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Using HEPES buffered culture medium in human IVF |
By:
Dr Malpani, MD, Malpani Infertility Clinic, Colaba, Bombay
22 April 2003
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Many companies now manufacture formulations of culture medium which contains HEPES, which are designed to be used when handling eggs and embryos outside the CO2 incubator, so as to minimise pH changes. This medium also usually contains bicarbonate buffer.
My question is: how does one equilibrate this medium prior to use? Should it be equilibrated in a CO2 incubator for 12 hours prior to use? Or should it be equilibrated in a simple incubator? |
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I agree. If you use a Co2 incubator in order to warm an HEPES bufered medium you have to close cap. I tested pH value in Flushing medium (Medicult) warming open overnigth in the incubator and it was near 7,30.
Jos? Rosales, LIVV (Venezuela) - 10 October 2004
We warm our HEPES buffered medium overnight in a room air 37 degree C dry oven. Since it is HEPES buffered it should not be exposed to increased CO2 conditions because it will alter the pH of the media. We try to minimize the amount of HEPES media that is transferred with our sperm or oocytes; again due to pH alteration. If you use oil overlay you should be able to manipulate the zygotes and embryos successfully without the use of HEPES if you only have them out of the incubator for a short period of time.
KELLY HANSHEW, BRYN MAWR HOSPITAL - 10 October 2004
I do not think that you need to equilibrate any media which contains HEPES in it. It will be acidic if one tries with CO2. HEPES containing media should be warmed before used.
Levent Keskintepe, Seer Institute - 10 October 2004
Any media which is buffered by bicarbonate must be equilibrated by + 37 deg C + 5 % CO2. even though the media's pH is further stabilized by HEPES.
The gamete handling meda ( bicarbonate bufferd ) are added with HEPES may be due to absorb the shock of pH disturbance due to its prolong exposure to
atmospheric air .
The actual IVF media are not stabilized by HEPES because it may cause ionic changes in embryos and the chances of exposure to atmosphere is less.
Thanks
Dr.D'Pankar Banerji, Ideal Fertility and IVF Center,jabalpur,Ind - 10 October 2004
I believe it should be equilbrated in a standard incubator. The pH has been adjusted to 7.4 with HEPES and usually 5mM Bicarbonate.If you put it in a 5% CO2 incubator the pH will fall below 7.0
Best wishes.
James Stanger, Hunter IVF/FertAid - 10 October 2004
Only need 37 ?C , not CO2 , if you use CO2 incubator close cap.
Xavier Blum, Innaifest-Ecuador - 10 October 2004
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