DISAGGREGATION OF EXPLANT

Nós do fluxograma

• DISAGGREGATION OF EXPLANT

• MECHANICAL DISAGGREGATION

• ENZYMATIC DISAGGREGATION

• EDTA TREATMENT

• PRIMARY EXPLANT TECHNIQUES

• DISSECTION

• FINE CHOPPING

• WASH BY SETTLING

• TRANSFER TO BSS

• REMOVE BSS

• ADD THIN FILM OF MEDIUM

• INCUBATE FOR 2-3 WEEKS

• EXPLANT OUT GROWTH

• TISSUE SAMPLE

• CELL+DEBRIS

• MORE CELL + DERBIS

• VIABLE CELL+DERBIS

• DILUTE THE CELLS

• INCUBATE 

• PRIMARY ADHERENT CELLS CULTURE  

• SUB CULTURE

• CELL LINE

• CHOPPED 

• WASHED

• COLD TRYPSINIZATION

• WARM TRYPSINIZATION

• TISSUE IN VIALS

• TISSUE PIECES+100ml TRYPSIN 

• PIECES+TRYPSIN

• PLACE ON ICE FOR 1.6 hr

• DISAGGREGATE CELLS +TISSUE RESIDUES

• DISPERSED CELLS IN MEDIUM

• INCUBATE 35.6 DEG C 20-30 MIN

• INCUBATE 35.6 DEG C 48-72 HR

• DISPERSED CELLS

• PRIMARY CELL CULTURE

• COLLAGENASE

• CENTRIFUGE

• RESUSPEND

• WASH 

• PRIMARY CELL CULTURE

• STEPS INVOLVED IN CELL CULTURE