1. Isolation and mechanical disaggregation of explants

2. Isolation of explant : Avoid contamination Retain cell survival
Explant dissected out
Blunt dissection or by using forceps
Avoid contamination
Sterlized with 70% alcohol
Retain cell survival
Placed in HBSS or EBSS +antibiotics

3. Precautions while extracting explant :
Strictly aseptic conditions
Under laminar flow cabinets or in sterile rooms
care should be taken not to violate ethical issues or legal boundaries

4. Mechanical disaggregation Enzymatic disaggregation
EDTA treatment

5. Methods of mechanical disaggregation
MINCE method
SCRAPE method
homogenization
Grinding
sieving
Methods of mechanical disaggregation

6. Mince method: Scrape method: Mincing tissue in culture media.
Scraping the surface with scalpel blade.

7. Homogenization: Grinding: Sieving:
Encouraging cells to lyse to release their contents.
Grinding:
Creating friction by sandwiching the sample between two hard surfaces that slide against each other.
Sieving:
Forcefully passage through screens

8. Effective method of mechanical disaggregation:
Slicing tissue in 0.5mm thickness.
Brief mincing in ice cold culture media with 10% PBS.
Cells thus freed were separated from tissue fragments by pouring mince over a stainless screen.
Remaining tissue gently returned to petri-dish and further minced in fresh media.
Process is repeated until cell release is no longer significant.
Cells obtained were pooled and designated as mechanically released cells.