Hello my fellow coursemates! How are you guys doing? Hope all of you are doing fine for your SIP and MP. This is the 4th week of SIP/MP and it is my pleasure to share my attachment experience.
I am attached to a research laboratory which study on the effect of polyphenolic chemosensitisers and anticancer drugs on tumour cells. Some examples of anticancer drugs include melphalan, chlorambucil and etc. Both chemotherapy drugs belong to the same class of nitrogen mustard alkylating agents. Melphalan is commonly used to treat multiple myeloma (cancer of plasma cells) and ovarian carcinoma. Chlorambucil is used primarily to treat chronic lymphocytic leukemia (cancer of lymphocytes, with particular to B cells). Some examples of polyphenolic chemosensitisers include 2,2'-dihydroxychalcone and 2'-hydroxy-4-methylchalcone (I shall elaborate more on polyphenolic chemosensitisers in my next post).
In the world today, there are 3 main types of cancer therapies which are chemotherapy, radiotherapy and surgery. Usually, they are used in combination to achieve greater effectiveness in treating tumours. My main focus will be on chemotherapy.
Chemotherapy is decreasing in its effectiveness mainly due to chemoresistance of tumour cells. This is primarily due to the gluthatione (GSH)-related detoxification system which allows tumour cells to be resistant to chemotherapy drugs. I shall elaborate further on this system in my next post.
In my study, I shall be focusing on the effect of 2,2'-dihydroxychalcone and chlorambucil in inducing DNA interstrand cross-links on human colon adenocarcinoma cells (HCACs) grown in-vitro. The HCACs are obtained from ATCC (American Type Culture Collection) and designated by COLO 320 HSR. These cells are grown in RPMI (Roswell Park Memorial Institute) medium and they are loosely adherent to the culture surface. In addition, they appear round and refractile under the inverted microscope. Since they are tumour cells, they divide very rapidly, and thus have high energy requirement.
During the course of my research, I am required to maintain these cells in peak condition so that I will have enough cells to continue my experiments. Hence, I will be applying what I have learnt from Mammalian Cell Technology (MCT) which I have taken as elective subject to part of my research. Without further delay, let me start off with the most fundamental, which is the preparation of the RPMI-1640 medium.
Subject: MCT
Tests:
1)Preparation of RPMI-1640 medium
2)Subculturing of cells
3)Changing of medium
1) Preparation of RPMI-1640 medium
Composition
· 1% sodium pyruvate
· 1% non-essential amino acids
· 1% antibiotics
· 10% fetal bovine serum (FBS)
Materials
· 500ml RPMI-1640 medium (ready-to-use)*
· Fetal Bovine Serum (FBS)*
· Antibiotics*
· Sodium pyruvate*
· Non-essential amino acids (NeAA)*
· BSC 2
· 70% ethanol
· Paper towels
· Pipettorˆ
· Falcon™ pipette tubes (5ml)ˆ
Methods
A. Preparation of BSC 2
1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.
B. Preparation of RPMI-1640 medium
1. Place the materials* in 37ºC waterbath.
2. Place the materials* into the BSC 2.
3. Pipette 5ml of sodium pyruvate into the pre-made 500ml RPMI-1640 medium.
4. Pipette 5ml of NeAA into the pre-made 500ml RPMI-1640 medium.
5. Pipette 5ml of antibiotics into the pre-made 500ml RPMI-1640 medium.
6. Pipette 50ml of FBS into the pre-made 500ml RPMI-1640 medium.
7. Store the medium at 2-8 ºC and warm up in 37ºC waterbath before any usage.
Function of components:
· Sodium pyruvate- Provide additional source of energy for rapidly-growing cells.
· NeAA- Provides source of amino acid (energy source for cells).
· Antibiotics- Minimises bacterial contamination of tissue culture.
· FBS- Provides essential nutrients to promote optimal cell growth and proliferation
2) Subculturing of cells
Principle
As cells grow and multiply, they tend to crowd together, which is termed as confluency. This can be observed under the inverted microscope where cells are in close proximity. A confluent tissue culture flask suggests the need for subculturing. Subculturing is defined as the inoculation of cells from a confluent flask into a new sterile flask with fresh medium. It allows cells to have more growth surface so as to minimize the competition for growth surface and nutrients. Subculturing is especially important for maintaining the viability, growth and proliferation of anchorage-dependent cells as these cells need to adhere to the surface of the tissue culture flask before they can start growing.
Materials
· 75cm² tissue culture flask (containing HCACs of 80% confluency)
· New 75cm² tissue culture flaskˆ
· RPMI-1640 medium*
· 0.0067M phosphate buffered saline (PBS)*
· Trypsin*
· Inverted light microscope
· BSC 2
· CO2 incubator
· Pipettorˆ
· Sterile 50ml Falcon™ tubeˆ
· Falcon™ pipette tubes (25ml and 5ml)ˆ
· Waste beakerˆ
· Biohazard bag
· Clorox (Bleach)
· 70% ethanol
· Paper towels
Methods
A. Preparation of BSC 2
1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.
B. Preparation of reagents
1. Incubate trypsin and RPMI-1640 medium in 37ºC water bath.
C. Washing of HCACs
1. Remove the 75cm² tissue culture flask (containing HCACs of 80% confluency) from the CO2 incubator.
2. Observe cell confluency (80%) under inverted light microscope.
3. Swab the 75cm² tissue culture flask with 70% ethanol before placing it into the BSC 2.
4. Swab the materials* before placing them into the BSC 2.
5. Discard the spent RPMI-1640 medium from the 75cm² tissue culture flask into the waste beaker.
6. Pipette 10ml of 0.0067M PBS into the 75cm² tissue culture flask to wash the HCACs.
7. Swirl the 75cm² tissue culture flask gently to facilitate the washing of HCACs.
8. Discard 10ml of PBS into the waste beaker.
D. Trypsinisation
9. Pipette 2ml of trypsin into the 75cm² tissue culture flask to detach the HCACs from the tissue culture flask surface.
10. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2 for 2-3 minutes.
11. Observe the 75cm² tissue culture flask under the inverted light microscope to ensure the HCACs are detached.
E. Subculturing of cells
12. Pipette 8ml of fresh RPMI-1640 medium into the 75cm² tissue culture flask to neutralise trypsin to prevent damage to the HCACs.
13. Mix the RPMI-1640 medium with trypsin well to ensure all the HCACs can be transferred with the RPMI-1640 medium into a sterile 50ml Falcon tube.
14. Pipette 10ml of mixture (8ml of RPMI-1640 medium and 2ml of trypsin) from the 75cm² tissue culture flask into a sterile 50ml Falcon tube.
15. Centrifuge the sterile 50ml Falcon tube at 20ºC, 1500rpm for 3 minutes.
16. Discard the supernatant into the waste beaker, leaving the cell pellet.
17. Resuspend cell pellet in 10ml of PBS.
18. Centrifuge the sterile 50ml Falcon tube at 20ºC, 1500rpm for 3 minutes.
19. Discard the supernatant into the waste beaker, leaving the cell pellet.
20. Resuspend cell pellet in 10ml of RPMI-1640 medium.
21. Pipette 19ml of fresh RPMI-1640 medium into a new 75cm² tissue culture flask.
22. Pipette 1ml of cell suspension from the sterile 50ml Falcon tube into a new 75cm² tissue culture flask.
23. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2.
F. End of experiment
1. Deactivate the waste solution in the waste beaker with Clorox.
2. Drain the waste solution into the sink after it turns from violet to colourless and run running tap water for 5-15 minutes.
3. Dispose all the used Falcon™ pipette tubes and paper towels and into the biohazard bag.
4. Swab the BSC 2 with 70% ethanol.
5. Return all the reagents to their appropriate storage areas.
3) Changing of medium
Principle
As cells grow and multiply in culture medium, they produce metabolic toxic waste products and use up nutrients in the medium. Essentially, we want to maintain the viability of cells and thus, the changing of medium is necessary as it restores back the level of nutrients and removes metabolic toxic waste products. An indication of the need to change medium is by observing the colour of the medium. Phenol red is added in the medium to act as a pH indicator. A yellow culture medium indicates the accumulation of metabolic toxic waste products and depletion of nutrients, prompting the need to change medium. A cloudy culture medium suggests possible bacterial contamination and the best action would be to discard the tissue culture flask.
Materials
· 75cm² tissue culture flask (containing yellow culture medium and cells)
· 0.0067M phosphate buffered saline (PBS)*
· RPMI-1640 medium*
· BSC 2
· CO2 incubator
· Pipettorˆ
· Falcon™ pipette tubes (10ml and 25ml)ˆ
· Waste beakerˆ
· Biohazard bag
· Clorox (Bleach)
· 70% ethanol
· Paper towels
Methods
A. Preparation of BSC 2
1. Place the materialsˆ into the BSC 2 and arrange them orderly.
2. Switch on the UV light for 15 minutes to ensure sterility of all materials.
3. Switch off the UV light, on the light, run the air circulation and swab the BSC 2 with 70% ethanol.
B. Changing of medium
1. Remove the 75cm² tissue culture flask (containing HCACs of 80% confluency) from the CO2 incubator.
2. Swab the 75cm² tissue culture flask with 70% ethanol before placing it into the BSC 2.
3. Swab the materials* before placing them into the BSC 2.
4. Discard the spent RPMI-1640 medium from the 75cm² tissue culture flask into the waste beaker.
5. Pipette 10ml of 0.0067M PBS into the 75cm² tissue culture flask to wash cells.
6. Swirl the 75cm² tissue culture flask gently to facilitate the washing of cells.
7. Discard 10ml of PBS into the waste beaker.
8. Pipette 20ml of fresh RPMI-1640 medium into the 75cm² tissue culture flask.
9. Incubate the 75cm² tissue culture flask at 37ºC in 5% CO2 for 2-3 minutes.
C. End of experiment
1. Deactivate the waste solution in the waste beaker with Clorox.
2. Drain the waste solution after it turns from violet to colourless.
3. Dispose all the used Falcon™ pipette tubes and paper towels and into the biohazard bag.
4. Swab the BSC 2 with 70% ethanol.
5. Return all the reagents to their appropriate storage areas.
In conclusion, subculturing of cells and replacement of medium is essential for maintaining the viability of cells. Alright, that is all for now! I hope my post is comprehensive and if there is any query, please feel free to ask.
Thankz! =)
Tan Han Yang
0606190G
TG01
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20 comments:
Hello Han Yang
Yo very glad you are enjoying your research... Cant wait to c u this friday lol
I got some questions to ask you .. don't worry i wont choke you :)
1) What are the mode of actions of melphalan, chlorambucil as anti-cancer drugs in the treatment of its respective cancers?
2) Can u provide a brief history of ATCC?
3) Is there any precautions you must take in handling tumor cells and what is the role of the BSC 2?
4) What is the specific type of antibiotics added to the medium? Will addition of antibiotics affect cell growth? Since good aseptic techniques are expected, is there really a need to add antibiotics in the media?
5) What are the principles of washing HCACS and Trypsinisation?
From: your friend
Ma Xianwei Benjamin
class: TG01
0606181F
Hey.
i kinda do similar things too. Just want to clarify something that you did pretty differently.
you mentioned that you prepared your own RPMI media. I prepare my own medias too but i use a filter and do it in the laminar flow hood BSL2, to ensure that there is no contamination. You didnt mention that you filter the RPMI. do you?
thanks
GLAD
Hello Benjamin,
1) What are the mode of actions of melphalan, chlorambucil as anti-cancer drugs in the treatment of its respective cancers?
Ans: Melphalan (ALKERAN®) is a bifunctional alkylating agent which works by causing alkylation of DNA in tumour cells and induce cross-linking of its double helix strands by binding to guanine. Ultimately, the above interferes and inhibits DNA replication and transcription which prevents the proliferation of tumour cells.
Chlorambucil (LEUKERAN®)is a bifunctional alkylating agent which targets both resting and rapidly dividing tumour cells. It causes alkylation of DNA and induces DNA interstrand cross-link. These result in disruption of DNA replication and transcription. DNA fragmentation can result and cell death occurs.
2) Can u provide a brief history of ATCC?
Ans: ATCC is established in 1914 and it is a world-class leading independent, private, non-profitable biological resource
center and research organisation.
Being a biological resource center, it validates microorganisms and cell lines and provide information on how to maintain the microorganisms and cell lines.
Being a research organisation, ATCC aims for continuous improvement as a biological resource center and generates new knowledge and technology.
For more information, please refer to www.atcc.org
3) Is there any precautions you must take in handling tumor cells and what is the role of the BSC 2?
Ans: Handling tumour cells is just as similar as how you handle normal mammalian cells in BSC. In addition, the necessary personal protective equipments (PPE) such as clean lab coat, covered shoes and gloves should be worn at all times when dealing with cells. According to the ATCC, this strain of cells is classified under biosafety level 1, which means they do not cause any disease in healthy individual. In short, there is no special precautions to take when handling tumour cells so long as the PPE are equipped and practice good aseptic techniques. You may be wondering why am I working in BSC 2 since this strain of cells is not pathogenic. The reason is simply because we do not have BSC 1 in the tissue culture lab. :)
Essentially, we do not want to introduce any contaminants into our cell culture, thus performing cell culture work in the BSC 2 provides the most sterile environment. Working in the BSC 2 serves to minimise the introduction of contaminants (particularly air-borne microbes)into my culture flask. In addition, it confers protection to the user, cells and environment.
4) What is the specific type of antibiotics added to the medium? Will addition of antibiotics affect cell growth? Since good aseptic techniques are expected, is there really a need to add antibiotics in the media?
Ans: The antibiotics added to the medium is a combination of 10.000 units of Penicillin, 10.000 µg of streptomycin, and 25 µg of Amphotericin B per mL. It is formulated in a way it can target a wide variety of Gram-positive and Gram-negative bacteria as well as possessing anti-fungal (antimycotic), anti-mold and anti-yeast activity.
In small concentrations(1%), the addition of antibiotics will not affect the growth of cells. As your aseptic techniques develop better, you can be less reliant on antibiotics as antibiotics are known to encourage the growth of antibiotic resistant organism. However, as a beginner, I do not want to take the risk of contaminating my cells.
5) What are the principles of washing HCACs and trypsinisation?
Washing cells with PBS help trypsin to work by removing any residual FBS (fetal bovine serum). It also serves to remove any waste products from the cells. Washing of cells with PBS is usually performed once or twice of your own preference.
Trypsin is a proteolytic enzyme which digests the fibronectin (a glycoprotein) adhering the cells to the surface of the culture flask. Since I am culturing anchorage-dependent cells, it is necessary for me to detach these cells with trypsin from the surface of the culture flask before I can perform subculture. Detached cells appear rounded up and float within trypsin under the inverted microscope. It is important to take note that cells should not be left with trypsin for too long as it may damage the cells. The addition of fresh medium which contains serum will inhibit trypsin activity.
I hope you have achieved enlightenment! =)
Han Yang
TG01
Hey GLAD,
I understand that culture medium cannot be autoclaved, thus filter sterilisation is the preferred way to ensure sterility of the medium without destroying some essential constituents of the medium such the buffer. However for myself, I do not filter sterilse my medium as the medium pruchased is already indicated as sterile. Hence, it can be used directly. In addition as mentioned in the post, I prepare my culture medium in the BSC 2, thus high standard of sterility is achieved. So far, I have used my medium to culture cells for 4 weeks and there has been no signs of contamination. =)
Perhaps, in cases whereby you need to prepare your medium manually by dissolving the constituents of the medium (poweder-formed), it will be good to filter sterilise the completed medium to ensure sterility.
I hope I have clarified your doubt. Please feel free to correct me if any part of my reply is wrong. =)
Han Yang
TG01
Hello Han Yang
W0w thanks i think ive achieved enlightenment =D
Hello Hanyang!!
Are there any possible errors that might occur in the subculturing of the cells? if there are, how will it affect the outcome of the experiment?
Another question: Why do have to incubate the tissue culture flask in a 5% CO2 incubator and not any other normal room temperature incubator?
oops i forget to say my name..
kk its AmiR
TG02
0607929G
Hello Amir,
Qn) Are there any possible errors that might occur in the subculturing of the cells? if there are, how will it affect the outcome of the experiment?
Ans: If the protocol is being followed well and good aseptic techniques such as performing cell culture work in BSC and good pipetting skills are practiced, there shouldn't be any problem with subculturing. However, if the above are not followed diligently, it will results in the contamination of cell line. When cells get contaminated, it is advisible to discard them rather than to salvage. When there are no cells left, I cannot proceed on with my experiments such as the seeding of cells and extraction of DNA.
Some possible errors may include:
1)Cell culture work is not performed in BSC.
2)Workplace in BSC is not swabbed with 70% ethanol before and after use.
3)Disorganised workplace in the BSC which can result in spills and contaminate cells.
4)The bottle of culture medium and other reagents such as PBS and trypsin are not capped immediately after use. This can introduce contaminants from sleeves of lab coat into these reagents and affect subsequent subcultures.
5)When pipette tip touches any non-sterile surfaces such as the opening of the bottle, it is not discarded but used to pipette reagents.
6)The cap of the bottle is placed facing down or up on the work surface in BSC. This can introduce contaminants onto the cap and eventually into reagents or culture flask. Instead, the last finger should be used to hold the cap while pipetting. It will takes some practice master it.
7)Cells are left with trypsin for too long and this will damage the cells. The addition of medium (with serum)will remedies this problem.
Remember, we cannot take it for granted that working in the BSC is 100% sterile and neglect good aseptic techniques. I cannot stress enough that good aseptic techniques are vital to perform proper subculture and any other cell culture work.
Qn) Why do we have to incubate the tissue culture flask in a 5% CO2 incubator and not any other normal room temperature incubator?
Ans: The RPMI-1640 medium is buffered by HEPES (N-2-hydroxyethylpiperazine-N'-2-ethylsulphonic acid) buffer which functions better than bicarbonate buffer in maintaining optimal pH (7.4-7.6)as it is more resistant to pH changes caued by CO2 flactuation. For the RPMI medium, the optimum CO2 concentration is 5%.
As cells grow, they produce CO2 and lactic acid as metabolic by-products. In medium which uses bicarbonate as buffer system, the bicarbonate compensates for the production of the CO2. It is important to take note of the concentration of CO2 produced by cells by monitoring the growth phase of the cells such as lag phase or when cell density is low. The above can result in low concentration of CO2 being produced and it may be insufficient to maintain optimal pH. As such, it is where the CO2 incubator comes in to restore the CO2 concentration.
The level of bicarbonate in medium determines the concentration of CO2 used to maintain an optimum pH, irregardless of cell type.
For example, the Minimum Essential Medium (MEM) with low bicarbonate levels requires 5% CO2 concentration whereas the Dulbecco's Modified Eagle's Medium (DMEM)with high levels of bicarbonate requires 10% CO2 concentration.
According to the ATCC, the human colon adenocarcinoma cells (COLO 320HSR) require to grow at 37°C for optimal survival and growth. The cells originate from human body (colon) and 37°C is the normal body temperature where cells thrive.
I hope I have answered your questions. =)
Thankz!
Han Yang
TG01
hey
i was just wondering, at the section: B. Changing of medium, step 4, how is it possible to discard the spent RPMI-1640 medium from the 75cm² tissue culture flask into the waste beaker without discarding the cells along? First and foremost, the cells are in the medium right?
i would appreciate it if you could explain it to me
thanks
rusydiana
Hello Rusydiana,
Qn) i was just wondering, at the section: B. Changing of medium, step 4, how is it possible to discard the spent RPMI-1640 medium from the 75cm² tissue culture flask into the waste beaker without discarding the cells along? First and foremost, the cells are in the medium right?
Ans) You are correct, the cells are immerged in the culture medium.
Before I answer your question, let me provide a brief description of my tissue culture flask.
The tissue culture flask I use is a disposable plastic (polystyrene) flask. It is constructed in a way it is optically clear for easy viewing of cells microscopically under the inverted microscope. The surface of the tissue culture flask is treated by gamma irradiation to ensure sterility and promote cell adhesion. It has also a flat growth surface to promote uniform and reproducible cultures. The neck of the flask is canted (tilt at an angle) so that medium does not overflow easily and thus minimises contamination. The screw cap is made up of polyethylene and is vented by 0.2um hydrophobic membrane which facilitates gaseous exchange while maintaining a sterile environment within the flask.
Having descibed the various features of the flask, now I shall answer your question.
After the inoculation of cells into the flask (with fresh medium) and a 24 hours incubation, cells enter the lag phase cycle where they will adhere to the surface of the flask via fibronectin (glycoprotein) secreted by the cells. The surface of the flask where cells adhere matches where the screw cap is tilted upwards. The flask must be positioned horizontally in the CO2 incubator, favouring the cells to attach to the treated surface. (Hope you can visualise what I say). As my cells are anchorage-dependent cells, they must adhere to the solid substrate (surface of flask) before they can start growing and proliferate. When cells are adhered to the substrate, trypsinisation (more information found in my reply to Benjamin's question) is the only way to detach the cells from the surface.
When I am discarding my spent medium into the waste beaker, I will always position the flask vertically and tilt the flask in a way the spent medium flows towards the opposite end (surface where cells do not adhere). Next, still in the tilted position, I will pipette the spent medium out of the flask and into the waste beaker. Rest assured the cells will not be pipetted out with the medium as they are already attach to the surface. The steps I mentioned above serve to minimise the chance of pipetting any cells out of the flask. =)
I hope my explanation proves some help in your understanding of the changing of medium.
Thankz!
Han Yang
TG01
hello.
how does the gluthatione (GSH)-related detoxification system help to make tumour cells resistant?
Thanks.
-Li Ping-
TG 02
Heyas Hanyang!
Really interesting stuffs you've got here eh! Now i'd be coming back here for sure.Can't wait for your next post.lOl.
Anyway, you mentioned that you'd be "focusing on the effect of 2,2'-dihydroxychalcone and chlorambucil in inducing DNA interstrand cross-links on human colon adenocarcinoma cells (HCACs) grown in-vitro." Were you refering to your major project? If so, what are the things that you might want to look out for that would indicate the effectiveness of the drug(s) against tumours in the experiment? Also, how might these differ in vivo?
Thanks lots mate!(=
Alexander Soo, TG02
0608122H
Hello,
What do you mean by nitrogen mustard alkylating agents? And how do 2,2'-dihydroxychalcone and chlorambucil induce DNA interstrand cross-links on human colon adenocarcinoma cells? Thanks!
GOh Shihui
0607135A
Hi all,
Since the questions asked are going to be discussed in my next post, I shall answer them here.
>Li Ping:
Qn)how does the gluthatione (GSH)-related detoxification system help to make tumour cells resistant?
I shall explain this system in a way it can be understood easily.
Ans)In tumour cells, they contain glutathione (GSH), GSH-related enzymes (in particular GSH S-transferase-GST) and GSH S-conjugate export pump (GS-X pump). When anticancer drug enters the tumour cell, GSH will bind to the anticancer drug, rendering it less toxic and more water-soluble, and this reaction is catalysed by GST. Lastly, the ATP-dependent GS-X pump will export the GSH conjugate of anticancer drug out of the tumour cells. Basically, this is the gist of how tumour cells acquire their resistance to anticancer drug.
>Alex:
Qn)Anyway, you mentioned that you'd be "focusing on the effect of 2,2'-dihydroxychalcone and chlorambucil in inducing DNA interstrand cross-links on human colon adenocarcinoma cells (HCACs) grown in-vitro." Were you refering to your major project? If so, what are the things that you might want to look out for that would indicate the effectiveness of the drug(s) against tumours in the experiment? Also, how might these differ in vivo?
Ans)Yes, I am refering to my MP. The higher the % of DNA cross-links, the higher the effectiveness of the drug. Chlorambucil works by alkylating the DNA and induces cross-links in DNA strands, thus preventing further replication and transcription of DNA. Eventually, these will result in cell death. Moreover, this also shows that 2,2'-dihydroxychalcone sensitises the tumour cells to Chlorambucil significantly, which is good.
I am sorry to tell u that I have not perform any in-vivo studies yet. Hence, I cannot state the differences between the effectiveness of the drug in-vitro and in-vivo. Of course, if I am permitted to perform in-vivo studies, my results will be much more reliable and significant than in-vitro.
Hope I have answered your doubts clearly.
Thankz!
Han Yang
TG01
heys
how do u keep the cells at the peak level? Does that mean they must be kept at optimum conditions coz of their rapid division?
hahaha..thx ah:D
Neela
TG02
Hi Neela,
Thanks for the concern for my cells!
Yes, they must be kept at optimum condition to reach the peak level which will be explained later.
First and foremost, the most important factor to take note in cell culture is to avoid any form of contamination. This can be achieved through strict aseptic techniques when pipetting and performing the culture work in BSC. Moreover, the RPMI medium prepared must be sterile so that the cell growth environment is sterile as well. Besides to avoid contamination, you must have a good knowledge on the growth requirements of your cell line. For example, tumour cells have high energy requirement in order to divide rapidly. Hence, to take care of this need, sodium pyruvate is added to supplement the additional energy needed. Of course, maintaining the optimum CO2 concentration (5%) in cell environment is essential too as it is part of the buffer system in maintaining the optimum pH. Regular observation of cell culture (e.g. a colour change in medium such as from red to yellow indicates a drop in pH) can aid you in making decision on whether to change medium, subculture or discard the whole flask (if contamination is present). Last but not least, subculturing (every 2-3 days) and changing of medium as mentioned in my post are steps which you do not want to neglect in cell culture as they help to prevent accumulation of metabolic waste products and ensure anchorage-dependent cells have ample growth surface to proliferate. If all the above are achieved, cells can proceed through their growth phase quickly and reach confluence in 2-3 days.
Summary:
-Strict aseptic techniques
-Incubation at 37°C and 5% CO2
-Sterile cell culture medium and reagents (e.g. trypsin, PBS)
-Good knowledge on cell line
-Regular observation of cell culture
-Prepare the cell culture medium that meets the needs of the cells.
-Subculture and change medium when needed
For more information, you can always refer to my earlier replies to my peers.
Thankz! =)
Han Yang
TG01
Hi Shihui,
Qn)What do you mean by nitrogen mustard alkylating agents? And how do 2,2'-dihydroxychalcone and chlorambucil induce DNA interstrand cross-links on human colon adenocarcinoma cells?
Ans)Nitrogen mustard alkylating agents is a class which Chlorambucil belongs to. As alkylating agent, Chlorambucil is able to alkylate DNA and induce cross-links in DNA strands, thus preventing replication and transcription of DNA in tumour cell, eventually leading to cell death. Besides its chemotherapeutic property, nitrogen mustard can be used in chemical warfare. Other examples of nitrogen mustard include melphalan and uramustine.
2,2'-dihydroxychalcone is a chemosensitiser which sensitises tumour cells (by reducing GST and GS-X pump activity) to Chlorambucil, effectively reducing chemoresistance of tumour cells. In this way, Chlorambucil can induce more DNA cross-links and results in increased death of tumour cells.
For more information, you can always refer to my reply to Li ping on the GSH-related detoxification system to understand the role of 2,2'-dihydroxychalcone.
Thankz!
Han Yang
TG01
Heyas Hanyang!
Hahas.'Course not in vivo yet. But was just wondering if you might know anything in that field course you said that the drug might not be effective for its intended use due to reisistence.
Anyway,so basically death of cells is all you'd be looking out for in the effectiveness of the drugs against the tumoric cells is it?
Thanks again mate(=
Alexander
Hi Alex,
I don't quite understand the first part of your question. Perhaps, my explanation below may fit your question.
Yes, due to drug resistance, Chlorambucil may not work as effective. Hence, researchers have discovered plant polyphenols such as 2,2'-dihydroxychalcone which sensitises tumour cells to Chlorambucil. In this way, Chlorambucil can remain in the tumour cell longer and induces more DNA crosslinks which results in more death of tumour cells and limit tumour cell proliferation.
In my study, I do not look out for the number of dead cells to interpret the effectiveness of the drug. In previous studies, they have performed MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay to determine the cytotoxicity of drug. Rather, I am focusing on whether the amount of DNA crosslinks will increase in the presence of sensitiser.
For your understanding, perhaps you may want to read up on my previous replies.
Thankz!
Han Yang
TG01
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