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 @ 37C

• Filtering Step• Suspension into Medium

Staining Cells

• Trypan BlueViability Assessment

• Turks Solution

Glacial Acetic Acid and crystal blue

Cell Counts Using Hemocytometer

X XD.F 10425

=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

CD4-FITC

foxp

3-P

E

foxp3-PE

CD

8-P

erC

P

PGE2

4

31

20

45

0

27

0

73

Viability

Medium

PGE2LPS+PGE2

LPS

9

4

85

9

5

85

5

2

91

5

4

90

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