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STEPS INVOLVED IN CELL CULTURE

Disaggregation of Explant

PRIMARY EXPLANT TECHNIQUE

MECHANICAL DISAGGREGATION

ENZYMATIC DISAGGREGATION

EDTA TREATMENT

Dissection

Fine chopping

Wash by settling

Tissue sample

Cell + debris

More cell +debris

Viable cell+debris

Chopped

Washed

Cold trypsinization

Tissues in vials

Collagenase

Centrifuge

Resuspend

Wash primary cell culture

Warm trypsinization

Transfer to BSS

Remove to BSS

Dilute the cells

Add thin film of medium

Incubate for 2-3 weeks

Incubate

Primary adherent cell culture

Explant out growth

Place on ice for 1.6 hrs

Disaggregate cells+ tissue residues

Incubate 335.6 degree 20-30 min

Tissue pieces+ 100ml trypsin

Pieces +trypsin

Dispersed cells in medium

Incubate 35.6 degree for 48-72 hrs

Dispersed cells

Primary cell

Subculture

Cell line