Primary culture ch:12

By : Saib al owini

Primary culture: steps

• 1- ISOLATION OF THE TISSUE• 2- Dissection and/or disaggregation : - Mechanically: sieving or pipetting - or chemically : crude or pure enzymes (trypsin alone or trypsin/EDTA ) , (glycanases, such

as hyaluronidase or heparinase,), (collagenase, dispase, pronase )

• Trypsin + pronase : complete but may damage • Collagenase + dispase : incomplete ,safe • Hyaluronidase + collagenase : for matrix

digestion • DNASE : for released DNA and support

reaggregation • 3- Culture

General requirements for 1ry culture

• Remove necrotic and lipids• Sharp instrument • Enzymes must removed • Begin with large # not all will survive • F12/DMEM with serum as beginning• Embryonic tissue is preferable ( soft,

proliferation rate)

ISOLATION OF THE TISSUE

• Ethical processes • Sterility • Bss • Where to dissect

Mouse Embryo

Protocol

• Induction of estrus• Dating the embryos• Kill the mouse by cervical dislocation• Tear the ventral skin transversely at

themedian line• Dissect out the uteri into a 25-mL or 50-mLscrew-capped vial containing 10 or 20 mL DBSS

Human biopsy

• Problems:-Many consents -Patent rights - The operation is performed by physicians - You will receive and record but it may has a

risk infection

PROTOCOL

Decontamination. Although most surgical speci-mens are sterile when removed,

PRIMARY EXPLANT

FINE CHOPED

3 DAY 7 DAY

Warm Trypsin

Enzymatic Disaggregation

EDTA or EGTAEGTA (ethylene glycol tetraacetic acid)(ethylenediaminetetraacetic acid )

Trypsinization with Cold Preexposure

SEPARATION OF VIABLE CELLS