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How to grow cancer cells on agar/ soft agar
#1
I am a biotech postgraduate working in a microbiology lab.I have tried a lot of techniques to grow the cancer cells.but was not sucessful.i have read many articles relating to this from my searches in google.
My query is that can i grow cancer cells on soft agar from the blood or wat other medium should i use to let them grow.
secondly wat conditions should i maintain for them to grow like temperature,etc.
lastly what is the incubation time.
REGARDS,

ARAOBig Grin
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#2
Cancer cells in culture

Growing cancer cells in culture is different that growing normal cells. Healthy cells are passing up to 70 division cycles before mitosis slows down and stops eventually. It’s believed that telomerase are shortening with each division cycle until they reach critical length preventing the cell from the further division. This phenomenon is known as replicative senescence. Other event that affect cell division is called contact inhibition: healthy cells are organized in one layer during mitosis and they are gradually populating the bottom of the Petri dish, gently touching each other; once they cover the dish – mitosis will cease.

Cancer cells act completely different. Unlike healthy cells, they could undergo indefinite number of divisions and they don’t “mind” cellular contact; after covering the surface of the Petri dish – they’ll continue dividing and piling up into mounds.They are usually grown on the much simpler medium compared to healthy cells.
This is what I found about in vitro cultivation of the cancer cell lines that might be interesting for you:

THP 1 cell line (human leukemic monocyte)

These cells are derived from peripheral blood of a 1 year old boy with acute monocytic leukemia. Suggested medium contains RPMI 1640, 2 mM Glutamine and 10% Fetal Bovine Serum (FBS). Thawed cells should be added to a conical based centrifuge tube. 4 ml of culture medium should be added to the tube. 100 ml of the suspension should be extracted for cell number counting. Centrifuge the tube at 100 - 150 x g for 5 minutes. Re-suspend the cells in fresh medium containing 20% FBS. Incubation in 5-7% carbon dioxide atmosphere at temperature of 37°C will result in exponential growth of the cells. Flask should be positioned vertically. This can take up to 7 days. When cell culture is established, FBS concentration could be reduced to 10%. 3-8x10⁵ cells/ml is optimal density for maintaining cells in exponential phase of growth.

ML-1 (human acute myelogenous leukemia derived cells) and HL-60 cell lines (human promyelocytic cell line)

ML-1 and HL-60 cell lines could be maintained in both the conventional serum-supplemented liquid cultures and in the serum-free liquid cultures.
In the serum supplemented cultures, 2.5 x 10⁶of both cells types should be suspended in 10 ml of RPMI 1640 (Roswell Park Memorial Institute medium) culture medium. 5% fetal calf serum (FCS) should be added. 25-sq cm tissue culture flask should be kept at 37°C in a humidified 5% carbon dioxide atmosphere. This procedure should be repeated every 5-6 days. At each subculture, cells should be harvested, washed, and placed in fresh medium supplied with 5% FCS. Trypan blue dye should be used for viable cell counts determination.

In the serum-free liquid cultures method, 2.5 x 10⁶ viable cells should be suspended in 10 ml of Iscove's Modified Dulbecco's Medium (IMDM) with human transferrin (5 M9/ml) and bovine insulin (5 microg/ml). 25-sq cm tissue culture flask should be conditioned as described above. This procedure should be repeated also every 5 to 6 days. At each subculture, cells should be harvested, washed, and placed in the fresh medium.

K562 cells (human chronic myelogenic leukemia cell line)

Cells should be grown and maintained in RPMI. It can be purchased from Invitrogen Corporation (Cat No. 23400-021). This medium is used for the expansion of human lymphoid cells. RPMI 1640 uses a bicarbonate buffering system and should be supplemented with 15 % fetal bovine serum (FBS) in 5% carbon dioxide atmosphere. It requires pH 8 and temperature of 37°C for two weeks.

RBL-2H3 cells (rat basophilic leukemia cell line)

RBL-2H3 are Wistar rat derived basophilic cells. Base medium used for these cells is ATCC-formulated Eagle's Minimum Essential Medium. 2.0 to 3.0 ml of Trypsin-EDTA solution should be added to flask and cells should be observed under an inverted microscope until cell layer is dispersed (5 to 15 minutes). Addition of the 6.0 to 8.0 ml of complete growth medium will result in appropriate cell growth. Culture should be kept in 5% carbon dioxide atmosphere and incubated at 37°C. Medium should be renewed every 2-3 days.

Each of these medium could be purchased easily. Detailed procedure is explained on the label. If you follow procedure according to manufacturer’s instruction, you’ll probably increase the chances for successful growth of cancer lines in culture.
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#3
Great information sharing about the cancer cell growth, generally many people are suffering from this diseases, today this is one of the topmost dangerous diseases. Medical technology is increasing more and more today, for all kind of diseases there is a solution for that.
Join With Envita Medical Center
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#4
Thanks for the information bojana i will revert to you back once i have got all the materials medias etc.

i have another query is that is it very expensive to maintain these cell lines and generally wat is the validity of these cell lines like how many passages or may be time frame etc.

sry for replying late as i was completing my research .
REGARDS,

ARAOBig Grin
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#5
Are cancer cells sensitive to the pH of the medium?
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How to grow cancer cells on agar/ soft agar12