1. Tissue Culture Methodology

2. 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

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 XX D.F 10 4 25 =0.50x10 6 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 CD4-FITC foxp3-PE CD8-PerCP PGE 2 4 31 20 45 0 27 0 73

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

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