Hello

Yes you can use , HEPES is chemical buffering using a zwitterion, HEPES, has a superior buffering capacity in the pH range 7.2-7.4 and does not require a controlled gaseous atmosphere. HEPES is relatively expensive and toxic at a higher concentration for some cell types. HEPES has also been shown to greatly increase the sensitivity of media to phototoxic effects induced by exposure to fluorescent light

As an example for use HEPES with Culture media

we use HEPES whith bone marrow-derived human mesenchymal stem cells (BM-hMSCs).

obtained the cells by withdrawing human bone marrow from bilateral punctures of the posterior iliac crest of the pelvic bone of healthy donors using syringes containing heparin sodium (1,000 units of heparin per ml bone marrow). The bone marrow was then diluted to 5-10 million nucleated cells per mL in Hank's Buffered Salt Solution (HBSS). Cells were layered over Ficoll-Paque (Amersham Pharmacia, cat #17-1440-03) and centrifuged at 400×g for 30 minutes at room temperature. The mononuclear cell layer was removed and washed twice in HBSS. Finally, the BM MNCs were cyropreserved in a cell cryopreservation medium comprising 86.5% IMDM, 7.5% DMSO, 4% Human Serum Albumin and 2% hydroxy-ethyl-starch. Upon arrival, we at PPRF stored the frozen cells in liquid nitrogen for later experimental use.

in attachment the article describes the effect of HEPES using . may help you

Good luck

thank you for your answer. But the research paper that you attached is saying we should avoid hepes usage in ivf. And, you didn't mention about hepes in your bone marrow derived human mesenchymal stem cell procedure. Kindly clarify me

Theoretically you can use HEPES if your aim is to lower the pH. However the pH is lowered as a direct result of the glucose consumed inside your media. Which means that your media composition will change anyway, only the pH will stay stable. I am not sure you want this. My suggestion would be to use more media or shorter media change intervals.

We have used HEPES-buffered media (usually alpha-medium or Iscove's medium) extensively for freshly explanted bone marrow cells as well as dozens of cell lines.  We use HEPES-buffered medium whenever cells need to be kept or manipulated outside the CO2 incubator for a significant length of time.  We usually keep the HEPES concentration low (10 mM) to stay clear of toxic effects.  Our HEPES-buffered media do not contain bicarbonate. 

@stoyanov, I will take your suggestion, I will use more media. But is it ok to add excess media to culture plate while doing media change?

@sieber, from your answer I need clarification that , after significant length of time when the culture plates return to incubator will it contains HEPES Medium or normal alpha MEM Medium? Shall we use that HEPES medium to cultivate cells throw-out the month?? Or should have to use only while working outside incubator? Because I am planing to cultivate cells without co2 incubator.. Is it possible??

You can grow cells in HEPES-buffered medium.  You can even do it in your incubator.  Just turn off the CO2, otherwise things get too acidic.

Sure, as far as the plate will take it...

I am clear now. Thank you so much 😊

This  paper like example , and i agree with researchers

In my experience working with cultures HEPES should be used in addition to, not instead of, sodium bicarbonate, as it is important to maintain sufficient bicarbonate in the medium for nutritional purposes. HEPES may be added to cell culture media at a final concentration of 10mM to 25mM to provide additional buffering capacity if required. Lower concentrations are not usually adequate to control fluctuations in pH, and higher concentrations are often toxic. Since the buffering capacity of HEPES is independent of the CO2 concentration, it is an ideal buffer for maintaining the pH of cultures when cell cultures require extended periods of manipulation outside of a CO2 incubator. HEPES buffered media are resistant to rapid, drastic pH changes, but will not prevent pH shifts entirely. Since high levels of HEPES can cause cytotoxicity, concentrations of this organic buffer should be reduced if toxicity is apparent for a specific cell line or primary cell culture. But be carefull with its use. For some cells HEPES in the presence of fluorescent lights can be toxic.

oh then we have to take more precautions to work with this HEPES. thank you alkarim for your time