disaggregation of explant

Flussdiagrammknoten

• DISAGGREAGATION OF EXPLANT

• Primary Explant Technique

• Mechanical Disaggregation

• Enzymatic Disaggregation

• Warm Trypisination

• Cold Trypisination

• Finely chopped

• Dissection of explant

• Washed by settling 

• Transfer in last wash

• Remove BSS

• Add thin film of medium

• Incubate 2 to 3 weeks

• Explant outgrowth

• TISSUE SAMPLE -  pass through 100um mesh petri dish  containing BSS

• CELL + DEBRIS - Repeat the above process through 50um mesh

• MORE CELLS + DEBRIS - Repeat & pass through 20um mesh

• VIABEL CELLS + DEBRIS -  Removal of debris

• DILUTE THE CELLS

• INCUBATE

• PRIMARY CELL CULTURE

• TISSUE SAMPLE

• CHOPPED TO 2 TO 3 MM PIECES

• TISSUE PIECES WASHED WITH DBSS

• TISSUE PIECES WITH WARM  TRYPSIN

• AFTER 30 MIN PIECES SETTLE

• SUPERNATANT

• REMOVE TRYPSIN & CENTRIFUGE

• DISPERSE CELL PELLET IN MEDIUM

• DISAGGREGATED CELLS ARE KEPT  VIAL ON ICE

• ADD FRESH TRYPSIN  & CONTINUOUS CENTRIFUGATION

• TISSUE PIECES IN A VIAL

• Vial kept on ice

• Soak tissue pieces in cold trypsin for 6 - 24 hr    

• Remove trypsin & incubate at 37 C for 20 - 30 min

• Disperse cells by pippeting

• Cells in medium

• Count cells

• Dilute in suitable medium

• Incubate

• Primary culture