Download Basics of Cell Culture

Tissue Cell Culture Technology:
Seeding, passaging, harvesting and
storing (cryopreservation) of cells
CEAC514
Assoc. Prof. Dr. Yasemin G. İşgör
VARIOUS APPLICATIONS OF TISSUE CULTURE TECHNIQUES
GENERAL CULTURE PARAMETERS -2
CELL CULTURE COLLECTIONS (available sources)
.
GENERAL METHODS AND CULTURE PARAMETERS -2
P R I M A RY C U L T U R E
 Cells when surgically or enzymatically removed from an organism and placed
in suitable culture environment will attach and grow are called as primary
culture where the Primary cells have a finite life span.
 Primary culture contains a very heterogeneous population of cells
(different types of cells present)
 Cells such as macrophages and neurons do not divide in vitro so can be
used as primary cultures
PRIMARY CULTURE (EXAMPLE)
 Stage of culture after the cells are isolated from the tissue and proliferated
under the appropriate conditions until they completely occupy the substrate
 Monolayer – Reach Confluence
 Then need to be subcultured (Passaged)
 Split & Move to fresh medium, new vessels
Chinese Hamster Ovary
Cells (CHO)
PREPARING PRIMARY CULTURE FROM MINCED TISSUE
(EXAMPLE)
PRIMARY CULTURE: BASIC METHOD
Chop the tissue in media
Use trypsin to
Separate primary
cells
Wash with buffer and
centrifuge, then
discard supernatant
Seed cells at low density
Digest w collagenase
/ incub @37ºC
Harvest cells before
confluence
ISOLATION OF CELLS FOR CULTURING
MONOLAYER PROPOGATION
THAWING FROZEN CELL STOCK (GENERAL PROTOCOL)
SUBCULTURING
HARVESTING CELLS WITH TRYPSIN-EDTA
FREEZING STOCKS
storing (cryopreservation) of cells
Liquid Nitrogen Cryogen:
The metal tank suitable to hold
cells at the vapor
Phase of liquid nitrogen