Primary culture ch:12 By : Saib al owini. Primary culture: steps 1- ISOLATION OF THE TISSUE 2- Dissection and/or disaggregation : -Mechanically: sieving.

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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-mL screw-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 EGTA EGTA (ethylene glycol tetraacetic acid) (ethylenediaminetetraacetic acid )

Trypsinization with Cold Preexposure

SEPARATION OF VIABLE CELLS

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