Hi Abhinav Kethiri

There could be multiple reasons, as follows:

• Collagen Matrix Integrity and Concentration: How consistent do you think your collagen coating has been? If there’s any variability in thickness or evenness, that might be part of the issue. Maybe experimenting with a higher collagen concentration could help reinforce the matrix, making it more durable and supportive for your cells throughout the week.
• Cellular Density and Metabolic Activity: Have you noticed any patterns related to cell density at which detachment occurs more frequently? High density often leads to enhanced matrix degradation due to increased enzymatic activity, like MMPs, from the cells. Adjusting the seeding density could be a key step. Perhaps incorporating some MMP inhibitors could also be a strategy to consider.
• Specific Cell Line-Matrix Interactions: Given the exact type of fibroblastic cells you're working with, do they have particular tendencies to degrade their substrate, perhaps more so than other cell types? If that's the case, exploring alternative substrates or collagen types resistant to enzymatic breakdown might be worth exploring. Have you considered using a different substrate or adjusting the matrix composition before?
• Optimisation of PBS Washing Protocol: How do you currently perform your PBS washes? Let's consider minimising the mechanical stress on the cells. For instance, pouring the PBS gently down the side of the well rather than directly over the cells might reduce the impact of the wash. It’s a minor tweak, but it could make a big difference.

It is not the cells that are detaching, this could be due to the coated collagen material being degrading. this could be solved be using the higher concentration of collagen along with the Hyaluronic acid.

You could consider washing with Ca2+ and Mg2+ supplemented PBS. Ion depleted PBS, as you are using it, might 'wash-out' necessary ion co-factors from adhesion molecules.

The answer its very simple.

Cell adhesion to collagens are predominantly mediated by integrins. Integrins require divalent cations (Ca++, Mg++) to fold correctly and to interact with binding motifs within the collagen fibrils.

This is also the reason one uses EDTA / EGTA when using trypsin to detach adherent cells.

I presume that you are washing with DPBS to remove serum and protein aggregates, that add background noise to your staining. Alternatively, you could use serum-free culture media (more expensive!) to wash your cells!

Solution? Use DPBS with Ca++/Mg++ (preferably without Phenol Red).

As most of them suggested addition of diavalent cations can solve your problem. Even after addition of Ions if you encounter the same problem, then employ fibronectin along with collagen or without collagen that would help you to overcome the problem.