Dear Mr. Bhaskar,

Try the following:

1. Do NOT heat inactivate the serum, because it destroys the essential growth factors and leads to improper cell adhesion and proliferation. If you are using the Gibco FBS south American origin, it already comes heat inactivated, you dont have to do it again.

2. Once your flask arrives, let it stay in your incubator atleast for 24 hours before sub culturing.

3. Antibiotics: please cross check the sensitivity of 3T3 cells to the antibiotic cocktail you are using. for example, some cell lines are sensitive to Amphotericin-B so if you are using an antibiotic and antimycotic solution, which has Amphoterecin-B in it,the cells will not survive. In such cases, just Penicillin-Streptomycin solution will do.

4. When you are sub culturing, after trypsinization, do NOT spin down the cells. Instead, you will add twice the volume of the medium to quench the action of trypsin right, then with out spinning the cells, distribute the suspension along with the trypsin into the two sub culturing flasks.

5. when you subculture, leave the cells in at least 8 ml medium, untouched for two days. Then according to the growth rate of your cells, you can change the medium or split them again.

Hope this is helpful

Hi Bhaskar,

I am agree with Mallu , do not use heat inactivated serum. Fibroblast like cells, also 3T3 cells should be culture in non-heat inactivated serum.

It is also important to know when you decide to sub culture them, normally 3T3 cells are easy to handle and proliferation rate is high, however, handling is always tricky. As bfar as I understood you do not have any contamination in your culture, am I right?

I am not agree with point 4, you can stop the action of trypsin by addition Medium +FCS, I usually spin them down and re suspend in new culture medium then divide them in proper flasks. Refresh the medium every 2-3 days, you do not need additional washing here.

BTW: If you have any picture of your cells ( healthy and dying) I would be happy to look at them, it will help to figure out what is going on.

Mojtaba

Earlier I also used to spin down after trypsinization but we lose a lot of cells during the process and the cells at the bottom of the pellet will be stressed and start dying.

The method I suggested works like a charm

If after following the above suggestions your cells are still dying, consider the autoclave. It can be a source of heavy metals, endotoxin and others. Also, some plastics break down to toxic products under heat and pressure.

In my experience, BALB/c 3T3 cells are easy to grow and quite robust.

My suggestions: Establish some cultures using exclusively supplies (medium, serum, trypsin-EDTA, disposable plasticware) from commercial sources. Buy all reagents as sterile solutions. Don't use anything that has been washed, autoclaved, fumigated or filtered in your lab. Don't add any antibiotics.

If cells grow well under these conditions, you know that there is something wrong with your home-grown reagents or with your recycled equipment. If your cells still fail to survive passaging, I would have a closer look at the trypsinization procedure.

By the way, what are you using to fumigate equipment or supplies?

Thank you for the reply frnds. Lab is fumigated and incubator is heat sterilized along with 70% ethanol. Biosafety cabinet by UV irradiation and 70% ethanol. All reagents filtered using 0.22micron filter sterile packed. Reagents stored in dedicated -20 C and 4C refrigerators for cell supplies.

Also would'nt it be a problem if complement is not inactivated before use

UV breaks down plastic to toxic (to some cells) substances. Do not keep plastics in the hood exposed to UV light.

When you replate the cells after trypsinization, do they initially attach, spread, and divide?

They do adhere but they do not divide and confluency is not seen even after 2-3 days.

Also can anyone please send me the cell culture if they have it. because I think the origin of the cells might be a problem

Tissue culture plastic has a shelf life. Try comparing plastic from another source with yours. Also, are these cells glutamine/pyruvate dependant? If so, these reagents can go bad.

As a first (simple) step I would make certain that ALL your plasticware (test tubes, culture dishes, pipettes, etc.) is certified for tissue culture work. Tissue culture-quality plasticware comes off a clean assembly line or is sterilized by ionizing radiation. Furthermore, the surface of culture dishes is treated to make it "adhesive".

By contrast, plasticware for bacteriological work is often sterilized with ethylene oxide. Small amounts of ethylene oxide can remain associated with the plastic and are released into the culture medium when you place the dishes into the incubator. Ethylene oxide is very toxic to cells and can easily kill cultures. If culture dishes are sold as "Petri dishes", they are almost certainly NOT meant to be used for tissue culture work.

Note that dishes for bacteriological work are less adhesive than tissue culture dishes. However, most brands still allow cells to attach, albeit not very strongly. However, they don't allow cells to spread. That's why it is important to know if cells spread after plating.

Is your culture medium ever coming into contact with recycled glassware? If it does, do you use a dishwashing detergent that is recommended for tissue culture glassware?

If your plasticware and your glassware look OK, you need to have a closer look at your culture medium and the handling of your cells.

Dear Mr.Bhaskar, I'm having the similar problems with my MC3T3 cells...it start to die after subculture..after 2 or 3 day. Did you try suggestions form Mallu Abhiram Charan Tej..is it working? If do so...i would like to try the suggested methods...

I suspected Mycoplasma contamination and discarded the culture. Also I bought new culture and started the procedure all over. Presently I am able to culture 3T3 even without antibiotic