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Tissue Culture Methodology

Tissue Culture Methodology. Historical Perspective. Early 20 th Century Technique Antibiotics and Laminar Flow Hoods Allow for Tissue Culturing to Grow Exponentially Unfortunately NOT commonly taught in a classroom setting. Outline. Primary Cells vs Cell lines Acquisition of Specimens

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Tissue Culture Methodology

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  1. Tissue Culture Methodology

  2. Historical Perspective • Early 20th Century Technique • Antibiotics and Laminar Flow Hoods Allow for Tissue Culturing to Grow Exponentially • Unfortunately NOT commonly taught in a classroom setting

  3. Outline • Primary Cells vs Cell lines • Acquisition of Specimens • Processing • Quantitation and Culturing • Propagating Cells For Long Periods • Cryopreservation • Conclusion

  4. Cell Lines • American Type Culture Collection (ATCC) • Collaborators • Easy To Grow • Option To Cryopreserve For Future

  5. Human Cells • Monocytes • T-cells • B-cells • NK Cells • Dendritic Cells • Neutrophils • Biopsies

  6. Human Blood • Density Gradient Centrifugation • Extraction of PBMCs • Magnetic Bead Separation

  7. Primary Cells From Non-Humans

  8. Non-Human Cells • Payers Patches • Spleen • Alveolar M

  9. Dissociation Of Cells • Mechanical Digestion (Syringe, Frosted Slides) • Collagenase D • 1 hour @ 37C • Filtering Step • Suspension into Medium

  10. Staining Cells • Trypan Blue Viability Assessment • Turks Solution • Glacial Acetic Acid and crystal blue

  11. Cell Counts Using Hemocytometer X X D.F 25 104 =0.50x106 cells/mL

  12. Media Formulations • RPMI 1640 • Antibiotics • FBS/Human AB • -ME • L-glutamine • Filtering Flasks

  13. Culturing • Enhancing Proliferation IL-2, -CD3/ -CD28 • Limiting Proliferation FBS Reduction to 2% CFSE

  14. Flow Cytometry 0 0 20 4 foxp3-PE CD8-PerCP 73 31 27 45 foxp3-PE CD4-FITC PGE2

  15. Viability LPS Medium 9 9 85 85 4 5 LPS+PGE2 PGE2 5 5 91 90 2 4

  16. Cryopreservation • Collect Cells, Spin, Count • Resuspend in 90% FBS+10 DMSO • DMSO/FBS 4°C • Store @–150 °C

  17. Contamination Issues • Sterile Incubator • Thawing Step Source of Contamination • Spray with 70% Ethanol, gloves • Sterile Hood • Sterile Hands • Minimize Exposure to Open Air

  18. 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

  19. CFSE

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