I want to add different ECM components to my biomaterial and mix it with cells to see in which way it supports the cells.
The problem is that collagen II and IV are solved in 25% acetic acid (> 4 M) and collagen I in 0.1 M acetic acid. I heard that people mix it with 10X PBS and adjust pH with NaOH, but I have concerns that this change in the ion concentration could also harm the cells.
Would be happy for any advice or protocol you have.
Many thanks!
Hi Ruben Scheuring,
Usually, the collagen solution would be neutralized first, and then used the neutralized solution to encapsulate cells. I know your concerns, but as far as I know, it is the final ion concentration of the solution not the original concentration of buffer matters. So to know cells whether the cells would survive, you need to calculate the final ion concentration of your solution and compare it with the 1X PBS or other buffers for cells.
By the way, one of my colleague used 10X PBS with NaOH, it was OK for cells. There were other ways to prepare hydrogels. I did it as the following steps: the pH of collagen solution was adjusted to neutral by addition of 0.1M NaOH, also NaCl solution was added to increase ionic strength, and then put them into the 37C water bath to form gel. The ion concentration of solution was similar to HEPES buffer, so cells were quite happy:)
Hope it’s useful! Good luck.
Hey Lu,
thank you for your post! Ok, then I will check the ion concentration in my media or HEPES buffer or PBS and try to calculate how much I would have to add. Actually, I thought that the ion strength of my final solution will be too high. Especially in the col II and IV solutions. But if you or anybody else might have a (different) protocol with more detailed instructions, please feel free to share it. I would really appreciate that ☺
In case you are considering the sodium acetate – acetic acid buffer, as it seems from the enunciate, please note that its buffering capacity is generally expected to decrease noticeably outside the pH range 3.7–5.7. You may want to check my post at the following related discussion, in case you are seeking for (stable) nearly neutral pH: https://www.researchgate.net/post/Can_anyone_suggest_me_the_buffer_conditions_or_methods_for_solubilising_collagen_powder_other_than_acidic_condition
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