Dr Siti Suri Lecture 6 Primary Cell Culture

TYPES OF PRIMARY CELL CULTURE •Mouse embryos •Chick embryos •Human biopsy materials •Transplantable animal tumour •Chick embryo organ rudiments (brain, heart, lungs, liver, gizzard, kidney, spinal cord, skin, muscle)

ISOLATION OF TISSUES •Must comply with local legislation and medical ethical rules. •Sterilize the site with 70% alcohol. •Remove tissue aseptically. •Transfer to the laboratory in transport medium •If delay in transporting to lab, keep at 4C for up to 72hour.

PRIMARY CULTURE Is a stage of the culture after first isolation of the cells but before the first subculture. 4 stages: 1) acquisition of samples, 2) isolation of tissues, 3) disaggregation, 4) culture seeding into culture vessel.   After isolation, a primary cell culture is obtained by allowing cells to migrate out from the fragment of tissue adhering to a suitable substrate by disaggregation.     PRIMARY EXPLANT •Suitable for small amount of tissues example skin biopsy. •Attachment on the substrate by using plasma clots, or fibrinogen and trombin. •Disaggregation by mechanical and enzymatic.

Mouse, mammals, Primary Embryo Eggs (best: for TC : embryo, young) because stage of differentiation)

explant

Finely cut

Finely cut tissue or explant explant

organ

Grow in media •Explants •Explants with outgrowth

•Mouse, mammals, Cell •Embryo •Embryonated Eggs (best: for TC : embryo, young) because stage of differentiation) Finely cut

Finely cut tissue or explant

explant

organ Enzymic digestion

culture

Grow in media -monolayer -suspension cells

Primary Explant Culture

1. Enzymatic disaggregation •Warm trypsin, 37C for 30 mins, cell damaged if too long exposure. •Cold preexposure, soak at 4C overnight and 37C for less 30 mins. Advantage: higher yield of viable cells, preserve more cell types •Other enzyme -collagenase benefit for connective tissues and muscle (fibrous tissue) - pronase, dipase, DNase, hyaluronidase   2. Mechanical disaggregation (prevent proteolytic damage) •Scrapping or spillage •Sieving •Syringes •Trituration by pipette

Warm trypsin  disaggregation

Add to stirrer flask

Cold trypsin  disaggregation

Collect the supernate containing cells, centrifuge, resuspend in medium, and store on ice

Mechanical  disaggregation