Primary culture ch:12

1. Primary culturech:12 By : Saib al owini

2. 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 )

3. Trypsin + pronase : complete but may damage • Collagenase + dispase : incomplete ,safe • Hyaluronidase + collagenase : for matrix digestion • DNASE : for released DNA and support reaggregation • 3- Culture

4. 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)

5. ISOLATION OF THE TISSUE • Ethical processes • Sterility • Bss • Where to dissect

6. Mouse Embryo

8. 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

13. 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

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

16. PRIMARY EXPLANT

17. FINE CHOPED

19. 7 DAY 3 DAY

21. Enzymatic Disaggregation Warm Trypsin EDTA or EGTA EGTA (ethylene glycol tetraacetic acid) (ethylenediaminetetraacetic acid )

25. Trypsinization with Cold Preexposure

30. SEPARATION OF VIABLE CELLS