Tissue Culture Methodology

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
 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

 Human Cells

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

• Density Gradient
Centrifugation
• Extraction of PBMCs
• Magnetic Bead
Separation
Primary Cells From Non-Humans
Non-Human Cells

• Payers Patches
• Spleen
• Alveolar M
 Dissociation Of Cells

• Mechanical Digestion
(Syringe, Frosted Slides)
• Collagenase D
– 1 hour @ 37C
• Filtering Step
• Suspension into Medium
 Staining Cells

• Trypan Blue
Viability Assessment

• Turks Solution
Glacial Acetic Acid and crystal blue
Cell Counts Using Hemocytometer

D.F X 25 X 104

=0.50x106 cells/mL
 Media Formulations

• RPMI 1640
• Antibiotics
• FBS/Human AB
 -ME
• L-glutamine
• Filtering Flasks
 Culturing

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

CFSE
 Flow Cytometry

20 4 0 0

CD8-PerCP
foxp3-PE

31 73
45 27

CD4-FITC foxp3-PE

PGE2
 Viability
Medium LPS

9 9

85 85

4 5

PGE2 LPS+PGE2
5 5

91 90

2 4
 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
CFSE