Cell Culture Q&A: Min. Cell Amount, Trypsin Inhib., pH & Osmolarity

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Discussion Overview

The discussion revolves around cell culture techniques, specifically addressing the minimum cell amount required for optimal growth, the use of trypsin and trypsin inhibitors, the role of bicarbonate in culture media, and the importance of maintaining pH and osmolarity within specific ranges. The scope includes practical applications and conceptual clarifications relevant to laboratory practices in cell culture.

Discussion Character

  • Technical explanation
  • Conceptual clarification
  • Debate/contested
  • Experimental/applied

Main Points Raised

  • One participant inquires about determining the minimum number of cells needed for a specific cell line to enter log phase growth quickly.
  • Another suggests plating different dilutions of the cell line to identify a threshold for normal growth.
  • Regarding trypsin inhibitors in serum, one participant proposes using trypsin after washing cells with PBS, while another mentions using a plastic scraper for non-sensitive cells.
  • There is a discussion on the necessity of bicarbonate in the medium, with one participant stating it acts as a buffer and energy source.
  • Participants discuss the appropriate pH range for cell culture, with one asserting that it should be between 7.0 and 7.4, while another emphasizes the importance of osmolarity matching that of the body.
  • A participant questions the rationale behind incubating cells in a CO2 environment, considering its effect on pH, prompting a response about mimicking in vivo conditions and the role of CO2 in buffering.

Areas of Agreement / Disagreement

Participants express various viewpoints on the use of trypsin and bicarbonate, as well as the implications of CO2 in cell culture. There is no consensus on some of the more nuanced aspects, such as the exact pH and osmolarity requirements, indicating that multiple competing views remain.

Contextual Notes

Some discussions involve assumptions about the sensitivity of cells to trypsin and the specific conditions under which they thrive, which may not be universally applicable across all cell lines. The relationship between CO2 levels and pH changes in culture media is also noted as a complex interaction that requires careful management.

sobored
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# How do I know the minimum of cells for a specific cell line that give a quick entry into log phase growth?

#If medium with serum contains trypsin inhibitor. What should I use to dislodge my cells?

#Should I always have HCO3- in the medium and what for?

# Should pH always be between 7.0 to 7.4 in the medium? What about the osmolarity?

Hope for any ideas.

Thanks.
 
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sobored said:
# How do I know the minimum of cells for a specific cell line that give a quick entry into log phase growth?

Plate different dilutions of your cell line and see were the cut off point is, usually there's a threshold were the cells are suddenly to sparsely plated to give rise to normal growth.

sobored said:
#If medium with serum contains trypsin inhibitor. What should I use to dislodge my cells?

Trypsin, just wash your cells once with PBS be4 adding the trypsin and you;ll be fine

sobored said:
#Should I always have HCO3- in the medium and what for?

Its a buffer to keep the CO2 from the incubator to change the pH of the culture medium, it can also serve as an energy source for your cells

sobored said:
# Should pH always be between 7.0 to 7.4 in the medium? What about the osmolarity?

Yes your cells won't have it any other way unless there bacterial :wink: osmolarity has to be the same as in the body like PBS, that's around 150 milimolars if I am not mistaken
 
Thank you. :smile:
 
Sho'Nuff said:
Its a buffer to keep the CO2 from the incubator to change the pH of the culture medium, it can also serve as an energy source for your cells


I forgot to ask why should we incubate our cells in the CO2 incubator when we know that the CO2 will change the pH of our medium? Won't this create more problems?

Any ideas?

Thanks.
 
We try to mimick the inside of the body ad good as we can in an incubator to obtain data that is more relevant to the situation in vivo. That means less oxygen (which is a stress factor to the cells) and more CO2, cells won't grow without it. CO2 is also part of the buffering system of the culture medium. Its a delicate balance...
 
I see. :smile: :cool:
 
sobored said:
#If medium with serum contains trypsin inhibitor. What should I use to dislodge my cells?
It is good to know that fcs (fetal calfs serum) contains a trypsin inhibitor. I wash my cells with some PBS and then do the digestion in some PBS with trypsin. Just don't leave the PBS on the cells too long: they don't like that much.

You can also dislodge your cells with a plastic scraper when cells are not sensitive to trypsin digestion.