The student worked on evaluating antitumor agents in vitro through cell culture and MTT assays. For cell culture, the student subcultured adherent A549 cells and suspension Raji cells, and resuscitated frozen cells. For MTT assays, the student tested the antitumor drug BEZ235 on A549 cells at different concentrations and measured optical density values. Though the results were abnormal, the student gained experience in carrying out cell culture techniques and MTT assay procedures.

1. Internship Report
1. Description of the project/ field/ domain the student has worked
in.
The project I have worked in is the evaluation of antitumor agents in vitro,
which consists of two main parts including cell culture and MTT assay.
Cell culture is the process used to offer abundant materials (i.e. cancer cells) to
subsequent MTT assay. Cell culture refers to the collect of cells from an animal
or plant and subsequently the cells are cultured in a favorable artificial
environment. The cells may be removed from the tissue directly and
disaggregated by enzymatic or mechanical means before cultivation, or they
may be derived from a cell line or cell strain that has already been established.
Subculturing, also called passaging, is the removal of the medium and transfer
of cells from a previous culture into fresh growth medium, a procedure that
enables the further propagation of the cell line or cell strain. The growth of cells
in culture proceeds from the lag phase following seeding to the log phase,
where the cells proliferate exponentially. When the cells in adherent cultures
occupy all the available substrate and have no room left for expansion, or when
the cells in suspension cultures exceed the capacity of the medium to support
further growth, cell proliferation is greatly reduced or ceases entirely. To keep
them at an optimal density for continued growth and to stimulate further
proliferation, the culture has to be divided and fresh medium supplied.
Moreover, if a surplus of cells are available from subculturing, they should be
treated with the appropriate protective agent (e.g., DMSO or glycerol) and
stored at temperatures below –130°C (cryopreservation) until they are needed.
Conversely, resuscitation of frozen cells is the procedure to thaw the frozen
cells which are stored at -80°C or liquid nitrogen and then culture them again.
In comparison with cryopreservation, resuscitation of cells requires a rapid
temperature-rise period, which prevents water from entering cells and causing
ice crystal, leading to influence the cellular survival.
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay
is a colorimetric assay for assessing cell metabolic activity. The mechanism is

2. that NAD (P) H-dependent cellular oxidoreductase may, under defined
conditions, reflect the number of viable cells. These enzymes are capable of
reducing MTT to its insoluble formazan, which displays a purple color and can
dissolve in DMSO. Then the OD value of the DMSO solution may be measured
at 490 nm (or 570 nm) wavelength. This method is commonly used to evaluate
the activity of antitumor agents in vitro.
2. Description of tasks and responsibilities which the student has
been assigned to.
(1) Subculturing Adherent Cells (A549)
Materials Needed
• Culture vessels containing your adherent cells
• Tissue-culture treated flasks, plates or dishes
• Complete growth medium, pre-warmed to 37°C
• Disposable, sterile 15-mL tubes
• 37°C incubator with humidified atmosphere of 5% CO2
• Balanced salt solution such as Phosphate Buffered Saline (PBS)
• Dissociation reagent such as trypsin
• Hemacytometer
• Inverted phase contrast microscope
• Pipettes
Protocol for Passaging Adherent Cells
1. Remove and discard the spent cell culture media from the culture vessel.
2. Wash cells using PBS. Gently add wash solution to the side of the vessel
opposite the attached cell layer to avoid disturbing the cell layer, and rock
the vessel back and forth several times. (Note: The wash step removes any

3. traces of serum, calcium, and magnesium that would inhibit the action of
the dissociation reagent.)
3. Remove and discard the wash solution from the culture vessel
4. Add trypsin to the side of the flask; use enough reagent to cover the cell
layer. Gently rock the container to get complete coverage of the cell layer.
5. Incubate the culture vessel at room temperature for approximately 2
minutes.
6. Observe the cells under the microscope for detachment. If cells are less
than 90% detached, increase the incubation time a few more minutes,
checking for dissociation every 30 seconds. You may also tap the vessel to
expedite cell detachment.
7. When ≥ 90% of the cells have detached, tilt the vessel for a minimal length
of time to allow the cells to drain. Add the equivalent of 2 volumes of
pre-warmed complete growth medium. Disperse the medium by pipetting
over the cell layer surface several times.
8. Transfer the cells to a 15-mL conical tube and centrifuge then at 200 × g
for 5 to 10 minutes.
9. Resuspend the cell pellet in a minimal volume of pre-warmed complete
growth medium and remove a sample for counting.
10. Determine the total number of cells and percent viability using a
hemacytometer.
11. Dilute cell suspension to the seeding density recommended for the cell line,
and pipet the appropriate volume into new cell culture vessels, and return
the cells to the incubator.
(2) Subculturing Suspension Cells (Raji)
Materials Needed
• Culture vessels containing Raji cells

4. • Conventional culture flasks
• Complete growth medium, pre-warmed to 37°C
• 37°C incubator with humidified atmosphere of 5% CO2
• Hemacytometer
• Inverted phase contrast microscope; Pipettes
Protocol for Passaging Suspension Cells
1. When the cells are ready for passaging, remove the flask from the
incubator and take a small sample from the culture flask using a sterile
pipette. If cells have settled down before taking the sample, swirl the flask
to evenly distribute the cells in the medium.
2. From the sample, determine the total number of cells and percent viability
using a hemacytometer.
3. Calculate the volume of media that you need to add to dilute the culture
down to the recommended seeding density.
4. Aseptically add the appropriate volume of pre-warmed growth medium into
the culture flask. You may split the culture to multiple flasks if needed.
5. Loosen the caps of the culture flasks one full turn to allow for proper gas
exchange, and return the flasks to the incubator.
(3) Resuscitation of Frozen Cells
Materials Needed:
Cryovial containing frozen cells
• Complete growth medium, pre-warmed to 37°C
• Disposable, sterile centrifuge tubes
• Water bath at 37°C
• 70% ethanol
• Tissue-culture treated flasks, plates, or dishes

5. Protocol for Resuscitation of Frozen Cells:
1. Remove the cryovial containing the frozen cells from liquid nitrogen storage
and immediately place it into a 37°C water bath.
2. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C
water bath until there is just a small bit of ice left in the vial.
3. Transfer the vial it into a laminar flow hood. Before opening, wipe the
outside of the vial with 70% ethanol.
4. Transfer the thawed cells dropwise into the centrifuge tube containing the
desired amount of pre-warmed complete growth medium appropriate for
your cell line.
5. Centrifuge the cell suspension at approximately 200 × g for 5–10 minutes.
The actual centrifugation speed and duration varies depending on the cell
type.
6. After the centrifugation, check the clarity of supernatant and visibility of a
complete pellet. Aseptically decant the supernatant without disturbing the
cell pellet.
7. Gently resuspend the cells in complete growth medium, and transfer them
into the appropriate culture vessel and into the recommended culture
environment.
(4) Freezing adherent cells (A549)
Materials Needed:
• Culture vessels containing cultured cells in log-phase of growth
• Fetal bovine serum (FBS)
• Complete growth medium
• Cryoprotective agent such as DMSO (use a bottle set aside for cell culture;
open only in a laminar flow hood)
• Disposable, sterile 15-mL or 50-mL conical tubes

6. • Reagents and equipment to determine viable and total cell counts (e.g.
hemacytometer)
• Sterile cryogenic storage vials (i.e., cryovials)
• Controlled rate freezing apparatus or isopropanol chamber
• Liquid nitrogen storage container
• Balanced salt solution such as Dulbecco’s Phosphate Buffered Saline (D-PBS),
containing no calcium, magnesium, or phenol red
• Dissociation reagent such as trypsin without phenol red
Protocol for Cryopreserving Cultured Cells
1. Prepare freezing medium (5：4：1=compete growth：FBS：DMSO) and store
at 2° to 8°C until use.
2. For adherent cells, gently detach cells from the tissue culture vessel
following the procedure used during the subculture. Resuspend the cells in
complete medium required for that cell type.
3. Determine the total number of cells and percent viability using a
hemacytometer. According to the desired viable cell density, calculate the
required volume of freezing medium.
4. Centrifuge the cell suspension at approximately 100–200 × g for 5 to 10
minutes. Aseptically decant supernatant without disturbing the cell pellet.
5. Resuspend the cell pellet in cold freezing medium at the recommended
viable cell density for the specific cell type.
6. Dispense aliquots of the cell suspension into cryogenic storage vials. As you
aliquot them, frequently and gently mix the cells to maintain a
homogeneous cell suspension.
7. Place the cryovials containing the cells in an ice box, then put them at
-20°C for 2h, and store them at –80°C overnight.
8. Transfer frozen cells to liquid nitrogen, and store them in the gas phase
above the liquid nitrogen.

7. (5) MTT assay
Materials Needed
• Culture vessels containing A549 cells in log-phase of growth
• Positive antitumor drug (BEZ235)
• 5 mg. mL-1 MTT solution
• DMSO
• 96-well plate
• Thermo Election Corporation
• 37°C incubator with humidified atmosphere of 5% CO2
• Needle and syringe
• Shaking platform
Protocol for MTT assay
1. Collect A549 cells and regulate the concentration of cell suspension. Each
well of 96-well plate was added with 100 L and make sure 103-104 cell/
well (marginal wells were filled with PBS).
2. When the cells overspread the bottom of each well, add a concentration
gradient of BEZ235 (i.e. 10-5, 10-6, 10-7, 10-8 M). Add 100 L into each well.
Set up 3-5 wells for each concentration.
3. The 96-well plate was incubated in a 37°C incubator with humidified
atmosphere of 5% CO2 for 48 h.
4. Add 20 µL of MTT solution to each well containing cells.
5. Incubate the plate at 37℃ for 4-5 h.
6. Remove media with needle and syringe.
7. Add 150 µL (or 200 µL) of DMSO to each well
8. Put plate one the shaking platform for 5 min to dissolve formazan crystals.

8. 9. Transfer to Thermo Election Corporation and measure OD at 490 nm (or
570 nm) wavelength.
3. Description of achievements, contributions or outcomes.
A. Cell culture
This figure shows the growth situation of A549 cells subcultured in June 30
after 24h of incubation. A549 cell is a type of epithelial-like cells which are
polygonal in shape with more regular dimensions, and grow attached to a
substrate in discrete patches. The number of the cells is relatively few, which
means that it is unnecessary to continue subculturing.

9. This figure shows the growth situation of A549 cells subcultured in June 27
after 4 days of incubation. There are a great number of cells, which have no
more room to growth. Therefore, it is urgently required to subculture them in
several new culture vessels.
B. MTT assay
(1) My First practice of MTT assay

10. These two figures show the 96-well plate only incubated with DMSO for 72h,
which represents negative control. Actually, this experiment mainly aims to
practice inoculating the same number of cells into each well. As might be
expected, according to apparent purple color (formazan), cells can maintain
rapid growth (E3 well is empty, which is due to an accident when adding
DMSO).
Results: OD values (after blank subtraction) at 490nm by Thermo Election
Corporation
According to this table, it is clear to observe the OD values which reflect the
number of viable cells present. The majority of OD values are approximately
identical, which indicates that I successfully inoculated the same number of
cells into each well, which prepared for my second and more complicated MTT
assay. Meanwhile, it was my first experience that I roughly carried out the
procedure of MTT assay in person.

11. (2) My second practice of MTT assay
The upper figure reveals the 96-well plate incubated with four concentrations
of BEZ235, a positive drug, (10-5, 10-6, 10-7, 10-8 ) for 48h. The lower figure
shows the 96-well plate after adding MTT and DMSO (purple formazan).
The arrangement of wells:

12. B 2, 3,4,5,6,8,9,10,11(10-8); C 2, 3,4,6,7,8,9,10,11 (10-7); D 2,
3,4,5,6,7,9,10,11(10-6); E 2,3,4,5,6,7,8,9,11 (10-5)
F 2, 4, 5, 6 (10-8); F 7, 8, 9, 10, 11 (10-7); G 2, 3,4,5,6 (10-6); G 7, 8, 10, 11
(10-5)
Negative control wells: B7, C5, D8, E10, F3, G9 (add DMSO which replaces
BEZ235)
Blank wells: H 11, 12 (only add PBS)
Results: OD values (after blank subtraction) at 490nm by Thermo Election
Corporation
(Some blank positions are the deviation values; some numbers in red font
represent negative control groups)
Average OD values
Inhibition rate of BEZ235 towards A549 cells
Compd. 10-8 10-7 10-6 10-5
BEZ235 37.84 40.73 62.38 55.78
BEZ235 50.82 45.35 56.97 32.27
Inhibition ratio = (OD negative controlgroup OD medicated group) / OD negative controlgroup
In general, the inhibition ratio should increase with the concentration of
BEZ235. And there should be a linear relationship between them. Unfortunately,
my data are abnormal. I think there are two main reasons to explain this
phenomenon. Firstly, due to insufficient trypsinization, I pipetted over the cell
Compd. 10-8 10-7 10-6 10-5
BEZ235 0.253 0.241 0.153 0.180
BEZ235 0.200 0.222 0.175 0.275
Value 1 2 3 4 5 6 7 8 9 10 11 12
A -0.06934 -0.06823 -0.07028 -0.06546 -0.07391 -0.06435 -0.05783 -0.06041 -0.06001 -0.06508 -0.05678 -0.06289
B -0.06652 0.215095 0.359964 0.307804 0.319015 0.212845 0.474904 0.252934 0.220645 0.293744 0.124955 -0.05475
C -0.05959 0.242415 0.237125 0.260685 0.489775 0.242765 0.234605 0.270885 0.242685 0.194635 -0.06273
D -0.05642 0.182735 0.231045 0.178145 0.036465 0.544735 0.185105 0.120075 0.142115 -0.0739
E -0.07045 0.123455 0.190755 0.215605 0.244165 0.219315 0.231915 0.172405 0.346435 0.160315 -0.07436
F -0.05434 0.364115 0.189475 0.245025 0.165965 0.231185 0.196665 0.199395 0.244315 0.240605 -0.05699
G -0.06102 0.307025 0.149255 0.213715 0.191725 0.145775 0.302944 0.272394 0.360965 0.271214 0.256095 -0.05587
H -0.05961 -0.05898 -0.05747 -0.06372 -0.05979 -0.06021 -0.05664 -0.00872 -0.05391 -0.04158 -0.03127 0.031275

13. layer surface many times after stopping trypsinization in order to mechanically
expedite cell detachment, which caused damage of cells and influenced their
normal growth. Secondly, when I add drugs into wells, I should have
continuously shaken the cell suspension to prevent cell sedimentation, which
guarantees that each well can be incubated with the same number of cells.
After overcoming these two mistakes, there should be a proportional
relationship between inhibition rate and concentration of antitumor drug. It is
easy to calculate the IC50, which refers to the concentration of a drug that is
required for 50% inhibition in vitro. This experiment is merely a practice;
actually, MTT assay is commonly used to test whether the IC50 of certain
unknown drugs are superior to ones of some positive drugs like BEZ235. In
other words, this technique is helpful to discover new antitumor drugs.
4. Description of benefits and learning outcomes for the student.
I basically master the main steps of cell culture including t thawing frozen cells,
subculturing adherent and suspension cells, and freezing cells. Moreover, I also
understand the procedure of MTT assay and the method of how to analyze the
results. There are some notes I have summarized during my internship, which
are helpful to enhance my experiment skills in the future.
(1) Freezing cells
1. Before freezing cells, it is indispensable to observe the situation of cells,
which directly influences the cell growth after resuscitation.
2. It is better to exchange the spent complete growth medium with fresh one
in the previous day before freezing, which guarantees that cells can acquire
abundant nutrient for the best state of proliferation.

14. 3. In the course of preparing freezing medium, I found that using complete
growth medium containing 10% or 20% fetal bovine serum and pure
serum have no obvious difference on cells. However, if the content of
DMSO is higher than 10% of the whole freezing medium, it would cause
severe cytotoxicity.
4. My senior have told me that if the cells you want to freeze are unused for a
short period (within three months), it is appropriate to store them at -70℃
refrigerator. If unused for a long period, it is recommended to store them
at liquid nitrogen.
(2) Resuscitation of Frozen Cells
1. The addition of the thawed cell suspension to culture medium effectively
dilutes the cryoprotectant (e.g. DMSO) reducing the toxicity of the
cryoprotectant. That is why it is important to add the thawed cell
suspension to a larger volume of culture medium immediately.
2. In the course of resuscitation of frozen cells, the speed of temperature
increasing must be large. As a result, after taking frozen cells out of liquid
nitrogen or -70℃ refrigerator they must be immediately placed into a 37°C
water bath.
3. When taking frozen cells out of liquid nitrogen, experimenters should
ensure personal protection and avoid spilling liquid nitrogen to frostbite
skins.
4. In general, each cryogenic storage box has been filled with many types of
cells; it is required to take out cells quickly and accurately. Moreover, if
cryogenic storage box is placed at room temperature, it would the growth of
other cells. Therefore, experimenters should mark their own cryogenic
storage vials in order to select cells conveniently.
(3) Subculturing adherent cells

15. 1. Trypsin is inactivated in the presence of serum. Therefore, it is essential to
remove all traces of serum from the culture medium by washing the
monolayer of cells with PBS without Ca2+/Mg2+
2. Cells should only be exposed to trypsin/EDTA long enough to detach cells.
Prolonged exposure could damage surface receptors and influence cell
growth.
3. Trypsin should be neutralized with serum prior to seeding cells into new
flasks otherwise cells will not attach.
4. During subculturing, hands should take the bottom of culture flask instead
of bottleneck, which prevents cells from external contamination.
5. After removing caps of culture vessels, experimenters should avoid moving
their hands over these culture vessels, which can prevent contaminants
from falling down from hands.
6. When resuspending the cell pellet in a centrifuge tube, it is better to press
the pipette before stretching into fluid and then repeat blowing, which can
remove the air in the tip. Otherwise, it would produce a lot of bubbles,
which is harmful to cell growth.
(4) MTT assay
1. MTT is a type of cancerogen, which means that experimenters should be
careful to add MTT to a 96-well plate.
2. MTT is easy to decompose under the light. Therefore, experimenters ought
to swiftly add MTT to a 96-well plate when turning off the light of clean
bench.
3. 96-well plates are incubated at 37℃ incubator, which cannot completely
guarantee a constant humidity. Evaporation would happen at the marginal
wells of a 96-well plate, which leads to increase the concentrations of
various constituents, causing different states of cells. Therefore, it is

16. recommended to add sterile PBS to 36 marginal wells in order to inhibit
evaporation of central 60 wells.
4. It is important to maintain cell suspension uniform in order to prevent cells
from precipitation, which makes sure that each well is inoculated with a
same number of cells. Hence, we can mix cell suspension after inoculating
several wells.
5. Before adding DMSO, it is necessary to remove supernatant from each well
of the 96-well plate. Otherwise, formazan crystal is difficult to dissolve.
Most important, it should be careful to avoid absorbing any crystal when
removing supernatant.
5. Personal conclusion and impression of the work placement.
First of all, this internship gives me a chance to dabble in a new aspect of
biotechnology including cell culture and MTT assay. Since I have never learned
them in school, I was so curious and active that I frequently asked questions to
my amicable senior during the whole internship. Although I encountered some
failures, my senior always ask me to patiently analyze and find out the reasons.
This helped me to get into a habit of tackling problems step by step. For
instance, when I detected that the cells subcutured in the first day of my
internship were contaminated by bacteria, I was almost hopeless and
frustrated due to this ruthless fiasco. My senior consoled me and guided me to
check all facilities and materials that were possible to cause cell contamination.

17. Finally, we found that the bacterium in my contaminated culture vessels was
same as the one discovered in the complete growth medium I used. Just in
case, we also autoclaved other facilities such as pipettes and tips. Accordingly,
we successfully solved this problem. Most important, this process taught me
how to confront with unexpected outcomes and deal with them with a clear and
calm head. Moreover, thanks to this experience, I became more patient and
scrupulous to treat my tasks. Specifically, when carrying out subculturing cells,
I must focus my attention on every step. For example, if the tip of a pipette
contacts with hands or other irrelevant facilities, I should dispose this
contaminated tip without hesitation. In addition, with regard to inoculating
96-well plate with cell suspension, it is relatively easy for to inoculate all wells
one by one because the pink cell suspension helps me recognize which wells
have been inoculated. Nevertheless, as for colorless and transparent drug
solution, I had no choice but to bear in mind those wells I had already added
drug into. Therefore, this process indeed sharpened my perseverance, which is
beneficial to my work and study in the future. In addition, I also understood the
difficulty in terms of some aspects except doing experiment itself. Researchers
have to contact sellers in person to purchase all reagents and consumables
they use and then submit an expense account to the treasurer's office. For the
purpose of cost savings, researchers wash and sterilize some used vessels so as
to recycle them. Due to limited number of clean bench, every researcher
obliges to order a position in advance for own experiments. Compared to
experimental lessons in my university, everything is already prepared by
instructors; therefore, I just go there and complete experimental tasks. In
other words, I do not need to be concerned with other tiring things. However,
as for those real researchers, doing research is their work and career; therefore,
they have to overcome and adapt to these daily matters. Spontaneously, I start
thinking about myself. If I plan to engage in doing research in the future, it
would be inevitable to prepare myself mentally in advance. In a word, by
means of this internship, I am familiar with my future vocation and have more
thoughts in regard to my career planning. Meanwhile, I am capable of carrying
out cell culture and MTT assay more proficiently in my future work and study.

18. This experience improves my attitude toward science and makes me more
patient, calm and rigorous, which indeed benefit all aspects of my career and
life. At last, I want to thank my senior and professional supervisor for spending
the time in helping and guiding me to satisfactorily complete my summer
internship.