Lecture 1 intro to tissue culture
Transcript of Lecture 1 intro to tissue culture
Outline
• Primary Cells vs Cell lines
• Acquisition of Specimens
• Processing
• Quantitation and Culturing
• Propagating Cells For Long Periods
• Cryopreservation
• Conclusion
Cell Lines
American Type Culture Collection (ATCC)
Collaborators
Easy To Grow
Option To Cryopreserve For Future
Dissociation Of Cells
• Mechanical Digestion
(Syringe, Frosted Slides)• Collagenase D
– 1 hour @ 37°C
• Filtering Step• Suspension into Medium
Staining Cells
• Trypan BlueViability Assessment
• Turks Solution
Glacial Acetic Acid and crystal blue
Culturing
• Enhancing Proliferation IL-2, α-CD3/ α-CD28
• Limiting Proliferation
FBS Reduction to 2%
CFSE
Cryopreservation
• Collect Cells, Spin, Count
• Resuspend in 90% FBS+10 DMSO
• DMSO/FBS 4°C• Store @–150 °C
Contamination Issues
• Sterile Incubator
• Thawing Step Source of Contamination
• Spray with 70% Ethanol, gloves
• Sterile Hood
• Sterile Hands
• Minimize Exposure to Open Air
Conclusions
• Tissue Culturing Training Is An Essential Skill• Biological Safety Cabinets Are The limiting Step• Labor Intensive Class With Focus On Practical
Training
• Greatest Challenge Is The Capital Investment and Commitment A University Must Make