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 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• PipettesProtocol 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 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 • 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; PipettesProtocol 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

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• 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.

(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.

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.

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

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.

(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: 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 55.78BEZ235 50.82 45.35 56.97 32.27

Inhibition ratio = (OD negative control group OD medicated group) / OD negative control group

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

Compd. 10-8 10-7 10-6 10-5

BEZ235 0.253 0.241 0.153 0.180BEZ235 0.200 0.222 0.175 0.275

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 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.

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

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 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. 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. 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.