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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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Presentation Transcript
historical perspective
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
Outline
  • Primary Cells vs Cell lines
  • Acquisition of Specimens
  • Processing
  • Quantitation and Culturing
  • Propagating Cells For Long Periods
  • Cryopreservation
  • Conclusion
slide4

Cell Lines

  • American Type Culture Collection (ATCC)
  • Collaborators
  • Easy To Grow
  • Option To Cryopreserve For Future
human cells
Human Cells
  • Monocytes
  • T-cells
  • B-cells
  • NK Cells
  • Dendritic Cells
  • Neutrophils
  • Biopsies
human blood
Human Blood
  • Density Gradient Centrifugation
  • Extraction of PBMCs
  • Magnetic Bead Separation
non human cells
Non-Human Cells
  • Payers Patches
  • Spleen
  • Alveolar M
dissociation of cells
Dissociation Of Cells
  • Mechanical Digestion

(Syringe, Frosted Slides)

  • Collagenase D
    • 1 hour @ 37C
  • Filtering Step
  • Suspension into Medium
staining cells
Staining Cells
  • Trypan Blue

Viability Assessment

  • Turks Solution
  • Glacial Acetic Acid and crystal blue
cell counts using hemocytometer
Cell Counts Using Hemocytometer

X

X

D.F

25

104

=0.50x106 cells/mL

media formulations
Media Formulations
  • RPMI 1640
  • Antibiotics
  • FBS/Human AB
  • -ME
  • L-glutamine
  • Filtering Flasks
culturing
Culturing
  • Enhancing Proliferation IL-2, -CD3/ -CD28
  • Limiting Proliferation

FBS Reduction to 2%

CFSE

flow cytometry
Flow Cytometry

0

0

20

4

foxp3-PE

CD8-PerCP

73

31

27

45

foxp3-PE

CD4-FITC

PGE2

viability
Viability

LPS

Medium

9

9

85

85

4

5

LPS+PGE2

PGE2

5

5

91

90

2

4

cryopreservation
Cryopreservation
  • Collect Cells, Spin, Count
  • Resuspend in 90% FBS+10 DMSO
  • DMSO/FBS 4°C
  • Store @–150 °C
contamination issues
Contamination Issues
  • Sterile Incubator
  • Thawing Step Source of Contamination
  • Spray with 70% Ethanol, gloves
  • Sterile Hood
  • Sterile Hands
  • Minimize Exposure to Open Air
conclusions
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