Hi Shrouk,

I agree that it will be important to have the correct growth factors and media for long-term survival and to establish a cell line. However, I think you need to start with your culture conditions.

I have found that depending on the tumors, there are vastly different conditions for dissociating them. Three (3) hours is quite excessive for any tumors I have dealt with (mainly breast & colon). Also, there are different collagenases; our best luck was with a mixture of collagenase types 2 & 4. Whenever we were looking at a new tumor, we did a curve of different digestion times vs. viability and dissociability to determine what was optimal. Also, be sure to "stop" the digestion with FBS-containing media! Additionally, some tumor cells are quite sensitive to how high the RPMs are on the centrifugatiom step(s). Finally, we would simply plate (in appropriate media of course) at an average of 0.5 viable cells/well (24-well plate) and wash after an overnight culture then allow single cells to grow - which sometimes took up to 2 wks.

I hope this helps!

Jennifer

Dear Shrouk, probably this could be due to several factors. One of these is the composition media and the other is the time of digestion. I think that 3 hours could result in some times in an overdigestion, hampering the viability of the cells. There are several papers referring to this type of digestion, however I recommend you to check under the microscope the digestion sample every 30 min. When you see a lot of single cells and also organoids the digestion is OK, If you are unable to obtain organoids in your digestion sample, probably you have a sample overdigested, compromising the cells viability.

The FBS has to be heat-inactivated, check the specifications of the manufacturer.

Good luck,

thank you Gemma and Thomas

Can you help me with any other protocol to establish the primary cell line ?

I was trying to find the suitable protocol for this issue But i couldn't

i'm new in this field

Hello Shrouk, we have established some breasat cancer cell lines. I send you the paper in which we described them. But I certainly agree with Tomas and Gemma that growth factors are very important.

Many Thanks Isabel

Dear Shrouk,

I tried before to establish ovarian and breast cancer cells from tumor in Egypt and I tried different protocols but without the growth factors because they are expensive but it did not work. I have to say that I got adherent cells but they die after one or two days due to absence of GFs. I contacted a company in Cairo and discussed this issue with them, they are sponsor for Lonza, and they offered 25% off any product that I need for primary culture cells. I recommend that you call them and tell Mr. Yehia that you heard about the offer and discuss it with him. his contact information is Mohamed Yehia

Senior Sales Specialist

Maadi Medical Supplies

45, Road 77, Maadi, Cairo, Egypt

Cell +201223155099

Tel. (202) 2378 1946

(202) 2751 3975

Facsimile (202) 2380 3028

E-Mail [email protected]

[email protected]

[email protected]

Website www.maadimed.net

www.facebook.com/Maadimedicalsupplies

I got this offer from a representative from Lonza company who came to Egypt and gave a presentation about the products from Lonza.

good luck and do not hesitate to contact me if you need anything

Many thanks Dr Fardous

i bought the media i'm using from Lonza dealing with Maadi med , thanks for advice

i'll call them inshaallah

ensha Allah

you may remind him about that offer and best wishes

Dr Fardous

Could you please send me your protocol for breast cancer primary cell culture establishment , if you still have it ?

Hi Shrouk,

I agree that it will be important to have the correct growth factors and media for long-term survival and to establish a cell line. However, I think you need to start with your culture conditions.

I have found that depending on the tumors, there are vastly different conditions for dissociating them. Three (3) hours is quite excessive for any tumors I have dealt with (mainly breast & colon). Also, there are different collagenases; our best luck was with a mixture of collagenase types 2 & 4. Whenever we were looking at a new tumor, we did a curve of different digestion times vs. viability and dissociability to determine what was optimal. Also, be sure to "stop" the digestion with FBS-containing media! Additionally, some tumor cells are quite sensitive to how high the RPMs are on the centrifugatiom step(s). Finally, we would simply plate (in appropriate media of course) at an average of 0.5 viable cells/well (24-well plate) and wash after an overnight culture then allow single cells to grow - which sometimes took up to 2 wks.

I hope this helps!

Jennifer

Dear Shrouk

The establishment of long term cancer cell line is very difficult and sometimes it depends on a chance. Short term culture for few day living cell line is easiest than long term culture. Breast cancer cell culture is one of the most difficult type of culturing. It depends on many things, and the most important is the texture of the solid tumour, how much it is soft and fragile how much it produces much cancer cells. In case of hard type you can use collaginase II enzyme to facilitate the disintegration of the tumour mass to release more cancer cells. Try to use minimum amount of a medium just to cover the pieces of the tumour but not try to put high amount of medium. RPMI 1640 is the best culture medium for beast cancer culture. However you have to repeat the procedure so many times in order to get one successful primary culture. I have tried tens of times in order to obtain two colon cancer cell lines.

Hi,

I'm currently working with primary human breast cancer and normal cells.

I have used the miltenyi biotec enzyme cocktail but collagenase iii is also effective.

I use HAMs F10 with 9% FBS,100U ml-' penicillin, 100ugml-' streptomycin, 2mm glutamine, 0.5ugml-'hydrocortisone,5ugml-'insulin and 5ngml-1 epidermal growth factor(EGF). I have managed to establish cells growing for several weeks and they even survive after they have been frozen in liqN2. However, i cannot say for sure if they are established cell lines. I have tested my cells for positivity to CD326 marker. I have to say that the appearance of adherent cells in either normal or tumour samples varied from 1 day to 1 week. I tend to put my first cell isolates in a small volume to allow the cells to adhere and keep them concentrated and observe them daily. I then increase the volume gradually by adding 0.5-1ml from fresh medium every day which allows natural growth factors produced by the cells themselves not to be diluted too much but also have some fresh nutrients. I'm attaching a paper that i found very useful to optimise my protocol. I hope that helps

I would like to add that in certain established commercially available lines such as MCF10A they also add cholera toxin which allows the cells to proliferate faster (also present in the protocol in the pdf i sent you. however i have not used this in my protocol. good luck again

I have grown both primary tumor (liposarcoma) and primary stromovascular cells from adipose tissue. A few things that I have found helpful: I use DMEM:Ham's F12 1:1 with 10% serum initially. Supplementing this medium with insulin can be beneficial (I know of at least 1 breast ca line that grows best with extra insulin (T47D). Also, coating your culture dishes with collagen or fibronectin can improve your success. I have had better luck plating the cells at a fairly high density initially as many can produce their own growth factors if there are enough present. For cancer cell lines, you should expect your culture to go through one or more crisis periods where nearly all the cells die off to occur. If it looks like the culture is crashing keep changing the medium regularly and keep it in the incubator. Most of all have patience, keep trying and good luck.

I would like to add that it is very important to understand that in tumor tissue, other non-tumor cells exist and some of them might grow with the same conditions as your cancer cells. The most challenging is fibroblasts which will overgrow your tumor cells if you supplement your culture medium with fair percentage of serum. On the other hand in serum free media normal epithelial cells (which might also exist in the tumor tissue) might overgrow the tumor cells. Researchers solved these issues either by selectively removing fibroblasts by MACS or keeping epithelial cells then avoid growth factors. The idea is that you have to keep track of the type of cells growing for example by testing for Ep-CAM or Cytokeratin expression to ensure that you have epithelial cell growing. You can also test to loss of heterozygosity to ensure that you have tumor cells and normal epithelial cells. I support advises given above like paying attension to centrifugation speed (I think 4000 rpm is too fast but I am not sure what is the diameter of the centrifuge and therefore how this translate into g instead of rpm). Also addition of collagen and fibronectin is a very good idea. Finally you need a lot of luck as this is not an easy task.

Note 1: Adenocarcinoma cells (like breast, colon..etc) are difficult to grow in vitro in opposite to other types of tumors like sarcomas, gliomas..etc. This supports the importance of micro-environment which is disrupted when cells grow in vitro.

Note 2: Stem Cell Technologies and Miltenyi biotec have kits/protocol for dissociation of tumor cells, you can contact them for technical information.

When you extract cells from tissue, the primary cell culture might turn benign over long term.

I highly recommend looking up the link below. And if possible passage your isolates as xenografts in NOD/SCID mice or nude mice. The tumor after xenografting can then be minced and stored in liquid N2 with DMSO (5-10%)

http://www.ncbi.nlm.nih.gov/pubmed/?term=Wicha+M

Wicha lab isolates breast cancer samples atleast a few times a week. They have a strong team for stemcell studies in breast cancer.

Try and google Hassan KA, he is good in isolation techniques.

This paper I followed during the isolation of the ovarian cancer cells from biopsy and it works really but after few days the cells die. as they mentioned above to establish long term tumor cells it is difficult and in order to keep them in culture you have to do retro or lenti viral transduction and characterization of the cells that you had isolated to make sure that you have the right and desired cells.

Shrouk, can you send for me your email so I can send for you my hand writting

this is mine

[email protected]

[email protected]

Hi,

I would like to suggest you to please initiates the procedure of culturing immediately on receiving the tissue sample. For better adherence you can coat the flask with 0.2% gelatin in PBS for 1h. Cut the tissue into small pieces ( very small) and try to keep in the 24 well or 6 well plate in minimal amount of media ( DMEM+ 20% FBS, trust me it works best). Take care that the explant is properly attached and the media just enough to prevent drying of the explant without floating. The other thing you can do is to partially digest with trypsin -EDTA ( 0.25% trypsin and 0.02% EDTA in HBSS buffer) for short period say 10 min, followed by deactivation by FBS and spinning to remove the unwanted material and the explant on the coated wells with minimum amount of media. Do not disturb for 48 h and change the media after every 2 days. I hope this protocol works fine.

Good luck

Shrouk,

Are you using mouse tumor or human post-op tissues? For mouse tumors, you may even do a very brief collagenase treatment or skip that step and just chop the tissue with sterile razor, starin the big chunks and add media before plating. Also, do not disturb the plate for at least two to three days, which helps adherence. Use 2X antibiotics for first few days to avoid contamination. Once the cells start to grow out of the small clusters, keep changing the media every 2-3 days. When the cells are showing a mixture of epithelial and fibroblastic population, start doing sequential (but gentle and brief, 30 seconds to a minute depending on the density of fibroblssts) trypsinization. Only the fibroblast will start to peel off, so just aspirate those and add fresh media to continue culturing in the same flask. It will take a few rounds to have epithelial population. I wouldn't rcommend adding collagen or gelatin at this stage, as it will be diffcult to isolate the fibroblasts. You can use any cell specific markers to sort out the epithelial cells once you have high number of cells for sorting. For human tumors, you may have to add some special nutrients in the media to stimulate growth, depending on the tumor type. Good luck!!

Still I say that establishment of a primary breast cancer cell remains difficult. However repeating the process of establishment with fine mincing the tumour mass will give fruitful result. Usually not all sample give rise to primary culture but specific type of tumour mass can yield long term culture.

Can anyone please provide me complete protocol for primary breast cancer culture! I am confused about growth factor. As I want to culture breast cancer cells, why should I add growth factor!

I think one of the most successful methods in establishing new breast cancer cell lines was that used by Stephen Ethier, who generated the SUM lines. He used implantation in vivo in immunodeficient mice and the successful xenografts were then dissociated and cultivated in a variety of conditions in vitro. He used HAM F12 mostly, with 5% serum and various combinations of growth factors or serum free medium with a combination of serum replacement factors and growth factors. His papers describe the protocols in detail.

Cells from different tumours will grow in different conditions. Expanding the tumour in vivo will allow you to try more in vitro conditions. Not all tumours that grew in vivo established lines in vitro. Also about 10-20% of tumours grow in animals, although NSG mice were reported to have a xenotransplantation success of up to 30%. This applies to breast cancer only, colon cancers have a better take in vivo and pancreatic, head and neck cancers, melanoma have really high xenotransplanation success.

In our in vivo xenotransplantation studies we had best results in dissociating tumours from patient samples or tumours passaged in animals with a protocol using collagenase from STEM Cell Technologies and short dissociation time (1-1.5h).