Tissue Cell Culture Technology Seeding passaging harvesting and

Tissue Cell Culture Technology: Seeding, passaging, harvesting and storing (cryopreservation) of cells CEAC 514 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 PRIMARY CULTURE 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