Exercise 3: Cell Culture
Purpose: To compare the growth of cells in vitro under different conditions.
Introduction:
Cell Growth Curve
Techniques for growing cells in the laboratory were originally developed by
microbiologists. The rapid generation time, autonomous growth and relatively few
nutritional requirements of most microorganisms made it easy to provide the cells with
an optimal environment and avoid contamination by other organisms. In the past 50
years, these techniques have been extended to the culture of cells from higher plants
and animals. Using cell culture, one can address questions relating not only to cell
growth per se, but also to nutritional requirements, metabolic activity, gene expression
and regulation, motility, as well as features of differentiation and aging. The chief
advantage of animal or plant cell culture is the control the investigator can exert over
the cells' environment without any influences from other systems of the multicellular
organism. However, it is important to keep in mind that the laboratory culture cannot
reproduce every detail of the cell's natural environment, and some of the missing
properties may be significant to the process being studied. Therefore, cell culture is a
model system, a simplified version of what is understood as “real life”.
When growth is not limited by the composition of the medium, cells are said to be
in “exponential” or “logarithmic” phase. In each successive time period, the size of the
population increases by a constant factor. When represented mathematically,
dN
= kN,
dt
where N is the number of cells present at time t and k is a growth constant that
depends on that cell type and those growth conditions.
Rearranging as:
dN
= kdt
N
Integrating between (No, to) and (N,t):
In (N/No) = kt
The population doubling time is the value of t for which N/No = 2. Moreover, a plot
of ln (N/No) as a function of t will be a straight line whose slope is k. (Even simpler is
to use semi-logarithmic graph paper, plotting t along the evenly spaced axis and N/N o
on the axis perpendicular to it.) If you know the initial number of cells and the growth
rate, the population of the culture in the log phase can be predicted at any subsequent
time.
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The exponential phase is usually limited by the availability of nutrients or the
environment's carrying capacity for waste. At that point, the population growth slows
until the culture is in the stationary phase, when cell death balances cell division as
cell division becomes slower. Cells in adult organs of most higher animals are in the
stationary phase. If conditions become sufficiently adverse, a death phase may ensue.
A growth curve is the result of an experiment to determine various phases of a
population of a certain cell type under a defined set of conditions (Figure 1).
Figure 1. Growth curve of a culture with different growth phases
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Cell Culture Technique
When working with cell culture, it is essential to maintain sterility. Typical
contaminants like bacteria can double in number every 30 minutes and molds can
double every 1.5 hours. Several measures are generally taken to ensure sterility:
1. Sterilize everything likely to come into contact with the culture. Media can be filter
sterilized or autoclaved, whereas glassware and disposable plasticware can be
autoclaved (if heat resistant), sterilized by radiation or treated with gas, and remain
packed before use. Tools can be dipped in alcohol and flamed.
2. Work in a clean protected area whenever possible. The ideal situation is to use a
laminar flow hood, in which the air is filtered to remove potential contaminants.
3. Avoid opening sterile containers for longer than necessary to sample or inoculate.
4. In some cases, it is advisable to include antibiotics such as penicillin or
streptomycin in the culture medium.
Cell Number Measurement
Cell numbers in the culture can be assessed by hemocytometer, a device for
cell number counting (as described in Exercise 1), or protein assay to monitor cell
growth.
It is sometimes necessary to determine the amount of proteins a cell contains in
order to understand the characteristics of the cell or to compare one cell type to
another. Once that value is obtained, one can use the total amount of proteins in a
cell lysate to determine the number of cells lysed. On the other hand, one can
determine the amount of proteins per cell using the Bradford method, providing that
you have enough cells to produce more than 10 g of proteins and you know the
number of cells. Cells in suspension can be collected by centrifugation, lysed in
detergent, and eventually assayed for proteins calorimetrically.
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Procedure:
A. Construction of growth curve for HL-60 cell culture
A stock culture of HL-60 cells grown in a 37oC incubator with 5% CO2 to the log
phase will be provided. Each group is required to examine the growth of these cells
under two different conditions, IMDM with 10% fetal bovine serum (FBS) and IMDM
with 2% FBS.
1. Using glass pipette and aseptic technique, transfer 16 ml of IMDM with 10% FBS
to a sterile flask (labelled as “10%”) and 16 ml of IMDM with 2% FBS to another
flask (labelled as “2%”).
2. Using aseptic technique, transfer 4 ml of the stock culture to each flask.
3. Gently shake the flasks several times.
4. Using micropipette and aseptic technique, transfer 0.5 ml of sample from Flask
“10%” to a microfuge tube (labelled as “10%”) and 0.5 ml of sample from Flask
“2%” to another microfuge tube (labelled as “2%”).
5. Using hemocytometer to count the number of cells in each microfuge tube (as in
Exercise 1). These cells for counting do not need to be handled aseptically.
6. For the Datasheet, calculate the mean and standard deviation of your data from
the eight Square A of the hemocytometer. The concentration of cells in the sample
is given by the mean x 104 cells/ml. Remember to correct this concentration by
the dilution factor if needed, and convert the unit to x 106 cells/ml.
7. On Days 1, 4 and 6, repeat Step 3 to 5 to sample your two cultures and count
the number of cells. Dilution of the sample is needed if the cell number is >70.
Pay close attention to the aseptic technique when sampling to avoid
contaminating the cell cultures.
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B. Preparation of cell pellet for protein assay
1. Aseptically transfer 2 ml of sample from Flasks “10%” and “2%” to two 15ml
Falcon tubes (labelled as “10%, Day 0” and “2%, Day #”, respectively).
2. Using a swinging bucket rotor and centrifuge, spin down the cells at 1,000
revolutions per minute (rpm) for 5 minutes. (Done by technicians)
Note: Whenever the centrifuge is used, the rotor must be balanced with tubes on
opposite sides with equal weight.
3. Remove tubes from the rotor and carefully discard the supernatant. Keep the
pellet that contains the cells.
4. Resuspend the cell pellet in each tube with 1 ml phosphate-buffered saline (PBS).
Then repeat Steps 2 and 3.
5. Store the two tubes with cell pellet at -20oC for subsequent use in Part D. Based
on your calculations in Part A, you should know the number of cells in the pellet
from each culture.
6. On Day 1, 4 and 6, repeat Step 1 to 5 to keep the cell pellets from your two
cultures. Remember to label each tube with your group number, sample name
and sampling day.
Note: Part C and D will be done on the following week.
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C. Construction of protein standard curve
Bradford reagent can be used to determine the protein content of bovine serum
albumin (BSA) and the data can be used to plot a standard curve as a reference.
Bradford reagent contains a dye, Coomassie blue, which binds to protein. The
dye/protein complex gives a blue color whose absorbance is directly proportional to
the protein concentration.
1. Dilute 0.5 ml of 1 mg/ml BSA (prepared in PBS) with 4.5 ml of PBS to obtain 5 ml
of 100 g/ml BSA.
2. Label two sets (Set 1 and 2) of six microfuge tubes (altogether 12 tubes) from 1
to 6.
3. Fill the tubes of Set 1 with 100 µg/ml BSA (prepared in Step 1) and 0.01%
SDS/EDTA as below. Tap the tubes to mix the content well.
Tube
100 g/ml BSA (ml)
0.01% SDS/EDTA (ml)
1
(Blank)
0
1.0
2
0.2
0.8
3
0.4
0.6
4
0.6
0.4
5
0.8
0.2
6
1.0
0
4. Add 1 ml Bradford reagent to the tubes of Set 2.
5. Add 100 l of sample from Set 1 Tube 1 to Tube 6 into the corresponding tubes
with Bradford reagent (Set 2). Mix the contents well. Color development should
occur within 3 minutes.
Note:
• It is important to mix the sample rapidly and thoroughly with the Bradford
reagent immediately, one tube at a time.
• As the Bradford reagent contains phosphoric acid, avoid contact with the skin.
6. After 3 minutes, transfer the contents of Set 2 to a cuvette. Measure the
absorbance at 595 nm using a spectrophotometer and record the data on the
Datasheet.
Note:
⚫ Before measuring Tube 2 to 6, autozero the spectrophotometer with Tube 1 of
Set 2 (Blank).
⚫ Wash the cuvette with alcohol twice and then distilled water twice between
each measurement.
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A595 (AU)
7. For the Laboratory Write-up, plot a graph with protein amount on the X-axis and
A595 on the Y-axis (e.g. Figure 2). Calculate the coefficient of determination and
the slope of the regression line. The coefficient should be >0.9.
0
2
4
6
8
10
Protein (µg/100µl)
Figure 2. A typical standard curve for the protein assay
D. Cell lysis and protein assay
1. For each of the eight tubes from Part B, add 0.5 ml of 0.1% SDS/EDTA. Vortex
the tubes vigorously to break up the cell clumps for proper cell lysis.
Note: Do not mix up with the 0.01% SDS/EDTA for diluting BSA (Part C Step 3).
Think: How does SDS lyse cells and solubilize proteins?
2. Immerse the tube in boiling water for one to two minutes to help solubilizing the
proteins.
3. Once the pellet is solubilized, add 4.5 ml of water to each tube. Mix the content
well. The sample is now regarded as “cell lysate”.
Think: What is the concentration of SDS/EDTA now?
4. Add 1 ml of Bradford reagent to eight microfuge tubes. Then transfer 100 l of
each cell lysate to each tube. Mix the contents well and wait for 3 minutes.
5. After 3 minutes, transfer the contents to a cuvette. Measure the absorbance at
595 nm with the spectrophotometer and record the data.
Note: If the absorbance exceeds the upper limit of the standard curve (i.e. the
absorbance of Set 2 Tube 6), you should dilute 0.2 ml of the sample with 1.8 ml
of 0.01% SDS/EDTA (not 0.1% SDS/EDTA for cell lysis), then use 100 µl of the
diluted sample for Step 4 and 5. Record the dilution factor the Datasheet.
6. For the Laboratory Write-up, calculate the amount of protein per tube. Correct
for dilutions if needed, calculate the total amount of protein per ml in each lysate.
Knowing the number of cells that were collected to obtain each lysate, calculate
the amount of protein per cell in terms of nanograms (ng) of protein per cell.
Show all calculations in your laboratory report.
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Experimental Datasheet of Exercise 3
Part A. Cell counting with the hemocytometer
Sampling
day
Raw cell count
2%
Dilution factor Average no. of
(if any)
cells
4
(x 10 cells/ml)
10%
2%
10%
2%
10%
Standard
deviation
2%
10%
0
1
4
6
In the laboratory report, plot a graph with two growth curves according to your result.
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Part C. Protein standard curve
Tube
100 g/ml BSA
(ml)
0.01%
SDS/EDTA (ml)
Protein amount
(µg) in 1 ml
Protein amount
(µg) in 100 l
1
(Blank)
0
1.0
0
0
2
0.2
0.8
20
2
3
0.4
0.6
4
0.6
0.4
5
0.8
0.2
6
1.0
0
A595
In the laboratory report, plot a standard curve and show the coefficient of
determination, slope, Y-intercept and linear equation.
Note: The intercept of the trendline does not need to be set as (0,0).
Part D. Protein assay on cell pellets
Day
A595
Protein
Protein
amount (µg) concentration
in 100 µl
(µg/ml)
Dilution
factor
Additional
Protein
Dilution amount (µg)
from the
factor
procedure
(if any)
No. of cell
Protein
per ml
amount (ng)
per ml
per cell
10%
2%
10%
2%
10%
2%
10%
2%
10%
2%
10%
2%
10%
2%
0
1
2.5*
4
6
* As 2 ml of cells (Part B Step 1) were diluted to 5 ml in total (Part D Step 1 to 3).
In the laboratory report, show the calculation steps and plot the growth curves using
the protein amount per cell.
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