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DISAGGREGATION OF EXPLANTS

PRIMARY EXPLANT TECHNIQUE

MECHANICAL DISAGGREGATION

ENZYMATIC DISAGGREGATION

dissection of explant

finely chopped

washed by settling

trnasfer in last wash

remove BSS

​​​​​​add thin film of medium

incubate 2 to 3 weeks

explant outgrowth

tissue sample:pass through 100 um petri dish containing BSS

cell debris:repeat the above process with 50um mesh

more cells + debris: repeat the process with 20um mesh 20um mesh

viable cells+ debris: removal of debris

diluthe the cells​​​​​​

incubate

primary cell culture

tissue sample

cut in to 2 to 3 mm pieces

tissue pieces washed with dbss

warm trypsination

cold trypsination

after 30 minutes pieces settle

tissue pieces with warm trypsin

supernatant

remove trypsin and centrifuge

disperse cel pellet in medium

disaggregated cells are kept via on ice

add fresh trypsin and continuous centrifugation

tissue pieces in a vial

vial kept on ice

soak tissue pieces in cold trypsin for 6 to 24 hrs

remove trypsin and incubate @ 37 c for 20 to 30 mins

disperse cells by pippetting

cells in medium

count cells

dilute in suitable medium

incubate

primary cell culture