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1. Tissue Culture Methodology

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

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

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

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

6. Primary Cells From Non-Humans

7. Non-Human Cells
• Payers Patches
• Spleen
• Alveolar MΦ

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

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

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

11. Media Formulations
• RPMI 1640
• Antibiotics
• FBS/Human AB
∀ β-ME
• L-glutamine
• Filtering Flasks

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

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

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

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

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

17. 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

18. CFSE