I have been working on developing an static in vitro digestion procedure with downstream application to Caco-2 cell cultures directly. When reviewing the literature many papers utilize dialysis membranes to "protect the cells from the digestion enzymes". However there are also many papers that do not note any adverse effects from the digestion enzymes. Some people alternatively choose to heat shock the digestant to denature the proteins. I would prefer not to use enzyme inhibitors as they sometimes negatively affect cell viability. What are the pros and cons of these alternatives?
Are dialysis membranes still necessary/preferred for in vitro digestion to protect Caco-2 cells from digestion enzymes?
I would expect the digestive enzymes to have digested themselves after a while, but if not, a fast way to get rid of them would be to use a centrifugal ultrafiltration device with a suitable molecular weight cutoff at least 3-fold lower than the molecular weight of the smallest digestive enzyme), allowing the passage of small peptides but not whole proteins. An advantage of this method, besides speed, is that the sample is not diluted.
