solid tumor culture question

[mcchromo]

Hello all,

I was wondering what people thought about when normal cell growth is
seen in solid tumor sample in situ setups, ie. harvest should be
picked before day 5 or day 7 or day 14, etc?

Any help would be much appreciated.

Sincerely,
mcchromo

[Random lawce]

Hi McChromo: I think the emphasis on harvesting tumor cultures at a
certain time is kind of odd. Each tumor type is different. I feel
from my own experience (18 years culturing and harvesting tumors) that
the abnormal cells in neuroblastomas quit dividing within 48 hours of
COLLECTION (not setup) while for kidney tumors I found some still
showing tumor cells at 28 days. One kidney tumor showed only normal
karyotypes from day 3-10, so we put it in "the morgue", a shelf in the
incubator that does not get fed. After 3 weeks, the fibroblasts were
all dead from the horrible conditions but the tumor was doing great
and we got nice karyograms. Germ cell tumors seem to do very well for
long culture times, while plasmacytomas, tumors of plasma cells, is
very short lived in culture. So are many brain tumors short lived.

Some would say that you need to worry about tumor karyogram changes in
culture but the kidney tumor did have the deletion of 3p that we were
looking for so it was clinically useful and I can't remember seeing
anything that I deemed in vivo artifact in a tumor (e.g., confined to
one culture vessel and different from the other vessels).

My rule of thumb is to harvest one or more cultures as soon as mitotic
cells are visualized in an inverted phase microscope (usually 3-8
days), and then more are harvested if needed. It is not unusual for
certain types of tumor to take 10 days to finally be mitotic enough
for harvest. Just observe the cultures carefully. Harvest them when
they start showing growth. Remember that they have circadian rhythms
that were set up in vivo and some are active only at night. Overnight
colcemid works really well for most of the tumor types.

Tumor cultures are the most challenging but the most rewarding to work
with when you "figure them out" like a good detective.

Helen

[Marjori Leiva Camparoto]

Good morning!
I had worked with tumor cells of the gliomas. My question about
colcemid concenytration and exposure time. Colcemi concentrations
reported in published methodologies of recent years range from 0.015
ug/ml to 0.2ug/ml.

What is concentration and time better (gliomas)?

Marjori

2012/1/25, Random lawce <helen...@yahoo.com>:

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[arundhati sharma]

Hi Helen,

you've mentioned that the neuroblastoma tumors stop dividing after 48 hrs. Could you tell me with ur experience on how to culture these cells?

thanx 

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[Random lawce]

We would use 15 ul of the 10 ug/ml stock Colcemid for a 5 ml culture (.
03 ug/ml final concentration). Helen

On Jan 26, 5:17 am, Marjori Leiva Camparoto <marjor...@gmail.com>
wrote:

> Good morning!
> I  had worked with tumor cells of the gliomas. My question about
> colcemid concenytration and exposure time. Colcemi concentrations
> reported in published methodologies of recent years range from 0.015
> ug/ml to 0.2ug/ml.
>
> What is concentration and time better (gliomas)?
>
> Marjori
>

> 2012/1/25, Random lawce <helenla...@yahoo.com>:

> >http://groups.google.com/group/cytogenetics-methods-and-trouble-shoot....

[Random lawce]

Hi Arundhati: These cells do best in suspension culture and treated
like a bone marrow. The cells that attach are usually all normal
stomal cells and it is the floating cells you want to try to harvest.
They do very well with long Colcemid times. One of the prettiest ones
I ever saw was one that yielded normal metaphases on a 0 hr direct
culture, and i decided to try overnight Colcemid; however, a big ice
storm hit our city and I could not get to work for 3 days. I
harvested it after the 3 days in Colcemid and it was abnormal, long
chromosomes, and very nice quality. Helen

On Jan 27, 12:22 am, arundhati sharma <arundhatishar...@gmail.com>
wrote:

> >http://groups.google.com/group/cytogenetics-methods-and-trouble-shoot...
> > .

[suhaip]

Hi Helen
First I would like to express my appreciation and my hard thanking for your effort in solid tumor topics
Hellen
I am very happy to be one of our group
I just want to know if there's specific medium u used to different type of tissue
Dose u add any additive to the culture in order to support the growth of tissue
Can you provide me by protocols u usually used in order to get cells and
Metaphases from the harvest

If u can sent me any article or any links cab help me yo improve my skills and to develop my experience

Sent from my iPhone

> For more options, visit this group at http://groups.google.com/group/cytogenetics-methods-and-trouble-shooting?hl=en-GB.
>

[Random lawce]

Hi Suhaip: we just use RPMI 1640 with 15% defined FBS (Hyclone), L-
glutamine, and gentamicin. It seems to work for the majority of cell
types. Harvest is standard with 0.075 M KCl and 3:1 fix. I am not
aware of a tissue culture link. Best wishes, Helen

On Jan 28, 4:39 am, suhaip <alnaser1983_suh...@yahoo.com> wrote:
> Hi Helen
> First I would like to express my appreciation and my hard thanking for your effort in solid tumor topics
> Hellen
> I am very happy to be one of our group
> I just want to know if there's specific medium u used to different type of tissue
> Dose u add any additive to the culture in order to support the growth of tissue
> Can you provide me by protocols u usually used in order to get cells and
> Metaphases from the harvest
>
> If u can sent me any article or any links cab help me yo improve my skills and to develop my experience
>
> Sent from my iPhone
>

> > For more options, visit this group athttp://groups.google.com/group/cytogenetics-methods-and-trouble-shoot....

[arundhati sharma]

 thanx Helen, please also tell me which media U use for these cultures. Have U ever tried culturing cells derived from  fine needle aspiration?

arundhati

For more options, visit this group at http://groups.google.com/group/cytogenetics-methods-and-trouble-shooting?hl=en-GB.

[Random lawce]

Dr. Sharma: we just use RPMI 1640 with 15% defined FBS (Hyclone), L-

glutamine, and gentamicin. It seems to work for the majority of cell
types.

Fine needle aspiration is pretty difficult for us. I would say that
about half of them yield metaphases. The trick is getting the right
aspirates. If they are very cellular and contain tumor (both iffy)
they can yield one small culture perhaps. Helen

On Jan 29, 9:10 pm, arundhati sharma <arundhatishar...@gmail.com>
wrote:

[arundhati sharma]

That's right. Even our FNA derived cultures have a success rate of  about 58 -60%.

thanks 

For more options, visit this group at http://groups.google.com/group/cytogenetics-methods-and-trouble-shooting?hl=en-GB.