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

Finely cut

Finely cut
tissue or explant

explant
Grow in media
•Explants
•Explants with outgrowth

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

Grow in media
-monolayer
Finely cut -suspension cells
tissue or explant

explant

organ
Enzymic digestion
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