Chapter 5 Microbial Nutrition and Culture Siti Sarah Jumali (ext 2123) Room 3/14
Dr Siti Suri Lecture 6 Primary Cell Culture
-
Upload
biotechnology-iium-kuantan -
Category
Documents
-
view
667 -
download
1
description
Transcript of Dr Siti Suri Lecture 6 Primary Cell Culture
![Page 1: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/1.jpg)
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.
![Page 2: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/2.jpg)
![Page 3: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/3.jpg)
PRIMARY CULTUREIs 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.
![Page 4: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/4.jpg)
Mouse, mammals,EmbryoEggs(best: for TC : embryo, young) because stage of differentiation)
organ
explantGrow in media•Explants •Explants with outgrowth
Primary explant
Finely cut
Finely cut tissue or explant
![Page 5: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/5.jpg)
•Mouse, mammals,•Embryo•Embryonated Eggs(best: for TC : embryo, young) because stage of differentiation)
organ
explant
Grow in media-monolayer-suspension cells
Cell culture
Finely cut
Finely cut tissue or explant
Enzymic digestion
![Page 6: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/6.jpg)
![Page 7: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/7.jpg)
Primary Explant Culture
![Page 8: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/8.jpg)
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
![Page 9: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/9.jpg)
Warm trypsin disaggregation
![Page 10: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/10.jpg)
Add to stirrer flask
Collect the supernate containing cells, centrifuge, resuspend in medium, and store on ice
Cold trypsin disaggregation
![Page 11: Dr Siti Suri Lecture 6 Primary Cell Culture](https://reader033.fdocuments.us/reader033/viewer/2022061118/5468957fb4af9f0e518b477f/html5/thumbnails/11.jpg)
Mechanical disaggregation