26 replies to this topic

[Bita]

What freezing medium is better for ADSCs (Adipose derived stem cells)?Should freezing medium be contained antibiotic or not?
helps are appreciated in advance

[Brahmavidhya Arun]

I have a basic question. whenever we prepare cell seed, we inoculate FTM for checking the sterility of cell seed.

Within 24 h. FTM changes to yellow with a thin line of pink ring at the top............i don't think it is contamination........... I inoculated FTM with serum alone, freezing medium and DMSO separately....but FTM resembles control.(no color change)......
please clarify my doubts..........


Thanks
Brahmavidhya K.V.

[bob1]

Brahmavidhya Arun, on 14 March 2012 - 12:33 AM, said:

Within 24 h. FTM changes to yellow with a thin line of pink ring at the top............i don't think it is contamination........... I inoculated FTM with serum alone, freezing medium and DMSO separately....but FTM resembles control.(no color change)......
please clarify my doubts..........

Can you please describe your procedure for this, that will help us to know what you have done and the likely causes of any problems.

[pzenatti]

Hi. I need help to culture cell line that I brought from USA to Brazil. When I thaw the cell, the cells are very good, but into one/two days all cells die. I have put growth factors, more FSB, but nothing worked. What is supposed I do? This cell is IL-7 dependent.

[spurnima]

Hi,

Can anyone please tell me if I can use Pen/strep in the freezing medium?

[rhombus]

spurnima, on 17 July 2012 - 12:56 AM, said:

Hi,

Can anyone please tell me if I can use Pen/strep in the freezing medium?

Yes you can BUT you should not be using ANY antibiotics AT ANY TIME under normal conditions

KIndest regards

Uncle Rhombus

[spurnima]

rhombus, on 17 July 2012 - 01:39 AM, said:

spurnima, on 17 July 2012 - 12:56 AM, said:

Hi,

Can anyone please tell me if I can use Pen/strep in the freezing medium?

Yes you can BUT you should not be using ANY antibiotics AT ANY TIME under normal conditions

KIndest regards

Uncle Rhombus

But we do add Pen/strep in the normal cell culture media, Please help me understand your reply.

[rhombus]

spurnima, on 17 July 2012 - 07:51 AM, said:

rhombus, on 17 July 2012 - 01:39 AM, said:

spurnima, on 17 July 2012 - 12:56 AM, said:

Hi,

Can anyone please tell me if I can use Pen/strep in the freezing medium?

Yes you can BUT you should not be using ANY antibiotics AT ANY TIME under normal conditions

KIndest regards

Uncle Rhombus

But we do add Pen/strep in the normal cell culture media, Please help me understand your reply.

Dear Spurnima,

The reason(s) for not adding antibiotics are:-

They are very dirty compounds that will affect the cells you are growing

They cover up CRYPTIC CONTAMINATIONS i.e. background levels of contamination that will again affect your cells

If you are using your cells for experimental purposes ......the antibiotics MAY interact in some way with the compounds that you are adding

It is an expensive addition THAT YOU DO NOT NEED TO ADD IN THE FIRST PLACE


Hope this explains some of the good reason not to add antibiotics to your cells in culture

KIndest regards

Uncle Rhombus

[spurnima]

Dear Rhombus,

Thank you very much Indeed.

Regards,
Purnima

[jeanny1]

hi, yesterday I want to freeze my cells (CHO from Invitrogen). Usually, I placed them at -20°C for 2 hours, then at -80°C overnight and then into liquid nitrogen. But, I forgave them at -20°C overnight (~20 hours). Then I placed them at -80°C. How long they should be there before placing them in nitrogen? (few hours/ day and overnight/??) And can I use them for transfection lately or are they completely degradeted?
thanks very much for reply

[rhombus]

jeanny1, on 18 July 2012 - 11:21 PM, said:

hi, yesterday I want to freeze my cells (CHO from Invitrogen). Usually, I placed them at -20°C for 2 hours, then at -80°C overnight and then into liquid nitrogen. But, I forgave them at -20°C overnight (~20 hours). Then I placed them at -80°C. How long they should be there before placing them in nitrogen? (few hours/ day and overnight/??) And can I use them for transfection lately or are they completely degradeted?
thanks very much for reply

Dear Jeanny1,

Please read the information below:-

Cryogenic storage at very low temperatures is presumed to provide an indefinite, if not near infinite, longevity to cells, although the actual “shelf life” is rather difficult to prove. In experiments with dried seeds, researchers found that there was noticeable variability in deterioration when samples were kept at different temperatures–even ultra cold temperatures. Temperatures below the glass transition point (Tg) of polyol's water solutions, around −136 °C (137 K; −213 °F), appear to be accepted as the range where biological activity very substantially slows down, and −196 °C (77 K; −321 °F), the boiling point of liquid nitrogen, is the preferred temperature for storing important specimens. While refrigerators, deep freezers and extra cold deep freezers are used for many items, generally the ultra cold of liquid nitrogen at −196 °C (77 K; −321 °F) is required for successful preservation of the more complex biological structures to virtually stop all biological activity


I organised a seminar at my university where the Health Protection Agency (HPA at Porton Down) came in and talked about Cell Authentication AND basic cell culture protocols. The lecturer, who manages the HPA cell facility, presented plenty of data about freezing cells and their storage. He stated that cells frozen, even with a Mr. Frosty, could only be stored for 2 weeks at - 80oC, before difficulties with propogation and growing could be observed. So with a Mr.Frosty the cells go into the -80 from the cell culture suite. They then stay there overnight AND THEN TRANSFERED TO LIQUID NITROGEN.

So.....with your cells ......SOME MAY SURVIVE but you have put a massive selection pressure upon them and SELECTED a sub population of cells that can survive and grow.

Hope this is useful

Kindest regards

Uncle Rhombus

[jeanny1]

Uncle Rhombus: thank you very much for your answer