به نام پروردگار

1. به نام پروردگار

2. بیوتکنولوژی جلسه سوم سلول

3. ارشد کلاس: هماهنگی ساعات جبرانی • گروه تشخیص بیماریهای ژنتیکی؟ • گروه طراحی پرایمر؟ • گروه های جدید؟ • ایمیل: Madjidm@hotmail.com

4. سطوح مختلف مطالعات • In vivo • a whole, living organism • In vitro • (a) cells derived from multicellular organisms (cell culture or tissue culture), (b) subcellular components (e.g. mitochondria or ribosomes), (c) cellular or subcellular extracts (e.g. wheat germ or reticulocyte extracts), or (d) purified molecules in the test tube (often proteins, DNA, or RNA, either individually or in combination). • In silico • performed on computer or via computer simulation • In situ • something intermediate between in vivo and in vitro

5. In vitro Healthy Volunteer Animal model Primary cells Cell line تحقیقات In vivo Patient

6. Cell Culture • رشد سلول های مورد نیاز در شرایط کاملا کنترل شده آزمایشگاهی • سلول پروکاریوت • باکتری ها • سلول یوکاریوت • سلول جانوری • سلول گیاهی • مخمرها

7. Animal cell culture • مزایا • محیط رشد قابل کنترل • مطالعه رفتار با تغییرات کمتر نسبت به موجود زنده • تعیین هویت دقیق سلول های مورد مطالعه • بررسی دقیق مواد و تیمارهای مورد آزمایش • عدم منع اخلاقی

8. Animal cell culture • معایب • نیاز به تعریف استانداردهای دقیق مطالعاتی جهت تکرار پذیری • نیاز به شرایط استریل کامل • تغییر ماهیت سلول در طول زمان • هزینه بالا

9. انواع سلول ها در کشت • Primary cultures • Secondary cultures • Normal • Immortalized • Transfected with some sort of oncogene; SV40 (Simian virus)Large T antigen وTumor cells (e.g.: Human cervix carcinomas: HeLa) • Cell lines • Adherent • Suspension

10. Cell number Life span Reproducibility Hard to work Primary phenotype Application, new assay-tech In many cases confirmation Primary cells Cell line Vs.

11. Cell Culture Growth Passage Aging Liquid nitrogen Senescence • Cell growth • Passage number (cell senescence)

12. مرجع • Cells from ATCC • American Type Culture Collection • http://www.atcc.org/ • انستیتو پاستور ایران • http://pasteur.ac.ir/ • http://ncbi.pasteur.ac.ir/

13. reagents • Basic medium • Medium constituents are nutrients (amino acids, glucose), vitamins, inorganic salts, and buffer substances • Fetal calf serum • an extremely complex mixture of albumins, globulins, growth promoters, and growth inhibitors. The quantity and quality of these components are affected by the age, nutrition, and health of the animals from which the serum is obtained. • Additive • growth factors, trace elements, essential metabolites, and proteins.

14. آلودگیهای کشت سلول • باکتریها • گرم مثبت • گرم منفی • مخمرها • قارچها • مایکوپلاسما • دیگر رده های سلولی

16. CO2-Incubator Airflow Solutions Dishes Freezers Liquid nitrogen Centrifuges Autoclave ovens Cryotubes Microscopes تجهیزات اتاق کشت سلول

17. CO2 Incubators • Cells are grown at +37°C in a CO2-incubator at 100% relative humidity. • CO2 is needed to control pH. • Cell physiology is highly sensitive to pH variations. • Some media need a concentration of 5% CO2, other media have to be run • at 10% CO2. • Inconsistent conditions within an incubator may cause variances between • culture plates. • Pollution, chemicals, and fungal or bacterial contamination from the incubator • may affect cell physiology.

18. Laminar Flow Box • HEPA filter rated at 99.99% efficient for 0.3 micron particulates.

19. Dishes • Dishes • Multiwell plates • Flasks • Flasks on slide

20. Freezers

21. Centrifuges

22. Autoclaves

23. Microscopes

24. FACSfluorescence activated cell sorting

25. موفق باشین

26. Why do we need cell culture? • To investigate the normal physiology or biochemistry of cells. • To identify the molecular mechanism of disease using primary cells obtained from patients or cell disease model. • To test the effect of various chemical compounds or drugs on specific cell types (normal or cancerous cells, for example). [DRUG SCREENING] • To generate artificial tissues, which technique is known as “tissue engineering”. • To synthesize or extract valuable biologicals from large scale cell cultures. For example, hormone, cytokine, vaccine (keep virus), monoclonal antibody

27. Type of cell culture Primary cell culture Continuouscell culture Cancer cell line Cells from body Immortalized Cell line

28. Cell Culture Growth Passage Aging Liquid nitrogen Senescence • Cell growth • Passage number (cell senescence)

29. Transfer x2 Doubling time Cell Growth Confluent Experiment 60-80% confluency Transfection Days (hours) in culture

30. Why don't cells live forever? Replicative senescence: telomere shortning, a tumor suppressor mechanism After a series of population doublings, cells enter a state where they no longer divide. Mature phenotype: end of differentiation

31. Telomerase

32. ابزار کشت سلول • اتاق کشت • هودها • ظروف کشت • محیط کشت سلول • تانک نیتروژن و فریزرها