHK 2%FCS 80BHK 10% FCS 30HeLa 2% FCS 48

HeLa 10% FCS 44


Mammalian cell culture growth response to animal serum

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