hi Afnan Saleem

Check the attached papers,

Reviews papers included cells recovery methods while a research article has used enzymatic digestion for cell retrieval.

Article Engineering and Functionalization of Gelatin Biomaterials: F...

Article Preparation of Gelatin and Gelatin/Hyaluronic Acid Cryogel S...

Article Gelatin‐Polysaccharide Composite Scaffolds for 3D Cell Cultu...

Regards

Saurabh

Suggest using an enzyme that specifically targets the gelatin, rather than a non-specific enzyme that can target both gelatin and the cell plasma membranes. For example, we use type-1 collagenase (Worthington) to release our cells from a type-1 collagen matrix, with >98% viability of the cells. Most other labs use trypsin, which cleaves between lysine and arginine residues (also present in cell membranes), with viability recovery ranging from 50-60%. Cells bind to their matrices through two different binding sites, calcium dependent and RGD. To release cells from calcium dependent binding sites we first use EGTA (specific calcium chelator) rather than EDTA (which will chelate divalent cations). After 5 minute incubation at ambient temperature one can start to see the cells detaching from their matrices. The EDTA solution is removed and a solution of collagenase is use to complete the process at the RGD binding sites, which in our hands takes from 30-60 seconds. Cells are removed by trituration (rather than scraping), and immediately immersed in a neutralizing solution containing 0.01% collagen in base medium as the buffer.

Agreed with Henry E Young, well-explained. However, the 3D-cell culture here is not really clear, are you talking about pre-seeding on the gelatin surface or pre-mixed prior to forming 3D-scaffold, especially hydrogel form. Using an enzymatic approach thru collagenase might work but the time of incubation is really critical to avoid over-digestion that at last may cause cell deterioration..Best of luck!