Culture of Mammalian Cells The Basic Considerations

1. H. Reza Soleimanpour (PhD) Culture of Mammalian Cells The Basic Considerations

2. Outline of the lecture What is tissue culture? Equipments Aseptic technique Cell culture nutrients and supplemets Tips

3. What is tissue culture? A means to study biological systems in vitro Doesn�t really mean microorganisms Refers mainly to mammalian cells and tissues Cell lines Primary cells

4. Brief history of tissue culture Late 19th century: Methods established for the cyropreservation of semen for selective breeding of livestock. 1907: Experiments published showing frog embryo nerve fibre growth in vitro. 1912: Connective tissue cells cultured for extended periods. 1948:First clone of cell line; L-cells (mouse). 1952:HeLa cells established from cervical carcinoma, the first human cell line. 1955:Defined media developed 1961 to present: Experiments using tissue culture methods expands exponentially. Absolute necessity for biological experimentation.

5. Equipments Tissue culture hood CO2 incubator Refrigerator or cold cabinet Freezer Centrifuge Microscope

6. Biological Containment Cabinets (Hood) Class I Class II, type A Class II, type B1 Class II, type B2 Class II, type B3 Class III

7. Class I hood Protects worker No product protection Inward airflow away from operator Should have HEPA filter Similar to a fume hood

8. Class II hood Personal protection Product protection Environmental protection Inward airflow HEPA filtered supply and exhaust air 4 designs Recirculates most of the air � Type A Exhausts most of the air � Type B1 Provides total exhaust � Type B2 � Level 3 containment Cabinet can be a combination � Type B3, MOST COMMON

9. Class II hood Used with low to moderate risk biological agents, i.e., Level 2 and 3 pathogens HBV HIV Tuberculosis Toxic chemicals and trace quantities of radionuclides

10. Class III hood Totally enclosed Gas-tight Negative air pressure Supply air is HEPA filtered Exhaust air is double HEPA filtered Can be used with Level 4 pathogens Ebola Hantavirus

11. The 10 commandments of working with Biological Safety Cabinet Preparation � turn on, check air pressure, allow air to purge workspace for at least 5 min. Disinfection-spray or swab all interior with appropriate disinfectant � 70% Ethanol. Assemble material �only material required, everything sterilized. Purge-continue air purge for a few minutes before working. Personal procedures-gloves, protective clothing (lab coat), etc.

12. The 10 commandments of working with Biological Safety Cabinet Perform procedures-introduce hands into work area, try not to remove hands from work area until all or most procedures complete, remove gloves into designated contaminated material container. Purge-allow air purge with no activity inside for a few minutes. Personal procedures-wash hands. Terminal disinfection-put on new gloves, remove tissue flasks to incubator, biohazard bag, etc. Spray or swab all interior surfaces with appropriate disinfectant; 70% ethanol. Shutdown-turn off blower and fluorescent lamp. Turn on UV lamp if applicable.

13. Incubator Temperature 37 C Gas phase CO2 � usually 5% Maintains pH (7.2-7.4) Calibrate regularly Humidity Water tray

14. Centrifuge Spin down cells Bench top Temperature controlled Usually 15 and 50 ml tubes Balance!!!

15. Microscope Inverted/Stereo Phase contrast Photo capabilities Regular � cell stains, cell counting (hemocytometer) Fluorescent Electron

16. Fridges and freezers Refrigerator for media storage, solutions -20C Freezer for FBS, antibiotics -80C Freezer for growth factors, labile reagents � Liquid Nitrogen for cell lines

17. Water bath Warming media Thawing frozen cells Enzyme reactions 37C Clean regularly Dry bath not as efficient

18. Aseptic TechniquesProtect Your Experiments Gloves!!!!! Sterilized media and serum, i.e., FBS. Autoclave capabilities Sterilized materials, i.e., pipettes, tips Appropriate Hood Swab all surfaces with 70% ethanol before and after working! Don�t Touch Anything to fingers!! Don�t Touch Anything to openings of containers!! Clean up spills immediately!! Don�t put tube or flask caps on to hood surface! at least not inner side down!! If you think you contaminated a pipette or tip or flask � YOU DID!!! get another one before you contaminate everything. Don�t bake bread with viable yeast the night before working!!

19. Cell Culture Nutrients and Supplements Sterile media Many different media for different cultures RPMI, IMDM, McCoy�s, Eagles,etc. Serum requirements FBS, Horse serum, Human serum Screen small aliquots of various lots Antibiotics Check special requirements L-glutamine beta-mercaptoethanol Growth factors

20. Tips Hoods Certify each year Clean regularly Incubators Check CO2 levels regularly Check humidity Clean regularly Aseptic Technique Common sense Cell Culture Don�t use antibiotics in stock cultures Check cultures daily, look for clear supernatant. Yellow color may mean the needs for passage or contamination. Use microscope to check cultures. Check all cell lines for mycoplasma contamination routinely. Balance centrifuge. If yeast found, CLEAN EVERYTHING immediately!